CN121443743A

CN121443743A - Tandem fusion agents and related lipid particles

Info

Publication number: CN121443743A
Application number: CN202480045052.2A
Authority: CN
Inventors: P·A·克鲁特; M·斯托帕托; J·V·沙; L·比亚斯科
Original assignee: Saina Biotechnology Co
Current assignee: Saina Biotechnology Co
Priority date: 2023-05-23
Filing date: 2024-05-22
Publication date: 2026-01-30
Also published as: AU2024274792A1; WO2024243340A1

Abstract

Provided herein are lipid particles comprising at least two attachment proteins derived from paramyxovirus envelope attachment proteins and at least one paramyxovirus fusion (F) protein. Lipid particles, such as lentiviral vectors or lentiviral-like particles, are also provided as viral vectors. Also provided are producer cells and compositions containing such lipid particles, and methods of making and using the lipid particles.

Description

Tandem fusion agents and related lipid particles

Cross Reference to Related Applications

The present application claims priority from U.S. provisional patent application No. 63/468,525, 2023, month 6, and 22, entitled "Tandem Fusogens AND RELATED LIPID PARTICLES (in-line fluxing agent and related lipid particles)", U.S. provisional patent application No. 63/522,722, 2023, month 9, and 20, entitled "Tandem Fusogens AND RELATED LIPID PARTICLES (in-line fluxing agent and related lipid particles)", U.S. provisional patent application No. 63/539,547, 2023, month 10, and 20, entitled "Tandem Fusogens AND RELATED LIPID PARTICLES (in-line fluxing agent and related lipid particles)", each of which is hereby incorporated by reference in its entirety.

Reference to electronic sequence Listing

The present application is submitted in electronic format along with the sequence listing. The sequence listing is provided in a file created by 22 days 5, 2024, under the name 18615_200940. Xml, which is 1,090,341 bytes in size. The information in the sequence listing in electronic format is incorporated by reference in its entirety.

Technical Field

The present disclosure relates to lipid particles comprising at least one attachment protein derived from a paramyxovirus envelope attachment protein and at least one paramyxovirus fusion (F) protein. In some embodiments, the attachment protein is re-targeted, such as by two or more targeting moieties. In some embodiments, the lipid particle is a viral vector, such as a lentiviral vector or a lentiviral-like particle. Also provided are producer cells and compositions containing such lipid particles, and methods of making and using the lipid particles.

Background

Lipid particles (including virus-based particles such as virus-like particles and viral vectors such as lentiviral particles) are commonly used to deliver exogenous agents to cells. For various particles, such as lentiviral vector particles, the host range may be altered by pseudotyping with at least one re-targeting attachment protein that is or comprises two or more targeting moieties. Thus, there is a need to efficiently prepare and produce particles with certain re-targeted pseudotyped envelope proteins to produce target cells of higher titer and with efficient transduction efficiency. The present disclosure addresses this need.

Disclosure of Invention

Provided herein is a lipid particle comprising (a) a re-targeting attachment protein comprising a paramyxovirus envelope attachment protein linked to (i) a first targeting moiety directed against a first target molecule expressed on the surface of a target cell, and (ii) a second targeting moiety directed against a second target molecule expressed on the surface of a target cell, and (b) at least one paramyxovirus fusion (F) protein, and wherein the proteins in (a) and (b) are exposed outside of the lipid bilayer.

In some of any of the provided embodiments, the paramyxovirus attachment protein is a variant paramyxovirus envelope attachment protein comprising one or more mutations to reduce natural tropism relative to a wild-type paramyxovirus envelope attachment protein not comprising said one or more mutations. In some of any of the provided embodiments, the paramyxovirus envelope attachment protein is a first paramyxovirus envelope attachment protein, and the lipid particle further comprises a second paramyxovirus envelope attachment protein, wherein the second paramyxovirus envelope attachment protein is a variant paramyxovirus envelope attachment protein comprising one or more mutations to reduce natural tropism relative to a wild-type paramyxovirus envelope attachment protein not comprising said one or more mutations, wherein the second paramyxovirus envelope attachment protein is exposed outside of the lipid bilayer. In some embodiments of any of the provided embodiments, targeting one or both of the first and second target molecules does not activate or inhibit the target cell, induces a phenotypic change (e.g., maturation and/or differentiation) of the target cell, induces proliferation of the target cell, and/or induces apoptosis of the target cell.

Provided herein is a lipid particle comprising (a) a re-targeting attachment protein comprising a first paramyxovirus envelope attachment protein operably linked to (i) a first targeting moiety directed against a first target molecule expressed on the surface of a target cell, and (ii) a second targeting moiety directed against a second target molecule expressed on the surface of a target cell, (b) a second paramyxovirus envelope attachment protein that is a variant paramyxovirus envelope attachment protein comprising one or more mutations to reduce natural tropism relative to a wild-type paramyxovirus envelope attachment protein not comprising said one or more mutations, and (c) at least one paramyxovirus fusion (F) protein, and wherein the proteins in (a), (b) and (c) are exposed outside of the lipid bilayer. Provided herein is a lipid particle comprising (a) a re-targeting attachment protein comprising a first paramyxovirus envelope attachment protein operably linked to (i) a first targeting moiety directed against a first target molecule expressed on the surface of a target cell, and (ii) a second targeting moiety directed against a second target molecule expressed on the surface of a target cell, (b) a second paramyxovirus envelope attachment protein, which is a variant paramyxovirus envelope attachment protein comprising one or more amino acid substitutions, to reduce natural tropism relative to a wild-type paramyxovirus envelope attachment protein comprising no one or more mutations, and (c) at least one paramyxovirus fusion (F) protein, and wherein the proteins in (a), (b) and (c) are exposed on the outside of the lipid bilayer.

In some embodiments of any of the provided embodiments, the first paramyxovirus envelope attachment protein is a variant paramyxovirus envelope attachment protein. In some of any of the provided embodiments, the variant paramyxovirus envelope attachment protein comprises one or more mutations that reduce natural tropism relative to a wild-type paramyxovirus envelope attachment protein that does not comprise the one or more mutations. In some of any of the provided embodiments, the first paramyxovirus envelope attachment protein and the second paramyxovirus envelope attachment protein are the same. In some of any of the provided embodiments, the second paramyxovirus envelope attachment protein is not linked or fused to a non-viral heterologous moiety that is a cell-specific targeting domain or functional domain. In some embodiments of any of the provided embodiments, neither the first targeting moiety nor the second targeting moiety is selected from the group consisting of a cytokine, a growth factor, a hormone, a neurotransmitter, an apoptotic ligand, and combinations thereof.

In some embodiments of any of the provided embodiments, targeting one or both of the first target molecule and the second target molecule does not modulate or induce a signal in the target cell. In some embodiments of any of the provided embodiments, the first targeting moiety and the second targeting moiety each bind to a cell surface molecule present on the target cell. In some embodiments of any of the provided embodiments, the first targeting moiety binds to a cell surface molecule present on a first target cell and the second targeting moiety binds to a surface molecule present on a second target cell. In some embodiments of any of the provided embodiments, the first target molecule and the second target molecule are different target molecules. In some embodiments of any of the provided embodiments, the first target molecule and the second target molecule are the same target molecule. In some embodiments of any of the provided embodiments, the first targeting moiety and the second targeting moiety bind different epitopes of the same target molecule.

In some embodiments of any of the provided embodiments, the cell surface molecule is a protein, glycan, or lipid. In some embodiments of any of the provided embodiments, the target cell is selected from the group consisting of a tumor-infiltrating lymphocyte, a T cell, a neoplastic or tumor cell, a virus-infected cell, a stem cell, a Central Nervous System (CNS) cell, a Hematopoietic Stem Cell (HSC), and a liver cell. In some of any of the provided embodiments, the target cell is selected from the group consisting of a cd3+ T cell, a cd4+ T cell, a cd8+ T cell, a liver cell, a hematopoietic stem cell, a cd34+ hematopoietic stem cell, a cd105+ hematopoietic stem cell, a cd117+ hematopoietic stem cell, a cd105+ endothelial cell, a B cell, a cd20+ B cell, a cd19+ B cell, a cancer cell, a cd133+ cancer cell, an epcam+ cancer cell, a cd19+ cancer cell, a Her2/neu+ cancer cell, a glua2+ neuron, a glua4+ neuron, a nkg2d+ natural killer cell, a slc1a3+ astrocyte, a slc7a10+ adipocyte, or a cd30+ lung epithelial cell. In some embodiments of any of the provided embodiments, the target cell is a liver cell. In some embodiments of any of the provided embodiments, the cell surface molecule is selected from the group consisting of CD34, CD117, and CD133. In some embodiments of any of the provided embodiments, the target cell is a T cell. In some embodiments of any of the provided embodiments, the cell surface molecule is selected from the group consisting of CD3, CD4, CD7, CD8, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, and ITGA3.

In some of any of the provided embodiments, the second paramyxovirus envelope attachment protein as a variant paramyxovirus envelope attachment protein comprises one or more amino acid substitutions corresponding to the amino acid substitutions set forth in reference SEQ ID No. 1 numbered selected from the group consisting of E501A, W504A, Q a and E533A.

Provided herein is a lipid particle comprising (a) a first re-targeting attachment protein comprising (i) a first paramyxovirus envelope attachment protein, and (ii) a first targeting moiety for CD133, and a second targeting moiety for CD133, and (b) at least one paramyxovirus fusion (F) protein, and the proteins in (a) and (b) are exposed outside of the lipid bilayer.

In some of any of the provided embodiments, (i) the first targeting moiety is an scFv and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 516, 525, 534, 543, and 552, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and/or (ii) the second targeting moiety is an scFv and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 516, 525, 534, 543, and 552, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

In some of any of the provided embodiments, (i) the first targeting moiety comprises (a) a CD133 binding agent comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS 536, 537 and 538, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NOS 540, 541 and 542, respectively, (b) a CD133 binding agent comprising CDR-H1 comprising the amino acid sequences of SEQ ID NOS 545, 546 and 547, respectively, CDR-H2 and CDR-H3, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NO. 549, 550 and 551, respectively, (c) CD133 binders comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO. 518, 519 and 520, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NO. 522, 523 and 524, respectively, (d) CD133 binders comprising a polypeptide comprising polypeptide sequences of SEQ ID NO. 527, respectively, 528 and 529, and CDR-L1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS: 531, 532 and 533, respectively, or (e) a CD133 binding agent comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS: 554, 555 and 556, respectively, and CDR-L1 comprising the amino acid sequences of SEQ ID NOS: 558, 559 and 560, respectively, CDR-L2 and CDR-L3, and/or (ii) a second targeting moiety comprising (a) a CD133 binding agent comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS 536, 537 and 538, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NOS 540, 541 and 542, respectively, (b) a CD133 binding agent comprising CDR-H1 comprising the amino acid sequences of SEQ ID NOS 545, 546 and 547, respectively, CDR-H2 and CDR-H3, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NO. 549, 550 and 551, respectively, (c) CD133 binders comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO. 518, 519 and 520, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NO. 522, 523 and 524, respectively, (d) CD133 binders comprising a polypeptide comprising polypeptide sequences of SEQ ID NO. 527, respectively, 528 and 529, and CDR-L1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS: 531, 532 and 533, respectively, or (e) a CD133 binding agent comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS: 554, 555 and 556, respectively, and CDR-L1 comprising the amino acid sequences of SEQ ID NOS: 558, 559 and 560, respectively, CDR-L2 and CDR-L3.

In some of any of the provided embodiments, (i) the first targeting moiety comprises (a) a CD133 binding agent comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS: 289, 565 and 538, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NOS: 540, 541 and 542, respectively, (b) a CD133 binding agent comprising CDR-H1 comprising the amino acid sequences of SEQ ID NOS: 566, 567 and 547, respectively, CDR-H2 and CDR-H3, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NO. 549, 550 and 551, respectively, (c) CD133 binders comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO. 568, 569 and 520, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NO. 522, 523 and 524, respectively, (d) CD133 binders comprising a polypeptide comprising the amino acid sequences of SEQ ID NO. 570, 570, 571 and 529, and CDR-L1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS: 531, 532 and 533, respectively, or (e) a CD133 binding agent comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS: 572, 573 and 556, respectively, and CDR-L1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS: 558, 559 and 560, respectively, CDR-L2 and CDR-L3, and/or (ii) a second targeting moiety comprising (a) a CD133 binding agent comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS 289, 565 and 538, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NOS 540, 541 and 542, respectively, (b) a CD133 binding agent comprising CDR-H1 comprising the amino acid sequences of SEQ ID NOS 566, 567 and 547, respectively, CDR-H2 and CDR-H3, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NO. 549, 550 and 551, respectively, (c) CD133 binders comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO. 568, 569 and 520, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NO. 522, 523 and 524, respectively, (d) CD133 binders comprising a polypeptide comprising the amino acid sequences of SEQ ID NO. 570, 570, 571 and 529, and CDR-L1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS: 531, 532 and 533, respectively, or (e) a CD133 binding agent comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS: 572, 573 and 556, respectively, and CDR-L1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS: 558, 559 and 560, respectively, CDR-L2 and CDR-L3.

In some of any of the provided embodiments, (i) the first targeting moiety comprises (a) a CD133 binding agent comprising a heavy chain Variable (VH) region comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 535, and a light chain Variable (VL) region comprising or having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 539, An amino acid sequence that is 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, (b) a CD133 binding agent comprising a VH region comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO 544, and a VL region comprising or having at least 90%, 91%, 92% of the amino acid sequence of SEQ ID NO 548, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence of SEQ ID No. 517 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a VL region comprising an amino acid sequence of SEQ ID No. 521 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94% or 99% sequence identity thereto, An amino acid sequence of 95%, 96%, 97%, 98% or 99% sequence identity, (d) a CD133 binding agent comprising a VH region comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 526, and a VL region comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 530, 97%, 98% or 99% sequence identity to an amino acid sequence, or (e) a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 553 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a VL region comprising the amino acid sequence of SEQ ID NO: 557 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and/or (ii) a second targeting moiety comprising (a) a CD133 binding agent comprising a heavy chain Variable (VH) region comprising the amino acid sequence of SEQ ID NO: 535 or an amino acid sequence having at least 90%, a heavy chain Variable (VH) region comprising the amino acid sequence of SEQ ID NO: 535 or a nucleic acid sequence having at least 90% sequence identity thereto, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence of SEQ ID No. 539, and a light chain Variable (VL) region comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence of SEQ ID No. 539, (b) a CD133 binding agent comprising a VH region comprising or having at least 90%, a VH region comprising or having at least 90% amino acid sequence of SEQ ID No. 544, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence of SEQ ID No. 548, and a VL region comprising the amino acid sequence of SEQ ID No. 548 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (c) a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID No. 517 or an amino acid sequence having at least 90%, 91%, 92%, a VH region comprising the amino acid sequence of SEQ ID No. 517, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO. 521, and a VL region comprising the amino acid sequence of SEQ ID NO. 521 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (d) a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO. 526 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94% amino acid sequence identity thereto, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence of SEQ ID NO: 530, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or (e) a CD133 binding agent comprising a VH region comprising an amino acid sequence of SEQ ID NO: 553, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96% sequence identity thereto, 97%, 98% or 99% sequence identity, and a VL region comprising the amino acid sequence of SEQ ID NO: 557 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

In some of any of the provided embodiments, (a) the first targeting moiety comprises a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO 517 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and (b) the second targeting moiety comprises a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO 526 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some of any of the provided embodiments, (a) the first targeting moiety comprises a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID No. 526 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and (b) the second targeting moiety comprises a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID No. 517 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments of any of the provided embodiments, wherein the first targeting moiety and the second targeting moiety bind to different epitopes on CD 133.

In some of any of the provided embodiments, the lipid particle further comprises a second paramyxovirus envelope attachment protein that is a variant paramyxovirus envelope attachment protein comprising one or more amino acid substitutions to reduce natural tropism relative to a wild-type paramyxovirus envelope attachment protein that does not comprise said one or more amino acid substitutions. In some of any of the provided embodiments, the second paramyxovirus envelope attachment protein as a variant paramyxovirus envelope attachment protein comprises one or more amino acid substitutions corresponding to the amino acid substitutions set forth in reference SEQ ID No. 1 numbered selected from the group consisting of E501A, W504A, Q a and E533A.

Provided herein is a lipid particle comprising (a) a first re-targeting attachment protein comprising (i) a first paramyxovirus envelope attachment protein, and (ii) a first targeting moiety for CD117, and a second targeting moiety for CD117, and (b) at least one paramyxovirus fusion (F) protein, and the proteins in (a), (b) are exposed outside of the lipid bilayer.

In some of any of the provided embodiments, (a) the first targeting moiety comprises a CD117 binding agent comprising a VHH single domain antibody comprising CDR-H1, CDR-H2 and CDR-H3 comprised within an amino acid sequence selected from the group consisting of SEQ ID NOS: 512-515, and/or wherein (b) the second targeting moiety comprises a CD117 binding agent comprising a VHH single domain antibody comprising CDR-H1, CDR-H2 and CDR-H3 comprised within an amino acid sequence selected from the group consisting of SEQ ID NOS: 512-515. In some of any of the provided embodiments, (a) the first targeting moiety comprises a VHH single domain antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 512-515 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and/or (b) the second targeting moiety comprises a VHH single domain antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 512-515 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some of any of the provided embodiments, (a) the first targeting moiety comprises a VHH comprising the amino acid sequence set forth in any of SEQ ID NOS: 512-515, and/or (b) the second targeting moiety comprises a VHH comprising the amino acid sequence set forth in any of SEQ ID NOS: 512-515. In some embodiments of any of the provided embodiments, the first targeting moiety and the second targeting moiety bind different epitopes on CD 117.

Provided herein is a lipid particle comprising (a) a first re-targeting attachment protein comprising (i) a first paramyxovirus envelope attachment protein, and (ii) a first targeting moiety for CD8, and a second targeting moiety for CD8, and (b) at least one paramyxovirus fusion (F) protein, and the proteins in (a) and (b) are exposed outside of the lipid bilayer.

In some of any of the provided embodiments, (i) the first targeting moiety is an scFv and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 489, 496, 501 or 508 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and/or (ii) the second targeting moiety is an scFv and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 489, 496, 501 or 508 or an amino acid sequence having at least 90%, an amino acid sequence having at least 90% sequence identity thereto, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some of any of the provided embodiments, (a) the first targeting moiety comprises a CD8 binding agent comprising a VHH single domain antibody comprising CDR-H1, CDR-H2 and CDR-H3 comprised within the amino acid sequence of SEQ ID NO: 377 and/or wherein (b) the second targeting moiety comprises a CD8 binding agent comprising a VHH single domain antibody comprising CDR-H1, CDR-H2 and CDR-H3 comprised within the amino acid sequence of SEQ ID NO: 377. in some of any of the provided embodiments, (a) the first targeting moiety comprises a VHH comprising the amino acid sequence set forth in SEQ ID NO. 377, and/or (b) the second targeting moiety comprises a VHH comprising the amino acid sequence set forth in SEQ ID NO. 377. In some of any of the provided embodiments, the first targeting moiety comprises a VHH comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 377, and/or the second targeting moiety is a scFv and comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% amino acid sequence selected from the group consisting of SEQ ID NO: 501, amino acid sequence of 97%, 98% or 99% sequence identity. In some of any of the provided embodiments, the first targeting moiety is a scFv and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 501 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and the second targeting moiety comprises a VHH comprising the amino acid sequence shown as SEQ ID NO: 377 or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% sequence identity thereto, amino acid sequence of 98% or 99% sequence identity.

In some embodiments of any of the provided embodiments, the first targeting moiety and the second targeting moiety bind different epitopes on CD 8.

In some of any of the provided embodiments, the lipid particle further comprises a second paramyxovirus envelope attachment protein that is a variant paramyxovirus envelope attachment protein comprising one or more mutations, the variant paramyxovirus envelope attachment protein reducing natural tropism relative to a wild-type paramyxovirus envelope attachment protein not comprising said one or more mutations. In some of any of the provided embodiments, the different epitopes are non-overlapping. In some embodiments of any of the provided embodiments, the first targeting moiety and the second targeting moiety bind different epitopes in a non-competitive manner.

In some embodiments of any of the provided embodiments, each of the first targeting moiety and the second targeting moiety is independently selected from the group consisting of an antibody or antigen binding fragment, a DARPin, an aptamer, affimer, an affibody, a desmin, avimer, a monomer, ANTICALIN, FYNOMER, and a targeting peptide. In some embodiments of any of the provided embodiments, the first targeting moiety and the second targeting moiety are independently selected from the group consisting of a single domain antibody or a single chain variable fragment (scFv).

Provided herein is a lipid particle comprising a) a re-targeting attachment protein comprising (i) a first paramyxovirus envelope attachment protein, and (ii) a first targeting moiety and a second targeting moiety for CD133, (b) a second paramyxovirus envelope attachment protein that is a variant paramyxovirus envelope attachment protein comprising one or more mutations, which reduces natural tropism relative to a wild-type paramyxovirus envelope attachment protein not comprising said one or more mutations, and (c) at least one paramyxovirus fusion (F) protein, and wherein the proteins in (a), (b) and (c) are exposed outside the lipid bilayer.

Provided herein is a lipid particle comprising (a) a re-targeting attachment protein comprising (i) a first paramyxovirus envelope attachment protein, and (ii) a first targeting moiety and a second targeting moiety for CD117, (b) a second paramyxovirus envelope attachment protein that is a variant paramyxovirus envelope attachment protein comprising one or more mutations, which variant paramyxovirus envelope attachment protein reduces natural tropism relative to a wild-type paramyxovirus envelope attachment protein not comprising said one or more mutations, and (c) at least one paramyxovirus fusion (F) protein, and wherein the proteins in (a), (b) and (c) are exposed outside the lipid bilayer.

Provided herein is a lipid particle comprising (a) a re-targeting attachment protein comprising (i) a first paramyxovirus envelope attachment protein, and (ii) a first targeting moiety and a second targeting moiety for CD8, (b) a second paramyxovirus envelope attachment protein that is a variant paramyxovirus envelope attachment protein comprising one or more mutations, which variant paramyxovirus envelope attachment protein reduces natural tropism relative to a wild-type paramyxovirus envelope attachment protein not comprising said one or more mutations, and (c) at least one paramyxovirus fusion (F) protein, and wherein the proteins in (a), (b) and (c) are exposed outside the lipid bilayer.

In some embodiments of any of the provided embodiments, the first paramyxovirus envelope attachment protein is a variant paramyxovirus envelope attachment protein. In some of any of the provided embodiments, the variant paramyxovirus envelope attachment protein comprises one or more mutations that reduce natural tropism relative to a wild-type paramyxovirus envelope attachment protein that does not comprise the one or more mutations. In some of any of the provided embodiments, the second paramyxovirus envelope attachment protein is a variant paramyxovirus envelope attachment protein. In some of any of the provided embodiments, the variant paramyxovirus envelope attachment protein comprises one or more mutations that reduce natural tropism relative to a wild-type paramyxovirus envelope attachment protein that does not comprise the one or more mutations. In some embodiments of any of the provided embodiments, the first targeting moiety is selected from the group consisting of a single domain antibody or a single chain variable fragment (scFv). In some embodiments of any of the provided embodiments, the second targeting moiety is selected from the group consisting of a single domain antibody or a single chain variable fragment (scFv). In some embodiments of any of the provided embodiments, the single domain antibody is a VHH.

In some embodiments of any of the provided embodiments, the first variant paramyxovirus envelope attachment protein and the second variant paramyxovirus envelope attachment protein are the same. In some of any of the provided embodiments, the first paramyxovirus envelope attachment protein and the second paramyxovirus envelope attachment protein are different. In some of any of the provided embodiments, the first paramyxovirus envelope attachment protein is an envelope attachment protein from a nipah virus, a hendra virus, or a measles virus, or a variant of any of the foregoing, or a biologically active portion thereof. In some of any of the provided embodiments, the first paramyxovirus envelope attachment protein is a wild-type paramyxovirus G protein, H protein or HN protein, or a variant of any of the foregoing, or a biologically active portion thereof. In some of any of the provided embodiments, the first paramyxovirus envelope attachment protein is a wild-type nipah virus G (NiV-G) protein, or is a variant or biologically active portion of NiV-G.

In some of any of the provided embodiments, the first paramyxovirus envelope attachment protein is a variant NiV-G, which is a variant or biologically active portion of wild-type NiV-G. In some of any of the provided embodiments, the second paramyxovirus envelope attachment protein is an envelope attachment protein from a nipah virus, a hendra virus, or a measles virus, or is a variant or biologically active portion of any of the foregoing. In some of any of the provided embodiments, the second paramyxovirus envelope attachment protein is a wild-type paramyxovirus G protein, H protein or HN protein, or a variant of any of the foregoing, or a biologically active portion thereof. In some of any of the provided embodiments, the second paramyxovirus envelope attachment protein is a wild-type nipah virus G (NiV-G) protein, or is a variant or biologically active portion of NiV-G. In some of any of the provided embodiments, the second paramyxovirus envelope attachment protein is a variant NiV-G, which is a variant or biologically active portion of wild-type NiV-G. In some of any of the provided embodiments, the second paramyxovirus envelope attachment protein is a variant paramyxovirus envelope glycoprotein from nipah virus, hendra virus, or measles virus, or a biologically active portion thereof. In some of any of the provided embodiments, the second paramyxovirus envelope attachment protein is a variant of a wild-type paramyxovirus G protein, H protein or HN protein or a biologically active portion thereof.

In some embodiments of any of the provided embodiments, the variant is a variant NiV-G that is a variant of a wild-type nipah virus G (NiV-G) protein or a biologically active portion thereof. In some of any of the provided embodiments, the variant NiV-G is truncated by up to 40 consecutive amino acids at or near the N-terminus of the wild-type NiV-G shown in SEQ ID NO. 1. In some of any of the provided embodiments, the variant NiV-G has a truncation of amino acids 2-34 of the wild-type NiV-G shown in SEQ ID NO. 1. In some of any of the provided embodiments, the variant NiV-G exhibits reduced binding to ephrin B2 or ephrin B3. In some of any of the provided embodiments, the variant NiV-G comprises one or more amino acid substitutions corresponding to amino acid substitutions shown with reference to SEQ ID NO. 1 numbered selected from the group consisting of E501A, W504A, Q A and E533A. In some of any of the provided embodiments, referring to the numbering shown in SEQ ID NO. 1, variant NiV-G comprises amino acid substitutions E501A, W504A, Q A and E533A. In some of any of the provided embodiments, the variant NiV-G has the amino acid sequence shown as SEQ ID NO:228 or an amino acid sequence having equal to or about 80%, at least equal to or about 81%, at least equal to or about 82%, at least equal to or about 83%, equal to or about 84%, at least equal to or about 85%, at least equal to or about 86%, or at least equal to or about 87%, at least equal to or about 88%, or at least equal to or about 89%, at least equal to or about 90%, at least equal to or about 91%, at least equal to or about 92%, at least equal to or about 93%, at least equal to or about 94%, at least equal to or about 95%, equal to or about 96%, at least equal to or about 97%, at least equal to or about 98%, or at least equal to or about 99% sequence identity to SEQ ID NO: 228. In some of any of the provided embodiments, the variant NiV-G has the amino acid sequence shown in SEQ ID NO. 228. In some of any of the provided embodiments, the at least one paramyxovirus fusion (F) protein is an F protein from henipav, or a biologically active portion thereof or a variant thereof.

In some embodiments of any of the provided embodiments, the henipa virus is hendra virus. In some embodiments of any of the provided embodiments, the henipa virus is a nipah virus. In some embodiments of any of the provided embodiments, the paramyxovirus F protein is a wild-type NiV-F protein or a variant or biologically active portion thereof. In some of any of the provided embodiments, the paramyxovirus F protein is a variant NiV-F, which is a variant or biologically active portion of a wild-type NiV-F protein. In some of any of the provided embodiments, the variant NiV-F is truncated by up to 22 consecutive amino acids at the C-terminus of the wild-type NiV-F shown in SEQ ID NO. 235, optionally excluding the initiating methionine. In some of any of the provided embodiments, the variant NiV-F protein is a truncated NiV-F lacking amino acids 525-546 of SEQ ID NO. 235. In some of any of the provided embodiments, the variant NiV-F has the amino acid sequence shown as SEQ ID NO:227 or an amino acid sequence having equal to or about 80%, at least equal to or about 81%, at least equal to or about 82%, at least equal to or about 83%, equal to or about 84%, at least equal to or about 85%, at least equal to or about 86%, or at least equal to or about 87%, at least equal to or about 88%, or at least equal to or about 89%, at least equal to or about 90%, at least equal to or about 91%, at least equal to or about 92%, at least equal to or about 93%, at least equal to or about 94%, at least equal to or about 95%, equal to or about 96%, at least equal to or about 97%, at least equal to or about 98%, or at least equal to or about 99% sequence identity to SEQ ID NO: 227. In some of any of the provided embodiments, the variant NiV-F has the amino acid sequence shown in SEQ ID NO: 227.

In some of any of the provided embodiments, the paramyxovirus F protein is an F0 precursor, or a proteolytically cleaved form thereof comprising F1 and F2 subunits. In some embodiments of any of the provided embodiments, the proteolytic cleavage form is a cathepsin L cleavage product. In some embodiments of any of the provided embodiments, the paramyxovirus envelope attachment protein and the first targeting moiety and the second targeting moiety are linked via one or more linkers.

In some embodiments of any of the provided embodiments, the one or more linkers are one or more peptide linkers. In some of any of the provided embodiments, the re-targeting attachment protein comprises, in order, a paramyxovirus attachment protein-first linker-first targeting moiety-second linker-second targeting moiety.

In some embodiments of any of the provided embodiments, the first linker and the second linker are independently peptide linkers. In some of any of the provided embodiments, the first linker and the second linker are the same. In some of any of the provided embodiments, the first linker and the second linker are different. In some of any of the provided embodiments, the peptide linker is 2 to 65 amino acids in length. In some of any of the provided embodiments, the peptide linker is a flexible linker comprising GS, GGS, GGGGS, GGGGGS or a combination thereof. In some of any of the provided embodiments, the peptide linker is selected from (GGS) n, wherein n is from 1 to 10, (GGGGS) n, wherein n is from 1 to 10, or (GGGGGS) n, wherein n is from 1 to 6. In some of any of the provided embodiments, the peptide linker is selected from the group consisting of SEQ ID NOs 589-592.

In some embodiments of any of the provided embodiments, the lipid particle further comprises one or more additional paramyxovirus envelope attachment glycoproteins embedded in the lipid bilayer. In some embodiments of any of the provided embodiments, the one or more additional paramyxovirus envelope attachment glycoproteins are re-targeting attachment proteins comprising a paramyxovirus envelope attachment protein and an additional targeting moiety. In some of any of the provided embodiments, the at least one paramyxovirus fusion (F) protein exhibits fusion activity with a target cell when the at least one paramyxovirus envelope attachment protein binds to the target molecule on the target cell.

In some embodiments of any of the provided embodiments, the lipid particle comprises viral nucleic acid. In some of any of the provided embodiments, the viral nucleic acid comprises one or more (e.g., all) of the following nucleic acid sequences, 5 'LTR (e.g., comprising U5 and lacking a functional U3 domain), psi packaging element (Psi), central polypurine tract (cPPT)/Central Termination Sequence (CTS) (e.g., DNA flap), poly-a tail sequence, post transcriptional regulatory element (e.g., WPRE), rev Response Element (RRE), and 3' LTR (e.g., comprising U5 and lacking a functional U3).

In some embodiments of any of the provided embodiments, the lipid particle is a viral vector. In some embodiments of any of the provided embodiments, it is a retroviral vector. In some embodiments of any of the provided embodiments, it is a lentiviral vector. In some embodiments of any of the provided embodiments, the lipid particle is free of viral genomic DNA. In some embodiments of any of the provided embodiments, the lipid particle is a virus-like particle. In some embodiments of any of the provided embodiments, the lipid particle is a retroviral-like particle. In some embodiments of any of the provided embodiments, the lipid particle is a virus-like particle. In some embodiments of any of the provided embodiments, the lipid particle is a lentiviral-like particle.

In some of any of the provided embodiments, the lipid particle is produced as a formulation with increased titer compared to a reference lipid particle formulation that is similarly produced but has only the first re-targeted attachment protein. In some of any of the provided embodiments, the lipid particle is produced as a formulation having increased titer as compared to a reference lipid particle formulation that is similarly produced but does not contain a second paramyxovirus envelope attachment protein, which is a variant paramyxovirus envelope attachment protein comprising one or more substitutions, to reduce natural tropism relative to a wild-type paramyxovirus envelope attachment protein not comprising the one or more substitutions. In some of any of the provided embodiments, the second paramyxovirus envelope attachment protein as a variant paramyxovirus envelope attachment protein comprises one or more amino acid substitutions corresponding to the amino acid substitutions set forth in reference SEQ ID No. 1 numbered selected from the group consisting of E501A, W504A, Q a and E533A. In some of any of the provided embodiments, the lipid particles are produced in suspension culture as a formulation having increased titer as compared to a reference lipid particle formulation that is similarly produced but has only the first re-targeted attachment protein.

In some embodiments of any of the provided embodiments, the titer increase is equal to or greater than 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more.

In some embodiments of any of the provided embodiments, the lipid particle further comprises an exogenous agent for delivery to the target cell. In some embodiments of any of the provided embodiments, the exogenous agent is present in the lumen. In some embodiments of any of the provided embodiments, the exogenous agent is a protein or a nucleic acid, optionally wherein the nucleic acid is DNA or RNA. In some embodiments of any of the provided embodiments, the exogenous agent is a nucleic acid encoding a cargo for delivery to the target cell. In some embodiments of any of the provided embodiments, the exogenous agent is or encodes a therapeutic agent, a diagnostic agent, or a genome modification enzyme. In some of any of the provided embodiments, the exogenous agent encodes a membrane protein, optionally wherein the membrane protein is an antigen receptor for targeting a cell expressed by or associated with a disease or disorder. In some of any of the provided embodiments, the membrane protein is a Chimeric Antigen Receptor (CAR). In some embodiments of any of the provided embodiments, the exogenous agent is a nucleic acid comprising a payload gene for correcting a genetic defect (optionally a genetic defect in a target cell), optionally wherein the genetic defect is associated with a liver cell or a liver cell.

In some of any of the provided embodiments, the binding of the paramyxovirus envelope attachment protein or biologically active portion thereof to the target molecule expressed on the surface of the target cell mediates fusion of the particle with the target cell and delivery of the exogenous agent to the target cell. In some embodiments of any of the provided embodiments, the exogenous agent is delivered to equal to or greater than 10%, 20%, 30%, 40%, 50%, 60% of the target cells. In some of any of the provided embodiments, delivery of the exogenous agent to the target cell is increased as compared to a reference particle formulation that is similarly produced but has only the first re-targeted attachment protein. In some embodiments of any of the provided embodiments, the increase in delivery to the target cell is equal to or greater than 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more.

Provided herein is a producer cell comprising (a) a nucleic acid encoding a re-targeting attachment protein comprising a paramyxovirus envelope attachment protein linked to (i) a first targeting moiety directed against a first target molecule expressed on the surface of a target cell and (ii) a second targeting moiety directed against a second target molecule expressed on the surface of a target cell, and (b) a nucleic acid encoding at least one paramyxovirus fusion (F) protein.

In some of any of the provided embodiments, the producer cell further comprises a nucleic acid encoding a second paramyxovirus envelope attachment protein that is a variant paramyxovirus envelope attachment protein comprising one or more amino acid substitutions to reduce natural tropism relative to a wild-type paramyxovirus envelope attachment protein that does not comprise said one or more amino acid substitutions.

In some embodiments of any of the provided embodiments, the cell further comprises a viral nucleic acid. In some embodiments of any of the provided embodiments, the viral nucleic acid is a lentiviral nucleic acid. In some embodiments of any of the provided embodiments, the cell is a mammalian cell. In some of any of the provided embodiments, the production cell is selected from the group consisting of a CHO cell, a BHK cell, a MDCK cell, a C3H 10T1/2 cell, a FLY cell, a Psi-2 cell, a BOSC 23 cell, a PA317 cell, a WEHI cell, a COS cell, a BSC 1 cell, a BSC 40 cell, a BMT 10 cell, a VERO cell, a W138 cell, a MRC5 cell, an A549 cell, a HT1080 cell, a 293T cell, a B-50 cell, a 3T3 cell, a NIH3T3 cell, a HepG2 cell, a Saos-2 cell, a Huh7 cell, a HeLa cell, a W163 cell, a 211 cell, and a 211A cell. In some embodiments of any of the provided embodiments, the producer cell comprises a 293T cell. In some embodiments of any of the provided embodiments, the viral nucleic acid lacks one or more genes involved in viral replication. In some embodiments of any of the provided embodiments, the viral nucleic acid comprises a nucleic acid encoding a viral packaging protein selected from one or more of Gag, pol, rev and Tat. In some of any of the provided embodiments, the viral nucleic acid comprises one or more (e.g., all) of the following nucleic acid sequences, 5 'LTR (e.g., comprising U5 and lacking a functional U3 domain), psi packaging element (Psi), central polypurine tract (cPPT)/Central Termination Sequence (CTS) (e.g., DNA flap), poly A tail sequence, post transcriptional regulatory element (e.g., WPRE), rev Response Element (RRE), and 3' LTR (e.g., comprising U5 and lacking a functional U3).

Provided herein is a method of preparing a lipid particle comprising a) providing a producer cell, such as any provided producer cell, b) culturing the cell under conditions that allow production of the lipid particle, and c) separating, enriching or purifying the lipid particle from the cell, thereby preparing the lipid particle. In some embodiments of any of the provided embodiments, the lipid particle is a pseudotyped lentiviral vector.

Provided herein is a lipid particle produced by any of the provided methods. Also provided herein is a composition comprising any provided lipid particle and/or a plurality of any provided lipid particles. Provided herein is a method of transducing a cell, the method comprising contacting the cell with any provided lipid particle and/or composition. Provided herein is a method of delivering an exogenous agent into a target cell, the method comprising contacting the cell with any provided lipid particle and/or composition with the target cell.

In some embodiments of any of the provided embodiments, the contacting is in vitro or ex vivo. In some embodiments of any of the provided embodiments, the contacting is in the subject.

Provided herein is a method of delivering an exogenous agent to a cell of a subject, the method comprising administering any provided lipid particle and/or composition to the subject. In some embodiments of any of the provided embodiments, the exogenous agent is or encodes a therapeutic agent for treating a disease or disorder in a subject.

Provided herein is a method of treatment comprising administering any provided lipid particle and/or composition to a subject. In some embodiments of any of the provided embodiments, the exogenous agent is or encodes a membrane protein, optionally a chimeric antigen receptor, for targeting an antigen associated with a disease or disorder in a subject. In some embodiments of any of the provided embodiments, the exogenous agent is used in gene therapy to correct a genetic defect or substitution defect or a deleted gene in the subject. In some embodiments of any of the provided embodiments, the subject is a human subject.

In some of any of the provided embodiments, the method delivers particles (e.g., lentiviral particles), including particles comprising an exogenous agent, to Hematopoietic Stem Cells (HSCs). In some of any such provided embodiments, the method further comprises administering to the subject one or more agents that stimulate mobilization of bone marrow cells from bone marrow to peripheral blood. In some of any of the provided embodiments, the subject has previously been administered one or more agents that stimulate mobilization of bone marrow cells from bone marrow to peripheral blood. In some of any of the provided embodiments, the one or more agents that stimulate mobilization are selected from the group consisting of Stem Cell Factor (SCF), small molecule VLA-4 inhibitor BI05192, BOP (N- (benzenesulfonyl) -L-prolyl-L-0- (1-pyrrolidinylcarbonyl) tyrosine), heparin, granulocyte colony-stimulating factor (G-CSF), MGTA-145, and plexafu (AMD 3100). In some of any of the provided embodiments, the one or more agents that stimulate mobilization comprises G-CSF. In some embodiments of any of the provided embodiments, the one or more agents that stimulate mobilization comprises pleshafu.

Drawings

FIG. 1A depicts viral vector titers of an exemplary tandem CD117 VHH binding agent and a single CD117 VHH binding agent. Viral titers of vectors pseudotyped with CD 117-targeted, tandem fusion agents and NiV-F proteins with and without additional blind fusion agents (±gm) are shown for CD117 overexpressing cells (fig. 1B) and cd34+ cells (fig. 1C). FIG. 1D shows primary cell transduction of an exemplary tandem binding agent.

Figure 2 depicts transduction of cd34+ primary cells via two exemplary CD133 tandem binding agents.

Fig. 3A and 3B illustrate transduction of the fusion agent shown. Fig. 3A depicts transduction of SupT 1T cells via two exemplary CD8 tandem binding agents. Figure 3B depicts transduction of pan T cells comparing a single binding agent to a tandem binding agent.

Fig. 4A shows an exemplary protocol for assessing transduction efficiency in long-term humanized mice. Fig. 4B shows the percent transduction in bone marrow compartments using tandem fusion agents.

The model study design for non-human primates is shown in fig. 5.

Detailed Description

Provided herein is a lipid particle comprising a fusion agent protein comprising two or more targeting moieties (also referred to as "tandem fusion agents") on the outer surface of the lipid particle. In some embodiments, the fusion agent is entrapped in the lipid bilayer of the lipid particle. In some embodiments, the fusion agent contains a viral envelope attachment protein linked to two or more targeting moieties. In some embodiments, at least two targeting moieties are directed against one or more target molecules on the cell surface, thereby re-targeting the attachment protein to the one or more target molecules. In some embodiments, the viral envelope attachment protein contains a first targeting moiety directed against a first target molecule and a second targeting moiety directed against a second target molecule. In some embodiments, the first target molecule and the second target molecule are the same. In some embodiments, the first target molecule and the second target molecule are different. In some embodiments, the attachment protein is fused to two or more targeting moieties, wherein one or more peptide linkers connect the attachment protein and the two or more targeting moieties. In some embodiments, the fusion agent facilitates fusion of the lipid particle with plasma cell membranes of one or more target cells.

In some embodiments, provided herein is a lipid particle, wherein the re-targeting attachment protein comprises at least two targeting moieties that are directed against one or more target molecules on the surface of the cell, and the lipid particle further comprises a second attachment protein that is a fusion protein (F protein). In some embodiments, the fusion agent consists of a viral attachment protein comprising two or more targeting moieties and a fusion protein (F protein), which together are the fusion agent. In some embodiments, the fusion agent may be derived from a paramyxovirus. In some embodiments, the viral attachment protein is a paramyxovirus envelope attachment protein, such as G, H or HN protein, and the F protein is at least one paramyxovirus fusion (F) protein. In some embodiments, the attachment protein is a variant attachment protein that contains one or more mutations (e.g., amino acid substitutions) to reduce or eliminate natural tropism relative to a wild-type viral envelope attachment protein (e.g., paramyxovirus envelope attachment protein) that does not contain the one or more mutations.

In some aspects, lipid particles provided herein comprise (a) a re-targeting attachment protein having (i) a first targeting moiety directed to a first target molecule expressed on the surface of a target cell, and (ii) a second targeting moiety directed to a second target molecule expressed on the surface of a target cell. In some embodiments, the lipid particle, such as a viral vector or virus-like particle, contains one or more attachment proteins (e.g., fusion agents). In some embodiments, the lipid particle, e.g., viral vector or virus-like particle, contains an exogenous or overexpressed attachment protein (e.g., a fusion agent). In some embodiments, the attachment protein is disposed in a lipid bilayer. In some embodiments, the attachment protein (e.g., a fusion agent) facilitates fusion of the lipid particle to the membrane. In some embodiments, the membrane is a plasma cell membrane of the target cell. In some embodiments, lipid particles, such as viral or non-viral vectors, comprising an attachment protein (e.g., a fusion agent) are integrated into the membrane into the lipid bilayer of the target cell. In some embodiments, the attachment protein (e.g., fusion agent) results in a mixture between the lipids in the lipid particle and the lipids in the target cell. In some embodiments, the attachment protein (e.g., fusion agent) results in the formation of one or more pores between the interior of the non-cellular particles and the cytosol of the target cell. In some embodiments, the attachment protein (e.g., fusion agent) may include a non-mammalian protein, such as a viral protein. In some embodiments, the viral fusion agent is a class I viral membrane fusion protein, a class II viral membrane protein, a class III viral membrane fusion protein, a viral membrane glycoprotein, or other viral fusion protein, or a homolog, fragment, variant, or protein fusion comprising one or more proteins or fragments thereof.

In some aspects, lipid particles provided herein comprise (a) a re-targeting attachment protein comprising a paramyxovirus envelope attachment protein operably fused in series with (i) a first targeting moiety directed against a first target molecule expressed on the surface of a target cell and (ii) a second targeting moiety directed against a second target molecule expressed on the surface of a target cell, and (b) a paramyxovirus fusion protein. In some embodiments, the re-targeting attachment protein comprises a variant paramyxovirus envelope attachment protein comprising one or more mutations to reduce natural tropism relative to a wild-type paramyxovirus envelope attachment protein that does not comprise the one or more mutations.

In some of any of the embodiments, the lipid particle further comprises a second viral envelope attachment protein embedded in the lipid bilayer of the lipid particle, the second viral envelope attachment protein not being attached to the targeting moiety. In some embodiments, the second viral envelope attachment protein is a paramyxovirus envelope attachment protein, such as G, H or HN protein. In some embodiments, the first viral envelope attachment protein and the second viral envelope attachment protein are the same. In certain embodiments, the second viral envelope adhesion protein is a variant paramyxovirus envelope adhesion protein comprising one or more mutations to reduce natural tropism relative to a wild-type paramyxovirus envelope adhesion protein not comprising said one or more mutations. And a second coating attachment protein. In such embodiments, the second viral envelope attachment protein is not linked or fused to a non-viral heterologous moiety, such as a targeting moiety.

In some aspects, lipid particles provided herein comprise (a) a re-targeting attachment protein comprising a first paramyxovirus envelope attachment protein operably fused in tandem with (i) a first targeting moiety directed against a first target molecule expressed on the surface of a target cell and (ii) a second targeting moiety directed against a second target molecule expressed on the surface of the target cell, (b) a second paramyxovirus envelope attachment protein, and (c) a paramyxovirus fusion protein. In some embodiments, the first paramyxovirus envelope attachment protein and the second paramyxovirus envelope attachment protein are the same. In some embodiments, each of the first and second paramyxovirus envelope attachment proteins is a variant paramyxovirus envelope attachment protein comprising one or more mutations to reduce natural tropism relative to a wild-type paramyxovirus envelope attachment protein that does not comprise the one or more mutations. In such embodiments, the second paramyxovirus envelope attachment protein is not linked or fused to a non-viral heterologous moiety, such as a targeting moiety.

In some embodiments, at least two targeting moieties (e.g., a first targeting moiety and a second targeting moiety) bind to a target molecule on the same target cell. In some embodiments, at least two targeting moieties (e.g., a first targeting moiety and a second targeting moiety) are identical. In some embodiments, at least two targeting moieties (e.g., a first targeting moiety and a second targeting moiety) are different. In some embodiments, at least two targeting moieties (e.g., a first targeting moiety and a second targeting moiety) bind different epitopes of the same target molecule.

In some embodiments, at least two targeting moieties (e.g., a first targeting moiety and a second targeting moiety) bind target molecules on the surface of different target cells.

Exemplary target cells include cells present in a blood sample from a subject. In some embodiments, the cells comprise a leukocyte component. In some embodiments, the target cells comprise polymorphonuclear cells (also known as PMN, PML, PMNL or granulocytes), and in some embodiments, the target cells comprise lymphocytes, monocytes, macrophages, dendritic cells, natural killer cells, T cells (e.g., CD4 or CD 8T cells, including cytotoxic T lymphocytes), or B cells. In some embodiments, the target cells comprise Hematopoietic Stem Cells (HSCs).

In some embodiments, the re-targeting attachment protein comprises a paramyxovirus envelope attachment protein and (i) a first targeting moiety directed against a first target molecule expressed on the surface of a T cell, and (ii) a second targeting moiety directed against a second target molecule expressed on the surface of a T cell. In some embodiments, the target molecule on a T cell is CD3, CD4, or CD8. In some embodiments, the first target molecule and the second target molecule are the same. In some embodiments, the first target molecule and the second target molecule are CD3. In some embodiments, the first target molecule and the second target molecule are CD4. In some embodiments, the first target molecule and the second target molecule are CD8. In some embodiments, the first targeting moiety and the second targeting moiety are the same. In some embodiments, the first targeting moiety and the second targeting moiety are different. In some embodiments, the first targeting moiety and the second targeting moiety bind to different epitopes of the same target molecule.

In some embodiments, the re-targeting attachment protein comprises a paramyxovirus envelope attachment protein and (i) a first targeting moiety directed against a first target molecule expressed on the surface of a T cell, and (ii) a second targeting moiety directed against a second target molecule expressed on the surface of a Hematopoietic Stem Cell (HSC). In some embodiments, the target molecule on the HSC is CD117 or CD133. In some embodiments, the first target molecule and the second target molecule are the same. In some embodiments, the first target molecule and the second target molecule are CD117. In some embodiments, the first target molecule and the second target molecule are CD133. In some embodiments, the first targeting moiety and the second targeting moiety are the same. In some embodiments, the first targeting moiety and the second targeting moiety are different. In some embodiments, the first targeting moiety and the second targeting moiety bind to different epitopes of the same target molecule.

In some embodiments, the targeting moiety (such as each of the first targeting moiety and the second targeting moiety) is independently selected from the group consisting of an antibody or antigen binding fragment, an engineered binding domain, a nanobody, DARPin, an aptamer, affimer, an affibody, a desmin, avimer, a monomer, ANTICALIN, FYNOMER, and a targeting peptide.

In some embodiments, any of the provided lipid particles further comprise at least one paramyxovirus fusion (F) protein or biologically active portion thereof embedded in the lipid bilayer. In some embodiments, the F protein is from paramyxovirus, henipav (e.g., hendra virus (HeV), nipah virus (NiV), cedar henipav virus (CedV), kesmash Virus (KV), mexican virus (MojV), or langa virus), or a biologically active portion thereof, or a variant or mutant thereof. In a particular embodiment, the F protein is from Nipah (NiV) virus.

In naturally occurring paramyxoviruses, fusion (F) and attachment (G, H or HN) glycoproteins mediate cell entry by paramyxoviruses such as nipah virus. In some embodiments, a combination of an F protein (such as a NiV-F protein) and a variant NiV-G protein as provided herein is capable of mediating cellular entry of provided lipid particles (e.g., lentiviral vectors).

F proteins (such as Nipah virus F protein, also known as NiV-F) are class I fusion proteins that share structural and functional features with fusion proteins of many families (e.g., HIV-1 gp41 or influenza virus hemagglutinin [ HA ]), such as extracellular domains with hydrophobic fusion peptides and two seven residue repeat regions (White JM et al 2008. Crit Rev Biochem Mol Biol 43:189-219). The F protein is synthesized as an inactive precursor F ₀ and activated by proteolytic cleavage into two disulfide-linked subunits F ₁ and F ₂ (Moll M. Et al, 2004. J. Virol. 78 (18): 9705-9712).

In some embodiments, the lipid particle comprises a heavy-targeting paramyxovirus envelope attachment protein comprising a G protein. The G protein is an attachment protein of a henipav virus (e.g., nipah virus or hendra virus), which is a type II transmembrane glycoprotein containing an N-terminal cytoplasmic tail, a transmembrane domain, an extracellular stem, and a globular head (Liu, Q. Et al, 2015. Journal of Virology, 89 (3): 1838-1850). NiV-G, an attachment protein of NiPavirus, recognizes the receptors hepatin B2 and hepatin B3. Binding of the receptor to NiV-G triggers a series of conformational changes that ultimately lead to triggering of NiV-F, which exposes the NiV-F fusion peptide, allowing for another series of conformational changes that lead to virus-cell membrane fusion (Stone J.A. et al 2016. J Virol.90 (23): 10762-10773). Ephrin B2 was previously identified as the primary NiV receptor (Negrete et al, 2005), and ephrin B3 was identified as the surrogate receptor (Negrete et al, 2006). In fact, wild-type NiV-G has a high affinity for ephrins B2 and B3, with an affinity binding constant (Kd) in the picomolar range (Negrete et al, 2006) (kd=0.06 nM and 0.58 nM for cell surface expressed ephrins B2 and B3, respectively). In some embodiments, the G protein is from paramyxovirus, henipav (e.g., hendra virus (HeV), nipah virus (NiV), cedar henipav (CedV), kesmash Virus (KV), mexican virus (MojV), or langa virus), or a biologically active portion thereof, or a variant or mutant thereof. In particular embodiments, the G protein is from Nipah (NiV) virus.

In some embodiments, the lipid particle may be a virus-like particle, a virus, or a viral vector (such as a lentiviral vector).

In some embodiments, a heavy targeting paramyxovirus envelope attachment protein comprising a G protein may be further linked to at least two targeting moieties as heavy targeting attachment proteins to facilitate specific targeting of the lipid particle to one or more target molecules for fusion with one or more desired target cells. Thus, in the embodiments provided, envelope adhesion proteins comprising G proteins can be re-targeted as re-targeted adhesion proteins to any desired cell type for specific targeting of lipid particles (e.g., lentiviral vectors), and in some cases, specific delivery of the transgene or heterologous protein contained therein to the target cell.

Thus, also provided herein are lipid particles comprising a lipid bilayer surrounding a lumen or cavity and a paramyxovirus envelope attachment protein that is re-targeted by two or more targeting moieties comprising an antigen binding domain or biologically active portion thereof, such as a single domain antibody (sdAb) variable domain, wherein the re-targeted G glycoprotein (e.g., tandem G protein) is embedded in the lipid bilayer of the lipid particle. In certain embodiments, the binding domain is an antibody that has the ability to bind (such as specifically bind) a desired target molecule. Exemplary binding domains are described in section II.

Transduction efficiency of lipid particles can be improved by engineering mutations in one or both of NiV-F and NiV-G. Several such mutations have been previously described (see, e.g., lee et al, 2011, trends in Microbiology). This can be used, for example, to maintain the specificity and picomolar affinity of NiV-G for ephrin B2 and/or B3. In addition, mutations in NiV-G that completely abrogate the binding of ephrin B2 and B3, but do not affect the association of the NiV-G with NiV-F, have been identified (Aguilar et al, J Biol chem 2009;284 (3): 1628-1635.; weise et al, J Virol 2010;84 (15): 7634-764; negrete et al, J Virol 2007;81 (19): 10804-10814; negrete et al, PLoS Pathe 2006; guillaume et al, J. Virol 2006, 80 (15) 7546-7554) in some cases, can be achieved by fusing a binding molecule to a G protein (e.g., niV-G, including a mutant having the ability to abrogate the binding of ephrin B2 and ephrin B3), which does allow for altered G protein to be targeted by the addition of a binding molecule to a different cell surface molecule that is not the desired type of ephrin B2, thus allowing for the binding of a variant of the human liver B533 to be the mutant, and thus the mutant type of the human B35-B35 can be reduced or more than one of the mutant types shown in the mutant, 35-B35 and 35-B-35 can be provided.

In some embodiments, the lipid particle (e.g., viral vector) is pseudotyped with a re-targeting attachment protein comprising a re-targeting paramyxovirus attachment protein as described herein, such as a tandem NiV-G protein.

The henipav F protein from various species has been reported to exhibit compatibility with G proteins from other species to trigger fusion (Brandel-TRETHEWAY et al, journal of virology 2019.93 (13): e 00577-19). In some aspects of the provided lipid particles (e.g., lentiviral vectors), the F protein is heterologous to the G protein, i.e., the F and G proteins or biologically active portions are from different henipav virus species. For example, the G protein is from Hendela virus and the F protein is the described NiV-F. In other aspects, the F and/or G proteins may be re-targeted F and/or G proteins containing regions of F and/or G proteins from different henipa virus species. In some embodiments, converting the region of amino acid residues of the F protein from one species of henipa virus to another may result in a fusion with the G protein of the species comprising the amino acid insertion. (Brandel-TRETHEWAY et al, 2019). In some cases, the chimeric F and/or G proteins contain an extracellular domain from one henipa virus species and a transmembrane and/or cytoplasmic domain from a different henipa virus species. For example, the F protein contains the extracellular domain of hendra virus and the transmembrane/cytoplasmic domain of nipah virus.

The lipid particles provided, such as lentiviral vectors, exhibit advantages over available envelope pseudotyped particles. For example, VSV-G is the most common envelope glycoprotein used for pseudotyping, but its broad tropism is often not ideal or desired for specific target cell delivery, such as for gene therapy or exogenous protein delivery. Furthermore, while alternative envelope proteins may exhibit reduced tropism or may be suitable for linking to a binding domain to redirect targeting to a desired target cell, the titer of lentiviral vector formulations containing such envelope proteins may be too low to allow efficient transduction. Thus, alternative methods are needed. It was found herein that certain repetitive proteins exhibit high titers when pseudotyped on lentiviral vectors.

Lipid particles, such as targeted lipid particles, that additionally contain one or more exogenous agents, such as for delivering a diagnostic or therapeutic agent to a cell, including after in vivo administration to a subject, are also provided. Also provided herein are methods and uses of the lipid particles, such as in diagnostic and therapeutic methods. Also provided are polynucleotides, methods for engineering, preparing, and producing lipid non-cellular particles, compositions containing the particles, and kits and devices containing and for using, producing, and administering the particles.

All publications (including patent documents, scientific articles, and databases) mentioned in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in a patent, application, published application and other publication, which is incorporated by reference herein, the definition set forth herein takes precedence over the definition set forth herein.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Definition of the definition

Unless defined otherwise, all technical, symbolic and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ease of reference, and inclusion of such definitions herein should not be construed to represent a substantial difference over what is commonly understood in the art. Abbreviations and symbols for chemical and biochemical names are according to IUPAC-IUB nomenclature unless otherwise indicated. Unless otherwise indicated, all numerical ranges include values defining the ranges as well as all integer values in between.

As used herein, the article "a" or "an" refers to a grammatical object of the article of manufacture of one or more than one (i.e., at least one). For example, "an element" means one element or more than one element.

As used herein, the term "about" will be understood by those of ordinary skill in the art and will vary to some extent in the context of use. As used herein, "about" when referring to a measurable value, such as an amount, short duration, etc., is intended to encompass variations from the specified value of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1%, as such variations are suitable for performing the disclosed methods.

As used herein, "lipid particle" refers to any biological or synthetic particle comprising an amphiphilic lipid bilayer surrounding a lumen or cavity. Typically, the lipid particle does not contain a core. Such lipid particles include, but are not limited to, viral particles (e.g., lentiviral particles), virus-like particles, viral vectors (e.g., lentiviral vectors), exosomes, enucleated cells, various vesicles (such as microvesicles, membrane vesicles, extracellular membrane vesicles, plasma membrane vesicles, giant plasma membrane vesicles), apoptotic bodies, mitotic particles (mitoparticle), nuclear erythrocytes (pyrenocyte), or lysosomes. In some embodiments, the lipid particle may be a fusion (fusosome). In some embodiments, the lipid particle is not a platelet. In some embodiments, the fusion is derived from a source cell. The lipid particle may also comprise an exogenous agent or nucleic acid encoding an exogenous agent, which may be present in the inner cavity of the lipid particle.

The terms "viral vector particle" and "viral vector" are used interchangeably herein and refer to a vector for transferring an exogenous agent (e.g., a non-virus or exogenous nucleic acid) into a recipient or target cell and which contains one or more viral structural proteins in addition to at least one non-structural viral genome component or functional fragment thereof (i.e., a polymerase, integrase, protease, or other non-structural component). Thus, the viral vector contains an exogenous agent to be transferred into the cell, such as a heterologous nucleic acid comprising a non-viral coding sequence. Examples of viral vectors are retroviral vectors, such as lentiviral vectors.

The term "retroviral vector" refers to a viral vector containing a retroviral nucleic acid or derived from a retrovirus. Retroviral vector particles comprise the components of a vector genome (retroviral nucleic acid), a nucleocapsid encapsulating the nucleic acid, and a membrane envelope surrounding the nucleocapsid. Typically, retroviral vectors contain sufficient retroviral genetic information to allow packaging of the RNA genome into viral particles capable of infecting target cells in the presence of packaging components. Infection of the target cell may include reverse transcription and integration into the target cell genome. The retroviral vector may be a recombinant retroviral vector which is replication defective and lacks genes necessary for replication, such as functional gag-pol and/or env genes and/or other genes necessary for replication. The retroviral vector may also be a self-inactivating (SIN) vector.

As used herein, "lentiviral vector" or LV refers to a viral vector containing lentiviral nucleic acid or derived from a lentivirus. Lentiviral vector particles comprise the components of a vector genome (lentiviral nucleic acid), a nucleocapsid encapsulating the nucleic acid, and a membrane surrounding the nucleocapsid. Typically, lentiviral vectors contain sufficient lentiviral genetic information to allow packaging of the RNA genome in the presence of packaging components into viral particles capable of infecting target cells. Infection of the target cell may include reverse transcription and integration into the target cell genome. The lentiviral vector may be a recombinant lentiviral vector that is replication defective and lacks genes necessary for replication, such as functional gag-pol and/or env genes and/or other genes necessary for replication. The lentiviral vector may also be a self-inactivating (SIN) vector.

As used herein, "retroviral nucleic acid" refers to a nucleic acid containing at least the minimum sequence requirements packaged into a retroviral vector, alone or in combination with a helper cell, helper virus, or helper plasmid. In the context of "lentiviral nucleic acid", nucleic acid refers to at least the minimal sequence requirements of packaging into a lentiviral vector, alone or in combination with a helper cell, helper virus, or helper plasmid. In some embodiments, the viral nucleic acid comprises one or more (e.g., all) of a 5 'LTR (e.g., to facilitate integration), a U3 (e.g., to activate viral genomic RNA transcription), R (e.g., tat binding region), a U5, 3' LTR (e.g., to facilitate integration), a packaging site (e.g., psi (ψ)), an RRE (e.g., to bind Rev and facilitate nuclear export). Viral nucleic acids may comprise RNA (e.g., when part of a virion) or DNA (e.g., when introduced into a source cell or after reverse transcription in a recipient cell). In some embodiments, the viral nucleic acid is packaged using helper cells, helper viruses, or helper plasmids comprising one or more (e.g., all) of gag, pol, and env.

As used herein, "fusion" refers to a lipid particle that contains an amphiphilic lipid bilayer surrounding a lumen or cavity and a fusion agent that interacts with the amphiphilic lipid bilayer. In some embodiments, the fusion is a membrane-enclosed formulation. In some embodiments, the fusion is derived from a source cell. The fusion may also comprise an exogenous agent or nucleic acid encoding an exogenous agent, which may be present in the lumen of the fusion.

As used herein, "fusion composition" refers to a composition comprising one or more fusion.

As used herein, "fusion agent" refers to an agent or molecule that creates an interaction between the lumens enclosed by two membranes. In embodiments, the fusion agent facilitates fusion of the membrane. In other embodiments, the fusion agent creates a junction, such as a pore, between two lumens (e.g., the lumen of a retroviral vector and the cytoplasm of a target cell). In some embodiments, the fusion agent comprises a complex of two or more proteins, e.g., wherein neither protein alone has fusion activity. In some embodiments, the fusion agent comprises a targeting domain. Examples of fusion agents include paramyxovirus F and G proteins, such as those from nipah virus (NiV), and biologically active portions or variants thereof, including any of the described.

As used herein, "re-targeted fusion agent" (such as a re-targeted G protein) refers to a fusion agent that comprises a targeting moiety having a sequence that is not part of the naturally occurring form of the fusion agent, wherein the targeting moiety targets or binds to a molecule on a desired cell type. In embodiments, the fusion agent comprises a targeting moiety that is different relative to the targeting moiety in the naturally occurring form of the fusion agent. In embodiments, the naturally occurring form of the fusion agent lacks a targeting domain, and the re-targeted fusion agent comprises a targeting moiety that is not present in the naturally occurring form of the fusion agent. In embodiments, the fusion agent is modified to comprise a targeting moiety. In some such embodiments, attachment of the targeting moiety to the fusion agent (e.g., G protein) may be performed directly or indirectly via a linker (such as a peptide linker). In embodiments, the fusion agent comprises one or more sequence alterations located outside the targeting moiety relative to the naturally occurring form of the fusion agent, e.g., in the transmembrane domain, domain with fusion activity, or cytoplasmic domain.

As used herein, "target cell" refers to a cell of a type to which a lipid particle (such as a targeted lipid particle) is desired to deliver an exogenous agent. In embodiments, the target cell is a cell of a particular tissue type or class, such as an immune effector cell, e.g., a T cell. In some embodiments, the target cell is a diseased cell, such as a cancer cell. In some embodiments, the fusion agent (e.g., a re-targeted fusion agent) results in preferential delivery of the exogenous agent to the target cell over the non-target cell.

As used herein, "non-target cells" refers to cells of a type to which lipid particles are not desired to deliver exogenous agents. In some embodiments, the non-target cells are cells of a particular tissue type or class. In some embodiments, the non-target cells are non-diseased cells, such as non-cancerous cells. In some embodiments, the fusion agent (e.g., a re-targeted fusion agent) results in less delivery of the exogenous agent to non-target cells than to target cells.

As used herein, a "biologically active portion" (such as in reference to a protein such as a G protein or F protein) refers to a portion of a protein that exhibits or retains the activity or properties of a full-length protein. For example, the biologically active portion of the F protein retains fusion activity to bind to the G protein when each is embedded in a lipid bilayer. For example, the biologically active portion of the G protein retains fusion activity to bind to the F protein when each is embedded in a lipid bilayer. The retained activity may include 10% -150% or more of the activity of the full length or wild type F protein or G protein. Examples of biologically active portions of F and G proteins include proteins having a cytoplasmic domain truncated, such as any of the described variants NiV-F having a truncated cytoplasmic tail.

As used herein, "percent (%) amino acid sequence identity" and "homology" with respect to a peptide, polypeptide, or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percent sequence identity and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining the percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared.

Amino acid substitutions may include, but are not limited to, substitution of one amino acid for another amino acid in a polypeptide. Exemplary substitutions are shown in table 1. Amino acid substitutions may be introduced into the antibody of interest and the products screened for desired activity, e.g., retention/improved binding.

Amino acids can be grouped according to common side chain characteristics:

(1) Hydrophobicity, norleucine Met, ala, val, leu, ile;

(2) Neutral hydrophilicity Cys, ser, thr, asn, gln;

(3) Acid, asp, glu;

(4) Basicity His, lys, arg;

(5) Residues affecting chain orientation, gly, pro;

(6) Aromatic Trp, tyr, phe.

Non-conservative substitutions will require the exchange of members of one of these classes for another class.

The term "corresponding to" with respect to the position of a protein, such as reciting a nucleotide or amino acid position "corresponding to" a nucleotide or amino acid position in a disclosed sequence (such as shown in the sequence listing), refers to a nucleotide or amino acid position identified upon alignment of the disclosed sequence based on structural sequence or using standard alignment algorithms (such as the GAP algorithm). For example, corresponding residues of similar sequences (e.g., fragments or species variants) can be determined by structural alignment methods with reference sequences. By aligning the sequences, the person skilled in the art can identify the corresponding residues, for example using conserved and identical amino acid residues as guidance.

As used herein, the term "isolated" refers to a molecule that has been separated from at least some of the components typically found or produced in nature. For example, a polypeptide is said to be "isolated" when it is separated from at least some of the components of the cell from which it is derived. Physically isolating a supernatant containing a polypeptide from a cell that produces the polypeptide is considered to be "isolating" the polypeptide in the event that the polypeptide is secreted by the cell after expression. Similarly, a polynucleotide is said to be "isolated" when it is not part of a larger polynucleotide (e.g., genomic DNA or mitochondrial DNA in the case of DNA polynucleotides) that it is typically found in nature or is separated from at least some components of the cell that produces the polynucleotide (e.g., in the case of RNA polynucleotides). Thus, a DNA polynucleotide contained in a vector within a host cell may be referred to as "isolated".

As used herein, the term "effective amount" means an amount of the pharmaceutical composition sufficient to significantly and positively alter the symptom and/or condition to be treated (e.g., provide a positive clinical response). The effective amount of the active ingredient for use in the pharmaceutical composition will vary with the particular condition being treated, the severity of the condition, the duration of treatment, the nature of concurrent therapy, the particular active ingredient or ingredients used, the particular pharmaceutically acceptable excipient or excipients used, and/or the carrier or carriers and similar factors within the knowledge and expertise of the attending physician.

As used herein, "exogenous agent" with respect to a lipid particle (such as a viral vector) refers to an agent that is neither contained nor encoded by the corresponding wild-type virus or by a fusion made from the corresponding wild-type source cell. In some embodiments, the exogenous agent is not naturally occurring, such as a protein or nucleic acid having a sequence that is altered (e.g., by an insertion, deletion, or substitution) relative to the naturally occurring protein. In some embodiments, the exogenous agent is not naturally present in the source cell. In some embodiments, the exogenous agent is naturally present in the source cell, but is exogenous to the virus. In some embodiments, the exogenous agent is not naturally present in the recipient cell. In some embodiments, the exogenous agent is naturally present in the recipient cell, but is not present at the desired level or at the desired time. In some embodiments, the exogenous agent comprises RNA or protein.

As used herein, "promoter" refers to a cis-regulatory DNA sequence that, when operably linked to a gene coding sequence, drives transcription of a gene. The promoter may comprise a transcription factor binding site. In some embodiments, the promoter cooperates with one or more enhancers distal to the gene.

As used herein, a composition refers to any mixture of two or more products, substances, or compounds (including cells). It may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

As used herein, the term "pharmaceutically acceptable" refers to materials that do not abrogate the biological activity or properties of the compound and that are relatively non-toxic (such as carriers or diluents), i.e., that can be administered to an individual without causing an undesirable biological effect or interacting in a deleterious manner with any of the components of the composition in which they are comprised.

As used herein, the term "pharmaceutical composition" refers to a mixture of at least one compound of the present invention with other chemical components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners and/or excipients. The pharmaceutical compositions facilitate administration of the compounds to an organism. There are a variety of techniques in the art for administering compounds including, but not limited to, intravenous, oral, aerosol, parenteral, ocular, pulmonary and topical administration.

As used herein, "disease" or "disorder" refers to a condition that is needed and/or desired to be treated.

As used herein, the term "treating (treat, treating or treatment)" refers to ameliorating a disease or disorder, e.g., slowing or preventing or reducing the progression of the disease or disorder or reducing at least one clinical symptom thereof. For the purposes of this disclosure, ameliorating a disease or disorder may include obtaining beneficial or desired clinical results including, but not limited to, any one or more of alleviating one or more symptoms, reducing the extent of the disease, preventing or delaying the spread of the disease (e.g., metastasis, e.g., to the lung or lymph node), preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, ameliorating the disease state, inhibiting the disease or progression of the disease, inhibiting or slowing the disease or progression thereof, preventing its progression, and alleviating (whether partially or wholly).

The terms "individual" and "subject" are used interchangeably herein to refer to an animal, such as a mammal. The term patient includes both human and veterinary subjects. In some embodiments, methods of treating mammals including, but not limited to, humans, rodents, apes, felines, dogs, equines, cattle, pigs, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian sports animals, and mammalian pets are provided. The subject may be male or female, and may be of any suitable age, including infant, juvenile, adolescent, adult and geriatric subjects. In some examples, an "individual" or "subject" refers to an individual or subject in need of treatment for a disease or disorder. In some embodiments, the subject receiving treatment may be a patient, indicating the fact that the subject has been identified as having or at sufficient risk of developing a treatment-related disorder. In particular embodiments, the subject is a human, such as a human patient.

As used herein, the term "nuclear export sequence" (NES) or "nuclear export signal" (NES) refers to a nuclear export signal or other sequence or domain that is present in a protein and is capable of targeting the protein to export from the nucleus to the cytoplasm through the nuclear pore complex using nuclear transport. The nuclear export domain may be fused (e.g., in-frame) to a polypeptide.

As used herein, the term "nuclear localization sequence" (NLS) or "nuclear localization sequence" (NLS) refers to a nuclear localization signal or other sequence or domain that is present in a protein and is capable of targeting the protein to import the nucleus from the cytoplasm through the nuclear pore complex using nuclear transport. Nuclear localization can be fused (e.g., in-frame fusion) to a polypeptide.

I. Lipid particles comprising a tandem fusion agent

In some embodiments, the lipid particle, such as a viral vector or virus-like particle, contains one or more attachment proteins (e.g., fusion agents). In some embodiments, the lipid particle, e.g., viral vector or virus-like particle, contains an exogenous or overexpressed attachment protein (e.g., a fusion agent). In some embodiments, the fusion agent is disposed in a lipid bilayer. In some embodiments, the fusion agent facilitates fusion of the lipid particle with the membrane.

In some embodiments, the lipid particle comprises a re-targeted attachment protein, such as an attachment protein that is linked to at least two targeting moieties (such as a first targeting moiety and a second targeting moiety).

In some embodiments, the attachment protein (e.g., fusion agent) may include a non-mammalian protein, such as a viral protein. In some embodiments, the viral fusion agent is a class I viral membrane fusion protein, a class II viral membrane protein, a class III viral membrane fusion protein, a viral membrane glycoprotein, or other viral fusion protein, or a homolog, fragment, variant, or protein fusion comprising one or more proteins or fragments thereof.

In some embodiments, the class I viral membrane fusion proteins include, but are not limited to, baculovirus F proteins, such as Nuclear Polyhedrosis Virus (NPV) genus F proteins, such as MNPV asparagus caterpillar (SeMNPV) F protein and gypsy moth MNPV (LdMNPV) and paramyxovirus F protein.

In some embodiments, the class II viral membrane proteins include, but are not limited to, tick encephalitis E (TBEV E), semliki forest virus E1/E2.

In some embodiments, the class III viral membrane fusion proteins include, but are not limited to, rhabdovirus G (e.g., fusion protein G of vesicular stomatitis virus (VSV-G)), herpes virus glycoprotein B (e.g., herpes simplex virus 1 (HSV-1) gB)), epstein-barr virus glycoprotein B (EBV gB), tol Gao Tu virus G, baculovirus gp64 (e.g., nyctada medicago multiple NPV (AcMNPV) gp 64), baboon endogenous retrovirus envelope glycoprotein (BaEV), and vitronectin virus (Borna disease virus, BDV) glycoprotein (BDV G).

Examples of other viral attachment proteins (e.g., as fusion agents for membrane glycoproteins and viral fusion proteins) include, but are not limited to, viral syncytial proteins, such as influenza Hemagglutinin (HA) or mutants, or fusion proteins thereof; human immunodeficiency virus type 1 envelope protein (HIV-1 ENV), gp120 from HIV that binds LFA-1 to form lymphocyte syncytia, HIV gp41, HIV gp160, or HIV trans-activator of transcription (TAT); viral glycoproteins VSV-G, viral glycoproteins from Rhabdoviridae, the glycoproteins gB and gH-gL of Varicella Zoster Virus (VZV), murine Leukemia Virus (MLV) -10A1, the G-glycoprotein of gibbon ape leukemia virus glycoprotein (GaLV), rabies, mokola virus, vesicular stomatitis virus and togavirus, murine hepatitis JHM surface raised proteins, porcine respiratory coronavirus spike and membrane glycoprotein, avian infectious bronchitis spike glycoprotein and precursors thereof, bovine enterocoronavirus spike protein, F and H, HN or G genes of measles virus, canine distemper virus, newcastle disease virus, human parainfluenza virus 3, simian virus 41, sendai virus and human respiratory syncytial virus, the gH of human herpesvirus type 1 and simian varicella virus, and chaperone proteins gL, human, bovine and cynomolgus virus gB, frd murine leukemia virus and Mamason virus envelope proteins, and the neuritic adenovirus of the mumps adenovirus, and glycoproteins F1 and F2, membrane glycoproteins from Venezuelan equine encephalomyelitis, paramyxovirus F protein, SIV gp160 protein, ebola virus G protein, or Sendai virus fusion proteins or homologs, fragments, variants thereof, and protein fusions comprising one or more proteins or fragments thereof.

Non-mammalian attachment proteins (e.g., fusion agents) include viral fusion agents, homologs thereof, fragments thereof, and fusion proteins comprising one or more proteins or fragments thereof. Viral fusion agents include class I fusion agents, class II fusion agents, class III fusion agents, and class IV fusion agents. In embodiments, a class I fusion agent, such as Human Immunodeficiency Virus (HIV) gp41, has a characteristic post-fusion conformation, has a characteristic trimer of α -helical hairpins with a central coiled-coil structure. Class I viral fusion proteins include proteins with a central post-fusion six-helix bundle. Class I viral fusion proteins include influenza HA, parainfluenza F, HIV Env, ebola GP, hemagglutinin from orthomyxoviruses, F proteins from paramyxoviruses (e.g., measles, (Katoh et al BMC Biotechnology 2010, 10: 37)), env proteins from retroviruses, and fusion agents of filoviruses and coronaviruses. In embodiments, a class II viral fusion agent such as dengue E glycoprotein has the structural feature of a β -sheet forming an elongated extracellular domain that refolding produces a hairpin trimer. In embodiments, the class II viral fusion agent lacks a central coiled coil. Class II viral fusion agents can be found in alphaviruses (e.g., E1 proteins) and flaviviruses (e.g., E glycoproteins). Class II viral fusions include fusions from semliki forest virus, xin Bisi virus, rubella virus and dengue virus. In embodiments, a class III viral fusion agent such as vesicular stomatitis virus G glycoprotein combines structural features found in class I and class II. In embodiments, the class III viral fusion agent comprises an alpha helix (e.g., forming a six-helix bundle to fold the protein as with the class I viral fusion agent) and a beta sheet having an amphiphilic fusion peptide at its end, reminiscent of the class II viral fusion agent. Class III viral fusion agents are found in rhabdoviruses and herpesviruses. In embodiments, the class IV viral fusion agent is a fusion related small transmembrane (FAST) protein (doi:10.1038/sj.emboj.7600767, Nesbitt, Rae L., "Targeted Intracellular Therapeutic Delivery Using Liposomes Formulated with Multifunctional FAST proteins" (2012).Electronic Thesis and Dissertation Repository. paper 388) encoded by a non-enveloped reovirus. In embodiments, the class IV viral fusion agents are small enough that they do not form hairpins (doi: 10.1146/annurev-cellbio-101512-122122, doi:10.1016/j. Devcel. 2007.12.008).

Additional exemplary attachment proteins, such as fusion agents, are disclosed in US 9,695,446、US 2004/0028687、US 6,416,997、US 7,329,807、US 2017/0112773、US 2009/0202622、WO 2006/027202 and US 2004/0009604, the entire contents of all of which are hereby incorporated by reference.

In some embodiments, the attachment protein (e.g., fusion agent) is WO2017/182585; WO2022/164935; WO2021/076788; hamilton et al, bioRxiv, 2022.08.24.505504; nikolic et al, nat Commun 9, 1029 (2018), dobson et al, nat. Methods, 19, 449-460 (2022), and any fusion moiety described in Yu et al, bioRxiv 2021.12.13.472464, e.g., any of the VSV or variant VSV glycoproteins described therein, such as VSV glycoproteins that bind reduced to a natural receptor.

In some embodiments, the attachment protein (e.g., fusion agent) is a poxviridae fusion agent.

In some embodiments, the lipid particle comprises an attachment protein that is a re-targeted first paramyxovirus attachment protein, such as a paramyxovirus envelope attachment protein linked to at least two targeting moieties (such as a first targeting moiety and a second targeting moiety). In some embodiments, the lipid particle further comprises at least one paramyxovirus fusion protein.

In some embodiments, a paramyxovirus envelope attachment protein and/or a re-targeting attachment protein provided herein exhibits fusion activity to a target cell, such as to deliver an exogenous agent or a nucleic acid exogenous agent to the target cell.

In some embodiments, the paramyxovirus attachment protein is or comprises hemagglutinin-neuraminidase (HN) from a respiratory paramyxovirus. In some embodiments, the respiratory paramyxovirus is sendai virus. The function of the HN glycoprotein of sendai virus is to attach to sialic acid via HN protein and mediate cell fusion via F protein to enter cells. In some embodiments, the paramyxovirus attachment protein is or comprises HN protein from murine parainfluenza virus type 1 (see, e.g., U.S. patent No. 10704061).

In some embodiments, the paramyxovirus attachment protein is or comprises nipah virus protein G, measles protein H, tree shrew paramyxovirus H protein, paramyxovirus G protein, paramyxovirus H protein, paramyxovirus HN protein, measles virus H protein, respiratory tract virus HN protein, sendai virus HN protein, mumps virus HN protein, avian mumps virus HN protein, or derivatives thereof. In some embodiments, the paramyxovirus attachment protein is or comprises a sequence selected from the group consisting of a Nippa virus G protein, a measles virus H protein, a tree shrew paramyxovirus H protein, paramyxovirus G and H proteins, an HN protein, a Hendela virus G protein, a Henlapavirus G protein, a measles virus H protein, a respiratory tract virus HN protein, a Sendai virus HN protein, a mumps virus HN protein, or an avian mumps virus HN protein, or derivatives thereof, or any combination thereof.

A. paramyxovirus attachment proteins

In some embodiments, the lipid particles provided herein comprise a paramyxovirus envelope attachment protein, a first paramyxovirus envelope attachment protein, and/or a second paramyxovirus envelope attachment protein. In some embodiments, the paramyxovirus attachment protein is re-targeted. In some embodiments, the paramyxoviral envelope attachment protein may be an envelope glycoprotein G, H and/or HN of the paramyxoviridae family.

In some embodiments, the lipid particle provided herein comprises a first paramyxovirus envelope attachment protein, a second paramyxovirus envelope attachment protein, and a third paramyxovirus envelope attachment protein. In some embodiments, each of the first paramyxovirus envelope attachment protein, the second paramyxovirus envelope attachment protein, and the third paramyxovirus envelope attachment protein may independently be an envelope glycoprotein G, H and/or HN of the paramyxoviridae family.

In some embodiments, the lipid particles provided herein comprise a first paramyxovirus envelope attachment protein, a second paramyxovirus envelope attachment protein, a third paramyxovirus envelope attachment protein, and one or more additional paramyxovirus envelope attachment proteins, such as a fourth paramyxovirus envelope attachment protein, or a fourth paramyxovirus envelope attachment protein and a fifth paramyxovirus envelope attachment protein, or a fourth paramyxovirus envelope attachment protein, a fifth paramyxovirus envelope attachment protein and a sixth paramyxovirus envelope attachment protein, or more. In some embodiments, each paramyxoviral envelope attachment protein may independently be an envelope glycoprotein G, H and/or HN of the paramyxoviridae family.

1. G protein

In some embodiments, the paramyxovirus envelope attachment protein, the first paramyxovirus envelope attachment protein and/or the second paramyxovirus envelope attachment protein and/or the third paramyxovirus envelope attachment protein and/or the fourth paramyxovirus envelope attachment protein and/or the fifth paramyxovirus envelope attachment protein and/or the sixth paramyxovirus envelope attachment protein and/or any additional paramyxovirus envelope attachment protein is an attachment glycoprotein G (G protein) or a biologically active portion thereof. In some embodiments, the re-targeted attachment protein comprises a first paramyxovirus envelope attachment protein G.

In some embodiments, the lipid particle comprises a re-targeted attachment protein, a first re-targeted attachment protein, and/or a second re-targeted attachment protein exposed on the surface of the targeted lipid particle. In some embodiments, the lipid particle further comprises a third re-targeted attachment protein exposed on the surface of the targeted lipid particle. In some embodiments, the lipid particle further comprises a third and fourth re-targeted attachment protein exposed on the surface of the targeted lipid particle. In some embodiments, the lipid particle further comprises a third, fourth, and fifth re-targeted attachment protein exposed on the surface of the targeted lipid particle. In some embodiments, the lipid particle further comprises a third re-targeted attachment protein, a fourth re-targeted attachment protein, a fifth re-targeted attachment protein, and one or more additional re-targeted attachment proteins exposed on the surface of the targeted lipid particle. In some embodiments, the re-targeted attachment protein is or comprises a paramyxovirus attachment protein, wherein the paramyxovirus attachment protein is an attachment glycoprotein G (G protein) or a biologically active portion thereof. In some embodiments, the re-targeting attachment protein is or comprises a paramyxovirus attachment protein, wherein the paramyxovirus attachment protein is an attachment glycoprotein G (G protein) or a biologically active portion thereof, and comprises a targeting moiety, such as a binding domain or binding agent, for a target molecule expressed on the surface of a target cell.

Envelope-attaching G proteins are type II transmembrane glycoproteins which contain an N-terminal cytoplasmic tail (e.g., amino acids 1-49 corresponding to SEQ ID NO: 1), a transmembrane domain (e.g., amino acids 50-70 corresponding to SEQ ID NO: 1), and an extracellular domain containing an extracellular stem (e.g., amino acids 71-187 corresponding to SEQ ID NO: 1) and a globular head (amino acids 188-602 corresponding to SEQ ID NO: 1). The N-terminal cytoplasmic domain is in the internal lumen of the lipid bilayer, and the C-terminal portion is the extracellular domain exposed outside of the lipid bilayer. The region in the C-terminal region of the stem (e.g., amino acids 71-187 corresponding to SEQ ID NO: 1) has been shown to be involved in interactions with and triggering of fusion of the F protein (Liu et al 2015J of Virology 89:1838). In wild type G protein, globular heads mediate binding of the receptor to Hennipa virus entry receptors, hepataxin B2 and hepataxin B3, but not essential for membrane fusion (Brandel-TRETHEWAY et al, journal of virology 2019.93 (13) e 00577-19).

In some embodiments herein, the tropism of a G protein is altered by the linkage of the G protein or a biologically active fragment thereof (e.g., a cytoplasmic truncation) to an sdAb variable domain. Binding of the G protein to the binding partner may trigger fusion mediated by a compatible paramyxovirus fusion protein (e.g., F protein) or a biologically active portion thereof (such as any of the F proteins described in ii.b below). The G protein sequences disclosed herein are primarily disclosed as expression sequences comprising an N-terminal methionine necessary for translation initiation. Since such N-terminal methionine is typically co-translated or post-translationally cleaved, the mature protein sequences of all G protein sequences disclosed herein are also considered to be devoid of an N-terminal methionine.

G glycoprotein is highly conserved among Huntiepaviras species. For example, the G proteins of NiV and HeV viruses share 79% amino acid identity. Studies have shown a high degree of compatibility between G protein and F protein of different species as demonstrated by heterotypic fusion activation (Brandel-TRETHEWAY et al, journal of virology 2019). As described above, the lipid particle may comprise at least two envelope-attaching proteins (e.g., co-fusion agents). In certain embodiments, the F protein, or functionally active variant or biologically active portion thereof, retains fusion activity associated with at least two of the provided envelope attachment proteins (e.g., co-fusion agents that are paramyxovirus attachment protein G), such as any of those described below. Fusion activity includes the activity of a paramyxovirus fusion protein (e.g., F protein) that binds to a G protein to facilitate or aid in cytoplasmic fusion of two membrane lumens (e.g., the lumens of lipid particles provided herein), such as at least two G proteins and F proteins that are embedded in their lipid bilayer, such as exposed on their surface, and a target cell (e.g., a cell containing a surface receptor or molecule that is recognized or bound by the G protein).

Exemplary henipav viral protein G sequences are provided in table 2

Table 2. Hennapaviras protein G sequence clusters. Column 1, genbank ID includes Genbank ID of viral entire genome sequence as centroid sequence of cluster. Column 2, nucleotide of CDS provides a nucleotide corresponding to CDS of genes in the whole genome. Column 3, complete gene name, provides complete name of the gene, including Genbank ID, virus seed, strain and protein name. Column 4, sequence, provides the amino acid sequence of the gene. Column 5, sequence/cluster # provides the number of sequences clustered with the centroid sequence. Column 6 provides the SEQ ID numbers of the sequences described.

In some embodiments, at least one G protein has the sequence shown in any one of SEQ ID NOs 1, 561, 562, 563, or 564, or is a functionally active variant or biologically active portion thereof having a sequence that is at least equal to or about 80%, at least equal to or about 81%, at least equal to or about 82%, at least equal to or about 83%, at least equal to or about 84%, at least equal to or about 85%, at least equal to or about 86%, at least equal to or about 87%, at least equal to or about 88%, at least equal to or about 89%, at least equal to or about 90%, at least equal to or about 91%, at least equal to or about 92%, at least equal to or about 93%, at least equal to or about 94%, at least equal to or about 95%, at least equal to or about 96%, at least equal to or about 97%, at least equal to or about 98%, or at least equal to or about 99% identical to any one of SEQ ID NOs 1, 561, 562, 563, or 564.

In certain embodiments, a paramyxovirus envelope attachment protein (e.g., G protein) or functionally active variant or biologically active moiety is a protein that retains fusion activity that binds to a paramyxovirus fusion protein (e.g., F protein) such as the NiV-F protein described herein. Fusion activity includes the activity of a paramyxovirus envelope attachment protein (e.g., G protein) that binds to a paramyxovirus fusion protein (e.g., F protein) to promote or aid cytoplasmic fusion of two membrane lumens, such as a lumen of a targeted lipid particle having embedded in its lipid bilayer a paramyxovirus fusion protein (e.g., F protein) and a paramyxovirus envelope attachment protein (e.g., G protein), and a target cell (e.g., a cell containing a surface receptor or molecule recognized or bound by the targeted envelope protein). In some embodiments, the paramyxovirus fusion protein (e.g., F protein) and paramyxovirus envelope attachment protein (e.g., G protein) are from the same paramyxovirus species (e.g., the same henipav virus species, such as NiV-G and NiV-F).

In some embodiments, at least one G protein or functionally active variant or biologically active portion thereof binds to ephrin B2 or ephrin B3. In some embodiments, the G protein is a variant G protein, such as the truncated G protein described, and retains binding to ephrin B2 or B3. References to retaining binding to ephrin B2 or B3 include binding similar to the level or extent of binding (such as at least 50%, at least 60%, at least 70%, at least 80% or at least 90%) of the corresponding wild-type G protein (such as shown in SEQ ID NOs: 1, 561, 562, 563 or 564).

In some embodiments, the paramyxovirus envelope attachment protein, the first paramyxovirus envelope attachment protein and/or the second paramyxovirus envelope attachment protein is a variant G protein that exhibits reduced binding to a natural binding partner of the wild-type G protein. In some embodiments, the first paramyxovirus envelope attachment protein and/or the second paramyxovirus envelope attachment protein and/or the third paramyxovirus envelope attachment protein is a variant G protein that exhibits reduced binding to a natural binding partner of the wild-type G protein. In some embodiments, the first paramyxovirus envelope attachment protein and/or the second paramyxovirus envelope attachment protein and/or the third paramyxovirus envelope attachment protein and/or the fourth paramyxovirus envelope attachment protein is a variant G protein that exhibits reduced binding to a natural binding partner of the wild-type G protein. In some embodiments, the first paramyxovirus envelope attachment protein and/or the second paramyxovirus envelope attachment protein and/or the third paramyxovirus envelope attachment protein and/or the fourth paramyxovirus envelope attachment protein and/or the fifth paramyxovirus envelope attachment protein and/or the one or more additional paramyxovirus envelope attachment proteins are variant G proteins that exhibit reduced binding to a natural binding partner of the wild-type G protein. In some embodiments, the variant G protein or biologically active portion thereof is a variant of wild-type NiV-G and exhibits reduced binding to one or both of the natural binding partners ephrin B2 or ephrin B3. In some embodiments, the variant G protein or biologically active moiety (such as the variant NiV-G protein) exhibits reduced binding to a natural binding partner. In some embodiments, the reduced binding to ephrin B2 or ephrin B3 is reduced by greater than 5% or about 5%, 10% or about 10%, 15% or about 15%, 20% or about 20%, 25% or about 25%, 30% or about 30%, 40% or about 40%, 50% or about 50%, 60% or about 60%, 70% or about 70%, 80% or about 80%, 90% or about 90%, or 100% or about 100%.

In some embodiments, mutations (e.g., amino acid substitutions) may improve transduction efficiency. In some embodiments, the mutation allows for specific targeting of other desired cell types that are not ephrin B2 or ephrin B3. In some embodiments, the mutation results in at least partial failure to bind to at least one natural receptor, which reduces binding to at least one of ephrin B2 or ephrin B3. In some embodiments, the mutations described herein interfere with natural receptor recognition.

In some embodiments, at least one G protein contains one or more amino acid substitutions in residues involved in interactions with one or both of ephrin B2 and ephrin B3. In some embodiments, referring to the numbering shown in SEQ ID NO. 1, the amino acid substitutions correspond to mutations E501A, W, A, Q A and E533A. In some embodiments, at least one G protein is a variant G protein comprising one or more amino acid substitutions selected from the group consisting of E501A, W504A, Q A and E533A, with reference to the numbering shown in SEQ ID NO. 1. In some embodiments, at least one G protein is a variant G protein that contains one or more amino acid substitutions selected from the group consisting of E501A, W504A, Q A and E533A with reference to SEQ ID NO. 1, and is a biologically active moiety that contains an N-terminal truncation.

In some embodiments, described herein are lipid particles comprising a paramyxovirus envelope attachment protein that is not re-targeted and comprises one or more amino acid substitutions to reduce natural tropism relative to a wild-type paramyxovirus envelope attachment protein that does not comprise the one or more amino acid substitutions. In one embodiment, the paramyxovirus envelope attachment protein is a variant paramyxovirus envelope attachment protein comprising one or more amino acid substitutions to reduce natural tropism relative to a wild-type paramyxovirus envelope attachment protein not comprising said one or more amino acid substitutions. In one embodiment, the paramyxovirus envelope attachment protein is a variant paramyxovirus envelope attachment protein comprising one or more amino acid substitutions corresponding to the amino acid substitutions numbered selected from the group consisting of E501A, W504A, Q A and E533A as set forth with reference to SEQ ID NO. 1.

In some embodiments, niV-G is a variant NiV-G protein containing an altered cytoplasmic tail as compared to native NiV-G (e.g., SEQ ID NO: 5), which is incorporated or can be incorporated into a lipid particle, such as a viral particle, including a lentiviral particle or lentiviral-like particle. The cytoplasmic tail of NiV-G corresponds to amino acids 1-45 of SEQ ID NO. 5. In some cases, it will be appreciated that the N-terminal methionine of NiV-G or variant NiV-G as described herein may be cleaved and the cytoplasmic tail is devoid of an initial N-terminal methionine. For example, in some embodiments, the cytoplasmic tail of wild type NiV-G can correspond to amino acids 2-45 of SEQ ID NO. 5, and the variant NiV-G protein contains an altered cytoplasmic tail as compared to amino acids 2-45 of SEQ ID NO. 5. In some embodiments, the variant NiV-G contains a modified cytoplasmic tail wherein the native cytoplasmic tail is truncated or replaced with a heterologous cytoplasmic tail.

Non-limiting examples of variant NiV-G proteins (including truncated NiV-G or NiV-G with altered or modified cytoplasmic tail) are described in WO2013148327, WO2017182585 or PCT/US 2022/081872. Additional exemplary variant NiV-G proteins are described in Bender et al 2016 PLoS Pathol 12 (6): e1005641

In some embodiments, at least one G protein is a variant G protein that is a functionally active variant or biologically active portion that contains one or more amino acid mutations (such as one or more amino acid insertions, deletions, substitutions, or truncations). In some embodiments, the mutations described herein involve amino acid insertions, deletions, substitutions, or truncations of amino acids compared to a reference G protein sequence. In some embodiments, the reference G protein sequence is a wild-type sequence of a G protein or a biologically active portion thereof. In some embodiments, the at least one functionally active variant or biologically active portion thereof is a variant of a wild-type hendra (HeV) viral G protein, a wild-type Nipah (NiV) viral G protein (NiV-G), a wild-type cedar (CedPV) viral G protein, a wild-type mejianovirus G protein, a wild-type bat paramyxovirus G protein, or a biologically active portion thereof. In some embodiments, the wild-type G protein has a sequence set forth in any one of SEQ ID NOs 1, 561, 562, 563, or 564.

In some embodiments, at least one G protein is a variant G protein that is a biologically active moiety that is an N-terminal and/or C-terminal truncated fragment of a wild-type hendra (HeV) viral G protein, a wild-type Nipah (NiV) viral G protein (NiV-G), a wild-type cedar (CedPV) viral G protein, a wild-type mejianovirus G protein, a wild-type bat paramyxovirus G protein. In particular embodiments, the truncation is an N-terminal truncation of all or part of the cytoplasmic domain. In some embodiments, at least one variant G protein is a biologically active portion that is truncated and lacks up to 49 consecutive amino acid residues at or near the N-terminus of a wild-type G protein, such as the wild-type G protein shown in any one of SEQ ID NOs: 1, 561, 562, 563, or 564. In some embodiments, at least one variant G protein is truncated and lacks up to 49 consecutive amino acids, such as up to 49、48、47、46、45、44、43、42、41、40、39、38、37、36、35、34、33、32、31、30、29、28、27、26、25、24、23、22、21、20、19、18、17、16、15、14、13、12、11、10、9、8、7、6、5、4、3、2 or 1 consecutive amino acids, at the N-terminus of the wild-type G protein.

In some embodiments, at least one G protein is a wild-type nipah virus G (NiV-G) protein or a hendra virus G protein, or a functionally active variant or biologically active portion thereof. In some embodiments, at least one G protein is a NiV-G protein having the sequence set forth in SEQ ID NO. 1, or a functional variant or biologically active portion thereof having an amino acid sequence with at least equal to or about 80%, at least equal to or about 81%, at least equal to or about 82%, at least equal to or about 83%, at least equal to or about 84%, at least equal to or about 85%, at least equal to or about 86%, at least equal to or about 87%, at least equal to or about 88%, at least equal to or about 89%, at least equal to or about 90%, at least equal to or about 91%, at least equal to or about 92%, at least equal to or about 93%, at least equal to or about 94%, at least equal to or about 95%, equal to or about 96%, at least equal to or about 97%, at least equal to or about 98%, at least equal to or about 99% sequence identity to SEQ ID NO. 1.

In some embodiments, the variant NiV-G comprises a modified cytoplasmic tail comprising a truncated cytoplasmic tail of a glycoprotein from the same nipah virus. In some embodiments, the variant NiV-G comprises a modified cytoplasmic tail wherein at least a portion of the native cytoplasmic tail (e.g., amino acids 1-45 corresponding to SEQ ID NO: 5) is a truncated portion of its glycoprotein from Nipah virus. In some embodiments, the cytoplasmic tail is a truncated portion having a length of at least 5 amino acids (from or about 5 to 44, from or about 5 to 40, from or about 5 to 30, from or about 5 to 20, from or about 5 to 10, from or about 10 to 44, from or about 10 to 40, from or about 10 to 30, from or about 10 to 20, from or about 20 to 44, from or about 20 to 40, from or about 20 to 30, from or about 30 to 44, from or about 30 to 40, from or about 40 to 44 amino acids). In some embodiments, the truncated portion is 5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43 or 44 amino acids in length. In some embodiments, the variant NiV-G has a cytoplasmic tail which is a truncated NiV-G cytoplasmic tail.

In some embodiments, the truncated NiV-G cytoplasmic tail has a deletion of up to 40, up to 35, up to 30, up to 29, up to 28, up to 27, up to 26, up to 25, up to 24, up to 23, up to 22, up to 21, up to 20, up to 19, up to 18, up to 17, up to 16, up to 15, or up to 14 consecutive amino acid residues at or near the N-terminus of the wild type NiV-G cytoplasmic tail shown in SEQ ID NO. 28. In some embodiments, the truncated NiV-G cytoplasmic tail has a deletion of up to 40, up to 35, up to 30, up to 29, up to 28, up to 27, up to 26, up to 25, up to 24, up to 23, up to 22, up to 21, up to 20, up to 19, up to 18, up to 17, up to 16, up to 15, or up to 14 consecutive amino acid residues at or near the N-terminus of the wild type NiV-G cytoplasmic tail shown in SEQ ID NO. 4.

In some embodiments, the cytoplasmic tail of NiV-G is shown in SEQ ID NO. 4. In some embodiments, the variant NiV-G has a deletion of 5 to 41 consecutive amino acid residues at or near the N-terminus of the cytoplasmic tail of the wild type NiV-G protein shown in SEQ ID NO. 4. In some embodiments, the variant NiV-G has a deletion of 26 to 40 consecutive amino acid residues at or near the N-terminus of the cytoplasmic tail of the wild type NiV-G protein shown in SEQ ID NO. 4.

In some embodiments, at least one G protein is a variant NiV-G protein that is a biologically active portion of wild-type NiV-G. In some embodiments, the biologically active moiety is an N-terminally truncated fragment. In some embodiments, the variant NiV-G protein is truncated and lacks up to 5 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein, such as compared to the wild-type NiV-G shown in SEQ ID NO. 1. In some embodiments, the variant NiV-G protein is truncated and lacks up to 10 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein, such as compared to the wild-type NiV-G shown in SEQ ID NO. 1. In some embodiments, the variant NiV-G protein is truncated and lacks up to 15 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein, such as compared to the wild-type NiV-G shown in SEQ ID NO. 1. In some embodiments, the variant NiV-G protein is truncated and lacks up to 20 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein, such as compared to the wild-type NiV-G shown in SEQ ID NO. 1. In some embodiments, the variant NiV-G protein is truncated and lacks up to 25 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein, such as compared to the wild-type NiV-G shown in SEQ ID NO. 1. In some embodiments, the variant NiV-G protein is truncated and lacks up to 30 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein, such as compared to the wild-type NiV-G shown in SEQ ID NO. 1. In some embodiments, the variant NiV-G protein is truncated and lacks up to 35 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein, such as compared to the wild-type NiV-G shown in SEQ ID NO. 1. In some embodiments, the variant NiV-G protein (also referred to as variant NiV-G) contains an N-terminal methionine.

In some embodiments, the variant NiV-G has a deletion of amino acid residue 2-41、2-40、2-39、2-38、2-37、2-36、2-35、2-34、2-33、2-32、2-31、2-30、2-29、2-28、2-27、2-26、2-25、2-22、2-21、2-16、2-11 of SEQ ID NO. 4 or cytoplasmic tail of 2-5. In some embodiments, the cytoplasmic tail is a truncated portion of the cytoplasmic tail of Nipah virus shown in any one of SEQ ID NOs 6-28. In some embodiments, the cytoplasmic tail is a truncated portion of the cytoplasmic tail of Nipah virus as shown in any one of SEQ ID NOS: 6-28, which lacks an N-terminal methionine. In some embodiments, the variant NiV-G has a sequence in which the cytoplasmic tail, such as shown in any one of SEQ ID NOS: 6-28, is directly linked to the N-terminus of the sequence shown in SEQ ID NO: 2. In some embodiments, the variant NiV-G has a sequence in which the cytoplasmic tail shown in any one of SEQ ID NOS: 6-28 is directly linked to the N-terminus of the sequence shown in SEQ ID NO: 3. In some embodiments, the cytoplasmic tail is shown as SEQ ID NO. 7, 13 or 19.

In some embodiments, the variant NiV-G comprises the amino acid sequence set forth in SEQ ID NO: 211, 220, or 221, or an amino acid sequence that exhibits at least 85% sequence identity, at least 86% sequence identity, at least 87% sequence identity, at least 88% sequence identity, at least 89% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity with any of SEQ ID NO: 211, 220, or 221. In some embodiments, the truncated NiV-G cytoplasmic tail is the amino acid sequence shown as SEQ ID NO. 211, 220, or 221.

In some embodiments, the variant NiV-G comprises a modified cytoplasmic tail comprising a heterologous cytoplasmic tail of a glycoprotein from another virus, or a truncated portion thereof. In some embodiments, the other virus is a member of the kingdom positive RNA virus. In some embodiments, the other virus is a member of the Paramyxoviridae, rhabdoviridae, arenaviridae, or retrovirus families. In some embodiments, the other virus is a member of the Paramyxoviridae family.

In some embodiments, the variant NiV-G comprises a modified cytoplasmic tail wherein at least a portion of the native cytoplasmic tail (e.g., amino acids 1-45 corresponding to SEQ ID NO: 5) is replaced with a heterologous cytoplasmic tail from another viral glycoprotein or virus-associated protein, or a truncated portion thereof. In some embodiments, the alternative cytoplasmic tail is a heterologous cytoplasmic tail of at least 5 amino acids in length or a truncated portion thereof. In some embodiments, the length of the substituted heterologous cytoplasmic tail or truncated portion thereof is from or about 5 to 180 amino acids, such as from or about 5 to 150, from or about 5 to 100, from or about 5 to 75, from or about 5 to 50, from or about 5 to 40, from or about 5 to 30, from or about 5 to 20, from or about 5 to 10, from or about 10 to 150, from or about 10 to 100, from or about 10 to 75, from or about 10 to 50, from or about 10 to 40, from or about 10 to 30, from or about 10 to 20, from or about 20 to 150, from or about 20 to 100, from or about 20 to 75, from or about 20 to 50, from or about 20 to 40, from or about 30 to 150, from or about 30 to 100, from or about 30 to 75, from or about 30 to 150, from or about 40 to about 40, from or about 40 to about 50, from or about 50 to about 50, from or about 40 to 75, from or about 50 to about 50, from or about 50 to 75. In some embodiments, the length of the substituted heterologous cytoplasmic tail or truncated portion thereof is 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids. In some embodiments, the heterologous cytoplasmic tail or a truncated portion thereof is directly linked to the N-terminus of the sequence shown in SEQ ID NO. 2.

In some embodiments, the heterologous cytoplasmic tail is a cytoplasmic tail of a glycoprotein from another virus (such as paramyxovirus, retrovirus, filovirus, rhabdovirus, or arenavirus), or a truncated portion thereof. In some embodiments, the virus is a paramyxovirus other than nipah virus. For example, the virus is measles virus, bats paramyxovirus, cedar virus, canine distemper virus, sendai virus, hendra virus, human parainfluenza virus, or newcastle disease virus. In some embodiments, the replacement heterologous cytoplasmic tail is a native cytoplasmic tail of another virus or a truncated portion of a native cytoplasmic tail, such as the truncated portion of the cytoplasmic tail shown in any one of SEQ ID NOs 40-166. In some embodiments, the variant NiV-G has a sequence in which the heterologous cytoplasmic tail as set forth in any one of SEQ ID NOS: 40-166, or a truncated portion thereof, is directly linked to the N-terminus of the sequence set forth in SEQ ID NO. 2. In some embodiments, the variant NiV-G contains a mutation in the extracellular domain that reduces or eliminates binding to ephrin B2 or B3 corresponding to one or more of E501A, W504A, Q A and E533A, with the residue number shown in SEQ ID NO: 1. In some embodiments, the variant NiV-G has a sequence in which the heterologous cytoplasmic tail as set forth in any one of SEQ ID NOS: 40-166, or a truncated portion thereof, is directly linked to the N-terminus of the sequence set forth in SEQ ID NO: 3. In some embodiments, it will be appreciated that the heterologous cytoplasmic tail or truncated portion thereof can comprise any of the sequences shown in any of SEQ ID NOs 40-166 lacking an N-terminal methionine.

In some embodiments, the virus is a retrovirus. For example, the virus may be baboon endogenous virus (BaEV), gibbon ape leukemia virus (GaLV), murine leukemia virus, or human immunodeficiency virus 1 (HIV-1). In some embodiments, the replacement heterologous cytoplasmic tail is a native cytoplasmic tail or a truncated portion of a native cytoplasmic tail of another virus, such as shown in any one of SEQ ID NOs 167-168, 174-177, 179-182, or 185-199. In some embodiments, variant NiV-G wherein the heterologous cytoplasmic tail as shown in any one of SEQ ID NOS: 167-168, 174-177, 179-182, or 185-199, or a truncated portion thereof, is directly linked to the sequence N-terminal to the sequence shown in SEQ ID NO: 2. In some embodiments, the variant NiV-G contains a mutation in the extracellular domain that reduces or eliminates binding to ephrin B2 or B3 corresponding to one or more of E501A, W504A, Q A and E533A, with the residue number shown in SEQ ID NO: 1. In some embodiments, variant NiV-G wherein the heterologous cytoplasmic tail as set forth in any one of SEQ ID NOS: 167-168, 174-177, 179-182, or 185-199, or a truncated portion thereof, is directly linked to the sequence N-terminal to the sequence set forth in SEQ ID NO: 3. In some embodiments, it will be appreciated that the heterologous cytoplasmic tail or truncated portion thereof may comprise any of the sequences shown in any of SEQ ID NOs 167-168, 174-177, 179-182 or 185-199 lacking an N-terminal methionine.

In some embodiments, the virus is a filovirus. For example, the virus may be ebola virus (EboV). In some embodiments, the replacement heterologous cytoplasmic tail is a native cytoplasmic tail or a truncated portion of a native cytoplasmic tail of another virus, such as shown in any one of SEQ ID NOs 172 or 173. In some embodiments, the variant NiV-G has a sequence in which the heterologous cytoplasmic tail as shown in either SEQ ID NO. 172 or 173, or a truncated portion thereof, is directly linked to the N-terminus of the sequence shown in SEQ ID NO. 2. In some embodiments, the variant NiV-G contains a mutation in the extracellular domain that reduces or eliminates binding to ephrin B2 or B3 corresponding to one or more of E501A, W504A, Q A and E533A, with the residue number shown in SEQ ID NO: 1. In some embodiments, the variant NiV-G has a sequence in which the heterologous cytoplasmic tail as shown in either SEQ ID NO. 172 or 173, or a truncated portion thereof, is directly linked to the N-terminus of the sequence shown in SEQ ID NO. 3. In some embodiments, it is understood that the heterologous cytoplasmic tail or truncated portion thereof can comprise any sequence shown in any one of SEQ ID NOs 172 or 173 lacking an N-terminal methionine.

In some embodiments, the virus is a rhabdovirus. For example, the virus may be a Cocal vesicle virus (Cocal) or a Vesicular Stomatitis Virus (VSV). In some embodiments, the replacement heterologous cytoplasmic tail is a native cytoplasmic tail of another virus or a truncated portion of a native cytoplasmic tail, such as shown in any one of SEQ ID NOs 170, 171, 183, or 184. In some embodiments, the variant NiV-G has a sequence in which the heterologous cytoplasmic tail as shown in any one of SEQ ID NOS: 70, 171, 183, or 184, or a truncated portion thereof, is directly linked to the N-terminus of the sequence shown in SEQ ID NO: 2. In some embodiments, the variant NiV-G contains a mutation in the extracellular domain that reduces or eliminates binding to ephrin B2 or B3 corresponding to one or more of E501A, W504A, Q A and E533A, with the residue number shown in SEQ ID NO: 1. In some embodiments, the variant NiV-G has a sequence in which the heterologous cytoplasmic tail as shown in any one of SEQ ID NOS: 70, 171, 183, or 184, or a truncated portion thereof, is directly linked to the N-terminus of the sequence shown in SEQ ID NO: 3. In some embodiments, it is understood that the heterologous cytoplasmic tail or truncated portion thereof can comprise any sequence shown in any one of SEQ ID NOs 70, 171, 183, or 184 that lacks an N-terminal methionine.

In some embodiments, the virus is an arenavirus. For example, the virus may be lymphocytic choriomeningitis virus (LCMV). In some embodiments, the alternative heterologous cytoplasmic tail is the native cytoplasmic tail of another virus or a truncated portion of the native cytoplasmic tail, such as shown in SEQ ID NO. 178. In some embodiments, the variant NiV-G has a sequence in which the heterologous cytoplasmic tail shown in SEQ ID NO. 178, or a truncated portion thereof, is directly linked to the N-terminus of the sequence shown in SEQ ID NO. 2. In some embodiments, the variant NiV-G contains a mutation in the extracellular domain that reduces or eliminates binding to ephrin B2 or B3 corresponding to one or more of E501A, W504A, Q A and E533A, with the residue number shown in SEQ ID NO: 1. In some embodiments, the variant NiV-G has a sequence in which the heterologous cytoplasmic tail shown in SEQ ID NO. 178, or a truncated portion thereof, is directly linked to the N-terminus of the sequence shown in SEQ ID NO. 3. In some embodiments, it will be appreciated that the heterologous cytoplasmic tail or truncated portion thereof can comprise any sequence shown in any of SEQ ID NOs 178 lacking an N-terminal methionine.

In some embodiments, at least one variant NiV-G protein is truncated and lacks up to amino acid 34 at or near the N-terminus of the wild-type NiV-G protein, such as compared to the wild-type NiV-G shown in SEQ ID NO. 1. In some embodiments, the variant NiV-G protein (also referred to as variant NiV-G) contains an N-terminal methionine. In some embodiments, the variant NiV-G protein lacks amino acids 2-34 as compared to the wild-type NiV-G shown in SEQ ID NO. 1. In some embodiments, niV-G is as shown in SEQ ID NO: 228.

In a particular embodiment, at least one G protein has the amino acid sequence shown in SEQ ID NO. 228, or is a functionally active variant thereof or a biologically active portion thereof that retains binding and/or fusion activity. In some embodiments, functionally active variants comprise an amino acid sequence having at least equal to or about 80%, at least equal to or about 85%, at least equal to or about 90%, at least equal to or about 91%, at least equal to or about 92%, at least equal to or about 93%, at least equal to or about 94%, at least equal to or about 95%, equal to or about 96%, at least equal to or about 97%, at least equal to or about 98%, or at least equal to or about 99% sequence identity to SEQ ID No. 228, and retain fusion activity to bind to the variant NiV-F protein described. In some embodiments, at least one G protein is a variant G protein comprising the amino acid sequence of SEQ ID NO: 228.

In some embodiments, the variant NiV-G contains a heterologous cytoplasmic tail that is a cytoplasmic tail of a glycoprotein from CD63, or a truncated portion thereof. In some embodiments, the heterologous cytoplasmic tail replaces at least a portion of the native cytoplasmic tail of NiV-G (e.g., amino acids 1-45 corresponding to SEQ ID NO: 5). In some embodiments, the heterologous tail is a contiguous sequence of 5,6, 7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25N-terminal amino acids of the native cytoplasmic tail of CD 63. In some embodiments, the natural cytoplasmic tail of CD63 is set forth in SEQ ID NO. 200, 201, or 202. In some embodiments, the heterologous cytoplasmic tail is a truncated portion of the CD63 cytoplasmic tail as shown in any one of SEQ ID NOs 200-205. In some embodiments, the variant NiV-G has a sequence in which the heterologous cytoplasmic tail as set forth in any one of SEQ ID NOS: 200-205 is directly linked to the N-terminus of the sequence set forth in SEQ ID NO. 2. In some embodiments, the variant NiV-G has a sequence in which the heterologous cytoplasmic tail as set forth in any one of SEQ ID NOS: 200-205 is directly linked to the N-terminus of the sequence set forth in SEQ ID NO: 3.

In some embodiments, the variant NiV-G comprises a modified cytoplasmic tail comprising a mutant cytoplasmic tail of a glycoprotein from the same nipah virus. In some embodiments, the variant NiV-G comprises a modified cytoplasmic tail, wherein at least a portion of the native cytoplasmic tail (e.g., amino acids 1-45 corresponding to SEQ ID NO: 5) is a mutant portion thereof from a glycoprotein of Nipah virus. In some embodiments, the cytoplasmic tail is a mutant portion of the cytoplasmic tail of Nipah virus shown in any one of SEQ ID NOs 29-38. In some embodiments, it will be appreciated that the truncated NiV-G cytoplasmic tail may comprise the sequence shown in any one of SEQ ID NOs 29-38 lacking an N-terminal methionine. In some embodiments, the variant NiV-G has a sequence in which the cytoplasmic tail as set forth in any one of SEQ ID NOS: 29-38 is directly linked to the N-terminus of the sequence set forth in SEQ ID NO: 2. In some embodiments, the variant NiV-G has a sequence in which the cytoplasmic tail as set forth in any one of SEQ ID NOS: 29-38 is directly linked to the N-terminus of the sequence set forth in SEQ ID NO: 3.

In some embodiments, any of the provided lipid particles (lentiviral vectors) may further comprise an F protein, such as a NiV-F protein, such as a full-length NiV-F protein or a biologically active portion thereof, or a variant thereof. Also provided herein, for example, are viral particles or virus-like particles, such as lentiviral particles or lentiviral-like particles, pseudotyped with any provided variant NiV-G proteins and NiV-F proteins, such as full-length NiV-F proteins, or biologically active portions or variants thereof. Exemplary NiV-F proteins are further described in section II.B.

In certain embodiments, the paramyxovirus envelope attachment protein, the first paramyxovirus envelope attachment protein and/or the second paramyxovirus envelope attachment protein (such as at least one G protein or functionally active variant or biologically active portion thereof) is a protein that retains fusion activity in combination with other re-targeting attachment proteins (such as more than one G protein expressed as a multimer on a lipid bilayer). In certain embodiments, the first paramyxovirus envelope attachment protein and/or the second paramyxovirus envelope attachment protein and/or the third paramyxovirus envelope attachment protein (such as at least one G protein or functionally active variant or biologically active portion thereof) is a protein that retains fusion activity in combination with other re-targeting attachment proteins (such as more than one G protein expressed as a multimer on a lipid bilayer). In certain embodiments, the first paramyxovirus envelope attachment protein and/or the second paramyxovirus envelope attachment protein and/or the third paramyxovirus envelope attachment protein and/or the fourth paramyxovirus envelope attachment protein (such as at least one G protein or functionally active variant or biologically active portion thereof) is a protein that retains fusion activity in combination with other re-targeting attachment proteins (such as more than one G protein expressed as a multimer on a lipid bilayer). In certain embodiments, the first paramyxovirus envelope attachment protein and/or the second paramyxovirus envelope attachment protein and/or the third paramyxovirus envelope attachment protein and/or the fourth paramyxovirus envelope attachment protein and/or one or more additional paramyxovirus envelope attachment proteins (such as at least one G protein or functionally active variant or biologically active portion thereof) are proteins that retain fusion activity in combination with other heavy targeting attachment proteins (such as more than one G protein expressed as a multimer on a lipid bilayer). Fusion activity includes the activity of binding of paramyxovirus envelope attachment proteins to proteins that are paramyxovirus fusion proteins (e.g., F proteins) to promote or aid cytoplasmic fusion of two membrane lumens, such as a lumen of a targeted lipid particle having at least two paramyxovirus envelope attachment proteins and paramyxovirus fusion proteins (e.g., F and G proteins) embedded in its lipid bilayer, and a target cell (e.g., a cell containing a surface receptor or molecule recognized or bound by the targeted envelope proteins).

Reference to retaining fusion activity includes activity of a lipid particle (e.g., a lentiviral vector) comprising at least two paramyxovirus envelope adhesion proteins and paramyxovirus fusion proteins (e.g., F and G proteins), which is similar (such as comprising the same variant NiV-F) but comprises a level or extent of binding of equal to or about 10% to equal to or about 150% or more of a reference lipid particle (e.g., a lentiviral vector) comprising a corresponding wild-type G protein (such as shown in SEQ ID NO: 1). For example, a lipid particle (e.g., lentiviral vector) that retains fusion activity has a similar (such as at least or at least about 10% of the fusion activity level or extent of a reference lipid particle that contains the same variant NiV-F) but contains the corresponding wild-type G protein, such as at least or at least about 15% of the fusion activity level or extent, at least or at least about 20% of the fusion activity level or extent, at least or at least about 25% of the fusion activity level or extent, at least or at least about 30% of the fusion activity level or extent, at least or at least about 35% of the fusion activity level or extent, at least or at least about 40% of the fusion activity level or extent, at least or at least about 45% of the fusion activity level or extent, at least or at least about 50% of the fusion activity level or extent, at least or at least about 55% of the fusion activity level or extent, at least or at least about 60% of the fusion activity level or extent, at least or at least about 65% of the fusion activity level or extent, at least or at least about 70% of the fusion activity level or extent, at least or at least about 75% of the fusion activity level or extent, at least or at least about 80% of the fusion activity level or extent, at least or at least about 85% of the fusion activity level or at least about 100% of the fusion activity level or extent.

Reference to retaining fusion activity includes activity of a lipid particle (e.g., lentiviral vector) comprising at least two paramyxovirus envelope adhesion proteins and paramyxovirus fusion proteins (e.g., F and G proteins), which is similar (such as comprising the same variant NiV-F) but comprises only one of the provided paramyxovirus envelope adhesion proteins (e.g., G proteins) at or about 10% to at or about 150% or more of the binding level or degree of the reference lipid particle (e.g., lentiviral vector). For example, a lipid particle (e.g., lentiviral vector) that retains fusion activity has a similar (such as at least or at least about 10% of the fusion activity level or extent of a reference lipid particle containing only one of the provided paramyxovirus envelope attachment proteins, such as at least or at least about 15%, at least or at least about 20%, at least or at least about 25%, at least or at least about 30%, at least or at least about 35%, at least or at least about 40%, at least or at least about 45%, at least or at least about 50%, at least or at least about 55%, at least or at least about 60%, at least or at least about 65%, at least or at least about 70%, at least or at least about 75%, at least or at least about 80%, at least about 85%, at least about 95%, at least about 100%, or at least about 100% of the fusion activity level or at least about 100%.

A. mutant paramyxovirus G protein

In some embodiments, the G protein is a paramyxovirus G glycoprotein (e.g., a variant paramyxovirus G glycoprotein) comprising one or more amino acid mutations that result in reduced glycosylation of the protein. The one or more amino acid mutations (also referred to as deglycosylation mutations) may be one or more amino acid substitutions (also referred to as mutations).

In some embodiments, the mutant paramyxovirus G glycoprotein comprises an amino acid substitution at one or more amino acid positions, which amino acid substitution reduces glycosylation of the G glycoprotein. In some embodiments, one or more amino acid substitutions disrupt the glycosylation site of the N-linkage. In some embodiments, one or more amino acid substitutions disrupt the glycosylation site of the O linkage.

In some embodiments, the mutant paramyxovirus G glycoprotein is derived from measles virus (e.g., measles virus (MeV), canine distemper virus, whale measles virus, peste des petits ruminants virus, seal distemper virus, rinderpest virus), henipav virus (e.g., hendra virus (HeV), nipah virus (NiV), cedar virus (CedPV), ink-river virus, langevirus, or bat paramyxovirus). In some embodiments, the mutant paramyxovirus G glycoprotein is a mutant of a paramyxovirus G glycoprotein derived from nipah virus or measles virus. In some embodiments, the mutant paramyxovirus G protein is a mutant of a paramyxovirus G protein selected from the group consisting of SEQ ID NOs: 1, 561-564, or a modified paramyxovirus G glycoprotein derived from any of 1, 5, 561-564 that contains an altered cytoplasmic tail. In some embodiments, the mutant paramyxovirus G protein has an amino acid sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95% sequence identity to any one of SEQ ID NOs 1, 5, 561-564, and contains an acid substitution at one or more amino acid positions that reduce glycosylation of the G glycoprotein as provided herein. In some embodiments, the mutant paramyxovirus G protein having one or more amino acid mutations that result in reduced glycosylation is a mutant of truncated NiV-G as shown in SEQ ID NO. 228.

The predicted position of the glycosylation site can be determined using the sequence of the protein. For example, N-glycosylation typically occurs at a site having the sequence N-X-S/T, where "X" is any amino acid other than P. Various algorithms and tools are available for predicting N-and O-linked glycosylation, including those described in SprintGly (http://sparks-lab.org/server/sprint-gly/)、NetNGlyc (https://services.healthtech.dtu.dk/service.phpNetNGlyc-1.0)、NetOGlyc (https://services.healthtech.dtu.dk/service.phpNetOGlyc-4.0) and GlycoMine ^struct (http:// glycomine. Erc. Monash. Edu/Lab/GlycoMine _Structure /), and Pitti et al, sci. Reports, 9:15975 (2019) and Pakhrin et al, molecules 26:7314 (2021). Any predicted glycosylation site may be substituted as described herein.

In some embodiments, the paramyxovirus G glycoprotein subjected to deglycosylation mutation is NiV-G shown in SEQ ID NO: 1 or a modified NiPa G glycoprotein (NiV-G) having an altered cytoplasmic tail as compared to native NiV-G (e.g., SEQ ID NO: 1). In some embodiments, the variant paramyxovirus G protein has an amino acid sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95% sequence identity to SEQ ID NO. 1 and contains an acid substitution at one or more amino acid positions that reduce glycosylation of the G glycoprotein as provided herein. In some embodiments, the paramyxovirus G glycoprotein subjected to deglycosylation mutation is NiV-G shown in SEQ ID NO. 5 or a modified NiPa G glycoprotein (NiV-G) having an altered cytoplasmic tail as compared to the native NiV-G (e.g., SEQ ID NO. 1). In some embodiments, the variant paramyxovirus G protein has an amino acid sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95% sequence identity to SEQ ID NO. 5 and contains an acid substitution at one or more amino acid positions that reduce glycosylation of the G glycoprotein as provided herein.

Exemplary modified NiV-G proteins with altered cytoplasmic tail are described in section ii.a.1, which may incorporate one or more amino acid substitutions for reducing glycosylation.

Amino acid positions for substitution are described herein with positions "corresponding to" the reference sequence. It will be appreciated that amino acid substitutions are not limited to being made only in the reference sequence, but may also be made in similar sequences by identifying residues that align or correspond to the reference position. For example, a position that "corresponds to" a protein position in a reference sequence may be identified after alignment of the similar sequence to the reference sequence based on structural sequence alignment or using a standard alignment algorithm (such as the GAP algorithm). By aligning the sequences, the person skilled in the art can identify the corresponding residues, for example using conserved and identical amino acid residues as guidance. For example, the mutated amino acid positions are described herein with reference to the exemplary truncated NiV-G sequence shown in SEQ ID NO. 5, however, by sequence alignment and identification of corresponding residues, similar amino acid positions for the described mutations can be generated in other modified NiV-G sequences, such as any of the sequences described in section II.A.1.

In some embodiments, one or more amino acid mutations are at positions corresponding to positions 39, 126, 128, 273, 345, 384, 448 and 496 of SEQ ID NO. 5. In some embodiments, the variant paramyxovirus G glycoprotein comprises an amino acid mutation at any one of positions 39, 126, 128, 273, 345, 384, 448 and 496 of SEQ ID NO. 5. In some embodiments, the variant paramyxovirus G glycoprotein comprises two or more amino acid mutations at any of positions 39, 126, 128, 273, 345, 384, 448 and 496 corresponding to SEQ ID NO. 5, such as mutations at 2,3, 4, 5, 7 or 8 of said positions.

In some embodiments, one or more amino acid mutations is at a position corresponding to position 39 of SEQ ID NO. 5. In some embodiments, one or more amino acid mutations is at a position corresponding to position 126 of SEQ ID NO. 5. In some embodiments, one or more amino acid mutations is at a position corresponding to position 128 of SEQ ID NO. 5. In some embodiments, one or more amino acid mutations is at a position corresponding to position 273 of SEQ ID NO. 5. In some embodiments, one or more amino acid mutations is at a position corresponding to position 345 of SEQ ID NO. 5. In some embodiments, one or more amino acid mutations is at a position corresponding to position 384 of SEQ ID NO. 5. In some embodiments, one or more amino acid mutations is at a position corresponding to position 448 of SEQ ID NO. 5. In some embodiments, one or more amino acid mutations is at a position corresponding to position 496 of SEQ ID NO. 5.

In some embodiments, the natural amino acid at the position comprising the amino acid mutation is asparagine or serine. In some embodiments, the amino acid mutation is an amino acid substitution. In some embodiments, the mutation is an asparagine-to-glutamine substitution. In some embodiments, the mutation is a serine to alanine substitution.

In some embodiments, the mutation is an asparagine-to-glutamine substitution (N39Q) at a position corresponding to position 39 of SEQ ID NO. 5. In some embodiments, the mutation is an asparagine-to-glutamine substitution (N126Q) at a position corresponding to position 126 of SEQ ID NO. 5. In some embodiments, the mutation is an asparagine-to-glutamine substitution (N273Q) at a position corresponding to position 273 of SEQ ID No. 5. In some embodiments, the mutation is an asparagine-to-glutamine substitution (N345Q) at a position corresponding to position 345 of SEQ ID No. 5. In some embodiments, the mutation is an asparagine-to-glutamine substitution (N384Q) at a position corresponding to position 384 of SEQ ID NO. 5. In some embodiments, the mutation is an asparagine-to-glutamine substitution (N448Q) at a position corresponding to position 448 of SEQ ID NO. 5. In some embodiments, the mutation is an asparagine-to-glutamine substitution (N496Q) at a position corresponding to position 496 of SEQ ID No. 5.

In some embodiments, the mutation is a serine to alanine substitution at a position corresponding to position 128 of SEQ ID NO. 5 (S128A).

In some embodiments, the G glycoprotein is derived from nipah virus G protein, and the one or more amino acid substitutions is at a position corresponding to a position selected from the group consisting of 39, 126, 128, 273, 345, 384, 448, and 496 of SEQ ID No. 5. In some embodiments, the one or more amino acid substitutions are selected from N39Q, N126Q, S A, N273Q, N345Q, N384Q, N448Q, N496Q or any combination thereof. In some embodiments, the G glycoprotein is a mutant NiV-G that contains one amino acid substitution from any of N39Q, N126Q, S, A, N273Q, N345Q, N384Q, N448Q, N496Q. In some embodiments, the G glycoprotein is a mutant NiV-G that contains two amino acid substitutions from any two of N39Q, N126Q, S, A, N273Q, N345Q, N384Q, N448Q, N496Q. In some embodiments, the G glycoprotein is a mutant NiV-G that contains three amino acid substitutions from any three of N39Q, N126Q, S, A, N273Q, N345Q, N384Q, N448Q, N496Q. In some embodiments, the G glycoprotein is a mutant NiV-G that contains four amino acid substitutions from any of N39Q, N126,126 126Q, S128, 128A, N273Q, N345Q, N384Q, N448Q, N Q. In some embodiments, the G glycoprotein is a mutant NiV-G that contains five amino acid substitutions from any of N39Q, N126,126 126Q, S128, 128A, N273Q, N345Q, N384Q, N448Q, N Q. In some embodiments, the G glycoprotein is a mutant NiV-G that contains six amino acid substitutions from any of N39Q, N126,126 126Q, S128, 128A, N273Q, N345Q, N384Q, N448Q, N Q. In some embodiments, the G glycoprotein is a mutant NiV-G that contains seven amino acid substitutions from any of N39Q, N126,126 126Q, S128, 128A, N273Q, N345Q, N384Q, N448Q, N Q. In some embodiments, the G glycoprotein is a mutant NiV-G that contains eight amino acid substitutions from any of N39Q, N126,126 126Q, S128, 128A, N273Q, N345Q, N384Q, N448Q, N Q. In some embodiments, one or more amino acid substitutions is in SEQ ID NO. 5 or in a modified NiPa G glycoprotein (NiV-G) having an altered cytoplasmic tail as compared to the native NiV-G (e.g., SEQ ID NO. 5). In some embodiments, the amino acid substitutions are in a modified NiV-G protein described in section ii.a. In some embodiments, the amino acid substitution is in NiV-G as set forth in SEQ ID NO. 5.

In some embodiments, the variant nipag protein comprises at least three amino acid substitutions. In some embodiments, amino acid substitutions are at positions 273, 384 and 496 of SEQ ID NO. 5. In some embodiments, amino acid substitutions are at positions 273, 345 and 496 of SEQ ID NO. 5. In some embodiments, the amino acid substitutions are at positions 39, 126, and 128 of SEQ ID NO. 5. In some embodiments, amino acid substitutions are at positions 39, 273, and 345 of SEQ ID NO. 5. In some embodiments, amino acid substitutions are at positions 39, 384 and 448 of SEQ ID NO. 5. In some embodiments, the amino acid substitutions are at positions 39, 448 and 496 of SEQ ID NO. 5. In some embodiments, amino acid substitutions are at positions 39, 128 and 273 of SEQ ID NO. 5. In some embodiments, amino acid substitutions are at positions 39, 345 and 384 of SEQ ID NO. 5. In some embodiments, amino acid substitutions are at positions 39, 384 and 448 of SEQ ID NO. 5.

In some embodiments, the variant nipag protein comprises at least two amino acid substitutions. In some embodiments, the amino acid substitutions are at positions 273 and 496 of SEQ ID NO. 5. In some embodiments, the amino acid substitutions are at positions 345 and 496 of SEQ ID NO. 5. In some embodiments, the amino acid substitutions are at positions 39 and 128 of SEQ ID NO. 5. In some embodiments, the amino acid substitutions are at positions 39 and 345 of SEQ ID NO. 5. In some embodiments, the amino acid substitutions are at positions 39 and 448 of SEQ ID NO. 5. In some embodiments, the amino acid substitutions are at positions 39 and 496 of SEQ ID NO. 5. In some embodiments, the amino acid substitutions are at positions 39 and 273 of SEQ ID NO. 5. In some embodiments, the amino acid substitutions are at positions 39 and 384 of SEQ ID NO. 5. In some embodiments, amino acid substitutions are at positions 384 and 448 of SEQ ID NO. 5.

In some embodiments, the amino acid substitution is at position 39 of SEQ ID NO. 5. In some embodiments, the amino acid substitution is at position 126 of SEQ ID NO. 5. In some embodiments, the amino acid substitution is at position 128 of SEQ ID NO. 5. In some embodiments, the amino acid substitution is at position 273 of SEQ ID NO. 5. In some embodiments, the amino acid substitution is at position 345 of SEQ ID NO. 5. In some embodiments, the amino acid substitution is at position 384 of SEQ ID NO. 5. In some embodiments, the amino acid substitution is at position 448 of SEQ ID NO. 5. In some embodiments, the amino acid substitution is at position 496 of SEQ ID NO. 5.

In some embodiments, the mutant nipag protein comprises an asparagine-to-glutamine substitution at position 39 of SEQ ID No. 5. In some embodiments, the mutant nipag protein comprises an asparagine-to-glutamine substitution at position 126 of SEQ ID No. 5. In some embodiments, the mutant nipag protein comprises an asparagine-to-glutamine substitution at position 273 of SEQ ID No. 5. In some embodiments, the mutant nipag protein comprises an asparagine-to-glutamine substitution at position 345 of SEQ ID NO: 5. In some embodiments, the mutant nipag protein comprises an asparagine-to-glutamine substitution at position 384 of SEQ ID No. 5. In some embodiments, the mutant nipag protein comprises an asparagine-to-glutamine substitution at position 448 of SEQ ID No. 5. In some embodiments, the mutant nipag protein comprises an asparagine-to-glutamine substitution at position 496 of SEQ ID No. 5. In some embodiments, the mutant nipag protein comprises a serine to alanine substitution at position 128 of SEQ ID No. 5.

In some embodiments, the mutant nipag proteins comprise a sequence selected from the group consisting of any one of SEQ ID NOs 640-766, such as any of the exemplary mutant nipag proteins shown in table 2A below. In some embodiments, the mutant Nipag G protein comprises the sequence of SEQ ID NO. 660. In some embodiments, the variant Nipag G protein comprises the sequence of SEQ ID NO: 663. In some embodiments, the variant Nipag protein comprises the sequence of SEQ ID NO: 667.

In some embodiments, the paramyxovirus G glycoprotein subjected to deglycosylation mutation is measles virus H (Mev-H) protein or a modified MeV-H protein having an altered cytoplasmic tail as compared to native MeV-H (e.g., SEQ ID NO: 769). In some embodiments, a mutant paramyxovirus G protein has an amino acid sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95% sequence identity to SEQ ID No. 769 and contains an acid substitution at one or more amino acid positions that reduce glycosylation of the G glycoprotein as provided herein.

In some embodiments, the G glycoprotein is derived from measles virus H (Mev-H) protein and the one or more amino acid substitutions is at a position corresponding to a position selected from the group consisting of 168, 187, 200, 215, 238 of SEQ ID No. 769. In some embodiments, the mutant Mev-H protein comprises at least two amino acid substitutions, such as 2, 3,4, or 5 substitutions at positions 168, 187, 200, 215, 238 of SEQ ID No. 769.

In some embodiments, the paramyxovirus G glycoprotein subjected to deglycosylation mutation is a canine distemper virus H (CDV-H) protein or a modified CDV-H protein having an altered cytoplasmic tail as compared to native CDV-H (e.g., SEQ ID NO: 770). In some embodiments, a mutant paramyxovirus G protein has an amino acid sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95% sequence identity to SEQ ID NO 770 and contains an acid substitution at one or more amino acid positions that reduce glycosylation of the G glycoprotein as provided herein.

In some embodiments, the G glycoprotein is derived from a canine distemper virus H (CDV-H) protein, and one or more amino acid substitutions are at a position corresponding to a position selected from the group consisting of 19, 149, 422 of SEQ ID NO. 770. In some embodiments, the variant CDV-H protein comprises at least two amino acid substitutions, such as 2 or 3 substitutions at positions 19, 149, 422 of SEQ ID NO. 770.

2. Heavy targeting attachment proteins

In some embodiments, a paramyxovirus envelope attachment protein such as a G protein (e.g., niV-G) is further attached or linked in series with at least two binding domains that bind the first and second target molecules, respectively, to comprise a re-targeting attachment protein. For example, in some aspects, a lipid particle is provided comprising a targeted paramyxovirus envelope attachment protein (e.g., chimeric attachment G protein) comprising any of the G proteins provided above attached (e.g., operably fused in tandem) to a first binding domain and a second binding domain, wherein a heavy targeted attachment protein (e.g., heavy targeted G protein) is exposed on the surface of the targeted lipid particle (e.g., lentiviral vector). In some of any of the provided embodiments, the lipid particle comprises a re-targeting attachment protein comprising (i) a paramyxovirus envelope attachment protein, and (ii) a first targeting moiety directed against a first target molecule expressed on the surface of a target cell, and (iii) a second targeting moiety directed against a second target molecule expressed on the surface of a target cell.

In some embodiments, each of the one or more paramyxovirus envelope attachment proteins, such as a G protein (e.g., niV-G), is further attached or linked in series to a first targeting moiety and a second targeting moiety (e.g., first and second binding domains or binding agents) that are directed against a first target molecule and a second target molecule expressed on the surface of a target cell. The binding domain or binding agent may be independently selected from any of the binding domains or binding agents described herein (e.g., in section II). Thus, in some embodiments, the lipid particle comprises one or more re-targeting attachment proteins, wherein each of the one or more re-targeting attachment proteins independently comprises (i) a paramyxovirus envelope attachment protein, and (ii) a first targeting moiety for a first target molecule expressed on the surface of a target cell, and (iii) a second targeting moiety for a second target molecule expressed on the surface of a target cell. The targeting moiety may be a binding domain or a binding agent, such as any of the binding domains or any binding agents described herein (e.g., in section II).

In some embodiments, the envelope attachment protein is a re-targeted attachment protein comprising henipav viral G protein or a biologically active portion thereof. In some embodiments, the envelope adhesion protein (e.g., G protein) can be re-targeted, such as a re-targeted adhesion protein, by being linked in tandem to a targeting moiety, such as a binding molecule (e.g., an antibody or antigen binding fragment, e.g., sdAb or scFv) that binds to a target cell. In some embodiments, the re-targeted attachment protein and the paramyxovirus fusion protein (e.g., the G protein and NiV-F protein provided herein) together exhibit fusion activity against a target cell, such as to deliver an exogenous agent or a nucleic acid exogenous agent to the target cell.

In some embodiments, the lipid particle comprises at least two re-targeting attachment proteins comprising a paramyxovirus envelope attachment protein (e.g., a G protein), wherein at least one re-targeting attachment protein is re-targeted by being serially connected (e.g., operably serially fused) to a first targeting moiety and a second targeting moiety, such as a binding molecule (e.g., an antibody or antigen binding fragment, e.g., sdAb or scFv) that binds to a target cell.

In some embodiments, the re-targeting attachment protein is re-targeted by tandem connection to a first targeting moiety and a second targeting moiety, wherein the targeting moiety is directed against a target molecule expressed on the surface of a target cell. In some embodiments, the re-targeting attachment protein is re-targeted by being serially linked to a first targeting moiety and a second targeting moiety, wherein the first targeting moiety and the second targeting moiety are directed against the same target molecule expressed on the surface of the target cell. In some embodiments, the re-targeting attachment protein is re-targeted by being serially linked to a first targeting moiety and a second targeting moiety, wherein the first targeting moiety and the second targeting moiety are directed against different first target molecules and second target molecules expressed on the surface of the target cell. In some embodiments, targeting one or both of the first and second target molecules does not activate or inhibit the target cell, induce a phenotypic change (e.g., maturation and/or differentiation) of the target cell, induce proliferation of the target cell, and/or induce apoptosis of the target cell.

In some embodiments, the lipid particle comprises at least three re-targeting attachment proteins comprising a paramyxovirus envelope attachment protein (e.g., a G protein), wherein at least one re-targeting attachment protein is re-targeted by being serially linked to a first targeting moiety and a second targeting moiety, such as a binding molecule (e.g., an antibody or antigen binding fragment, e.g., sdAb or scFv) that binds to a target cell. In some embodiments, the lipid particle comprises at least three re-targeting attachment proteins comprising an envelope attachment protein (e.g., a G protein), wherein at least two re-targeting attachment proteins are re-targeted by being serially linked to a first targeting moiety and a second targeting moiety, such as a binding molecule (e.g., an antibody or antigen binding fragment, e.g., sdAb or scFv) that binds to a target cell. In some embodiments, the lipid particle comprises at least three re-targeting attachment proteins comprising an envelope attachment protein (e.g., a G protein), wherein the at least three re-targeting attachment proteins are re-targeted by being serially linked to a first targeting moiety and a second targeting moiety, such as a binding molecule (e.g., an antibody or antigen binding fragment, e.g., sdAb or scFv) that binds to a target cell.

In some embodiments, the first, second, and third re-targeting attachment proteins are re-targeted by being serially linked to a first targeting moiety and a second targeting moiety, wherein the targeting moiety is independently directed against a first target molecule and a second target molecule expressed on the surface of the target cell. In some embodiments, the first, second, and third heavy targeting attachment proteins are retargeted by being serially connected to the first, second, and third targeting moieties, wherein the first and second targeting moieties, or a second targeting moiety and a third targeting moiety, or a first targeting moiety, a second targeting moiety and a third targeting moiety, are directed against the same target molecule expressed on the surface of the target cell. In some embodiments, the first, second, and third re-targeting attachment proteins are re-targeted by being serially connected to the first, second, and third targeting moieties, wherein the first, second, and third targeting moieties are directed against different first, second, and third target molecules expressed on the surface of the target cell. In some embodiments, targeting one, two, or three of the first, second, and third target molecules does not activate or inhibit the target cell, induce a phenotypic change (e.g., maturation and/or differentiation) of the target cell, induce proliferation of the target cell, and/or induce apoptosis of the target cell.

In some embodiments, the lipid particle comprises at least four or at least five re-targeting attachment proteins comprising paramyxovirus envelope attachment proteins (e.g., G proteins), wherein at least one, at least two, at least three, or at least four re-targeting attachment proteins are re-targeted by being serially linked to a first targeting moiety and a second targeting moiety such as a binding molecule (e.g., an antibody or antigen binding fragment, e.g., sdAb or scFv) that binds to a target cell. In some embodiments, the lipid particle comprises at least four or at least five re-targeting attachment proteins comprising an envelope attachment protein (e.g., a G protein), wherein at least two or at least three re-targeting attachment proteins are re-targeted by being serially linked to a first targeting moiety and a second targeting moiety such as a binding molecule (e.g., an antibody or antigen binding fragment, e.g., sdAb or scFv) that binds to a target cell. In some embodiments, the lipid particle comprises at least four or at least five re-targeting attachment proteins comprising an envelope attachment protein (e.g., a G protein), wherein at least four or at least five re-targeting attachment proteins are re-targeted by being linked in tandem to a targeting moiety such as a binding molecule (e.g., an antibody or antigen binding fragment, e.g., sdAb or scFv) that binds to a target cell.

In some embodiments, the paramyxovirus re-targeting attachment protein is a targeting envelope protein provided herein that contains a G protein. In some embodiments, the paramyxovirus re-targeting attachment protein comprises at least one envelope attachment protein (e.g., a G protein) that is any of those provided in section ii.a, including a NiV-G protein having a cytoplasmic domain modification, a truncated NiV-G cytoplasmic tail, or a modified NiV-G cytoplasmic tail.

In some of any of the embodiments, the re-targeting attachment protein comprises (a) a re-targeting attachment protein comprising a first paramyxovirus envelope attachment protein operably fused in tandem with (i) a first targeting moiety directed against a first target molecule expressed on the surface of a target cell and (ii) a second targeting moiety directed against a second target molecule expressed on the surface of a target cell. In some embodiments, the targeting moiety is a binding domain, such as any of the binding domains or binders described in section ii.a.2 herein, e.g., a T cell binding domain or a HSC binding domain. In some embodiments, the binding domain can be any agent that binds to a cell surface molecule on a target cell. In some embodiments, the binding domain may be an antibody or antibody portion or fragment. In some embodiments, the binding domain is a single domain antibody (sdAb). In some embodiments, the binding domain is a single chain variable fragment (scFv). The binding domain may be directly or indirectly linked to the G protein. In certain embodiments, the binding domain is linked to the C-terminus (C-terminal amino acid) of the G protein or biologically active portion thereof. The ligation may be performed by a peptide linker (such as a flexible peptide linker).

The re-targeting attachment protein comprising a first paramyxovirus envelope attachment protein operably fused in tandem with (i) a first targeting moiety directed against a first target molecule expressed on the surface of a target cell and (ii) a second targeting moiety can be modulated to have different binding strengths. For example, scFv and antibodies with various binding strengths can be used to alter the fusion activity of a re-targeted attachment protein to cells exhibiting high or low amounts of target antigen. For example, DARPin with different affinities can be used to alter fusion activity on cells exhibiting high or low amounts of target antigen. The binding domain can also be modulated to target different regions on the target ligand, which will affect the rate of fusion with the cells displaying the target.

The binding domain may comprise a humanized antibody molecule, a full IgA, igG, igE or IgM antibody, a bispecific or multispecific antibody (e.g., zybodies, etc.), an antibody fragment, such as a Fab fragment, fab ' fragment, F (ab ') 2 fragment, fd ' fragment, fd fragment, and isolated CDRs or collections thereof, a single chain Fv, a polypeptide-Fc fusion, a single domain antibody (e.g., a shark single domain antibody, such as IgNAR or fragment thereof), a camelidae antibody, a masking antibody (e.g., probodies "), small Modular ImmunoPharmaceuticals (" SMIPsTM "), a single chain or tandem diabody (TandAb), VHH, ANTICALINS, nanobodies, minibody (minibodies), biTE, ankyrin repeat protein or DARPINs, avimers, DART, TCR-like antibody, ADNECTINS, affilins, trans-bodies, affibodies, trimerX, microProteins, fynomers, CENTYRINS, and KALBITOR. The targeting moiety may also include an antibody or antigen binding fragment thereof (e.g., fab ', F (ab') 2, fv fragment, scFv antibody fragment, disulfide-linked Fv (sdFv), fd fragment consisting of VH and CH1 domains, linear antibody, single domain antibody such as sdAb (VL or VH), nanobody, or camelidae VHH domain), antigen binding fibronectin type III (Fn 3) scaffold such as fibronectin polypeptide miniantibody, ligand, cytokine, chemokine, or T Cell Receptor (TCR).

The binding domain may comprise a humanized antibody molecule, a full IgA, igG, igE or IgM antibody, a bispecific or multispecific antibody (e.g., zybodies, etc.), an antibody fragment, such as a Fab fragment, fab ' fragment, F (ab ') 2 fragment, fd ' fragment, fd fragment, and isolated CDRs or collections thereof, a single chain Fv, a polypeptide-Fc fusion, a single domain antibody (e.g., a shark single domain antibody, such as IgNAR or fragment thereof), a camelidae antibody, a masking antibody (e.g., probodies "), small Modular ImmunoPharmaceuticals (" SMIPsTM "), a single chain or tandem diabody (TandAb), VHH, ANTICALINS, nanobodies, minibody (minibodies), biTE, ankyrin repeat protein or DARPINs, avimers, DART, TCR-like antibody, ADNECTINS, affilins, trans-bodies, affibodies, trimerX, microProteins, fynomers, CENTYRINS, and KALBITOR. The targeting moiety may also include an antibody or antigen binding fragment thereof (e.g., fab ', F (ab') 2, fv fragment, scFv antibody fragment, disulfide-linked Fv (sdFv), fd fragment consisting of VH and CH1 domains, linear antibodies, single domain antibodies such as sdAb (VL or VH), nanobody, or camelidae VHH domain), antigen binding fibronectin type III (Fn 3) scaffold such as fibronectin polypeptide minibody, or T Cell Receptor (TCR). In some embodiments, the binding domain does not comprise a ligand, cytokine or chemokine,

In some embodiments, the binding domain is a single chain molecule. In some embodiments, the binding domain is a single domain antibody. In some embodiments, the binding domain is a single-chain variable fragment. In particular embodiments, the binding domain comprises a human or humanized antibody variable sequence.

In some embodiments, the binding domain is a single domain antibody. In some embodiments, single domain antibodies may be human or humanized. In some embodiments, the single domain antibody or portion thereof is naturally occurring. In some embodiments, the single domain antibody or portion thereof is synthetic.

In some embodiments, a single domain antibody is an antibody whose complementarity determining region is part of a single domain polypeptide. In some embodiments, the single domain antibody is a heavy chain-only antibody variable domain. In some embodiments, the single domain antibody does not comprise a light chain.

In some embodiments, heavy chain antibodies lacking a light chain are referred to as VHHs. In some embodiments, the single domain antibody has a molecular weight of 12-15 kDa. In some embodiments, the single domain antibody comprises a camelidae antibody or a shark antibody. In some embodiments, the single domain antibody molecule is derived from an antibody produced in a species in the family camelidae, such as camel, llama, dromedary, alpaca, camel, and alpaca. In some embodiments, the single domain antibody is referred to as an immunoglobulin neoantigen receptor (IgNAR) and is derived from cartilaginous fish. In some embodiments, the single domain antibody is produced by cleaving the dimeric variable domain of human or mouse IgG into monomers and camelizing the critical residues.

In some embodiments, single domain antibodies can be generated from a display library (e.g., a phage display library). In some embodiments, the display library is generated from a library of VHHs from the family Camelidae immunized with various antigens as described in Arbabi et al, FEBS Letters, 414, 521-526 (1997), lauwereys et al, EMBO J., 17, 3512-3520 (1998), DECANNIERE et al, structures, 7, 361-370 (1999). In some embodiments, a display library is generated comprising fragments of antibodies of the non-immunized camelidae. In some embodiments, a single domain antibody human single domain antibody library is synthetically generated by introducing diversity into one or more scaffolds.

In some embodiments, the binding domain is a single domain antibody (sdAb). In some embodiments, the binding domain is a single chain variable fragment (scFv). The binding domain may be directly or indirectly linked to a paramyxovirus envelope attachment protein, a first paramyxovirus envelope attachment protein and/or a second paramyxovirus envelope attachment protein (e.g., a G protein and/or a re-targeting attachment protein). In certain embodiments, the binding domain is linked to the C-terminus (C-terminal amino acid) of the G protein or biologically active portion thereof. The ligation may be performed by a peptide linker (such as a flexible peptide linker). In certain embodiments, the first targeting moiety and/or the second targeting moiety is operably fused in tandem with a paramyxovirus envelope attachment protein.

In some embodiments, the C-terminus of the binding domain is attached to the C-terminus of the G protein or biologically active portion thereof. In some embodiments, the N-terminus of the binding domain is exposed on the outer surface of the lipid bilayer. In some embodiments, the N-terminus of the binding domain binds to a cell surface molecule of a target cell. In some embodiments, the binding domain specifically binds to a cell surface molecule present on a target cell. In some embodiments, the cell surface molecule is a protein, glycan, lipid, or low molecular weight molecule. In some embodiments, the binding domain is any of the binding domains as described above.

In some embodiments, a binding domain (e.g., an sdAb or one of any of the binding domains as described herein) binds to a cell surface antigen of a cell. In some embodiments, the cell surface antigen is characteristic of a type of cell. In some embodiments, the cell surface antigen is characteristic of more than one type of cell.

In some embodiments, the cell surface molecule of the target cell is an antigen or a portion thereof. In some embodiments, a single domain antibody or portion thereof is an antibody having a monomeric single domain antigen binding/recognition domain capable of selectively binding to a particular antigen. In some embodiments, the single domain antibody binds to an antigen present on a target cell.

Exemplary cells include polymorphonuclear cells (also known as PMN, PML, PMNL or granulocytes), stem cells, embryonic stem cells, neural stem cells, mesenchymal Stem Cells (MSCs), hematopoietic Stem Cells (HSCs), human myogenic stem cells, muscle-derived stem cells (MuStem), embryonic stem cells (ES or ESCs), limbal epithelial stem cells, cardiomyocytes, progenitor cells, immune effector cells, lymphocytes, macrophages, dendritic cells, natural killer cells, T cells, cytotoxic T lymphocytes, allogeneic cells, resident cardiomyocytes, induced pluripotent stem cells (iPS), adipose-derived or phenotypically modified stem or progenitor cells, cd133+ cells, aldehyde dehydrogenase positive cells (aldh+), umbilical Cord Blood (UCB) cells, peripheral Blood Stem Cells (PBSCs), neurons, neural progenitor cells, pancreatic beta cells, glial cells, or hepatocytes.

In some embodiments, the target cell is a cell of a target tissue. The target tissue may include liver, lung, heart, spleen, pancreas, gastrointestinal tract, kidney, testis, ovary, brain, reproductive organs, central nervous system, peripheral nervous system, skeletal muscle, endothelium, inner ear or eye.

In some embodiments, the target cell is a muscle cell (e.g., skeletal muscle cell), a kidney cell, a liver cell (e.g., liver cell), or a cardiac muscle cell (CARDIAC CELL) (e.g., cardiac muscle cell (cardiomyocyte)). In some embodiments, the target cell is a cardiac cell (e.g., cardiomyocyte (e.g., resting cardiomyocyte)), a hepatoblast (e.g., bile duct hepatoblast), an epithelial cell, a T cell (e.g., naive T cell), a macrophage (e.g., tumor infiltrating macrophage), or a fibroblast (e.g., cardiac fibroblast).

In some embodiments, the target cell is a tumor-infiltrating lymphocyte, T cell, neoplastic or tumor cell, virus-infected cell, stem cell, central Nervous System (CNS) cell, hematopoietic Stem Cell (HSC), hepatocyte, or fully differentiated cell. In some embodiments, the target cell is a cd3+ T cell, a cd4+ T cell, a cd8+ T cell, a liver cell, a hematopoietic stem cell, a cd34+ hematopoietic stem cell, a cd105+ hematopoietic stem cell, a cd117+ hematopoietic stem cell, a cd105+ endothelial cell, a B cell, a cd20+ B cell, a cd19+ B cell, a cancer cell, a cd133+ cancer cell, an epcam+ cancer cell, a cd19+ cancer cell, a Her2/neu+ cancer cell, a glua2+ neuron, a glua4+ neuron, a nkg2d+ natural killer cell, a slc1a3+ astrocyte, a slc7a10+ adipocyte, or a cd30+ lung epithelial cell.

In some embodiments, the target cell is an antigen presenting cell, MHC class ii+ cell, professional antigen presenting cell, atypical antigen presenting cell, macrophage, dendritic cell, bone marrow dendritic cell, plasmacytoid dendritic cell, cd11c+ cell, cd11b+ cell, spleen cell, B cell, liver cell, endothelial cell, or non-cancerous cell. In some embodiments, the first target molecule and the second target molecule are present on the same target cell. In some embodiments, the first target molecule and the second target molecule are present on different cells.

In some embodiments, the binding domain (e.g., sdAb) variable domain binds to a cell surface molecule or antigen. In some embodiments, the cell surface molecule is ASGR1, ASGR2, TM4SF5, CD8, CD4, or low density lipoprotein receptor (LDL-R). In some embodiments, the cell surface molecule is ASGR1. In some embodiments, the cell surface molecule is ASGR2. In some embodiments, the cell surface molecule is TM4SF5. In some embodiments, the cell surface molecule is CD8. In some embodiments, the cell surface molecule is CD4. In some embodiments, the cell surface molecule is LDL-R.

In some embodiments, the target cell is a cell of the hematopoietic lineage. Reference to "hematopoietic cells" includes blood cells from the myeloid and lymphoid lineages. In particular, the term "hematopoietic cells" includes undifferentiated or poorly differentiated cells, such as hematopoietic stem cells and progenitor cells, as well as differentiated cells, such as T lymphocytes, B lymphocytes, or dendritic cells. In some embodiments, the hematopoietic cells are Hematopoietic Stem Cells (HSCs), cd34+ progenitor cells, specifically peripheral blood cd34+ cells, very early progenitor cd34+ cells, B cell cd19+ progenitor cells, myeloid progenitor cd13+ cells, T lymphocytes, B lymphocytes, monocytes, dendritic cells, cancer B cells, specifically B cell chronic lymphocytic leukemia (BCLL) cells, and Marginal Zone Lymphoma (MZL) B cells or thymocytes.

As known to those skilled in the art, many hematopoietic cells are produced from bone marrow hematopoietic stem cells.

In some embodiments, the hematopoietic cells are Hematopoietic Stem Cells (HSCs), which are cells capable of replenishing all blood cell types and self-renewing. Hematopoietic stem cells can be specifically defined as cells that, when injected into the circulation of a recipient mouse with a depleted hematopoietic system, maintain levels of bone marrow, T cells, and B cells at robustly detectable levels (typically greater than 1% of peripheral blood cells) for 16 weeks (Schroeder (2010) CELL STEM CELL 6:203-207).

In some embodiments, the hematopoietic cells are "cd34+ progenitor cells," which are heterogeneous populations of cells comprising HSCs, pluripotent stem cells, and a subpopulation of cells at an early stage of lineage commitment. In normal adult animals, cd34+ progenitor cells continually migrate to and from the bone marrow. They can differentiate to produce all hematopoietic cell lineages found in the circulation. In some embodiments, the hematopoietic cells are very early progenitor cd34+ cells, which are a subpopulation of cd34+ progenitor cells enriched from HSCs.

In some embodiments, the hematopoietic cells are "peripheral blood cd34+ cells," which are cd34+ cells present in the blood.

In some embodiments, the hematopoietic cells are B cell cd19+ progenitor cells, which are B lineage cell populations that express cell surface CD10, CD34, and CD 19.

In some embodiments, the hematopoietic cells are myeloid progenitor cd13+ cells, which are a population of myeloid lineage cells that express cell surface CD34 and CD13, and in some cases also CD 33.

In some embodiments, the target cell is selected from the group consisting of a bone marrow-lymphoid balancing hematopoietic lineage cell, a bone marrow-biased hematopoietic lineage cell, a lymphoid-biased hematopoietic lineage cell, a platelet-bone marrow-biased hematopoietic lineage cell, a long-term hematopoietic lineage cell, a metaphase hematopoietic lineage cell, or a short-term hematopoietic lineage cell. In some embodiments, the target cell is selected from the group consisting of monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes, and platelets. In some embodiments, the target cell is selected from the group consisting of a T cell, a B cell, a Natural Killer (NK) cell, and an innate lymphocyte.

In some embodiments, the target cell is an effector cell, e.g., an immune system cell that expresses one or more Fc receptors and mediates one or more effector functions. In some embodiments, the target cells may include one or more of monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, large granular lymphocytes, langerhans cells, natural Killer (NK) cells, T lymphocytes (e.g., T cells), γδ T cells, B lymphocytes (e.g., B cells), and may be from any organism, including humans, mice, rats, rabbits, and monkeys.

In some embodiments, the hematopoietic cell is a T cell. In some embodiments, the T cell is a naive T cell. In some embodiments, the T cell is a memory T cell.

In some embodiments, the hematopoietic cell is a B cell. In some embodiments, the target cell is a resting B cell, such as a naive or memory B cell. In some embodiments, the target cell is a cancer B cell, such as a B cell chronic lymphocytic leukemia (BCLL) cell or a Marginal Zone Lymphoma (MZL) B cell.

In some embodiments, the target cell is a thymocyte. In some embodiments, the target cell is a Natural Killer (NK) cell. In some embodiments, the thymocytes express CD4 or CD8. In some embodiments, the thymocytes do not express CD4 or CD8. In some embodiments, the Natural Killer (NK) cell is a CD56 expressing cell.

In some embodiments, the target cell is a cd3+ T cell, a cd4+ T cell, or a cd8+ T cell.

In some embodiments, the target cell is an antigen presenting cell, MHC class ii+ cell, professional antigen presenting cell, atypical antigen presenting cell, macrophage, dendritic cell, bone marrow dendritic cell, plasmacytoid dendritic cell, cd11c+ cell, cd11b+ cell, or B cell.

In some embodiments, the binding domain (e.g., sdAb) variable domain binds to a cell surface molecule or antigen. In some embodiments, the cell surface molecule is ASGR1, ASGR2, TM4SF5, CD3, CD8, CD4, CD7, or low density lipoprotein receptor (LDL-R). In some embodiments, the cell surface molecule is ASGR1. In some embodiments, the cell surface molecule is ASGR2. In some embodiments, the cell surface molecule is TM4SF5. In some embodiments, the cell surface molecule is CD3. In some embodiments, the cell surface molecule is CD8. In some embodiments, the cell surface molecule is CD4. In some embodiments, the cell surface molecule is LDL-R. In some embodiments, the cell surface molecule is ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, or ITGA3.

In some embodiments, the re-targeted attachment protein comprises a paramyxovirus envelope attachment protein (e.g., a G protein or functionally active variant or biologically active portion thereof) directly linked to a binding domain and/or variable domain thereof. In some embodiments, the targeting envelope protein is a fusion protein having the structure (N '-single domain antibody-C') - (C '-G protein-N').

In some embodiments, the re-targeted attachment protein comprises a paramyxovirus envelope attachment protein (e.g., a G protein or functionally active variant or biologically active portion thereof) indirectly linked to its binding domain and/or variable domain via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a chemical linker.

In some embodiments, the linker is a peptide linker and the targeting envelope protein is a fusion protein comprising a paramyxovirus envelope attachment protein (e.g., a G protein or functionally active variant or biologically active portion thereof) linked to the sdAb variable domain via a peptide linker. In some embodiments, the targeting envelope protein is a fusion protein having the structure (N '-single domain antibody-C') -linker- (C '-G protein-N').

In some embodiments, the linker is a polypeptide linker. The polypeptide linker may be a flexible linker or a rigid linker or a combination of both. In some aspects, the linker is a short, medium, or long linker. In some embodiments, the peptide linker is up to 65 amino acids in length. In some embodiments, the peptide linker comprises or is from about 2 to 65 amino acids, 2 to 60 amino acids, 2 to 56 amino acids, 2 to 52 amino acids, 2 to 48 amino acids, 2 to 44 amino acids, 2 to 40 amino acids, 2 to 36 amino acids, 2 to 32 amino acids, 2 to 28 amino acids, 2 to 24 amino acids, 2 to 20 amino acids, 2 to 18 amino acids, 2 to 14 amino acids, 2 to 12 amino acids, 2 to 10 amino acids, 2 to 8 amino acids, 2 to 6 amino acids, 6 to 65 amino acids, 6 to 60 amino acids, 6 to 56 amino acids, 6 to 52 amino acids, 6 to 48 amino acids, 6 to 44 amino acids, 6 to 40 amino acids, 6 to 36 amino acids, 6 to 32 amino acids, 6 to 28 amino acids, 6 to 24 amino acids, 6 to 20 amino acids, 6 to 18 amino acids, 6 to 14 amino acids, 6 to 12 amino acids, 6 to 10 amino acids, 6 to 8 amino acids, 8 to 65 amino acids, 8 to 60 amino acids, 8 to 56 amino acids, 8 to 52 amino acids, 8 to 48 amino acids, 8 to 44 amino acids, 8 to 40 amino acids, 8 to 36 amino acids, 8 to 32 amino acids, 8 to 28 amino acids, 8 to 24 amino acids, 8 to 20 amino acids, 8 to 18 amino acids, 8 to 14 amino acids, 8 to 12 amino acids, 8 to 10 amino acids, 10 to 65 amino acids, 10 to 60 amino acids, 10 to 56 amino acids, 10 to 52 amino acids, 10 to 48 amino acids, 10 to 44 amino acids, 10 to 40 amino acids, 10 to 36 amino acids, 10 to 32 amino acids, 10 to 28 amino acids, 10 to 24 amino acids, 10 to 20 amino acids, 10 to 18 amino acids, 10 to 14 amino acids, 10 to 12 amino acids, 12 to 65 amino acids, 12 to 60 amino acids, 12 to 56 amino acids, 12 to 52 amino acids, 12 to 48 amino acids, 12 to 44 amino acids, 12 to 40 amino acids, 12 to 36 amino acids, 12 to 32 amino acids, 12 to 28 amino acids, 12 to 24 amino acids, 12 to 20 amino acids, 12 to 18 amino acids, 12 to 14 amino acids, 14 to 65 amino acids, 14 to 60 amino acids, 14 to 56 amino acids, 14 to 52 amino acids, 14 to 48 amino acids, 14 to 44 amino acids, 14 to 40 amino acids, 14 to 36 amino acids, 14 to 32 amino acids, 14 to 28 amino acids, 14 to 24 amino acids, 14 to 20 amino acids, 14 to 18 amino acids, 18 to 65 amino acids, 18 to 60 amino acids, 18 to 56 amino acids, 18 to 52 amino acids, 18 to 48 amino acids, 18 to 44 amino acids, 18 to 40 amino acids, 18 to 36 amino acids, 18 to 32 amino acids, 18 to 28 amino acids, 18 to 24 amino acids, 18 to 20 amino acids, 20 to 65 amino acids, 20 to 60 amino acids, 20 to 56 amino acids, 20 to 52 amino acids, 20 to 48 amino acids, 20 to 44 amino acids, 20 to 40 amino acids, 20 to 36 amino acids, 20 to 32 amino acids, 20 to 28 amino acids, 20 to 26 amino acids, 20 to 24 amino acids, 24 to 65 amino acids, 24 to 60 amino acids, 24 to 56 amino acids, 24 to 52 amino acids, 24 to 48 amino acids, 24 to 44 amino acids, 24 to 40 amino acids, 24 to 36 amino acids, 24 to 32 amino acids, 24 to 30 amino acids, 24 to 28 amino acids, 28 to 65 amino acids, 28 to 60 amino acids, 28 to 56 amino acids, 28 to 52 amino acids, 28 to 48 amino acids, 28 to 44 amino acids, 28 to 40 amino acids, 28 to 36 amino acids, 28 to 34 amino acids, 28 to 32 amino acids, 32 to 65 amino acids, 32 to 60 amino acids, 32 to 56 amino acids, 32 to 52 amino acids, 32 to 48 amino acids, 32 to 44 amino acids, 32 to 40 amino acids, 32 to 38 amino acids, 32 to 36 amino acids, 36 to 65 amino acids, 36 to 60 amino acids, 36 to 56 amino acids, 36 to 52 amino acids, 36 to 48 amino acids, 36 to 44 amino acids, 36 to 40 amino acids, 40 to 65 amino acids, 40 to 60 amino acids, 40 to 56 amino acids, 40 to 52 amino acids, 40 to 48 amino acids, 40 to 44 amino acids, 44 to 65 amino acids, 44 to 60 amino acids, 44 to 56 amino acids, 44 to 52 amino acids, 44 to 48 amino acids, 48 to 65 amino acids, 48 to 60 amino acids, 48 to 56 amino acids, 48 to 52 amino acids, 50 to 65 amino acids, 50 to 60 amino acids, 50 to 56 amino acids, 50 to 52 amino acids, 54 to 65 amino acids, 54 to 60 amino acids, 54 to 56 amino acids, 58 to 65 amino acids, 58 to 60 amino acids, or 60 to 65 amino acids. In some embodiments, the peptide linker is 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 amino acid polypeptides.

The linker may be naturally occurring, synthetic, or a combination of both. Natural linkers can be flexible or limiting (e.g., structured) and can be very diverse in amino acid sequence and composition. Their degree of resistance to proteolysis depends on which proteins they originate from and the biological environment (extracellular, intracellular, prokaryotic, eukaryotic, etc.) to which these proteins are exposed in nature. In some embodiments, the linker is a linker based on a peptide found in a human protein. Examples of natural linkers are (i) KES GSVS SEQL AQFRSLD (see Bird et al, (1988) Science, 242, 423-426), (ii) sequences corresponding to the hinge domain of immunoglobulins without light chains (see Hamers-Casterman et al, (1993) Nature, 363, 446-448 and PCT International publication No. WO 096/34103). Examples of linkers for use with anti-albumin domain antibodies (e.g., human, humanized, camelized human or camelidae VHH domain antibodies) are EPKIPQPQPKPQPQPQPQPKPQPKPEPECTCPKCP and TNEVCKCPKCP. Other linkers derived from human and camelidae hinges are disclosed in EPO656946, which is incorporated herein by reference. The hinge-derived linker may have a variable length, for example, 0 to about 50 amino acids, including 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48 or 49 amino acids.

Particularly suitable linker polypeptides mainly comprise amino acid residues selected from glycine (Gly), serine (Ser), alanine (Ala) and threonine (Thr). For example, the linker may contain at least 75% (based on the total number of residues present in the peptide linker), such as at least 80%, at least 85%, or at least 90% of the amino acid residues selected from Gly, ser, ala and Thr. The linker may also consist of only Gly, ser, ala and/or Thr residues. In some embodiments, the linker contains 1-25 glycine residues, 5-20 glycine residues, 5-15 glycine residues, or 8-12 glycine residues. In some aspects, suitable peptide linkers typically contain at least 50% glycine residues, such as at least 75% glycine residues. In some embodiments, the peptide linker comprises only glycine residues. In some embodiments, the peptide linker comprises only glycine and serine residues.

In particular embodiments, the linker is a flexible peptide linker. The flexible linker is designed not to adopt a stable secondary structure when linking the two polypeptide moieties, allowing a range of conformations in the fusion protein. These linkers are preferably hydrophilic in nature to prevent them from interacting with one or both fusion polypeptides. In general, small polar residues such as glycine and serine are prevalent in these linkers to increase the flexibility and hydrophilicity characteristics of the peptide backbone, respectively. The length of these joints is variable and is preferably determined empirically or by means of 3D calculation methods. In general, the preferred linker length will be the minimum length compatible with good expression, good solubility, and complete restoration of the native function and structure of interest. Due to its flexible nature, the flexible linker may constitute a good substrate for endogenous proteases. In general, unless a desired feature, the flexible linker is free of amino acids that are readily recognized by endogenous proteases with broad substrate specificity, such as charged amino acids or large hydrophobic/aromatic amino acids.

In some embodiments, these linkers consist essentially of the amino acids glycine and serine, denoted herein as GS linkers. In some such embodiments, the linker is 1-20 amino acids, such as 1-20 amino acids consisting essentially of glycine. In some embodiments, the linker is 1-20 amino acids, such as 1-20 amino acids consisting essentially of glycine and serine. In some embodiments, the linker is a flexible peptide linker comprising the amino acids glycine and serine, referred to as a GS-linker. In some embodiments, the peptide linker comprises the sequence GS, GGS, GGGGS, GGGGGS or a combination thereof. In some embodiments, the polypeptide linker has the sequence (GGS) n, wherein n is 1 to 10. In some embodiments, the polypeptide linker has the sequence (GGGGS) n, wherein n is 1 to 10. In some embodiments, the polypeptide linker has the sequence (GGGGGS) n, where n is 1 to 6. In some embodiments, the polypeptide linker has or comprises the amino acid sequence of SEQ ID NO. 405 (GGGGSGGGGSGGGGS).

In some cases, it is desirable to provide some rigidity to the peptide linker. This can be achieved by including proline residues in the amino acid sequence of the peptide linker. Thus, in some embodiments, the linker comprises at least one proline residue in the amino acid sequence of the peptide linker. For example, the peptide linker can have an amino acid sequence in which at least 25% (e.g., at least 50% or at least 75%) of the amino acid residues are proline residues. In a particular embodiment, the peptide linker comprises only proline residues.

In some aspects, the peptide linker comprises at least one cysteine residue, such as one cysteine residue. For example, in some embodiments, the linker comprises at least one cysteine residue and an amino acid residue selected from the group consisting of Gly, ser, ala and Thr. In some such embodiments, the linker comprises a glycine residue and a cysteine residue, such as only a glycine residue and a cysteine residue. Typically, each peptide linker will contain only one cysteine residue. One example of a specific linker comprising a cysteine residue includes a peptide linker having the amino acid sequence Glym-Cys-Glyn, where n and m are each integers from 1 to 12, such as 3 to 9, 4 to 8, or 4 to 7.

In some embodiments, the linker of the fusion protein is a structured linker. Restriction and/or structural linkers are designed to adopt a stable secondary structure when linking two polypeptide moieties, thereby limiting the conformational range in the fusion protein. Such joints typically employ a helical structure spanning several turns. Also, the length of these joints is variable and is preferably determined empirically or by means of computational methods. In some embodiments, the restriction and/or structuring linker maintains the longest distance between each polypeptide of the fusion. This is particularly relevant when both polypeptides have a tendency to form heteroaggregates. Due to their structure, the restriction linkers may also be more resistant to proteolytic degradation, providing advantages when injected in vivo. Examples of restriction linkers are cited from PCT International publication No. WO 00/24884 (e.g., SSSASASSA, GSPGSPG or ATTTGSSPGPT), US 6,132,992 (e.g., helical peptide linkers).

In particular embodiments, the structured linker contains the sequence (AP) n or (EAAAK) n, where n is 2 to 20, preferably 4 to 10, including but not limited to AS- (AP) n-GT or AS- (EAAAK) n-GT, where n is 2 to 20, such AS 2,3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In other embodiments, the linker comprises the sequence (GGGGA) n, (PGGGS) n, (AGGGS) n, or GGS- (EGKSSGSGSESKST) n-GGS (wherein n is 2 to 20), (ADAAP) n-G (wherein n is 2 to 20), (GEPQG) n (wherein n is 2 to 20), (GEPQG) n-G (wherein n is 2 to 20), (AGGEP) n (wherein n is 2 to 20), (AGGEP) n-G (wherein n is 2 to 20), (AGSEP) n (wherein n is 2 to 20), (AGSEP) n-G (wherein n is 2 to 20), (GGGEQ) n (wherein n is 2 to 20), (GGGEQ) n-G (wherein n is 2 to 20). In some embodiments, the linker is SSSASASSA, GSPGSPG, ATTTGSSPGPT, ADAAPADAAPG, GEPQGGEPQGG, AGGEPAGGEPG, AGSEPAGSEPG or GGGEQGGGEQG.

In some embodiments, the polypeptide linker has or comprises the amino acid sequence of SEQ ID NO 589 (AHHSED). In some embodiments, the polypeptide linker has or comprises the amino acid sequence of SEQ ID NO 590 (EPKTPKPQPQPQPQPQPNPTTE).

In some embodiments, the re-targeted attachment protein comprising a binding domain, a first binding domain, and/or a second binding domain linked to at least one paramyxovirus envelope attachment protein may comprise an engineered binding domain, such as an artificially generated binding domain. The binding domain may comprise nanobodies, darpins, aptamers, affimer, affibodies, desmin, avimer, monomers, ANTICALIN, FYNOMER. Any engineered binding domain known in the art and suitable for use in the present invention may be used, such as, for example, olaleye et al, biomolecules,2021, month 12; 11 (12): 1791.

A. T cell binding domains

In some embodiments, the lipid particles disclosed herein comprise one or more re-targeting attachment proteins that each independently comprise (i) a paramyxovirus envelope attachment protein, and (ii) a targeting moiety for a target molecule expressed on the surface of a target cell. In some embodiments, the targeting moiety is a T cell binding domain, e.g., a T cell binding agent, such as any of those disclosed herein.

In some embodiments, the lipid particles disclosed herein comprise one or more T cell binding domains (e.g., T cell binding agents) that target a viral vector to a T cell. In some embodiments, the T cell binding agent binds to a molecule expressed on the surface of a T cell. The cell surface molecule may be a receptor, co-receptor or GPI-anchored protein. In some embodiments, the T cell binding agent binds CD3, CD4, or CD8.

In some embodiments, the T cell binding agent may be fused to or incorporated into a protein fusion agent or lipid particle envelope attachment protein (e.g., a re-targeting attachment protein). In some embodiments, the T cell binding agent may be incorporated into the viral envelope by fusion with a transmembrane domain. In some embodiments, the T cell binding agent targets the lipid particle to a T cell.

In certain embodiments, the T cell binding agent may be fused or incorporated with a protein fusion agent or attachment protein to re-target the lipid particle to the T cell. In some embodiments, for re-targeting, the T cell binding agent is fused to a protein fusion agent or envelope adhesion protein that is mutated to reduce binding to the natural binding partner of the fusion agent or viral envelope protein. In some embodiments, the fusion agent is or comprises a mutant G protein, or biologically active portion thereof, which is a mutant of wild-type NiV-G and exhibits reduced binding to one or both of the natural binding partners ephrin B2 or ephrin B3, including any of the above. Thus, in some aspects, the fluxing agent may be re-targeted to exhibit altered tropism. In some embodiments, the binding confers a re-targeted binding compared to the binding of a wild-type surface glycoprotein in which a new or different binding activity is conferred. In certain embodiments, the binding confers a re-targeted binding compared to binding of a wild-type G protein in which a new or different binding activity is conferred. In some embodiments, the fusion agent is randomly mutated. In some embodiments, the fusion agent is rationally mutated. In some embodiments, the fusion agent undergoes directed evolution. In some embodiments, the fusion agent is truncated and only a subset of peptides are used in the viral vector. In some embodiments, the amino acid residues in the measles hemagglutinin protein may be mutated to alter the binding properties of the protein, thereby redirecting fusion (doi: 10.1038/nbt942, volume Molecular Therapy, 8 th, 1427-1436 2008, 8 th month) ,doi:10.1038/nbt1060, DOI: 10.1128/JVI.76.7.3558–3563.2002, DOI: 10.1128/JVI.75.17.8016–8020.2001, doi: 10.1073pnas.0604993103).

In some embodiments, the protein fusion agent may be re-targeted by covalently conjugating the T cell binding agent to the attachment protein. In some embodiments, the fusion agent and T cell binding agent are covalently conjugated by expression of a chimeric protein (e.g., a re-targeted attachment protein) comprising the fusion agent linked to the T cell binding agent. The T cell binding agent may comprise any targeting protein capable of conferring specific binding to a target molecule expressed on the surface of a T cell. In some embodiments, the targeting protein may also include antibodies or antigen binding fragments thereof (e.g., fab ', F (ab') 2, fv fragments, scFv antibody fragments, disulfide-linked Fv (sdFv), fd fragments consisting of VH and CH1 domains, linear antibodies, single domain antibodies such as sdAb (VL or VH), nanobodies, or camelidae VHH domains), antigen binding fibronectin type III (Fn 3) scaffolds such as fibronectin polypeptide miniantibodies, ligands, cytokines, chemokines, or T Cell Receptors (TCRs). In some embodiments, the T cell binding agent is an antibody or antigen binding fragment thereof. In some embodiments, the fusion protein may be engineered to bind to the Fc region of an antibody targeting an antigen on a target cell, thereby redirecting fusion activity to cells displaying the antibody target (DOI: 10.1128/JVI.75.17.8016-8020.2001, DOI:10.1038/nm 1192). In some embodiments, the altered and unaltered fusion agent may be displayed on the same retroviral vector or VLP (doi: 10.1016/j. Biological.2014.01.051).

In some embodiments, single chain variable fragments (scFvs) may be conjugated to a fusion agent to redirect fusion activity to T cells (doi:10.1038/nbt1060, DOI 10.1182/blood-2012-11-468579, doi:10.1038/nmeth.1514, doi:10.1006/mthe.2002.0550, HUMAN GENE THERAPY 11:817– 826, doi:10.1038/nbt942, doi:10.1371/journal.pone.0026381, DOI 10.1186/s12896-015-0142-z). displaying scFv binding targets, in some embodiments, engineered ankyrin repeat proteins (DARPin) may be conjugated to a fusion agent to redirect fusion activity to T cells displaying DARPin binding targets (doi: 10.1038/mt.2013.16, doi:10.1038/mt.2010.298, doi: 10.4049/jimmunol.1500956) and combinations of different DARPin (doi: 10.1038/mto.2016.3). In some embodiments, a single domain antibody (e.g., VHH) can be conjugated to a fusion agent to redirect fusion activity to T cells that display sdAb binding targets. In some embodiments, receptor ligands and antigens may be conjugated to a fusion agent to redirect fusion activity to T cells displaying the target receptor (DOI: 10.1089/hgtb.2012.054, DOI: 10.1128/JVI.76.7.3558-3563.2002).

I. CD3 binding agents

In some embodiments, the lipid particles disclosed herein comprise one or more re-targeting attachment proteins that each independently comprise (i) a paramyxovirus envelope attachment protein, and (ii) a targeting moiety for a target molecule expressed on the surface of a target cell, wherein the target molecule is CD3. In some embodiments, the targeting moiety is a CD3 binding domain, e.g., a CD3 binding agent, such as any of those disclosed herein.

In some embodiments, the lipid particles disclosed herein comprise one or more CD3 binding agents. For example, the CD3 binding agent may be fused to or incorporated into the heavy target attachment protein. In another embodiment, the CD3 binding agent may be incorporated into the lipid particle envelope by fusion with a transmembrane domain.

Exemplary CD3 binding agents include antibodies and fragments thereof (e.g., scFv, VHH) that bind CD 3. Such antibodies may be derived from any species and may be, for example, mouse, rabbit, human, humanized or camelid antibodies.

Exemplary antibodies include OKT3, CRIS-7, I2C, bostuzumab, carboxithromab, morotuzumab -CD3、A-319、AFM11、AMG 199、AMG 211、AMG 424、AMG 427、AMG 562、AMG 564、APVO436、CC-93269、ERY974、GBR1302、GEM333、GEM2PSCA、GNC-035、HPN424、IGM-2323、JNJ-63709178、JNJ-63898081、JNJ-75348780、JNJ-78306358、M701、M802、MGD007、MOR209/ES414、PF-06671008、REGN5459、RO7283420、SAR442257、SAR443216、TNB-383B、TNB-486、TNB-585、Y150、 Ai Kapa tomab, west Wo Si tomab, sibizumab, rituximab, E3932 s mab (Lu Weike), lu Weike mab, lu Weike tomab, gefeitumumab, grasonitumumab, obulin dabigamab (Lu Weike), pa3932 Tamab, paramotuzumab, sorituximab, talatuzumab, taprituzumab (Lu Weike), taprituzumab, tentuzumab velocimab, arnusemimab (Lu Weike), lu Weike, panoramab (Lu Weike), pertuzumab, lu Weike mab (Lu Weike), nivaltuzumab (Lu Weike), henatuzumab, ertuzumab, futuzumab, ornitumumab, taquasimab, terlipressizumab, velitumumab, elcatuzumab, oxtuzumab Lu Weike, U.S. Pat. Nos. Lu Weike, and Lu Weike; U.S. patent application nos. US Lu Weike, US Lu Weike and US Lu Weike, and anti-CD 3 antibodies disclosed in PCT application nos. WO Lu Weike, WO Lu Weike and WO Lu Weike. Other exemplary binding agents include engineered ankyrin repeat protein (DARPin) and fibronectin type III (Fn 3) scaffold-based binding agents.

In some embodiments, the CD3 binding agent comprises a heavy chain Variable (VH) region comprising CDR-H1, CDRH-2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 474, 475 and 476, respectively, and a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 477, 478 and 479, respectively. In some embodiments, the CD3 binding agent comprises a VH region comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO. 480, and a VL region comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO. 481. In some embodiments, the CD3 binding agent comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO. 480 and a VL region comprising the amino acid sequence set forth in SEQ ID NO. 481. In some embodiments, the CD3 binding agent is an scFv. In some embodiments, the CD3 binding agent comprises the amino acid sequence shown as SEQ ID NO. 482. In some embodiments, the CD3 binding agent is OKT3.

In some embodiments, the CD3 binding agent is activated (e.g., the CD3 binding agent activates T cells). In some embodiments, the CD3 binding agent is non-activated (e.g., it does not activate T cells).

In some embodiments, the CD3 binding agent comprises a humanized antibody molecule, an intact IgA, igG, igE or IgM antibody, a bispecific or multispecific antibody (e.g., zybodies @, etc.), an antibody fragment, such as a Fab fragment, fab ' fragment, F (ab ') ₂ fragment, fd ' fragment, fd fragment, and isolated CDR, or a collection thereof, a single chain Fv, a polypeptide-Fc fusion, a single domain antibody (e.g., a shark single domain antibody, such as IgNAR or a fragment thereof), a camelid antibody, a masking antibody (e.g., probodies;) Small Modular ImmunoPharmaceuticals ("smitm"), a single chain or tandem diabody (TandAb; VHH; ANTICALINS;, nanobodies; minibody, biTE;, ankyrin repeat protein or DARPINs;, avimers; DART, TCR-like antibody, ADNECTINS;, affilins; trans-bodies;, affibodies;, trimerX;, microProteins, fynomers;, CENTYRINS;, and KALBITOR.

In some embodiments, the CD3 binding agent is a peptide. In some embodiments, the CD3 binding agent is an antibody, such as a single chain variable fragment (scFv). In some embodiments, the CD3 binding agent is an antibody, such as a single domain antibody. In some embodiments, the antibody may be human or humanized. In some embodiments, the CD3 binding agent is a VHH. In some embodiments, the antibody or portion thereof is naturally occurring. In some embodiments, the antibody or portion thereof is synthetic.

In some embodiments, antibodies may be generated from phage display libraries to have specificity for a desired target ligand. In some embodiments, phage display libraries are generated from a library of VHHs from the family Camelidae immunized with various antigens, as described in Arbabi et al, FEBS Letters, 414, 521-526 (1997), lauwereys et al, EMBO J., 17, 3512-3520 (1998), DECANNIERE et al, structures, 7, 361-370 (1999). In some embodiments, phage display libraries comprising non-immunized camelidae antibody fragments are generated. In some embodiments, libraries of human single domain antibodies are synthetically generated by introducing diversity into one or more scaffolds.

In some embodiments, the C-terminus of the CD3 binding agent is attached to the C-terminus of a G protein (e.g., a fusion agent) or biologically active portion thereof. In some embodiments, the N-terminus of the CD3 binding agent is exposed on the outer surface of the lipid bilayer.

In some embodiments, the CD3 binding agent is the only surface displayed non-viral sequence of the viral vector. In some embodiments, the CD3 binding agent is the only membrane-bound non-viral sequence of the viral vector. In some embodiments, the viral vector does not contain a molecule that binds or stimulates T cells other than a CD3 binding agent. In some embodiments, the viral vector contains a non-activated CD3 binding agent.

In some embodiments, the viral vector may display CD3 binding agent that is not conjugated to a protein fusion agent in order to redirect fusion activity to cells bound by the targeting moiety, or to affect homing.

CD4 binding agents

In some embodiments, the lipid particles disclosed herein comprise one or more re-targeting attachment proteins that each independently comprise (i) a paramyxovirus envelope attachment protein, and (ii) a targeting moiety for a target molecule expressed on the surface of a target cell, wherein the target molecule is CD4. In some embodiments, the targeting moiety is a CD4 binding domain, e.g., a CD4 binding agent, such as any of those disclosed herein.

In some embodiments, the lipid particles disclosed herein comprise one or more CD4 binding agents. For example, the CD4 binding agent may be fused or incorporated with a protein fusion agent or attachment protein. In another embodiment, the CD4 binding agent may be incorporated into the lipid particle envelope by fusion with a transmembrane domain.

In some embodiments of any of the provided embodiments, the CD4 binding agent is exposed on the surface of the lipid particle. In some embodiments, the CD4 binding agent is fused to a transmembrane domain incorporated into the lipid particle envelope.

Exemplary CD4 binding agents include antibodies and fragments thereof (e.g., scFv, VHH) that bind to CD 4. Such antibodies may be derived from any species and may be, for example, mouse, rabbit, human, humanized or camelid antibodies. Exemplary antibodies include WO2002102853、WO2004083247、WO2004067554、WO2007109052、WO2008134046、WO2010074266、WO2012113348、WO2013188870、WO2017104735、WO2018035001、WO2018170096、WO2019203497、WO2019236684、WO2020228824、US 5,871,732、US 7,338,658、US 7,722,873、US 8,399,621、US 8,911,728、US 9,005,963、US 9,587,022、US 9,745,552、 U.S. provisional application No. 63/326,269, ibalizumab (ibalizumab) disclosed in U.S. provisional application No. 63/341,681, zanonomumab (zanolimumab), qu Jiazhu mab (tregalizumab), prizetimab (priliximab), celecoxib (cedelizumab), clenbuterab (clenoliximab), clenbuterab (keliximab) and anti-CD 4 antibodies, and other exemplary binding agents for antibody B486A1、RPA-T4、CE9.1 (Novus Biologicals);GK1.5、RM4-5、RPA-T4、OKT4、4SM95、S3.5、N1UG0 (ThermoFisher);GTX50984、ST0488、10B5、EP204 (GeneTex);GK1.3、5A8、10C12、W3/25、8A5、13B8.2、6G5 (Absolute Antibody);VIT4、M-T466、M-T321、REA623, (Miltenyi);MEM115、MT310 (Enzo Life Sciences);H129.19、5B4、6A17、18-46、A-1、C-1、OX68 (Santa Cruz);EP204、D2E6M (Cell Signaling Technology). include engineered anchor protein repeat proteins (DARPin) (e.g., anti-CD 4 DARPin disclosed in WO 2017182585) and binding agents based on fibronectin type III (Fn 3) scaffolds. Each of U.S. 9,005,963, U.S. provisional application No. 63/326,269, and U.S. provisional application No. 63/341,681 is incorporated by reference herein in its entirety.

In some embodiments, the protein fusion or attachment protein may be re-targeted by mutating amino acid residues in a fusion or targeting protein (e.g., a hemagglutinin (H) protein or a G protein). In certain embodiments, the fusion agent (e.g., G protein) is mutated to reduce binding to the natural binding partner of the fusion agent. In some embodiments, the fusion agent is or comprises a mutant G protein, or biologically active portion thereof, which is a mutant of wild-type NiV-G and exhibits reduced binding to one or both of the natural binding partners ephrin B2 or ephrin B3, including any of the above. Thus, in some aspects, the fluxing agent may be re-targeted to exhibit altered tropism. In some embodiments, the binding confers a re-targeted binding compared to the binding of a wild-type surface glycoprotein in which a new or different binding activity is conferred. In certain embodiments, the binding confers a re-targeted binding compared to binding of a wild-type G protein in which a new or different binding activity is conferred. In some embodiments, the fusion agent is randomly mutated. In some embodiments, the fusion agent is rationally mutated. In some embodiments, the fusion agent undergoes directed evolution. In some embodiments, the fusion agent is truncated and only a subset of peptides are used in the viral vector. In some embodiments, the amino acid residues in the measles hemagglutinin protein may be mutated to alter the binding properties of the protein, thereby redirecting fusion (doi: 10.1038/nbt942, volume Molecular Therapy, 8 th, 1427-1436 2008, 8 th month) ,doi:10.1038/nbt1060, DOI: 10.1128/JVI.76.7.3558–3563.2002, DOI: 10.1128/JVI.75.17.8016–8020.2001, doi: 10.1073pnas.0604993103).

In some embodiments, the protein fusion agent can be re-targeted by covalently conjugating the CD4 binding agent to a fusion protein or attachment protein (e.g., a re-targeted attachment protein). In some embodiments, the fusion agent and CD4 binding agent are covalently conjugated by expression of a chimeric protein (e.g., a re-targeted attachment protein) comprising the fusion agent linked to the CD4 binding agent. In some embodiments, single chain variable fragments (scFvs) may be conjugated to a fusion agent to redirect fusion activity to cells (doi:10.1038/nbt1060, DOI 10.1182/blood-2012-11-468579, doi:10.1038/nmeth.1514, doi:10.1006/mthe.2002.0550, HUMAN GENE THERAPY 11:817– 826, doi:10.1038/nbt942, doi:10.1371/journal.pone.0026381, DOI 10.1186/s12896-015-0142-z). displaying scFv binding targets, in some embodiments, engineered anchor protein repeat proteins (DARPin) may be conjugated to a fusion agent to redirect fusion activity to cells displaying DARPin binding targets (doi: 10.1038/mt.2013.16, doi:10.1038/mt.2010.298, doi: 10.4049/jimmunol.1500956) and combinations of different DARPin (doi: 10.1038/mto.2016.3). In some embodiments, receptor ligands and antigens may be conjugated to a fusion agent to redirect fusion activity to cells displaying the target receptor (DOI: 10.1089/hgtb.2012.054, DOI: 10.1128/JVi.76.7.3558-3563.2002). In some embodiments, the targeting protein may also include antibodies or antigen binding fragments thereof (e.g., fab ', F (ab') 2, fv fragments, scFv antibody fragments, disulfide-linked Fv (sdFv), fd fragments consisting of VH and CH1 domains, linear antibodies, single domain antibodies such as sdAb (VL or VH), nanobodies, or camelidae VHH domains), antigen binding fibronectin type III (Fn 3) scaffolds such as fibronectin polypeptide miniantibodies, ligands, cytokines, chemokines, or T Cell Receptors (TCRs). In some embodiments, the protein fusion agent can be re-targeted by non-covalent conjugation of the CD4 binding agent to a fusion protein or targeting protein (e.g., a re-targeting attachment protein). In some embodiments, the fusion protein may be engineered to bind to the Fc region of an antibody targeting an antigen on a target cell, thereby redirecting fusion activity to cells displaying the antibody target (DOI: 10.1128/JVI.75.17.8016-8020.2001, DOI:10.1038/nm 1192). In some embodiments, the altered and unaltered fusion agent may be displayed on the same retroviral vector or VLP (doi: 10.1016/j. Biological.2014.01.051).

In some embodiments, the CD4 binding agent comprises a humanized antibody molecule, an intact IgA, igG, igE or IgM antibody, a bispecific or multispecific antibody (e.g., zybodies @, etc.), an antibody fragment, such as a Fab fragment, fab ' fragment, F (ab ') ₂ fragment, fd ' fragment, fd fragment, and isolated CDR, or a collection thereof, a single chain Fv, a polypeptide-Fc fusion, a single domain antibody (e.g., a shark single domain antibody, such as IgNAR or a fragment thereof), a camelid antibody, a masking antibody (e.g., probodies;) Small Modular ImmunoPharmaceuticals ("smitm"), a single chain or tandem diabody (TandAb; VHH; ANTICALINS;, nanobodies; minibody, biTE;, ankyrin repeat protein or DARPINs;, avimers; DART, TCR-like antibody, ADNECTINS;, affilins; trans-bodies;, affibodies;, trimerX;, microProteins, fynomers;, CENTYRINS;, and KALBITOR.

In some embodiments, the CD4 binding agent is a peptide. In some embodiments, the CD4 binding agent is an antibody, such as a single chain variable fragment (scFv). In some embodiments, the CD4 binding agent is an antibody, such as a single domain antibody. In some embodiments, the antibody may be human or humanized. In some embodiments, the CD4 binding agent is a VHH. In some embodiments, the antibody or portion thereof is naturally occurring. In some embodiments, the antibody or portion thereof is synthetic.

In some embodiments, antibodies may be generated from phage display libraries to have specificity for a desired target ligand. In some embodiments, phage display libraries are generated from a library of VHHs from the family Camelidae immunized with various antigens, as described in Arbabi et al, FEBS Letters, 414, 521-526 (1997), lauwereys et al, EMBO J., 17, 3512-3520 (1998), DECANNIERE et al, structures, 7, 361-370 (1999). In some embodiments, phage display libraries comprising fragments of antibodies of the non-immunized camelidae are generated. In some embodiments, libraries of human single domain antibodies are synthetically generated by introducing diversity into one or more scaffolds.

In some embodiments, the C-terminus of the CD4 binding agent is attached to the C-terminus of a G protein (e.g., a fusion agent) or biologically active portion thereof. In some embodiments, the N-terminus of the CD4 binding agent is exposed on the outer surface of the lipid bilayer.

In some embodiments, the CD4 binding agent is the only surface displayed non-viral sequence of the lipid particle. In some embodiments, the CD4 binding agent is the only membrane-bound non-viral sequence of the lipid particle. In some embodiments, the lipid particle does not contain molecules other than CD4 binding agents that bind or stimulate T cells.

In some embodiments, the lipid particle may display CD4 binding agent that is not conjugated to a protein fusion agent in order to redirect fusion activity to cells bound by the targeting moiety, or to affect homing.

In some embodiments, protein fusion agents derived from viruses or organisms that do not infect humans do not have a native fusion target in the patient, and thus have high specificity.

In some of any of the provided embodiments, the CD4 binding agent is an anti-CD 4 antibody or antigen binding fragment. In some of any of the provided embodiments, the anti-CD 4 antibody or antigen-binding fragment is mouse, rabbit, human, or humanized. In some embodiments, the antigen binding fragment is a single chain variable fragment (scFv). In some embodiments, the antigen binding fragment is an anti-CD 4 scFv.

In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS: 260, 261 and 262, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 263, 264 and 265, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS: 260, 261 and 262, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS: 263, 264 and 265, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 266, 267 and 268, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 269, 270 and 265, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 266, 267 and 268, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 269, 270 and 265, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 271, 272 and 268, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 269, 270 and 265, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS: 271, 272 and 268, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS: 269, 270 and 265, respectively. In some embodiments, the anti-CD 4 scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO. 273. In some embodiments, the anti-CD 4 scFv comprises a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 274. In some embodiments, the anti-CD 4 scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO. 273, and a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 274. In some embodiments, VH and VL are connected by a linker. in some embodiments, the linker comprises the amino acid sequence shown as SEQ ID NO 275. In some embodiments, the anti-CD 4 scFv comprises the amino acid sequence shown as SEQ ID NO 276.

In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 277, 278 and 279, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 280, 281 and 282, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 277, 278 and 279, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 280, 281 and 282, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 283, 284 and 285, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 286, 287 and 288, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 283, 284 and 285, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 286, 287 and 288, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 289, 290 and 285, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 286, 287 and 282, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 289, 290 and 285, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 286, 287 and 282, respectively. In some embodiments, the anti-CD 4 scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO 291. In some embodiments, the anti-CD 4 scFv comprises a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO. 292. In some embodiments, the anti-CD 4 scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO. 291, and a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 292. In some embodiments, VH and VL are connected by a linker. in some embodiments, the linker comprises the amino acid sequence shown as SEQ ID NO 275. In some embodiments, the anti-CD 4 scFv comprises the amino acid sequence shown as SEQ ID NO. 293.

In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 294, 295 and 296, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 297, 298 and 299, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 294, 295 and 296, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 297, 298 and 299, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NO 300, 301, 302, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 303, 304 and 299, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NO: 300, 301, 302, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NO: 303, 304 and 299, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS: 305, 306, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 303, 304 and 299, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS: 305, 306, 302, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS: 224, 225 and 172, respectively. In some embodiments, the anti-CD 4 scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO. 307. In some embodiments, the anti-CD 4 scFv comprises a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO. 308. In some embodiments, the anti-CD 4 scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO. 307, and a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 308. In some embodiments, VH and VL are connected by a linker. In some embodiments, the linker comprises the amino acid sequence shown as SEQ ID NO 275. In some embodiments, the anti-CD 4 scFv comprises the amino acid sequence shown as SEQ ID NO. 309.

In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 310, 311 and 312, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 313, 314 and 315, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS: 310, 311 and 312, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS: 313, 314 and 315, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NO 316, 317, 318, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 319, 320 and 315, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 316, 317, 318, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 319, 320 and 315, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 321, 322, 318, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 319, 321 and 315, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 321, 322, 318, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 319, 320 and 323, respectively. In some embodiments, the anti-CD 4 scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO. 323. In some embodiments, the anti-CD 4 scFv comprises a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO. 324. In some embodiments, the anti-CD 4 scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO. 323, and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO. 324. In some embodiments, VH and VL are connected by a linker. In some embodiments, the linker comprises the amino acid sequence shown as SEQ ID NO 275. In some embodiments, the anti-CD 4 scFv comprises the amino acid sequence shown as SEQ ID NO. 325.

In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 326, 327 and 328, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 329, 330 and 331, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 326, 327 and 328, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 329, 330 and 331, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 332, 333 and 334, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 335, 336 and 331, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS: 332, 333 and 334, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS: 335, 336 and 331, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 337, 338 and 334, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 335, 336 and 331, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 337, 338 and 334, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 335, 336 and 331, respectively. In some embodiments, the anti-CD 4 scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO. 339. In some embodiments, the anti-CD 4 scFv comprises a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 340. In some embodiments, the anti-CD 4 scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO. 339, and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO. 340. In some embodiments, VH and VL are connected by a linker. In some embodiments, the linker comprises the amino acid sequence shown as SEQ ID NO 275. In some embodiments, the anti-CD 4 scFv comprises the amino acid sequence shown as SEQ ID NO. 341.

In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 342, 343 and 344, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 345, 346 and 347, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 342, 343 and 344, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 345, 346 and 347, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 348, 349 and 350, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 351, 352 and 347, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 242, 243 and 244, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 245, 246 and 198, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 353, 354 and 350, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 351, 353 and 347, respectively. In some embodiments, the anti-CD 4 scFv comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 353, 354 and 350, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 351, 352 and 347, respectively. in some embodiments, the anti-CD 4 scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 355. In some embodiments, the anti-CD 4 scFv comprises a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO. 356. In some embodiments, the anti-CD 4 scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:355, and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 356. In some embodiments, VH and VL are connected by a linker. In some embodiments, the linker comprises the amino acid sequence shown as SEQ ID NO 275. In some embodiments, the anti-CD 4 scFv comprises the amino acid sequence shown as SEQ ID NO. 357.

In some embodiments, the anti-CD 4 antibody or antigen-binding fragment is a single domain antibody. In some embodiments, the anti-CD 4 antibody or antigen-binding fragment is a camelidae (e.g., llama, alpaca, camel) anti-CD 4 antibody or antigen-binding fragment (e.g., VHH). In some embodiments, the anti-CD 4 antibody or antigen-binding fragment is an anti-CD 4 VHH. In some embodiments, the anti-CD 4 VHH comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 358, 359 and 360, respectively. In some embodiments, the anti-CD 4 VHH comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 361, 362 and 363, respectively. In some embodiments, the anti-CD 4 VHH comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 364, 365 and 363, respectively. In some embodiments, the anti-CD 4 VHH comprises the amino acid sequence shown in SEQ ID NO 366.

CD7 binding agents

In some embodiments, the lipid particles disclosed herein comprise one or more re-targeting attachment proteins that each independently comprise (i) a paramyxovirus envelope attachment protein, and (ii) a targeting moiety for a target molecule expressed on the surface of a target cell, wherein the target molecule is CD7. In some embodiments, the targeting moiety is a CD7 binding domain, e.g., a CD7 binding agent, such as any of those disclosed herein.

In some embodiments, the lipid particles disclosed herein comprise one or more CD7 binding agents. For example, the CD7 binding agent may be fused or incorporated with a protein fusion agent or attachment protein. In another embodiment, the CD7 binding agent may be incorporated into the lipid particle envelope by fusion with a transmembrane domain.

Exemplary CD7 binding agents include antibodies and fragments thereof (e.g., scFv, VHH) that bind CD 7. Such antibodies may be derived from any species and may be, for example, mouse, rabbit, human, humanized or camelid antibodies. Exemplary antibodies include those disclosed in grsinilimab, SPV-T3a and WO 2015/184941;US10106609;WO2017/213979;WO2018/098306;WO2019086534;US11447548;WO2019/102234;WO2022/136887;WO2022/136888;WO2020/212710;WO2021/160267;WO2022/095803;WO2022/151851. Additional exemplary anti-CD 7 binding agents and G proteins are described in U.S. provisional application No. 63/172,518, which is incorporated herein by reference. Other exemplary binding agents include engineered ankyrin repeat protein (DARPin) and fibronectin type III (Fn 3) scaffold-based binding agents.

In some embodiments, the protein fusion agent or attachment protein can be re-targeted by mutating amino acid residues in the fusion protein or targeting protein (e.g., re-targeting attachment protein). In certain embodiments, the fusion agent (e.g., G protein) is mutated to reduce binding to the natural binding partner of the fusion agent. In some embodiments, the fusion agent is or comprises a mutant G protein, or biologically active portion thereof, which is a mutant of wild-type NiV-G and exhibits reduced binding to one or both of the natural binding partners ephrin B2 or ephrin B3, including any of the above. Thus, in some aspects, the fluxing agent may be re-targeted to exhibit altered tropism. In some embodiments, the binding confers a re-targeted binding compared to the binding of a wild-type surface glycoprotein in which a new or different binding activity is conferred. In certain embodiments, the binding confers a re-targeted binding compared to binding of a wild-type G protein in which a new or different binding activity is conferred. In some embodiments, the fusion agent is randomly mutated. In some embodiments, the fusion agent is rationally mutated. In some embodiments, the fusion agent undergoes directed evolution. In some embodiments, the fusion agent is truncated and only a subset of peptides are used in the viral vector. In some embodiments, the amino acid residues in the measles hemagglutinin protein may be mutated to alter the binding properties of the protein, thereby redirecting fusion (doi: 10.1038/nbt942, volume Molecular Therapy, 8 th, 1427-1436 2008, 8 th month) ,doi:10.1038/nbt1060, DOI: 10.1128/JVI.76.7.3558–3563.2002, DOI: 10.1128/JVI.75.17.8016–8020.2001, doi: 10.1073pnas.0604993103).

In some embodiments, the protein fusion agent (e.g., attachment protein) can be re-targeted by covalently conjugating the CD7 binding agent to the fusion protein or attachment protein (e.g., re-targeting attachment protein). In some embodiments, the fusion agent and CD7 binding agent are covalently conjugated by expression of a chimeric protein comprising the fusion agent linked to a CD8 binding agent. In some embodiments, single chain variable fragments (scFvs) may be conjugated to a fusion agent to redirect fusion activity to cells (doi:10.1038/nbt1060, DOI 10.1182/blood-2012-11-468579, doi:10.1038/nmeth.1514, doi:10.1006/mthe.2002.0550, HUMAN GENE THERAPY 11:817– 826, doi:10.1038/nbt942, doi:10.1371/journal.pone.0026381, DOI 10.1186/s12896-015-0142-z). displaying scFv binding targets, in some embodiments, engineered anchor protein repeat proteins (DARPin) may be conjugated to a fusion agent to redirect fusion activity to cells displaying DARPin binding targets (doi: 10.1038/mt.2013.16, doi:10.1038/mt.2010.298, doi: 10.4049/jimmunol.1500956) and combinations of different DARPin (doi: 10.1038/mto.2016.3). In some embodiments, receptor ligands and antigens may be conjugated to a fusion agent to redirect fusion activity to cells displaying the target receptor (DOI: 10.1089/hgtb.2012.054, DOI: 10.1128/JVi.76.7.3558-3563.2002). In some embodiments, the targeting protein may also include antibodies or antigen binding fragments thereof (e.g., fab ', F (ab') 2, fv fragments, scFv antibody fragments, disulfide-linked Fv (sdFv), fd fragments consisting of VH and CH1 domains, linear antibodies, single domain antibodies such as sdAb (VL or VH), nanobodies, or camelidae VHH domains), antigen binding fibronectin type III (Fn 3) scaffolds such as fibronectin polypeptide miniantibodies, ligands, cytokines, chemokines, or T Cell Receptors (TCRs). In some embodiments, the protein fusion agent may be re-targeted by non-covalent conjugation of the CD7 binding agent to a fusion protein or targeting protein (e.g., a hemagglutinin protein). In some embodiments, the fusion protein may be engineered to bind to the Fc region of an antibody targeting an antigen on a target cell, thereby redirecting fusion activity to cells displaying the antibody target (DOI: 10.1128/JVI.75.17.8016-8020.2001, DOI:10.1038/nm 1192). In some embodiments, the altered and unaltered fusion agent may be displayed on the same retroviral vector or VLP (doi: 10.1016/j. Biological.2014.01.051).

In some embodiments, the CD7 binding agent comprises a humanized antibody molecule, an intact IgA, igG, igE or IgM antibody, a bispecific or multispecific antibody (e.g., zybodies @, etc.), an antibody fragment, such as a Fab fragment, fab ' fragment, F (ab ') ₂ fragment, fd ' fragment, fd fragment, and isolated CDR, or a collection thereof, a single chain Fv, a polypeptide-Fc fusion, a single domain antibody (e.g., a shark single domain antibody, such as IgNAR or a fragment thereof), a camelid antibody, a masking antibody (e.g., probodies;) Small Modular ImmunoPharmaceuticals ("smitm"), a single chain or tandem diabody (TandAb; VHH; ANTICALINS;, nanobodies; minibody, biTE;, ankyrin repeat protein or DARPINs;, avimers; DART, TCR-like antibody, ADNECTINS;, affilins; trans-bodies;, affibodies;, trimerX;, microProteins, fynomers;, CENTYRINS;, and KALBITOR.

In some embodiments, the CD7 binding agent is a peptide. In some embodiments, the CD7 binding agent is an antibody, such as a single chain variable fragment (scFv). In some embodiments, the CD7 binding agent is an antibody, such as a single domain antibody. In some embodiments, the CD7 binding agent is a VHH. In some embodiments, the antibody may be human or humanized. In some embodiments, the antibody or portion thereof is naturally occurring. In some embodiments, the antibody or portion thereof is synthetic.

In some embodiments, the C-terminus of the CD7 binding agent is attached to the C-terminus of a G protein (e.g., a fusion agent) or biologically active portion thereof. In some embodiments, the N-terminus of the CD7 binding agent is exposed on the outer surface of the lipid bilayer.

In some embodiments, the CD7 binding agent is the only surface displayed non-viral sequence of the viral vector. In some embodiments, the CD7 binding agent is the only membrane-bound non-viral sequence of the viral vector. In some embodiments, the viral vector does not contain a molecule that binds or stimulates T cells other than a CD7 binding agent.

In some embodiments, the viral vector may display CD7 binding agent that is not conjugated to a protein fusion agent in order to redirect fusion activity to cells bound by the targeting moiety, or to affect homing.

CD8 binding agents

In some embodiments, the lipid particles disclosed herein comprise one or more re-targeting attachment proteins that each independently comprise (i) a paramyxovirus envelope attachment protein, and (ii) a targeting moiety for a target molecule expressed on the surface of a target cell, wherein the target molecule is CD8. In some embodiments, the targeting moiety is a CD8 binding domain, e.g., a CD8 binding agent, such as any of those disclosed herein.

In some embodiments, the lipid particles disclosed herein comprise one or more CD8 binding agents. For example, the CD8 binding agent may be fused or incorporated with a protein fusion agent or attachment protein. In another embodiment, the CD8 binding agent may be incorporated into the lipid particle envelope by fusion with a transmembrane domain.

Exemplary CD8 binding agents include antibodies and fragments thereof (e.g., scFv, VHH) that bind to one or more of CD8 a and CD8 β. Such antibodies may be derived from any species and may be, for example, mouse, rabbit, human, humanized or camelid antibodies. Exemplary antibodies include those disclosed in WO2014025828、WO2014164553、WO2020069433、WO2015184203、US20160176969、WO2017134306、WO2019032661、WO2020257412、WO2018170096、WO2020060924、US10730944、US20200172620, and additional exemplary anti-CD 8 binding agents and G proteins of non-human antibody OKT8;RPA-T8、12.C7 (Novus);17D8、3B5、LT8、RIV11、SP16、YTC182.20、MEM-31、MEM-87、RAVB3、C8/144B (Thermo Fisher);2ST8.5H7、Bu88、3C39、Hit8a、SPM548、CA-8、SK1、RPA-T8 (GeneTex);UCHT4 (Absolute Antibody);BW135/80 (Miltenyi);G42-8 (BD Biosciences);C8/1779R、mAB 104 (Enzo Life Sciences);B-Z31 (Sapphire North America);32-M4、5F10、MCD8、UCH-T4、5F2 (Santa Cruz);D8A8Y、RPA-T8 (Cell Signaling Technology). are described in U.S. provisional application No. 63/172,518, which is incorporated herein by reference. Other exemplary binding agents include engineered ankyrin repeat protein (DARPin) and fibronectin type III (Fn 3) scaffold-based binding agents.

In some embodiments, the CD8 binding agent comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NO: 483, 484 and 485, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NO: 486, 487 and 488, respectively. In some embodiments, the CD8 binding agent comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO. 369, and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO. 370. In some embodiments, the CD8 binding agent comprises the sequence set forth in SEQ ID NO 489.

In some embodiments, the CD8 binding agent comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 490, 491 and 492, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 493, 494 and 495, respectively. In some embodiments, the CD8 binding agent comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO. 371, and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO. 372. In some embodiments, the CD8 binding agent comprises the sequence set forth in SEQ ID NO. 496.

In some embodiments, the CD8 binding agent comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 497, 498 and 499, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS 486, 487 and 500, respectively. In some embodiments, the CD8 binding agent comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO. 373 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO. 374. In some embodiments, the CD8 binding agent comprises the sequence set forth in SEQ ID NO. 501.

In some embodiments, the CD8 binding agent comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS: 502, 503 and 504, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences shown in SEQ ID NOS: 505, 506 and 507, respectively. In some embodiments, the CD8 binding agent comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO. 375, and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO. 376. In some embodiments, the CD8 binding agent comprises the sequence set forth in SEQ ID NO. 508.

In some embodiments, the CD8 binding agent comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences shown in SEQ ID NOS 509, 510 and 511, respectively. In some embodiments, the CD8 binding agent comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO. 377. In some embodiments, the CD8 binding agent comprises the sequence set forth in SEQ ID NO 377.

In some embodiments, the CD8 binding agent comprises any CD8 binding agent as described in US 2019/0144885, which is incorporated herein by reference in its entirety.

In some embodiments, the CD8 binding agent is an scFv comprising a VH and a VL as set forth in any one of the following, wherein the VH and VL are separated by a linker. In some embodiments, the CD8 binding agent is a VHH having the following sequence. In some embodiments, the CD8 binding agent is linked to the C-terminus of truncated NiV-G as shown in SEQ ID NO. 19 to provide a re-targeted NiV-G. In some embodiments, the re-targeted NiV-G is pseudotyped with NiV-F (e.g., as shown in SEQ ID NO: 227) on a lentiviral vector. In some embodiments, the lentiviral vector further comprises a payload gene encoding an anti-CD 19 CAR. In some embodiments, the anti-CD 19 CAR comprises an anti-CD 19 FMC63 scFv binding domain shown as SEQ ID NO. 239, a CD8 hinge shown as SEQ ID NO. 367, a CD8 transmembrane domain shown as SEQ ID NO. 368, a 4-1bb signaling domain shown as SEQ ID NO. 248, and a CD3 zeta signaling domain shown as SEQ ID NO. 249.

CD8_1

VH (SEQ ID NO.: 369):

QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGIIDPSDGNTNYAQNFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKERAAAGYYYYMDVWGQGTTVTVSS

VL (SEQ ID NO.: 370):

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKR

CD8_2

VH (SEQ ID NO.:371):

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYIQWVRQAPGQGLEWMGWINPNSGGTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKEGDYYYGMDAWGQGTMVTVSS

VL (SEQ ID NO.:372):

DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQTPHTFGQGTKVEIKR

CD8_3

VH (SEQ ID NO.:373):

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGGFDPEDGETIYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDQGWGMDVWGQGTTVTVSS

VL(SEQ ID NO.:374):

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSTPYTFGQGTKLEIKR

CD8_4

VH (SEQ ID NO.:375):

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNHYMHWVRQAPGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASSESGSDLDYWGQGTLVTVSS

VL (SEQ ID NO.:376):

DIQMTQSPSSLSASVGDRVTITCRASQTIGNYVNWYQQKPGKAPKLLIYGASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSAPLTFGGGTKVEIKR

In some embodiments, the CD8 binding agent is a VHH as shown below:

VHH (SEQ ID NO.:377):

QVQLVESGGGLVQAGGSLRLSCAASGRTFSGYVMGWFRQAPGKQRKFVAAISRGGLSTSYADSVKGRFTISRDNAKNTVFLQMNTLKPEDTAVYYCAADRSDLYEITAASNIDSWGQGTLVTVSS

In some embodiments, the protein fusion agent (e.g., attachment protein) can be re-targeted by covalently conjugating the CD8 binding agent to the fusion protein or attachment protein (e.g., re-targeting attachment protein). In some embodiments, the fusion agent and CD8 binding agent are covalently conjugated by expression of a chimeric protein comprising the fusion agent linked to a CD8 binding agent. In some embodiments, single chain variable fragments (scFvs) may be conjugated to a fusion agent to redirect fusion activity to cells (doi:10.1038/nbt1060, DOI 10.1182/blood-2012-11-468579, doi:10.1038/nmeth.1514, doi:10.1006/mthe.2002.0550, HUMAN GENE THERAPY 11:817– 826, doi:10.1038/nbt942, doi:10.1371/journal.pone.0026381, DOI 10.1186/s12896-015-0142-z). displaying scFv binding targets, in some embodiments, engineered anchor protein repeat proteins (DARPin) may be conjugated to a fusion agent to redirect fusion activity to cells displaying DARPin binding targets (doi: 10.1038/mt.2013.16, doi:10.1038/mt.2010.298, doi: 10.4049/jimmunol.1500956) and combinations of different DARPin (doi: 10.1038/mto.2016.3). In some embodiments, receptor ligands and antigens may be conjugated to a fusion agent to redirect fusion activity to cells displaying the target receptor (DOI: 10.1089/hgtb.2012.054, DOI: 10.1128/JVi.76.7.3558-3563.2002). In some embodiments, the targeting protein may also include antibodies or antigen binding fragments thereof (e.g., fab ', F (ab') 2, fv fragments, scFv antibody fragments, disulfide-linked Fv (sdFv), fd fragments consisting of VH and CH1 domains, linear antibodies, single domain antibodies such as sdAb (VL or VH), nanobodies, or camelidae VHH domains), antigen binding fibronectin type III (Fn 3) scaffolds such as fibronectin polypeptide miniantibodies, ligands, cytokines, chemokines, or T Cell Receptors (TCRs). In some embodiments, the protein fusion agent may be re-targeted by non-covalent conjugation of the CD8 binding agent to a fusion protein or targeting protein (e.g., a hemagglutinin protein). In some embodiments, the fusion protein may be engineered to bind to the Fc region of an antibody targeting an antigen on a target cell, thereby redirecting fusion activity to cells displaying the antibody target (DOI: 10.1128/JVI.75.17.8016-8020.2001, DOI:10.1038/nm 1192). In some embodiments, the altered and unaltered fusion agent may be displayed on the same retroviral vector or VLP (doi: 10.1016/j. Biological.2014.01.051).

In some embodiments, the CD8 binding agent comprises a humanized antibody molecule, an intact IgA, igG, igE or IgM antibody, a bispecific or multispecific antibody (e.g., zybodies @, etc.), an antibody fragment, such as a Fab fragment, fab ' fragment, F (ab ') ₂ fragment, fd ' fragment, fd fragment, and isolated CDR, or a collection thereof, a single chain Fv, a polypeptide-Fc fusion, a single domain antibody (e.g., a shark single domain antibody, such as IgNAR or a fragment thereof), a camelid antibody, a masking antibody (e.g., probodies;) Small Modular ImmunoPharmaceuticals ("smitm"), a single chain or tandem diabody (TandAb; VHH; ANTICALINS;, nanobodies; minibody, biTE;, ankyrin repeat protein or DARPINs;, avimers; DART, TCR-like antibody, ADNECTINS;, affilins; trans-bodies;, affibodies;, trimerX;, microProteins, fynomers;, CENTYRINS;, and KALBITOR.

In some embodiments, the CD8 binding agent is a peptide. In some embodiments, the CD8 binding agent is an antibody, such as a single chain variable fragment (scFv). In some embodiments, the CD8 binding agent is an antibody, such as a single domain antibody. In some embodiments, the CD8 binding agent is a VHH. In some embodiments, the antibody may be human or humanized. In some embodiments, the antibody or portion thereof is naturally occurring. In some embodiments, the antibody or portion thereof is synthetic.

In some embodiments, the C-terminus of the CD8 binding agent is attached to the C-terminus of a G protein (e.g., a fusion agent) or biologically active portion thereof. In some embodiments, the N-terminus of the CD8 binding agent is exposed on the outer surface of the lipid bilayer.

In some embodiments, the CD8 binding agent is the only surface displayed non-viral sequence of the viral vector. In some embodiments, the CD8 binding agent is the only membrane-bound non-viral sequence of the viral vector. In some embodiments, the viral vector does not contain a molecule that binds or stimulates T cells other than a CD8 binding agent.

In some embodiments, the viral vector may display CD8 binding agent that is not conjugated to a protein fusion agent in order to redirect fusion activity to cells bound by the targeting moiety, or to affect homing.

Non-limiting examples of re-targeted fusion agents comprising CD8 binding agents are described in US 11,535,869, the entire contents of which are incorporated herein by reference.

B. HSC binding domains

In some embodiments, the lipid particles disclosed herein comprise one or more re-targeting attachment proteins that each independently comprise (i) a paramyxovirus envelope attachment protein, and (ii) a targeting moiety for a target molecule expressed on the surface of a target cell. In some embodiments, the targeting moiety is a HSC binding domain, e.g., a HSC binding agent, such as any of those disclosed herein.

In some embodiments, the lipid particles disclosed herein comprise one or more HSC binding domains (e.g., HSC binders) that target the viral vector to cells of HSCs. In some embodiments, the HSC binding agent binds to a molecule expressed on the surface of the HSC. The cell surface molecule may be a receptor, co-receptor or GPI-anchored protein. In some embodiments, the HSC binding agent binds ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, or ITGA3. In some embodiments, the HSC binding agent may be fused to or incorporated into a protein fusion agent or lipid particle envelope adhesion protein (e.g., a re-targeting adhesion protein). In some embodiments, the HSC binding agent may be incorporated into the viral envelope by fusion with a transmembrane domain. In some embodiments, the HSC binding agent targets the lipid particle to HSC.

In particular embodiments, the HSC binding agent may be fused or incorporated with a protein fusion or attachment protein, thereby re-targeting the lipid particle to the HSC. In some embodiments, for re-targeting, the HSC binder is fused to a protein fusion agent or envelope adhesion protein that is mutated to reduce binding to the natural binding partner of the fusion agent or viral envelope protein. In some embodiments, the fusion agent is or comprises a mutant G protein, or biologically active portion thereof, which is a mutant of wild-type NiV-G and exhibits reduced binding to one or both of the natural binding partners ephrin B2 or ephrin B3, including any of the above. Thus, in some aspects, the fluxing agent may be re-targeted to exhibit altered tropism. In some embodiments, the binding confers a re-targeted binding compared to the binding of a wild-type surface glycoprotein in which a new or different binding activity is conferred. In certain embodiments, the binding confers a re-targeted binding compared to binding of a wild-type G protein in which a new or different binding activity is conferred. In some embodiments, the fusion agent is randomly mutated. In some embodiments, the fusion agent is rationally mutated. In some embodiments, the fusion agent undergoes directed evolution. In some embodiments, the fusion agent is truncated and only a subset of peptides are used in the viral vector. In some embodiments, the amino acid residues in the measles hemagglutinin protein may be mutated to alter the binding properties of the protein, thereby redirecting fusion (doi: 10.1038/nbt942, volume Molecular Therapy, 8 th, 1427-1436 2008, 8 th month) ,doi:10.1038/nbt1060, DOI: 10.1128/JVI.76.7.3558–3563.2002, DOI: 10.1128/JVI.75.17.8016–8020.2001, doi: 10.1073pnas.0604993103).

In some embodiments, the protein fusion agent may be re-targeted by covalently conjugating the HSC binding agent to the attachment protein. In some embodiments, the fusion agent and HSC binder are covalently conjugated by expression of a chimeric protein (e.g., a re-targeted attachment protein) comprising the fusion agent linked to the HSC binder. The HSC binding agent may comprise any targeting protein capable of conferring specific binding to a target molecule expressed on the HSC surface. In some embodiments, the targeting protein may also include antibodies or antigen binding fragments thereof (e.g., fab ', F (ab') 2, fv fragments, scFv antibody fragments, disulfide-linked Fv (sdFv), fd fragments consisting of VH and CH1 domains, linear antibodies, single domain antibodies such as sdAb (VL or VH), nanobodies, or camelidae VHH domains), antigen binding fibronectin type III (Fn 3) scaffolds such as fibronectin polypeptide miniantibodies, ligands, cytokines, chemokines, or T Cell Receptors (TCRs). In some embodiments, the HSC binding agent is an antibody or antigen binding fragment thereof. In some embodiments, the fusion protein may be engineered to bind to the Fc region of an antibody targeting an antigen on a target cell, thereby redirecting fusion activity to cells displaying the antibody target (DOI: 10.1128/JVI.75.17.8016-8020.2001, DOI:10.1038/nm 1192). In some embodiments, the altered and unaltered fusion agent may be displayed on the same retroviral vector or VLP (doi: 10.1016/j. Biological.2014.01.051).

In some embodiments, a single chain variable fragment (scFv) can be conjugated to a fusion agent to redirect fusion activity to HSC (doi:10.1038/nbt1060, DOI 10.1182/blood-2012-11-468579, doi:10.1038/nmeth.1514, doi:10.1006/mthe.2002.0550, HUMAN GENE THERAPY 11:817– 826, doi:10.1038/nbt942, doi:10.1371/journal.pone.0026381, DOI 10.1186/s12896-015-0142-z). displaying scFv binding targets, in some embodiments, a designed ankyrin repeat protein (DARPin) can be conjugated to a fusion agent to redirect fusion activity to HSCs displaying DARPin binding targets (doi: 10.1038/mt.2013.16, doi:10.1038/mt.2010.298, doi: 10.4049/jimmunol.1500956) and combinations of different DARPins (doi: 10.1038/mto.2016.3). In some embodiments, a single domain antibody (e.g., VHH) can be conjugated to a fusion agent to redirect fusion activity to HSCs that display sdAb binding targets. In some embodiments, receptor ligands and antigens may be conjugated to a fusion agent to redirect fusion activity to HSCs displaying target receptors (DOI: 10.1089/hgtb.2012.054, DOI: 10.1128/JVI.76.7.3558-3563.2002).

In some embodiments, the target cell is a cd34+ progenitor cell. In some embodiments, the target cell molecule is expressed on at least a subset of cd34+ progenitor cells.

In some embodiments, the cell surface molecule is expressed on HSCs. In some embodiments, the cell surface molecule is expressed on MPP. In some embodiments, the cell surface molecule is expressed on MLP. In some embodiments, the cell surface molecule is expressed on ETP. In some embodiments, the cell surface molecule is expressed on a MEP. In some embodiments, the cell surface molecule is expressed on CMP. In some embodiments, the cell surface molecule is expressed on GMP. In some embodiments, the cell surface molecule is expressed on any combination of the aforementioned cd34+ progenitor cell subsets. In some embodiments, the cell surface molecule is expressed on HSCs and MPPs. In some embodiments, the cell surface molecule is expressed on myeloid progenitor cells. In some embodiments, the cell surface molecule is expressed on a lymphoid progenitor cell. In some embodiments, the cell surface molecule is expressed on myeloid progenitor cells. In some embodiments, the cell surface molecule is expressed on HSC, MPP, MEP, CMP and GMP.

In some embodiments, the cell surface molecule is ASCT2. In some embodiments, the target cell is asct2+.

In some embodiments, the cell surface molecule is CD105. In some embodiments, the target cell is cd105+.

In some embodiments, the cell surface molecule is CD110. In some embodiments, the target cell is cd110+.

In some embodiments, the cell surface molecule is CD117. In some embodiments, the target cell is cd117+.

In some embodiments, the cell surface molecule is CD133. In some embodiments, the target cell is cd133+.

In some embodiments, the cell surface molecule is CD146. In some embodiments, the target cell is cd146+.

In some embodiments, the cell surface molecule is CD164. In some embodiments, the target cell is cd164+.

In some embodiments, the cell surface molecule is CD34. In some embodiments, the target cell is cd34+.

In some embodiments, the cell surface molecule is CD46. In some embodiments, the target cell is cd46+.

In some embodiments, the cell surface molecule is CD49f. In some embodiments, the target cell is cd49f+.

In some embodiments, the cell surface molecule is CD90. In some embodiments, the target cell is cd90+.

In some embodiments, the cell surface molecule is EPCR. In some embodiments, the target cell is epcr+.

In some embodiments, the cell surface molecule is ITGA3. In some embodiments, the target cell is itga3+.

In some embodiments, the target molecule is CD133. In some embodiments, the target cell is cd133+. In some embodiments, the targeting agent is an anti-CD 133 antibody. Exemplary anti-CD 133 antibodies include CART133, AC133, 293C3-SDIE, CMab-43, RW03, 293C3H9 (293C 3) and W6B3H10 (W6B 3), U.S. Pat. Nos. US8722858, US9249225, US9624303, US10106623, US10711068, US11098109, US11214628, US11352435 and US11220551, U.S. Pat. No. US20130224202, PCT application No. WO200901840、WO2011089211、WO2011149493、WO2014128185、WO2015121383、WO2016154623、WO2018045880、WO2018072025 and WO2022022718, and anti-CD 133 antibodies disclosed in Canadian patent application No. CA 2962157.

In some embodiments, the lipid particles disclosed herein comprise one or more re-targeting attachment proteins that each independently comprise (i) a paramyxovirus envelope attachment protein, and (ii) a targeting moiety for a target molecule expressed on the surface of a target cell, wherein the target molecule is CD133. In some embodiments, the targeting moiety is a CD133 binding domain, e.g., a CD133 binding agent, such as any of those disclosed herein.

In some embodiments, the lipid particle comprises one or more HSC binding domains that are CD133 binding agents that target the viral vector to cells of HSCs. In some embodiments, the lipid particle comprises two or more HSC binding domains that are each CD133 binding agents that target the viral vector to cells of HSCs. In some embodiments, each HSC binding domain of two or more HSC binding domains, each of which is a CD133 binding agent, binds a different epitope of the same target molecule (CD 133). In some embodiments, the lipid particle comprises two or more (e.g., two, three, four, or five or more) CD 133-binding agents.

In some embodiments, the lipid particle comprises one or more targeting moieties, such as a HSC binding domain, selected from (a) a CD133 binding agent comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS 536, 537 and 538, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NOS 540, 541 and 542, respectively; (b) a CD133 binder comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO: 545, 546 and 547, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NO: 549, 550 and 551, respectively, (c) a CD133 binder comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO: 518, 519 and 520, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NO: 522, 523 and 524, respectively, (d) a CD133 binder comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO: 527, 528 and 529, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NO: 531, 532 and 533, respectively, and (e) a CD133 binder comprising CDR-L1, CDR-L2 and CDR-L133 comprising the amino acid sequences of SEQ ID NO: 522, 523 and 524, respectively, and CDR-H560, and CDR-H3 comprising the amino acid sequences of SEQ ID NO: 527, 528 and 529, respectively, and amino acid sequences of amino acids of 55 and 554 and 553, respectively, and 553, respectively CDR-L2 and CDR-L3.

In some embodiments, the lipid particle comprises one or more targeting moieties, such as a HSC binding domain, selected from (a) a CD133 binding agent comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS 289, 565 and 538, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NOS 540, 541 and 542, respectively; (b) a CD133 binder comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOs 566, 567 and 547, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NOs 549, 550 and 551, respectively; (c) a CD133 binder comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO: 568, 569 and 520, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NO: 522, 523 and 524, respectively, (d) a CD133 binder comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO: 570, 571 and 529, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NO: 531, 532 and 533, respectively, and (e) a CD133 binder comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO: 572, 573 and 556, respectively, and CDR-L1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO: 558, 559 and 560, respectively CDR-L2 and CDR-L3.

In some embodiments, the lipid particle comprises one or more (e.g., one, two, three, or more) targeting moieties independently selected from (a) a CD133 binding agent comprising a heavy chain Variable (VH) region comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 535, and an amino acid sequence comprising or having at least 90%, a, A light chain Variable (VL) region comprising an amino acid sequence of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, (b) a CD133 binding agent comprising a VH region comprising an amino acid sequence of SEQ ID NO: 544 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and an amino acid sequence of SEQ ID NO: 548 or an amino acid sequence having at least 90%, 91%, 92%, 93% sequence identity thereto, A VL region comprising an amino acid sequence of 94%, 95%, 96%, 97%, 98% or 99% sequence identity, (c) a CD133 binding agent comprising a VH region comprising an amino acid sequence of SEQ ID NO 517 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a VL region comprising an amino acid sequence of SEQ ID NO 521 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, A VL region comprising an amino acid sequence of 98% or 99% sequence identity, (d) a CD133 binding agent comprising a VH region comprising an amino acid sequence of SEQ ID NO: 526 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a VL region comprising an amino acid sequence of SEQ ID NO: 530 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and (e) a CD133 binding agent comprising a VL region comprising an amino acid sequence of SEQ ID NO: 553 or an amino acid sequence having at least 90%, a, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and a VL region comprising the amino acid sequence of SEQ ID NO: 557 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

In some embodiments, the lipid particle comprises one or more targeting moieties, e.g., a HSC binding domain, comprising an amino acid sequence independently selected from the group consisting of SEQ ID NOs 516, 525, 534, 543, and 552, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, each of the one or more CD133 binding agents is independently an scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 516, 525, 534, 543, and 552, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

In some embodiments, the lipid particle comprises a first CD133 binding agent that is a scFv comprising the amino acid sequence of SEQ ID NO: 516 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a second CD133 binding agent that is a scFv comprising the amino acid sequence of SEQ ID NO: 525 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

In some embodiments, the target molecule is CD105. In some embodiments, the target cell is cd105+. In some embodiments, the targeting agent is an anti-CD 105 antibody. Exemplary anti-CD 105 antibodies include card Luo Tuo mab, TRC105, huRH, and TCR205, as well as U.S. patent nos. US8221753, US8609094, US9150652, US95181212, US9926375, US9944714, US10155820, and US10336831, U.S. patent application nos. US20100098692, US20100196398, US20170007714, and US20220233591, PCT application nos. WO2010039873、WO2011041441、WO2016077451、WO2018067819、WO2010032059、WO2012149412、WO2015118031、WO2021118955 and WO2021118957, and anti-CD 105 antibodies disclosed in korean patent No. KR101398707B 1.

In some embodiments, the lipid particle disclosed herein comprises one or more re-targeting attachment proteins that each independently comprise (i) a paramyxovirus envelope attachment protein, and (ii) a targeting moiety for a target molecule expressed on the surface of a target cell, wherein the target molecule is CD117. In some embodiments, the targeting moiety is a CD117 binding domain, e.g., a CD117 binding agent, such as any of those disclosed herein.

In some embodiments, the target molecule is CD117. In some embodiments, the target cell is cd117+. In some embodiments, the targeting agent is an anti-CD 117 antibody. In some embodiments, the lipid particle comprises one or more HSC binding domains that are CD117 binding agents that target the viral vector to cells of HSCs. In some embodiments, the lipid particle comprises one or more targeting moieties, e.g., a HSC binding domain, that is a CD117 binding agent comprising an amino acid sequence independently selected from the group consisting of SEQ ID NOs 512-515 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 593-639, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

In some embodiments, the CD117 binding agent is a VHH comprising CDR-H1, CDR-H2 and CDR-H3 contained within an amino acid sequence selected from the group consisting of SEQ ID NOS 512-515, 593-639, wherein the target molecule is CD117.

In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 593 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 594 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 595 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 596 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 597 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 598 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 599 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 600 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 601 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 602 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. in some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO 603 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 512 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 604 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 605 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 606 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 607 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 608 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 609 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 610 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO 611 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 612 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 613 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 614 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 615 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 616 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO 617 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 618 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 619 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 620 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO 353 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. in some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 622 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO 623 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 624 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 513 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 625 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 626 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 627 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 628 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 629 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 630 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. in some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 514 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 631 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 632 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 633 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO 634 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 515 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 635 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 636 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 637 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 638 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID No. 639 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

In some embodiments, the lipid particle comprises one or more targeting moieties, such as a HSC binding domain, selected from (a) a CD117 binding agent comprising an amino acid sequence comprising SEQ ID NO: 512 or a VHH having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a CD117 binding agent comprising an amino acid sequence comprising SEQ ID NO: 513 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (c) a CD117 binding agent comprising an amino acid sequence comprising SEQ ID NO: 514 or a VHH having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and (d) a CD117 binding agent comprising an amino acid sequence comprising SEQ ID NO: 515 or a VHH having at least 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98% or 99% sequence identity thereto.

In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 593. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 594. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 595. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 596. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 597. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 598. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 599. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 600. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 601. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 602. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 603. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 512. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 604. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 605. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 606. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 607. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 608. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 609. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 610. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO 611. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 612. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 613. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 614. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 615. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 616. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 617. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 618. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 619. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 620. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 621. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 622. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 623. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 624. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 513. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 625. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 626. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO: 627. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 628. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 629. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 630. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 514. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 631. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 632. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 633. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 634. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 515. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 635. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 636. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 637. In some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO 638. in some embodiments, the CD117 binding agent is a VHH comprising the amino acid sequence of SEQ ID NO. 639.

In some embodiments, the target molecule is EPCR. In some embodiments, the target cell is epcr+. In some embodiments, the targeting agent is an anti-EPCR antibody. Exemplary anti-EPCR antibodies include JRK1494, JRK1535, and U.S. patent application nos. US20210355231 and US20220127374, and anti-EPCR antibodies disclosed in PCT application nos. WO2020051277 and WO 2020161478.

In some embodiments, the target molecule is CD34. In some embodiments, the target cell is cd34+. In some embodiments, the targeting agent is an anti-CD 34 antibody. Exemplary anti-CD 34 antibodies include h4C8, 9C5, and U.S. patent nos. US8399249, US8927696, and US10106623, and anti-CD 34 antibodies disclosed in U.S. patent application No. US20090221003、US20130143238、US20100311955、US20130172533、US20170320966、US20170298148、US20180169177、US20190135945; and PCT application nos. WO2009079922 and WO 2015121383.

In some embodiments, the target molecule is ASCT2. In some embodiments, the target cell is asct2+. In some embodiments, the targeting agent is an anti-ASCT 2 antibody. Exemplary anti-ASCT 2 antibodies include idastuzumab, MEDI7247, KM4008, KM4012, KM4018, and U.S. patent nos. US8268592, US8501180, US8945870, US8673592, and US10829554, U.S. patent application nos. US20180273617, US20190367605, US20210024629, and anti-ASCT 2 antibodies disclosed in PCT application nos. WO2017083451, WO 2018089393.

In some embodiments, the target molecule is CD90. In some embodiments, the target cell is cd90+. In some embodiments, the targeting agent is an anti-CD 90 antibody. Exemplary anti-CD 90 antibodies include EPR3133, CL1028, CL1040, AF-9, JF10-09, 5E10, 7E1B11, as well as U.S. patent application No. US20210054068, and anti-CD 90 antibodies disclosed in PCT application No. WO 2017214050.

In some embodiments, the target molecule is CD164. In some embodiments, the target cell is cd164+. In some embodiments, the targeting agent is an anti-CD 164 antibody. Exemplary anti-CD 164 antibodies include 67D2, H-4, 32G1, EML2058, 5C5, N6B6, 4B4, and 15-11-14, as well as PCT application number WO2006002438, and anti-CD 164 antibodies disclosed in German patent numbers DE19727813C1 and DE19727815C 1.

In some embodiments, the target molecule is CD49f. In some embodiments, the target cell is cd49f+. In some embodiments, the targeting agent is an anti-CD 49f antibody. Exemplary anti-CD 49F antibodies include CL6957, goH3, SR45-00, and MP4F10, as well as U.S. patent nos. US5538725, US10030071, U.S. patent application nos. US20110301227, US20160194400, US20160280789, and anti-CD 49F antibodies disclosed in PCT application nos. WO2015034052 and WO 2018127655.

In some embodiments, the target molecule is CD146. In some embodiments, the target cell is cd146+. In some embodiments, the targeting agent is an anti-CD 146 antibody. Exemplary anti-CD 146 antibodies include the anti-CD 146 antibodies disclosed in Ipri Li Shan, PRX003, ABX-MA1, huAA98, M2H-1, M2J-1 and JM1-24-3, and U.S. Pat. No. US6924360、US7067131、US709844、US9447190、US9782500、US10407506、US10414825、US10407507、US10584177、US10905771、US11427648;, U.S. Pat. No. US20030147809、US20040115205、US20060246077、US20140314744、US20150239980、US20140227292、US20160206764、US20190192573、US20170002089、US20150259419、US20170037144、US20170129954、US20170101470、US20180271994、US20180105602、US20200010563、US20200165336、US20200216560、US20200262929、US20200100838、US20220041748、US20220041749; and PCT application No. WO2003057006、WO2003057837、WO2003057838、WO2012170071、WO2014000338、WO2015044218、WO2015136469、WO2015136470、WO2017046776、WO2017046774、WO2017149513、WO2017153953、WO2017208210、WO2018033630、WO2018220467、WO2018223140、WO2019068842、WO2019133639、WO2019137309、WO2020132190、WO2020132232 and WO 2022082073.

Additional exemplary targeting agents and corresponding target molecules are described in the following table. The targeting agent may be any targeting agent that binds a related target molecule described in the cited related documents.

B. F protein

In some embodiments, the lipid particle comprises one or more paramyxovirus fusion (F) proteins. In some embodiments, the lipid particle contains exogenous or overexpressed paramyxovirus fusion (F) proteins. In some embodiments, the paramyxovirus fusion (F) protein is disposed in a lipid bilayer. In some embodiments, the paramyxovirus fusion (F) protein (e.g., a fusion agent) facilitates fusion of the lipid particle to the membrane. In some embodiments, the membrane is a plasma cell membrane. In some embodiments, the paramyxovirus fusion (F) protein binds to a binding partner on the surface of the target cell. In some embodiments, the paramyxovirus fusion (F) protein comprises a protein having a hydrophobic fusion peptide domain.

In some embodiments, the paramyxovirus fusion (F) protein is or comprises nipah virus protein F, measles virus F protein, tree shrew paramyxovirus F protein, hendra virus F protein, hennipah virus F protein, measles virus F protein, respiratory tract virus F protein, sendai virus F protein, mumps virus F protein, or avian mumps virus F protein.

In some embodiments, the paramyxovirus fusion (F) protein comprises a henipav F protein molecule or a biologically active portion thereof. In some embodiments, the henipav virus F protein is hendra (HeV) virus F protein, nipah (NiV) virus F protein, cedar (CedPV) virus F protein, mexican virus F protein, langa virus F protein, or bata paramyxovirus F protein, or biologically active portions thereof.

Table 4 provides a non-limiting example of F protein. In some embodiments, the N-terminal hydrophobic fusion peptide domain of the F protein molecule, or a biologically active portion thereof, is exposed outside of the lipid bilayer.

In some embodiments, the paramyxovirus fusion (F) protein is a variant NipaF protein (NiV-F). In some embodiments, the variant NiV-F protein exhibits fusion activity. In some embodiments, the variant NiV-F promotes fusion of the lipid particle (e.g., lentiviral vector) to the membrane. The F protein of henipav virus (including NiV-F) is encoded as an F0 precursor containing a signal peptide (e.g., corresponding to amino acid residues 1-26 below). After cleavage of the signal peptide, mature F0 (SEQ ID NO:235, i.e., SEQ ID NO: 256) is transported to the cell surface and then endocytosed and cleaved by cathepsin L (e.g., between amino acids 109-110 of NiV-F corresponding to the amino acids shown in SEQ ID NO: 235) into mature fusion subunits F1 (e.g., amino acids 110-546 of NiV-F corresponding to SEQ ID NO: 235) and F2 (e.g., amino acid residues 27-109 of NiV-F corresponding to SEQ ID NO: 235). F1 and F2 subunits bind through disulfide bonds and circulate back to the cell surface. The F1 subunit contains a fusion peptide domain (e.g., corresponding to amino acids 110-129 of NiV-F shown below, e.g., SEQ ID NO: 235) at the N-terminus of the F1 subunit, where it is capable of inserting into the cell membrane to drive fusion. In certain cases, the fusion activity is blocked by the association of the F protein with the G protein until the G binds to the target molecule, causing it to dissociate from F and expose the fusion peptide to mediate membrane fusion.

The sequence and activity of the F protein is highly conserved among different henipa virus species. For example, the F proteins of NiV and HeV viruses share 89% amino acid sequence identity. Furthermore, in some cases, the Huntipa virus F protein exhibits compatibility with G proteins from other species to trigger fusion (Brandel-TRETHEWAY et al, journal of virology 2019.93 (13): e 00577-19). In some aspects of the provided lipid particles, the F protein is heterologous to the G protein, i.e., the F and G proteins or biologically active portions are from different henipav virus species. For example, the F protein is from hendra virus and the G protein is from nipah virus. In other aspects, the F protein may be a chimeric F protein comprising regions of F proteins from different henipa virus species. In some embodiments, converting the region of amino acid residues of the F protein from one species of henipa virus to another may result in a fusion with the G protein of the species comprising the amino acid insertion. (Brandel-TRETHEWAY et al, 2019). In some cases, the chimeric F protein contains an extracellular domain from one henipa virus species and a transmembrane and/or cytoplasmic domain from a different henipa virus species. For example, the F protein contains the extracellular domain of hendra virus and the transmembrane/cytoplasmic domain of nipah virus. The F protein sequences disclosed herein are disclosed primarily as expression sequences comprising an N-terminal signal sequence. Since such N-terminal signal sequences are typically co-translated or post-translationally cleaved, the mature protein sequences of all F protein sequences disclosed herein are also considered to be devoid of an N-terminal signal sequence.

In some embodiments, the F protein or biologically active portion thereof is a wild-type nipah virus F (NiV-F) protein or a hendra virus F protein, or a functionally active variant or biologically active portion thereof. For example, in some embodiments, the F protein or biologically active portion thereof is a wild-type NiV-F protein or functionally active variant or biologically active portion thereof.

In some embodiments, the F protein has the amino acid sequence shown as SEQ ID NO. 234, SEQ ID NO. 235, SEQ ID NO. 236, SEQ ID NO. 237 or SEQ ID NO. 238, or is a functionally active variant thereof or a biologically active portion thereof that retains fusion activity. In some embodiments, functionally active variants comprise an amino acid sequence that has at least equal to or about 80%, at least equal to or about 85%, at least equal to or about 90%, at least equal to or about 91%, at least equal to or about 92%, at least equal to or about 93%, at least equal to or about 94%, at least equal to or about 95%, equal to or about 96%, at least equal to or about 97%, at least equal to or about 98%, or at least equal to or about 99% sequence identity to SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237, or SEQ ID NO:238, and retains fusion activity to bind to a G protein (such as a variant NiV-G provided herein). In some embodiments, the biologically active portion has an amino acid sequence that has at least equal to or about 80%, at least equal to or about 85%, at least equal to or about 90%, at least equal to or about 91%, at least equal to or about 92%, at least equal to or about 93%, at least equal to or about 94%, at least equal to or about 95%, equal to or about 96%, at least equal to or about 97%, at least equal to or about 98%, or at least equal to or about 99% sequence identity to SEQ ID NO. 234, SEQ ID NO. 235, SEQ ID NO. 236, SEQ ID NO. 237, or SEQ ID NO. 238.

In particular embodiments, the F protein has the amino acid sequence shown as SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:259, or is a functionally active variant thereof or a biologically active portion thereof that retains fusion activity. In some embodiments, functionally active variants comprise an amino acid sequence that has at least equal to or about 80%, at least equal to or about 85%, at least equal to or about 90%, at least equal to or about 91%, at least equal to or about 92%, at least equal to or about 93%, at least equal to or about 94%, at least equal to or about 95%, equal to or about 96%, at least equal to or about 97%, at least equal to or about 98%, or at least equal to or about 99% sequence identity to SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258, or SEQ ID NO:259, and retains fusion activity to bind to a G protein (such as variant NiV-G provided herein). In some embodiments, the biologically active portion has an amino acid sequence that has at least equal to or about 80%, at least equal to or about 85%, at least equal to or about 90%, at least equal to or about 91%, at least equal to or about 92%, at least equal to or about 93%, at least equal to or about 94%, at least equal to or about 95%, equal to or about 96%, at least equal to or about 97%, at least equal to or about 98%, or at least equal to or about 99% sequence identity to SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258, or SEQ ID NO: 259.

Fusion activity includes the activity of a paramyxovirus fusion (F) protein binding to paramyxovirus envelope proteins (e.g., one or more G proteins) to promote or aid cytoplasmic fusion of two membrane lumens, such as a lumen of a targeted lipid particle having henipav F and at least two G proteins embedded in its lipid bilayer, and a target cell, such as a cell containing a surface receptor or molecule recognized or bound by the targeted envelope proteins. In some embodiments, the F protein and the at least one G protein are from the same Huntiepa virus species (e.g., niV-G and NiV-F). In some embodiments, the F protein and at least one G protein are from different henipa virus species (e.g., niV-G and HeV-F). In certain embodiments, the F protein of the functionally active variant or biologically active portion retains a cleavage site (e.g., a cleavage site between amino acids 109-110 corresponding to SEQ ID NO: 235) that is cleaved by cathepsin L.

Reference to retaining fusion activity includes activity (in combination with a G protein such as a variant G protein provided herein) that is equal to or about 10% to equal to or about 150% or greater of the level or extent of binding of a corresponding wild-type F protein such as SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237 or SEQ ID NO:238, SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:259 or a cathepsin L cleaved form thereof containing F1 and F2 subunits. In some embodiments, the fusion activity is at least or at least about 10% of the fusion activity level or extent of the corresponding wild-type F protein, such as at least or at least about 15% of the fusion activity level or extent of the corresponding wild-type F protein, such as at least or at least about 20% of the fusion activity level or extent of the corresponding wild-type F protein, such as at least or at least about 25% of the fusion activity level or extent of the corresponding wild-type F protein, such as at least or at least about 30% of the fusion activity level or extent of the corresponding wild-type F protein, such as at least or at least about 35% of the fusion activity level or extent of the corresponding wild-type F protein, such as at least or at least about 40% of the fusion activity level or extent of the corresponding wild-type F protein, such as at least or at least about 45% of the fusion activity level or extent of the corresponding wild-type F protein, such as at least or at least about 50% of the fusion activity level or at least about, such as at least about 55% of the fusion activity level or extent of the corresponding wild-type F protein, such as at least about 65% of the fusion activity level or at least about 75% of the fusion activity level or at least about F protein, such as at least about 75% of the fusion activity level or at least about the fusion activity of the corresponding wild-type F protein, such as at least or at least about 95% of the level or extent of fusion activity corresponding to wild-type F protein, such as at least or at least about 100% of the level or extent of fusion activity corresponding to wild-type F protein, or such as at least or at least about 120% of the level or extent of fusion activity corresponding to wild-type F protein.

In some embodiments, the paramyxovirus fusion (F) protein is a mutant F protein, which is a functionally active fragment or biologically active portion containing one or more amino acid mutations (such as one or more amino acid insertions, deletions, substitutions or truncations). In some embodiments, the mutations described herein involve amino acid insertions, deletions, substitutions, or truncations of amino acids compared to the reference F protein sequence. In some embodiments, the reference F protein sequence is a wild-type sequence of an F protein or a biologically active portion thereof. In some embodiments, the mutant F protein or biologically active portion thereof is a mutant of a wild-type hendra (Hev) viral F protein, nipah (NiV) viral F protein, cedar (CedPV) viral F protein, a mevinoviral F protein, or a bata paramyxovirus F protein. In some embodiments, the wild-type F protein is encoded by a nucleotide sequence encoding any one of SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237 or SEQ ID NO:238, SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:259, or a cathepsin L cleaved form thereof containing F1 and F2 subunits.

In some embodiments, the mutant F protein is a truncated biologically active portion and lacks up to 22 consecutive amino acid residues at or near the C-terminus of the wild-type F protein, such as the wild-type F protein shown in any one of SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237 or SEQ ID NO:238, SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO: 259. In some embodiments, the mutant F protein is truncated and lacks up to 22 contiguous amino acids, such as up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 contiguous amino acids at the C-terminus of the wild-type F protein.

In some embodiments, provided lipid particles of NiV-F such as mutant or truncated NiV-F contain F0 precursor or its containing F1 and F2 subunit proteolytic cleavage form, such as in the cleavage site (for example with SEQ ID NO:235 between amino acids corresponding to amino acids 109-110) protein cleavage, to produce two chain can be connected by disulfide bond. In some embodiments, a NiV-F such as a wild-type NiV-F or a truncated or mutated NiV-F protein is produced or encoded as a F ₀ precursor, which can then be proteolytically cleaved to yield an F protein containing F1 and F2 subunits linked by disulfide bonds. Thus, it should be understood that references herein to a particular sequence of NiV-F (SEQ ID NO) generally refer to the F ₀ precursor sequence, but should also be understood to include proteolytic cleavage forms or sequences thereof that contain two cleavage chains F1 and F2. For example, niV-F such as mutant or truncated NiV-F contains the F1 subunit corresponding to amino acids 110-546 of NiV-F shown in SEQ ID NO. 235 or a truncated or mutant sequence thereof, and F2 corresponding to amino acid residues 27-109 of NiV-F shown in SEQ ID NO. 235.

In some embodiments, the mutant F protein is a truncated biologically active portion and lacks up to 22 consecutive amino acid residues at or near the C-terminus of a wild-type NiV-F protein, such as the wild-type NiV-F protein shown in SEQ ID NO:235 or SEQ ID NO: 256. In some embodiments, the mutant F protein is truncated and lacks up to 22 contiguous amino acids, such as up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 contiguous amino acids at the C-terminus of a wild-type NiV-F protein, such as the wild-type NiV-F protein shown in SEQ ID NO:235 or SEQ ID NO: 256. In some embodiments, the mutant F protein contains an F1 subunit and an F2 subunit, wherein (1) the F1 subunit is truncated and lacks up to 22 contiguous amino acids at or near the C-terminus of the wild-type F1 subunit, such as lacks up to 22 contiguous amino acids at or near the C-terminus of the wild-type F1 subunit corresponding to amino acids 110-546 of NiV-F shown in SEQ ID NO:235, and (2) the F2 subunit has a sequence corresponding to amino acid residues 27-109 of NiV-F shown in SEQ ID NO: 235.

In some embodiments, the paramyxovirus fusion (F) protein is a mutant NiV-F protein, which is a biologically active portion thereof comprising a 22 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein (SEQ ID NO:235 or SEQ ID NO: 256). In some embodiments, the NiV-F protein is encoded by a nucleotide sequence encoding the sequence set forth in SEQ ID NO: 226. In some embodiments, the NiV-F protein is encoded by a nucleotide sequence encoding a sequence having at least equal to or about 90%, at least equal to or about 91%, at least equal to or about 92%, at least equal to or about 93%, at least equal to or about 94%, at least equal to or about 95%, equal to or about 96%, at least equal to or about 97%, at least equal to or about 98%, or at least equal to or about 99% sequence identity to SEQ ID NO: 226. In a particular embodiment, the variant F protein is a mutant NiV-F protein having the amino acid sequence shown in SEQ ID NO. 227. In some embodiments, the NiV-F protein is encoded by a sequence having at least equal to or about 90%, at least equal to or about 91%, at least equal to or about 92%, at least equal to or about 93%, at least equal to or about 94%, at least equal to or about 95%, equal to or about 96%, at least equal to or about 97%, at least equal to or about 98%, or at least equal to or about 99% sequence identity to SEQ ID NO 227. In some embodiments, the F protein molecule or biologically active portion thereof comprises the sequence set forth in SEQ ID NO: 227.

In some embodiments, the mutant F protein contains an F1 subunit and an F2 subunit, wherein (1) the F1 subunit is shown as amino acids 110-524 of SEQ ID NO:226 and (2) the F2 subunit is shown as amino acids 27-109 of SEQ ID NO: 226.

In some embodiments, the mutant F protein contains an F1 subunit and an F2 subunit, wherein (1) the F1 subunit is shown as amino acids 84-498 of SEQ ID NO:227 and (2) the F2 subunit is shown as amino acids 1-83 of SEQ ID NO: 227.

C. Polynucleotide

Provided herein are polynucleotides comprising nucleic acid sequences encoding re-targeted attachment proteins. Polynucleotides encoding at least two re-targeted attachment proteins are also provided herein. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a G protein, an F protein, or a biologically active portion thereof. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a first G protein and a second G protein or biologically active portions thereof. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a first G protein, a second G protein, an F protein, or a biologically active portion thereof. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a first G protein, a second G protein, a third G protein, an F protein, or a biologically active portion thereof. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a first G protein, a second G protein, a third G protein, a fourth G protein, an F protein, or biologically active portions thereof. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a first G protein, a second G protein, a third G protein, a fourth G protein, a fifth G protein, an F protein, or biologically active portions thereof. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a first G protein, a second G protein, a third G protein, a fourth G protein, a fifth G protein, one or more additional G proteins, an F protein, or biologically active portions thereof. In some embodiments, the polynucleotide further comprises a nucleic acid sequence encoding a binding domain, such as a single domain antibody (sdAb) variable domain or biologically active portion thereof. The polynucleotide may comprise a nucleotide sequence encoding any of the chimeric attachment proteins described above. The polynucleotide may be a synthetic nucleic acid. Expression vectors comprising any of the provided polynucleotides are also provided.

In some any embodiment, expression of the natural or synthetic nucleic acid is typically achieved by operably linking the nucleic acid encoding the gene of interest to a promoter and integrating the construct into an expression vector. In some embodiments, the vector may be suitable for replication and integration in eukaryotes. In some embodiments, the cloning vector contains transcriptional and translational terminators, initiation sequences, and promoters useful for expression of the desired nucleic acid sequences. In some of any of the embodiments, the plasmid comprises a promoter suitable for expression in a cell.

In some embodiments, the polynucleotide contains at least one promoter operably linked to control the expression of the targeted re-targeted attachment protein and/or G protein and/or F protein. For the expression of the re-targeted adhesion protein, at least one module in each promoter serves to locate the initiation site of RNA synthesis. The most notable examples are TATA boxes, but the lack of TATA boxes in some promoters, such as promoters of mammalian terminal deoxynucleotidyl transferase genes and promoters of SV40 genes, discrete elements covering the initiation site itself help to fix the start position.

In some embodiments, additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. In some embodiments, additional promoter elements are located in the region 30-110 bp upstream of the start site, but many promoters have recently been shown to also contain functional elements downstream of the start site. In some embodiments, the spacing between promoter elements is generally flexible such that promoter function is maintained when the elements are inverted or moved relative to each other. In some embodiments, the thymidine kinase (tk) promoter, the spacing between promoter elements may be increased to 50 bp a before the activity begins to decrease. In some embodiments, individual elements may act synergistically or independently to activate transcription, depending on the promoter.

The promoter may be one naturally associated with the gene or polynucleotide sequence, which may be obtained by isolating 5' non-coding sequences located upstream of the coding segment and/or exon. Such promoters may be referred to as "endogenous". Similarly, an enhancer may be an enhancer naturally associated with a polynucleotide sequence, downstream or upstream of the sequence. Alternatively, certain advantages will be obtained by placing the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment. Recombinant or heterologous enhancer also refers to an enhancer that is not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, as well as non-naturally occurring "promoters or enhancers, i.e., different elements containing different transcriptional regulatory regions, and/or mutations that alter expression. In addition to synthetically producing nucleic acid sequences of promoters and enhancers, recombinant cloning and/or nucleic acid amplification techniques (including PCR) can be used in combination with the compositions disclosed herein to produce sequences (U.S. Pat. nos. 4,683,202 and 5,928,906).

In some embodiments, a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. In some embodiments, the promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked thereto. In some embodiments, a suitable promoter is an extended growth factor-la (EF-l a). In some embodiments, a suitable promoter is human PGK (hPGK). In some embodiments, a suitable promoter is RPBSA. In some embodiments, other constitutive promoter sequences may also be used, including, but not limited to, simian virus 40 (SV 40) early promoter, mouse Mammary Tumor Virus (MMTV), human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, moMuLV promoter, avian leukemia virus promoter, epstein-Barr virus (Epstein-Barr virus) immediate early promoter, rous sarcoma virus (Rous sarcoma virus) promoter, and human gene promoters such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter.

In some embodiments, the promoter is a T cell-associated promoter (e.g., a T cell promoter). In some embodiments, the promoter is a promoter that drives gene expression in T cells. In some embodiments, the promoter sequence is a T cell promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked thereto. In some embodiments, the promoter is dLck or a CD3 delta promoter. In some embodiments, the promoter is an ELAM promoter, hTNFAIP1, hVCAM1, hCCL2, or pA20 promoter.

In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter provides a molecular switch that can turn on expression of its operably linked polynucleotide sequence when expression is desired or turn off expression when expression is not desired. In some embodiments, the inducible promoter comprises a metallothionein promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

In some embodiments, exogenously controlled inducible promoters can be used to regulate expression of heavy target attachment proteins, G proteins, F proteins, and/or antigen binding domains such as single domain antibodies (sdabs) variable domains. For example, radiation-inducible promoters, thermally-inducible promoters, and/or drug-inducible promoters may be used to selectively drive transgene expression in, for example, a targeting region. In such embodiments, the location, duration, and level of transgene expression can be modulated by the administration of an exogenous induction source.

In some embodiments, the expression of the re-targeted attachment protein is regulated using a drug-inducible promoter. For example, in some cases, the promoter, enhancer, or transactivator comprises a Lac operator sequence, a tetracycline operator sequence, a galactose operator sequence, a doxycycline operator sequence, a rapamycin operator sequence, a tamoxifen operator sequence, or a hormone-responsive operator sequence, or an analog thereof. In some cases, the inducible promoter comprises a Tetracycline Responsive Element (TRE). In some embodiments, the inducible promoter comprises an Estrogen Responsive Element (ERE) that can activate gene expression in the presence of tamoxifen. In some cases, a drug-inducible element such as a TRE may be combined with a selected promoter to enhance transcription in the presence of a drug such as doxycycline. In some embodiments, the drug-inducible promoter is a small molecule inducible promoter.

Any of the provided polynucleotides can be modified to remove CpG motifs and/or to optimize codons for translation in a particular species (such as human, canine, feline, equine, ovine, bovine, etc. species). In some embodiments, the polynucleotide is optimized for human codon usage (i.e., human codon optimized). In some embodiments, the polynucleotide is modified to remove CpG motifs. In other embodiments, the provided polynucleotides are modified to remove CpG motifs and to perform codon optimization, such as human codon optimization. Methods for codon optimisation and CpG motif detection and modification are well known. In general, polynucleotide optimization enhances transgene expression, increases transgene stability and retains the amino acid sequence of the encoded polypeptide.

To assess the expression of the targeted envelope protein, the expression vector to be introduced into the cell may also contain a selectable marker gene or a reporter gene or both to facilitate identification and selection of expression particles, e.g., viral particles. In other embodiments, the selectable marker may be carried on a separate DNA fragment and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences so as to be capable of expression in a host cell. Useful selectable markers are known in the art and include, for example, antibiotic resistance genes, such as neo and the like.

Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Reporter genes encoding readily determinable proteins are well known in the art. Typically, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and encodes a protein whose expression is evidenced by some readily detectable property, such as enzymatic activity. The expression of the reporter gene is determined at a suitable time after introduction of the DNA into the recipient cell.

Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or green fluorescent protein genes (see, e.g., ui-Tei et al, 2000, FEBS Lett. 479:79-82). Suitable expression systems are well known and may be prepared or commercially available using well known techniques. Unique internal restriction sites may be used or internal deletion constructs may be created by partial digestion of non-unique restriction sites. The constructs may then be transfected into cells that exhibit high levels of desired polynucleotide and/or polypeptide expression. Typically, constructs with minimal 5' flanking regions that show the highest levels of reporter gene expression are identified as promoters. Such promoter regions may be linked to a reporter gene and used to evaluate the ability of an agent to modulate promoter-driven transcription.

Lipid particles and methods of producing the same

Provided herein is a lipid particle comprising a lipid bilayer, a lumen surrounded by the lipid bilayer, and a re-targeted attachment protein, such as any one described, wherein the re-targeted attachment protein is embedded within the lipid bilayer. In some embodiments, the provided lipid particles preferentially target hematopoietic cells (e.g., T cells), which is mediated by the tropism of a re-targeted attachment protein, such as a G protein. In some embodiments, the lipid particle may additionally contain an exogenous agent (e.g., a therapeutic agent) for delivery to the cell. In some embodiments, the lipid particle is introduced into a cell of the subject. Methods of delivering any provided lipid particle to a cell are also provided.

In some embodiments, the provided lipid particles exhibit fusion activity mediated by a re-targeted attachment protein (such as a G protein and/or any provided F protein) that facilitates the merger or fusion of the two lumens of the lipid particles and the target cell membrane. Thus, there is fusion in the lipid particles provided. In some embodiments, the fusion comprises a naturally derived amphiphilic lipid bilayer with a re-targeting attachment protein as a fusion agent. In some embodiments, the fusion comprises (a) a lipid bilayer, (b) a lumen (e.g., comprising cytosol) surrounded by the lipid bilayer, and (c) a fusion agent that is exogenous or overexpressed relative to the source cell. In some embodiments, the re-targeted attachment protein is disposed in a lipid bilayer. In some embodiments, the fusion comprises several different types of lipids, e.g., amphiphilic lipids, such as phospholipids.

In some embodiments, the lipid particle comprises a bilayer of a naturally derived amphiphilic lipid surrounding a lumen or cavity. In some embodiments, the lipid particle comprises a lipid bilayer as the outermost surface. In some embodiments, the lipid bilayer surrounds the lumen. In some embodiments, the lumen is aqueous. In some embodiments, the inner lumen is in contact with a hydrophilic head group inside the lipid bilayer. In some embodiments, the lumen is cytoplasmic. In some embodiments, the cytoplasm contains a cellular component present in the source cell. In some embodiments, the cytoplasm does not contain a component present in the source cell. In some embodiments, the lumen is a cavity. In some embodiments, the cavity contains an aqueous environment. In some embodiments, the cavity does not contain an aqueous environment.

In some aspects, the lipid bilayer is derived from a source cell during the production of the lipid-containing particle. Exemplary methods for producing lipid-containing particles are provided in section i.e. In some embodiments, the lipid bilayer comprises a membrane component of a cell that produces the lipid bilayer, e.g., a phospholipid, a membrane protein, and the like. In some embodiments, the lipid bilayer comprises a cytoplasm comprising components found in microvesicle-producing cells, such as solutes, proteins, nucleic acids, etc., but not all components of the cell, such as their lack of nuclei. In some embodiments, the lipid bilayer is considered exosome-like. The size of the lipid bilayer may vary and in some cases has a diameter ranging from 30 to 300 nm (such as from 30 to 150 nm and including from 40 to 100 nm).

In some embodiments, the lipid bilayer is a viral envelope. In some embodiments, the viral envelope is obtained from a source cell. In some embodiments, the viral envelope is obtained from a viral capsid from a source cytoplasmic membrane. In some embodiments, the lipid bilayer is obtained from a membrane other than the plasma membrane of the host cell. In some embodiments, the viral envelope lipid bilayer is embedded with a re-targeting attachment protein that is a viral protein, including a viral glycoprotein as described herein, such as a G protein, and in some aspects, also an F protein.

In other aspects, the lipid bilayer comprises a synthetic lipid complex. In some embodiments, the synthetic lipid complex is a liposome. In some embodiments, the lipid bilayer is a vesicular structure characterized by a phospholipid bilayer membrane and an internal aqueous medium. In some embodiments, the lipid bilayer has a plurality of lipid layers separated by an aqueous medium. In some embodiments, lipid bilayers spontaneously form when phospholipids are suspended in an excess of aqueous solution. In some examples, the lipid component undergoes self-rearrangement prior to forming the closed structure, and entraps water and dissolved solutes between the lipid bilayers.

In some embodiments, the targeted envelope protein and the fusion agent (such as any of those described above, including any of those that are exogenous or overexpressed relative to the source cell) are placed in a lipid bilayer.

In some embodiments, the lipid particle comprises several different types of lipids. In some embodiments, the lipid is an amphiphilic lipid. In some embodiments, the amphiphilic lipid is a phospholipid. In some embodiments, the phospholipid comprises phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine. In some embodiments, the lipid comprises a phospholipid such as phosphorylcholine and phosphoinositide. In some embodiments, the lipid comprises DMPC, DOPC, and DSPC.

In some embodiments, the bilayer may be composed of one or more lipids of the same or different types. In some embodiments, the source cell is selected from the group consisting of HEK293 cells, CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, hepG2 cells, saos-2 cells, huh7 cells, heLa cells, W163 cells, 211 cells, and 211A cells.

In some embodiments, the lipid particle may be a viral particle, a virus-like particle, a nanoparticle, a vesicle, an exosome, a dendrimer, a lentivirus, a viral vector, a enucleated cell, a microvesicle, a membrane vesicle, an extracellular membrane vesicle, a plasma membrane vesicle, a giant plasma membrane vesicle, an apoptotic body, a mitotic particle, a nuclear erythrocyte, a lysosome, another membrane-enclosed vesicle, or a lentiviral vector, a virus-based particle, a virus-like particle (VLP), or a cell-based particle.

In particular embodiments, the lipid particle is of viral origin. In some embodiments, the lipid particle may be a virus-based particle, such as a viral vector particle (e.g., a lentiviral vector particle) or a virus-like particle (e.g., a lentiviral-like particle). In some embodiments, the lipid bilayer is a viral envelope. In some embodiments, the viral envelope is obtained from a host cell. In some embodiments, the viral envelope is obtained from a viral capsid from a source cytoplasmic membrane. In some embodiments, the lipid bilayer is obtained from a membrane other than the plasma membrane of the host cell. In some embodiments, the viral envelope lipid bilayer is embedded with a viral protein, including a viral glycoprotein.

In particular embodiments, the lipid particle is not of viral origin. In some embodiments, the lipid particle may be a nanoparticle, a vesicle, an exosome, a dendrimer, a enucleated cell, a microvesicle, a membrane vesicle, an extracellular membrane vesicle, a plasma membrane vesicle, a giant plasma membrane vesicle, an apoptotic body, a mitotic particle, a nuclear erythrocyte, a lysosome, another membrane-enclosed vesicle, or a cell-derived particle.

In some embodiments, the lipid bilayer comprises a membrane component of the host cell from which the lipid bilayer is derived, e.g., a phospholipid, a membrane protein, and the like. In some embodiments, the lipid bilayer comprises cytosol including components found in cells from which the vehicle is derived, such as solutes, proteins, nucleic acids, etc., but not all components of the cell, such as lack of a nucleus. In some embodiments, the lipid bilayer is considered exosome-like. The size of the lipid bilayer may vary and in some cases has a diameter ranging from 30 to 300nm (such as from 30 to 150 nm and including from 40 to 100 nm).

In certain embodiments, an exogenous agent such as a polynucleotide or polypeptide is encapsulated in the inner cavity of the lipid particle. Embodiments of the provided lipid particles can have a variety of characteristics that facilitate delivery of a payload (e.g., a desired transgene or exogenous agent) to a target cell. The exogenous agent may be a polynucleotide or a polypeptide. In some embodiments, the lipid particles provided herein are administered to a subject, e.g., a mammal, e.g., a human. In such embodiments, the subject may be at risk for a particular disease or disorder, may have symptoms of a particular disease or disorder, or may be diagnosed or identified as having a particular disease or disorder. In one embodiment, the subject has cancer. In one embodiment, the subject has an infectious disease. In some embodiments, the lipid particle contains a nucleic acid sequence (polynucleotide) encoding an exogenous agent or polypeptide exogenous agent for use in treating a disease or disorder.

The lipid particles may comprise spherical particles or may comprise particles of elongate or irregular shape.

In some embodiments, one or more characteristics of the particle composition that are related to its size may be evaluated, including diameter, its range of variation above and below the mean (mean) or median of the diameters, coefficient of variation, polydispersity index, or other measure of particle size in the composition. Various methods for particle characterization may be used, including but not limited to laser diffraction, dynamic light scattering (DLS; also known as photon correlation spectroscopy), or image analysis, such as microscopy or automated image analysis.

In some embodiments, the lipid particles provided have a diameter or mean (average) diameter of the particles in the composition of less than about 3 μm, less than about 2 μm, less than about 1 μm, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500m, less than about 400 nm, less than about 300, less than about 200 nm, less than about 150nm, less than about 100nm, less than about 50nm, or less than about 20 nm. In some embodiments, the diameter of the lipid particle or the average (mean) diameter of the particles in the composition is less than about 400 nm. In another embodiment, the diameter of the lipid particle or the average (mean) diameter of the particles in the composition is less than about 150 nm. In some embodiments, the diameter of the lipid particle or the average (mean) diameter of the particles in the composition is between equal to or about 2 μm and equal to or about 1 μm, between equal to or about 1 μm and equal to or about 900 nm, between equal to or about 900 nm and equal to or about 800 nm, between equal to or about 800 and equal to or about 700 nm, between equal to or about 700 nm and equal to or about 600 nm, between equal to or about 600 nm and equal to or about 500 nm, between equal to or about 500 nm and equal to or about 400 nm, between equal to or about 400 nm and equal to or about 300 nm, between equal to or about 300 nm and equal to or about 200 nm, between equal to or about 200 and equal to or about 100nm, between equal to or about 100 and equal to or about 50nm, or between equal to or about 20nm and equal to or about 50 nm.

In some embodiments, the median particle diameter in the particle composition is between equal to or about 10nm and equal to or about 1000 nM, between equal to or about 25 nm and equal to or about 500 nm, between equal to or about 40 nm and equal to or about 300 nm, between equal to or about 50nm and equal to or about 250 nm, between equal to or about 60 nm and equal to or about 225 nm, between equal to or about 70 nm and equal to or about 200 nm, between equal to or about 80 nm and equal to or about 175 nm, or between equal to or about 90 nm and equal to or about 150 nm.

In some embodiments, 90% of the lipid particles in the composition fall within 50% of the median diameter of the lipid particles. In some embodiments, 90% of the lipid particles in the composition fall within 25% of the median diameter of the lipid particles. In some embodiments, 90% of the lipid particles in the composition fall within 20% of the median diameter. In some embodiments, 90% of the lipid particles in the composition fall within 15% of the median diameter of the lipid particles. In some embodiments, 90% of the lipid particles in the composition fall within 10% of the median diameter of the lipid particles.

In some embodiments, 75% of the lipid particles in the composition fall within +/-2 or +/-1 St Dev standard deviations (St Dev) of the average diameter of the lipid particles. In some embodiments, 80% of the lipid particles in the composition fall within +/-2 St Dev or +/-1 St Dev of the average diameter of the lipid particles. In some embodiments, 85% of the lipid particles in the composition fall within +/-2 St Dev or +/-1 St Dev of the average diameter of the lipid particles. In some embodiments, 90% of the lipid particles in the composition fall within +/-2 St Dev or +/-1 St Dev of the average diameter of the lipid particles. In some embodiments, 95% of the lipid particles in the composition fall within +/-2 St Dev or +/-1 St Dev of the average diameter of the lipid particles.

In some embodiments, the lipid particles have an average hydrodynamic radius of about 100 nm to about two microns, as determined, for example, by DLS. In some embodiments, the lipid particle has an average hydrodynamic radius of between or about 2 μm and or about 1 μm, between or about 1 μm and or about 900 nm, between or about 900 nm and equal or about 800 nm, between or about 800 and equal or about 700 nm, between or about 700 nm and equal or about 600 nm, between or about 600 nm and equal or about 500 nm, between or about 500 nm and equal or about 400 nm, between or about 400 nm and equal or about 300 nm, between or about 300 nm and equal or about 200 nm, between or about 200 and equal or about 100 nm, between or about 100 and equal or about 50 nm, or between or about 20 nm and equal or about 50 nm.

In some embodiments, the lipid particles have an average geometric radius of about 100nm to about two microns, as determined, for example, by multi-angle light scattering. In some embodiments, the lipid particle has an average geometric radius of between or about 2 μm and or about 1 μm, between or about 1 μm and or about 900 nm, between or about 900 nm and equal or about 800 nm, between or about 800 and equal or about 700 nm, between or about 700 nm and equal or about 600 nm, between or about 600 nm and equal or about 500 nm, between or about 500 nm and equal or about 400 nm, between or about 400 nm and equal or about 300 nm, between or about 300 nm and equal or about 200 nm, between or about 200 and equal or about 100nm, between or about 100 and equal or about 50 nm, or between or about 20 nm and equal or about 50 nm.

In some embodiments, the lipid particle composition has a coefficient of variation (COV) (i.e., standard deviation divided by mean) of less than 30% or less than about 30%, less than 25% or less than about 25%, less than 20% or less than about 20%, less than 15% or less than about 15%, less than 10% or less than about 10% or less than 5% or less than about 5%.

In some embodiments, the provided lipid particle composition is characterized by its polydispersity index, which is a measure of the size distribution of the particles, with values between 1 (maximum dispersion) and 0 (same size for all particles) being possible. In some embodiments of the present invention, in some embodiments, the compositions of lipid particles provided herein have a polydispersity index of between equal to or about 0.05 and equal to or about 0.7, between equal to or about 0.05 and equal to or about 0.6, between equal to or about 0.05 and equal to or about 0.5, between equal to or about 0.05 and equal to or about 0.4, between equal to or about 0.05 and equal to or about 0.3, between equal to or about 0.05 and equal to or about 0.2, between equal to or about 0.05 and equal to or about 0.1, between equal to or about 0.1 and equal to or about 0.7, between equal to or about 0.1 and equal to or about 0.5, between equal to or about 0.1 and equal to or about 0.4, between equal to or about 0.1 and about 0.3, between equal to or about 0.1 and about 0.2, between equal to or about 0.2 and about 0.2, between equal to or about 0.1 and about 0.5, between equal to or about 0.1 and about 0.1, and about 0.5. In some embodiments, the polydispersity index is less than 0.05 or less than about 0.05, less than 0.1 or less than about 0.1, less than 0.15 or less than about 0.15, less than 0.2 or less than about 0.2, less than 0.25 or less than about 0.25, less than 0.3 or less than about 0.3, less than 0.4 or less than about 0.4, less than 0.5 or less than about 0.5, less than 0.6 or less than about 0.7. Various lipid particles are known, any of which may be produced according to the provided embodiments. Non-limiting examples of lipid particles include any one as described in or contain features as described in International published PCT application No. WO 2017/095946;WO 2017/095944;WO 2017/095940;WO 2019/157319;WO 2018/208728;WO 2019/113512;WO 2019/161281;WO 2020/102578;WO 2019/222403;WO 2020/014209;WO 2020/102485;WO 2020/102499;WO 2020/102503;WO 2013/148327;WO 2017/182585;WO 2011/058052; or WO 2017/068077, each of which is incorporated by reference in its entirety.

The characteristics of the lipid particles provided are described in the following subsections.

A. virus-based particles

Provided herein are virus-based particles derived from viruses, including those derived from retroviruses or lentiviruses, that contain a re-targeted attachment protein, such as described in section II. In some embodiments, the amphiphilic lipid bilayer of the lipid particle is or comprises a viral envelope. In some embodiments, the amphiphilic lipid bilayer of the lipid particle is or comprises a lipid derived from a producer cell. In some embodiments, the viral envelope may comprise a fluxing agent, for example, a fluxing agent endogenous to the virus or a pseudofluxing agent. In some embodiments, the lumen or cavity of the lipid particle comprises a viral nucleic acid, e.g., a retroviral nucleic acid, e.g., a lentiviral nucleic acid. In some embodiments, the viral nucleic acid may be a viral genome. In some embodiments, the lipid particle further comprises one or more viral nonstructural proteins, for example in a cavity or lumen thereof. In some embodiments, the virus-based particle is or comprises a virus-like particle (VLP). In some embodiments, the VLP does not comprise any viral genetic material. In some embodiments, the virus-based particles do not comprise any virus-derived nucleic acids or viral proteins, such as viral structural proteins.

Biological methods for introducing exogenous agents into host cells include the use of DNA and RNA vectors. DNA and RNA vectors can also be used to house and deliver polynucleotides and polypeptides. Viral vectors and virus-like particles, and in particular retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors and virus-like particles may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. See, for example, U.S. patent nos. 5,350,674 and 5,585,362. Methods for producing cells comprising vectors and/or exogenous acids are well known in the art. See, e.g., sambrook et al, 2001, molecular Cloning: A Laboratory Manual, cold Spring Harbor Laboratory, new York.

In some embodiments, the viral particle or virus-like particle bilayer of amphiphilic lipids is or comprises lipids derived from an infected host cell. In some embodiments, the lipid bilayer is a viral envelope. In some embodiments, the viral particle or virus-like particle envelope is obtained from a host cell. In some embodiments, the viral particle or virus-like particle envelope is obtained from a viral capsid from a source cytoplasmic membrane. In some embodiments, the lipid bilayer is obtained from a membrane other than the plasma membrane of the host cell. In some embodiments, the viral particle or virus-like particle envelope lipid bilayer is embedded with one or more re-targeted attachment proteins, the one or more proteins being viral proteins, including viral glycoproteins, including G protein and/or F protein.

In some embodiments, a viral particle or virus-like particle, e.g., a retroviral particle or one or more transduction units of a retroviral-like particle, is administered to a subject. In some embodiments, at least 1, 10, 100, 1000, 104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, or 1014 transduction units/kg are administered to the subject. In some embodiments, at least 1, 10, 100, 1000, 104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, or 1014 transduction units per target cells per ml of blood are administered to the subject.

1. Viral vector particles

In some embodiments, the lipid particle is or comprises a virus or viral vector, such as a retrovirus or retrovirus vector, such as a lentivirus or lentivirus vector. In some embodiments, the virus or viral vector is recombinant. For example, the viral particles may be referred to as recombinant viruses and/or recombinant viral vectors, which are used interchangeably. In some embodiments, the lipid particle is a recombinant lentiviral vector particle.

In some embodiments, the lipid particle comprises a lipid bilayer comprising a retroviral vector comprising an envelope. For example, in some embodiments, the amphiphilic lipid bilayer is or comprises a viral envelope. The viral envelope may comprise a fluxing agent, for example a heavy targeting attachment protein fluxing agent, which is endogenous to the virus or which is a pseudofluxing agent. In some embodiments, the lumen or cavity of the viral vector comprises a viral nucleic acid, e.g., a retroviral nucleic acid, e.g., a lentiviral nucleic acid. The viral nucleic acid may be a viral genome. In some embodiments, the viral vector may also comprise one or more viral nonstructural proteins, for example in a cavity or lumen thereof. In some embodiments, the virus-based vector particle is a lentivirus. In some embodiments, the lentiviral vector particle is human immunodeficiency virus-1 (HIV-1).

In some aspects, the viral vector particles are limited in the number of polynucleotides that can be packaged. In some embodiments, the nucleotides encoding the polypeptide to be packaged may be modified such that they retain functional activity with fewer nucleotides in the coding region than the nucleotides encoding the wild-type peptide. Such modifications may include truncations or other deletions. In some embodiments, more than one polypeptide may be expressed from the same promoter, such that they are fusion polypeptides. In some embodiments, the insert size to be packaged (i.e., the viral genome or portion thereof; or heterologous polynucleotide as described) may be between 500-1000, 1000-2000, 2000-3000, 3000-4000, 4000-5000, 5000-6000, 6000-7000, or 7000-8000 nucleotides in length. In some embodiments, the insert may be more than 8000 nucleotides in length, such as 9000, 10,000, or 11,000 nucleotides.

In some embodiments, a viral vector particle, such as a retroviral vector particle, comprises one or more of gag polyprotein, polymerase (e.g., pol), integrase (e.g., functional or nonfunctional variant), protease, and fusion agent. In some embodiments, the lipid particle further comprises rev. In some embodiments, one or more of the above-described proteins are encoded in the retroviral genome (i.e., an insert as described above), and in some embodiments, one or more of the above-described proteins are provided in trans, e.g., by a helper cell, helper virus, or helper plasmid. In some embodiments, the lipid particle nucleic acid (e.g., retroviral nucleic acid) comprises one or more of the following nucleic acid sequences 5 'LTR (e.g., comprising U5 and lacking a functional U3 domain), psi packaging element (Psi, ψ), central polypurine region (cPPT) promoter operably linked to a payload gene, payload gene (optionally comprising introns prior to open reading frame), poly A tail sequence, WPRE, and 3' LTR (e.g., comprising U5 and lacking a functional U3). In some embodiments, the lipid particle nucleic acid further comprises a retroviral cis-acting RNA packaging element and a cPPT/CTS element. In some embodiments, the lipid particle nucleic acid further comprises one or more insulator elements. In some embodiments, the recognition site is located between the Poly a tail sequence and WPRE.

In some embodiments, the lipid particle comprises a supramolecular complex formed from viral proteins that self-assemble into capsids. In some embodiments, the lipid particle is a viral particle derived from a viral capsid. In some embodiments, the lipid particle is a viral particle derived from a viral nucleocapsid. In some embodiments, the lipid particle comprises a nucleocapsid derivative that retains the properties of the packaged nucleic acid.

In some embodiments, the lipid particle packages nucleic acid from a host cell carrying one or more viral nucleic acids (e.g., retroviral nucleic acids) during expression. In some embodiments, the nucleic acid does not encode any genes involved in viral replication. In a particular embodiment, the lipid particle is a virus-based particle, e.g., a retroviral particle such as a lentiviral particle, which is replication defective.

In some cases, the lipid particle is a viral particle that is morphologically indistinguishable from the wild-type infectious virus. In some embodiments, the viral particles present the entire viral proteome as antigen. In some embodiments, the viral particles present only a portion of the proteome as antigen.

In some embodiments, the retroviral nucleic acid comprises one or more (e.g., all) of a 5' promoter (e.g., for controlling expression of the entire packaged RNA), a 5' LTR (e.g., comprising R (polyadenylation tail signal) and/or U5 comprising a primer activation signal), a primer binding site, a psi packaging signal, an RRE element for nuclear export, a promoter located directly upstream of the transgene to control expression of the transgene, a transgene (or other exogenous element), a polypurine region, and a 3' LTR (e.g., comprising mutated U3, R, and U5). In some embodiments, the retroviral nucleic acid further comprises one or more of cPPT, WPRE, and/or an insulator element.

Retroviruses typically replicate by reverse transcribing their genomic RNA into linear double-stranded DNA copies and subsequently covalently integrating their genomic DNA into the host genome. Illustrative retroviruses suitable for use in particular embodiments include, but are not limited to, moloney murine leukemia virus (M-MuLV), moloney murine sarcoma virus (MoMSV), harv murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline Leukemia Virus (FLV), foamy virus (spumavirus), friedel murine leukemia virus, murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV), and lentiviruses.

In some embodiments, the retrovirus is a gamma retrovirus. In some embodiments, the retrovirus is epsilon retrovirus. In some embodiments, the retrovirus is an alpha retrovirus. In some embodiments, the retrovirus is a beta retrovirus. In some embodiments, the retrovirus is a delta retrovirus. In some embodiments, the retrovirus is a lentivirus. In some embodiments, the retrovirus is a foamy retrovirus. In some embodiments, the retrovirus is an endogenous retrovirus.

Exemplary lentiviruses include, but are not limited to, HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2), vicina-Meidi virus (VMV) virus, caprine arthritis-encephalitis virus (CAEV), equine Infectious Anemia Virus (EIAV), feline Immunodeficiency Virus (FIV), bovine Immunodeficiency Virus (BIV), and Simian Immunodeficiency Virus (SIV). In some embodiments, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is used.

Viral vectors may comprise nucleic acid molecules (e.g., transfer plasmids) that include viral-derived nucleic acid elements that generally facilitate transfer or integration of the nucleic acid molecules (e.g., including nucleic acids encoding exogenous agents) into the cell genome or into viral particles that mediate nucleic acid transfer. Viral vector particles typically include various viral components, sometimes including host cell components in addition to nucleic acids. The viral vector may comprise a virus or viral particle capable of transferring nucleic acid into a cell (e.g., nucleic acid encoding an exogenous agent) or into a transferred nucleic acid (e.g., as naked DNA). Viral vectors and transfer plasmids may comprise structural and/or functional genetic elements derived primarily from viruses. Retroviral vectors may comprise viral vectors or plasmids containing structural and functional genetic elements derived primarily from retroviruses or parts thereof. Lentiviral vectors may comprise viral vectors or plasmids containing structural and functional genetic elements or portions thereof, including LTRs derived primarily from lentiviruses.

In embodiments, a lentiviral vector (e.g., a lentiviral expression vector) may comprise a lentiviral transfer plasmid (e.g., as naked DNA) or an infectious lentiviral particle. With respect to elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc., it is understood that the sequences of these elements may be present in the form of RNA in lentiviral particles and in the form of DNA in DNA plasmids.

In some vectors described herein, there may be no at least a portion of one or more protein coding regions that are conducive to replication or are necessary for replication, as compared to the corresponding wild-type virus. This makes viral vector replication defective. In some embodiments, the vector is capable of transducing a target non-dividing host cell and/or integrating its genome into the host genome.

The structure of the wild-type retroviral genome typically comprises 5 'Long Terminal Repeats (LTRs) and 3' LTRs with a packaging signal between or within them that enables the genome to be packaged, a primer binding site, integration sites to enable integration into the host cell genome, and gag, pol and env genes encoding packaging components that facilitate viral particle assembly. More complex retroviruses have additional features such as rev and RRE sequences in HIV that enable efficient export of RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of the infected target cells. In provirus, the viral gene is flanked at both ends by regions called Long Terminal Repeats (LTRs). LTRs are involved in proviral integration and transcription. The LTR also acts as an enhancer-promoter sequence and can control the expression of viral genes. Encapsidation of retroviral RNA occurs through the psi sequence located at the 5' end of the viral genome.

The LTRs themselves are typically similar (e.g., identical) sequences, which can be divided into three elements, designated U3, R, and U5. U3 is derived from a sequence unique to the 3' end of RNA. R is derived from sequences repeated at both ends of the RNA, and U5 is derived from sequences unique to the 5' end of the RNA. The size of these three elements can vary considerably among different retroviruses.

For viral genomes, the transcription initiation site is typically at the boundary between U3 and R in one LTR, while the poly (A) addition (termination) site is at the boundary between R and U5 in the other LTR. U3 contains most of the transcriptional control elements of provirus, including promoters and multiple enhancer sequences that are responsive to the cellular and, in some cases, viral transcriptional activator proteins. Some retroviruses contain any one or more of the following genes tot, rev, tax and rex, which encode proteins involved in regulating gene expression. Regarding the structural genes gag, pol and env themselves, gag encodes the internal structural proteins of the virus. Gag proteins are proteolytically processed into the mature proteins MA (matrix), CA (capsid) and NC (nucleocapsid). The pol gene encodes a Reverse Transcriptase (RT) which contains a DNA polymerase that mediates genome replication, a related RNase H and an Integrase (IN). The env gene encodes the Surface (SU) glycoprotein and Transmembrane (TM) protein of virions, which form complexes that interact specifically with cellular receptor proteins. This interaction promotes infection, for example, by fusing the proviral membrane with the cell membrane.

In the replication-defective retroviral vector genome gag, pol and env may be absent or non-functional. The R regions at both ends of the RNA are typically repetitive sequences. U5 and U3 represent unique sequences at the 5 'and 3' ends of the RNA genome, respectively.

The retrovirus may contain additional genes encoding proteins other than gag, pol, and env. Examples of additional genes include one or more of vif, vpr, vpx, vpu, tat, rev and nef (in HIV). EIAV has, inter alia, an additional gene S2. The proteins encoded by the additional genes have a variety of functions, some of which may be identical to those provided by cellular proteins. For example, in EIAV, tat acts as a transcriptional activator of the viral LTR (Derse and Newbold 1993 Virology 194:530-6; maury et al, 1994 Virology 200:632-42). It binds to a stable stem-loop RNA secondary structure called TAR. Rev regulates and coordinates expression of viral genes via the Rev Response Element (RRE) (Martarano et al, 1994J. Virol. 68:3102-11). The mechanism of action of these two proteins is thought to be substantially similar to that in primate viruses. In addition, an EIAV protein Ttm has been identified which is encoded by the first exon of tat spliced into the start of the transmembrane protein of the env coding sequence.

In addition to proteases, reverse transcriptases and integrases, non-primate lentiviruses contain a fourth pol gene product encoding a dUTP enzyme. This may play a role in the ability of these lentiviruses to infect certain non-dividing or slowly dividing cell types.

In embodiments, a Recombinant Lentiviral Vector (RLV) is a vector with sufficient retroviral genetic information to allow packaging of an RNA genome into a viral particle capable of infecting a target cell in the presence of a packaging component. Infection of the target cell may include reverse transcription and integration into the target cell genome. RLVs typically carry a non-viral coding sequence to be delivered by the vector to a target cell, such as a nucleic acid encoding an exogenous agent described herein. In embodiments, the RLV is unable to replicate independently within the target cell to produce infectious retroviral particles. In general, RLV lacks functional gag-pol and/or env genes and/or other genes involved in replication. The vector may be configured as a split intron vector, for example, as described in PCT patent application WO 99/15683, which is incorporated herein by reference in its entirety.

In some embodiments, the lentiviral vector comprises a minimal viral genome, e.g., the viral vector has been manipulated to remove non-essential elements and retain essential elements, thereby providing the functionality required to infect, transduce, and deliver a nucleotide sequence of interest to a target host cell, e.g., as described in WO 98/17815, which is incorporated herein by reference in its entirety.

The minimal lentiviral genome may comprise, for example, (5 ') R-U5-one or more first nucleotide sequences-U3-R (3'). However, the plasmid vector used to produce the lentiviral genome within the source cell may also include transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in the source cell. These regulatory sequences may comprise native sequences associated with the transcribed retroviral sequence, e.g., the 5' U3 region, or they may comprise a heterologous promoter, e.g., another viral promoter, e.g., the CMV promoter. Some lentiviral genomes contain additional sequences that promote efficient viral production. For example, in the case of HIV, rev and RRE sequences may be included. Alternatively or in combination, codon optimisation may be used, for example, the gene encoding the exogenous agent may be codon optimised, for example as described in WO 01/79518, which is incorporated herein by reference in its entirety. Alternative sequences that perform similar or identical functions to the rev/RRE system may also be used. For example, functional analogues of the rev/RRE system are found in the Meissen-Feishan monkey virus (Mason Pfizer monkey virus). This is called CTE and comprises RRE-type sequences in the genome that are thought to interact with factors in the infected cells. Cytokines may be considered rev analogs. Thus, CTE may be used as a substitute for rev/RRE systems. In addition, the Rex protein of HTLV-I can functionally replace the Rev protein of HIV-I. Rev and Rex have similar effects on IRE-BP.

In some embodiments, a retroviral nucleic acid (e.g., a lentiviral nucleic acid, such as a primate or non-primate lentiviral nucleic acid) (1) comprises a deleted gag gene wherein the deletion in gag removes one or more nucleotides downstream of about nucleotide 350 or 354 of the gag coding sequence, (2) there are no one or more auxiliary genes in the retroviral nucleic acid, (3) lacks a tat gene but comprises a leader sequence located between the end of the 5' LTR and the ATG of gag, and (4) a combination of (1), (2) and (3). In one embodiment, the lentiviral vector comprises all of features (1) and (2) and (3). Such a strategy is described in more detail in WO 99/32646, which is incorporated herein by reference in its entirety.

In some embodiments, primate lentivirus minimal systems do not require HIV/SIV additional genes vif, vpr, vpx, vpu, tat, rev and nef for vector production or for transduction of dividing and non-dividing cells. In some embodiments, the EIAV minimal vector system does not require S2 for vector production or for transduction of dividing and non-dividing cells.

The deletion of additional genes may allow the generation of vectors that are devoid of genes associated with lentiviral (e.g., HIV) infectious diseases. In particular, tat is associated with diseases. Second, the deletion of additional genes allows the vector to package more heterologous DNA. Third, genes of unknown function such as S2 may be omitted, thereby reducing the risk of causing adverse effects. Examples of minimal lentiviral vectors are disclosed in WO 99/32646 and WO 98/17815.

In some embodiments, the retroviral nucleic acid lacks at least tat and S2 (if it is an EIAV vector system), and may also lack vif, vpr, vpx, vpu and nef. In some embodiments, the retroviral nucleic acid further lacks rev, RRE, or both.

In some embodiments, the retroviral nucleic acid comprises vpx. The Vpx polypeptide binds to and induces degradation of SAMHD1 restriction factors that degrade free dntps in the cytoplasm. Thus, as Vpx degradation SAMHD1 and reverse transcription activity increases, the concentration of free dntps in the cytoplasm increases, thereby facilitating reverse transcription and integration of the retroviral genome into the target cell genome.

Different cells differ in the use of specific codons. This codon preference corresponds to a preference for the relative abundance of a particular tRNA in a cell type. By altering codons in the sequence, matching them to the relative abundance of the corresponding tRNA, it is possible to increase expression. For the same reason, it is possible to reduce expression by deliberately selecting codons for which the corresponding tRNA is known to be very rare in a particular cell type. Thus, an additional degree of translational control is available. Additional description of codon optimisation is found, for example, in WO 99/41397, which is incorporated herein by reference in its entirety.

Many viruses, including HIV and other lentiviruses, use a large number of rare codons, and by altering these codons to correspond to commonly used mammalian codons, the expression of packaging components in mammalian producer cells can be enhanced.

In some embodiments, codon optimization has many other advantages. In some embodiments, due to their sequence changes, the nucleotide sequences encoding the packaging components may reduce or eliminate RNA instability sequences (INS) from them. At the same time, the amino acid sequence coding sequence of the packaging component is preserved, such that the viral components encoded by said sequence remain the same or at least sufficiently similar such that the function of the packaging component is not compromised. In some embodiments, codon optimization also overcomes the output Rev/RRE requirement such that the optimized sequence is Rev independent. In some embodiments, codon optimization also reduces homologous recombination between different constructs within the vector system (e.g., between overlapping regions in the gag-pol and env open reading frames). In some embodiments, codon optimization results in increased viral titers and/or increased safety.

In some embodiments, only codons that are associated with INS are codon optimized. In other embodiments, the sequences are all codon optimized except for the sequence comprising the gag-pol frameshift site.

The gag-pol gene comprises two overlapping reading frames encoding the gag-pol protein. The expression of both proteins depends on the frameshift during translation. This frame shift occurs due to ribosome "sliding" during translation. This slippage is thought to be caused at least in part by ribosome-arrest RNA secondary structure. This secondary structure is present downstream of the frameshift site in the gag-pol gene. For HIV, the overlap region extends from nucleotide 1222 downstream of the start of gag (where nucleotide 1 is a of the gag ATG) to the end of gag (nt 1503). Thus, the overlap region of the 281 bp fragment and the two reading frames spanning the frameshift site is preferably not codon optimized. In some embodiments, retaining this fragment will be able to more efficiently express the gag-pol protein. For EIAV, the start of overlap is at nt 1262 (where nucleotide 1 is a of gag ATG). The end of the overlap is at nt 1461. To ensure that the frameshift site and gag-pol overlap are preserved, wild-type sequences from nt 1156 to 1465 may be preserved.

In some embodiments, for example to accommodate convenient restriction sites, derivatization may be performed from optimal codon usage, and conservative amino acid changes may be introduced into the gag-pol protein.

In some embodiments, codon optimization is based on codons with poor codon usage in mammalian systems. The third base can be changed, and sometimes the second and third bases can also be changed.

In some embodiments, due to the degeneracy of the genetic code, it will be appreciated that many gag-pol sequences are available to the skilled artisan. Furthermore, a number of retroviral variants are described which can be used as starting points for the generation of codon optimised gag-pol sequences. Lentiviral genomes may be quite variable. For example, there are many quasi-classes of HIV-I that remain functional. This is also the case for EIAV. These variants may be used to enhance specific parts of the transduction process. Examples of HIV-I variants can be found in the HIV database maintained by Los Alamos national laboratory. Details of EIAV clones can be found in the NCBI database maintained by the national institutes of health (National Institutes of Health).

It is within the level of skill in the art to empirically determine the appropriate codon optimization for a viral sequence. The strategy of codon optimized sequences including gag-pol sequences can be used for any retrovirus, such as EIAV, FIV, BIV, CAEV, VMR, SIV, HIV-I and HIV-2. In addition, the method can be used to increase the expression of genes from HTLV-1, HTLV-2, HFV, HSRV and Human Endogenous Retroviruses (HERV), MLV and other retroviruses.

In embodiments, the retroviral vector comprises a packaging signal comprising 255 to 360 nucleotides of gag in the vector that still retains the env sequence, or about 40 nucleotides of gag in a specific combination of splice donor mutant gag and env deletion. In some embodiments, the retroviral vector comprises a gag sequence comprising one or more deletions, e.g., the gag sequence comprises about 360 nucleotides that can be derived from the N-terminus.

In some embodiments, the retroviral vector, helper cell, helper virus, or helper plasmid may comprise a retroviral structural protein and a helper protein, such as gag, pol, env, tat, rev, vif, vpr, vpu, vpx or nef protein or other retroviral proteins. In some embodiments, the retroviral proteins are derived from the same retrovirus. In some embodiments, the retroviral protein is derived from more than one retrovirus, e.g., 2, 3, 4, or more retroviruses.

In some embodiments, the Gag and Pol coding sequences are generally organized as Gag-Pol precursors in a native lentivirus. The Gag sequence encodes a 55-kD Gag precursor protein, also known as p 55. p55 is cleaved during the maturation process by the virally encoded protease (product of the pol gene) into four smaller proteins designated MA (matrix [ p17 ]), CA (capsid [ p24 ]), NC (nucleocapsid [ p9 ]), and p6. The pol precursor protein is cleaved from Gag by the virally encoded protease and further digested to isolate protease (p 10), RT (p 50), RNase H (p 15) and integrase (p 31) activities.

In some embodiments, the lentiviral vector is integration defective. In some embodiments, pol is integrase-deficient, such as encoded by a mutation in the integrase gene. For example, the pol coding sequence may contain inactivating mutations in the integrase, such as by mutating one or more amino acids involved in catalytic activity, i.e., one or more of aspartic acid 64, aspartic acid 116, and/or glutamic acid 152. In some embodiments, the integrase mutation is a D64V mutation. In some embodiments, mutations in the integrase allow packaging of the viral RNA into lentiviruses. In some embodiments, mutations in the integrase allow packaging of the viral proteins into lentiviruses. In some embodiments, mutations in the integrase reduce the likelihood of insertional mutagenesis. In some embodiments, mutations in the integrase reduce the likelihood of producing Replication Competent Recombinants (RCR) (Wanisch et al 2009. Mol Ther. 1798): 1316-1332). In some embodiments, the native Gag-Pol sequence may be used in a helper vector (e.g., a helper plasmid or helper virus), or may be modified. Such modifications include chimeric Gag-Pol, wherein the Gag and Pol sequences are obtained from different viruses (e.g., different species, subspecies, strains, clades, etc.), and/or wherein the sequences have been modified to improve transcription and/or translation and/or reduce recombination.

In some embodiments, the retroviral nucleic acid comprises a polynucleotide encoding a 150-250 (e.g., 168) nucleotide portion of the gag protein, which polynucleotide (i) comprises a mutant INS1 inhibitory sequence that reduces RNA nuclear export restriction relative to wild type INS1, (ii) contains two nucleotide insertions that result in frameshifting and premature termination, and/or (iii) does not comprise the INS2, INS3, and INS4 inhibitory sequences of gag.

In some embodiments, the vectors described herein are hybrid vectors comprising retroviral (e.g., lentiviral) sequences and non-lentiviral viral sequences. In some embodiments, the hybrid vector comprises retroviral (e.g., lentiviral) sequences for reverse transcription, replication, integration, and/or packaging.

According to certain embodiments, most or all of the viral vector backbone sequences are derived from lentiviruses, such as HIV-1. However, it is understood that many different sources of retroviral and/or lentiviral sequences may be used, or used in combination, and that many substitutions and alterations in certain lentiviral sequences may be accommodated without compromising the ability of the transfer vector to perform the functions described herein. A variety of lentiviral vectors are described in Naldini et al, (l 996a, l996b and 1998), zufferey et al, (1997), dull et al, 1998, U.S. Pat. Nos. 6,013,516 and 5,994,136, many of which may be suitable for the production of retroviral nucleic acids.

At each end of the provirus, long Terminal Repeats (LTRs) are typically found. The LTR typically comprises a domain located at the end of a retroviral nucleic acid, which in its natural sequence context is a forward repeat and contains U3, R and U5 regions. The LTRs generally promote expression of retroviral genes (e.g., promotion of gene transcripts, initiation, and polyadenylation) and viral replication. The LTR may contain a number of regulatory signals including transcriptional control elements, polyadenylation signals, and sequences for viral genome replication and integration. The viral LTR is generally divided into three regions, designated U3, R and U5. The U3 region typically contains enhancer and promoter elements. The U5 region is typically the sequence between the primer binding site and the R region, and may contain polyadenylation sequences. The R (repeat) region may flank the U3 and U5 regions. LTRs are typically composed of U3, R and U5 regions and may occur at the 5 'and 3' ends of the viral genome. In some embodiments, adjacent to the 5' LTR is a sequence for reverse transcription of the genome (tRNA primer binding site) and for efficient packaging of the viral RNA into particles (Psi site).

In some embodiments, the packaging signal may comprise sequences located within the retroviral genome that mediate insertion of viral RNA into viral capsids or particles, see, e.g., clever et al, 1995. J. Of Virology, volume 69, stage 4; pages 2101-2109. Several retroviral vectors use the minimal packaging signal (psi sequence) for encapsidation of the viral genome.

In various embodiments, the retroviral nucleic acid comprises a modified 5 'LTR and/or 3' LTR. Either or both of the LTRs may comprise one or more modifications, including but not limited to one or more deletions, insertions, or substitutions. The 3' LTR is typically modified to improve the safety of the lentiviral or retroviral system by making the virus replication defective (e.g., a virus that cannot replicate completely and efficiently so as not to produce infectious virions (e.g., replication defective lentiviral progeny)).

In some embodiments, the vector is a self-inactivating (SIN) vector, such as a replication defective vector, e.g., a retrovirus or lentivirus vector, wherein the right (3') LTR enhancer-promoter region (referred to as the U3 region) has been modified (e.g., by deletion or substitution) to prevent transcription of the virus beyond the first round of viral replication. In some aspects, provided herein are replication-incompetent (also referred to herein as replication-defective) vector particles that are unable to participate in replication in the absence of packaging cells (i.e., the viral vector particles are not produced by transduced cells). In some aspects, this is because the right (3 ') LTR U3 region can serve as a template for the left (5') LTR U3 region during viral replication, and thus the absence of the U3 enhancer-promoter inhibits viral replication. In embodiments, the 3' LTR is modified such that the U5 region is removed, altered, or replaced, e.g., with an exogenous poly (a) sequence. The 3 'LTR, 5' LTR, or both 3 'and 5' LTR may be modified LTRs. Other modifications to viral vectors (i.e., retroviral or lentiviral vectors) that render the vector replication incompetent are known in the art.

In some embodiments, the U3 region of the 5' LTR is replaced with a heterologous promoter to drive transcription of the viral genome during viral particle production. Examples of heterologous promoters that may be used include, for example, the viral simian virus 40 (SV 40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), moloney murine leukemia virus (MoMLV), rous Sarcoma Virus (RSV), and Herpes Simplex Virus (HSV) (thymidine kinase) promoters. In some embodiments, the promoter is capable of driving high levels of transcription in a Tat-independent manner. In certain embodiments, heterologous promoters have additional advantages in the manner in which transcription of the viral genome is controlled. For example, the heterologous promoter may be inducible such that transcription of all or part of the viral genome occurs only in the presence of an inducing factor. The induction factor includes, but is not limited to, one or more chemical compounds or physiological conditions such as temperature or pH of the cultured host cell.

In some embodiments, the viral vector comprises a TAR (trans-activation reaction) element, e.g., located in the R region of the lentiviral (e.g., HIV) LTR. This element interacts with lentiviral transactivator (tat) genetic elements to enhance viral replication. However, such an element is not necessary, for example, in embodiments in which the U3 region of the 5' LTR is replaced with a heterologous promoter.

The R region, e.g., a region within the retroviral LTR beginning at the start of the capping group (i.e., transcription start) and ending before the start of the poly A region, may flank the U3 region and the U5 region. The R region functions during reverse transcription that transfers nascent DNA from one end of the genome to the other.

Retroviral nucleic acids may also comprise FLAP elements, for example nucleic acids whose sequences include the central polypurine region and the central termination sequences (cPPT and CTS) of retroviruses (e.g., HIV-1 or HIV-2). Suitable FLAP elements are described in U.S. Pat. No. 6,682,907 and in Zennou et al, 2000, cell, 101:173, which are incorporated herein by reference in their entirety. During HIV-1 reverse transcription, the initiation of positive strand DNA centrally in the central polypurine region (cPPT) and termination centrally in the Central Termination Sequence (CTS) can lead to the formation of a triple strand DNA structure, the HIV-1 central DNA flap. In some embodiments, the retroviral or lentiviral vector backbone comprises one or more FLAP elements upstream or downstream of a gene encoding an exogenous agent. For example, in some embodiments, the transfer plasmid comprises a FLAP element, such as a FLAP element derived from or isolated from HIV-L.

In embodiments, the retroviral or lentiviral nucleic acid comprises one or more export elements, e.g., cis-acting post-transcriptional regulatory elements that regulate the transport of RNA transcripts from the nucleus to the cytoplasm. Examples of RNA export elements include, but are not limited to, human Immunodeficiency Virus (HIV) Rev Response Elements (RRE) (see, e.g., cullen et al, 1991. J. Virol. 65:1053; and Cullen et al, 1991. Cell 58:423), and hepatitis B virus post-transcriptional regulatory elements (HPRE), which are incorporated herein by reference in their entirety. Typically, the RNA export element is placed within the 3' UTR of the gene and may be inserted as one or more copies.

In some embodiments, expression of a heterologous sequence (e.g., a nucleic acid encoding an exogenous agent) is increased by incorporating one or more (e.g., all) of a post-transcriptional regulatory element, a polyadenylation site, and a transcription termination signal into the vector. A variety of post-transcriptional regulatory elements may increase expression of heterologous nucleic acids in proteins, such as woodchuck hepatitis virus post-transcriptional regulatory elements (WPRE; zufferey et al, 1999, J. Virol., 73:2886), post-transcriptional regulatory elements present in hepatitis B virus (HPRE) (Huang et al, mol. Cell. Biol., 5:3864), and the like (Liu et al, 1995, genes Dev., 9:1766), each of which is incorporated herein by reference in its entirety. In some embodiments, a retroviral nucleic acid described herein comprises a post-transcriptional regulatory element, such as WPRE or HPRE

In some embodiments, a retroviral nucleic acid described herein lacks or does not comprise a post transcriptional regulatory element such as WPRE or HPRE.

Elements that direct termination and polyadenylation of heterologous nucleic acid transcripts may be included, for example, to increase expression of the exogenous agent. The transcription termination signal may be present downstream of the polyadenylation signal. In some embodiments, the vector comprises a polyadenylation sequence 3' to the polynucleotide encoding the exogenous agent. The poly a site may comprise a DNA sequence that directs RNA polymerase II termination and polyadenylation of the nascent RNA transcript. Polyadenylation sequences can promote mRNA stability by adding polya tails to the 3' end of the coding sequence and thus help increase translation efficiency. Illustrative examples of poly a signals that can be used in retroviral nucleic acids include AATAAA, ATT AAA, AGTAAA, bovine growth hormone poly a sequence (BGHpA), rabbit b globin poly a sequence (rPgpA), or another suitable heterologous or endogenous poly a sequence.

In some embodiments, the retroviral or lentiviral vector further comprises one or more insulator elements, such as the insulator elements described herein.

In various embodiments, the vector comprises a promoter operably linked to a polynucleotide encoding an exogenous agent. The vector may have one or more LTRs, any of which comprises one or more modifications, such as one or more nucleotide substitutions, additions or deletions. The vector may also contain one or more helper elements (e.g., cPPT/FLAP) that increase transduction efficiency, viral packaging (e.g., psi (ψ) packaging signal, RRE), and/or other elements that increase expression of the exogenous gene (e.g., poly (a) sequences), and may optionally contain WPRE or HPRE.

In some embodiments, the lentiviral nucleic acid comprises, e.g., from 5 'to 3', one or more (e.g., all) of a promoter (e.g., CMV), an R sequence (e.g., comprising TAR), a U5 sequence (e.g., for integration), a PBS sequence (e.g., for reverse transcription), a DIS sequence (e.g., for genomic dimerization), a psi packaging signal, a portion of a gag sequence, an RRE sequence (e.g., for nuclear export), a cPPT sequence (e.g., for nuclear import), a promoter driving expression of an exogenous agent, a gene encoding an exogenous agent, a WPRE sequence (e.g., for efficient transgene expression), a PPT sequence (e.g., for reverse transcription), an R sequence (e.g., for polyadenylation and termination), and a U5 signal (e.g., for integration).

2. Virus-like particles

In some embodiments, the virus-based particle is a virus-like lipid particle (VLP) derived from a virus. In some embodiments, the viral envelope may comprise a fluxing agent, e.g., an endogenous fluxing agent or a pseudofluxing agent of the virus, e.g., a re-targeting attachment protein, such as the G or F proteins described in section II. VLPs include those derived from retroviruses or lentiviruses. While VLPs mimic the native virion structure, they lack the viral genome information necessary for independent replication within the host cell. Thus, in certain aspects, VLPs are non-infectious. In particular embodiments, the VLP does not contain a viral genome. In some embodiments, the VLP bilayer of amphiphilic lipids is or comprises a viral envelope. In some embodiments, the amphiphilic lipid bilayer of the lipid particle is or comprises a cell-derived lipid. In some embodiments, the VLP contains at least one type of structural protein from a virus. In most cases, such proteins form protein capsids. In some cases, the capsid will also be encapsulated in a lipid bilayer derived from cells that have released assembled VLPs (e.g., VLPs comprising human immunodeficiency virus structural proteins such as GAGs). In some embodiments, the VLP further comprises a targeting moiety as an envelope protein within the lipid bilayer.

In some embodiments, the carrier vehicle particles comprise supramolecular complexes formed from viral proteins that self-assemble into capsids. In some embodiments, the vector vehicle particle is a virus-like particle derived from a viral capsid protein. In some embodiments, the vector particles are virus-like particles derived from viral nucleocapsid proteins. In some embodiments, the vector vehicle particles comprise a nucleocapsid derived protein that retains the properties of the packaged nucleic acid. In some embodiments, the virus-based particles, such as virus-like particles, comprise only viral structural glycoproteins in proteins from the viral genome. In some embodiments, the vector particles do not contain a viral genome.

In some embodiments, the vector particles package nucleic acid from the host cell, such as nucleic acid encoding an exogenous agent, during expression. In some embodiments, the nucleic acid does not encode any genes involved in viral replication. In particular embodiments, the vector particles are virus-like particles, e.g., retrovirus-like particles, such as lentivirus-like particles, which are replication-defective.

In some embodiments, the vector particle is a virus-like particle comprising sequences that do not contain or lack viral RNA (which may be the result of removing or scavenging viral RNA from the sequences). In some embodiments, this may be achieved by using an endogenous packaging signal binding site on gag. In some embodiments, the endogenous packaging signal binding site is on pol. In some embodiments, the RNA to be delivered will contain a cognate packaging signal. In some embodiments, a heterologous binding domain located on the RNA to be delivered (which is heterologous to gag) and a homologous binding site located on gag or pol can be used to ensure packaging of the RNA to be delivered. In some embodiments, the heterologous sequence may be non-viral or it may be viral, in which case it may be derived from a different virus. In some embodiments, the carrier particles may be used to deliver therapeutic RNA, in which case no functional integrase and/or reverse transcriptase is required. In some embodiments, the carrier particles may also be used to deliver a therapeutic gene of interest, in which case pol is typically included.

In some embodiments, the VLP comprises a supramolecular complex formed by self-assembly of viral proteins into a capsid. In some embodiments, the VLP is derived from a viral capsid. In some embodiments, the VLP is derived from a viral nucleocapsid. In some embodiments, the VLP is nucleocapsid derived and retains the properties of the packaged nucleic acid. In some embodiments, the VLP comprises only viral structural glycoproteins. In some embodiments, the VLP does not contain a viral genome.

3. Method for producing virus-based particles

Large scale viral particle production is generally useful for achieving desired viral titers. Viral particles can be produced by transfection of a transfer vector into a packaging cell line comprising viral structures and/or helper genes, such as gag, pol, env, tat, rev, vif, vpr, vpu, vpx or nef genes or other retroviral genes.

In some embodiments, the viral vector particles may be produced in a variety of cell culture systems including bacterial, mammalian cell lines, insect cell lines, yeast, and plant cells. Methods of producing such virus-based particles can also be found in U.S. patent No. 10,316,295, incorporated herein by reference. Exemplary methods for producing viral vector particles are described.

In some embodiments, the element for producing a viral vector (i.e., a recombinant viral vector, such as a replication-incompetent lentiviral vector) is contained in or present on a packaging cell line. In some embodiments, the viral vector may comprise packaging elements rev, gag and pol delivered to the packaging cell line via one or more packaging vectors.

In embodiments, the packaging vector is an expression vector or viral vector lacking a packaging signal and comprising polynucleotides encoding one, two, three, four or more viral structures and/or helper genes. Typically, the packaging vector is contained in a packaging cell and introduced into the cell by transfection, transduction or infection. Retroviral (e.g., lentiviral) transfer vectors can be introduced into packaging cell lines by transfection, transduction, or infection to produce the source cells or cell lines. The packaging vector is introduced into the human cell or cell line by standard methods including, for example, calcium phosphate transfection, lipofection or electroporation. In some embodiments, the packaging vector is introduced into cells with a dominant selection marker such as neomycin, hygromycin, puromycin, blasticidin, bleomycin (zeocin), thymidine kinase, DHFR, gln synthase, or ADA, followed by selection and isolation of clones in the presence of the appropriate drug. The selectable marker gene may be physically linked to the gene encoded by the packaging vector, for example, by an IRES or a self-cleaving viral peptide. In some embodiments, the packaging vector is a packaging plasmid.

Producer cell lines (also referred to as packaging cell lines) include cell lines that do not contain packaging signals but stably or transiently express viral structural proteins and replicases (e.g., gag, pol, and env) that can package viral particles. Any suitable cell line may be used, such as mammalian cells, e.g. human cells. Suitable cell lines that may be used include, for example, CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, hepG2 cells, saos-2 cells, huh7 cells, heLa cells, W163 cells, 211 cells, and 211A cells. In embodiments, the packaging cell is a 293 cell, 293T cell, or a549 cell.

In some embodiments, the producer cell (i.e., source cell line) comprises a cell line capable of producing recombinant retroviral particles, the cell line comprising a producer cell line and a transfer vector construct comprising a packaging signal. Methods of preparing viral stocks are depicted, for example, by Y.Soneoka et al (1995) Nucl. Acids Res. 23:628-633 and N.R. Landau et al (1992) J. Virol. 66:5110-5113, which are incorporated herein by reference. Infectious viral particles may be collected from packaging cells, for example, by cell lysis or by collecting the supernatant of a cell culture. Optionally, the collected viral particles may be enriched or purified.

In some embodiments, the source cell comprises one or more plasmids encoding viral structural proteins and replicases (e.g., gag, pol, and env) that can package the viral particle (i.e., packaging the plasmid). In some embodiments, the sequences encoding at least two of the gag, pol, and env precursors are on the same plasmid. In some embodiments, the sequences encoding gag, pol, and env precursors are on different plasmids. In some embodiments, the sequences encoding gag, pol, and env precursors have the same expression signal, e.g., a promoter. In some embodiments, the sequences encoding the gag, pol, and env precursors have different expression signals, e.g., different promoters. In some embodiments, the expression of gag, pol, and env precursors is inducible. In some embodiments, plasmids encoding viral structural proteins and replicases are transfected at the same time or at different times. In some embodiments, the plasmid encoding the viral structural protein and replicase is transfected at the same time as the packaging vector or at a different time.

In some embodiments, the source cell line comprises one or more stably integrated viral structural genes. In some embodiments, expression of the stably integrated viral structural gene is inducible.

In some embodiments, expression of the viral structural gene is regulated at the transcriptional level. In some embodiments, expression of the viral structural gene is regulated at the translational level. In some embodiments, expression of the viral structural gene is regulated at a post-translational level.

In some embodiments, expression of the viral structural gene is regulated by a tetracycline (Tet) -dependent system, wherein a Tet-regulated transcriptional repressor (Tet-R) binds to the DNA sequence contained in the promoter and inhibits transcription by steric hindrance (Yao et al, 1998; jones et al, 2005). Upon addition of doxycycline (dox), tet-R is released, allowing transcription. A variety of other suitable transcription regulating promoters, transcription factors and small molecule inducers are suitable for use in regulating transcription of viral structural genes.

In some embodiments, the third generation lentiviral component, human immunodeficiency virus type 1 (HIV), rev, gag/Pol, and envelope, under the control of a Tet-regulated promoter and coupled to an antibiotic resistance cassette, are integrated into the source cell genome, respectively. In some embodiments, the source cell integrates only one copy of each of Rev, gag/Pol, and envelope proteins in the genome.

In some embodiments, a nucleic acid encoding an exogenous agent (e.g., a retroviral nucleic acid encoding an exogenous agent) is also integrated into the genome of the source cell. In some embodiments, the nucleic acid encoding the exogenous agent remains free. In some embodiments, the nucleic acid encoding the exogenous agent is transfected into a source cell having stably integrated Rev, gag/Pol, and envelope proteins in the genome. See, for example Milani et al EMBO Molecular Medicine, 2017, which is incorporated by reference herein in its entirety.

In some embodiments, a retroviral nucleic acid described herein is incapable of reverse transcription. In embodiments, such nucleic acids are capable of transiently expressing exogenous agents. The retrovirus or VLP may contain a null reverse transcriptase protein or may not contain a reverse transcriptase protein. In embodiments, the retroviral nucleic acid comprises a null Primer Binding Site (PBS) and/or att site. In embodiments, one or more viral accessory genes (including rev, tat, vif, nef, vpr, vpu, vpx and S2 or functional equivalents thereof) are null or deleted in the retroviral nucleic acid. In embodiments, one or more accessory genes selected from S2, rev and tat are null or deleted in the retroviral nucleic acid.

Typically, modern retroviral vector systems comprise a viral genome carrying cis-acting vector sequences for transcription, reverse transcription, integration, translation and packaging of viral RNA into viral particles, and (2) production cell lines expressing trans-acting retroviral gene sequences (e.g., gag, pol, and env) required for the production of viral particles. By completely isolating the cis-acting and trans-acting vector sequences, the virus cannot maintain replication for more than one infection cycle. The production of live viruses can be avoided by a number of strategies, such as avoiding recombination by minimizing overlap between cis-acting and trans-acting sequences.

A virus-like particle (VLP) comprising a sequence that does not contain or lacks viral RNA as described in section iii.a.2 may be the result of removal or elimination of viral RNA from the sequence. Similar to the viral vector particles disclosed in section iii.a.1, VLPs contain a viral outer envelope made up of a host cell (i.e., a producer cell or a source cell) lipid bilayer and at least one viral structural protein. In some embodiments, a viral structural protein refers to any viral protein or fragment thereof that contributes to the viral core or capsid structure.

In general, for viral vector particles, expression of the gag precursor protein alone mediates vector assembly and release. In some aspects, the gag protein or fragment thereof has been demonstrated to assemble into a structure similar to the viral core. In one embodiment, this can be achieved by using an endogenous packaging signal binding site on gag. Or the endogenous packaging signal binding site is on pol. In this embodiment, the RNA to be delivered will contain a cognate packaging signal. In another embodiment, a heterologous binding domain located on the RNA to be delivered (heterologous to gag) and a homologous binding site located on gag or pol can be used to ensure packaging of the RNA to be delivered. The heterologous sequence may be non-viral, or it may be viral, in which case it may be derived from a different virus. VLPs may be used to deliver therapeutic RNAs, in which case no functional integrase and/or reverse transcriptase is required. These VLPs may also be used to deliver therapeutic genes of interest, in which case pol is typically included.

In one embodiment, gag-pol is altered and the packaging signal is replaced by a corresponding packaging signal. In this embodiment, the particles may package RNA with a new packaging signal. The advantage of this approach is that RNA sequences lacking viral sequences, such as RNAi, can be packaged.

Another approach relies on overexpression of the RNA to be packaged. In one embodiment, the RNA to be packaged is overexpressed in the absence of any RNA containing a packaging signal. This may result in a large amount of therapeutic RNA being packaged and this amount is sufficient to transduce the cells and have a biological effect.

In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a viral gag protein or retroviral gag and pol proteins, wherein the gag protein or pol protein comprises a heterologous RNA binding domain capable of recognizing the corresponding sequence in the RNA sequence, to facilitate packaging of the RNA sequence into a viral vector particle. In some embodiments, the heterologous RNA binding domain comprises an RNA binding domain derived from a bacteriophage coat protein, rev protein, U1 microribonucleoprotein particle protein, nova protein, TF 111A protein, TIS 11 protein, trp RNA binding attenuation protein (TRAP), or pseudouridine synthase.

In some embodiments, assembly of the virus-based vector particle (i.e., VLP) is initiated by binding of the core protein to a unique encapsidation sequence (e.g., UTR with stem-loop structure) within the viral genome. In some embodiments, the interaction of the core with the encapsidation sequence promotes oligomerization.

In some embodiments, the source cells used to produce VLPs comprise one or more plasmids encoding viral structural proteins (e.g., gag, pol), which may be packaged into viral particles (i.e., packaging plasmids). In some embodiments, the sequences encoding at least two of the gag and pol precursors are on the same plasmid. In some embodiments, the sequences encoding the gag and pol precursors are on different plasmids. In some embodiments, the sequences encoding the gag and pol precursors have the same expression signal, e.g., a promoter. In some embodiments, the sequences encoding the gag and pol precursors have different expression signals, e.g., different promoters. In some embodiments, expression of gag and pol precursors is inducible.

In some embodiments, the formation of VLPs or any virus-based particles may be detected by any suitable technique known in the art, such as described in section III above. Examples of such techniques include, for example, electron microscopy, dynamic light scattering, selective chromatographic separation, and/or density gradient centrifugation.

B. exogenous agent

In some embodiments, a lipid particle or pharmaceutical composition comprising the lipid particle as described herein contains an exogenous agent. In some embodiments, a lipid particle described herein or a pharmaceutical composition comprising the lipid particle contains a nucleic acid encoding an exogenous agent. In some embodiments, the lipid particle contains an exogenous agent. In some embodiments, the lipid particle contains a nucleic acid encoding an exogenous agent. The coding sequence of a nucleic acid encoding an exogenous agent is also referred to herein as a payload gene. In some embodiments, the exogenous agent or nucleic acid encoding the exogenous agent is present in the lumen of the lipid particle.

In some embodiments, the exogenous agent is a protein or nucleic acid (e.g., DNA, chromosome (e.g., human artificial chromosome), RNA (e.g., mRNA or miRNA)). In some embodiments, the exogenous agent comprises or encodes a membrane protein. In some embodiments, the exogenous agent comprises or encodes a therapeutic agent. In some embodiments, the therapeutic agent is selected from one or more of a protein, such as an enzyme, transmembrane protein, receptor, or antibody, a nucleic acid, such as DNA, chromosome (e.g., human artificial chromosome), RNA, mRNA, siRNA, or miRNA, or a small molecule.

In some embodiments, the lipid particle or pharmaceutical composition delivers at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the exogenous agent (e.g., the exogenous agent comprising or encoding the therapeutic agent) contained in the lipid particle to the target cell. In some embodiments, a lipid particle (e.g., fusion) that is in contact with (e.g., fused to) a target cell delivers an exogenous agent (e.g., an exogenous agent comprising or encoding a therapeutic agent) to the target cell that comprises an average of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the lipid particle (e.g., fusion) that is in contact with (e.g., fused to) the target cell. In some embodiments, the lipid particle composition delivers at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the exogenous agent (e.g., the exogenous agent comprising or encoding the therapeutic agent) contained in the lipid particle composition to the target tissue.

In some embodiments, the exogenous agent is not naturally expressed in the cells from which the lipid particle is derived. In some embodiments, the exogenous agent is naturally expressed in the cells from which the lipid particle is derived. In some embodiments, the exogenous agent is loaded into the lipid particle by expression in the cell from which the lipid particle was derived (e.g., expression of DNA or mRNA introduced by transfection, transduction, or electroporation). In some embodiments, the exogenous source is expressed by DNA integrated into the genome or remains episomal. In some embodiments, the expression of the exogenous agent is constitutive. In some embodiments, expression of the exogenous agent is induced. In some embodiments, expression of the exogenous agent is induced immediately prior to the production of the lipid particle. In some embodiments, expression of the exogenous agent is induced simultaneously with expression of the fusion agent.

In some embodiments, the exogenous agent is loaded into the lipid particle by electroporation into the lipid particle itself or into cells from which the lipid particle is derived. In some embodiments, the exogenous agent is loaded into the lipid particle by transfection (e.g., transfection of DNA or mRNA encoding the exogenous agent) into the lipid particle itself or into cells from which the lipid particle is derived.

In some embodiments, the exogenous agent may include one or more nucleic acid sequences, one or more polypeptides, combinations of nucleic acid sequences and/or polypeptides, one or more organelles, and any combination thereof. In some embodiments, the exogenous agent may include one or more cellular components. In some embodiments, the exogenous agent comprises one or more cytoplasmic and/or nuclear components.

In some embodiments, the lipid particle contains an exogenous agent that is a nucleic acid or contains a nucleic acid encoding an exogenous agent. In some embodiments, the nucleic acid is operably linked to a "positive target cell-specific regulatory element" (or positive TCSRE). In some embodiments, positive TCSRE is a functional nucleic acid sequence. In some embodiments, positive TCSRE comprises a promoter or enhancer. In some embodiments TCSRE is a nucleic acid sequence that increases the level of exogenous agents in a target cell. In some embodiments, the positive target cell-specific regulatory element comprises a T cell-specific promoter, a T cell-specific enhancer, a T cell-specific splice site, a T cell-specific site that extends the half-life of an RNA or protein, a T cell-specific mRNA nuclear export promotion site, a T cell-specific translation enhancement site, or a T cell-specific post-translational modification site. In some embodiments, the T cell specific promoter is the promoter described in Immgen consortium, which is incorporated herein by reference in its entirety, e.g., the T cell specific promoter is the IL2RA (CD 25), LRRC32, FOXP3, or IKZF2 promoter. In some embodiments, the T cell specific promoter or enhancer is Schmidl et al, blood. 2014, 24; 123 (17): promoter or enhancer described in e68-78, which is incorporated herein by reference in its entirety. In some embodiments, the T cell specific promoter is a transcriptionally active fragment of any one of the foregoing. In some embodiments, the T cell specific promoter is a variant having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to any of the foregoing.

In some embodiments, the lipid particle contains an exogenous agent that is a nucleic acid or contains a nucleic acid encoding an exogenous agent. In some embodiments, the nucleic acid is operably linked to a "negative target cell-specific regulatory element" (or negative TCSRE). In some embodiments, negative TCSRE is a functional nucleic acid sequence. In some embodiments, negative TCSRE is a miRNA recognition site leading to inhibition of degradation of lipid particles in non-target cells. In some embodiments, the exogenous agent is operably linked to a "non-target cell-specific regulatory element" (or NTCSRE). In some embodiments NTCSRE comprises a nucleic acid sequence that reduces the level of an exogenous agent in a non-target cell as compared to in a target cell. In some embodiments NTCSRE comprises a non-target cell-specific miRNA recognition sequence, a non-target cell-specific protease recognition site, a non-target cell-specific ubiquitin ligase site, a non-target cell-specific transcriptional repression site, or a non-target cell-specific epigenetic repression site. In some embodiments NTCSRE comprises a tissue-specific miRNA recognition sequence, a tissue-specific protease recognition site, a tissue-specific ubiquitin ligase site, a tissue-specific transcriptional inhibition site, or a tissue-specific epigenetic inhibition site. In some embodiments NTCSRE comprises a non-target cell-specific miRNA recognition sequence, a non-target cell-specific protease recognition site, a non-target cell-specific ubiquitin ligase site, a non-target cell-specific transcriptional repression site, or a non-target cell-specific epigenetic repression site. In some embodiments, NTCSRE comprises a non-target cell-specific miRNA recognition sequence, and the miRNA recognition sequence is capable of being bound by one or more of miR 31, miR363, or miR29 c. In some embodiments NTCSRE is located or encoded within a transcribed region encoding an exogenous agent, optionally wherein the RNA produced by the transcribed region comprises a miRNA recognition sequence within the UTR or coding region.

1. Nucleic acid

In some embodiments, the exogenous agent may comprise a nucleic acid. For example, the exogenous agent may comprise an RNA that enhances expression of the endogenous protein, or an siRNA or miRNA that inhibits expression of the endogenous protein. For example, endogenous proteins can modulate a structure or function in a target cell. In some embodiments, the exogenous agent may include a nucleic acid encoding an engineered protein that modulates a structure or function in the target cell. In some embodiments, the exogenous agent is a nucleic acid that targets a transcriptional activator that modulates a structure or function in the target cell.

In some embodiments, the lipid particles described herein comprise a nucleic acid, such as RNA or DNA. In some embodiments, the nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, the nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product, such as RNA or a protein. In some embodiments, the nucleic acid comprises one or more introns. In some embodiments, the nucleic acid is prepared by one or more of isolation from a natural source, enzymatic synthesis (in vivo or in vitro) by complementary template-based polymerization, replication in a recombinant cell or system, and chemical synthesis. In some embodiments, the nucleic acid is at least 3、4、5、6、7、8、9、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、1 10、120、130、140、150、160、170、180、190、20、225、250、275、300、325、350、375、400、425、450、475、500、600、700、800、900、1000、1500、2000、2500、3000、3500、4000、4500、5000 or more residues in length. In some embodiments, the nucleic acid is partially or fully single stranded, and in some embodiments, the nucleic acid is partially or fully double stranded. In some embodiments, the nucleic acid has a nucleotide sequence comprising at least one element encoding a polypeptide or is a complement of a sequence encoding a polypeptide. Nucleic acids may include variants, e.g., having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% overall sequence identity to a reference nucleic acid. In some embodiments, the variant nucleic acid does not share at least one characteristic sequence element with the reference nucleic acid. In some embodiments, the variant nucleic acid has one or more biological activities of the reference nucleic acid. In some embodiments, the nucleic acid variant has the same nucleic acid sequence as the reference nucleic acid, but with a small sequence change at a particular position. In some embodiments, less than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in the variant are substituted, inserted, or deleted as compared to the reference. In some embodiments, the variant nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substitution residues as compared to the reference. In some embodiments, variant nucleic acids comprise few (e.g., less than about 5, about 4, about 3, about 2, or about 1) functional residues that are involved in a particular biological activity relative to a reference. In some embodiments, the variant nucleic acid comprises no more than about 15, about 12, about 9, about 3, or about 1 additions or deletions as compared to the reference, and in some embodiments, no additions or deletions. In some embodiments, the variant nucleic acid comprises less than about 27, about 24, about 21, about 18, about 15, about 12, about 9, about 6, about 3, or less than about 9, about 6, about 3, or about 2 additions or deletions as compared to the reference.

In some embodiments, the exogenous agent includes a nucleic acid, such as DNA, nDNA (nuclear DNA), mtDNA (mitochondrial DNA), protein-encoding DNA, gene, operon, chromosome, genome, transposon, retrotransposon, viral genome, intron, exon, modified DNA, mRNA (messenger RNA), tRNA (transfer RNA), modified RNA, microrna, siRNA (small interfering RNA), tmRNA (transfer messenger RNA), rRNA (ribosomal RNA), mtRNA (mitochondrial RNA), snRNA (microrna), micronucleolar RNA (snoRNA), smY RNA (mRNA trans-splicing RNA), gRNA (guide RNA), TERC (telomerase RNA component), aRNA (antisense RNA), cis-NAT (cis-natural antisense transcript), CRISPR RNA (crRNA), incRNA (long non-coding RNA), piRNA (piwi interacting RNA), shRNA (short hairpin RNA), tasiRNA (trans-acting siRNA), eRNA (enhancer RNA), satellite RNA, RNA (RNA encoding RNA), RNA (pc RNA), RNAi (circular RNA), and any combination thereof. In some embodiments, the nucleic acid is a wild-type nucleic acid. In some embodiments, the protein is a mutant nucleic acid. In some embodiments, the nucleic acid is a fusion or a chimera of multiple nucleic acid sequences.

In embodiments, the nucleic acid encodes one or more (e.g., two or more) inhibitory RNA molecules directed against one or more RNA targets. The inhibitory RNA molecule may be, for example, miRNA or shRNA. In some embodiments, the inhibitory molecule may be a precursor of a miRNA, such as a Pri-miRNA or Pre-miRNA, or a precursor of a shRNA. In some embodiments, the inhibitory molecule may be an artificially derived miRNA or shRNA. In other embodiments, the inhibitory RNA molecule may be dsRNA (transcribed or artificially introduced) that is processed into siRNA or the siRNA itself. In some embodiments, the inhibitory RNA molecule may be a miRNA or shRNA having a sequence that is not found in nature, or having at least one functional fragment that is not found in nature, or having a combination of functional fragments that are not found in nature. In exemplary embodiments, at least one or all of the inhibitory RNA molecules is miR-l55. In some embodiments, a retroviral vector described herein encodes two or more inhibitory RNA molecules directed against one or more RNA targets. In some embodiments, two or more inhibitory RNA molecules may be directed against different targets. In other embodiments, two or more inhibitory RNA molecules are directed against the same target. In some embodiments, the exogenous agent comprises shRNA. shRNA (short hairpin RNA) may comprise a double-stranded structure formed by a single self-complementary RNA strand. The shRNA construct may comprise a nucleotide sequence that is identical to a portion of a coding or non-coding sequence of a target gene. RNA sequences having insertions, deletions and single point mutations relative to the target sequence may also be used. Greater than 90% sequence identity, or even 100% sequence identity, between the inhibitory RNA and the target gene portion may be used. In certain embodiments, the duplex-forming portion of the shRNA is at least 20, 21, or 22 nucleotides in length, e.g., corresponding in size to an RNA product produced by Dicer-dependent cleavage. In certain embodiments, the shRNA construct is at least 25, 50, 100, 200, 300, or 400 bases in length. In certain embodiments, the shRNA construct is 400-800 bases in length. shRNA constructs are highly tolerant to variations in loop sequence and loop size. In embodiments, the retroviral vector encoding siRNA, miRNA, shRNA or ribozymes comprises one or more regulatory sequences, such as a strong constitutive pol III, e.g., a human U6 snRNA promoter, a mouse U6 snRNA promoter, a human and mouse H l RNA promoter, and a human tRNA-val promoter, or a strong constitutive pol II promoter.

A. Polypeptides

In some embodiments, the lipid particle contains a nucleic acid encoding a protein exogenous agent (also referred to as a "payload gene encoding an exogenous agent"). In some embodiments, the lipid particles described herein comprise an exogenous agent that is or comprises a protein.

In some embodiments, the protein may include portions other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and/or may be otherwise processed or modified. In some embodiments, a protein may sometimes include more than one polypeptide chain, e.g., linked by one or more disulfide bonds or otherwise associated.

In some embodiments, the protein may contain L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs. In some embodiments, the protein may comprise natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. In some embodiments, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and/or a characteristic portion thereof. In some embodiments, a polypeptide may include variants thereof, e.g., having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% total sequence identity to a reference polypeptide. In some embodiments, the variant polypeptide does not share at least one characteristic sequence element with the reference polypeptide. In some embodiments, the variant polypeptide has one or more biological activities of the reference polypeptide. In some embodiments, the polypeptide variant has the same amino acid sequence as the reference nucleic acid, but with a small sequence change at a particular position. In some embodiments, less than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in the variant are substituted, inserted, or deleted as compared to the reference. In some embodiments, the variant polypeptide comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substitution residues as compared to the reference. In some embodiments, variant polypeptides comprise a very small amount (e.g., less than about 5, about 4, about 3, about 2, or about 1) of substitution, insertion, or deletion functions that are involved in a particular biological activity relative to a reference. In some embodiments, the variant polypeptide comprises no more than about 5, about 4, about 3, about 2, or about 1 additions or deletions as compared to the reference, and in some embodiments, no additions or deletions. In some embodiments, variant polypeptides comprise less than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and typically less than about 5, about 4, about 3, or about 2 additions or deletions as compared to a reference. in some embodiments, the protein comprises a polypeptide, such as an enzyme, structural polypeptide, signaling polypeptide, regulatory polypeptide, transport polypeptide, sensory polypeptide, motor polypeptide, defensive polypeptide, storage polypeptide, transcription factor, antibody, cytokine, hormone, catabolic polypeptide, anabolic polypeptide, proteolytic polypeptide, metabolic polypeptide, kinase, transferase, hydrolase, lyase, isomerase, ligase, enzyme regulatory polypeptide, protein binding polypeptide, lipid binding polypeptide, membrane fusion polypeptide, cell differentiation polypeptide, epigenetic polypeptide, cell death polypeptide, nuclear transport polypeptide, nucleic acid binding polypeptide, reprogramming polypeptide, DNA editing polypeptide, protein binding polypeptide, lipid binding polypeptide, membrane fusion polypeptide, cell differentiation polypeptide, epigenetic polypeptide, cell death polypeptide, nuclear transport polypeptide, nucleic acid binding polypeptide, reprogramming polypeptide, DNA editing polypeptide, and/or a protein, DNA repair polypeptides, DNA recombination polypeptides, transposase polypeptides, DNA integration polypeptides, targeting endonucleases (e.g., zinc finger nucleases, transcription activator-like nucleases (TALENs), cas9 and homologs thereof), recombinases, and any combination thereof. In some embodiments, the protein targets a protein in the cell for degradation. In some embodiments, the protein targets the protein in the cell to degrade by localizing the protein to the proteasome. In some embodiments, the protein is a wild-type protein. In some embodiments, the protein is a mutant protein.

Exemplary protein exogenous agents are described in the following subsections. In some embodiments, the lipid particles provided herein may comprise any such exogenous agent. In particular embodiments, the lipid particle contains a nucleic acid encoding any such exogenous agent.

B. Cytoplasmic proteins

In some embodiments, the exogenous agent comprises a cytoplasmic protein, such as a protein that is produced in the recipient cell and is localized to the cytoplasm of the recipient cell. In some embodiments, the exogenous agent comprises a secreted protein, e.g., a protein produced and secreted by a recipient cell. In some embodiments, the exogenous agent comprises a nuclear protein, such as a protein that is produced in the recipient cell and is infused into the nucleus of the recipient cell. In some embodiments, the exogenous agent comprises an organelle protein (e.g., a mitochondrial protein), such as a protein that is produced in and infused into an organelle (e.g., a mitochondria) of a recipient cell. In some embodiments, the protein is a wild-type protein or a mutant protein. In some embodiments, the protein is a fusion protein or a chimeric protein.

C. Membrane proteins

In some embodiments, the exogenous agent comprises a membrane protein. In some embodiments, the membrane protein comprises a Chimeric Antigen Receptor (CAR), a T cell receptor, an integrin, an ion channel, a pore-forming protein, a Toll-like receptor, an interleukin receptor, a cell adhesion protein, or a transport protein.

1. Chimeric Antigen Receptor (CAR)

In certain embodiments, the payload gene can comprise an exogenous polynucleotide encoding a CAR. CARs (also known as chimeric immune receptors, chimeric T cell receptors, or artificial T cell receptors) are receptor proteins that have been engineered to confer new capabilities to a host cell (e.g., T cell) for targeting a particular protein. These receptors are chimeric in that they combine antigen binding and T cell activation functions in a single receptor. The polycistronic vectors of the present disclosure can be used to express one or more CARs in a host cell (e.g., T cell) for cell-based therapies against various target antigens. CARs expressed by one or more expression cassettes may be the same or different. In these embodiments, the CAR may comprise an extracellular binding domain (also referred to as a "binding agent"), a transmembrane domain, and an intracellular signaling domain that specifically bind to a target antigen. In certain embodiments, the CAR may further comprise one or more additional elements, including one or more signal peptides, one or more extracellular hinge domains, and/or one or more intracellular co-stimulatory domains. The domains may be directly adjacent to each other or may have one or more amino acid linking domains. The nucleotide sequence encoding the CAR may be derived from a mammalian sequence, such as a mouse sequence, a primate sequence, a human sequence, or a combination thereof. Where the nucleotide sequence encoding the CAR is non-human, the sequence of the CAR may be humanized. The nucleotide sequence encoding the CAR may also be codon optimized for expression in mammalian cells, e.g., human cells. In any of these embodiments, the nucleotide sequence encoding the CAR can be at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any of the nucleotide sequences disclosed herein. Sequence variations may be due to codon optimization, humanization, restriction enzyme-based cloning scars, and/or additional amino acid residues linking functional domains, etc.

In certain embodiments, the CAR may comprise a signal peptide at the N-terminus. Non-limiting examples of signal peptides include CD8 a signal peptide, igK signal peptide, and granulocyte-macrophage colony-stimulating factor receptor subunit a (GMCSFR-a, also known as colony-stimulating factor 2 receptor subunit a (CSF 2 RA)) signal peptide, and variants thereof, the amino acid sequences of which are provided in table 5 below.

In certain embodiments, the extracellular binding domain of the CAR may comprise one or more antibodies specific for a target antigen or multiple target antigens. The antibody may be an antibody fragment, such as an scFv, or a single domain antibody fragment, such as a VHH. In certain embodiments, scFv may comprise the heavy chain variable region (V _H) and the light chain variable region (V _L).V_H and V _L) of an antibody linked by a linker, which may be linked in either order, i.e., V _H -linker-V _L or V _L -linker-V _H non-limiting examples of linkers include Whitlow linkers, (G ₄S)_n (n may be a positive integer, e.g., 1,2,3,4, 5,6, etc.) linkers and variants thereof; CS1/SLAMF7, CD38, CD138, GPRC5D, TACI and BCMA (associated with myeloma), GD2, HER2, EGFR, EGFRvIII, B H3, PSMA, PSCA, CAIX, CD171, CEA, CSPG4, EPHA2, FAP, FR alpha, IL-13R alpha, mesothelin, MUC1, MUC16 and ROR1 (associated with solid tumors), in any of these embodiments, the extracellular binding domain of the CAR may be codon optimized for expression in a host cell, or have a variant sequence to increase the function of the extracellular binding domain.

In certain embodiments, the CAR may comprise a hinge domain, also referred to as a spacer region. The terms "hinge" and "spacer" may be used interchangeably throughout this disclosure. Non-limiting examples of hinge domains include the CD8 a hinge domain, CD28 hinge domain, igG4 hinge-CH 2-CH3 domain, and variants thereof, the amino acid sequences of which are provided in table 6 below.

In certain embodiments, the transmembrane domain of a CAR may comprise the transmembrane region of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or functional variants thereof, including the human form of each of these sequences. In other embodiments, the transmembrane domain may comprise transmembrane region ：CD8α、CD8β、4-1BB/CD137、CD28、CD34、CD4、FcεRIγ、CD16、OX40/CD134、CD3ζ、CD3ε、CD3γ、CD3δ、TCRα、TCRβ、TCRζ、CD32、CD64、CD64、CD45、CD5、CD9、CD22、CD37、CD80、CD86、CD40、CD40L/CD154、VEGFR2、FAS and FGFR2B or a functional variant thereof, including human versions of each of these sequences. Table 7 provides several exemplary transmembrane domain amino acid sequences.

In some embodiments of the present invention, in some embodiments, the intracellular signaling domain and/or intracellular co-stimulatory domain of the CAR may comprise one or more signaling domain ：B7-1/CD80、B7-2/CD86、B7-H1/PD-L1、B7-H2、B7-H3、B7-H4、B7-H6、B7-H7、BTLA/CD272、CD28、CTLA-4、Gi24/VISTA/B7-H5、ICOS/CD278、PD-1、PD-L2/B7-DC、PDCD6、4-1BB/TNFSF9/CD137、4-1BB ligand/TNFSF 9, BAFF/BLyS/TNFSF13B, BAFF R/TNFRSF13C, CD/TNFRSF 7, CD27 ligand/TNFSF 7, CD30/TNFRSF8, CD30 ligand/TNFSF 8, CD40/TNFRSF5, CD40/TNFSF5, CD40 ligand/TNFSF 5, DR3/TNFRSF25, GITR/TNFRSF18, GITR ligand/TNFSF 18, HVEM/TNFRSF14, LIGHT/TNFSF14, lymphotoxin-alpha/TNFbeta OX40/TNFRSF4, OX40 ligand /TNFSF4、RELT/TNFRSF19L、TACI/TNFRSF13B、TL1A/TNFSF15、TNFα、TNF RII/TNFRSF1B、2B4/CD244/SLAMF4、BLAME/SLAMF8、CD2、CD2F-10/SLAMF9、CD48/SLAMF2、CD58/LFA-3、CD84/SLAMF5、CD229/SLAMF3、CRACC/SLAMF7、NTB-A/SLAMF6、SLAM/CD150、CD2、CD7、CD53、CD82/Kai-1、CD90/Thy1、CD96、CD160、CD200、CD300a/LMIR1、HLA I, HLA-DR, ikaros, integrin alpha 4/CD49d, integrin alpha 4 beta 1, integrin α 4 β 7/LPAM-1、LAG-3、TCL1A、TCL1B、CRTAM、DAP12、Dectin-1/CLEC7A、DPPIV/CD26、EphB6、TIM-1/KIM-1/HAVCR、TIM-4、TSLP、TSLP R、 lymphocyte function-associated antigen-1 (LFA-1), NKG2C, CD3 zeta, immunoreceptor tyrosine-based activation motif (ITAM), CD27, CD28, 4-1BB, CD134/OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligand that specifically binds to CD83, and functional variants thereof, including the humanoid form of each of these sequences. In some embodiments, the intracellular signaling domain and/or intracellular co-stimulatory domain comprises one or more signaling domains selected from the group consisting of a CD3 zeta domain, ITAM, CD28 domain, 4-1BB domain or a functional variant thereof. Table 8 provides amino acid sequences of several exemplary intracellular co-stimulatory and/or signaling domains. In certain embodiments, as in the case of Texarensaine described below, the CD3 zeta signaling domain of SEQ ID NO:392 may have a mutation at amino acid position 14, such as a glutamine (Q) to lysine (K) mutation (see SEQ ID NO: 393).

In certain embodiments in which the polycistronic vector encodes two or more CARs, the two or more CARs may comprise the same functional domain, or one or more different functional domains, as described. For example, two or more CARs can comprise different signal peptides, extracellular binding domains, hinge domains, transmembrane domains, costimulatory domains, and/or intracellular signaling domains to minimize the risk of recombination due to sequence similarity. Or alternatively, two or more CARs may comprise the same domain. Where the same domain or domains and/or backbones are used, codon differences are optionally introduced at the nucleotide sequence level to minimize the risk of recombination.

1) CD19 CAR

In some embodiments, the CAR is a CD19 CAR ("CD 19-CAR"), and in these embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding the CD19 CAR. In some embodiments, a CD19 CAR may comprise a signal peptide, an extracellular binding domain that specifically binds CD19, a hinge domain, a transmembrane domain, an intracellular co-stimulatory domain, and/or an intracellular signaling domain in tandem.

In some embodiments, the signal peptide of the CD19 CAR comprises a CD8 a signal peptide. In some embodiments, the CD8 a signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:378 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 378. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the IgK signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO. 379 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence set forth in SEQ ID NO. 379. In some embodiments, the signal peptide comprises GMCSFR- α or CSF2RA signal peptide. In some embodiments, GMCSFR- α or CSF2RA signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID No. 380 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence set forth in SEQ ID No. 380.

In some embodiments, the extracellular binding domain of the CD19 CAR is specific for CD19 (e.g., human CD 19). The extracellular binding domain of a CD19 CAR may be codon optimized for expression in a host cell, or have variant sequences to increase the function of the extracellular binding domain. In some embodiments, the extracellular binding domain comprises an immunogenic active portion of an immunoglobulin molecule, e.g., an scFv.

In some embodiments, the extracellular binding domain of the CD19 CAR comprises an scFv derived from an FMC63 monoclonal antibody (FMC 63) comprising a heavy chain variable region (V _H) and a light chain variable region (V _L) of FMC63 linked by a linker. FMC63 and derived scFv have been described in Nicholson et al, mol. Immun. 34 (16-17): 1157-1165 (1997) and PCT application publication No. WO2018/213337, the entire contents of each of which are incorporated herein by reference. In some embodiments, the amino acid sequences of the complete FMC 63-derived scFv (also referred to as FMC63 scFv) and the different portions thereof are provided in table 9 below. In some embodiments, the CD 19-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID No. 394, 395 or 400 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to an amino acid sequence set forth in SEQ ID No. 40, 41 or 46. In some embodiments, the CD 19-specific scFv may comprise one or more CDRs having the amino acid sequences shown in SEQ ID NOS 42-44 and 48-50. In some embodiments, the CD 19-specific scFv may comprise a light chain having one or more CDRs having the amino acid sequence set forth in SEQ ID NOS 396-398. In some embodiments, a CD 19-specific scFv may comprise a heavy chain having one or more CDRs having the amino acid sequences shown in SEQ ID NOS 402-404. In any of these embodiments, the CD 19-specific scFv can comprise one or more CDRs comprising one or more amino acid substitutions, or comprise a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to any of the identified sequences. In some embodiments, the extracellular binding domain of a CD19 CAR comprises or consists of one or more CDRs as described herein.

In some embodiments, the linker connecting the V _H and V _L portions of the scFv is a Whitlow linker having the amino acid sequence shown in SEQ ID NO. 399. In some embodiments, the Whitlow linker may be replaced with a different linker, such as a 3xG ₄ S linker having the amino acid sequence shown in SEQ ID NO. 405, which results in a different FMC 63-derived scFv having the amino acid sequence shown in SEQ ID NO. 404. In certain of these embodiments, the CD 19-specific scFv comprises or consists of an amino acid sequence shown as SEQ ID NO. 404 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence shown as SEQ ID NO. 404.

In some embodiments, the extracellular binding domain of CD19 CAR is derived from CD 19-specific antibodies, including, for example, SJ25C1 (Bejcek et al, cancer Res. 55:2346-2351 (1995)), HD37 (Pezutto et al, J. Immunol. 138 (9): 2793-2799 (1987)), 4G7 (Meeker et al, hybrid 3:305-320 (1984)), B43 (Bejcek (1995)), BLY3 (Bejcek (1995)), B4 (Freedman et al, 70:418-427 (1987)), B4 HB12B (Kansas & Tedder, J. Immunol. 147:4094-4102 (1991)), yazawa et al, protocol Natl Acad. Sci. USA 102:15178-15183 (2005)), J. Pharmacol. Exp. Ther. 213-222 (2010), B4 (Callard:418-427), B4 (1987), B4 (1998), B4 (1987), B4:418-1989), and CD 381 (1989). In any of these embodiments, the extracellular binding domain of the CD19 CAR may comprise or consist of V _H、V_L and/or one or more CDRs of any of these antibodies.

In some embodiments, the hinge domain of the CD19 CAR comprises a CD 8a hinge domain, e.g., a human CD 8a hinge domain. In some embodiments, the CD 8a hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO. 381 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence set forth in SEQ ID NO. 381. In some embodiments, the hinge domain comprises a CD28 hinge domain, e.g., a human CD28 hinge domain. In some embodiments, the CD28 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:382 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 382. In some embodiments, the CD28 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:383 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 383. In some embodiments, the hinge domain comprises an IgG4 hinge domain, e.g., a human IgG4 hinge domain. In some embodiments, the IgG4 hinge domain comprises or consists of an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to an amino acid sequence set forth in SEQ ID NO:384 or SEQ ID NO: 385. In some embodiments, the hinge domain comprises an IgG4 hinge-Ch 2-Ch3 domain, e.g., a human IgG4 hinge-Ch 2-Ch3 domain. In some embodiments, the IgG4 hinge-Ch 2-Ch3 domain comprises or consists of an amino acid sequence shown as SEQ ID NO:386 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to an amino acid sequence shown as SEQ ID NO: 386.

In some embodiments, the transmembrane domain of the CD19 CAR comprises a CD8 a transmembrane domain, e.g., a human CD8 a transmembrane domain. In some embodiments, the CD8 a transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID No. 387 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to an amino acid sequence set forth in SEQ ID No. 387. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, e.g., a human CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises or consists of the amino acid sequence set forth in SEQ ID No. 388 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence set forth in SEQ ID No. 388. In some embodiments, the CD28 transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:389 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 389.

In some embodiments, the intracellular co-stimulatory domain of the CD19 CAR comprises a 4-1BB co-stimulatory domain. 4-1BB (also known as CD 137) transmits potent co-stimulatory signals to T cells, thereby promoting differentiation and enhancing long-term survival of T lymphocytes. In some embodiments, the 4-1BB costimulatory domain is human. In some embodiments, the 4-1BB costimulatory domain comprises or consists of an amino acid sequence depicted as SEQ ID NO:390 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence depicted as SEQ ID NO: 390. In some embodiments, the intracellular co-stimulatory domain comprises a CD28 co-stimulatory domain. CD28 is another costimulatory molecule on T cells. In some embodiments, the CD28 co-stimulatory domain is human. In some embodiments, the CD28 co-stimulatory domain comprises or consists of an amino acid sequence set forth in SEQ ID No. 391 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence set forth in SEQ ID No. 391. In some embodiments, the intracellular co-stimulatory domain of the CD19 CAR comprises a 4-1BB co-stimulatory domain and a CD28 co-stimulatory domain as described.

In some embodiments, the intracellular signaling domain of the CD19 CAR comprises a CD3 zeta (ζ) signaling domain. Cd3ζ binds to T Cell Receptor (TCR) generating a signal and contains an immunoreceptor tyrosine-based activation motif (ITAM). The CD3 zeta signaling domain refers to an amino acid residue from the zeta chain cytoplasmic domain that is sufficient to functionally transmit the initial signal necessary for T cell activation. In some embodiments, the CD3 zeta signaling domain is human. In some embodiments, the CD3 zeta signaling domain comprises or consists of an amino acid sequence set forth in SEQ ID No. 392 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence set forth in SEQ ID No. 392.

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD19 CAR, including, for example, a CD 19-specific scFv comprising a sequence set forth in SEQ ID No. 394 or SEQ ID No. 395, a CD8 a hinge domain of SEQ ID No. 381, a CD8 a transmembrane domain of SEQ ID No. 387, a 4-1BB costimulatory domain of SEQ ID No. 390, a CD3 zeta signaling domain of SEQ ID No. 392, and/or variants thereof (i.e., having a sequence at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 identical to the disclosed sequence). In any of these embodiments, the CD19 CAR can additionally comprise a signal peptide as described (e.g., a CD8 a signal peptide).

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD19 CAR, including, for example, a CD19 CAR comprising a CD 19-specific scFv having the sequence set forth in SEQ ID No. 394 or SEQ ID No. 395, an IgG4 hinge domain of SEQ ID No. 384 or SEQ ID No. 385, a CD28 transmembrane domain of SEQ ID No. 388, a 4-1BB costimulatory domain of SEQ ID No. 390, a CD3 zeta signaling domain of SEQ ID No. 392, and/or variants thereof (i.e., having a sequence at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99) to the disclosed sequence. In any of these embodiments, the CD19 CAR can additionally comprise a signal peptide as described (e.g., a CD8 a signal peptide).

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD19 CAR, including, for example, a CD 19-specific scFv comprising a sequence set forth in SEQ ID No. 394 or SEQ ID No. 395, a CD28 hinge domain of SEQ ID No. 383, a CD28 transmembrane domain of SEQ ID No. 388, a CD28 costimulatory domain of SEQ ID No. 391, a CD3 zeta signaling domain of SEQ ID NO 392, and/or variants thereof (i.e., a CD19 CAR having a sequence at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 identical to the disclosed sequence). In any of these embodiments, the CD19 CAR can additionally comprise a signal peptide as described (e.g., a CD8 a signal peptide).

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD19 CAR as set forth in SEQ ID No. 406 or being at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the nucleotide sequence set forth in SEQ ID No. 406 (see table 11). The encoded CD19 CAR has the corresponding amino acid sequence shown in SEQ ID No. 407 or is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence shown in SEQ ID No. 407, with the components CD 8a signal peptide, FMC63 scFv (V _L -Whitlow linker-V _H), CD 8a hinge domain, CD 8a transmembrane domain, 4-1BB costimulatory domain and CD3 zeta signaling domain.

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a commercially available embodiment of the CD19 CAR. Non-limiting examples of commercial embodiments of CD19 CARs expressed and/or encoded by T cells include temozolomide (tisagenlecleucel), li Jimai, and (lisocabtagene maraleucel), alopecide (axicabtagene ciloleucel), and briyl olmesate (brexucabtagene autoleucel).

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a texarenate or a portion thereof. Tixarence comprises a CD19 CAR having a CD8 alpha signal peptide, a FMC63 scFv (V _L-3xG₄ S linker-V _H), a CD8 alpha hinge domain, a CD8 alpha transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain. The nucleotide and amino acid sequences of CD19 CAR in texarensai are provided in table 10, and the comments of the sequences are provided in table 11.

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding Li Jimai th, or a portion thereof. Li Jimai the Lungnatore comprises a CD19 CAR having GMCSFR-alpha or CSF2RA signal peptide, FMC63 scFv (V _L -Whitlow linker-V _H), an IgG4 hinge domain, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain. The nucleotide and amino acid sequences of CD19 CAR in Li Jimai's rence are provided in table 10, and the comments of the sequences are provided in table 12.

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding alemtuquone or a portion thereof. The alopecie comprises a CD19 CAR having the following components GMCSFR-alpha or CSF2RA signal peptide, FMC63 scFv (V _L -Whitlow linker-V _H), CD28 hinge domain, CD28 transmembrane domain, CD28 co-stimulatory domain and CD3 zeta signaling domain. The nucleotide and amino acid sequences of CD19 CAR in alemtujopsis are provided in table 10, and the comments of the sequences are provided in table 13.

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding briyl alendronate or a portion thereof. The briyl olanexidine comprises a CD19 CAR having the following components GMCSFR-alpha signal peptide, FMC63 scFv, CD28 hinge domain, CD28 transmembrane domain, CD28 costimulatory domain, and CD3 zeta signaling domain.

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD19 CAR as set forth in SEQ ID No. 408, 410, or 412 or being at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the nucleotide sequence set forth in SEQ ID No. 408, 410, or 412. The encoded CD19 CAR has a corresponding amino acid sequence as set forth in SEQ ID No. 409, 411, or 413, respectively, or is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID No. 409, 411, or 413, respectively.

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD19 CAR as set forth in SEQ ID No. 408, 410, or 412 or being at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the nucleotide sequence set forth in SEQ ID No. 408, 410, or 412. The encoded CD19 CAR has a corresponding amino acid sequence as set forth in SEQ ID No. 409, 411, or 412, respectively, that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID No. 409, 411, or 412, respectively.

2) CD20 CAR

In some embodiments, the CAR is a CD20 CAR ("CD 20-CAR"), and in these embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding the CD20 CAR. CD20 is an antigen that is found on the surface of B cells early in the pre-B phase and at progressively increasing levels until the B cells mature, as well as on most B cell neoplasm cells. CD20 positive cells are sometimes also found in cases of hodgkin's disease, myeloma and thymoma. In some embodiments, a CD20 CAR may comprise a signal peptide in tandem, an extracellular binding domain that specifically binds CD20, a hinge domain, a transmembrane domain, an intracellular co-stimulatory domain, and/or an intracellular signaling domain.

In some embodiments, the signal peptide of the CD20 CAR comprises a CD8 a signal peptide. In some embodiments, the CD8 a signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:378 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 378. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the IgK signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO. 379 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence set forth in SEQ ID NO. 379. In some embodiments, the signal peptide comprises GMCSFR- α or CSF2RA signal peptide. In some embodiments, GMCSFR- α or CSF2RA signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID No. 380 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence set forth in SEQ ID No. 380.

In some embodiments, the extracellular binding domain of the CD20 CAR is specific for CD20 (e.g., human CD 20). The extracellular binding domain of CD20 CAR may be codon optimized for expression in a host cell, or have a variant sequence to increase the function of the extracellular binding domain. In some embodiments, the extracellular binding domain comprises an immunogenic active portion of an immunoglobulin molecule, e.g., an scFv.

In some embodiments, the extracellular binding domain of the CD20 CAR is derived from a CD20 specific antibody, including, for example, leu16, IF5, 1.5.3, rituximab, otouzumab, timox, ofatuzumab, tositumumab, ornitumumab, veltuzumab, rituximab, and ore Li Zhushan antibodies. In any of these embodiments, the extracellular binding domain of the CD20 CAR can comprise or consist of V _H、V_L and/or one or more CDRs of any antibody.

In some embodiments, the extracellular binding domain of the CD20 CAR comprises an scFv derived from a Leu16 monoclonal antibody comprising a heavy chain variable region (V _H) and a light chain variable region (V _L) of Leu16 linked by a linker. See Wu et al, protein engineering 14 (12): 1025-1033 (2001). In some embodiments, the linker is a 3xG ₄ S linker. In other embodiments, the linker is a Whitlow linker as described herein. In some embodiments, the amino acid sequences of the different portions of the complete Leu 16-derived scFv (also referred to as the Leu16 scFv) and the different portions thereof are provided in table 14 below. In some embodiments, the CD 20-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID No. 414, 415, or 419 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to an amino acid sequence set forth in SEQ ID No. 414, 415, or 419. In some embodiments, the CD 20-specific scFv may comprise one or more CDRs having the amino acid sequences shown in SEQ ID NOS 416-418, 420, 421 and 422. In some embodiments, the CD 20-specific scFv may comprise a light chain having one or more CDRs having the amino acid sequences shown in SEQ ID NOS 416-418. In some embodiments, a CD 20-specific scFv may comprise a heavy chain having one or more CDRs having the amino acid sequences shown in SEQ ID NOs 420, 421 and 422. In any of these embodiments, the CD 20-specific scFv can comprise one or more CDRs comprising one or more amino acid substitutions, or comprise a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to any of the identified sequences. In some embodiments, the extracellular binding domain of a CD20 CAR comprises or consists of one or more CDRs as described herein.

In some embodiments, the hinge domain of the CD20 CAR comprises a CD8 a hinge domain, e.g., a human CD8 a hinge domain. In some embodiments, the CD8 a hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:378 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 378. In some embodiments, the hinge domain comprises a CD28 hinge domain, e.g., a human CD28 hinge domain. In some embodiments, the CD28 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:382 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 382. In some embodiments, the hinge domain comprises an IgG4 hinge domain, e.g., a human IgG4 hinge domain. In some embodiments, the IgG4 hinge domain comprises or consists of an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to an amino acid sequence set forth in SEQ ID NO:384 or SEQ ID NO: 385. In some embodiments, the hinge domain comprises an IgG4 hinge-Ch 2-Ch3 domain, e.g., a human IgG4 hinge-Ch 2-Ch3 domain. In some embodiments, the IgG4 hinge-Ch 2-Ch3 domain comprises or consists of an amino acid sequence shown as SEQ ID NO:386 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to an amino acid sequence shown as SEQ ID NO: 386.

In some embodiments, the transmembrane domain of the CD20 CAR comprises a CD 8a transmembrane domain, e.g., a human CD 8a transmembrane domain. In some embodiments, the CD 8a transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID No. 387 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to an amino acid sequence set forth in SEQ ID No. 387. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, e.g., a human CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises or consists of the amino acid sequence set forth in SEQ ID No. 388 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence set forth in SEQ ID No. 388.

In some embodiments, the intracellular co-stimulatory domain of the CD20 CAR comprises a 4-1BB co-stimulatory domain, e.g., a human 4-1BB co-stimulatory domain. In some embodiments, the 4-1BB costimulatory domain comprises or consists of an amino acid sequence depicted as SEQ ID NO:390 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence depicted as SEQ ID NO: 390. In some embodiments, the intracellular co-stimulatory domain comprises a CD28 co-stimulatory domain, e.g., a human CD28 co-stimulatory domain. In some embodiments, the CD28 co-stimulatory domain comprises or consists of an amino acid sequence set forth in SEQ ID No. 391 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence set forth in SEQ ID No. 391.

In some embodiments, the intracellular signaling domain of the CD20 CAR comprises a CD3 zeta (ζ) signaling domain, e.g., a human CD3 ζ signaling domain. In some embodiments, the CD3 zeta signaling domain comprises or consists of an amino acid sequence set forth in SEQ ID No. 392 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence set forth in SEQ ID No. 392.

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD20 CAR, including, for example, a CD 20-specific scFv comprising a sequence set forth in SEQ ID No. 414, a CD8 a hinge domain of SEQ ID No. 381, a CD8 a transmembrane domain of SEQ ID No. 387, a 4-1BB costimulatory domain of SEQ ID No. 390, a CD3 zeta signaling domain of SEQ ID No. 392, and/or variants thereof (i.e., having a sequence at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99) to the disclosed sequence.

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD20 CAR, including, for example, a CD 20-specific scFv comprising a sequence set forth in SEQ ID No. 414, a CD28 hinge domain of SEQ ID No. 381, a CD8 a transmembrane domain of SEQ ID No. 387, a 4-1BB costimulatory domain of SEQ ID No. 390, a CD3 zeta signaling domain of SEQ ID No. 392, and/or variants thereof (i.e., a CD20 CAR having a sequence at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence).

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD20 CAR, including, for example, a CD20 CAR comprising a CD 20-specific scFv having the sequence set forth in SEQ ID No. 414, an IgG4 hinge domain of SEQ ID No. 384 or SEQ ID No. 385, a CD8 a transmembrane domain of SEQ ID No. 387, a 4-1BB costimulatory domain of SEQ ID No. 390, a CD3 zeta signaling domain of SEQ ID No. 392, and/or variants thereof (i.e., having a sequence at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 identical to the disclosed sequence).

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD20 CAR, including, for example, a CD 20-specific scFv comprising a sequence set forth in SEQ ID No. 414, a CD8 a hinge domain of SEQ ID No. 387, a CD28 transmembrane domain of SEQ ID No. 389, a 4-1BB costimulatory domain of SEQ ID No. 390, a CD3 zeta signaling domain of SEQ ID No. 392, and/or variants thereof (i.e., having a sequence at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99) to the disclosed sequence.

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD20 CAR, including, for example, a CD20 CAR comprising a CD 20-specific scFv having the sequence set forth in SEQ ID NO:414, a CD28 hinge domain of SEQ ID NO:382, a CD28 transmembrane domain of SEQ ID NO:388, a 4-1BB costimulatory domain of SEQ ID NO:390, a CD3 zeta signaling domain of SEQ ID NO:392, and/or variants thereof (i.e., having a sequence at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence).

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD20 CAR, including, for example, a CD20 CAR comprising a CD 20-specific scFv having the sequence set forth in SEQ ID No. 414, an IgG4 hinge domain of SEQ ID No. 384 or SEQ ID No. 385, a CD28 transmembrane domain of SEQ ID No. 388, a 4-1BB costimulatory domain of SEQ ID No. 390, a CD3 zeta signaling domain of SEQ ID No. 392, and/or variants thereof (i.e., having a sequence at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 identical to the disclosed sequence).

3) CD22 CAR

In some embodiments, the CAR is a CD22 CAR ("CD 22-CAR"), and in these embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding the CD22 CAR. CD22 is a transmembrane protein that is found predominantly on the surface of mature B cells and acts as an inhibitory receptor for B Cell Receptor (BCR) signaling. CD22 is expressed in 60% -70% of B cell lymphomas and leukemias (e.g., B-chronic lymphocytic leukemia, hairy cell leukemia, acute Lymphocytic Leukemia (ALL) and burkitt's lymphoma), and is absent on the cell surface or on stem cells at early stages of B cell development. In some embodiments, a CD22 CAR may comprise a signal peptide in tandem, an extracellular binding domain that specifically binds CD22, a hinge domain, a transmembrane domain, an intracellular co-stimulatory domain, and/or an intracellular signaling domain.

In some embodiments, the signal peptide of the CD22 CAR comprises a CD8 a signal peptide. In some embodiments, the CD8 a signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO. 450 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence set forth in SEQ ID NO. 450. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the IgK signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO. 379 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence set forth in SEQ ID NO. 379. In some embodiments, the signal peptide comprises GMCSFR- α or CSF2RA signal peptide. In some embodiments, GMCSFR- α or CSF2RA signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID No. 380 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence set forth in SEQ ID No. 380.

In some embodiments, the extracellular binding domain of the CD22 CAR is specific for CD22 (e.g., human CD 22). The extracellular binding domain of CD22 CAR may be codon optimized for expression in a host cell, or have a variant sequence to increase the function of the extracellular binding domain. In some embodiments, the extracellular binding domain comprises an immunogenic active portion of an immunoglobulin molecule, e.g., an scFv.

In some embodiments, the extracellular binding domain of the CD22 CAR is derived from a CD22 specific antibody, including, for example, SM03, oantituzumab, epalizumab, mositumomab, and pinatuzumab. In any of these embodiments, the extracellular binding domain of the CD22 CAR can comprise or consist of V _H、V_L and/or one or more CDRs of any of these antibodies.

In some embodiments, the extracellular binding domain of the CD22 CAR comprises an scFv derived from an m971 monoclonal antibody (m 971) comprising a heavy chain variable region (V _H) and a light chain variable region (V _L) of m971 linked by a linker. In some embodiments, the linker is a 3xG ₄ S linker. In other embodiments, a Whitlow linker may alternatively be used. In some embodiments, the amino acid sequences of the entire m 971-derived scFv (also referred to as the m971 scFv) and the different portions thereof are provided in table 15 below. In some embodiments, the CD 22-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID No. 423 or 432 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to an amino acid sequence set forth in SEQ ID No. 423 or 432. In some embodiments, the CD 22-specific scFv may comprise one or more CDRs having the amino acid sequences shown in SEQ ID NOS 425-427 and 429-431 and 434-436 and 438-440. In some embodiments, a CD 22-specific scFv may comprise a heavy chain having one or more CDRs having the amino acid sequences shown in SEQ ID NOS 425-427 or 434-436. In some embodiments, the CD 22-specific scFv may comprise a light chain having one or more CDRs having the amino acid sequence set forth in SEQ ID NOS 429-431 or 438-440. In any of these embodiments, the CD 22-specific scFv can comprise one or more CDRs comprising one or more amino acid substitutions, or comprise a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to any of the identified sequences. In some embodiments, the extracellular binding domain of a CD22 CAR comprises or consists of one or more CDRs as described herein.

In some embodiments, the extracellular binding domain of the CD22 CAR comprises an scFv derived from m971-L7, which is an affinity matured variant of m971 with significantly increased CD22 binding affinity (from about 2 nM up to less than 50 pM) compared to the parent antibody m 971. In some embodiments, the scFv derived from m971-L7 comprises V _H and V _L of m971-L7 linked by a 3xG ₄ S linker. In other embodiments, a Whitlow linker may alternatively be used. In some embodiments, the amino acid sequences of the complete m971-L7 derived scFv (also referred to as m971-L7 scFv) and the different portions thereof are provided in Table 15 below. In some embodiments, the CD 22-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID No. 423 or 432 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to an amino acid sequence set forth in SEQ ID No. 423 or 432. In any of these embodiments, the CD 22-specific scFv can comprise one or more CDRs comprising one or more amino acid substitutions, or comprise a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to any of the identified sequences. In some embodiments, the extracellular binding domain of a CD22 CAR comprises or consists of one or more CDRs as described herein.

In some embodiments, the extracellular binding domain of the CD22 CAR comprises immunotoxin HA22 or BL22. Immunotoxins BL22 and HA22 are therapeutic agents that comprise scFv specific for CD22 fused to a bacterial toxin, and thus can bind to the surface of and kill cancer cells expressing CD 22. BL22 comprises dsFv, RFB4 of an anti-CD 22 antibody fused to a truncated form of the Pseudomonas exotoxin A of 38-kDa (Bang et al Clin. Cancer Res., 11:1545-50 (2005)). HA22 (CAT 8015, mositumomab immunotoxin) is a mutated, higher affinity version of BL22 (Ho et al j. Biol. Chem., 280 (1): 607-17 (2005)). Suitable sequences of antigen binding domains of HA22 and BL22 specific for CD22 are disclosed, for example, in U.S. Pat. nos. 7,541,034, 7,355,012 and 7,982,011, which are hereby incorporated by reference in their entirety.

In some embodiments, the hinge domain of the CD22 CAR comprises a CD8 a hinge domain, e.g., a human CD8 a hinge domain. In some embodiments, the CD8 a hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO. 381 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence set forth in SEQ ID NO. 381. In some embodiments, the hinge domain comprises a CD28 hinge domain, e.g., a human CD28 hinge domain. In some embodiments, the CD28 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:382 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 382. In some embodiments, the hinge domain comprises an IgG4 hinge domain, e.g., a human IgG4 hinge domain. In some embodiments, the IgG4 hinge domain comprises or consists of an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to an amino acid sequence set forth in SEQ ID NO:384 or SEQ ID NO: 385. In some embodiments, the hinge domain comprises an IgG4 hinge-Ch 2-Ch3 domain, e.g., a human IgG4 hinge-Ch 2-Ch3 domain. In some embodiments, the IgG4 hinge-Ch 2-Ch3 domain comprises or consists of an amino acid sequence shown as SEQ ID NO:386 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to an amino acid sequence shown as SEQ ID NO: 386.

In some embodiments, the transmembrane domain of the CD22 CAR comprises a CD 8a transmembrane domain, e.g., a human CD 8a transmembrane domain. In some embodiments, the CD 8a transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID No. 387 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to an amino acid sequence set forth in SEQ ID No. 387. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, e.g., a human CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises or consists of the amino acid sequence set forth in SEQ ID No. 388 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence set forth in SEQ ID No. 388.

In some embodiments, the intracellular co-stimulatory domain of the CD22 CAR comprises a 4-1BB co-stimulatory domain, e.g., a human 4-1BB co-stimulatory domain. In some embodiments, the 4-1BB costimulatory domain comprises or consists of an amino acid sequence depicted as SEQ ID NO:390 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence depicted as SEQ ID NO: 390. In some embodiments, the intracellular co-stimulatory domain comprises a CD28 co-stimulatory domain, e.g., a human CD28 co-stimulatory domain. In some embodiments, the CD28 co-stimulatory domain comprises or consists of an amino acid sequence set forth in SEQ ID No. 391 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence set forth in SEQ ID No. 391.

In some embodiments, the intracellular signaling domain of the CD22 CAR comprises a CD3 zeta (zeta) signaling domain, e.g., a human CD3 zeta signaling domain. In some embodiments, the CD3 zeta signaling domain comprises or consists of an amino acid sequence set forth in SEQ ID No. 392 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence set forth in SEQ ID No. 392.

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD22 CAR, including, for example, a CD 22-specific scFv comprising a sequence set forth in SEQ ID No. 423 or SEQ ID No. 432, a CD8 a hinge domain of SEQ ID No. 381, a CD8 a transmembrane domain of SEQ ID No. 387, a 4-1BB costimulatory domain of SEQ ID No. 390, a CD3 zeta signaling domain of SEQ ID No. 392, and/or variants thereof (i.e., having a sequence at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 identical to the disclosed sequence).

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD22 CAR, including, for example, a CD 22-specific scFv comprising a sequence set forth in SEQ ID No. 423 or SEQ ID No. 432, a CD28 hinge domain of SEQ ID No. 382, a CD8 a transmembrane domain of SEQ ID No. 387, a 4-1BB costimulatory domain of SEQ ID No. 390, a CD3 zeta signaling domain of SEQ ID No. 392, and/or variants thereof (i.e., having a sequence at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 identical to the disclosed sequence).

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD22 CAR, including, for example, a CD22 CAR comprising a CD 22-specific scFv having the sequence set forth in SEQ ID No. 423 or SEQ ID No. 432, an IgG4 hinge domain of SEQ ID No. 384 or SEQ ID No. 385, a CD 8a transmembrane domain of SEQ ID No. 387, a 4-1BB costimulatory domain of SEQ ID No. 390, a CD3 zeta signaling domain of SEQ ID No. 392, and/or variants thereof (i.e., having a sequence that is at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 identical to the disclosed sequence).

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD22 CAR, including, for example, a CD 22-specific scFv comprising a sequence set forth in SEQ ID No. 423 or SEQ ID No. 432, a CD8 a hinge domain of SEQ ID No. 381, a CD28 transmembrane domain of SEQ ID No. 389, a 4-1BB costimulatory domain of SEQ ID No. 390, a CD3 zeta signaling domain of SEQ ID No. 391, and/or variants thereof (i.e., having a sequence at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 identical to the disclosed sequence).

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD22 CAR, including, for example, a CD 22-specific scFv comprising a sequence set forth in SEQ ID No. 423 or SEQ ID No. 432, a CD28 hinge domain of SEQ ID No. 383, a CD28 transmembrane domain of SEQ ID No. 389, a 4-1BB costimulatory domain of SEQ ID No. 390, a CD3 zeta signaling domain of SEQ ID No. 392, and/or variants thereof (i.e., a CD22 CAR having a sequence at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 identical to the disclosed sequence).

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD22 CAR, including, for example, a CD22 CAR comprising a CD 22-specific scFv having the sequence set forth in SEQ ID No. 423 or SEQ ID No. 432, an IgG4 hinge domain of SEQ ID No. 384 or SEQ ID No. 385, a CD28 transmembrane domain of SEQ ID No. 388, a 4-1BB costimulatory domain of SEQ ID No. 390, a CD3 zeta signaling domain of SEQ ID No. 392, and/or variants thereof (i.e., having a sequence at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99) to the disclosed sequence.

4) BCMA CAR

In some embodiments, the CAR is a BCMA CAR ("BCMA-CAR"), and in these embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding the BCMA CAR. BCMA is a member of the Tumor Necrosis Family Receptor (TNFR) expressed on cells of the B cell lineage, with highest expression on terminally differentiated B cells or mature B lymphocytes. BCMA is involved in mediating plasma cell survival to maintain long-term humoral immunity. Recently, BCMA expression has been associated with a variety of cancers such as multiple myeloma, hodgkin and non-hodgkin lymphomas, various leukemias and glioblastomas. In some embodiments, BCMA CARs may comprise a signal peptide, an extracellular binding domain that specifically binds BCMA, a hinge domain, a transmembrane domain, an intracellular co-stimulatory domain, and/or an intracellular signaling domain in tandem.

In some embodiments, the signal peptide of the BCMA CAR comprises a CD8 a signal peptide. In some embodiments, the CD8 a signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:378 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 378. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the IgK signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO. 379 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence set forth in SEQ ID NO. 379. In some embodiments, the signal peptide comprises GMCSFR- α or CSF2RA signal peptide. In some embodiments, GMCSFR- α or CSF2RA signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID No. 380 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence set forth in SEQ ID No. 380.

In some embodiments, the extracellular binding domain of a BCMA CAR is specific for BCMA (e.g., human BCMA). The extracellular binding domain of a BCMA CAR may be codon optimized for expression in a host cell or have variant sequences to increase the function of the extracellular binding domain.

In some embodiments, the extracellular binding domain comprises an immunogenic active portion of an immunoglobulin molecule, e.g., an scFv. In some embodiments, the extracellular binding domain of a BCMA CAR is derived from BCMA-specific antibodies, including, for example, bei Lan tamab, erlenmestat, terrisstat, LCAR-B38M, and sidase. In any of these embodiments, the extracellular binding domain of a BCMA CAR may comprise or consist of V _H、V_L and/or one or more CDRs of any of these antibodies.

In some embodiments, the extracellular binding domain of a BCMA CAR comprises an scFv derived from c11d5.3, which is a murine monoclonal antibody as described in Carpenter et al, clin.cancer res.19 (8): 2048-2060 (2013). See also PCT application publication No. WO 2010/104949. The c11d5.3 derived scFv may comprise a heavy chain variable region (V _H) and a light chain variable region (V _L) of c11d5.3 linked by a Whitlow linker, the amino acid sequences of which are provided in table 17. In some embodiments, the BCMA specific extracellular binding domain comprises or consists of an amino acid sequence shown as SEQ ID NO:441, 450, or 463 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to an amino acid sequence shown as SEQ ID NO:441, 450, or 463. In some embodiments, the BCMA specific extracellular binding domain may comprise one or more CDRs having the amino acid sequences set forth in SEQ ID NOS 443-445, 447-449, 452-454, 456-458, 460-432, 465-467, and 469-471. In some embodiments, the BCMA specific extracellular binding domain may comprise a light chain having one or more CDRs having the amino acid sequences set forth in SEQ ID NOS 443-445, 452-454, 465-467. In some embodiments, the BCMA specific extracellular binding domain may comprise a heavy chain having one or more CDRs having the amino acid sequences shown in SEQ ID NOS 447-449, 456-458, 469-471. In any of these embodiments, the BCMA-specific scFv can comprise one or more CDRs comprising one or more amino acid substitutions, or a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to any of the identified sequences. In some embodiments, the extracellular binding domain of a BCMA CAR comprises or consists of one or more CDRs as described herein.

In some embodiments, the extracellular binding domain of a BCMA CAR comprises an scFv derived from another murine monoclonal antibody c12a3.2 (as described in Carpenter et al, clin. Cancer res. 19 (8): 2048-2060 (2013) and PCT application publication No. WO 2010/104949), the amino acid sequences of which are also provided in table 16 below. In some embodiments, the BCMA specific extracellular binding domain comprises or consists of an amino acid sequence shown as SEQ ID NO:441, 450, 459, or 463 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to an amino acid sequence shown as SEQ ID NO:441, 450, 459, or 463. In any of these embodiments, the BCMA-specific scFv can comprise one or more CDRs comprising one or more amino acid substitutions, or a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to any of the identified sequences. In some embodiments, the extracellular binding domain of a BCMA CAR comprises or consists of one or more CDRs as described herein.

In some embodiments, the extracellular binding domain of a BCMA CAR comprises a murine monoclonal antibody having high specificity for human BCMA, designated BB2121 in Friedman et al, hum, gene ter 29 (5): 585-601 (2018). See also PCT application publication No. WO2012163805.

In some embodiments, the extracellular binding domain of a BCMA CAR comprises a single variable fragment of two heavy chains (VHH) that can bind to two epitopes of BCMA, as described in Zhao et al, j. See also PCT application publication No. WO2018/028647.

In some embodiments, the extracellular binding domain of a BCMA CAR comprises a fully human heavy chain variable domain (FHVH), as described in Lam et al, nat. Commun. 11 (1): 283 (2020), also referred to as FHVH. See also PCT application publication No. WO2019/006072. The amino acid sequences of FHVH and its CDRs are provided below in table 16. In some embodiments, the BCMA specific extracellular binding domain comprises or consists of an amino acid sequence shown in SEQ ID No. 164 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to an amino acid sequence shown in SEQ ID No. 164. In some embodiments, the BCMA specific extracellular binding domain may comprise one or more CDRs having the amino acid sequences set forth in SEQ ID NOS 165-167. In any of these embodiments, the BCMA specific extracellular binding domain can comprise one or more CDRs comprising one or more amino acid substitutions, or a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any of the identified sequences. In some embodiments, the extracellular binding domain of a BCMA CAR comprises or consists of one or more CDRs as described herein.

In some embodiments, the extracellular binding domain of a BCMA CAR comprises an scFv derived from CT103A (or CAR 0085) (as described in U.S. patent No. 11,026,975 B2), the amino acid sequence of which is provided in table 16 below. In some embodiments, the BCMA specific extracellular binding domain comprises or consists of an amino acid sequence shown in SEQ ID No. 463, 464 or 468 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to an amino acid sequence shown in SEQ ID No. 463, 464 or 468. In some embodiments, the BCMA specific extracellular binding domain may comprise one or more CDRs having the amino acid sequences shown in SEQ ID NOS 465-497 and 469-471. In some embodiments, the BCMA specific extracellular binding domain may comprise a light chain having one or more CDRs having the amino acid sequences shown in SEQ ID NOS: 465-497. In some embodiments, the BCMA specific extracellular binding domain may comprise a heavy chain having one or more CDRs having the amino acid sequences shown in SEQ ID NOS.469-471. In any of these embodiments, the BCMA-specific scFv can comprise one or more CDRs comprising one or more amino acid substitutions, or a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to any of the identified sequences. In some embodiments, the extracellular binding domain of a BCMA CAR comprises or consists of one or more CDRs as described herein.

Additionally, BCMA-directed CARs and binding agents have been described in U.S. application publication nos. 2020/0246681 A1 and 2020/0339699 A1, the entire contents of each of which are incorporated herein by reference.

In some embodiments, the hinge domain of a BCMA CAR comprises a CD8 a hinge domain, e.g., a human CD8 a hinge domain. In some embodiments, the CD8 a hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO. 381 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence set forth in SEQ ID NO. 381. In some embodiments, the hinge domain comprises a CD28 hinge domain, e.g., a human CD28 hinge domain. In some embodiments, the CD28 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:382 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 382. In some embodiments, the hinge domain comprises an IgG4 hinge domain, e.g., a human IgG4 hinge domain. In some embodiments, the IgG4 hinge domain comprises or consists of an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to an amino acid sequence set forth in SEQ ID NO:384 or SEQ ID NO: 385. In some embodiments, the hinge domain comprises an IgG4 hinge-Ch 2-Ch3 domain, e.g., a human IgG4 hinge-Ch 2-Ch3 domain. In some embodiments, the IgG4 hinge-Ch 2-Ch3 domain comprises or consists of an amino acid sequence shown as SEQ ID NO:386 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to an amino acid sequence shown as SEQ ID NO: 386.

In some embodiments, the transmembrane domain of a BCMA CAR comprises a CD 8a transmembrane domain, e.g., a human CD 8a transmembrane domain. In some embodiments, the CD 8a transmembrane domain comprises or consists of an amino acid sequence set forth in SEQ ID No. 387 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to an amino acid sequence set forth in SEQ ID No. 387. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, e.g., a human CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises or consists of the amino acid sequence set forth in SEQ ID No. 388 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence set forth in SEQ ID No. 388.

In some embodiments, the intracellular co-stimulatory domain of the BCMA CAR comprises a 4-1BB co-stimulatory domain, e.g., a human 4-1BB co-stimulatory domain. In some embodiments, the 4-1BB costimulatory domain comprises or consists of an amino acid sequence depicted as SEQ ID NO:390 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence depicted as SEQ ID NO: 390. In some embodiments, the intracellular co-stimulatory domain comprises a CD28 co-stimulatory domain, e.g., a human CD28 co-stimulatory domain. In some embodiments, the CD28 co-stimulatory domain comprises or consists of an amino acid sequence set forth in SEQ ID No. 391 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence set forth in SEQ ID No. 391.

In some embodiments, the intracellular signaling domain of a BCMA CAR comprises a CD3 zeta (ζ) signaling domain, e.g., a human CD3 ζ signaling domain. In some embodiments, the CD3 zeta signaling domain comprises or consists of an amino acid sequence set forth in SEQ ID No. 392 or an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence set forth in SEQ ID No. 392.

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a BCMA CAR, including, for example, a BCMA CAR comprising any of the described BCMA specific extracellular binding domains, the CD 8a hinge domain of SEQ ID NO 381, the CD 8a transmembrane domain of SEQ ID NO 387, the 4-1BB costimulatory domain of SEQ ID NO 390, the CD3 zeta signaling domain of SEQ ID NO 392, and/or variants thereof (i.e., having a sequence at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence). In any of these embodiments, the BCMA CAR can additionally comprise a signal peptide (e.g., a CD 8a signal peptide) as described.

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a BCMA CAR, including, for example, a BCMA CAR comprising any of the described BCMA-specific extracellular binding domains, CD 8a hinge domain of SEQ ID No. 381, CD 8a transmembrane domain of SEQ ID No. 387, CD28 co-stimulatory domain of SEQ ID No. 391, CD3 zeta signaling domain of SEQ ID No. 392, and/or variants thereof (i.e., having a sequence at least 80% identical, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identical to the disclosed sequence). In any of these embodiments, the BCMA CAR may additionally comprise a signal peptide as described.

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a BCMA CAR as set forth in SEQ ID No. 406 or being at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the nucleotide sequence set forth in SEQ ID No. 406 (see table 17). The encoded BCMA CAR has the corresponding amino acid sequence shown in SEQ ID No. 407 or is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to the amino acid sequence shown in SEQ ID No. 407, with the components CD8 a signal peptide, CT103A scFv (V _L -Whitlow linker-V _H), CD8 a hinge domain, CD8 a transmembrane domain, 4-1BB costimulatory domain and CD3 zeta signaling domain.

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a commercially available BCMA CAR embodiment, including, for example, ai Jiwei am (ide-cel, also known as bb 2121). In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding Ai Jiwei th, or a portion thereof. Ai Jiwei the pharmaceutical composition comprises a BCMA CAR having a BB2121 binding agent, a CD8 a hinge domain, a CD8 a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3 zeta signaling domain.

3. Gene editing enzyme

In some embodiments, the exogenous agent is or comprises a genome editing technique. In some embodiments, the exogenous agent is or comprises a heterologous protein associated with genomic editing techniques. Any of a variety of agents associated with gene editing techniques may be included as exogenous agents and/or heterologous proteins, such as for delivering a gene editing mechanism to a cell. In some embodiments, the gene editing techniques may include systems involving nuclease, nicking enzyme, homing, integrase, transposase, recombinase and/or reverse transcriptase activities. In some embodiments, gene editing techniques may be used for knockout or knockdown of genes. In some embodiments, gene editing techniques may be used to knock-in or integrate DNA into regions of the genome. In some embodiments, the exogenous agent and/or heterologous protein mediates Single Strand Breaks (SSBs). In some embodiments, the exogenous agent and/or heterologous protein mediates double-strand breaks (DSBs), including double-strand breaks associated with non-homologous end joining (NHEJ) or Homology Directed Repair (HDR). In some embodiments, the exogenous agent and/or heterologous protein does not mediate SSB. In some embodiments, the exogenous agent and/or heterologous protein does not mediate DSB. In some embodiments, the exogenous agent and/or heterologous protein may be used for DNA base editing or lead editing. In some embodiments, the exogenous agent and/or heterologous protein may be used for programmable addition via a site-specific targeting element (PASTE). In some embodiments, the payload agent is a programmable DNA binding polypeptide.

In some embodiments, the exogenous agent is a programmable DNA binding polypeptide and/or nuclease for use in a gene editing method. In some embodiments, the nuclease is a Zinc Finger Nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a CRISPR-associated protein nuclease (Cas). In some embodiments, the programmable DNA-binding polypeptide is a CRISPR-associated protein nuclease (Cas). In some embodiments, the Cas protein is selected from the group consisting of Cas3, cas9, cas10, cas12, and Cas 13. In some embodiments, the Cas is Cas12a (also referred to as cpf) from Prevotella (Prevotella), franciscensis novica (FRANCISELLA NOVICIDA), amino acid coccus (Acidaminococcus sp.), chaetocerida (Lachnospiraceae bacterium) or Franciscensis (FRANCISELLA) bacteria. In some embodiments, the Cas is Cas9 from streptococcus pyogenes (Streptococcus pyogenes). In some embodiments, the Cas is Cas9 (SpCas) from streptococcus pyogenes (Streptococcus pyogenes). In some embodiments, cas9 is from staphylococcus aureus (Staphylococcus aureus) (SaCas 9). In some embodiments, cas9 is from neisseria meningitidis (NEISSERIA MENINGITIDIS) (NmeCas 9). In some embodiments, cas9 is from campylobacter jejuni (Campylobacter jejuni) (CjCas) 9. In some embodiments, cas9 is from streptococcus thermophilus (Streptococcus thermophilis) (StCas) 9. In some embodiments, the Cas is Cas12a (also referred to as Cpf 1) from a prasuvorexa or franciscensis bacterium, or Cas12b from a bacillus, optionally bacillus exovilla (Bacillus hisashii). In some embodiments, the Cas is Cas12a (also referred to as cpf) from Prevotella (Prevotella), franciscensis novica (FRANCISELLA NOVICIDA), amino acid coccus (Acidaminococcus sp.), chaetocerida (Lachnospiraceae bacterium) or Franciscensis (FRANCISELLA) bacteria. In some embodiments, the nuclease is MAD7 or CasX. In some of any of the embodiments, the Cas is Cas3, cas13, casMini, or any other Cas protein known in the art. See, e.g., wang et al Biosensors and Bioelectronics (165) 1:2020, and Wu et al Nature REVIEWS CHEMISTRY (4) 441:2020). In some embodiments, the Cas9 nuclease may be Cas9 or a functional fragment thereof from any bacterial species. See, e.g., makarova et al, nature Reviews, microbiology, 9:467-477 (2011), incorporated herein by reference in its entirety, including supplemental information.

In some embodiments, delivery of the nuclease is by a provided vector encoding a programmable DNA-binding polypeptide and/or nuclease (e.g., cas). In some embodiments, the delivery of the programmable DNA-binding polypeptide and/or nuclease is by a provided vector comprising RNA encoding the programmable DNA-binding polypeptide and/or nuclease (e.g., cas nickase, catalytically inactivated Cas). In some embodiments, delivery of the programmable DNA-binding polypeptide and/or nuclease is by a provided VLP comprising RNA encoding the programmable DNA-binding polypeptide and/or nuclease (e.g., cas nickase, catalytically inactivated Cas). In some embodiments, the Cas is a catalytically active Cas. In some embodiments, cas is a catalytically inactive Cas (also known as dead Cas, dCas), which is a Cas containing one or more mutations that inactivate the catalytic activity of the domain. In some embodiments, the Cas is a Cas nickase.

In some embodiments, the provided viral vector particles contain a nuclease protein, and the programmable DNA-binding polypeptide and/or the nuclease protein is delivered directly to the target cell. Methods of delivering programmable DNA binding polypeptides and/or nuclease proteins include, for example, those described in Cai et al, elife, 2014, 3:e01911 and international patent publication No. WO 2017068077. For example, provided viral vector particles comprise one or more Cas proteins, such as Cas9. In some embodiments, the programmable DNA-binding polypeptide and/or nuclease protein (e.g., cas, such as Cas 9) is engineered as a chimeric nuclease protein with a viral structural protein (e.g., GAG) for packaging into a viral vector particle (e.g., a lentiviral vector particle). For example, a chimeric Cas9 protein fused to a structural GAG protein may be packaged within a lentiviral vector particle. In some embodiments, the fusion protein is a cleavable fusion protein between (i) a viral structural protein (e.g., GAG) and (ii) a nuclease protein (e.g., cas protein, such as Cas 9).

In some cases, the cleavable fusion protein comprising a viral structural protein (e.g., GAG protein (e.g., MLV-GAG or HIV-GAG)) and (ii) a programmable DNA binding protein and/or a nuclease protein (e.g., cas protein (e.g., any Cas protein described herein)) comprises a Nuclear Export Sequence (NES). In some embodiments, NES promotes localization of the fusion protein to the cytosol.

In some embodiments, the cleavable fusion protein comprises at least one NES sequence (e.g., 2 or more, 3 or more, 4 or more, or 5 or more NES sequences). In some embodiments, one or more NES sequences (2 or more, 3 or more, 4 or more, or 5 or more NES sequences) are located at or near (e.g., within 50 amino acids of) the N-terminus and/or C-terminus of the cleavable fusion protein. In one embodiment, the cleavable fusion protein comprises a NES sequence located at the N-terminus and a NES sequence located at the C-terminus of the fusion protein.

In some embodiments, the cleavable fusion protein comprises at least one NES sequence (e.g., 2 or more, 3 or more, 4 or more, or 5 or more NES sequences) located at or near (e.g., within 50 amino acids of) the N-terminus and/or C-terminus of the programmable DNA binding protein and/or nuclease protein. In one embodiment, the cleavable fusion protein comprises a NES sequence located at the N-terminus and/or a NES sequence located at the C-terminus of the programmable DNA binding protein and/or nuclease protein.

In some cases, the cleavable fusion protein comprising a viral structural protein (e.g., GAG protein (e.g., MLV-GAG or HIV-GAG)) and (ii) a programmable DNA binding protein and/or a nuclease protein (e.g., cas protein (e.g., any Cas protein described herein)) comprises a Nuclear Localization Signal (NLS). In some embodiments, the NLS facilitates delivery of the fusion protein or a therapeutic polypeptide released from the fusion protein (or polynucleotide encoding a therapeutic polypeptide) (e.g., a polypeptide released from the fusion protein (or polynucleotide encoding a polypeptide) after cleavage of the cleavable linker) into the nucleus of the target cell.

In some embodiments, the cleavable fusion protein comprises at least one NLS sequence (e.g., 2 or more, 3 or more, 4 or more, or 5 or more NLS sequences). In some embodiments, one or more NLS sequences (2 or more, 3 or more, 4 or more, or 5 or more NLS sequences) are located at or near the N-terminus and/or C-terminus of the cleavable fusion protein (e.g., within 50 amino acids thereof). In one embodiment, the cleavable fusion protein comprises an NLS sequence at the N-terminus and an NLS sequence at the C-terminus of the fusion protein.

In some embodiments, the cleavable fusion protein comprises at least one NLS sequence (e.g., 2 or more, 3 or more, 4 or more, or 5 or more NLS sequences) located at or near (e.g., within 50 amino acids of) the N-terminus and/or C-terminus of the programmable DNA binding protein and/or nuclease protein. In one embodiment, the cleavable fusion protein comprises an NLS sequence located at the N-terminus and/or an NLS sequence located at the C-terminus of the nuclease protein.

In some embodiments, the cleavable fusion protein comprises one NES sequence and one NLS sequence. In some embodiments, the viral structural protein, NES sequence, NLS sequence, and therapeutic polypeptide (e.g., programmable DNA binding protein and/or nuclease protein) sequences are located from N-terminus to C-terminus of the viral structural protein-NES-NLS-therapeutic polypeptide or viral structural protein-NES-therapeutic polypeptide-NLS. In some embodiments, the viral structural protein, NES sequence, NLS sequence, and therapeutic polypeptide sequence are located from N-terminus to C-terminus of the viral structural protein-NES ⁿ -NLS-therapeutic polypeptide or the viral structural protein-NES ⁿ -therapeutic polypeptide-NLS, where N is equal to or greater than 2. In such embodiments, the cleavable linker is located before the therapeutic polypeptide (e.g., immediately before the NLS that precedes the therapeutic polypeptide).

In some embodiments, the cleavable fusion protein comprises one NES sequence and two NLS sequences. In some embodiments, the viral structural protein, NES sequence, NLS sequence, and therapeutic polypeptide (e.g., programmable DNA binding protein and/or nuclease protein) sequences are located from N-terminus to C-terminus of the viral structural protein-NES-NLS-therapeutic polypeptide-NLS. In some embodiments, the viral structural protein, NES sequence, NLS sequence, and therapeutic polypeptide sequence are located from N-terminus to C-terminus of the viral structural protein-NES ⁿ -NLS-therapeutic polypeptide-NLS, where N is equal to or greater than 2. In such embodiments, the cleavable linker is located before the therapeutic polypeptide (e.g., immediately before the NLS that precedes the therapeutic polypeptide). In one embodiment, the cleavable fusion protein comprises, from N-terminus to C-terminus, a viral structural protein-NES-cleavable linker-NLS-therapeutic polypeptide-NLS.

In some embodiments, the cleavable fusion protein has a configuration selected from the group consisting of a gag-cleavage site-NLS-therapeutic polypeptide (e.g., cas protein) -NLS, a gag-NES (3 x) -cleavage site-NLS-therapeutic polypeptide (e.g., cas protein) -NLS, and a gag-cleavage site-NLS-therapeutic polypeptide (e.g., cas protein) -NLS-cleavage site-NES.

In some embodiments, cas is a wild-type Cas9, which can site-specifically cleave double-stranded DNA, resulting in activation of a double-strand break (DSB) repair mechanism. DSB can be repaired by the non-homologous end joining (NHEJ) pathway of the cell (Overballe-Petersen et al, 2013, proc NATL ACAD SCI USA, vol. 110: 19860-19865), resulting in disruption of the insertion and/or deletion (indel) of the target locus. Alternatively, if a donor template with homology to the target locus is provided, the DSB can be repaired by the Homology Directed Repair (HDR) pathway, allowing for precise substitution mutations (Overballe-Petersen et al, 2013, proc NATL ACAD SCI USA, vol. 110: 19860-19865; gong et al, 2005, nat. Struct Mol Biol, vol. 12:304-312). In some embodiments, cas is a mutant form, referred to as Cas9D10A, having only nickase activity. This means that Cas9D10A cleaves only one DNA strand and does not activate NHEJ. In contrast, when homologous repair templates are provided, DNA repair proceeds only through the high fidelity HDR pathway, resulting in reduced indel mutations (Cong et al, 2013, science, volume 339: 819-823; jinek et al, 2012, science, volume 337: 816-821; qi et al, 2013 Cell, volume 152: 1173-1183). When the loci are designed to generate paired Cas9 complex targeting of adjacent DNA nicks, cas9D10A is even more attractive in terms of target specificity (Ran et al, 2013, cell, volume 154: 1380-1389). In some embodiments, the Cas is nuclease-deficient Cas9 (Qi et al, 2013 Cell, volume 152: 1173-1183). For example, mutation H840A in the HNH domain and mutation D10A in the RuvC domain inactivate cleavage activity, but do not prevent DNA binding. Thus, the variants can be used to target any region of the genome in a sequence-specific manner without cleavage. Conversely, dCas9 can be used as a gene silencing or activating tool by fusion with various effector domains. Furthermore, by coupling the guide RNA or Cas9 protein with a fluorophore or fluorescent protein, it can be used as a visualization tool.

In some embodiments, the Cas protein comprises one or more mutations that allow the Cas protein to be converted into a nickase capable of cleaving only one strand of a double-stranded DNA molecule (e.g., SSB). For example, a Cas9 that is generally capable of inducing a double strand break may be converted to a Cas9 that is capable of inducing a single strand break by mutating one of two Cas9 catalytic domains, a RuvC domain comprising RuvC I, ruvC II and RuvC III motifs, or a NHN domain. In some embodiments, the Cas protein comprises one or more mutations in the RuvC catalytic domain or the HNH catalytic domain. In some embodiments, the genome-modified protein is a recombinant nuclease that has been modified to have nickase activity. In some embodiments, the recombinant nuclease cleaves a strand that hybridizes to a guide RNA (e.g., sgRNA), but does not cleave a strand that is complementary to the strand that hybridizes to the guide RNA (e.g., sgRNA). In some embodiments, the recombinant nuclease does not cleave a strand that hybridizes to the guide RNA (e.g., sgRNA), but cleaves a strand that is complementary to the strand that hybridizes to the guide RNA (e.g., sgRNA).

In some embodiments, the Cas protein is selected from the group ：Cas3、Cas4、Cas5、Cas8a、Cas8b、Cas8c、Cas9、Cas10、Cas12、Cas12a (Cpf1)、Cas12b (C2c1)、Cas12c (C2c3)、Cas12d (CasY)、Cas12e (CasX)、Cas12f (C2c10)、Cas12g、Cas12h、Cas12i、Cas12k (C2c5)、Cas13、Cas13a (C2c2)、Cas13b、Cas13c、Cas13d、C2c4、C2c8、C2c9、Cmr5、Cse1、Cse2、Csf1、Csm2、Csn2、Csx10、Csx11、Csy1、Csy2、Csy3 and Mad7 consisting of. In some embodiments, the Cas protein is Cas9. In some embodiments, cas9 is from a bacterium selected from the group consisting of streptococcus pyogenes, staphylococcus aureus, neisseria meningitidis, campylobacter jejuni, and streptococcus thermophilus. In some embodiments, cas9 is from streptococcus pyogenes. In some embodiments, cas9 is from streptococcus pyogenes and comprises one or more mutations in the RuvC I, ruvC II, or RuvC III motif. In some embodiments, cas9 is from streptococcus pyogenes and comprises a D10A mutation in the RuvC I motif. In some embodiments, cas9 is from streptococcus pyogenes and comprises one or more mutations in the HNH catalytic domain. In some embodiments, cas9 is from streptococcus pyogenes and comprises one or more mutations in the HNH catalytic domain selected from the group consisting of H840A, H854A and H863A. In some embodiments, cas9 is from streptococcus pyogenes and comprises the H840A mutation in the HNH catalytic domain. In some embodiments, cas9 is from streptococcus pyogenes and comprises a mutation selected from the group consisting of D10A, H840A, H854A and H863A.

In some embodiments, the Cas protein is selected from the group consisting of Cas3, cas9, cas10, cas12, and Cas 13. In particular embodiments, the nuclease is a Cas nuclease, such as Cas9. In some embodiments, the delivery of CRISPR/Cas can be used to introduce single point mutations (deletions or insertions) in a particular target gene via a single gRNA. In contrast, using a pair of gRNA-guided Cas9 nucleases, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations. In some embodiments, the one or more agents capable of inducing DSBs (e.g., heterologous proteins) comprise Cas9 or a functional fragment thereof, as well as a first guide RNA (e.g., a first sgRNA) and a second guide RNA (e.g., a second sgRNA). In some embodiments, the guide RNA (e.g., the first guide RNA or the second guide RNA) binds to the recombinant nuclease and targets the recombinant nuclease to a specific location within the target gene, such as a location within the sense strand or the antisense strand of the target gene, which is or comprises a cleavage site. In some embodiments, the recombinant nuclease is a Cas protein from any bacterial species, or is a functional fragment thereof. In some embodiments, the Cas protein is a Cas9 nuclease. In some embodiments, cas9 may be Cas9 or a functional fragment thereof from any bacterial species. See, e.g., makarova et al, nature Reviews, microbiology, 9:467-477 (2011), incorporated herein by reference in its entirety, including supplemental information. In some embodiments, cas9 is from streptococcus pyogenes (SpCas 9). In some embodiments, cas9 is from staphylococcus aureus (Staphylococcus aureus) (SaCas 9). In some embodiments, cas9 is from neisseria meningitidis (NEISSERIA MENINGITIDIS) (NmeCas 9). In some embodiments, cas9 is from campylobacter jejuni (Campylobacter jejuni) (CjCas) 9. In some embodiments, cas9 is from streptococcus thermophilus (Streptococcus thermophilis) (StCas) 9.

In some embodiments, cas9 is from streptococcus pyogenes (SpCas 9) and comprises one or more mutations in the RuvC catalytic domain or the HNH catalytic domain. In some embodiments, one or more mutations in the RuvC catalytic domain or the HNH catalytic domain inactivate the catalytic activity of the domain. In some embodiments, the recombinant nuclease has RuvC activity, but no HNH activity. In some embodiments, the recombinant nuclease does not have RuvC activity, but has HNH activity. In some embodiments, cas9 is from streptococcus pyogenes (SpCas 9) and comprises one or more mutations selected from the group consisting of D10A, H840A, H854A and H863A. In some embodiments, cas9 is from streptococcus pyogenes (SpCas 9) and comprises one or more mutations in the RuvC I, ruvC II, or RuvC III motif. In some embodiments, cas9 is from streptococcus pyogenes (SpCas 9) and comprises a mutation in the RuvC I motif. In some embodiments, cas9 is from streptococcus pyogenes (SpCas 9) and comprises a D10A mutation in the RuvC I motif. In some embodiments, cas9 is from streptococcus pyogenes (SpCas 9) and comprises one or more mutations in the HNH catalytic domain. In some embodiments, the one or more mutations in the HNH catalytic domain are selected from the group consisting of H840A, H854A and H863A. In some embodiments, cas9 is from streptococcus pyogenes (SpCas 9) and comprises the H840A mutation in the HNH catalytic domain. In some embodiments, cas9 is from streptococcus pyogenes (SpCas 9) and comprises the H840A mutation. In some embodiments, cas9 is from streptococcus pyogenes (SpCas 9) and comprises the D10A mutation. In some embodiments, cas9 is from streptococcus pyogenes (SpCas 9) and comprises one or more mutations selected from the group consisting of N497A, R661A, Q695A and Q926A. In some embodiments, cas9 is from streptococcus pyogenes (SpCas 9) and comprises one or more mutations selected from the group consisting of R780A, K810A, K855A, H982A, K1003A, R1060A and K848A. In some embodiments, cas9 is from streptococcus pyogenes (SpCas 9) and comprises one or more mutations selected from the group consisting of N692A, M694A, Q695A and H698A. In some embodiments, cas9 is from streptococcus pyogenes (SpCas 9) and comprises one or more mutations selected from the group consisting of M495V, Y515N, K526E and R661Q. In some embodiments, cas9 is from streptococcus pyogenes (SpCas 9) and comprises one or more mutations selected from the group consisting of F539S, M763I and K890N. In some embodiments, cas9 is from streptococcus pyogenes (SpCas 9) and comprises one or more mutations selected from the group consisting of E480K, E543D, E1219V, A262T, S409I, M694I, E108G, S a.

In some embodiments, cas9 is from streptococcus pyogenes (SaCas 9). In some embodiments, saCas9 is wild-type SaCas9. In some embodiments, saCas9 comprises one or more mutations in the REC3 domain. In some embodiments, saCas9 comprises one or more mutations in the REC1 domain. In some embodiments, saCas9 comprises one or more mutations selected from the group consisting of N260D, N260Q, N260E, Q414A, Q414L. In some embodiments, saCas9 comprises one or more mutations in the recognition leaf. In some embodiments, saCas9 comprises one or more mutations selected from the group consisting of R245A, N413A, N a. In some embodiments, saCas9 comprises one or more mutations in the RuvC-III domain. In some embodiments, saCas9 comprises an R654A mutation.

In some embodiments, the Cas protein is Cas12. In some embodiments, the Cas protein is Cas12a (i.e., cpf a 1). In some embodiments, cas12a is selected from the group consisting of francisco novacus U112 (FnCas a), amino acid coccus species BV3L6 (AsCas a), moraxella nivea (Moraxella bovoculi) aax11—00205 (Mb 3Cas12 a), chaetoceraceae bacteria ND2006 (LbCas a), sulfur micro-spirobacteria species (Thiomicrospira sp.) Xs5 (TsCas a), moraxella nivea aax08_00205 (Mb 2Cas12 a), and vibrio butyricum species (Butyrivibrio sp.) NC3005 (BsCas a). In some embodiments, cas12a recognizes a T-rich 5' proto-spacer adjacent motif (PAM). In some embodiments, cas12a processes its own crRNA without transactivation crRNA (tracrRNA). In some embodiments, cas12a processes both rnase and dnase activity. In some embodiments, cas12a is a split Cas12a platform, consisting of the N-terminal and C-terminal fragments of Cas12 a. In some embodiments, the split Cas12a platform is from a chaetoceros bacterium.

In some embodiments, the particles containing a Cas nuclease (e.g., cas 9) further comprise one or more CRISPR-Cas system guide RNAs for targeting a desired target gene. In some embodiments, the CRISPR guide RNA is effectively encapsulated in CAS-containing particles. In some embodiments, the provided particles (e.g., lentiviral particles) further comprise a targeting nucleic acid.

In some embodiments, the lipid particle further comprises the polynucleotide itself, i.e., a polynucleotide that does not encode a heterologous protein. In some embodiments, the polynucleotide itself is associated with a gene editing system. For example, the lipid particle may comprise a guide RNA (gRNA), such as a single guide RNA (sgRNA).

In some embodiments, the one or more agents (e.g., the one or more exogenous agents and/or the heterologous protein) comprise or are used in combination with a guide RNA (e.g., a single guide RNA (sgRNA)) for inducing a DSB at the cleavage site. In some embodiments, the one or more agents comprise or are used in combination with more than one guide RNA (e.g., a first sgRNA and a second sgRNA) for inducing DSBs at the cleavage site by SSBs on each strand. In some embodiments, one or more agents (e.g., heterologous proteins) may be used in combination with a donor template (e.g., single stranded DNA oligonucleotides (ssODN)) for HDR-mediated integration of the donor template into a target gene, such as at a targeting sequence. In some embodiments, one or more agents (e.g., one or more exogenous agents and/or heterologous proteins) can be used in combination with a donor template (e.g., ssODN) and a guide RNA (e.g., sgRNA) for HDR-mediated integration of the donor template into a target gene, such as at a targeting sequence. In some embodiments, one or more agents (e.g., one or more exogenous agents and/or heterologous proteins) can be used in combination with a donor template (e.g., ssODN) and a first guide RNA (e.g., a first sgRNA) and a second guide RNA (e.g., a second sgRNA) for HDR-mediated integration of the donor template into a target gene, such as at a targeting sequence.

In certain embodiments, the genome modifier is a Cas protein, such as Cas9. In some embodiments, the delivery of CRISPR/Cas can be used to introduce single point mutations (deletions or insertions) in a particular target gene via a single gRNA. In contrast, using a pair of gRNA-guided Cas9 nucleases, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations. In some embodiments, the dCas9 version of the CRISPR/Cas9 system can be used to target protein domains for transcriptional regulation, epigenetic modification, and microscopic visualization of specific genomic loci.

In some embodiments, the genomic modifier (e.g., cas 9) targets the cleavage site by interacting with a guide RNA (e.g., sgRNA) that hybridizes to a DNA sequence immediately preceding the Protospacer Adjacent Motif (PAM) sequence. In general, a guide RNA (e.g., sgRNA) is any nucleotide sequence that comprises a sequence (e.g., crRNA sequence) that has sufficient complementarity to a target gene sequence to hybridize to the target gene sequence at a cleavage site and direct the specific binding of a recombinant nuclease to the sequence of the portion of the target gene that comprises the cleavage site. Complete complementarity (100%) is not necessarily required, provided that sufficient complementarity exists to cause hybridization and promote formation of a complex (e.g., CRISPR complex) comprising a recombinant nuclease (e.g., cas 9) and a guide RNA (e.g., sgRNA). In some embodiments, the cleavage site is located at a site within the target gene that is homologous to the sequence of the guide RNA (e.g., sgRNA). In some embodiments, the cleavage site is located about 3 nucleotides upstream of the PAM sequence. In some embodiments, the cleavage site is located about 3 nucleotides upstream of the junction between the guide RNA and PAM sequence. In some embodiments, the cleavage site is located 3 nucleotides upstream of the PAM sequence. In some embodiments, the cleavage site is located 4 nucleotides upstream of the PAM sequence.

In some embodiments, one or more agents capable of inducing a DSB (e.g., one or more exogenous agents and/or heterologous proteins) comprise a fusion protein comprising a DNA binding domain and a DNA cleavage domain. In some embodiments, the DNA cleavage domain is or comprises a recombinant nuclease. In some embodiments, the fusion protein is a TALEN comprising a DNA binding domain and a DNA cleavage domain. In some embodiments, the DNA binding domain is a transcription activator-like (TAL) effector DNA binding domain. In some embodiments, the TAL effector DNA binding domain is from a Xanthomonas (Xanthomonas) bacterium. In some embodiments, the DNA cleavage domain is a Fokl nuclease domain. In some embodiments, TAL effector DNA binding domains are engineered to target a specific target sequence, such as a portion of a target gene that comprises a cleavage site.

In some embodiments, the fusion protein is a Zinc Finger Nuclease (ZFN) comprising a zinc finger DNA binding domain and a DNA cleavage domain. In some embodiments, the DNA cleavage domain is a Fokl nuclease domain. In some embodiments, the zinc finger DNA binding domain is engineered to target a specific target sequence, e.g., a portion of a target gene comprising a cleavage site, such as a targeting sequence.

In some embodiments, the provided lipid particles can be used in methods of delivering an exogenous agent that involve introducing one or more agents (e.g., one or more exogenous agents and/or heterologous proteins) into a cell, the one or more agents being capable of inducing SSB at a cleavage site within the sense strand of an endogenous target gene and inducing SSB at a cleavage site within the antisense strand in the cell.

In some embodiments, the cleavage site distance in the sense strand and the nucleotide complementary to the cleavage site in the antisense strand are less than 400, less than 350, less than 300, less than 250, less than 200, less than 175, less than 150, less than 125, less than 100, less than 90, less than 80, less than 75, less than 70, less than 65, less than 60, less than 55, less than 50, less than 45, less than 40, or less than 35 nucleotides apart. In some embodiments, the cleavage site distance in the antisense strand and the nucleotide complementary to the cleavage site in the sense strand are less than 400, less than 350, less than 300, less than 250, less than 200, less than 175, less than 150, less than 125, less than 100, less than 90, less than 80, less than 75, less than 70, less than 65, less than 60, less than 55, less than 50, less than 45, less than 40, or less than 35 nucleotides apart. In some embodiments, the cleavage site distance in the sense strand and the nucleotide complementary to the cleavage site in the antisense strand are 20 to 400, 20 to 350, 20 to 300, 20 to 250, 20 to 200, 20 to 150, 20 to 125, 20 to 100,20 to 90, 20 to 80, 20 to 70, 30 to 400, 30 to 350, 30 to 300, 30 to 250, 30 to 200, 30 to 150, 30 to 125, 30 to 100, 30 to 90, 30 to 80, 30 to 70, 40 to 400, 40 to 350, 40 to 300, 40 to 250, 40 to 200, 40 to 150, 40 to 125, 40 to 100, 40 to 90, 40 to 80, or 40 to 70 nucleotides apart. In some embodiments, the cleavage site in the antisense strand is 20 to 400, 20 to 350, 20 to 300, 20 to 250, 20 to 200, 20 to 150, 20 to 125, 20 to 100,20 to 90, 20 to 80, 20 to 70, 30 to 400, 30 to 350, 30 to 300, 30 to 250, 30 to 200, 30 to 150, 30 to 125, 30 to 100, 30 to 90, 30 to 80, 30 to 70, 40 to 400, 40 to 350, 40 to 300, 40 to 250, 40 to 200, 40 to 150, 40 to 125, 40 to 100, 40 to 90, 40 to 80, or 40 to 70 nucleotides apart from the nucleotide complementary to the cleavage site in the sense strand.

In some embodiments, the one or more agents (e.g., one or more exogenous agents and/or heterologous proteins) capable of inducing SSB at a cleavage site within the sense strand and inducing SSB at a cleavage site within the antisense strand comprise a recombinant nuclease. In some embodiments, the recombinant nucleases include those that induce SSB in the sense strand and those that induce SSB in the antisense strand, and both are referred to as recombinant nucleases. Thus, in some embodiments, the methods involve introducing into the cell one or more agents (e.g., one or more exogenous agents and/or heterologous proteins) comprising a recombinant nuclease for inducing SSB at a cleavage site in the sense strand and SSB at a cleavage site in the antisense strand within an endogenous target gene in the cell. Although "one," "the" recombinant nucleases are described in some embodiments as inducing SSB in the antisense strand and SSB in the sense strand, it is to be understood that this includes the case where two identical recombinant nucleases are used, such that one recombinant nuclease induces SSB in the sense strand and the other recombinant nuclease induces SSB in the antisense strand. In some embodiments, the recombinant nuclease that induces SSB lacks the ability to induce DSB by cleaving both strands of double-stranded DNA.

In some embodiments, the one or more agents capable of inducing SSB comprise a recombinant nuclease and a first guide RNA (e.g., a first sgRNA) and a second guide RNA (e.g., a second sgRNA).

In some embodiments, the genome modifier is a Cas protein, a transcription activator-like effector nuclease (TALEN), or a Zinc Finger Nuclease (ZFN). In some embodiments, the recombinant nuclease is a Cas nuclease. In some embodiments, the recombinant nuclease is a TALEN. In some embodiments, the recombinant nuclease is a ZFN.

In some embodiments, the one or more agents capable of inducing SSB at a cleavage site within the sense strand and inducing SSB at a cleavage site within the antisense strand comprise a fusion protein comprising a DNA binding domain and a DNA cleavage domain. In some embodiments, the DNA cleavage domain is or comprises a recombinant nuclease. In some embodiments, the fusion protein is a TALEN comprising a DNA binding domain and a DNA cleavage domain. In some embodiments, the DNA binding domain is a transcription activator-like (TAL) effector DNA binding domain. In some embodiments, the TAL effector DNA binding domain is from a Xanthomonas (Xanthomonas) bacterium. In some embodiments, the DNA cleavage domain is a Fokl nuclease domain. In some embodiments, TAL effector DNA binding domains are engineered to target a specific target sequence, such as a portion of a target gene that comprises a cleavage site. In some embodiments, the fusion protein is a Zinc Finger Nuclease (ZFN) comprising a zinc finger DNA binding domain and a DNA cleavage domain. In some embodiments, the DNA cleavage domain is a Fokl nuclease domain. In some embodiments, the zinc finger DNA binding domain is engineered to target a specific target sequence, e.g., a portion of a target gene comprising a cleavage site, such as a targeting sequence.

In some embodiments, the one or more agents capable of inducing SSB at a cleavage site within the sense strand and inducing SSB at a cleavage site within the antisense strand involve the use of a CRISPR/Cas gene editing system. In some embodiments, the one or more agents comprise a recombinant nuclease.

In some embodiments, the genome modifier is a Cas protein. In some embodiments, the Cas protein comprises one or more mutations that cause the Cas protein to be converted to a nickase that lacks the ability to cleave both strands of a double-stranded DNA molecule. In some embodiments, the Cas protein comprises one or more mutations that allow the Cas protein to be converted into a nickase capable of cleaving only one strand of a double-stranded DNA molecule. For example, a Cas9 that is generally capable of inducing a double strand break may be converted to a Cas9 that is capable of inducing a single strand break by mutating one of two Cas9 catalytic domains, a RuvC domain comprising RuvC I, ruvC II and RuvC III motifs, or a NHN domain. In some embodiments, the Cas protein comprises one or more mutations in the RuvC catalytic domain or the HNH catalytic domain. In some embodiments, the genome-modified protein is a recombinant nuclease that has been modified to have nickase activity. In some embodiments, the recombinant nuclease cleaves a strand that hybridizes to a guide RNA (e.g., sgRNA), but does not cleave a strand that is complementary to the strand that hybridizes to the guide RNA (e.g., sgRNA). In some embodiments, the recombinant nuclease does not cleave a strand that hybridizes to the guide RNA (e.g., sgRNA), but cleaves a strand that is complementary to the strand that hybridizes to the guide RNA (e.g., sgRNA).

In some embodiments, the lipid particle further comprises a guide RNA (gRNA), such as a single guide RNA (sgRNA). Thus, in some embodiments, the heterologous agent comprises a guide RNA (gRNA). In some embodiments, the gRNA is a single guide RNA (sgRNA).

In some embodiments, the genome modification protein (e.g., cas 9) targets the cleavage site by interacting with a guide RNA (e.g., a first guide RNA, such as a first sgRNA, or a second guide RNA, such as a second sgRNA) that hybridizes to a DNA sequence on the sense strand or the antisense strand immediately preceding the proto-spacer adjacent motif (PAM) sequence.

In some embodiments, the genomic modifier (e.g., cas 9) targets the cleavage site on the sense strand by interacting with a first guide RNA (e.g., a first sgRNA) that hybridizes to a sequence on the sense strand immediately preceding the PAM sequence. In some embodiments, the genome modifier (e.g., cas 9) targets the cleavage site on the antisense strand by interacting with a second guide RNA (e.g., a second sgRNA) that hybridizes to a sequence on the antisense strand immediately preceding the PAM sequence.

In some embodiments, a first guide RNA (e.g., first sgNA) specific to the sense strand of the target gene of interest is used to target a recombinant nuclease (e.g., cas 9) to induce SSB at a cleavage site within the sense strand of the target gene. In some embodiments, a first guide RNA (e.g., first sgNA) specific for the antisense strand of the target gene of interest is used to target a recombinant nuclease (e.g., cas 9) to induce SSB at a cleavage site within the antisense strand of the target gene.

In some embodiments, a second guide RNA (e.g., second sgNA) specific for the sense strand of the target gene of interest is used to target a recombinant nuclease (e.g., cas 9) to induce SSB at a cleavage site within the sense strand of the target gene. In some embodiments, a second guide RNA (e.g., second sgNA) specific for the antisense strand of the target gene of interest is used to target a recombinant nuclease (e.g., cas 9) to induce SSB at a cleavage site within the antisense strand of the target gene.

In some embodiments, a first guide RNA (e.g., first sgNA) specific for the sense strand of the target gene of interest is used to target a recombinant nuclease (e.g., cas 9) to induce SSB at a cleavage site within the sense strand of the target gene, and a second guide RNA (e.g., second sgNA) specific for the antisense strand of the target gene of interest is used to target a recombinant nuclease (e.g., cas 9) to induce SSB at a cleavage site within the antisense strand of the target gene.

In some embodiments, a first guide RNA (e.g., first sgNA) specific for the antisense strand of the target gene of interest is used to target a recombinant nuclease (e.g., cas 9) to induce SSB at a cleavage site within the antisense strand of the target gene, and a second guide RNA (e.g., second sgNA) specific for the sense strand of the target gene of interest is used to target a recombinant nuclease (e.g., cas 9) to induce SSB at a cleavage site within the sense strand of the target gene. In general, a guide RNA (e.g., a first guide RNA, such as a first sgRNA, or a second guide RNA, such as a second sgRNA) is any nucleotide sequence that comprises a sequence (e.g., crRNA sequence) that has sufficient complementarity to a target gene sequence to hybridize to the target gene sequence at a cleavage site and direct the specific binding of a recombinant nuclease to the sequence of the portion of the target gene that comprises the cleavage site. Complete complementarity (100%) is not necessarily required, provided that sufficient complementarity exists to cause hybridization and promote formation of a complex (e.g., a CRISPR complex) comprising a recombinant nuclease (e.g., cas 9) and a guide RNA (e.g., a first guide RNA, such as a first sgRNA, or a second guide RNA, such as a second sgRNA).

In some embodiments, the cleavage site is located at a site within the target gene that is homologous to a sequence contained within the guide RNA (e.g., sgRNA). In some embodiments, the cleavage site of the sense strand is located within the sense strand of the target gene at a site homologous to a sequence contained within the first guide RNA (e.g., the first sgRNA). In some embodiments, the cleavage site of the antisense strand is located within the antisense strand of the target gene at a site homologous to a sequence contained within the first guide RNA (e.g., the first sgRNA). In some embodiments, the cleavage site of the sense strand is located within the sense strand of the target gene at a site homologous to a sequence contained within the second guide RNA (e.g., the second sgRNA). In some embodiments, the cleavage site of the antisense strand is located within the antisense strand of the target gene at a site homologous to a sequence contained within the second guide RNA (e.g., the second sgRNA). In some embodiments, the cleavage site of the sense strand is located within the sense strand of the target gene at a site homologous to a sequence contained within a first guide RNA (e.g., a first sgRNA), and the cleavage site of the antisense strand is located within the antisense strand of the target gene at a site homologous to a sequence contained within a second guide RNA (e.g., a second sgRNA). In some embodiments, the cleavage site of the antisense strand is located within the antisense strand of the target gene at a site homologous to a sequence contained within a first guide RNA (e.g., a first sgRNA), and the cleavage site of the sense strand is located within the sense strand of the target gene at a site homologous to a sequence contained within a second guide RNA (e.g., a second sgRNA). In some embodiments, the cleavage site of the antisense strand is located within the antisense strand of the target gene at a site homologous to a sequence contained within the second guide RNA (e.g., the second sgRNA), and the cleavage site of the sense strand is located within the sense strand of the target gene at a site homologous to a sequence contained within the first guide RNA (e.g., the first sgRNA).

In some embodiments, the sense strand comprises a targeting sequence, and the targeting sequence comprises a SNP and Protospacer Adjacent Motif (PAM) sequence. In some embodiments, the sense strand comprises a targeting sequence and the targeting sequence comprises a SNP and a Protospacer Adjacent Motif (PAM) sequence, and the antisense strand comprises a sequence complementary to the targeting sequence and comprises a PAM sequence. In some embodiments, the antisense strand comprises a targeting sequence, and the targeting sequence comprises a SNP and Protospacer Adjacent Motif (PAM) sequence. In some embodiments, the antisense strand comprises a targeting sequence and the targeting sequence comprises a SNP and a Protospacer Adjacent Motif (PAM) sequence, and the sense strand comprises a sequence complementary to the targeting sequence and comprises a PAM sequence.

In some embodiments, the cleavage site on the sense strand and/or the antisense strand is located about 3 nucleotides upstream of the PAM sequence. In some embodiments, the cleavage site on the sense strand and/or the antisense strand is located about 3 nucleotides upstream of the junction between the guide RNA and PAM sequence. In some embodiments, the cleavage site on the sense strand and/or the antisense strand is located 3 nucleotides upstream of the PAM sequence. In some embodiments, the cleavage site on the sense strand and/or the antisense strand is located 4 nucleotides upstream of the PAM sequence.

In some embodiments, the PAM sequence recognized by the recombinant nuclease is in the sense strand. In some embodiments, the PAM sequence recognized by the recombinant nuclease is in the antisense strand. In some embodiments, the PAM sequence recognized by the recombinant nuclease is in the sense strand and in the antisense strand. In some embodiments, the PAM sequence on the sense strand and the PAM sequence on the antisense strand are directed outward. In some embodiments, the PAM sequence on the sense strand and the PAM sequence on the antisense strand comprise the same nucleic acid sequence, which may be any PAM sequence disclosed herein. In some embodiments, the PAM sequence on the sense strand and the PAM sequence on the antisense strand each comprise a different nucleic acid sequence, each of which can be any PAM sequence disclosed herein.

In some embodiments, the PAM sequence recognized by a recombinant nuclease (e.g., cas 9) varies depending on the particular recombinant nuclease and the bacterial species from which it is derived.

Methods for designing guide RNAs (e.g., sgrnas) and exemplary targeting sequences thereof (e.g., crRNA sequences) may include those described in, for example, international PCT publication nos. WO2015/161276, WO2017/193107, and WO 2017/093969. Exemplary guide RNA structures (including specific domains) are described in WO2015/161276, for example in figures 1A to 1G therein. Since the guide RNA is an RNA molecule, it will contain the base uracil (U), whereas any DNA encoding the guide RNA molecule will contain the base thymine (T). In some embodiments, the guide RNAs (e.g., sgrnas) comprise CRISPR-targeting RNA sequences (crrnas) and transactivating crRNA sequences (tracrrnas). In some embodiments, the first guide RNA (e.g., first sgRNA) and the second guide RNA (e.g., second sgRNA) each comprise crRNA and tracrRNA. In some embodiments, the guide RNA (e.g., sgRNA) is an RNA comprising, from 5 'to 3', a crRNA sequence and a tracrRNA sequence. In some embodiments, each of the first guide RNA (e.g., first sgRNA) and the second guide RNA (e.g., second sgRNA) is an RNA that includes, from 5 'to 3', a crRNA sequence and a tracrRNA sequence. In some embodiments, the crRNA and tracrRNA are not naturally present together in the same sequence.

In some embodiments, the crRNA comprises a nucleotide sequence that is homologous (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous, or 100% homologous) to a portion of the target gene comprising the cleavage site. In some embodiments, the crRNA comprises a nucleotide sequence that is 100% homologous to the portion of the target gene comprising the cleavage site. In some embodiments, the portion of the target gene comprising the cleavage site is a portion of the sense strand of the target gene comprising the cleavage site. In some embodiments, the portion of the target gene comprising the cleavage site is a portion of the antisense strand of the target gene comprising the cleavage site.

In some embodiments, the sgrnas comprise crRNA sequences homologous to sequences comprising cleavage sites in the target gene. In some embodiments, the first sgRNA comprises a crRNA sequence that is homologous to a sequence comprising a cleavage site in the sense strand of the target gene, and/or the second sgRNA comprises a crRNA sequence that is homologous to a sequence comprising a cleavage site in the antisense strand of the target gene. In some embodiments, the first sgRNA comprises a crRNA sequence that is homologous to a sequence comprising a cleavage site in the antisense strand of the target gene, and/or the second sgRNA comprises a crRNA sequence that is homologous to a sequence comprising a cleavage site in the sense strand of the target gene.

In some embodiments, the crRNA sequence has 100% sequence identity to a sequence comprising a cleavage site in the target gene. In some embodiments, the crRNA sequence of the first sgRNA has 100% sequence identity to a sequence comprising a cleavage site in the sense strand of the target gene and/or the crRNA sequence of the second sgRNA has 100% sequence identity to a sequence comprising a cleavage site in the antisense strand of the target gene. In some embodiments, the crRNA sequence of the first sgRNA has 100% sequence identity to a sequence comprising a cleavage site in the antisense strand of the target gene and/or the crRNA sequence of the second sgRNA has 100% sequence identity to a sequence comprising a cleavage site in the sense strand of the target gene.

Guidance on crRNA sequence selection can be found, for example, in Fu Y et al, nat Biotechnol 2014 (doi: 10.1038/nbt.2808) and Sternberg SH et al, nature 2014 (doi: 10.1038/Nature 13011). Examples of the location of crRNA sequences within a guide RNA (e.g., sgRNA) structure include those described in WO2015/161276, e.g., in fig. 1A-1G therein.

References to "crrnas" should be understood to also include references to crrnas of the first and second sgrnas, each independently. Thus, references to embodiments of "crrnas" should be understood to refer to embodiments of (i) crrnas, (ii) crrnas of the first sgrnas, and (iii) crrnas of the second sgrnas independently. In some embodiments, the crRNA is 15-27 nucleotides in length, i.e., the crRNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides in length. In some embodiments, the crRNA is 18-22 nucleotides in length. In some embodiments, the crRNA is 19-21 nucleotides in length. In some embodiments, the crRNA is 20 nucleotides in length.

In some embodiments, the crRNA is homologous to a portion of the target gene comprising a cleavage site. In some embodiments, the crRNA is homologous to a portion of the sense strand of the target gene comprising a cleavage site. In some embodiments, the crRNA is homologous to a portion of the antisense strand of the target gene comprising a cleavage site. In some embodiments, the crRNA of the first sgRNA is homologous to a portion of the sense strand of the target gene that comprises a cleavage site, and the crRNA of the second sgRNA is homologous to a portion of the antisense strand of the target gene that comprises a cleavage site.

In some embodiments, the crRNA is homologous to a portion of the antisense strand of the target gene comprising a cleavage site. In some embodiments, the crRNA is homologous to a portion of the sense strand of the target gene comprising a cleavage site. In some embodiments, the crRNA of the first sgRNA is homologous to a portion of the antisense strand of the target gene that comprises a cleavage site, and the crRNA of the second sgRNA is homologous to a portion of the sense strand of the target gene that comprises a cleavage site.

In some embodiments, the crRNA is homologous to a portion of the target gene comprising a cleavage site and is 15-27 nucleotides in length, i.e., the crRNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides in length. In some embodiments, the portion of the target gene comprising the cleavage site is on the sense strand. In some embodiments, the portion of the target gene comprising the cleavage site is on the antisense strand.

In some embodiments, the crRNA is homologous to a portion (i.e., sequence) of the sense or antisense strand of the target gene that contains a cleavage site and immediately upstream of the PAM sequence.

In some embodiments, the tracrRNA sequence can be or comprise any sequence of tracrRNA used in any CRISPR/Cas9 system known in the art. References to "tracrRNA" should be understood to also include references to tracrRNA of the first sgRNA and tracrRNA of the second sgRNA, each independently. Thus, references to embodiments of "tracrRNA" should be understood to refer to embodiments of (i) tracrRNA, (ii) tracrRNA of the first sgRNA, and (iii) tracrRNA of the second sgRNA independently. Exemplary CRISPR/Cas9 systems, sgrnas, crrnas, and tracrRNA, and methods of making and using the same, include those described, for example, in international PCT publication nos. WO2015/161276, WO2017/193107, and WO2017/093969, and those described, for example, in U.S. patent application publication nos. 20150232882、20150203872、20150184139、20150079681、20150073041、20150056705、20150031134、20150020223、20140357530、20140335620、20140310830、20140273234、20140273232、20140273231、20140256046、20140248702、20140242700、20140242699、20140242664、20140234972、20140227787、20140189896、20140186958、20140186919、20140186843、20140179770、20140179006、20140170753、20140093913 and 20140080216.

In some embodiments, the heterologous protein is associated with base editing. Base Editors (BE) are typically fusions of Cas ("CRISPR-associated") domains and nucleobase modification domains (e.g., natural or evolved deaminase, such as cytidine deaminase, which includes apodec 1 ("apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1"), CDA ("cytidine deaminase"), and AID ("activation-induced cytidine deaminase") domains). In some cases, the base editor may also contain proteins or domains that alter cellular DNA repair processes to increase the efficiency and/or stability of the resulting single nucleotide changes.

In some aspects, currently available base editors include a cytidine base editor (e.g., BE 4) that converts target C.G to T.A and an adenine base editor (e.g., ABE 7.10) that converts A.T to g.c. In some aspects, cas9 targeted deamination was demonstrated for the first time to BE associated with a Base Editor (BE) system designed to induce base changes without introducing double-stranded DNA breaks. Further, the rat deaminase apodec 1 (rAPOBEC 1) fused to the inactivated Cas9 (dCas 9) was used to successfully convert cytidine upstream of PAM of sgRNA to thymidine. In some aspects, this first BE system is optimized by changing dCas9 to a "nickase" Cas 9D 10A that nicks on the deaminated cytidine opposing strand. Without being bound by theory, this is expected to initiate long patch Base Excision Repair (BER), where deaminated chains are preferentially used for template repair to produce U.A base pairs, which are then converted to T.A during DNA replication.

In some embodiments, the exogenous agent and/or heterologous protein is or encodes a base editor (e.g., a nucleobase editor). In some embodiments, the exogenous agent and/or heterologous protein is a nucleobase editor comprising a catalytically inactive first DNA-binding protein domain, a domain having base editing activity, and a second DNA-binding protein domain having nicking enzyme activity, wherein the DNA-binding protein domains are expressed on a single fusion protein or expressed separately (e.g., on separate expression vectors). In some embodiments, the base editor is a fusion protein comprising a domain having base editing activity (e.g., cytidine deaminase or adenosine deaminase) and two nucleic acid programmable DNA binding protein domains (napDNAbp), the first having nickase activity, and the second napDNAbp being catalytically inactive, wherein at least two napDNAbp are linked by a linker. In some embodiments, the base editor is a fusion protein comprising a DNA domain of a CRISPR-Cas (e.g., cas 9) having nickase activity (nCas; nCas 9), a catalytically inactive domain of a CRISPR-Cas protein (e.g., cas 9) (dCas; e.g., dCas 9) having nucleic acid programmable DNA binding activity, and a deaminase domain, wherein dCas is linked to nCas by a linker and dCas is immediately adjacent to the deaminase domain. In some embodiments, the base editor is an adenine to thymine or "ATBE" (or thymine to adenine or "TABE") transversion base editor. Exemplary base editors and base editor systems include any one as described in patent publication number US20220127622、US20210079366、US20200248169、US20210093667、US20210071163、WO2020181202、WO2021158921、WO2019126709、WO2020181178、WO2020181195、WO2020214842、WO2020181193, which is hereby incorporated by reference in its entirety.

In some embodiments, the exogenous agent and/or heterologous protein is an exogenous agent and/or heterologous protein for target-directed reverse transcription (TPRT) or "directed editing". In some embodiments, the lead editing mediates targeted insertions, deletions, all 12 possible base-to-base conversions, and combinations thereof in human cells without the need for DSBs or donor DNA templates.

Leader editing is a genomic editing method that uses a nucleic acid programmable DNA binding protein ("napDNAbp") working in conjunction with a polymerase (i.e., in the form of a fusion protein supplied in trans with napDNAbp or otherwise) to write new genetic information directly to a designated DNA site, where the leader editing system is programmed with a leader editing (PE) guide RNA ("PEgRNA") that both designates the target site and templates the synthesis of the desired editing by engineering onto the guide RNA (e.g., at the 5 'or 3' end, or at the interior portion of the guide RNA) in the form of a replacement DNA strand. The replacement strand containing the desired edit (e.g., a single nucleobase substitution) shares the same sequence with the endogenous strand of the target site to be edited (except that it contains the desired edit). The endogenous strand of the target site is replaced by a newly synthesized replacement strand containing the desired editing, through DNA repair and/or replication mechanisms. In some cases, lead editing may be considered a "search and replace" genome editing technique in that the lead editor searches for and locates the desired target site to be edited and encodes a replacement strand containing the desired edit, which simultaneously installs a strand of endogenous DNA that replaces the corresponding target site. For example, the lead editing can be adapted to perform precise CRISPR/Cas-based genome editing to avoid double strand breaks. In some embodiments, the heterologous protein is or encodes a Cas protein-reverse transcriptase fusion or related system to target a particular DNA sequence with a guide RNA, create a single stranded nick at the target site, and use the nicked DNA as a primer for reverse transcription of an engineered reverse transcriptase template integrated with the guide RNA. In some embodiments, the leader editor protein is paired with two leader editing guide RNAs (pegrnas) that template the synthesis of complementary DNA flaps on opposite strands of genomic DNA, resulting in replacement of endogenous DNA sequences between PE-induced nick sites with those encoded by pegRNA.

In some embodiments, the exogenous agent and/or heterologous protein is or encodes a leader editor that is a reverse transcriptase or any DNA polymerase known in the art. Thus, in one aspect, the leader editor may comprise Cas9 (or equivalently napDNAbp) programmed to target a DNA sequence by associating the DNA sequence with a specific guide RNA (i.e., PEgRNA) containing a spacer sequence that anneals to a complementary proto-spacer in the target DNA. Such methods include Anzalone et al (doi.org/10.1038/s 41586-019-1711-4) or any of the methods disclosed in PCT publication Nos. WO2020191248, WO2021226558 or WO2022067130, which are hereby incorporated by reference in their entirety.

In some embodiments, the exogenous agent and/or heterologous protein is used for programmable addition via a site-specific targeting element (PASTE). In some aspects, a PASTE is a platform in which genomic insertion is guided via a CRISPR-Cas9 nickase fused to both a reverse transcriptase and a serine integrase. As described in Ioannidi et al (doi.org/10.1101/2021.11.01.466786), PASTE does not create a double strand break, but allows integration of sequences up to about 36 kb. In some embodiments, the serine integrase may be any serine integrase known in the art. In some embodiments, the serine integrase has sufficient orthogonality that the PASTE can be used for multiple gene integration, integrating at least two different genes at least two genomic loci simultaneously. In some embodiments, the PASTE has editing efficiency comparable to or superior to integration based on homology-directed repair or non-homologous end joining, has activity in non-dividing cells, and fewer detectable off-target events.

In some embodiments, the exogenous agent and/or heterologous protein is or encodes one or more polypeptides having an activity selected from the group consisting of nuclease activity (e.g., programmable nuclease activity), nicking enzyme activity (e.g., programmable nicking enzyme activity), homing activity (e.g., programmable DNA binding activity), nucleic acid polymerase activity (e.g., DNA polymerase or RNA polymerase activity), integrase activity, recombinase activity, or base editing activity (e.g., cytidine deaminase or adenosine deaminase activity).

In some embodiments, delivery of the nuclease is by a provided vector (e.g., cas) encoding the nuclease.

In some embodiments, the provided lipid particles contain a nuclease protein, and the nuclease protein is delivered directly to the target cell. Methods of delivering nuclease proteins include, for example, those described in Cai et al Elife, 2014, 3:e01911 and international patent publication No. WO 2017068077. For example, the provided lipid particles comprise one or more Cas proteins, such as Cas9. In some embodiments, a nuclease protein (e.g., cas, such as Cas 9) is engineered as a chimeric nuclease protein with a viral structural protein (e.g., GAG) for packaging into a lipid particle (e.g., lentiviral vector particle, VLP, or gesicle). For example, a chimeric Cas9 protein fused to a structural GAG protein may be packaged within a lipid particle. In some embodiments, the fusion protein is a cleavable fusion protein between (i) a viral structural protein (e.g., GAG) and (ii) a nuclease protein (e.g., cas protein, such as Cas 9). In some embodiments, the fusion protein is a cleavable fusion protein between (i) a viral Matrix (MA) protein and (ii) a nuclease protein (e.g., a Cas protein, such as Cas 9). In some embodiments, the particle contains a nuclease protein (e.g., a Cas protein, such as Cas 9) immediately downstream of the gag start codon.

In some embodiments, provided lipid particles contain mRNA encoding a Cas nuclease (e.g., cas 9). In some embodiments, the provided lipid particles contain a guide RNA (gRNA), such as a single guide RNA (sgRNA).

In some embodiments, the dCas9 version of the CRISPR/Cas9 system can be used to target protein domains for transcriptional regulation, epigenetic modification, and microscopic visualization of specific genomic loci.

In some embodiments, provided viral particles (e.g., lentiviral particles) containing a Cas nuclease (e.g., cas 9) further comprise or are further complexed with one or more CRISPR-Cas system guide RNAs to target a desired target gene. In some embodiments, the CRISPR guide RNA is effectively encapsulated in a viral particle comprising CAS. In some embodiments, provided viral particles (e.g., lentiviral particles) further comprise or are further complexed with a targeting nucleic acid.

4. Small molecules

In some embodiments, the exogenous agent comprises a small molecule, such as an ion (e.g., ca ²⁺、C1-、Fe²⁺), a carbohydrate, a lipid, an active oxygen species, an active nitrogen species, an isoprenoid, a signaling molecule, heme, a polypeptide cofactor, an electron-accepting compound, an electron-donating compound, a metabolite, a ligand, and any combination thereof. In some embodiments, the small molecule is a drug that interacts with a target in a cell. In some embodiments, the small molecule targets a protein in the cell for degradation. In some embodiments, the small molecule targets a protein in the cell to degrade by localizing the protein to the proteasome. In some embodiments, the small molecule is a proteolytically targeted chimeric molecule (PROTAC).

In some embodiments, the exogenous agent comprises a mixture of proteins, nucleic acids, or metabolites, such as a plurality of polypeptides, a plurality of nucleic acids, a plurality of small molecules, a combination of nucleic acids, polypeptides, and small molecules, a ribonucleoprotein complex (e.g., cas9-gRNA complex), a plurality of transcription factors, a plurality of epigenetic factors, reprogramming factors (e.g., oct4, sox2, cMyc, and Klf 4), a plurality of regulatory RNAs, and any combination thereof.

III pharmaceutical composition

Provided herein are compositions comprising the lipid particles herein, including lipid particles comprising a re-targeting attachment protein comprising (i) a paramyxovirus envelope attachment protein, and (ii) a targeting moiety for a first target molecule expressed on the surface of a target cell, and at least one paramyxovirus fusion protein. The pharmaceutical composition may comprise any of said lipid particles.

Also provided herein are compositions comprising any of the lipid particles described herein.

Also provided herein are compositions comprising the lipid particles herein, including lipid particles comprising a re-targeting attachment protein, the lipid particles comprising (a) a first paramyxovirus envelope attachment protein, and a first targeting moiety to a target molecule expressed on the surface of a target cell, (b) a second paramyxovirus envelope attachment protein, and a second targeting moiety to a target molecule expressed on the surface of a target cell, and (c) at least one paramyxovirus fusion protein.

Also provided herein are compositions comprising the lipid particles herein, including lipid particles comprising a re-targeting attachment protein, the lipid particles comprising (a) a first paramyxovirus envelope attachment protein, and a first targeting moiety directed against a target molecule expressed on the surface of a target cell, (b) a second paramyxovirus envelope attachment protein, and a second targeting moiety directed against a target molecule expressed on the surface of a target cell, (c) a third paramyxovirus envelope attachment protein, wherein the third paramyxovirus envelope attachment protein is a variant paramyxovirus envelope attachment protein comprising one or more mutations to reduce natural tropism relative to a wild-type paramyxovirus envelope attachment protein not comprising said one or more mutations, and (d) at least one paramyxovirus fusion protein.

Also provided herein are compositions comprising lipid particles herein, including lipid particles comprising a re-targeting attachment protein, the lipid particles comprising (a) a first paramyxovirus envelope attachment protein, and a first targeting moiety directed against a target molecule expressed on the surface of a target cell, (b) a second paramyxovirus envelope attachment protein, and a second targeting moiety directed against a target molecule expressed on the surface of a target cell, (c) a third paramyxovirus envelope attachment protein, wherein the third paramyxovirus envelope attachment protein is a variant paramyxovirus envelope attachment protein comprising one or more mutations to reduce natural tropism relative to a wild-type paramyxovirus envelope attachment protein not comprising said one or more mutations, and (d) at least one paramyxovirus fusion protein, and optionally one or more additional paramyxovirus envelope attachment proteins and one or more additional targeting moieties directed against a target molecule expressed on the surface of a target cell.

The pharmaceutical compositions provided herein may comprise any of the lipid particles.

In some aspects, the present disclosure also provides pharmaceutical compositions comprising the compositions described herein and a pharmaceutically acceptable carrier.

The term "pharmaceutical formulation" refers to a formulation in a form that allows for the biological activity of the active ingredient contained therein to be effective, and which does not contain additional components that have unacceptable toxicity to the subject to whom the formulation is to be administered.

By "pharmaceutically acceptable carrier" is meant an ingredient other than the active ingredient in a pharmaceutical formulation that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

In some aspects, the choice of carrier is determined in part by the particular lipid particle and/or by the method of administration. Thus, there are a variety of suitable formulations. For example, the pharmaceutical composition may contain a preservative. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride (benzalkonium chloride). In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of from about 0.0001% to about 2% by weight of the total composition. The vehicle is described, for example, by Remington's Pharmaceutical Sciences, 16 th edition, osol, a. Code (1980). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, hexamethyldiammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol, or benzyl alcohol, alkyl p-hydroxybenzoates such as methyl or propyl p-hydroxybenzoate, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, sugar, or sorbitol, salt forming ions such as sodium, metal complexes (e.g., zn-complexes), and non-ionic counter-surfactants such as PEG.

In some embodiments, the lipid particle meets the pharmaceutical or pharmaceutical manufacturing quality control practice (GMP) standard. In some embodiments, the lipid particles are prepared according to the Good Manufacturing Practice (GMP). In some embodiments, the lipid particle has a pathogen level below a predetermined reference value, e.g., is substantially free of pathogens. In some embodiments, the lipid particle has a contaminant level below a predetermined reference value, e.g., is substantially free of contaminants. In some embodiments, the lipid particle has low immunogenicity.

In some embodiments, the formulation of the pharmaceutical compositions described herein may be prepared by any method known in the pharmacological arts or developed hereafter. In some embodiments, the method of manufacture includes the steps of associating the active ingredient with a carrier or one or more other auxiliary ingredients and then shaping or packaging the product into the desired single or multi-dose unit, if necessary or desired.

In some embodiments, a "unit dose" is a discrete amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient. In some embodiments, the amount of active ingredient is generally equal to the dose of active ingredient to be administered to the subject or a convenient portion of such dose (e.g., one half or one third of such dose). In some embodiments, the unit dosage form may be used in a single daily dose or in one of a plurality of daily doses (e.g., about 1 to 4 or more times per day). In some embodiments, when multiple daily doses are used, the unit dosage form may be the same or different for each dose.

In some embodiments, the lipid particle containing the variant NiV-G is a viral vector or a virus-like particle (e.g., section III). In some embodiments, the compositions provided herein can be formulated in dosage units of Genomic Copies (GC). Suitable Methods for determining GC are described and include, for example, qPCR or digital droplet PCR (ddPCR), as described, for example, in M.Lock et al, hu GENE THERAPY Methods, hum Gene Ther Methods (2): 115-25.2014, which is incorporated herein by reference. In some embodiments, the viral vector or virus-like particle is administered at a dose of about 10 ⁴ to about 10 ¹⁰ GC units, inclusive. In some embodiments, the viral vector or virus-like particle is administered at a dose of about 10 ⁹ to about 10 ¹⁵ GC units, inclusive. In some embodiments, the viral vector or virus-like particle is administered at a dose of about 10 ⁵ to about 10 ⁹ GC units, inclusive. In some embodiments, the viral vector or virus-like particle is administered at a dose of about 10 ⁶ to about 10 ⁹ GC units, inclusive. In some embodiments, the viral vector or virus-like particle is administered at a dose of about 10 ¹² to about 10 ¹⁴ GC units, inclusive. In some embodiments, the administered dose is 1.0X10 ⁹ GC units, 5.0X10 ⁹ GC units, 1.0X10 ¹⁰ GC units, 5.0X10 ¹⁰ GC units, 1.0X10 ¹¹ GC units, 5.0X10 ¹¹ GC units, 1.0X10 ¹² GC units, 5.0X10 ¹² GC units, Or 1.0X10 ¹³ GC units, 5.0X10 ¹³ GC units, 1.0X10 ¹⁴ GC units, 5.0X10 ¹⁴ GC units or 1.0X10 ¹⁵ GC units.

In some embodiments, the viral vector or virus-like particle is administered at a dose of about 10 ⁴ to about 10 ¹⁰ infectious units, inclusive. In some embodiments, the viral vector or virus-like particle is administered at a dose of about 10 ⁹ to about 10 ¹⁵ infectious units, inclusive. In some embodiments, the viral vector or virus-like particle is administered at a dose of about 10 ⁵ to about 10 ⁹ infectious units. In some embodiments, the viral vector or virus-like particle is administered at a dose of about 10 ⁶ to about 10 ⁹ infectious units. In some embodiments, the viral vector or virus-like particle is administered at a dose of about 10 ¹² to about 10 ¹⁴ infectious units, inclusive. In some embodiments, the dosage is administered in a range of 1.0X10 ⁹ units ^、5.0×10⁹ units, 1.0X10 ¹⁰ units, 5.0X10 ¹⁰ units, 1.0X10 ¹¹ units, 5.0X10 ¹¹ units, 1.0X10 ¹² units, 5.0X10 ¹² units, or 1.0X10 ¹³ units, 5.0X10 ¹³ units, 1.0X10 ¹⁴ units, 5.0X10 ¹⁴ units or 1.0X10 ¹⁵ units. Techniques that can be used to quantify the infectious units are conventional in the art and include viral particle count assays, fluorescence microscopy, and plaque titer assays. For example, the number of adenovirus particles can be determined by measuring the absorbance of a 260. Similarly, the infectious units can also be determined by quantitative immunofluorescence of vector-specific proteins using monoclonal antibodies or by plaque assay.

In some embodiments, the method of calculating the infection unit comprises a plaque assay, wherein titration of the virus is performed on a cell monolayer, and the number of plaques is counted after several days to several weeks. For example, the infection titer is determined, such as by a plaque assay, e.g., an assay that evaluates cytopathic effect (CPE). In some embodiments, CPE assay is performed by serial dilution of virus on agarose covered monolayer cells (such as HFF cells). After a period of incubation to achieve a cytopathic effect, such as about 3to 28 days, typically 7 to 10 days, the cells can be fixed and foci of missing cells that appear as plaques can be determined. In some embodiments, the unit of infection may be determined using the end point dilution (TCID 50) method, which determines the viral dilution at which 50% of the cell culture is infected, and thus, generally, a range of titers, such as one log, may be determined.

In some embodiments, the viral vector or virus-like particle is administered at a dose of about 10 ⁴ to about 10 ¹⁰ plaque forming units (pfu), inclusive. In some embodiments, the viral vector or virus-like particle is administered at a dose of about 10 ⁹ to about 10 ¹⁵ pfu, inclusive. In some embodiments, the viral vector or virus-like particle is administered at a dose of about 10 ⁵ to about 10 ⁹ pfu. In some embodiments, the viral vector or virus-like particle is administered at a dose of about 10 ⁶ to about 10 ⁹ pfu. In some embodiments, the viral vector or virus-like particle is administered at a dose of about 1012 to about 10 ¹⁴ pfu, inclusive. In some embodiments, the dose administered is 1.0×10⁹ pfu、5.0×10⁹ pfu、1.0×10¹⁰ pfu、5.0×10¹⁰pfu、1.0×10¹¹ pfu、5.0×10¹¹ pfu、1.0×10¹² pfu、5.0×10¹² pfu, or 1.0X10 ¹³ pfu、5.0×10¹³ pfu、1.0×10¹⁴ pfu、5.0×10¹⁴ pfu or 1.0X10 ¹⁵ pfu.

In some embodiments, the subject will receive a single injection. In some embodiments, the administration may be repeated at daily/weekly/monthly intervals for an indefinite period of time and/or until the efficacy of the treatment has been determined. As described herein, treatment efficacy may be determined by assessing the symptoms and clinical parameters described herein and/or by detecting a desired response.

The exact amount of lipid particles provided by the desired carrier will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the particular polynucleic acid, polypeptide or carrier used, its mode of administration, and the like. The appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation with the teachings presented herein.

In some embodiments, the compositions are provided in the form of a sterile liquid formulation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which in some aspects may be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Furthermore, the liquid composition is more convenient to administer, in particular by injection. On the other hand, the adhesive composition may be formulated within a suitable viscosity range to provide longer contact times with specific tissues. The liquid or viscous composition may comprise a carrier, which may be a solvent or dispersion medium containing, for example, water, brine, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

Sterile injectable solutions may be prepared by incorporating the lipid particles in a solvent, such as with a suitable carrier, diluent or excipient, such as sterile water, physiological saline, dextrose and the like. The composition may also be lyophilized. Depending on the route of administration and the desired formulation, the composition may contain auxiliary substances such as wetting, dispersing or emulsifying agents (e.g. methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavouring agents, colouring agents and the like. In some aspects, reference may be made to standard text for preparing suitable formulations.

The injectable formulation may be prepared in conventional form (liquid solutions or suspensions), solid form of suspension suitable for dissolution in a liquid prior to injection, or as an emulsion. As used herein, "parenteral administration" includes intradermal, intranasal, subcutaneous, intramuscular, intraperitoneal, intravenous and intratracheal routes, as well as sustained release or sustained release systems such that a constant dose is maintained.

Various additives may be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffering agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, and the like). The absorption of injectable pharmaceutical forms may be prolonged by the use of delayed absorption agents, such as aluminum monostearate and gelatin.

Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

In some embodiments, the vehicle formulation may comprise an antifreeze agent. As used herein, the term "antifreeze" refers to one or more agents that, when combined with a given substance, help reduce or eliminate damage to the substance upon freezing. In some embodiments, cryoprotectants are combined with carrier vehicles to stabilize them during freezing. In some aspects, cryopreserving RNA between-20 ℃ and-80 ℃ may be advantageous for long-term (e.g., 36 months) stability of the polynucleotide. In some embodiments, the RNA species is mRNA. In some embodiments, cryoprotectants are included in the vehicle formulation to stabilize the polynucleotide through freeze/thaw cycles and under frozen storage conditions. The cryoprotectant of the provided embodiments may include, but is not limited to, sucrose, trehalose, lactose, glycerol, dextrose, raffinose, and/or mannitol. Trehalose is listed by the U.S. food and drug administration (Food and Drug Administration) as a generally recognized safe substance (GRAS) and is commonly used in commercial pharmaceutical formulations.

Formulations to be used for in vivo administration are typically sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

IV method of use

In some embodiments, the lipid particles provided herein or pharmaceutical compositions containing the lipid particles can be administered to a subject, e.g., a mammal, e.g., a human. In such embodiments, the subject may be at risk for a particular disease or disorder, may have symptoms of a particular disease or disorder, or may be diagnosed or identified as having a particular disease or disorder. In one embodiment, the subject has cancer. In one embodiment, the subject has an infectious disease. In some embodiments, the lipid particle contains a nucleic acid sequence encoding an exogenous agent for treating a disease or disorder in a subject. For example, the exogenous agent is a neoplastic cell-targeted protein or an exogenous agent specific thereto, and the lipid particle is administered to the subject to treat the tumor or cancer of the subject. In another example, the exogenous agent is an inflammatory mediator or immune molecule, such as a cytokine, and the lipid particle is administered to the subject to treat any disorder in which it is desirable to modulate (e.g., increase) an immune response, such as cancer or an infectious disease. In some embodiments, the lipid particles are administered in an amount or dose effective to effect treatment of a disease, condition, or disorder. Provided herein are uses of any provided lipid particle in such methods and treatments, and in the manufacture of a medicament to perform such methods of treatment. In some embodiments, the method is performed by administering a lipid particle or a composition comprising the lipid particle to a subject suffering from, having suffered from, or suspected of suffering from the disease or condition or disorder. In some embodiments, the method thereby treats a disease or condition or disorder in a subject. Also provided herein is the use of any composition (such as the pharmaceutical compositions provided herein) for treating a disease, condition, or disorder associated with a particular gene or protein targeted or provided by an exogenous agent.

In some embodiments, the provided methods or uses relate to administration of a pharmaceutical composition, including oral, inhalation, transdermal or parenteral (including intravenous, intratumoral, intraperitoneal, intramuscular, intracavity, intranodal and subcutaneous) administration. In some embodiments, the lipid particles may be administered alone or formulated as a pharmaceutical composition. In some embodiments, the lipid particles or compositions described herein may be administered to a subject, e.g., a mammal, e.g., a human. In some any embodiment, the subject may be at risk for a particular disease or disorder (e.g., a disease or disorder described herein), may have symptoms of a particular disease or disorder (e.g., a disease or disorder described herein), or may be diagnosed or identified as having a particular disease or disorder (e.g., a disease or disorder described herein). In some embodiments, the disease is a disease or disorder.

In some embodiments, the lipid particles may be administered in the form of a unit dose composition, such as a unit dose oral, parenteral, transdermal or inhalation composition. In some embodiments, the compositions are prepared by mixing and are suitable for oral, inhalation, transdermal or parenteral administration, and thus may be in the form of tablets, capsules, oral liquid preparations, powders, granules, lozenges, reconstitutable powders, injectable and infusable solutions or suspensions or suppositories or aerosols.

In some embodiments, the administration regimen may affect the composition of the effective amount. In some embodiments, the therapeutic formulation may be administered to the subject either before or after disease diagnosis. In some embodiments, several divided doses and staggered doses may be administered daily or sequentially, or doses may be infused continuously, or may be bolus injections. In some embodiments, the dosage of the therapeutic formulation may be increased or decreased proportionally as indicated by the degree of urgency of the therapeutic or prophylactic condition.

In some embodiments, administration of the compositions of the invention to a subject (preferably a mammal, more preferably a human) may be performed using known procedures at dosages and for periods of time effective to prevent or treat the disease. In some embodiments, the effective amount of therapeutic compound necessary to achieve a therapeutic effect may vary depending on factors such as the activity of the particular compound used, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the state, age, sex, weight, condition, general health and past medical history of the disease or disorder in the subject being treated, and like factors well known in the medical arts. In some embodiments, the dosage regimen may be adjusted to provide the optimal therapeutic response. In some embodiments, several divided doses may be administered daily, or the doses may be proportionally reduced as indicated by the urgency of the treatment situation. In some embodiments, an effective dose range of the therapeutic compounds of the present invention is about 1 to 5,000 mg/kg body weight/day. One of ordinary skill in the art will be able to study the relevant factors and determine the effective amount of the therapeutic compound without undue experimentation.

In some embodiments, the compound may be administered to the subject several times daily, or it may be administered less frequently, such as once daily, once weekly, once every two weeks, once monthly, or even less frequently (such as once every several months or even once annually or less frequently). In some embodiments, in non-limiting examples, the amount of the compound administered daily may be administered daily, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. In some embodiments, for every other day, a dose of 5 mg/day may be started on monday, a first subsequent dose of 5 mg/day on wednesday, a second subsequent dose of 5 mg/day on friday, and so on. The frequency of dosage will be apparent to those skilled in the art and will depend on many factors such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, and the like.

In some embodiments, the dosage level of the active ingredient in the pharmaceutical compositions of the present invention may be varied in order to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, while being non-toxic to the subject.

A physician (e.g., a physician or veterinarian) of ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. In some embodiments, the physician or veterinarian may begin the dosage of the compound of the invention employed in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In some embodiments, it is particularly advantageous to formulate the compounds in dosage unit form for ease of administration and uniformity of dosage. In some embodiments, dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for subjects to be treated, each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect, in association with the desired pharmaceutical vehicle. In some embodiments, the dosage unit form of the present invention is specified by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) limitations inherent in the art of formulating/formulating such therapeutic compounds for use in treating a disease in a subject.

In some embodiments, the term "container" includes any receptacle for holding a pharmaceutical composition. In some embodiments, the container is a package containing the pharmaceutical composition. In other embodiments, the container is not a package containing the pharmaceutical composition, i.e., the container is a receptacle, such as a box or vial containing the packaged pharmaceutical composition or the unpackaged pharmaceutical composition and instructions for use of the pharmaceutical composition. It will be appreciated that instructions for use of the pharmaceutical composition may be contained on a package containing the pharmaceutical composition and that the instructions thus form an increased functional relationship with the packaged product. In some embodiments, the instructions may include information regarding the ability of the compound to perform its intended function (e.g., treating or preventing a disease in a subject or delivering an imaging agent or diagnostic agent to a subject).

In some embodiments, routes of administration of any of the compositions disclosed herein include oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (per) buccal, (per) urethral, vaginal (e.g., vaginal and perivaginal), nasal (intra) and (per) rectal), intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation and topical administration.

In some of any of the embodiments, suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, lozenges, dispersions, suspensions, solutions, syrups, particles, beads, transdermal patches, gels, powders, pellets, emulsions, lozenges, creams, pastes, plaster, lotions, raw tablets, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration, and the like.

In some embodiments, lipid particle compositions comprising exogenous agents or cargo may be used to deliver such exogenous agents or cargo to a tissue or subject. In some embodiments, the cellular protein expression level can be altered by administering a lipid particle composition described herein to deliver cargo. In certain embodiments, the administered composition directs up-regulation (by expression in a cell, delivery in a cell, or induction within a cell) of one or more cargo (e.g., polypeptide or mRNA) that provides functional activity that is substantially absent or reduced in the cell in which the polypeptide is delivered. In some embodiments, the deleted functional activity may be enzymatic activity, structural activity, or regulatory activity in nature. In some embodiments, the administered composition directs up-regulation of one or more polypeptides that (e.g., synergistically) increase the functional activity present but substantially absent in the cells in which the polypeptide is up-regulated. In some of any of the embodiments, the administered composition directs down-regulation (by expression in, delivery in, or induction within a cell) of one or more cargo (e.g., polypeptide, siRNA, or miRNA) that inhibits functional activity present or up-regulated in the cell delivering the polypeptide, siRNA, or miRNA. In some of any of the embodiments, the functional activity that is up-regulated may be enzymatic activity, structural activity, or regulatory activity in nature. In some embodiments, the administered composition directs down-regulation of one or more polypeptides that (e.g., synergistically) reduce the functional activity present or up-regulated in the cells in which the polypeptide is down-regulated. In some embodiments, the composition administered directs the up-regulation of certain functional activities and down-regulation of other functional activities.

In some of any of the embodiments, the lipid particle composition (e.g., a lipid particle composition comprising mitochondria or DNA) mediates an effect on the target cells, and the effect lasts for at least 1, 2,3, 4, 5, 6, or 7 days, 2,3, or 4 weeks, or 1, 2,3, 6, or 12 months. In some embodiments (e.g., wherein the lipid particle composition comprises an exogenous protein), the effect lasts less than 1, 2,3, 4, 5, 6, or 7 days, 2,3, or 4 weeks, or 1, 2,3, 6, or 12 months.

In some of any of the embodiments, the lipid particle composition described herein is delivered ex vivo to a cell or tissue, such as a human cell or tissue. In embodiments, the composition improves a function of an ex vivo cell or tissue, such as improving cell viability, respiration, or other function (e.g., another function described herein).

In some embodiments, the composition is delivered to an ex vivo tissue in a damaged state (e.g., from a wound, disease, hypoxia, ischemia, or other damage).

In some embodiments, the composition is delivered to an ex vivo graft (e.g., a tissue explant or tissue for transplantation, e.g., a human vein, a musculoskeletal graft such as bone or tendon, cornea, skin, heart valve, nerve, or an isolated or cultured organ, e.g., an organ to be transplanted into a human, e.g., a human heart, liver, lung, kidney, pancreas, intestine, thymus, eye). In some embodiments, the composition is delivered to the tissue or organ before, during, and/or after transplantation.

In some embodiments, the composition is delivered with, administered with, or contacted with a cell (e.g., a cell preparation). In some embodiments, the cell preparation may be a cell therapeutic preparation (a cell preparation intended for administration to a human subject). In embodiments, the cell preparation comprises cells that express a Chimeric Antigen Receptor (CAR) (e.g., express a recombinant CAR). The CAR-expressing cells can be, for example, T cells, natural Killer (NK) cells, cytotoxic T Lymphocytes (CTLs), regulatory T cells. In embodiments, the cell preparation is a neural stem cell preparation. In embodiments, the cell formulation is a Mesenchymal Stem Cell (MSC) formulation. In embodiments, the cell formulation is a Hematopoietic Stem Cell (HSC) formulation. In embodiments, the cell formulation is an islet cell formulation.

In some embodiments, the lipid particle compositions described herein may be administered to a subject, e.g., a mammal, e.g., a human. In such embodiments, the subject may be at risk for a particular disease or disorder (e.g., a disease or disorder described herein), may have symptoms of a particular disease or disorder (e.g., a disease or disorder described herein), or may be diagnosed or identified as having a particular disease or disorder (e.g., a disease or disorder described herein).

In some embodiments, the source of the lipid particle is from the same subject to whom the lipid particle composition is administered. In other embodiments, they are different. In some embodiments, the source of the lipid particle and the recipient tissue may be autologous (from the same subject) or heterologous (from a different subject). In some embodiments, the donor tissue of the lipid particle compositions described herein can be a different tissue type than the recipient tissue. In some embodiments, the donor tissue may be muscle tissue and the recipient tissue may be connective tissue (e.g., adipose tissue). In other embodiments, the donor tissue and the recipient tissue may be of the same or different types, but from different organ systems.

In some embodiments, the lipid particle compositions described herein can be administered to a subject having cancer, an autoimmune disease, an infectious disease, a metabolic disease, a neurodegenerative disease, or a genetic disease (e.g., enzyme deficiency). In some embodiments, the subject is in need of regeneration.

In some embodiments, the lipid particle is co-administered with an inhibitor of membrane fusion of the protein. For example, inhibin (Suppressyn) is a human protein that inhibits cell-cell fusion (Sugimoto et al , "A novel human endogenous retroviral protein inhibits cell-cell fusion" Scientific Reports 3: 1462 (DOI: 10.1038/srep01462))., in some embodiments, lipid particles are co-administered with inhibitors of inhibin (e.g., siRNA or inhibitory antibodies).

In some embodiments, provided herein are also methods of mobilizing hematopoietic cells (such as hematopoietic stem cells) in connection with the provided methods of delivering particles (e.g., lentiviral particles), including particles comprising an exogenous agent for a target cell. In some embodiments, the target cells targeted for delivery by the provided particles (e.g., lentiviral particles) are Hematopoietic Stem Cells (HSCs). In some embodiments, the delivery methods comprise administering an mobilizing agent, e.g., a mobilization regimen, to the subject, and administering particles (e.g., lentiviral particles) to the subject according to the provided methods. In some embodiments, the mobilizing agent is administered to the subject prior to introducing or administering the particles (e.g., lentiviral particles) to the subject. In some embodiments, the mobilizing agent comprises a mobilizing regimen that renders the therapeutically inaccessible hematopoietic cells therapeutically accessible. In some embodiments, the mobilizing agent increases the number of hematopoietic cells in the peripheral blood, allowing for a more accessible source of hematopoietic cells targeted by the particles (e.g., lentiviral particles) according to the described methods. In some embodiments, the mobilizing agent stimulates mobilization of bone marrow cells from the subject's bone marrow to the peripheral blood.

In some embodiments, provided herein are also methods involving administering to a subject (i) an agent that stimulates bone marrow cell mobilization from the subject's bone marrow to peripheral blood and (II) particles (e.g., any of the particles described in section II), with or without the use of any of the targeting agents described herein.

In some embodiments, the mobilizing agent increases the number of stem cells in peripheral blood, allowing a more accessible source of stem cells for use in the described methods. In some embodiments, the mobilizing agent increases the number of hematopoietic cells in the circulation of the subject. In some embodiments, the mobilizing agent is a mobilizer of hematopoietic stem cells or progenitor cells. In some embodiments, the mobilizing agent induces hematopoietic cells to leave bone marrow.

In some embodiments, the hematopoietic cells are cd34+ and may include cd34+ progenitor cells. In some embodiments, the hematopoietic cells are HSCs. In some aspects, the mobilizing agent is a stem cell mobilizing agent.

As used herein, "mobilizing" and "mobilizing hematopoietic cells" are used interchangeably to refer to the act of inducing the migration of hematopoietic cells (such as cd34+ cells, including progenitor and/or hematopoietic stem cells) from a first location (e.g., stem cell niche, e.g., bone marrow) to a second location (e.g., tissue (e.g., peripheral blood) or organ (e.g., spleen)). In some embodiments, the process of mobilizing hematopoietic cells involves recruiting stem cells from their resident tissue or organ to the peripheral blood after treatment with a mobilizing agent, such as using mobilizing agents known to those skilled in the art, including any cytokines and chemotherapeutic drugs (e.g., G-CSF) known in the art for this purpose. In some aspects, the process mimics the physiological release of stem cells from tissues or organs in response to stress signals during injury and inflammation. In some embodiments, the one or more mobilizing agents act as agonists or antagonists that prevent the attachment of hematopoietic cells to the cells or tissues of their microenvironment. In some embodiments, the one or more mobilizing agents induce release of proteases that cleave adhesion molecules or support structures between the hematopoietic cells and their attachment sites. In some embodiments, the mobilizing agent is capable of mobilizing any hematopoietic cells, such as stem cells and/or progenitor cells, wherein the heparan sulfate proteoglycans are responsible for maintaining the adhesion of the cells in their cell niches. In one aspect, a method of mobilizing hematopoietic cells in a subject comprises administering to the subject an effective amount of an agent that inhibits the level or activity of heparan sulfate proteoglycans, thereby mobilizing the hematopoietic cells in the subject.

In some embodiments, the mobilizing agent increases circulation of hematopoietic cells and/or mobilizes hematopoietic cells sequestered in bone marrow away from the bone marrow into their accessible compartments, e.g., accessible for transduction by lipid particles and/or viral vectors. For example, administration of mobilization therapy to a subject can increase circulation of hematopoietic cells and/or mobilize hematopoietic cells sequestered in bone marrow from the bone marrow into compartments that they can access, such as peripheral blood.

In some embodiments, the mobilizing agent is administered prior to the particle (e.g., lentiviral particle or other viral particle). In some embodiments, the mobilizing agent is administered to the subject within 7 days prior to administration of the particle (e.g., lentiviral particle), such as within 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day prior to administration of the particle (e.g., lentiviral particle). In some embodiments, the mobilizing agent is administered twice daily, once daily, or twice or three times a week. In some embodiments, the mobilizing agent is administered once a day for several consecutive days prior to administration of the particle (e.g., lentiviral particle). In some embodiments, at least one dose of the mobilizing agent is administered to the subject on the same day as the provided method of contacting the target cells with the particles (e.g., lentiviral particles). In some embodiments, at least one dose of mobilizing agent is administered to the subject within 12 hours prior to administration of the lipid particle (e.g., lentiviral vector), such as within 10 hours, 8 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or 1 hour prior to administration of the particle (e.g., lentiviral particle).

Exemplary mobilizing agents include Stem Cell Factor (SCF), small molecule VLA-4 inhibitor BI05192, BOP (N- (benzenesulfonyl) -L-prolyl-L-0- (1-pyrrolidinylcarbonyl) tyrosine), heparin, granulocyte colony-stimulating factor (G-CSF), and plexafu (also known as AMD 3100). In some embodiments, the mobilizing agent of the method is selected from the group consisting of granulocyte-macrophage colony-stimulating factor (GM-CSF), fms-associated tyrosine kinase 3 (flt-3) ligand, stromal cell derived factor 1 (SDF-1), agonists of chemokine (C-C motif) receptor 1 (CCR 1) such as chemokine (C-C motif) ligand 3 (CCL 3, also known as macrophage inflammatory protein-1α (MIP-1α)), agonists of chemokine (C-X-C motif) receptor 1 (CXCR 1) and CXCR2 such as chemokine (C-X-C motif) ligand (CXCL 1), CXCL2 (also known as growth-associated oncogene protein- β (Gro- β)) and CXCL8 (also known as interleukin-8 (IL-8)), agonists of 4 such as CTCE-002, ATI-2341 and Met-SDF-1, very Late Antigen (VLA) -4, TG-4, and AMD-00565, and any combination thereof.

In some embodiments, the mobilizing agent is Stem Cell Factor (SCF), small molecule VLA-4 inhibitor BI05192, BOP (N- (benzenesulfonyl) -L-prolyl-L-0- (1-pyrrolidinylcarbonyl) tyrosine), heparin, granulocyte colony-stimulating factor (G-CSF), or plexafu (AMD 3100). In some embodiments, the mobilizing agent comprises a combination of G-CSF and pleshafu (AMD 3100). In some of any of the embodiments, the G-CSF is non-grastim (e.g., neupogen or Zarzio). In some embodiments, the G-CSF is a pegylated G-CSF, such as polyethylene glycol fegrastim (e.g., neulasta).

Any of a variety of known methods for mobilizing hematopoietic cells using mobilizing agents may be used, including, but not limited to, PCT publication No. WO2021211450, U.S. publication Nos. US20200268850 and US20170106021, and any of those described in U.S. patent Nos. 7,939,057 and 10,907,177.

In some embodiments, the mobilization regimen comprises administration of at least one mobilization agent. In various embodiments, (i) four days before administration of the first dose of particles (e.g., lentiviral particles), (ii) the day of administration of the first dose of particles (e.g., lentiviral particles), and (iii) the day of administration of one or more subsequent doses of particles (e.g., lentiviral particles), at least one mobilizing agent is administered to the subject. In some embodiments, the at least one mobilizing agent is administered to the subject (i) the day prior to administration of the first dose of particles (e.g., lentiviral particles) and (ii) the day of administration of the first dose of particles (e.g., lentiviral particles).

In some embodiments, the mobilization regimen comprises administration of one or both of G-CSF and pleshafu/AMD 3100. In various embodiments, (i) four days before administration of the first dose of particles (e.g., lentiviral particles), (ii) the day of administration of the first dose of particles (e.g., lentiviral particles), and (iii) the day of administration of one or more subsequent doses of particles (e.g., lentiviral particles), G-CSF is administered to the subject. In various embodiments, the subject is administered pleshafu/AMD 3100 (i) the day prior to administration of the first dose of particles (e.g., lentiviral particles) and (ii) the day of administration of the first dose of particles (e.g., lentiviral particles).

In some embodiments, at least one mobilizing agent is administered once daily at a dose equal to or at least 0.1, 1.0, 10, 20, 30, 40, 50, 75, 100, 150, or 200 μg/kg. In various embodiments, the daily dosage of at least one mobilizing agent ranges from a lower limit of 0.1 μg/kg/day, 1.0 μg/kg/day, 10 μg/kg/day, 20 μg/kg/day, 30 μg/kg/day, 40 μg/kg/day, 50 μg/kg/day, or 75 μg/kg/day and an upper limit of 100 μg/kg/day, 150 μg/kg/day, or 200 μg/kg/day. In various embodiments, at least one mobilizing agent is administered once daily at a dose equal to or at least 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 15 mg/kg, or 20 mg/kg. In various embodiments, the daily dosage of the at least one stem cell mobilizing agent ranges from a lower limit of 1 mg/kg/day, 2 mg/kg/day, 3 mg/kg/day, 4 mg/kg/day, 5 mg/kg/day, or 7.5 mg/kg/day and an upper limit of 10 mg/kg/day, 15 mg/kg/day, or 20 mg/kg/day.

In various embodiments, G-CSF is administered once daily at a dose equal to or at least 10 μg/kg, 20 μg/kg, 30 μg/kg, 40 μg/kg, 50 μg/kg, 75 μg/kg, 100 μg/kg, 150 μg/kg, or 200 μg/kg. In various embodiments, the daily dosage of G-CSF ranges from a lower limit of 10 μg/kg/day, 20 μg/kg/day, 30 μg/kg/day, 40 μg/kg/day, 50 μg/kg/day or 75 μg/kg/day and an upper limit of 100 μg/kg/day, 150 μg/kg/day or 200 μg/kg/day. In various embodiments, the pleshafu/AMD 3100 is administered once daily at a dose equal to or at least 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 15 mg/kg, or 20 mg/kg. In various embodiments, the daily dosage of G-CSF ranges from a lower limit of 1 mg/kg/day, 2 mg/kg/day, 3 mg/kg/day, 4 mg/kg/day, 5 mg/kg/day, or 7.5 mg/kg/day and an upper limit of 10 mg/kg/day, 15 mg/kg/day, or 20 mg/kg/day.

In some embodiments, G-CSF may be administered at a dose of 0.5-16 μg/kg (e.g., 5-16 μg/kg or 10-16 μg/kg) per day for 1-10 days (e.g., 1-7 days, or specifically 1-3 days). In another example, G-CSF may be administered to healthy donors at a dose of 10-16 μg/kg per day for up to seven days. When peripheral blood collection is combined with apheresis starting from, for example, day 4, three or four days of treatment may be sufficient. In another example, G-CSF may be administered at a dose of 10 μg/kg per day for four days, with apheresis starting on, for example, day 4. The most common dose of G-CSF in healthy donors is 10 μg/kg body weight per day, with white blood cell isolation starting on day 5 until a sufficient number of stem cells are collected (e.g., collection can be performed once or twice a day for 1 to 4 days, such as 1 or 2 days). G-CSF may be administered subcutaneously or intravenously. Methods and dosages of G-CSF administration are described in Juttner et al (Blood 89:2233-2258, 1997), kroschinsky et al (Haemallogic 90:1556-1671, 2005), U.S. Pat. No. 6,162,427, 2005/0186182, WO 2010051335, and WO 2005014023, all of which are incorporated herein by reference in their entirety. If cyclophosphamide is administered for mobilization, G-CSF typically begins 2-5 days after cyclophosphamide infusion is complete. Methods of administering a combination mobilizing agent of G-CSF and one or more chemotherapeutic agents are described in Andre et al (transfusions 43:50-57, 2003), ataergin et al (Am. J. Hematol. 83:644-648, 2008) and Demirer et al (Br. J. Haemaol. 116:468-474, 2002), all of which are incorporated herein by reference in their entirety.

Pleshafu can be administered at a dose of 1-300 μg/kg. At 240 μg/kg, the number of mobilized stem cells peaked about 4-10 hours after administration of pleshafu. In some examples, the pleshafu can be administered at a dose of about 1-300 μg/kg (e.g., 100-300 μg/kg or 200-300 μg/kg) once or twice daily for 1-10 days (e.g., 1-5 days).

The combination of pleshafu and G-CSF for stem cell mobilization was FDA approved in 2008 for patients with non-hodgkin's lymphoma and multiple myeloma. The combination therapy may also be used to mobilize stem cells. A typical combination therapy may include, for example, administration of about 0.5-16 μg/kg (e.g., 10 μg/kg) of G-CSF per day, with administration of 1-300 μg/kg (e.g., 240 μg/kg) of pleshafu a few days (e.g., 1-3 days) after administration of G-CSF. The two doses may be administered together for about 2-10 days (e.g., 4 days) or until sufficient hematopoietic cells are collected. Pleshafu can be administered subcutaneously or intravenously alone or in combination with G-CSF.

In some embodiments, a method of mobilizing hematopoietic cells in a subject comprises administering to the subject at least one mobilizing agent. In some embodiments, a method of mobilizing hematopoietic cells comprises administering to a subject at least one mobilizing agent comprising (i) at least one heparan sulfate inhibitor and (ii) at least one of a CXCR2 agonist and a CXCR4 antagonist.

In some embodiments, the mobilized hematopoietic cells include KLS-CD150+CD48-cells. In some embodiments, the mobilized hematopoietic cells include CD 34-CD133+ cells. In some embodiments, the mobilized hematopoietic cells and/or progenitor cells include common myeloid progenitor cells. In some embodiments, the mobilized hematopoietic cells and/or progenitor cells include granulocyte/monocyte progenitor cells. In some embodiments, the mobilized hematopoietic cells and/or progenitor cells include megakaryocyte/erythrocyte progenitor cells. In some embodiments, the mobilized hematopoietic cells and/or progenitor cells include committed lymphoid progenitor cells. In some embodiments, the mobilized hematopoietic cells and/or progenitor cells include a combination of common myeloid progenitor cells, granulocyte/monocyte progenitor cells, megakaryocyte/erythrocyte progenitor cells. In some embodiments, the hematopoietic progenitor cells comprise CD 150-CD 48-CD244+ cells. In some embodiments, the hematopoietic progenitor cells comprise CD 150-CD48+CD244+ cells. In some embodiments, the hematopoietic progenitor cells comprise Lin-SCA-1-c-kit+CD34+CD16/32 mid cells. In some embodiments, the hematopoietic progenitor cells comprise lin-SCA-1-c-kit+CD 34-CD 16/32low cells. In some embodiments, the mobilized hematopoietic cells include cd34+ peripheral blood stem cells.

V. exemplary embodiments

Exemplary embodiment A

1. A lipid particle comprising (a) a re-targeting attachment protein comprising a paramyxovirus envelope attachment protein linked to (i) a first targeting moiety directed against a first target molecule expressed on the surface of a target cell and (ii) a second targeting moiety directed against a second target molecule expressed on the surface of a target cell, and (b) at least one paramyxovirus fusion (F) protein, and wherein the proteins in (a) and (b) are exposed outside of a lipid bilayer.

2. The lipid particle of embodiment 1, wherein the paramyxovirus attachment protein is a variant paramyxovirus envelope attachment protein comprising one or more mutations to reduce natural tropism relative to a wild-type paramyxovirus envelope attachment protein not comprising said one or more mutations.

3. The lipid particle of embodiment 1 or 2, wherein the paramyxovirus envelope attachment protein is a first paramyxovirus envelope attachment protein, and the lipid particle further comprises a second paramyxovirus envelope attachment protein, wherein the second paramyxovirus envelope attachment protein is a variant paramyxovirus envelope attachment protein comprising one or more mutations to reduce natural tropism relative to the wild-type paramyxovirus envelope attachment protein not comprising the one or more mutations, wherein the second paramyxovirus envelope attachment protein is exposed on the outer side of the lipid bilayer.

4. The lipid particle of any one of embodiments 1-3, wherein targeting one or both of the first target molecule and the second target molecule does not activate or inhibit the target cell, induce a phenotypic change (e.g., maturation and/or differentiation) of the target cell, induce proliferation of the target cell, and/or induce apoptosis of the target cell.

5. A lipid particle comprising (a) a re-targeting attachment protein comprising a first paramyxovirus envelope attachment protein operably linked to (i) a first targeting moiety directed against a first target molecule expressed on the surface of a target cell, and (ii) a second targeting moiety directed against a second target molecule expressed on the surface of a target cell, (b) a second paramyxovirus envelope attachment protein, the second paramyxovirus envelope attachment protein being a variant paramyxovirus envelope attachment protein comprising one or more mutations to reduce natural tropism relative to a wild-type paramyxovirus envelope attachment protein not comprising the one or more mutations, and (c) at least one paramyxovirus fusion (F) protein, and wherein the proteins in (a), (b) and (c) are exposed on the outside of a lipid bilayer.

6. The lipid particle of embodiments 1-5, wherein the first paramyxovirus envelope attachment protein is a variant paramyxovirus envelope attachment protein.

7. The lipid particle of embodiment 6, wherein the variant paramyxovirus envelope attachment protein comprises one or more mutations that reduce natural tropism relative to the wild-type paramyxovirus envelope attachment protein that does not comprise the one or more mutations.

8. The lipid particle of any one of embodiments 1-8, wherein the first paramyxovirus envelope attachment protein and the second paramyxovirus envelope attachment protein are the same.

9. The lipid particle of any one of embodiments 1-8, wherein the second paramyxovirus envelope attachment protein is not linked or fused to a non-viral heterologous moiety, the non-viral heterologous moiety being a cell-specific targeting domain or a functional domain.

10. The lipid particle of embodiments 1-9, wherein neither the first targeting moiety nor the second targeting moiety is selected from the group consisting of cytokines, growth factors, hormones, neurotransmitters, apoptotic ligands, and combinations thereof.

11. The lipid particle of any one of embodiments 1-10, wherein targeting one or both of the first target molecule and the second target molecule does not modulate or induce a signal in the target cell.

12. The lipid particle of any one of embodiments 1-11, wherein the first targeting moiety and the second targeting moiety each bind a cell surface molecule present on a target cell.

13. The lipid particle of any one of embodiments 1-11, wherein the first targeting moiety binds to a cell surface molecule present on a first target cell and the second targeting moiety binds to a surface molecule present on a second target cell.

14. The lipid particle of any one of embodiments 1-13, wherein the first target molecule and the second target molecule are different target molecules.

15. The lipid particle of any one of embodiments 1 to 14, wherein the first target molecule and the second target molecule are the same target molecule.

16. The lipid particle of embodiment 15, wherein the first targeting moiety and the second targeting moiety bind different epitopes of the same target molecule.

17. The lipid particle of any one of embodiments 1 to 16, wherein the cell surface molecule is a protein, glycan, or lipid.

18. The lipid particle of any one of embodiments 1-17, wherein the target cell is selected from the group consisting of tumor infiltrating lymphocytes, T cells, neoplastic or tumor cells, virus-infected cells, stem cells, central Nervous System (CNS) cells, hematopoietic Stem Cells (HSCs), and liver cells.

19. The lipid particle of any one of embodiments 1-18, wherein the target cell is selected from the group consisting of a cd3+ T cell, a cd4+ T cell, a cd8+ T cell, a liver cell, a hematopoietic stem cell, a cd34+ hematopoietic stem cell, a cd105+ hematopoietic stem cell, a cd117+ hematopoietic stem cell, a cd105+ endothelial cell, a B cell, a cd20+ B cell, a cd19+ B cell, a cancer cell, a cd133+ cancer cell, an epcam+ cancer cell, a cd19+ cancer cell, a Her2/neu+ cancer cell, a glua2+ neuron, a glua4+ neuron, a nkg2d+ natural killer cell, a SLC1a3+ astrocyte, a SLC7a10+ adipocyte, or a cd30+ lung epithelial cell.

20. The lipid particle of any one of embodiments 1-19, wherein the target cell is a hepatocyte.

21. The lipid particle of embodiment 20, wherein the cell surface molecule is selected from the group consisting of CD34, CD117, and CD133.

22. The lipid particle of any one of embodiments 1-19, wherein the target cell is a T cell.

23. The lipid particle of embodiment 22, wherein the cell surface molecule is selected from the group consisting of CD3, CD4, CD7, CD8, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, and ITGA3.

24. A lipid particle comprising (a) a first re-targeting attachment protein comprising (i) a first paramyxovirus envelope attachment protein, and (ii) a first targeting moiety for CD133, and a second targeting moiety for CD133, and (b) at least one paramyxovirus fusion (F) protein, and the proteins in (a) and (b) being exposed outside of a lipid bilayer.

25. The lipid particle of any one of embodiments 1 to 24, wherein (i) the first targeting moiety is a scFv and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 516, 525, 534, 543, and 552 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and/or (ii) the second targeting moiety is a scFv and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 516, 525, 534, 543, and 552 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

26. The lipid particle of any one of embodiments 1 to 24, wherein (i) the first targeting moiety comprises (a) a CD133 binding agent comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO 536, 537 and 538, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NO 540, 541 and 542, respectively, (b) a CD133 binding agent comprising a polypeptide comprising SEQ ID NO 545, respectively, 546 and 547, and CDR-L1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS 549, 550 and 551, respectively, (c) a CD133 binding agent comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS 518, 519 and 520, respectively, and CDR-L1 comprising the amino acid sequences of SEQ ID NOS 522, 523 and 524, respectively, CDR-L2 and CDR-L3, (d) CD133 binders comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO: 527, 528 and 529, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NO: 531, 532 and 533, respectively, or (e) CD133 binders comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO: 554, 555 and 556, respectively, and comprising SEQ ID NO: 558, respectively, 559 and 560, and/or (ii) the second targeting moiety comprises (a) a CD133 binding agent comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS 536, 537 and 538, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NOS 540, 541 and 542, respectively, (b) a CD133 binding agent comprising a polypeptide comprising SEQ ID NOS 545, respectively, 546 and 547, and CDR-L1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS 549, 550 and 551, respectively, (c) a CD133 binding agent comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS 518, 519 and 520, respectively, and CDR-L1 comprising the amino acid sequences of SEQ ID NOS 522, 523 and 524, respectively, CDR-L2 and CDR-L3, (d) CD133 binders comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO: 527, 528 and 529, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NO: 531, 532 and 533, respectively, or (e) CD133 binders comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO: 554, 555 and 556, respectively, and comprising SEQ ID NO: 558, respectively, 559 and 560, CDR-L1, CDR-L2 and CDR-L3.

27. The lipid particle of any one of embodiments 1 to 24, wherein (i) the first targeting moiety comprises (a) a CD133 binding agent comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS 289, 565 and 538, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NOS 540, 541 and 542, respectively, (b) a CD133 binding agent comprising a polypeptide comprising SEQ ID NOS 566, respectively, 567 and 547, and CDR-L1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOs 549, 550 and 551, respectively, (c) a CD133 binding agent comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOs 568, 569 and 520, respectively, and CDR-L1 comprising the amino acid sequences of SEQ ID NOs 522, 523 and 524, respectively, CDR-L2 and CDR-L3, (d) CD133 binders comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO: 570, 571 and 529, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NO: 531, 532 and 533, respectively, or (e) CD133 binders comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO: 572, 573 and 556, respectively, and comprising SEQ ID NO: 558, respectively, 559 and 560, and/or (ii) the second targeting moiety comprises (a) a CD133 binding agent comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS 289, 565 and 538, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NOS 540, 541 and 542, respectively, (b) a CD133 binding agent comprising a polypeptide comprising SEQ ID NOS 566, respectively, 567 and 547, and CDR-L1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOs 549, 550 and 551, respectively, (c) a CD133 binding agent comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOs 568, 569 and 520, respectively, and CDR-L1 comprising the amino acid sequences of SEQ ID NOs 522, 523 and 524, respectively, CDR-L2 and CDR-L3, (d) CD133 binders comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO: 570, 571 and 529, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NO: 531, 532 and 533, respectively, or (e) CD133 binders comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO: 572, 573 and 556, respectively, and comprising SEQ ID NO: 558, respectively, 559 and 560, CDR-L1, CDR-L2 and CDR-L3.

28. The lipid particle of any one of embodiments 1 to 24, wherein (i) the first targeting moiety comprises (a) a CD133 binding agent comprising a heavy chain Variable (VH) region comprising the amino acid sequence of SEQ ID NO: 535 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and an amino acid sequence comprising or having at least 90%, 91%, 92%, 93%, 94%, 95% sequence identity thereto of SEQ ID NO: 539, A light chain Variable (VL) region of an amino acid sequence of 96%, 97%, 98% or 99% sequence identity; (b) a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO 544 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO 548 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (c) a CD133 binding agent comprising an amino acid sequence comprising SEQ ID NO 517 or an amino acid sequence having at least 90%, a polypeptide sequence having at least one of the amino acid sequences of SEQ ID NO 517 and a polypeptide sequence having at least one of the amino acid sequences of SEQ ID NO and the polypeptide sequence of SEQ ID NO, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO. 521, and a VL region comprising the amino acid sequence of SEQ ID NO. 521 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (d) a CD133 binding agent comprising an amino acid sequence comprising SEQ ID NO. 526 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94% thereof, A VH region comprising an amino acid sequence of 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO. 530 or a VL region comprising an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or (e) a CD133 binding agent comprising a VH region comprising an amino acid sequence of SEQ ID NO. 553 or an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a VH region comprising an amino acid sequence of SEQ ID NO. 557 or an amino acid sequence of at least 90%, a CD133 binding agent comprising a VH region comprising an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID No. 535, and/or (ii) the second targeting moiety comprises (a) a CD133 binding agent comprising a heavy chain Variable (VH) region comprising the amino acid sequence of SEQ ID No. 535 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a light chain Variable (VH) region comprising the amino acid sequence of SEQ ID No. 539 or an amino acid sequence having at least 90%, a light chain Variable (VH) sequence having at least 90% or a light chain Variable (VH) sequence having at least one or more amino acid sequences selected from the group consisting of SEQ ID NOs, A light chain Variable (VL) region comprising an amino acid sequence of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, (b) a CD133 binding agent comprising a VH region comprising an amino acid sequence of SEQ ID NO: 544 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and an amino acid sequence of SEQ ID NO: 548 or an amino acid sequence having at least 90%, 91%, 92%, 93% sequence identity thereto, A VL region comprising an amino acid sequence of 94%, 95%, 96%, 97%, 98% or 99% sequence identity, (c) a CD133 binding agent comprising a VH region comprising an amino acid sequence of SEQ ID NO 517 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a VL region comprising an amino acid sequence of SEQ ID NO 521 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, A VL region of an amino acid sequence of 98% or 99% sequence identity; (d) a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 526 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a VL region comprising the amino acid sequence of SEQ ID NO: 530 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or (e) a CD133 binding agent comprising an amino acid sequence comprising SEQ ID NO: 553 or a VL region having at least 90%, a, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and a VL region comprising the amino acid sequence of SEQ ID NO: 557 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

29. The lipid particle of any one of embodiments 1-28, wherein (a) the first targeting moiety comprises a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO 517 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and (b) the second targeting moiety comprises a CD133 binding agent comprising the amino acid sequence of SEQ ID NO 526 or a VH region comprising the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

30. The lipid particle of any one of embodiments 1-28, wherein (a) the first targeting moiety comprises a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID No. 526 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and (b) the second targeting moiety comprises a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID No. 517 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

31. The lipid particle of any one of embodiments 1-30, wherein the first targeting moiety and the second targeting moiety bind different epitopes on CD 133.

32. A lipid particle comprising (a) a first re-targeting attachment protein comprising (i) a first paramyxovirus envelope attachment protein, and (ii) a first targeting moiety for CD117, and a second targeting moiety for CD117, and (b) at least one paramyxovirus fusion (F) protein, and the proteins in (a), (b) being exposed outside of a lipid bilayer.

33. The lipid particle of embodiments 1-23 and 32, wherein (a) the first targeting moiety comprises a CD117 binding agent comprising a VHH single domain antibody comprising CDR-H1, CDR-H2 and CDR-H3 comprised within the amino acid sequence selected from the group consisting of SEQ ID NOs 512-515, and/or wherein (b) the second targeting moiety comprises a CD117 binding agent comprising a VHH single domain antibody comprising CDR-H1, CDR-H2 and CDR-H3 comprised within the amino acid sequence selected from the group consisting of SEQ ID NOs 512-515.

34. The lipid particle of any one of embodiments 1-23 and 32-33, wherein

(A) The first targeting moiety comprises a VHH single domain antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 512-515 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and/or (b) the second targeting moiety comprises a VHH single domain antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 512-515 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

35. The lipid particle of embodiment 33 or embodiment 34, wherein (a) the first targeting moiety comprises a VHH comprising an amino acid sequence set forth in any one of SEQ ID NOS: 512-515, and/or (b) the second targeting moiety comprises a VHH comprising an amino acid sequence set forth in any one of SEQ ID NOS: 512-515.

36. The lipid particle of any one of embodiments 1 to 23 and 32 to 35, wherein the first targeting moiety and the second targeting moiety bind different epitopes on CD 117.

37. A lipid particle comprising (a) a first re-targeting attachment protein comprising (i) a first paramyxovirus envelope attachment protein, and (ii) a first targeting moiety for CD8, and a second targeting moiety for CD8, and (b) at least one paramyxovirus fusion (F) protein, and the proteins in (a) and (b) being exposed outside of a lipid bilayer.

A lipid particle comprising (a) a first re-targeted attachment protein comprising (i) an amino acid sequence selected from the group consisting of SEQ ID NO 584 or 585, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and (b) at least one paramyxovirus fusion (F) protein, and wherein said proteins in (a), (b) are exposed outside of the lipid bilayer.

A lipid particle comprising (a) a first re-targeting attachment protein comprising (i) an amino acid sequence selected from SEQ ID NO 584 or 585, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) at least one paramyxovirus fusion (F) protein, and wherein said proteins of (a), (b) are exposed outside the lipid bilayer, and (c) a second paramyxovirus envelope attachment protein, said second paramyxovirus envelope attachment protein being a variant paramyxovirus envelope attachment protein comprising one or more amino acid substitutions to reduce natural tropism relative to a wild-type paramyxovirus envelope attachment protein not comprising said one or more amino acid substitutions, optionally wherein said second paramyxovirus envelope attachment protein as a variant paramyxovirus envelope attachment protein comprises one or more amino acid substitutions corresponding to amino acid number A, Q of SEQ ID NO 501 and amino acid number 3962 of group F-A-number 533.

38. The lipid particle of embodiment 37 or 37b or 37c, wherein (i) the first targeting moiety is an scFv and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 489, 496, 501 or 508 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and/or (ii) the second targeting moiety is an scFv and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 489, 496, 501 or 508 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

39. The lipid particle of embodiments 1-23 and 37 or 37c, wherein (a) the first targeting moiety comprises a CD8 binding agent comprising a VHH single domain antibody comprising CDR-H1, CDR-H2 and CDR-H3 comprised within the amino acid sequence of SEQ ID NO: 377, and/or wherein (b) the second targeting moiety comprises a CD8 binding agent comprising a VHH single domain antibody comprising CDR-H1, CDR-H2 and CDR-H3 comprised within the amino acid sequence of SEQ ID NO: 377.

40. The lipid particle of embodiment 37 or 37b or 37c or 39, wherein (a) the first targeting moiety comprises a VHH comprising the amino acid sequence shown in SEQ ID NO: 377 and/or (b) the second targeting moiety comprises a VHH comprising the amino acid sequence shown in SEQ ID NO: 377.

41. The lipid particle of any one of embodiments 1 to 23 and 37 to 40, wherein the first targeting moiety and the second targeting moiety bind different epitopes on CD 8.

42. The lipid particle of any one of embodiments 1-30, further comprising a second paramyxovirus envelope attachment protein, said second paramyxovirus envelope attachment protein being a variant paramyxovirus envelope attachment protein comprising one or more mutations, said variant paramyxovirus envelope attachment protein reducing natural tropism relative to said wild-type paramyxovirus envelope attachment protein not comprising said one or more mutations.

43. The lipid particle of any one of embodiments 16 to 42, wherein the different epitopes are non-overlapping.

44. The lipid particle of any one of embodiments 16 to 43, wherein the first targeting moiety and the second targeting moiety bind the different epitopes in a non-competitive manner.

45. The lipid particle of embodiments 1-44, wherein each of the first targeting moiety and the second targeting moiety is independently selected from the group consisting of an antibody or antigen binding fragment, DARPin, aptamer, affimer, affibody, desmin, avimer, monomer, ANTICALIN, FYNOMER, and targeting peptide.

46. The lipid particle of any one of embodiments 1-45, wherein the first targeting moiety and the second targeting moiety are independently selected from the group consisting of a single domain antibody or a single chain variable fragment (scFv).

47. A lipid particle comprising (a) a re-targeting attachment protein comprising (i) a first paramyxovirus envelope attachment protein, and (ii) a first targeting moiety and a second targeting moiety for CD133, (b) a second paramyxovirus envelope attachment protein, which is a variant paramyxovirus envelope attachment protein comprising one or more mutations, which reduces natural tropism relative to a wild-type paramyxovirus envelope attachment protein not comprising said one or more mutations, and (c) at least one paramyxovirus fusion (F) protein, and wherein the proteins in (a), (b) and (c) are exposed outside of the lipid bilayer.

48. A lipid particle comprising (a) a re-targeting attachment protein comprising (i) a first paramyxovirus envelope attachment protein, and (ii) a first targeting moiety and a second targeting moiety for CD117, (b) a second paramyxovirus envelope attachment protein, said second paramyxovirus envelope attachment protein being a variant paramyxovirus envelope attachment protein comprising one or more mutations, said variant paramyxovirus envelope attachment protein reducing natural tropism relative to a wild-type paramyxovirus envelope attachment protein not comprising said one or more mutations, and (c) at least one paramyxovirus fusion (F) protein, and wherein said proteins in (a), (b) and (c) are exposed outside of the lipid bilayer.

49. A lipid particle comprising (a) a re-targeting attachment protein comprising (i) a first paramyxovirus envelope attachment protein, and (ii) a first targeting moiety and a second targeting moiety for CD8, (b) a second paramyxovirus envelope attachment protein, said second paramyxovirus envelope attachment protein being a variant paramyxovirus envelope attachment protein comprising one or more mutations, said variant paramyxovirus envelope attachment protein reducing natural tropism relative to a wild-type paramyxovirus envelope attachment protein not comprising said one or more mutations, and (c) at least one paramyxovirus fusion (F) protein, and wherein said proteins in (a), (b) and (c) are exposed outside of the lipid bilayer.

50. The lipid particle of embodiments 24-49, wherein the first paramyxovirus envelope attachment protein is a variant paramyxovirus envelope attachment protein.

51. The lipid particle of embodiment 50, wherein the variant paramyxovirus envelope attachment protein comprises one or more mutations that reduce natural tropism relative to a wild-type paramyxovirus envelope attachment protein that does not comprise the one or more mutations.

52. The lipid particle of any one of embodiments 24-51, wherein the second paramyxovirus envelope attachment protein is a variant paramyxovirus envelope attachment protein.

53. The lipid particle of embodiment 52, wherein the variant paramyxovirus envelope attachment protein comprises one or more mutations that reduce natural tropism relative to a wild-type paramyxovirus envelope attachment protein that does not comprise the one or more mutations.

54. The lipid particle of any one of embodiments 1-53, wherein the first targeting moiety is selected from the group consisting of a single domain antibody or a single chain variable fragment (scFv).

55. The lipid particle of any one of embodiments 1-54, wherein the second targeting moiety is selected from the group consisting of a single domain antibody or a single chain variable fragment (scFv).

56. The lipid particle of embodiments 46 and 54 or 55, wherein the single domain antibody is a VHH.

57. The lipid particle of any one of embodiments 1-56, wherein the first variant paramyxovirus envelope attachment protein and the second variant paramyxovirus envelope attachment protein are the same.

58. The lipid particle of any one of embodiments 1-57, wherein the first paramyxovirus envelope attachment protein and the second paramyxovirus envelope attachment protein are different.

59. The lipid particle of any one of embodiments 1 to 58, wherein the first paramyxovirus envelope attachment protein is an envelope attachment protein from a nipah virus, a hendra virus, or a measles virus, or a variant of any one of the foregoing or a biologically active portion thereof.

60. The lipid particle of any one of embodiments 1-59, wherein the first paramyxovirus envelope attachment protein is a wild-type paramyxovirus G protein, H protein or HN protein, or a variant or biologically active portion of any one of the foregoing.

61. The lipid particle of embodiment 59 or embodiment 60, wherein the first paramyxovirus envelope attachment protein is a wild-type nipah virus G (NiV-G) protein, or is a variant or biologically active portion of NiV-G.

62. The lipid particle of any one of embodiments 59-61, wherein the first paramyxovirus envelope attachment protein is a variant NiV-G, which is a variant or biologically active portion of wild-type NiV-G.

63. The lipid particle of any one of embodiments 1-62, wherein the second paramyxovirus envelope attachment protein is an envelope attachment protein from a nipah virus, a hendra virus, or a measles virus, or is a variant or biologically active portion of any one of the foregoing.

64. The lipid particle of any one of embodiments 1-63, wherein the second paramyxovirus envelope attachment protein is a wild-type paramyxovirus G protein, H protein or HN protein, or a variant or biologically active portion of any one of the foregoing.

65. The lipid particle of embodiment 63 or embodiment 64, wherein the second paramyxovirus envelope attachment protein is a wild-type nipah virus G (NiV-G) protein, or is a variant or biologically active portion of NiV-G.

66. The lipid particle of any one of embodiments 1 to 65, wherein the second paramyxovirus envelope attachment protein is a variant NiV-G, which is a variant or biologically active portion of wild-type NiV-G.

67. The lipid particle of any one of embodiments 3-62, wherein the second paramyxovirus envelope attachment protein is a variant paramyxovirus envelope glycoprotein from nipah virus, hendra virus, or measles virus, or a biologically active portion thereof.

68. The lipid particle of any one of embodiments 3-62 and 67, wherein the second paramyxovirus envelope attachment protein is a variant of a wild-type paramyxovirus G protein, H protein or HN protein or a biologically active portion thereof.

69. The lipid particle of any one of embodiments 65 and 66, wherein the variant is a variant NiV-G that is a variant of a wild-type nipah virus G (NiV-G) protein or a biologically active portion thereof.

70. The lipid particle of embodiment 62, embodiment 66, or embodiment 69, wherein the variant NiV-G is truncated by up to 40 consecutive amino acids at or near the N-terminus of the wild-type NiV-G shown in SEQ ID No. 1.

71. The lipid particle of any one of embodiments 62, 66, 69 and 70, wherein the variant NiV-G has a truncation of amino acids 2-34 of the wild-type NiV-G shown in SEQ ID No. 1.

72. The lipid particle of any one of embodiments 62, 65 and 69-71, wherein the variant NiV-G exhibits reduced binding to ephrin B2 or ephrin B3.

73. The lipid particle of embodiment 72, wherein the variant NiV-G comprises one or more amino acid substitutions corresponding to amino acid substitutions numbered selected from the group consisting of E501A, W504, 504A, Q530A and E533A as set forth in reference to SEQ ID No. 1.

74. The lipid particle of embodiment 72 or embodiment 73, wherein the variant NiV-G comprises the numbered amino acid substitutions E501A, W504A, Q a and E533A as set forth in reference to SEQ ID No. 1.

75. The lipid particle of any one of embodiments 62, 66 and 69 to 74, wherein the variant NiV-G has the amino acid sequence shown in SEQ ID NO:228 or an amino acid sequence having equal or about 80%, at least equal or about 81%, at least equal or about 82%, at least equal or about 83%, equal or about 84%, at least equal or about 85%, at least equal or about 86%, or at least equal or about 87%, at least equal or about 88%, or at least equal or about 89%, at least equal or about 90%, at least equal or about 91%, at least equal or about 92%, at least equal or about 93%, at least equal or about 94%, at least equal or about 95%, equal or about 96%, at least equal or about 97%, at least equal or about 98%, or at least equal or about 99% sequence identity to SEQ ID NO: 228.

76. The lipid particle of any one of embodiments 62, 66 and 69-74, wherein the variant NiV-G has the amino acid sequence shown in SEQ ID No. 228.

77. The lipid particle of any one of embodiments 1-76, wherein the at least one paramyxovirus fusion (F) protein is an F protein from henipav, or a biologically active portion thereof or a variant thereof.

78. The lipid particle of embodiment 77, wherein the henipa virus is hendra virus.

79. The lipid particle of embodiment 77, wherein the henipa virus is a nipah virus.

80. The lipid particle of any one of embodiments 1-79, wherein the paramyxovirus F protein is a wild-type NiV-F protein or a variant or biologically active portion thereof.

81. The lipid particle of any one of embodiments 1-80, wherein the paramyxovirus F protein is a variant NiV-F, which is a variant or biologically active portion of a wild-type NiV-F protein.

82. The lipid particle of embodiment 81, wherein the variant NiV-F is truncated by up to 22 consecutive amino acids at the C-terminus of the wild-type NiV-F shown in SEQ ID No. 235, optionally excluding the initiating methionine.

83. The lipid particle of embodiment 81 or embodiment 82, wherein the variant NiV-F protein is a truncated NiV-F lacking amino acids 525-546 of SEQ ID No. 235.

84. The lipid particle of any one of embodiments 81-83, wherein the variant NiV-F has the amino acid sequence shown in SEQ ID NO:227 or an amino acid sequence having equal to or about 80%, at least equal to or about 81%, at least equal to or about 82%, at least equal to or about 83%, equal to or about 84%, at least equal to or about 85%, at least equal to or about 86%, or at least equal to or about 87%, at least equal to or about 88%, or at least equal to or about 89%, at least equal to or about 90%, at least equal to or about 91%, at least equal to or about 92%, at least equal to or about 93%, at least equal to or about 94%, at least equal to or about 95%, equal to or about 96%, at least equal to or about 97%, at least equal to or about 98%, or at least equal to or about 99% sequence identity to SEQ ID NO: 227.

85. The lipid particle of any one of embodiments 81 to 84, wherein the variant NiV-F has the amino acid sequence shown in SEQ ID No. 227.

86. The lipid particle of any one of embodiments 1-85, wherein the paramyxovirus F protein is an F0 precursor, or a proteolytically cleaved form thereof comprising F1 and F2 subunits.

87. The lipid particle of embodiment 86, wherein the proteolytically cleaved form is a cathepsin L cleavage product.

88. The lipid particle of any one of embodiments 1-87, wherein the paramyxovirus envelope attachment protein and the first targeting moiety and the second targeting moiety are linked via one or more linkers.

89. The lipid particle of embodiment 88, wherein the one or more linkers are one or more peptide linkers.

90. The lipid particle of any one of embodiments 1-89, wherein the re-targeting attachment protein comprises, in order, a paramyxovirus attachment protein-first linker-first targeting moiety-second linker-second targeting moiety.

91. The lipid particle of embodiment 90, wherein the first linker and the second linker are independently peptide linkers.

92. The lipid particle of embodiment 91, wherein the first linker and the second linker are the same.

93. The lipid particle of embodiment 91, wherein the first linker and the second linker are different.

94. The lipid particle of embodiment 89 or embodiment 91, wherein the peptide linker is 2 to 65 amino acids in length.

95. The lipid particle of any one of embodiments 91-94, wherein the peptide linker is a flexible linker comprising GS, GGS, GGGGS, GGGGGS or a combination thereof.

96. The lipid particle of any one of embodiments 91-95, wherein the peptide linker is selected from (GGS) n, wherein n is 1 to 10, (GGGGS) n, wherein n is 1 to 10, or (GGGGGS) n, wherein n is 1 to 6.

97. The lipid particle of any one of embodiments 1-94, wherein the peptide linker is selected from the group consisting of SEQ ID NOs 589-592.

98. The lipid particle of any one of embodiments 1-97, wherein the lipid particle further comprises one or more additional paramyxovirus envelope adhesion glycoproteins embedded in the lipid bilayer.

99. The lipid particle of embodiment 98, wherein the one or more additional paramyxovirus envelope attachment glycoproteins are re-targeted attachment proteins comprising a paramyxovirus envelope attachment protein and an additional targeting moiety.

100. The lipid particle of any one of embodiments 1-99, wherein the at least one paramyxovirus fusion (F) protein exhibits fusion activity with a target cell when at least one paramyxovirus envelope attachment protein binds to the target molecule on the target cell.

101. The lipid particle of any one of embodiments 1-100, wherein the lipid particle comprises a viral nucleic acid.

102. The lipid particle of embodiment 101, wherein the viral nucleic acid comprises one or more (e.g., all) of the following nucleic acid sequences, 5 'LTR (e.g., comprising U5 and lacking a functional U3 domain), psi packaging element (Psi), central polypurine tract (cPPT)/Central Termination Sequence (CTS) (e.g., DNA flap), poly-a tail sequence, post-transcriptional regulatory element (e.g., WPRE), rev Response Element (RRE), and 3' LTR (e.g., comprising U5 and lacking a functional U3).

103. The lipid particle of any one of embodiments 1-102, wherein the lipid particle is a viral vector.

104. The lipid particle of any one of embodiments 1-103, which is a retroviral vector.

105. The lipid particle of any one of embodiments 1-103, which is a lentiviral vector.

106. The lipid particle of any one of embodiments 1-102, wherein the lipid particle is free of viral genomic DNA.

107. The lipid particle of any one of embodiments 1 to 102 and 106, which is a virus-like particle.

108. The lipid particle of any one of embodiments 1 to 102, 106, and 107, which is a retrovirus-like particle.

109. The lipid particle of any one of embodiments 1 to 102, 106, and 107, which is a lentiviral-like particle.

110. The lipid particle of any one of embodiments 1-109, wherein the lipid particle is produced as a formulation with increased titer compared to a reference lipid particle formulation that is similarly produced but has only the first re-targeted attachment protein.

111. The lipid particle of embodiment 110, wherein the titer is increased equal to or greater than 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more.

112. The lipid particle of any one of embodiments 1-111, further comprising an exogenous agent for delivery to a target cell.

113. The lipid particle of embodiment 112, wherein the exogenous agent is present in the lumen.

114. The lipid particle of embodiment 112 or embodiment 113, wherein the exogenous agent is a protein or a nucleic acid, optionally wherein the nucleic acid is DNA or RNA.

115. The lipid particle of any one of embodiments 112-114, wherein the exogenous agent is a nucleic acid encoding a cargo for delivery to the target cell.

116. The lipid particle of any one of embodiments 112-115, wherein the exogenous agent is or encodes a therapeutic agent, a diagnostic agent, or a genome-modifying enzyme.

117. The lipid particle of any one of embodiments 112 to 116, wherein the exogenous agent encodes a membrane protein, optionally wherein the membrane protein is an antigen receptor for targeting a cell expressed by or associated with a disease or disorder.

118. The lipid particle of embodiment 117, wherein the membrane protein is a Chimeric Antigen Receptor (CAR).

119. The lipid particle of any one of embodiments 112 to 116, wherein the exogenous agent is a nucleic acid comprising a payload gene for correcting a genetic defect, optionally a genetic defect in the target cell, optionally wherein the genetic defect is associated with a liver cell or a hepatocyte.

120. The lipid particle of any one of embodiments 112 to 119, wherein binding of the paramyxovirus envelope attachment protein or biologically active portion thereof to a target molecule expressed on the surface of a target cell mediates fusion of the particle with the target cell and delivery of the exogenous agent to the target cell.

121. The lipid particle of any one of embodiments 112 to 120, wherein the exogenous agent is delivered to equal to or greater than 10%, 20%, 30%, 40%, 50%, 60% of the target cells.

122. The lipid particle of any one of embodiments 112-121, wherein delivery of the exogenous agent to the target cell is increased as compared to a reference particle formulation that is similarly produced but has only the first re-targeted attachment protein.

123. The lipid particle of embodiment 122, wherein the delivery to the target cell is increased by an amount equal to or greater than 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more.

124. A producer cell comprising (a) a nucleic acid encoding a re-targeting attachment protein comprising a paramyxovirus envelope attachment protein linked to (i) a first targeting moiety directed against a first target molecule expressed on the surface of a target cell and (ii) a second targeting moiety directed against a second target molecule expressed on the surface of a target cell, and (b) a nucleic acid encoding at least one paramyxovirus fusion (F) protein.

125. The production cell of embodiment 124, wherein the cell further comprises a viral nucleic acid.

126. The production cell of any one of embodiment 125 wherein the viral nucleic acid is a lentiviral nucleic acid.

127. The production cell of any one of embodiments 124-126, wherein the cell is a mammalian cell.

128. The production cell according to any one of embodiments 124-127, wherein the production cell is selected from the group consisting of CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, hepG2 cells, saos-2 cells, huh7 cells, heLa cells, W163 cells, 211 cells, and 211A cells.

129. The producer cell of any of embodiments 124-128, wherein the producer cell comprises a 293T cell.

130. The production cell of any one of embodiments 125-129 wherein the viral nucleic acid lacks one or more genes involved in viral replication.

131. The producer cell of any one of embodiments 125 to 130, wherein the viral nucleic acid comprises a nucleic acid encoding a viral packaging protein selected from one or more of Gag, pol, rev and Tat.

132. The producer cell of any one of embodiments 125-131, wherein the viral nucleic acid comprises one or more (e.g., all) of the following nucleic acid sequences, 5 'LTR (e.g., comprising U5 and lacking a functional U3 domain), psi packaging element (Psi), central polypurine tract (cPPT)/Central Termination Sequence (CTS) (e.g., DNA flap), poly-a tail sequence, post-transcriptional regulatory element (e.g., WPRE), rev Response Element (RRE), and 3' LTR (e.g., comprising U5 and lacking a functional U3).

133. A method of preparing a lipid particle comprising a) providing a production cell according to any one of embodiments 124 to 132, b) culturing the cell under conditions allowing production of the lipid particle, and c) isolating, enriching or purifying the lipid particle from the cell, thereby preparing the lipid particle.

134. The method of embodiment 133, wherein the lipid particle is a pseudotyped lentiviral vector.

135. A lipid particle produced by the method of embodiment 133 or embodiment 134.

136. A composition comprising a plurality of lipid particles of any one of embodiments 1 to 123 and 135.

137. The composition of embodiment 136, further comprising a pharmaceutically acceptable carrier.

138. A method of transducing a cell, the method comprising contacting the cell with the lipid particle of any one of embodiments 1 to 123 and 135 or the composition of embodiment 136 or embodiment 137.

139. A method of delivering an exogenous agent into a target cell, the method comprising contacting the lipid particle of any one of embodiments 112-123 and 135 or the composition of embodiment 136 or embodiment 137 with the target cell.

140. The method of embodiment 138 or embodiment 139, wherein the contacting is in vitro or ex vivo.

141. The method of embodiment 138 or embodiment 139, wherein the contacting is in the subject.

142. A method of delivering an exogenous agent to a cell of a subject, the method comprising administering to the subject the lipid particle of any one of embodiments 112-123 and 135 or the composition of embodiment 136 or embodiment 137.

143. The method of embodiment 142, wherein the exogenous agent is or encodes a therapeutic agent for treating a disease or disorder in the subject.

144. A method of treatment comprising administering to a subject the lipid particle of any one of embodiments 112-123 and 135 or the composition of embodiment 136 or embodiment 137.

145. The method of any one of embodiments 139-144, wherein the exogenous agent is or encodes a membrane protein, optionally a chimeric antigen receptor, for targeting an antigen associated with a disease or disorder in the subject.

146. The method of any one of embodiments 139-144, wherein the exogenous agent is used in gene therapy to correct a genetic defect or substitution defect or deleted gene in the subject.

147. The method of any one of embodiments 142-146, wherein the subject is a human subject.

148. The method of any one of embodiments 138 to 147, wherein the method further comprises administering to the subject one or more agents that stimulate mobilization of bone marrow cells from bone marrow to peripheral blood.

149. The method of any one of embodiments 138-148, wherein the subject has been previously administered one or more agents that stimulate bone marrow cell mobilization from bone marrow to peripheral blood.

150. The method of embodiment 148 or 149, wherein the one or more agents that stimulate mobilization are selected from the group consisting of Stem Cell Factor (SCF), small molecule VLA-4 inhibitor BI05192, BOP (N- (benzenesulfonyl) -L-prolyl-L-0- (1-pyrrolidinylcarbonyl) tyrosine), heparin, granulocyte colony-stimulating factor (G-CSF), MGTA-145, and plexafu (AMD 3100).

151. The method of any one of embodiments 148 to 150, wherein the one or more agents that stimulate mobilization comprises G-CSF.

152. The method of any one of embodiments 148 to 150, wherein the one or more agents that stimulate mobilization comprises plexafu.

Exemplary embodiment B

5. A lipid particle comprising (a) a re-targeting attachment protein comprising a first paramyxovirus envelope attachment protein operably linked to (i) a first targeting moiety directed against a first target molecule expressed on the surface of a target cell, and (ii) a second targeting moiety directed against a second target molecule expressed on the surface of a target cell, (b) a second paramyxovirus envelope attachment protein, the second paramyxovirus envelope attachment protein being a variant paramyxovirus envelope attachment protein comprising one or more amino acid substitutions to reduce natural tropism relative to a wild-type paramyxovirus envelope attachment protein not comprising the one or more amino acid substitutions, and (c) at least one paramyxovirus fusion (F) protein, and wherein the proteins in (a), (b) and (c) are exposed on the outside of a lipid bilayer.

6. The lipid particle of any one of embodiments 1 to 5, wherein the first paramyxovirus envelope attachment protein is a variant paramyxovirus envelope attachment protein.

7. The lipid particle of embodiment 6, wherein the variant paramyxovirus envelope attachment protein comprises one or more amino acid substitutions, reducing natural tropism relative to the wild-type paramyxovirus envelope attachment protein without said one or more amino acid substitutions.

8. The lipid particle of any one of embodiments 1-7, wherein the first paramyxovirus envelope attachment protein and the second paramyxovirus envelope attachment protein are the same.

9. The lipid particle of any one of embodiments 1-8, wherein the second paramyxovirus envelope attachment protein is not linked or fused to a non-viral heterologous moiety, the non-viral heterologous moiety being a cell-specific targeting domain or functional domain, optionally wherein the second paramyxovirus envelope attachment protein is not linked or fused to a cell-specific targeting domain, the cell-specific targeting domain being a targeting moiety directed against a target molecule expressed on the surface of a target cell.

13. The lipid particle of any one of embodiments 1-12, wherein the first targeting moiety binds to a cell surface molecule present on a first target cell and the second targeting moiety binds to a surface molecule present on a second target cell.

15. The lipid particle of any one of embodiments 1 to 13, wherein the first target molecule and the second target molecule are the same target molecule.

24. The lipid particle of any one of embodiments 5 to 23, wherein the second paramyxovirus envelope attachment protein as a variant paramyxovirus envelope attachment protein comprises one or more amino acid substitutions corresponding to the amino acid substitutions numbered selected from the group consisting of E501A, W, A, Q a and E533A as set forth in reference SEQ ID No. 1.

25. A lipid particle comprising (a) a first re-targeting attachment protein comprising (i) a first paramyxovirus envelope attachment protein, and (ii) a first targeting moiety for CD133, and a second targeting moiety for CD133, and (b) at least one paramyxovirus fusion (F) protein, and the proteins in (a) and (b) being exposed outside of a lipid bilayer.

26. The lipid particle of any one of embodiments 1 to 25, wherein (i) the first targeting moiety is a scFv and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 516, 525, 534, 543, and 552 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and/or (ii) the second targeting moiety is a scFv and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 516, 525, 534, 543, and 552 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

27. The lipid particle of any one of embodiments 1 to 25, wherein (i) the first targeting moiety comprises (a) a CD133 binding agent comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO 536, 537 and 538, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NO 540, 541 and 542, respectively, (b) a CD133 binding agent comprising a polypeptide comprising SEQ ID NO 545, respectively, 546 and 547, and CDR-L1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS 549, 550 and 551, respectively, (c) a CD133 binding agent comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS 518, 519 and 520, respectively, and CDR-L1 comprising the amino acid sequences of SEQ ID NOS 522, 523 and 524, respectively, CDR-L2 and CDR-L3, (d) CD133 binders comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO: 527, 528 and 529, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NO: 531, 532 and 533, respectively, or (e) CD133 binders comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO: 554, 555 and 556, respectively, and comprising SEQ ID NO: 558, respectively, 559 and 560, and/or (ii) the second targeting moiety comprises (a) a CD133 binding agent comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS 536, 537 and 538, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NOS 540, 541 and 542, respectively, (b) a CD133 binding agent comprising a polypeptide comprising SEQ ID NOS 545, respectively, 546 and 547, and CDR-L1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS 549, 550 and 551, respectively, (c) a CD133 binding agent comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS 518, 519 and 520, respectively, and CDR-L1 comprising the amino acid sequences of SEQ ID NOS 522, 523 and 524, respectively, CDR-L2 and CDR-L3, (d) CD133 binders comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO: 527, 528 and 529, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NO: 531, 532 and 533, respectively, or (e) CD133 binders comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO: 554, 555 and 556, respectively, and comprising SEQ ID NO: 558, respectively, 559 and 560, CDR-L1, CDR-L2 and CDR-L3.

28. The lipid particle of any one of embodiments 1 to 25, wherein (i) the first targeting moiety comprises (a) a CD133 binding agent comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS 289, 565 and 538, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NOS 540, 541 and 542, respectively, (b) a CD133 binding agent comprising a polypeptide comprising SEQ ID NOS 566, respectively, 567 and 547, and CDR-L1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOs 549, 550 and 551, respectively, (c) a CD133 binding agent comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOs 568, 569 and 520, respectively, and CDR-L1 comprising the amino acid sequences of SEQ ID NOs 522, 523 and 524, respectively, CDR-L2 and CDR-L3, (d) CD133 binders comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO: 570, 571 and 529, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NO: 531, 532 and 533, respectively, or (e) CD133 binders comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO: 572, 573 and 556, respectively, and comprising SEQ ID NO: 558, respectively, 559 and 560, and/or (ii) the second targeting moiety comprises (a) a CD133 binding agent comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOS 289, 565 and 538, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NOS 540, 541 and 542, respectively, (b) a CD133 binding agent comprising a polypeptide comprising SEQ ID NOS 566, respectively, 567 and 547, and CDR-L1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOs 549, 550 and 551, respectively, (c) a CD133 binding agent comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NOs 568, 569 and 520, respectively, and CDR-L1 comprising the amino acid sequences of SEQ ID NOs 522, 523 and 524, respectively, CDR-L2 and CDR-L3, (d) CD133 binders comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO: 570, 571 and 529, respectively, and CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NO: 531, 532 and 533, respectively, or (e) CD133 binders comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of SEQ ID NO: 572, 573 and 556, respectively, and comprising SEQ ID NO: 558, respectively, 559 and 560, CDR-L1, CDR-L2 and CDR-L3.

29. The lipid particle of any one of embodiments 1 to 25, wherein (i) the first targeting moiety comprises (a) a CD133 binding agent comprising a heavy chain Variable (VH) region comprising the amino acid sequence of SEQ ID NO 535 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and an amino acid sequence comprising or having at least 90%, 91%, 92%, 93%, 94%, 95% sequence identity thereto of SEQ ID NO 539, A light chain Variable (VL) region of an amino acid sequence of 96%, 97%, 98% or 99% sequence identity; (b) a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO 544 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; and a VL region comprising the amino acid sequence of SEQ ID NO 548 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (c) a CD133 binding agent comprising an amino acid sequence comprising SEQ ID NO 517 or an amino acid sequence having at least 90%, a polypeptide sequence having at least one of the amino acid sequences of SEQ ID NO 517 and a polypeptide sequence having at least one of the amino acid sequences of SEQ ID NO and the polypeptide sequence of SEQ ID NO, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO. 521, and a VL region comprising the amino acid sequence of SEQ ID NO. 521 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (d) a CD133 binding agent comprising an amino acid sequence comprising SEQ ID NO. 526 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94% thereof, A VH region comprising an amino acid sequence of 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO. 530 or a VL region comprising an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or (e) a CD133 binding agent comprising a VH region comprising an amino acid sequence of SEQ ID NO. 553 or an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a VH region comprising an amino acid sequence of SEQ ID NO. 557 or an amino acid sequence of at least 90%, a CD133 binding agent comprising a VH region comprising an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID No. 535, and/or (ii) the second targeting moiety comprises (a) a CD133 binding agent comprising a heavy chain Variable (VH) region comprising the amino acid sequence of SEQ ID No. 535 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a light chain Variable (VH) region comprising the amino acid sequence of SEQ ID No. 539 or an amino acid sequence having at least 90%, a light chain Variable (VH) sequence having at least 90% or a light chain Variable (VH) sequence having at least one or more amino acid sequences selected from the group consisting of SEQ ID NOs, A light chain Variable (VL) region comprising an amino acid sequence of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, (b) a CD133 binding agent comprising a VH region comprising an amino acid sequence of SEQ ID NO: 544 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and an amino acid sequence of SEQ ID NO: 548 or an amino acid sequence having at least 90%, 91%, 92%, 93% sequence identity thereto, A VL region comprising an amino acid sequence of 94%, 95%, 96%, 97%, 98% or 99% sequence identity, (c) a CD133 binding agent comprising a VH region comprising an amino acid sequence of SEQ ID NO 517 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a VL region comprising an amino acid sequence of SEQ ID NO 521 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, A VL region of an amino acid sequence of 98% or 99% sequence identity; (d) a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 526 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a VL region comprising the amino acid sequence of SEQ ID NO: 530 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or (e) a CD133 binding agent comprising an amino acid sequence comprising SEQ ID NO: 553 or a VL region having at least 90%, a, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and a VL region comprising the amino acid sequence of SEQ ID NO: 557 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

30. The lipid particle of any one of embodiments 1 to 29, wherein (a) the first targeting moiety comprises a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO 517 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and (b) the second targeting moiety comprises a CD133 binding agent comprising the amino acid sequence of SEQ ID NO 526 or a VH region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

31. The lipid particle of any one of embodiments 1 to 29, wherein (a) the first targeting moiety comprises a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 526 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and (b) the second targeting moiety comprises a CD133 binding agent comprising a VH region comprising the amino acid sequence of SEQ ID NO: 517 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

32. The lipid particle of any one of embodiments 1 to 31, wherein the first targeting moiety and the second targeting moiety bind different epitopes on CD 133.

33. The lipid particle of any one of embodiments 25-32, further comprising a second paramyxovirus envelope attachment protein, the second paramyxovirus envelope attachment protein being a variant paramyxovirus envelope attachment protein comprising one or more amino acid substitutions, to reduce natural tropism relative to the wild-type paramyxovirus envelope attachment protein not comprising the one or more amino acid substitutions.

34. The lipid particle of embodiment 33, wherein the second paramyxovirus envelope attachment protein as a variant paramyxovirus envelope attachment protein comprises one or more amino acid substitutions corresponding to the amino acid substitutions numbered selected from the group consisting of E501A, W504A, Q a and E533A as set forth in reference SEQ ID No. 1.

35. A lipid particle comprising (a) a first re-targeting attachment protein comprising (i) a first paramyxovirus envelope attachment protein, and (ii) a first targeting moiety for CD117, and a second targeting moiety for CD117, and (b) at least one paramyxovirus fusion (F) protein, and the proteins in (a), (b) being exposed outside of a lipid bilayer.

36. The lipid particle of embodiments 1 to 25 and 35, wherein (a) the first targeting moiety comprises a CD117 binding agent comprising a VHH single domain antibody comprising CDR-H1, CDR-H2 and CDR-H3 comprised within an amino acid sequence selected from the group consisting of SEQ ID NOS: 512-515, and/or wherein (b) the second targeting moiety comprises a CD117 binding agent comprising a VHH single domain antibody comprising CDR-H1, CDR-H2 and CDR-H3 comprised within an amino acid sequence selected from the group consisting of SEQ ID NOS: 512-515.

37. The lipid particle of any one of embodiments 1 to 25 and 35 to 36, wherein (a) the first targeting moiety comprises a VHH single domain antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 512-515 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and/or (b) the second targeting moiety comprises a VHH single domain antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 512-515 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

38. The lipid particle of embodiment 36 or embodiment 37, wherein (a) the first targeting moiety comprises a VHH comprising an amino acid sequence set forth in any one of SEQ ID NOS: 512-515, and/or (b) the second targeting moiety comprises a VHH comprising an amino acid sequence set forth in any one of SEQ ID NOS: 512-515.

39. The lipid particle of any one of embodiments 1-25 and 35-38, wherein the first targeting moiety and the second targeting moiety bind different epitopes on CD 117.

40. The lipid particle of any one of embodiments 35-39, further comprising a second paramyxovirus envelope attachment protein, said second paramyxovirus envelope attachment protein being a variant paramyxovirus envelope attachment protein comprising one or more amino acid substitutions, to reduce natural tropism relative to the wild-type paramyxovirus envelope attachment protein not comprising said one or more amino acid substitutions.

41. The lipid particle of embodiment 40, wherein the second paramyxovirus envelope attachment protein as a variant paramyxovirus envelope attachment protein comprises one or more amino acid substitutions corresponding to amino acid substitutions numbered selected from the group consisting of E501A, W504A, Q a and E533A as set forth in reference SEQ ID No. 1.

42. A lipid particle comprising (a) a first re-targeting attachment protein comprising (i) a first paramyxovirus envelope attachment protein, and (ii) a first targeting moiety for CD8, and a second targeting moiety for CD8, and (b) at least one paramyxovirus fusion (F) protein, and the proteins in (a) and (b) being exposed outside of a lipid bilayer.

43. The lipid particle of embodiment 42, wherein (i) the first targeting moiety is a scFv and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 489, 496, 501, or 508 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and/or (ii) the second targeting moiety is a scFv and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 489, 496, 501, or 508 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

44. The lipid particle of embodiments 1 to 25 and 42, wherein (a) the first targeting moiety comprises a CD8 binding agent comprising a VHH single domain antibody comprising CDR-H1, CDR-H2 and CDR-H3 comprised within the amino acid sequence of SEQ ID NO: 377, and/or wherein (b) the second targeting moiety comprises a CD8 binding agent comprising a VHH single domain antibody comprising CDR-H1, CDR-H2 and CDR-H3 comprised within the amino acid sequence of SEQ ID NO: 377.

45. The lipid particle of embodiments 42 or 44, wherein (a) the first targeting moiety comprises a VHH comprising the amino acid sequence shown in SEQ ID NO: 377 and/or (b) the second targeting moiety comprises a VHH comprising the amino acid sequence shown in SEQ ID NO: 377.

46. The lipid particle of any one of embodiments 42-45, wherein (a) the first targeting moiety comprises a VHH comprising the amino acid sequence shown as SEQ ID NO 377 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and/or (b) the second targeting moiety is a scFv and comprises the amino acid sequence selected from the group consisting of SEQ ID NO 501 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

47. The lipid particle of any one of embodiments 42-45, wherein (a) the first targeting moiety is a scFv and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 501 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and (b) the second targeting moiety comprises a VHH comprising the amino acid sequence shown as SEQ ID NO: 377 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

48. The lipid particle of any one of embodiments 1-24 and 42-47, wherein the first targeting moiety and the second targeting moiety bind different epitopes on CD 8.

49. The lipid particle of any one of embodiments 16 to 48, wherein the different epitopes are non-overlapping.

50. The lipid particle of any one of embodiments 16 to 49, wherein the first targeting moiety and the second targeting moiety bind the different epitopes in a non-competitive manner.

51. The lipid particle of embodiments 1-50, wherein each of the first targeting moiety and the second targeting moiety is independently selected from the group consisting of an antibody or antigen binding fragment, DARPin, aptamer, affimer, affibody, desmin, avimer, monomer, ANTICALIN, FYNOMER, and targeting peptide.

52. The lipid particle of any one of embodiments 1-51, wherein the first targeting moiety and the second targeting moiety are independently selected from the group consisting of a single domain antibody or a single chain variable fragment (scFv).

53. The lipid particle of any one of embodiments 42-52, further comprising a second paramyxovirus envelope attachment protein, the second paramyxovirus envelope attachment protein being a variant paramyxovirus envelope attachment protein comprising one or more amino acid substitutions, to reduce natural tropism relative to the wild-type paramyxovirus envelope attachment protein not comprising the one or more amino acid substitutions.

54. The lipid particle of embodiment 49, wherein the second paramyxovirus envelope attachment protein as a variant paramyxovirus envelope attachment protein comprises one or more amino acid substitutions corresponding to the amino acid substitutions numbered selected from the group consisting of E501A, W504A, Q a and E533A as set forth in reference SEQ ID No. 1.

55. A lipid particle comprising (a) a re-targeting attachment protein comprising (i) a first paramyxovirus envelope attachment protein, and (ii) a first targeting moiety and a second targeting moiety for CD133, (b) a second paramyxovirus envelope attachment protein, which is a variant paramyxovirus envelope attachment protein comprising one or more amino acid substitutions, which reduces natural tropism relative to a wild-type paramyxovirus envelope attachment protein not comprising said one or more amino acid substitutions, and (c) at least one paramyxovirus fusion (F) protein, and wherein the proteins in (a), (b) and (c) are exposed outside of the lipid bilayer.

56. A lipid particle comprising (a) a re-targeting attachment protein comprising (i) a first paramyxovirus envelope attachment protein, and (ii) a first targeting moiety and a second targeting moiety for CD117, (b) a second paramyxovirus envelope attachment protein, which is a variant paramyxovirus envelope attachment protein comprising one or more amino acid substitutions, which reduces natural tropism relative to a wild-type paramyxovirus envelope attachment protein not comprising said one or more amino acid substitutions, and (c) at least one paramyxovirus fusion (F) protein, and wherein the proteins in (a), (b) and (c) are exposed outside of the lipid bilayer.

57. A lipid particle comprising (a) a re-targeting attachment protein comprising (i) a first paramyxovirus envelope attachment protein, and (ii) a first targeting moiety and a second targeting moiety for CD8, (b) a second paramyxovirus envelope attachment protein, which is a variant paramyxovirus envelope attachment protein comprising one or more amino acid substitutions, which reduces natural tropism relative to a wild-type paramyxovirus envelope attachment protein not comprising said one or more amino acid substitutions, and (c) at least one paramyxovirus fusion (F) protein, and wherein the proteins in (a), (b) and (c) are exposed outside of the lipid bilayer.

58. The lipid particle of embodiments 24-57, wherein the first paramyxovirus envelope attachment protein is a variant paramyxovirus envelope attachment protein.

59. The lipid particle of embodiment 58, wherein the variant paramyxovirus envelope attachment protein comprises one or more amino acid substitutions, reducing natural tropism relative to the wild-type paramyxovirus envelope attachment protein without said one or more amino acid substitutions.

60. The lipid particle of any one of embodiments 25-59, wherein the second paramyxovirus envelope attachment protein is a variant paramyxovirus envelope attachment protein.

61. The lipid particle of embodiment 60, wherein the variant paramyxovirus envelope attachment protein comprises one or more amino acid substitutions, the natural tropism is reduced relative to the wild-type paramyxovirus envelope attachment protein not comprising the one or more amino acid substitutions.

62. The lipid particle of any one of embodiments 1-61, wherein the first targeting moiety is selected from the group consisting of a single domain antibody or a single chain variable fragment (scFv).

63. The lipid particle of any one of embodiments 1-62, wherein the second targeting moiety is selected from the group consisting of a single domain antibody or a single chain variable fragment (scFv).

64. The lipid particle of embodiment 54 and claim 62 or 63, wherein the single domain antibody is a VHH.

65. The lipid particle of any one of embodiments 1 to 64, wherein the first variant paramyxovirus envelope attachment protein and the second variant paramyxovirus envelope attachment protein are the same.

66. The lipid particle of any one of embodiments 1-65, wherein the first paramyxovirus envelope attachment protein and the second paramyxovirus envelope attachment protein are different.

67. The lipid particle of any one of embodiments 1 to 66, wherein the first paramyxovirus envelope attachment protein is an envelope attachment protein from a nipah virus, a hendra virus, or a measles virus, or a variant of any one of the foregoing or a biologically active portion thereof.

68. The lipid particle of any one of embodiments 1-67, wherein the first paramyxovirus envelope attachment protein is a wild-type paramyxovirus G protein, H protein or HN protein, or a variant or biologically active portion of any one of the foregoing.

69. The lipid particle of embodiment 67 or claim 68, wherein the first paramyxovirus envelope attachment protein is a wild-type nipah virus G (NiV-G) protein, or a variant or biologically active portion of NiV-G.

70. The lipid particle of any one of embodiments 67-69, wherein the first paramyxovirus envelope attachment protein is a variant NiV-G, which is a variant or biologically active portion of wild-type NiV-G.

71. The lipid particle of any one of embodiments 1-70, wherein the second paramyxovirus envelope attachment protein is an envelope attachment protein from nipah virus, hendra virus, or measles virus, or is a variant or biologically active portion of any one of the foregoing.

72. The lipid particle of any one of embodiments 1-71, wherein the second paramyxovirus envelope attachment protein is a wild-type paramyxovirus G protein, H protein or HN protein, or a variant or biologically active portion of any one of the foregoing.

73. The lipid particle of embodiment 71 or claim 72, wherein the second paramyxovirus envelope attachment protein is a wild-type nipah virus G (NiV-G) protein, or is a variant or biologically active portion of NiV-G.

74. The lipid particle of any one of embodiments 1 to 73, wherein the second paramyxovirus envelope attachment protein is a variant NiV-G, which is a variant or biologically active portion of wild-type NiV-G.

75. The lipid particle of any one of embodiments 3-70, wherein the second paramyxovirus envelope attachment protein is a variant paramyxovirus envelope glycoprotein from nipah virus, hendra virus, or measles virus, or a biologically active portion thereof.

76. The lipid particle of any one of embodiments 3-70 and 75, wherein the second paramyxovirus envelope attachment protein is a variant of a wild-type paramyxovirus G protein, H protein or HN protein or a biologically active portion thereof.

77. The lipid particle of embodiment 73 or 74, wherein the variant is a variant NiV-G that is a variant of a wild-type nipah virus G (NiV-G) protein or a biologically active portion thereof.

78. The lipid particle of embodiment 70, claim 74, or claim 77, wherein the variant NiV-G is truncated by up to 40 consecutive amino acids at or near the N-terminus of the wild-type NiV-G shown in SEQ ID No. 1.

79. The lipid particle of any one of embodiments 70, 74, 77 and 78, wherein the variant NiV-G has a truncation of amino acids 2-34 of the wild-type NiV-G shown in SEQ ID No. 1.

80. The lipid particle of any one of embodiments 70, 73 and 77-79, wherein the variant NiV-G exhibits reduced binding to ephrin B2 or ephrin B3.

81. The lipid particle of embodiment 80, wherein the variant NiV-G comprises one or more amino acid substitutions corresponding to amino acid substitutions numbered selected from the group consisting of E501A, W504, 504A, Q530A and E533A as set forth in reference to SEQ ID No. 1.

82. The lipid particle of embodiment 80 or claim 81, wherein the variant NiV-G comprises the numbered amino acid substitutions E501A, W504A, Q a and E533A as set forth in reference to SEQ ID No. 1.

83. The lipid particle of any one of embodiments 70, 74 and 77-79, wherein the variant NiV-G has an amino acid sequence having equal to or about 80%, at least equal to or about 81%, at least equal to or about 82%, at least equal to or about 83%, equal to or about 84%, at least equal to or about 85%, at least equal to or about 86%, or at least equal to or about 87%, at least equal to or about 88%, or at least equal to or about 89%, at least equal to or about 90%, at least equal to or about 91%, at least equal to or about 92%, at least equal to or about 93%, at least equal to or about 94%, at least equal to or about 95%, equal to or about 96%, at least equal to or about 97%, at least equal to or about 98%, or at least equal to or about 99% sequence identity to SEQ ID NO 228.

84. The lipid particle of any one of embodiments 70, 74 and 77-79, wherein the variant NiV-G has the amino acid sequence set forth in SEQ ID No. 228.

85. The lipid particle of any one of embodiments 1-84, wherein the at least one paramyxovirus fusion (F) protein is an F protein from henipav, or a biologically active portion thereof or a variant thereof.

86. The lipid particle of embodiment 85, wherein the henipav is hendra virus.

87. The lipid particle of embodiment 85, wherein the henipa virus is a nipah virus.

88. The lipid particle of any one of embodiments 1-87, wherein the paramyxovirus F protein is a wild-type NiV-F protein or a variant or biologically active portion thereof.

89. The lipid particle of any one of embodiments 1-88, wherein the paramyxovirus F protein is a variant NiV-F, the variant NiV-F being a variant or biologically active portion of a wild-type NiV-F protein.

90. The lipid particle of embodiment 89, wherein the variant NiV-F is truncated by up to 22 consecutive amino acids at the C-terminus of the wild-type NiV-F shown in SEQ ID No. 235, optionally excluding the initiating methionine.

91. The lipid particle of embodiment 89 or claim 90, wherein the variant NiV-F protein is a truncated NiV-F lacking amino acids 525-546 of SEQ ID No. 235.

92. The lipid particle of any one of embodiments 89-91, wherein the variant NiV-F has the amino acid sequence shown in SEQ ID NO:227 or an amino acid sequence having equal or about 80%, at least equal or about 81%, at least equal or about 82%, at least equal or about 83%, equal or about 84%, at least equal or about 85%, at least equal or about 86%, or at least equal or about 87%, at least equal or about 88%, or at least equal or about 89%, at least equal or about 90%, at least equal or about 91%, at least equal or about 92%, at least equal or about 93%, at least equal or about 94%, at least equal or about 95%, equal or about 96%, at least equal or about 97%, at least equal or about 98%, or at least equal or about 99% sequence identity to SEQ ID NO: 227.

93. The lipid particle of any one of embodiments 89-92, wherein the variant NiV-F has the amino acid sequence shown in SEQ ID NO: 227.

94. The lipid particle of any one of embodiments 1-93, wherein the paramyxovirus F protein is an F0 precursor, or a proteolytically cleaved form thereof comprising F1 and F2 subunits.

95. The lipid particle of embodiment 94, wherein the proteolytically cleaved form is a cathepsin L cleavage product.

96. The lipid particle of any one of embodiments 1-95, wherein the paramyxovirus envelope attachment protein and the first targeting moiety and the second targeting moiety are linked via one or more linkers.

97. The lipid particle of embodiment 96, wherein the one or more linkers are one or more peptide linkers.

98. The lipid particle of any one of embodiments 1-97, wherein the re-targeting attachment protein comprises, in order, a paramyxovirus attachment protein-first linker-first targeting moiety-second linker-second targeting moiety.

99. The lipid particle of embodiment 98, wherein the first linker and/or the second linker is independently a peptide linker.

100. The lipid particle of embodiment 99, wherein the first linker and the second linker are the same.

101. The lipid particle of embodiment 99, wherein the first linker and the second linker are different.

102. The lipid particle of embodiment 97 or claim 99, wherein the peptide linker is 2 to 65 amino acids in length.

103. The lipid particle of any one of embodiments 99-102, wherein the peptide linker is a flexible linker comprising GS, GGS, GGGGS, GGGGGS or a combination thereof.

104. The lipid particle of any one of embodiments 99-103, wherein the peptide linker is selected from (GGS) n, wherein n is 1 to 10, (GGGGS) n, wherein n is 1 to 10, or (GGGGGS) n, wherein n is 1 to 6.

105. The lipid particle of any one of embodiments 1-104, wherein the peptide linker is selected from the group consisting of SEQ ID NOs 589-592.

106. The lipid particle of any one of embodiments 1-105, wherein the lipid particle further comprises one or more additional paramyxovirus envelope adhesion glycoproteins embedded in the lipid bilayer.

107. The lipid particle of embodiment 106, wherein the one or more additional paramyxovirus envelope attachment glycoproteins are re-targeted attachment proteins comprising a paramyxovirus envelope attachment protein and an additional targeting moiety.

108. The lipid particle of any one of embodiments 1-107, wherein the at least one paramyxovirus fusion (F) protein exhibits fusion activity with a target cell when at least one paramyxovirus envelope attachment protein binds to the target molecule on the target cell.

109. The lipid particle of any one of embodiments 1-108, wherein the lipid particle comprises a viral nucleic acid.

110. The lipid particle of embodiment 109, wherein the viral nucleic acid comprises one or more (e.g., all) of the following nucleic acid sequences, 5 'LTR (e.g., comprising U5 and lacking a functional U3 domain), psi packaging element (Psi), central polypurine tract (cPPT)/Central Termination Sequence (CTS) (e.g., DNA flap), poly-a tail sequence, post-transcriptional regulatory element (e.g., WPRE), rev Response Element (RRE), and 3' LTR (e.g., comprising U5 and lacking a functional U3).

111. The lipid particle of any one of embodiments 1-110, wherein the lipid particle is a viral vector.

112. The lipid particle of any one of embodiments 1-111, which is a retroviral vector.

113. The lipid particle of any one of embodiments 1-111, which is a lentiviral vector.

114. The lipid particle of any one of embodiments 1-108 and 111-113, wherein the lipid particle is free of viral genomic nucleic acid.

115. The lipid particle of any one of embodiments 1-108 and 111-113, wherein the lipid particle is free of viral genomic DNA.

116. The lipid particle of any one of embodiments 1 to 110 and 115, which is a virus-like particle.

117. The lipid particle of any one of embodiments 1 to 110, 115, and 116, which is a retrovirus-like particle.

118. The lipid particle of any one of embodiments 1 to 110, 115, and 116, which is a lentiviral-like particle.

119. The lipid particle of any one of embodiments 1-118, wherein the lipid particle is produced as a formulation with increased titer compared to a reference lipid particle formulation that is similarly produced but has only the first re-targeted attachment protein.

120. The lipid particle of any one of embodiments 5 to 119, wherein the lipid particle is produced as a formulation having increased titer as compared to a reference lipid particle formulation that is similarly produced but does not comprise the second paramyxovirus envelope attachment protein, which is a variant paramyxovirus envelope attachment protein comprising one or more substitutions, to reduce natural tropism relative to the wild-type paramyxovirus envelope attachment protein not comprising the one or more substitutions.

121. The lipid particle of embodiment 120, wherein the second paramyxovirus envelope attachment protein as a variant paramyxovirus envelope attachment protein comprises one or more amino acid substitutions corresponding to the amino acid substitutions numbered selected from the group consisting of E501A, W504A, Q a and E533A as set forth in reference SEQ ID No. 1.

122. The lipid particle of any one of embodiments 1 to 119, wherein the lipid particle is produced in suspension culture as a formulation having increased titer compared to a reference lipid particle formulation that is similarly produced but has only the first re-targeted attachment protein.

123. The lipid particle of any one of embodiments 120-122, wherein the titer increase is equal to or greater than 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more.

124. The lipid particle of any one of embodiments 1-123, further comprising an exogenous agent for delivery to a target cell.

125. The lipid particle of embodiment 124, wherein the exogenous agent is present in the lumen.

126. The lipid particle of embodiments 124 or 125, wherein the exogenous agent is a protein or a nucleic acid, optionally wherein the nucleic acid is DNA or RNA.

127. The lipid particle of any one of embodiments 124-126, wherein the exogenous agent is a nucleic acid encoding a cargo for delivery to the target cell.

128. The lipid particle of any one of embodiments 124-127, wherein the exogenous agent is or encodes a therapeutic agent, a diagnostic agent, or a genome-modifying enzyme.

129. The lipid particle of any one of embodiments 124-128, wherein the exogenous agent encodes a membrane protein, optionally wherein the membrane protein is an antigen receptor for targeting a cell expressed by or associated with a disease or disorder.

130. The lipid particle of embodiment 129, wherein the membrane protein is a Chimeric Antigen Receptor (CAR).

131. The lipid particle of any one of embodiments 124-128, wherein the exogenous agent is a nucleic acid comprising a payload gene for correcting a genetic defect, optionally a genetic defect in the target cell, optionally wherein the genetic defect is associated with a liver cell or a hepatocyte.

132. The lipid particle of any one of embodiments 124-131, wherein binding of the paramyxovirus envelope attachment protein or biologically active portion thereof to a target molecule expressed on the surface of a target cell mediates fusion of the particle with the target cell and delivery of the exogenous agent to the target cell.

133. The lipid particle of any one of embodiments 124-132, wherein the exogenous agent is delivered to equal to or greater than 10%, 20%, 30%, 40%, 50%, 60% of the target cells.

134. The lipid particle of any one of embodiments 124-133, wherein delivery of the exogenous agent to the target cell is increased as compared to a reference particle formulation that is similarly produced but has only the first re-targeted attachment protein.

135. The lipid particle of embodiment 134, wherein the delivery to the target cell is increased by equal to or greater than 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more.

136. A producer cell comprising (a) a nucleic acid encoding a re-targeting attachment protein comprising a paramyxovirus envelope attachment protein linked to (i) a first targeting moiety directed against a first target molecule expressed on the surface of a target cell, and (ii) a second targeting moiety directed against a second target molecule expressed on the surface of a target cell, and (b) a nucleic acid encoding at least one paramyxovirus fusion (F) protein.

137. The producer cell of embodiment 136, further comprising a nucleic acid encoding a second paramyxovirus envelope attachment protein that is a variant paramyxovirus envelope attachment protein comprising one or more amino acid substitutions to reduce natural tropism relative to the wild-type paramyxovirus envelope attachment protein not comprising said one or more amino acid substitutions.

138. The production cell of embodiment 136 or 137 wherein the cell further comprises a viral nucleic acid.

139. The production cell of any one of embodiment 138 wherein the viral nucleic acid is a lentiviral nucleic acid.

140. The production cell of any one of embodiments 136 to 139 wherein the cell is a mammalian cell.

141. The production cell according to any one of embodiments 136 to 140, wherein the production cell is selected from the group consisting of CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, hepG2 cells, saos-2 cells, huh7 cells, heLa cells, W163 cells, 211 cells, and 211A cells.

142. The production cell of any one of embodiments 136-141 wherein the production cell comprises a 293T cell.

143. The production cell of any one of embodiments 137-142 wherein the viral nucleic acid lacks one or more genes involved in viral replication.

144. The production cell of any one of embodiments 137-143 wherein the viral nucleic acid comprises a nucleic acid encoding a viral packaging protein selected from one or more of Gag, pol, rev and Tat.

145. The producer cell of any of embodiments 138-144, wherein the viral nucleic acid comprises one or more (e.g., all) of the following nucleic acid sequences, 5 'LTR (e.g., comprising U5 and lacking a functional U3 domain), psi packaging element (Psi), central polypurine tract (cPPT)/Central Termination Sequence (CTS) (e.g., DNA flap), poly-a tail sequence, post-transcriptional regulatory element (e.g., WPRE), rev Response Element (RRE), and 3' LTR (e.g., comprising U5 and lacking a functional U3).

146. A method of preparing a lipid particle comprising a) providing a production cell as defined in any one of claims 136 to 145, b) culturing the cell under conditions that allow production of the lipid particle, and c) isolating, enriching or purifying the lipid particle from the cell, thereby preparing the lipid particle.

147. The method of embodiment 146, wherein the lipid particle is a pseudotyped lentiviral vector.

148. A lipid particle produced by the method of embodiment 146 or claim 147.

149. A composition comprising a plurality of lipid particles of any one of embodiments 1 to 135 and 148.

150. The composition of embodiment 149, further comprising a pharmaceutically acceptable carrier.

151. A method of transducing a cell, the method comprising contacting the cell with the lipid particle of any one of embodiments 1-135 and 148 or the composition of claim 149 or claim 150.

152. A method of delivering an exogenous agent into a target cell, the method comprising contacting the lipid particle of any one of embodiments 124-135 and 148 or the composition of claim 149 or claim 150 with the target cell.

153. The method of embodiment 151 or claim 152, wherein said contacting is in vitro or ex vivo.

154. The method of embodiment 151 or claim 152, wherein said contacting is in a subject.

155. A method of delivering an exogenous agent to a cell of a subject, the method comprising administering to the subject the lipid particle of any one of embodiments 124-135 and 148 or the composition of claim 149 or claim 150.

156. The method of embodiment 155, wherein the exogenous agent is or encodes a therapeutic agent for treating a disease or disorder in the subject.

157. A method of treatment comprising administering to a subject the lipid particle of any one of embodiments 124-135 and 148 or the composition of claim 149 or claim 150.

158. The method of any one of embodiments 152-157, wherein the exogenous agent is or encodes a membrane protein, optionally a chimeric antigen receptor, for targeting an antigen associated with a disease or disorder in the subject.

159. The method of any one of embodiments 152-157, wherein the exogenous agent is used in gene therapy to correct a genetic defect or substitution defect or deleted gene in the subject.

160. The method of any one of embodiments 155 to 159, wherein the subject is a human subject.

161. The method of any one of embodiments 151-160, wherein the method further comprises administering to the subject one or more agents that stimulate mobilization of bone marrow cells from bone marrow to peripheral blood.

162. The method of any one of embodiments 151-161, wherein the subject has been previously administered one or more agents that stimulate bone marrow cell mobilization from bone marrow to peripheral blood.

163. The method of embodiment 161 or 162, wherein the one or more agents that stimulate mobilization are selected from the group consisting of Stem Cell Factor (SCF), small molecule VLA-4 inhibitor BI05192, BOP (N- (benzenesulfonyl) -L-prolyl-L-0- (1-pyrrolidinylcarbonyl) tyrosine), heparin, granulocyte colony-stimulating factor (G-CSF), MGTA-145, and plexafu (AMD 3100).

164. The method of any one of embodiments 161 to 163, wherein the one or more agents that stimulate mobilization comprises G-CSF.

165. The method of any one of embodiments 161 to 164, wherein the one or more agents that stimulate mobilization comprises plexafu.

VI. Examples

The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1 transduction of CD117 cells

Studies were conducted to evaluate methods for re-targeting Lentiviral Vectors (LV) to specific proteins. In this example, a Lentiviral Vector (LV) is pseudotyped with a binding agent, such as a VHH peptide, to re-target the LV with an envelope protein (fusion agent) that targets CD117. CD117 is a single pass transmembrane protein (500 aa) present on at least the surface of Hematopoietic Stem Cells (HSCs), multipotent progenitor cells (MPPs), common myeloid progenitor Cells (CMP), and common lymphoid progenitor Cells (CLPs). To target CD117, the ability of exemplary CD117 VHH binding agents to transduce CD117 overexpressing cells was identified in an adherent LV production system. These binding agents, as shown in SEQ ID NOS.512-515, may be used to target CD117.

For targeted delivery of exemplary exogenous agents, a tandem fusion agent containing two VHH binders, each directed against CD117 and separated by a linker, mutated to eliminate its natural tropism was generated as a fusion to the C-terminus of the exemplary Nipag G (NiV-G) protein sequence GcΔ34 (Bender et al 2016 PLoS Pathol 12 (6): e 1005641). Blinded G comprises point mutations (e.g., amino acid substitutions) E501A, W, 504, A, Q A and E533A to disrupt binding to ephrin B2/B3 and has the amino acid sequence shown in SEQ ID NO: 228. In this example, a number of tandem fusion agents containing a first VHH and a second VHH were tested, as summarized in table E1. The first VHH and the second VHH are linked to a GS linker (e.g.SEQ ID NO: 275). A single binding agent fusion agent containing a VHH binding agent for CD117 was similarly fused to the C-terminus of gcΔ34 NiV-G, as described below. LV was also generated to contain an exemplary Nipag G (NiV-G) protein sequence GcΔ34, which contains point mutations E501A, W504, 504A, Q530A and E533A to eliminate its natural tropism, but is not fused to any binding agent (e.g., the shown binding agent-free NiV-G or Gm). LV was additionally pseudotyped with an exemplary NipaF (NiV-F) protein sequence NivFdel (SEQ ID NO:226; or SEQ ID NO:227 without signal sequence; bender et al, 2016 PLoS).

To generate lentiviral vectors, plasmids encoding tandem or single re-targeted NiV-G fusion proteins, binding agent-free NiV-G proteins and NiV-F proteins were transfected into the same HEK 293 cell population. Cells are also transfected with packaging plasmids (e.g., gag/pol, rev) for the production of viral vectors and a transfer plasmid encoding enhanced green fluorescent protein (eGFP). After viral vector production, the cell culture is centrifuged to pellet the cells, and the supernatant containing the crude virus is collected.

The titers of exemplary CD117 tandem VHH binding agent and single VHH binding agent in CD117 overexpressing cell lines were assessed (fig. 1A). The highest functional titers were observed for LV pseudotyped with fusion NiV-G comprising tandem binders. When used to transduce CD117 overexpressing cell lines, this was observed to correlate with a functional crude titer within 10 ⁶.

In a complementation experiment, LV pseudotyped with a re-targeting tandem fusion agent targeting CD117 and NiV-F proteins as described above was produced in squamous cell carcinoma line SCC3687 cells. LV was concentrated 100-fold by ultracentrifuge and tested for titers on CD117 overexpressing cells (FIG. 1B) and CD34+ cells (FIG. 1C). Some tandem binders were observed to be particularly effective in transducing cd34+ cells. For example, an exemplary tandem fusion agent, cd117_a (SEQ ID NO 575), showed a two-fold improvement in titer on CD117 overexpressing cells and a near 2-log improvement on cd34+ cells.

Next, primary cell transduction was assessed for both exemplary tandem binders compared to a single binder that also targets CD117 (fig. 1D). It was observed that the candidate CD117 tandem binding agent cd117_a (SEQ ID NO 575) showed efficacy in transducing unstimulated cd34+ primary cells. In contrast, despite high titers on CD117 overexpressing cell lines, single binders show little or no ability to transduce cd34+ primary cells.

These results support that pseudotyping lipid particles such as LV with tandem binding agents fused to viral attachment proteins significantly improves targeting of LV to desired target cells.

Example 2 CD133+CD transduction of 34+ cells

Similar to the above experiments, lentiviral Vectors (LV) were pseudotyped with binding agents, such as scFv peptides, to re-target LV with envelope proteins (fusion agents) targeting CD 133. To target CD133, exemplary CD133 scFv binding agents as shown in SEQ ID NOS 534, 543, 516, 525, 552 may be used.

For targeted delivery of exemplary exogenous agents, two tandem fusion agents containing two scFv binders, each directed against CD133 and separated by a linker, mutated to eliminate their natural tropism, were generated as fusions with the C-terminus of the exemplary Nipag G (NiV-G) protein sequence GcΔ34 (Bender et al 2016 PLoS Pathol 12 (6): e 1005641). Blinding G contained point mutations E501A, W, A, Q A and E533A to disrupt binding to ephrin B2/B3 and had the amino acid sequence shown in SEQ ID NO: 228. In this example, two tandem fusion agents containing a first scFv and a second scFv were tested, as summarized in table E2. The first scFv and the second scFv are linked to a GS linker (e.g., SEQ ID NO: 275). A single binding agent fusion agent comprising a scFv binding agent against CD133 was similarly fused to the C-terminus of gcΔ34 NiV-G, as described below. LV was also generated to contain an exemplary Nipag G (NiV-G) protein sequence GcΔ34, which contains point mutations E501A, W504, 504A, Q530A and E533A to eliminate its natural tropism, but is not fused to any binding agent (e.g., the shown binding agent-free NiV-G or Gm). LV was additionally pseudotyped with an exemplary NipaF (NiV-F) protein sequence NivFdel (SEQ ID NO:226; or SEQ ID NO:227 without signal sequence; bender et al, 2016 PLoS).

Cd34+ primary cell transduction of two exemplary CD133 tandem binders were tested against each construct as a single scFv binder (figure 2). It was observed that placing the exemplary CD133 binders in tandem resulted in higher cd34+ cell transduction compared to the single binders.

Example 3 transduction of CD8+ T cells

Similar to the experiments described above, lentiviral Vectors (LV) were pseudotyped with binding agents such as scFv or VHH peptides to re-target LV with envelope proteins (fusion agents) targeting CD 8. To target CD8, exemplary CD8 binding agents as shown in SEQ ID NOS 377, 489, 496, 501 or 508 may be used.

For targeted delivery of exemplary exogenous agents, two tandem fusion agents containing two binding agents each directed against CD8 and separated by a linker were generated as fusions with the C-terminus of the exemplary Nipag G (NiV-G) protein sequence GcΔ34, which were mutated to eliminate their natural tropism (Bender et al 2016 PLoS Pathol 12 (6): e 1005641). Blinding G contained point mutations E501A, W, A, Q A and E533A to disrupt binding to ephrin B2/B3 and had the amino acid sequence shown in SEQ ID NO: 228. In this example, a tandem fusion agent containing a first binding agent and a second binding agent was tested, as summarized in table E3. The first binding agent and the second binding agent are linked to a GS linker (e.g., SEQ ID NO: 275). A single binding agent comprising a scFv or VHH binding agent directed against CD8 was similarly fused to the C-terminus of gcΔ34 NiV-G, as described below. LV was also generated to contain an exemplary Nipag G (NiV-G) protein sequence GcΔ34, which contains point mutations E501A, W504, 504A, Q530A and E533A to eliminate its natural tropism, but is not fused to any binding agent (e.g., the shown binding agent-free NiV-G or Gm). LV was additionally pseudotyped with an exemplary NipaF (NiV-F) protein sequence NivFdel (SEQ ID NO:226; or SEQ ID NO:227 without signal sequence; bender et al, 2016 PLoS).

To generate lentiviral vectors, plasmids encoding tandem or single re-targeted NiV-G fusion proteins, binding agent-free (blinded) NiV-G proteins and NiV-F proteins were transfected into the same HEK 293 cell population. Cells are also transfected with packaging plasmids (e.g., gag/pol, rev) for the production of viral vectors and a transfer plasmid encoding enhanced green fluorescent protein (eGFP). After viral vector production, the cell culture is centrifuged to pellet the cells, and the supernatant containing the crude virus is collected.

SupT 1T cell transduction was tested for two exemplary CD8 tandem binders CD8_A and CD8_B for each binder construct as a single VHH or scFv binder (FIG. 3A). It was observed that placing the exemplary CD8 binding agents in tandem resulted in higher T cell transduction compared to the single binding agent. LV pseudotyped with tandem CD8 NiV-G binders was observed to show a 10-fold increase in titer when expressed on vectors with blinded G.

Resting pan T cell transduction of an exemplary CD8 tandem binding agent cd8_a was tested against a single binding agent construct (cd8_b2). For this experiment, frozen pan T cells were thawed and seeded in IL-2 (10 mg/mL) (or IL-7) and expanded. After expansion, 80,000-100,000 cells were seeded in 100 μ L Immunocult medium +pen/strep +il-2 in each well of a 96-well plate. Approximately 100. Mu.L of lentiviral preparation was added to the cells and the 96-well plate was centrifuged at 10000g for 90 minutes at room temperature. The cells were then activated using CD3/CD28 activation beads (25. Mu.L/1E 6 cells) and IL 7. After incubation at 37 ℃, cells were harvested for staining and/or VCN analysis to determine titers. Fig. 3B shows the pan-T titers of single binder (cd8_b2) and tandem binders (cd8_a). It was observed that including binderless NiV-G and re-targeting Ni-G during production resulted in increased titers of at least the exemplary CD8 binding agents in tandem. It was also observed that tandem binding agent CD8_A (SEQ ID NO: 584 comprising the CD8_B1 binding agent (SEQ ID NO: 377) and the CD8_B2 binding agent (SEQ ID NO: 501)) had a 5-fold increase over the single binding agent CD8_B2.

These results support that pseudotyping of lipid particles such as LV with tandem binding agents (scFv or VHH or combinations thereof) fused to viral attachment proteins significantly improves targeting of LV to desired target cells.

Example 4 Selective modulation of CD34+ cells in vivo

In a set of complementary experiments similar to example 1 above, lentiviral Vectors (LV) were pseudotyped with a binding agent, such as a VHH peptide, to re-target the LV with an envelope protein (fusion agent) targeting CD117. These binding agents (e.g., as shown in SEQ ID NOS: 512-515) may be used to target CD117.

For targeted delivery of exemplary exogenous agents, two tandem fusion agents containing two different VHH binders, each directed against CD117 and separated by a linker, mutated to eliminate their natural tropism, were generated as fusions with the C-terminus of the exemplary nipag (NiV-G) protein sequence Gc Δ34 (Bender et al 2016 PLoS Pathol 12 (6): e 1005641). Blinding G contained point mutations E501A, W, A, Q A and E533A to disrupt binding to ephrin B2/B3 and had the amino acid sequence shown in SEQ ID NO: 228. The first VHH and the second VHH are linked to a GS linker (e.g.SEQ ID NO: 275). LV was also generated to contain an exemplary Nipag G (NiV-G) protein sequence GcΔ34, which contains point mutations E501A, W504, 504A, Q530A and E533A to eliminate its natural tropism, but is not fused to any binding agent (e.g., the shown binding agent-free NiV-G or Gm). LV was additionally pseudotyped with an exemplary NipaF (NiV-F) protein sequence NivFdel (SEQ ID NO:226; or SEQ ID NO:227 without signal sequence; bender et al, 2016 PLoS). Control BaEVTR LV was generated using BaEVTR envelopes.

To generate lentiviral vectors, plasmids encoding tandem or single re-targeted NiV-G fusion proteins, binding agent-free NiV-G proteins and NiV-F proteins were transfected into the same HEK 293 cell population. Cells are also transfected with packaging plasmids (e.g., gag/pol, rev) for the production of viral vectors and a transfer plasmid encoding the GFP gene. After viral vector production, the cell culture is centrifuged to pellet the cells, and the supernatant containing the crude virus is collected.

The ability of lentiviral vectors pseudotyped with tandem fusion agents to specifically target a minority cell population in bone marrow was investigated. To assess in vivo delivery, human CD34+ cells (huCD34+) were infused into NBSGW mice (NOD.Cg-KitW-41J Tyr+ PRKDCSCID IL2rgtm1 Wjl/ThomJ). After 84 days of maintenance, the GFP-expressing lentiviral vector was delivered intravenously. Mice were sacrificed on day 96 and bone marrow cells were assessed for GFP expression.

Transduction efficiencies of mice equivalently dosed with CD117 LV were compared to BaEVTR LV, as shown in figure 4A. It was observed that when gfp+ cells in Bone Marrow (BM) were gated, only 0.2% of these cells were cd117+ when BaEVTR LV was used. This is in contrast to 60% observed after administration of CD117 LV (fig. 4B). Similarly, when measuring GFP% on hucd45+ cells, no significant difference was observed between the two vectors at equivalent doses. However, gating of the target cd117+ population instead resulted in CD117 LV targeting that population 175-fold greater than BaEVTR LV (fig. 4B).

These results demonstrate that exemplary lentiviral vectors pseudotyped with tandem fusion agents against CD117 are highly effective in editing cd34+ cells in vivo and demonstrate 178-fold targeting selectivity of the vector to target cells in vivo. Without wishing to be bound by theory, the localization of CD117 to bone marrow may explain the increased selectivity in vivo. Taken together, these data reflect the high specificity of the re-targeted CD117 LV and demonstrate the ability of the re-targeted CD117 LV to reach a homogenous target cell population while largely avoiding transduction of off-target cells.

Example 5 in vivo Gene editing in a non-human primate model

This example illustrates an exemplary design for evaluating various fusion agent-payload combinations for administration in a non-human primate (NHP). Virus-like particles (VLPs) are produced with tandem nipah virus (NiV) fusion agents that re-target HSPCs, which have specific binding agents to CD133 and/or CD 117. These binding agent re-targeted nipag fusion agents comprise two HSPC binding portions connected in series to the exemplary NiV-G sequence gcΔ34 shown in SEQ ID No. 228. In some embodiments, VLPs pseudotyped with tandem fusion agents are further pseudotyped with blinded NiV-G comprising amino acid substitutions E501A, W, A, Q A and E533A to eliminate binding to ephrin B2/B3 (e.g., as shown in SEQ ID NO: 228), but not re-targeted by fusion with a binding agent sequence or tandem binding agent sequence. For NiV fusion agents, VLPs are produced using a Nipaf protein expressed on the lipid envelope and a binding agent re-targeted tandem NipaG protein (see U.S. 2019/0144885, which is incorporated herein by reference, e.g., nivFdel (SEQ ID NO:226; or SEQ ID NO:227, which does not have a signal sequence; bender et al 2016 PLoS)).

VLPs are also packaged with a gene editing payload. After VLP production, the cell culture is centrifuged to pellet the cells and the supernatant containing the crude virus is collected.

The primary fusion agent was selected based in part on overall VLP performance, as measured by the percentage of long-term edits (i.e., edits produced by the payload) in the peripheral blood myeloid cells. The second fusion agent is selected based in part on HSPC-specific tropism, as measured, for example, by the percentage of editing (i.e., editing produced by the payload) relative to the target HSPC population in various mature hematopoietic lineages derived from HSPCs or in other cell lines.

Based on the OTA PCR-based analysis, the payload (e.g., such as a nuclease-based editor or a base-editing-based editor) is selected based on the in vitro performance of resting NHPCD34+ cells. The property may be based on a reporter gene or a functional reading (such as a change in mRNA or protein levels in a biological sample). A genomic target is selected characterized by a "neutral" locus or a "therapeutic" locus homologous to a potential therapeutic target for a disease indication (e.g., BCL 11A).

The model study design is shown in fig. 5. The mobilization protocol described above was first performed on NHP animals. Briefly, AMD3100 was administered 3 consecutive days, with VLPs administered at a controlled rate of dosing about 2 hours after each mobilization. Without wishing to be bound by theory, mobilization of cells from bone marrow to peripheral blood peaks typically 1.5-3 hours after AMD treatment. Peripheral blood was collected throughout the study, while bone marrow was collected by aspirate every two months. Additional needle aspiration of the liver was performed to evaluate off-target editing as a function of safety and biodistribution. Animals were sacrificed 9 months after the vector dose to assess permanent genetic modification of long term HSCs in bone marrow and potential editing in off-target tissues.

The present invention is not intended to limit the scope of the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the described compositions and methods will be apparent from the description and teachings herein. Such changes may be practiced without departing from the true scope and spirit of the disclosure, and are intended to fall within the scope of the disclosure.

VII sequence

Claims

1. A lipid particle, said lipid particle comprising:

(a) A retargeting attachment protein comprising a paramyxoviral envelope attachment protein, the paramyxoviral envelope attachment protein being linked to (i) a first targeting portion for a first target molecule expressed on the surface of a target cell, and (ii) a second targeting portion for a second target molecule expressed on the surface of a target cell; and

(b) at least one paramyxoviral fusion (F) protein; and

The proteins in (a) and (b) are exposed on the outside of the lipid bilayer.

2. The lipid particle of claim 1, wherein the paramyxovirus attachment protein is a variant paramyxovirus envelope attachment protein containing one or more mutations to reduce natural affinity relative to wild-type paramyxovirus envelope attachment proteins that do not contain said one or more mutations.

3. The lipid particle of claim 1 or 2, wherein the paramyxovirus envelope attachment protein is a first paramyxovirus envelope attachment protein, and the lipid particle further comprises a second paramyxovirus envelope attachment protein, wherein the second paramyxovirus envelope attachment protein is a variant paramyxovirus envelope attachment protein containing one or more mutations to reduce natural affinity relative to the wild-type paramyxovirus envelope attachment protein not containing the one or more mutations, wherein the second paramyxovirus envelope attachment protein is exposed on the outer side of the lipid bilayer.

4. The lipid particles of any one of claims 1 to 3, wherein targeting one or both of the first target molecule and the second target molecule does not activate or inhibit the target cells, induce phenotypic changes (e.g., maturation and/or differentiation) of the target cells, induce proliferation of the target cells, and/or induce apoptosis of the target cells.

5. A lipid particle, said lipid particle comprising:

(a) A retargeting attachment protein comprising a first paramyxoviral envelope attachment protein operatively linked to (i) a first targeting portion for a first target molecule expressed on the surface of a target cell, and (ii) a second targeting portion for a second target molecule expressed on the surface of a target cell.

(b) A second paramyxovirus envelope attachment protein, wherein the second paramyxovirus envelope attachment protein is a variant paramyxovirus envelope attachment protein containing one or more amino acid substitutions to reduce native affinity relative to wild-type paramyxovirus envelope attachment proteins that do not contain said one or more amino acid substitutions; and

(c) At least one paramyxovirus fusion (F) protein;

The proteins described in (a), (b) and (c) are exposed on the outer side of the lipid bilayer.

6. The lipid particle of any one of claims 1 to 5, wherein the first paramyxovirus envelope attachment protein is a variant paramyxovirus envelope attachment protein.

7. The lipid particle of claim 6, wherein the variant paramyxovirus envelope attachment protein comprises one or more amino acid substitutions, the one or more amino acid substitutions reducing the native orientation relative to the wild-type paramyxovirus envelope attachment protein not containing the one or more amino acid substitutions.

8. The lipid particle of any one of claims 1 to 7, wherein the first paramyxovirus envelope attachment protein and the second paramyxovirus envelope attachment protein are the same.

9. The lipid particle of any one of claims 1 to 8, wherein the second paramyxoviral envelope attachment protein is not connected to or fused to a non-viral heterologous portion, the non-viral heterologous portion being a cell-specific targeting domain or a functional domain, optionally wherein the second paramyxoviral envelope attachment protein is not connected to or fused to a cell-specific targeting domain, the cell-specific targeting domain being a targeting portion of a target molecule expressed on the surface of a target cell.

10. The lipid particle of claims 1 to 9, wherein the first targeting portion and the second targeting portion are not selected from the group consisting of free cytokines, growth factors, hormones, neurotransmitters, apoptosis ligands, and combinations thereof.

11. The lipid particle of any one of claims 1 to 10, wherein targeting one or both of the first target molecule and the second target molecule does not modulate or induce signaling in the target cells.

12. The lipid particle of any one of claims 1 to 11, wherein the first targeting portion and the second targeting portion each bind to a cell surface molecule present on a target cell.

13. The lipid particle of any one of claims 1 to 12, wherein the first targeting portion binds to cell surface molecules present on a first target cell, and the second targeting portion binds to surface molecules present on a second target cell.

14. The lipid particle of any one of claims 1 to 13, wherein the first target molecule and the second target molecule are different target molecules.

15. The lipid particle of any one of claims 1 to 13, wherein the first target molecule and the second target molecule are the same target molecule.

16. The lipid particle of claim 15, wherein the first targeting portion and the second targeting portion bind to different epitopes of the same target molecule.

17. The lipid particle of any one of claims 1 to 16, wherein the cell surface molecule is a protein, a polysaccharide, or a lipid.

18. The lipid particles of any one of claims 1 to 17, wherein the target cells are selected from the group consisting of: tumor-infiltrating lymphocytes, T cells, neoplastic or tumor cells, virus-infected cells, stem cells, central nervous system (CNS) cells, hematopoietic stem cells (HSCs), and liver cells.

19. The lipid particle of any one of claims 1 to 18, wherein the target cell is selected from the group consisting of: CD3+ T cells, CD4+ T cells, CD8+ T cells, hepatocytes, hematopoietic stem cells, CD34+ hematopoietic stem cells, CD105+ hematopoietic stem cells, CD117+ hematopoietic stem cells, CD105+ endothelial cells, B cells, CD20+ B cells, CD19+ B cells, cancer cells, CD133+ cancer cells, EpCAM+ cancer cells, CD19+ cancer cells, Her2/Neu+ cancer cells, GluA2+ neurons, GluA4+ neurons, NKG2D+ natural killer cells, SLC1A3+ astrocytes, SLC7A10+ adipocytes, or CD30+ lung epithelial cells.

20. The lipid particle of any one of claims 1 to 19, wherein the target cell is a hepatocyte.

21. The lipid particle of claim 20, wherein the cell surface molecule is selected from the group consisting of CD34, CD117 and CD133.

22. The lipid particle of any one of claims 1 to 19, wherein the target cell is a T cell.

23. The lipid particles of claim 22, wherein the cell surface molecules are selected from the group consisting of: CD3, CD4, CD7, CD8, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR and ITGA3.

24. The lipid particle of any one of claims 5 to 23, wherein the second paramyxovirus envelope attachment protein, as a variant paramyxovirus envelope attachment protein, comprises one or more amino acid substitutions, said one or more amino acid substitutions corresponding to amino acid substitutions numbered from the group consisting of E501A, W504A, Q530A and E533A as shown in SEQ ID NO:1.

25. A lipid particle, said lipid particle comprising:

(a) A first-level targeting attachment protein comprising (i) a first paramyxoviral envelope attachment protein; and (ii) a first targeting portion for CD133; and a second targeting portion for CD133; and

(b) at least one paramyxoviral fusion (F) protein; and

The proteins described in (a) and (b) are exposed on the outside of the lipid bilayer.

26. The lipid particles according to any one of claims 1 to 25, wherein:

(i) The first targeting portion is scFv and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 516, 525, 534, 543 and 552 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with it; and/or

(ii) The second targeting portion is scFv and contains an amino acid sequence selected from the group consisting of SEQ ID NO: 516, 525, 534, 543 and 552 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with it.

27. The lipid particles according to any one of claims 1 to 25, wherein:

(i) The first targeting portion comprises: (a) a CD133 binder comprising CDR-H1, CDR-H2, and CDR-H3 comprising amino acid sequences of SEQ ID NO: 536, 537, and 538, respectively, and CDR-L1, CDR-L2, and CDR-L3 comprising amino acid sequences of SEQ ID NO: 540, 541, and 542, respectively; (b) a CD133 binder comprising CDR-H1, CDR-H2, and CDR-H3 comprising amino acid sequences of SEQ ID NO: 545, 546, and 547, respectively, and CDR-L1, CDR-L2, and CDR-L3 comprising amino acid sequences of SEQ ID NO: 549, 550, and 551, respectively; and (c) a CD133 binder comprising amino acid sequences of SEQ ID NO: 545, 546, and 547, respectively. (d) A CD133 binder comprising CDR-H1, CDR-H2, and CDR-H3 containing amino acid sequences of SEQ ID NO: 527, 528, and 529, respectively, and CDR-L1, CDR-L2, and CDR-L3 containing amino acid sequences of SEQ ID NO: 531, 532, and 533, respectively; or (e) a CD133 binder comprising CDR-H1, CDR-H2, and CDR-H3 containing amino acid sequences of SEQ ID NO: 554, 555, and 556, respectively, and CDR-L1, CDR-L2, and CDR-L3 containing amino acid sequences of SEQ ID NO: 522, 523, and 524, respectively. The CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequences of 558, 559, and 560; and/or

(ii) The second targeting portion comprises: (a) a CD133 binder comprising CDR-H1, CDR-H2, and CDR-H3 comprising amino acid sequences of SEQ ID NO: 536, 537, and 538, respectively, and CDR-L1, CDR-L2, and CDR-L3 comprising amino acid sequences of SEQ ID NO: 540, 541, and 542, respectively; (b) a CD133 binder comprising CDR-H1, CDR-H2, and CDR-H3 comprising amino acid sequences of SEQ ID NO: 545, 546, and 547, respectively, and CDR-L1, CDR-L2, and CDR-L3 comprising amino acid sequences of SEQ ID NO: 549, 550, and 551, respectively; (c) a CD133 binder comprising amino acid sequences of SEQ ID NO: 545, 546, and 547, respectively, and CDR-L1, CDR-L2, and CDR-L3 comprising amino acid sequences of SEQ ID NO: 549, 550, and 551, respectively; (d) A CD133 binder comprising CDR-H1, CDR-H2, and CDR-H3 containing amino acid sequences of SEQ ID NO: 518, 519, and 520, and CDR-L1, CDR-L2, and CDR-L3 containing amino acid sequences of SEQ ID NO: 522, 523, and 524, respectively; or (e) a CD133 binder comprising CDR-H1, CDR-H2, and CDR-H3 containing amino acid sequences of SEQ ID NO: 527, 528, and 529, respectively, and CDR-L1, CDR-L2, and CDR-L3 containing amino acid sequences of SEQ ID NO: 531, 532, and 533, respectively; or (e) a CD133 binder comprising CDR-H1, CDR-H2, and CDR-H3 containing amino acid sequences of SEQ ID NO: 554, 555, and 556, respectively, and CDR-L1, CDR-L2, and CDR-L3 containing amino acid sequences of SEQ ID NO: 522, 523, and 524, respectively. CDR-L1, CDR-L2, and CDR-L3 of amino acid sequences 558, 559, and 560.

28. The lipid particles according to any one of claims 1 to 25, wherein:

(i) The first targeting portion comprises: (a) a CD133 binder comprising CDR-H1, CDR-H2, and CDR-H3 comprising amino acid sequences of SEQ ID NO: 289, 565, and 538, respectively, and CDR-L1, CDR-L2, and CDR-L3 comprising amino acid sequences of SEQ ID NO: 540, 541, and 542, respectively; (b) a CD133 binder comprising CDR-H1, CDR-H2, and CDR-H3 comprising amino acid sequences of SEQ ID NO: 566, 567, and 547, respectively, and CDR-L1, CDR-L2, and CDR-L3 comprising amino acid sequences of SEQ ID NO: 549, 550, and 551, respectively; and (c) a CD133 binder comprising amino acid sequences of SEQ ID NO: 540, 541, and 542, respectively. (d) A CD133 binder comprising CDR-H1, CDR-H2, and CDR-H3 containing amino acid sequences of SEQ ID NO: 570, 571, and 529, respectively, and CDR-L1, CDR-L2, and CDR-L3 containing amino acid sequences of SEQ ID NO: 531, 532, and 533, respectively; or (e) a CD133 binder comprising CDR-H1, CDR-H2, and CDR-H3 containing amino acid sequences of SEQ ID NO: 572, 569, and 520, respectively, and CDR-L1, CDR-L2, and CDR-L3 containing amino acid sequences of SEQ ID NO: 572, 569, and 520, respectively; and CDR-L1, CDR-L2, and CDR-L3 containing amino acid sequences of SEQ ID NO: 531, 532, and 533, respectively; or (e) a CD133 binder comprising CDR-H1, CDR-H2, and CDR-H3 containing amino acid sequences of SEQ ID NO: 572, 573, and 556, respectively, and CDR-L3 containing amino acid sequences of SEQ ID NO: 572, 573, and 556, respectively. The CDR-L1, CDR-L2, and CDR-L3 of the amino acid sequences of 558, 559, and 560; and/or

(ii) The second targeting portion comprises: (a) a CD133 binder comprising CDR-H1, CDR-H2, and CDR-H3 comprising amino acid sequences of SEQ ID NO: 289, 565, and 538, respectively, and CDR-L1, CDR-L2, and CDR-L3 comprising amino acid sequences of SEQ ID NO: 540, 541, and 542, respectively; (b) a CD133 binder comprising CDR-H1, CDR-H2, and CDR-H3 comprising amino acid sequences of SEQ ID NO: 566, 567, and 547, respectively, and CDR-L1, CDR-L2, and CDR-L3 comprising amino acid sequences of SEQ ID NO: 549, 550, and 551, respectively; (c) a CD133 binder comprising amino acid sequences of SEQ ID NO: 540, 541, and 542, respectively, and CDR-L3 comprising amino acid sequences of SEQ ID NO: 549, 550, and 551, respectively; (d) A CD133 binder comprising CDR-H1, CDR-H2, and CDR-H3 containing amino acid sequences of SEQ ID NO: 570, 571, and 529, respectively, and CDR-L1, CDR-L2, and CDR-L3 containing amino acid sequences of SEQ ID NO: 531, 532, and 533, respectively; or (e) a CD133 binder comprising CDR-H1, CDR-H2, and CDR-H3 containing amino acid sequences of SEQ ID NO: 572, 569, and 520, respectively, and CDR-L1, CDR-L2, and CDR-L3 containing amino acid sequences of SEQ ID NO: 572, 569, and 520, respectively; and CDR-L1, CDR-L2, and CDR-L3 containing amino acid sequences of SEQ ID NO: 531, 532, and 533, respectively; or (e) a CD133 binder comprising CDR-H1, CDR-H2, and CDR-H3 containing amino acid sequences of SEQ ID NO: 572, 573, and 556, respectively, and CDR-L3 containing amino acid sequences of SEQ ID NO: 572, 573, and 556, respectively. CDR-L1, CDR-L2, and CDR-L3 of amino acid sequences 558, 559, and 560.

29. The lipid particles according to any one of claims 1 to 25, wherein:

(i) The first targeting portion comprises: (a) a CD133 binder comprising a heavy chain variable (VH) region containing the amino acid sequence of SEQ ID NO: 535 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; and a light chain variable (VL) region containing the amino acid sequence of SEQ ID NO: 539 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; and (b) a CD133 binder comprising a VH region containing the amino acid sequence of SEQ ID NO: 544 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; and a heavy chain variable (VL) region containing the amino acid sequence of SEQ ID NO: 539 ...; and a heavy chain variable (VL) region containing the amino acid sequence of SEQ ID NO: 539; and a heavy chain variable (VL) region containing the amino acid sequence of SEQ ID NO: 539; and a heavy chain variable (VL) region containing the amino acid sequence of SEQ ID NO: 539; and a heavy chain variable (VL) region containing the amino acid sequence of SEQ ID NO: 539; and a heavy chain variable (VL) region containing the amino acid sequence of SEQ ID NO: 539; and a heavy chain variable (VL) region containing the amino acid (c) A CD133 binder comprising a VL region containing the amino acid sequence of SEQ ID NO: 548 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it; and a VH region containing the amino acid sequence of SEQ ID NO: 517 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it; and a VL region containing the amino acid sequence of SEQ ID NO: 521 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it; and (d) a CD133 binder comprising a VL region containing the amino acid sequence of SEQ ID NO: 548 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. The VH region containing the amino acid sequence of SEQ ID NO: 526 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it; and the VL region containing the amino acid sequence of SEQ ID NO: 530 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it; or (e) a CD133 binder comprising the VH region containing the amino acid sequence of SEQ ID NO: 553 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it; and the VL region containing the amino acid sequence of SEQ ID NO: 557 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it; and/or

(ii) The second targeting portion comprises: (a) a CD133 binder comprising a heavy chain variable (VH) region containing the amino acid sequence of SEQ ID NO: 535 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; and a light chain variable (VL) region containing the amino acid sequence of SEQ ID NO: 539 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; and (b) a CD133 binder comprising a VH region containing the amino acid sequence of SEQ ID NO: 544 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; and a light chain variable (VL) region containing the amino acid sequence of SEQ ID NO: 539 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; and a light chain variable (VL) region containing the amino acid sequence of SEQ ID NO: 539. (c) A CD133 binder comprising a VL region containing the amino acid sequence of SEQ ID NO: 517 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it; and a VL region containing the amino acid sequence of SEQ ID NO: 521 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it; and (d) a CD133 binder comprising a VH region containing the amino acid sequence of SEQ ID NO: 521 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. The VH region containing the amino acid sequence of SEQ ID NO: 526 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it; and the VL region containing the amino acid sequence of SEQ ID NO: 530 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it; or (e) a CD133 binder containing the VH region containing the amino acid sequence of SEQ ID NO: 553 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it; and the VL region containing the amino acid sequence of SEQ ID NO: 557 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

30. The lipid particle according to any one of claims 1 to 29, wherein...

(a) The first targeting portion comprises a CD133 binder, which includes a VH region containing the amino acid sequence of SEQ ID NO: 517 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it; and

(b) The second targeting portion comprises a CD133 binder comprising a VH region containing an amino acid sequence of SEQ ID NO: 526 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with it.

31. The lipid particles according to any one of claims 1 to 29, wherein:

(a) The first targeting portion comprises a CD133 binder, which includes a VH region containing the amino acid sequence of SEQ ID NO: 526 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it; and

(b) The second targeting portion comprises a CD133 binder comprising a VH region containing an amino acid sequence of SEQ ID NO: 517 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with it.

32. The lipid particle of any one of claims 1 to 31, wherein the first targeting portion and the second targeting portion bind to different epitopes on CD133.

33. The lipid particle of any one of claims 25 to 32, wherein the lipid particle further comprises a second paramyxovirus envelope attachment protein, the second paramyxovirus envelope attachment protein being a variant paramyxovirus envelope attachment protein comprising one or more amino acid substitutions to reduce nativeness relative to wild-type paramyxovirus envelope attachment protein not comprising said one or more amino acid substitutions.

34. The lipid particle of claim 33, wherein the second paramyxovirus envelope attachment protein, as a variant paramyxovirus envelope attachment protein, comprises one or more amino acid substitutions, said one or more amino acid substitutions corresponding to amino acid substitutions numbered from the group consisting of E501A, W504A, Q530A and E533A as shown in SEQ ID NO:1.

35. A lipid particle, said lipid particle comprising:

(a) A first-level targeting attachment protein comprising (i) a first paramyxoviral envelope attachment protein; and (ii) a first targeting portion for CD117; and a second targeting portion for CD117; and

(b) at least one paramyxoviral fusion (F) protein; and

36. The lipid particles according to claims 1 to 25 and 35, wherein...

(a) The first targeting portion comprises a CD117 binder comprising a VHH single-domain antibody, said VHH single-domain antibody comprising CDR-H1, CDR-H2 and CDR-H3 contained in the amino acid sequence selected from the group consisting of SEQ ID NO: 512-515; and/or wherein

(b) The second targeting portion comprises a CD117 binder comprising a VHH single-domain antibody comprising CDR-H1, CDR-H2 and CDR-H3 contained in the amino acid sequence selected from the group consisting of SEQ ID NO: 512-515.

37. The lipid particles according to any one of claims 1 to 25 and 35 to 36, wherein...

(a) The first targeting portion comprises a VHH single-domain antibody, the VHH single-domain antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 512-515 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it; and/or

(b)) The second targeting portion comprises a VHH single-domain antibody, the VHH single-domain antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 512-515 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with it.

38. The lipid particles of claim 36 or claim 37, wherein...

(a) The first targeting portion comprises VHH, said VHH comprising the amino acid sequence shown in any of SEQ ID NO: 512-515; and/or

(b) The second targeting portion comprises VHH, which comprises the amino acid sequence shown in any of SEQ ID NO: 512-515.

39. The lipid particle of any one of claims 1 to 25 and 35 to 38, wherein the first targeting portion and the second targeting portion bind to different epitopes on CD117.

40. The lipid particle of any one of claims 35 to 39, wherein the lipid particle further comprises a second paramyxovirus envelope attachment protein, the second paramyxovirus envelope attachment protein being a variant paramyxovirus envelope attachment protein comprising one or more amino acid substitutions to reduce nativeness relative to wild-type paramyxovirus envelope attachment protein not comprising said one or more amino acid substitutions.

41. The lipid particle of claim 40, wherein the second paramyxovirus envelope attachment protein, as a variant paramyxovirus envelope attachment protein, comprises one or more amino acid substitutions, said one or more amino acid substitutions corresponding to amino acid substitutions numbered from the group consisting of E501A, W504A, Q530A and E533A as shown in SEQ ID NO:1.

42. A lipid particle, said lipid particle comprising:

(a) A first-level targeting attachment protein comprising (i) a first paramyxoviral envelope attachment protein; and (ii) a first targeting portion for CD8; and a second targeting portion for CD8; and

(b) at least one paramyxoviral fusion (F) protein; and

43. The lipid particles of claim 42, wherein...

(i) The first targeting portion is scFv and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 489, 496, 501 or 508, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with it; and/or

(ii) The second targeting portion is scFv and contains an amino acid sequence selected from the group consisting of SEQ ID NO: 489, 496, 501 or 508 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with it.

44. The lipid particles according to claims 1 to 25 and 42, wherein...

(a) The first targeting portion comprises a CD8 binding agent comprising a VHH single-domain antibody, said VHH single-domain antibody comprising CDR-H1, CDR-H2 and CDR-H3 contained in the amino acid sequence of SEQ ID NO: 377; and/or wherein

(b) The second targeting portion comprises a CD8 binding agent comprising a VHH single-domain antibody comprising CDR-H1, CDR-H2 and CDR-H3 contained in the amino acid sequence of SEQ ID NO: 377.

45. The lipid particles of claim 42 or 44, wherein...

(a) The first targeting portion comprises VHH, which contains the amino acid sequence shown in SEQ ID NO: 377; and/or

(b) The second targeting portion comprises VHH, which contains the amino acid sequence shown in SEQ ID NO: 377.

46. The lipid particle according to any one of claims 42 to 45, wherein...

(a) The first targeting portion comprises VHH, said VHH comprising the amino acid sequence shown in SEQ ID NO: 377 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it; and/or

(b) The second targeting portion is scFv and contains an amino acid sequence selected from the group consisting of SEQ ID NO: 501 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with it.

47. The lipid particle according to any one of claims 42 to 45, wherein...

(a) The first targeting portion is scFv and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 501 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it; and

(b) The second targeting portion comprises VHH, which comprises the amino acid sequence shown in SEQ ID NO: 377 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with it.

48. The lipid particle of any one of claims 1 to 24 and 42 to 47, wherein the first targeting portion and the second targeting portion bind to different epitopes on CD8.

49. The lipid particle of any one of claims 16 to 48, wherein the different epitopes are non-overlapping.

50. The lipid particle of any one of claims 16 to 49, wherein the first targeting portion and the second targeting portion bind the different epitopes in a non-competitive manner.

51. The lipid particle of claims 1 to 50, wherein each of the first targeting portion and the second targeting portion is independently selected from the group consisting of: antibody or antigen-binding fragments, DARPin, aptamers, affimers, affinity molecules, desmin, Avimer, monomers, anticalin, fynomer, and targeting peptides.

52. The lipid particle of any one of claims 1 to 51, wherein the first targeting portion and the second targeting portion are independently selected from the group consisting of single-domain antibodies or single-chain variable fragments (scFv).

53. The lipid particle of any one of claims 42 to 52, wherein the lipid particle further comprises a second paramyxovirus envelope attachment protein, the second paramyxovirus envelope attachment protein being a variant paramyxovirus envelope attachment protein comprising one or more amino acid substitutions to reduce nativeness relative to wild-type paramyxovirus envelope attachment protein not comprising said one or more amino acid substitutions.

54. The lipid particle of claim 49, wherein the second paramyxovirus envelope attachment protein, as a variant paramyxovirus envelope attachment protein, comprises one or more amino acid substitutions, said one or more amino acid substitutions corresponding to amino acid substitutions numbered from the group consisting of E501A, W504A, Q530A and E533A as shown in reference SEQ ID NO: 1.

55. A lipid particle, said lipid particle comprising:

(a) A retargeting attachment protein comprising (i) a first paramyxoviral envelope attachment protein; and (ii) a first targeting portion and a second targeting portion targeting CD133.

(b) A second paramyxovirus envelope attachment protein, wherein the second paramyxovirus envelope attachment protein is a variant paramyxovirus envelope attachment protein containing one or more amino acid substitutions, the one or more amino acid substitutions reducing the native affinity relative to the wild-type paramyxovirus envelope attachment protein without the one or more amino acid substitutions; and

(c) at least one paramyxoviral fusion (F) protein; and

56. A lipid particle, said lipid particle comprising:

(a) A retargeting attachment protein comprising (i) a first paramyxoviral envelope attachment protein; and (ii) a first targeting portion and a second targeting portion targeting CD117.

(c) at least one paramyxoviral fusion (F) protein; and

57. A lipid particle, said lipid particle comprising:

(a) A retargeting attachment protein comprising (i) a first paramyxoviral envelope attachment protein; and (ii) a first targeting portion and a second targeting portion targeting CD8.

(c) at least one paramyxoviral fusion (F) protein; and

58. The lipid particle of claims 24 to 57, wherein the first paramyxovirus envelope attachment protein is a variant paramyxovirus envelope attachment protein.

59. The lipid particle of claim 58, wherein the variant paramyxovirus envelope attachment protein comprises one or more amino acid substitutions, the one or more amino acid substitutions reducing the native orientation relative to the wild-type paramyxovirus envelope attachment protein not containing the one or more amino acid substitutions.

60. The lipid particle of any one of claims 25 to 59, wherein the second paramyxovirus envelope attachment protein is a variant paramyxovirus envelope attachment protein.

61. The lipid particle of claim 60, wherein the variant paramyxovirus envelope attachment protein comprises one or more amino acid substitutions, the one or more amino acid substitutions reducing the native orientation relative to the wild-type paramyxovirus envelope attachment protein without the one or more amino acid substitutions.

62. The lipid particle of any one of claims 1 to 61, wherein the first targeting portion is selected from the group consisting of single-domain antibodies or single-chain variable fragments (scFv).

63. The lipid particle of any one of claims 1 to 62, wherein the second targeting portion is selected from the group consisting of single-domain antibodies or single-chain variable fragments (scFv).

64. The lipid particle of claim 54 and claim 62 or 63, wherein the single-domain antibody is VHH.

65. The lipid particle of any one of claims 1 to 64, wherein the first variant paramyxovirus envelope attachment protein and the second variant paramyxovirus envelope attachment protein are identical.

66. The lipid particle of any one of claims 1 to 65, wherein the first paramyxovirus envelope attachment protein and the second paramyxovirus envelope attachment protein are different.

67. The lipid particle of any one of claims 1 to 66, wherein the first paramyxoviral envelope attachment protein is an envelope attachment protein from Nipah virus, Hendra virus, or measles virus, or a variant of any of the foregoing or its biologically active portion.

68. The lipid particle of any one of claims 1 to 67, wherein the first paramyxovirus envelope attachment protein is a wild-type paramyxovirus G protein, H protein, or HN protein, or a variant or bioactive portion of any of the foregoing.

69. The lipid particle of claim 67 or claim 68, wherein the first paramyxoviral envelope attachment protein is wild-type Nipah virus G (NiV-G) protein, or a variant or bioactive portion of NiV-G.

70. The lipid particle of any one of claims 67 to 69, wherein the first paramyxoviral envelope attachment protein is a variant NiV-G, the variant NiV-G being a variant or bioactive portion of wild-type NiV-G.

71. The lipid particle of any one of claims 1 to 70, wherein the second paramyxoviral envelope attachment protein is an envelope attachment protein from Nipah virus, Hendra virus, or measles virus, or a variant or bioactive portion of any of the foregoing.

72. The lipid particle of any one of claims 1 to 71, wherein the second paramyxovirus envelope attachment protein is a wild-type paramyxovirus G protein, H protein, or HN protein, or a variant or bioactive portion of any of the foregoing.

73. The lipid particle of claim 71 or claim 72, wherein the second paramyxoviral envelope attachment protein is wild-type Nipah virus G (NiV-G) protein, or a variant or bioactive portion of NiV-G.

74. The lipid particle of any one of claims 1 to 73, wherein the second paramyxoviral envelope attachment protein is a variant NiV-G, the variant NiV-G being a variant or bioactive portion of wild-type NiV-G.

75. The lipid particle of any one of claims 3 to 70, wherein the second paramyxoviral envelope attachment protein is a variant paramyxoviral envelope glycoprotein or its biologically active portion derived from Nipah virus, Hendra virus, or measles virus.

76. The lipid particle of any one of claims 3 to 70 and 75, wherein the second paramyxovirus envelope attachment protein is a variant of wild-type paramyxovirus G protein, H protein, or HN protein, or a biologically active portion thereof.

77. The lipid particle of claim 73 or 74, wherein the variant is variant NiV-G, which is a variant of the wild-type Nipah virus G (NiV-G) protein or its biologically active portion.

78. The lipid particle of claim 70, claim 74 or claim 77, wherein the variant NiV-G is truncated by up to 40 consecutive amino acids at or near the N-terminus of the wild-type NiV-G shown in SEQ ID NO:1.

79. The lipid particle of any one of claims 70, 74, 77 and 78, wherein the variant NiV-G has a truncated amino acid 2-34 of the wild-type NiV-G shown in SEQ ID NO:1.

80. The lipid particles of any one of claims 70, 73, and 77 to 79, wherein the variant NiV-G exhibits reduced binding to hepatin B2 or hepatin B3.

81. The lipid particle of claim 80, wherein the variant NiV-G comprises: one or more amino acid substitutions, the one or more amino acid substitutions corresponding to amino acid substitutions numbered from the group consisting of E501A, W504A, Q530A and E533A as shown in SEQ ID NO:1.

82. The lipid particles of claim 80 or claim 81, wherein the variant NiV-G comprises amino acid substitutions E501A, W504A, Q530A and E533A as indicated by reference to SEQ ID NO:1.

83. The lipid particle of any one of claims 70, 74, and 77 to 79, wherein the variant NiV-G has an amino acid sequence that is equal to or about 80%, at least equal to or about 81%, at least equal to or about 82%, at least equal to or about 83%, equal to or about 84%, at least equal to or about 85%, at least equal to or about 86%, or at least equal to or about 87%, at least equal to or about 88%, or at least equal to or about 89%, at least equal to or about 90%, at least equal to or about 91%, at least equal to or about 92%, at least equal to or about 93%, at least equal to or about 94%, at least equal to or about 95%, equal to or about 96%, at least equal to or about 97%, at least equal to or about 98%, or at least equal to or about 99% sequence identity with SEQ ID NO:228.

84. The lipid particles of any one of claims 70, 74 and 77 to 79, wherein the variant NiV-G has the amino acid sequence shown in SEQ ID NO:228.

85. The lipid particle of any one of claims 1 to 84, wherein the at least one paramyxovirus fusion (F) protein is an F protein from Hennipa virus, or its biologically active portion or a variant thereof.

86. The lipid particle of claim 85, wherein the Hennipa virus is a Hendra virus.

87. The lipid particle of claim 85, wherein the Hennipa virus is a Nipah virus.

88. The lipid particle of any one of claims 1 to 87, wherein the paramyxovirus F protein is a wild-type NiV-F protein or a variant or biologically active portion thereof.

89. The lipid particle of any one of claims 1 to 88, wherein the paramyxovirus F protein is a variant NiV-F, the variant NiV-F being a variant or bioactive portion of the wild-type NiV-F protein.

90. The lipid particle of claim 89, wherein the variant NiV-F is truncated at the C-terminus of the wild-type NiV-F shown in SEQ ID NO:235 by up to 22 consecutive amino acids, optionally excluding the initiating methionine.

91. The lipid particle of claim 89 or claim 90, wherein the variant NiV-F protein is a truncated NiV-F lacking amino acids 525-546 of SEQ ID NO:235.

92. The lipid particle of any one of claims 89 to 91, wherein the variant NiV-F has the amino acid sequence shown in SEQ ID NO: 227 or an amino acid sequence having sequence identity with SEQ ID NO: 227 of 80% or more, at least 81% or more, at least 82% or more, at least 83% or more, at least 84% or more, at least 85% or more, at least 86% or more, or at least 87% or more, at least 88% or more, or at least 89% or more, at least 90% or more, at least 91% or more, at least 92% or more, at least 93% or more, at least 94% or more, at least 95% or more, at least 96% or more, at least 97% or more, at least 98% or more, or at least 99% of the sequence.

93. The lipid particle of any one of claims 89 to 92, wherein the variant NiV-F has the amino acid sequence shown in SEQ ID NO: 227.

94. The lipid particle of any one of claims 1 to 93, wherein the paramyxovirus F protein is an F0 precursor or a proteolytically cleaved form comprising F1 and F2 subunits.

95. The lipid particles of claim 94, wherein the protein hydrolysis cleavage form is a cathepsin L cleavage product.

96. The lipid particle of any one of claims 1 to 95, wherein the paramyxovirus envelope attachment protein and the first and second targeting portions are connected via one or more adapters.

97. The lipid particle of claim 96, wherein the one or more adapters are one or more peptide adapters.

98. The lipid particle of any one of claims 1 to 97, wherein the retargeting attachment protein comprises, in sequence: paramyxovirus attachment protein - first adapter - first targeting portion - second adapter - second targeting portion.

99. The lipid particle of claim 98, wherein the first connector and/or the second connector are independently peptide connectors.

100. The lipid particle of claim 99, wherein the first connector and the second connector are identical.

101. The lipid particle of claim 99, wherein the first connector and the second connector are different.

102. The lipid particle of claim 97 or claim 99, wherein the length of the peptide linker is 2 to 65 amino acids.

103. The lipid particle of any one of claims 99 to 102, wherein the peptide linker is a flexible linker comprising GS, GGS, GGGGS, GGGGGS, or a combination thereof.

104. The lipid particle of any one of claims 99 to 103, wherein the peptide linker is selected from: (GGS)n, wherein n is 1 to 10; (GGGGS)n, wherein n is 1 to 10; or (GGGGGS)n, wherein n is 1 to 6.

105. The lipid particle according to any one of claims 1 to 104, wherein the peptide linker is selected from SEQ ID NO:589-592.

106. The lipid particle of any one of claims 1 to 105, wherein the lipid particle further comprises one or more additional paramyxoviral envelope attachment glycoproteins embedded in the lipid bilayer.

107. The lipid particle of claim 106, wherein the one or more additional paramyxovirus envelope attachment glycoproteins are retargeting attachment proteins comprising paramyxovirus envelope attachment proteins and additional targeting portions.

108. The lipid particle of any one of claims 1 to 107, wherein the at least one paramyxovirus fusion (F) protein exhibits fusion activity with the target cell when at least one paramyxovirus envelope attachment protein binds to a target molecule on the target cell.

109. The lipid particle according to any one of claims 1 to 108, wherein the lipid particle comprises viral nucleic acid.

110. The lipid particle of claim 109, wherein the viral nucleic acid comprises one or more (e.g., all) of the following nucleic acid sequences: 5' LTR (e.g., containing U5 and lacking a functional U3 domain), Psi packaging element (Psi), central polypurine bundle (cPPT)/central termination sequence (CTS) (e.g., DNA flap), poly-A tail sequence, post-transcriptional regulatory element (e.g., WPRE), Rev response element (RRE), and 3' LTR (e.g., containing U5 and lacking a functional U3 domain).

111. The lipid particle according to any one of claims 1 to 110, wherein the lipid particle is a viral vector.

112. The lipid particle according to any one of claims 1 to 111, wherein the lipid particle is a retroviral vector.

113. The lipid particle according to any one of claims 1 to 111, wherein the lipid particle is a lentiviral vector.

114. The lipid particle according to any one of claims 1 to 108 and 111 to 113, wherein the lipid particle does not contain viral genomic nucleic acid.

115. The lipid particle according to any one of claims 1 to 108 and 111 to 113, wherein the lipid particle does not contain viral genomic DNA.

116. The lipid particle according to any one of claims 1 to 110 and 115, wherein the lipid particle is a virus-like particle.

117. The lipid particle according to any one of claims 1 to 110, 115 and 116, wherein the lipid particle is a retrovirus-like particle.

118. The lipid particle according to any one of claims 1 to 110, 115 and 116, wherein the lipid particle is a lentivirus-like particle.

119. The lipid particle of any one of claims 1 to 118, wherein the lipid particle is produced as a formulation having an increased titer compared to a reference lipid particle formulation similarly produced but having only the first targeting attachment protein.

120. The lipid particles of any one of claims 5 to 119, wherein the lipid particles are produced as a formulation having an increased titer compared to a reference lipid particle formulation similarly produced but without the second paramyxovirus envelope attachment protein, the second paramyxovirus envelope attachment protein being a variant paramyxovirus envelope attachment protein containing one or more substitutions to reduce native orientation relative to wild-type paramyxovirus envelope attachment proteins not containing said one or more substitutions.

121. The lipid particle of claim 120, wherein the second paramyxovirus envelope attachment protein, as a variant paramyxovirus envelope attachment protein, comprises one or more amino acid substitutions, said one or more amino acid substitutions corresponding to amino acid substitutions numbered from the group consisting of E501A, W504A, Q530A and E533A as shown in SEQ ID NO:1.

122. The lipid particles of any one of claims 1 to 119, wherein the lipid particles are generated in a suspension culture as a formulation having an increased titer compared to a reference lipid particle formulation similarly generated but having only the first targeting attachment protein.

123. The lipid particle of any one of claims 120 to 122, wherein the titer increase is equal to or greater than 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 2 times, 2.5 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more.

124. The lipid particle of any one of claims 1 to 123, wherein the lipid particle further comprises an exogenous agent for delivery to target cells.

125. The lipid particle of claim 124, wherein the exogenous agent is present in the lumen.

126. The lipid particles of claim 124 or 125, wherein the exogenous agent is a protein or nucleic acid, optionally wherein the nucleic acid is DNA or RNA.

127. The lipid particle of any one of claims 124 to 126, wherein the exogenous agent is a nucleic acid encoding cargo for delivery to the target cells.

128. The lipid particle of any one of claims 124 to 127, wherein the exogenous agent is or encodes a therapeutic agent, a diagnostic agent, or a genome-modifying enzyme.

129. The lipid particle of any one of claims 124 to 128, wherein the exogenous agent encodes a membrane protein, optionally wherein the membrane protein is an antigen receptor for targeting cells expressed by or associated with a disease or condition.

130. The lipid particle of claim 129, wherein the membrane protein is a chimeric antigen receptor (CAR).

131. The lipid particle of any one of claims 124 to 128, wherein the exogenous agent is a nucleic acid containing a payload gene for correcting a genetic defect, optionally a genetic defect in the target cell, optionally wherein the genetic defect is associated with a liver cell or hepatocyte.

132. The lipid particle of any one of claims 124 to 131, wherein the binding of the paramyxovirus envelope attachment protein or its bioactive portion to a target molecule expressed on the surface of a target cell mediates the fusion of the particle with the target cell and the delivery of the exogenous agent to the target cell.

133. The lipid particles of any one of claims 124 to 132, wherein the exogenous agent is delivered to 10%, 20%, 30%, 40%, 50%, or 60% of the target cells.

134. The lipid particle of any one of claims 124 to 133, wherein the delivery of the exogenous agent to the target cell is increased compared to a reference particle formulation that is similarly produced but only has a first-level targeting attachment protein.

135. The lipid particles of claim 134, wherein the delivery to the target cells is increased by 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 2 times, 2.5 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more.

136. A production cell comprising (a) a nucleic acid encoding a retargeting attachment protein, the retargeting attachment protein comprising a paramyxoviral envelope attachment protein, the paramyxoviral envelope attachment protein being linked to (i) a first targeting portion of a first target molecule expressed on the surface of a target cell, and (ii) a second targeting portion of a second target molecule expressed on the surface of a target cell; and

(b) Nucleic acid encoding at least one paramyxovirus fusion (F) protein.

137. The production cell of claim 136, further comprising nucleic acid encoding a second paramyxovirus envelope attachment protein, the second paramyxovirus envelope attachment protein being a variant paramyxovirus envelope attachment protein containing one or more amino acid substitutions to reduce native tropism relative to the wild-type paramyxovirus envelope attachment protein not containing said one or more amino acid substitutions.

138. The production cell as claimed in claim 136 or 137, wherein the cell further comprises viral nucleic acid.

139. The production cell of claim 138, wherein the viral nucleic acid is a lentiviral nucleic acid.

140. The production cell according to any one of claims 136 to 139, wherein the cell is a mammalian cell.

141. The production cells according to any one of claims 136 to 140, wherein the production cells are selected from the group consisting of: CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells.

142. The production cell according to any one of claims 136 to 141, wherein the production cell comprises 293T cells.

143. The production cell of any one of claims 137 to 142, wherein the viral nucleic acid lacks one or more genes involved in viral replication.

144. The production cell of any one of claims 137 to 143, wherein the viral nucleic acid comprises a nucleic acid encoding a viral packaging protein selected from one or more of Gag, Pol, Rev, and Tat.

145. The production cell of any one of claims 138 to 144, wherein the viral nucleic acid comprises one or more (e.g., all) of the following nucleic acid sequences: 5' LTR (e.g., containing U5 and lacking a functional U3 domain), Psi packaging element (Psi), central polypurine bundle (cPPT)/central termination sequence (CTS) (e.g., DNA flap), poly-A tail sequence, post-transcriptional regulatory element (e.g., WPRE), Rev response element (RRE), and 3' LTR (e.g., containing U5 and lacking a functional U3 domain).

146. A method for preparing lipid particles, the method comprising:

a) Providing production cells as described in any one of claims 136 to 145;

b) Culture the cells under conditions that allow for the generation of the lipid particles, and

c) Prepare the lipid particles by isolating, enriching, or purifying the lipid particles from the cells.

147. The method of claim 146, wherein the lipid particles are pseudotyped lentiviral vectors.

148. A lipid particle, said lipid particle being generated by the method of claim 146 or claim 147.

149. A composition comprising a plurality of lipid particles as described in any one of claims 1 to 135 and 148.

150. The composition of claim 149, wherein the composition further comprises a pharmaceutically acceptable carrier.

151. A method of transducing cells, the method comprising contacting the cells with lipid particles as described in any one of claims 1 to 135 and 148 or with a composition as described in claim 149 or claim 150.

152. A method of delivering an exogenous agent to target cells, the method comprising contacting a lipid particle as described in any one of claims 124 to 135 and 148 or a composition as described in claim 149 or claim 150 with the target cells.

153. The method of claim 151 or claim 152, wherein the contact is external or detached.

154. The method of claim 151 or claim 152, wherein the contact is within the subject's body.

155. A method of delivering an exogenous agent to the cells of a subject, the method comprising administering to the subject a lipid particle as described in any one of claims 124 to 135 and 148 or a composition as described in claim 149 or claim 150.

156. The method of claim 155, wherein the exogenous agent is or is encoded as a therapeutic agent for treating the disease or condition of the subject.

157. A method of treatment comprising administering to a subject lipid particles as described in any one of claims 124 to 135 and 148 or a composition as described in claim 149 or claim 150.

158. The method of any one of claims 152 to 157, wherein the exogenous agent is or encodes a membrane protein, optionally chimeric with an antigen receptor, for targeting an antigen associated with a disease or condition of the subject.

159. The method of any one of claims 152 to 157, wherein the exogenous agent is used in gene therapy to correct a genetic defect in the subject or to replace a defective or missing gene.

160. The method of any one of claims 155 to 159, wherein the subject is a human subject.

161. The method of any one of claims 151 to 160, wherein the method further comprises administering to the subject one or more agents that stimulate the mobilization of bone marrow cells from the bone marrow into the peripheral blood.

162. The method of any one of claims 151 to 161, wherein the subject has previously been administered one or more agents that stimulate bone marrow cells to be mobilized from the bone marrow into the peripheral blood.

163. The method of claim 161 or 162, wherein the one or more stimulating agents are selected from the group consisting of: stem cell factor (SCF), small molecule VLA-4 inhibitor BI05192, BOP (N-(benzenesulfonyl)-L-prolyl-L-O-(1-pyrrolidinylcarbonyl)tyrosine), heparin, granulocyte colony-stimulating factor (G-CSF), MGTA-145, and praxavir (AMD3100).

164. The method of any one of claims 161 to 163, wherein the one or more stimulating agents for mobilization include G-CSF.

165. The method of any one of claims 161 to 164, wherein the one or more stimulating agents for mobilization include plexafor.