WO2025240518A1

WO2025240518A1 - Modified lentiviral transfer plasmids

Info

Publication number: WO2025240518A1
Application number: PCT/US2025/029200
Authority: WO
Inventors: Raymond Liu; Justin TAFT; Lexus R. Johnson
Original assignee: Dispatch Biotherapeutics Inc
Current assignee: Dispatch Biotherapeutics Inc
Priority date: 2024-05-14
Filing date: 2025-05-13
Publication date: 2025-11-20
Anticipated expiration: 2026-11-14

Abstract

Provided herein are improved transfer plasmids that can be used to generate lentivirus and uses thereof, such as for producing lentivirus. Also provided are lentivirus and methods of transducing cells using same.

Description

MODIFIED LENTIVIRAL TRANSFER PLASMIDS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63/647,557 filed on May 14, 2024, entitled “MODIFIED LENTIVIRAL TRANSFER PLASMIDS”, the contents of which are incorporated by reference in their entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 307612001740SeqList.xml created May 13, 2025, which is 133,514 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

FIELD

[0003] The present disclosure provides a transfer plasmid that can be used to generate lentivirus with improved performance. Among provided embodiments, are methods of using the provided transfer plasmids, such as for producing lentivirus. Also provided are lentivirus and methods of transducing cells using same.

BACKGROUND

[0004] Expression of a polynucleotide encoding a nucleotide sequence of interest (NOI) (also referred to as “payload” or in some cases “transgene”) is important for many cell therapies. One method of introducing a NOI, such as a recombinant receptor and/or additional polypeptide, to a cell involves the use of a viral vector system, which involves transfection of a host cell with a viral transfer plasmid to generate viral vectors encoding the NOI. The viral vectors can then be used to transduce a cell (i.e., a target cell) to induce expression of the NOI. Viral vectors useful in such methods include lentiviruses, adenoviruses, and adeno-associated viruses.

[0005] Viral vector performance, including expression of a NOI, can be impacted by upstream processes of generating the viral vector, including vector size of the viral transfer plasmid. There exists a need for improved viral transfer plasmids suitable for rapid and efficient production of viral vectors that efficiently transduce target cells and express the NOI. Provided herein are embodiments that meet such needs. SUMMARY

[0006] In some aspects, provided herein is a transfer plasmid comprising a nucleic acid sequence comprising in order: (a) a polynucleotide sequence encoding a partial gag, wherein the polynucleotide sequence comprises a lentivirus 5’ Long Terminal Repeat (LTR) or a modified 5’ LTR; a y packaging signal, a Rev response element (RRE), and a nucleotide sequence encoding a gp41 peptide sequence; (b) a central polypurine tract/central termination sequence (cPPT/CTS); and (c) a 3' long terminal repeat (LTR) that is an inactivated 3' LTR or a self-inactivating 3' LTR; wherein the transfer plasmid is devoid of: a polynucleotide encoding a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE); and a polynucleotide sequence region between the polynucleotide sequence encoding the partial gag of (a) and the cPPT/CTS sequence of (b) that comprises a portion of the pol integrase polynucleotide comprising the core domain.

[0007] In some aspects, provided herein is a transfer plasmid comprising a nucleic acid sequence comprising in order: (a) a lentivirus 5’ Long Terminal Repeat (LTR) or a modified 5’ LTR; (b) a y packaging signal; (c) a Rev response element (RRE); (d) a nucleotide sequence encoding a gp41 peptide sequence; (e) a central polypurine tract/central termination sequence (cPPT/CTS); and (f) a 3' long terminal repeat (LTR) that is an inactivated 3' LTR or a self-inactivating 3' LTR; wherein the transfer plasmid is devoid of: a polynucleotide sequence region downstream of the nucleotide sequence encoding the gp41 peptide sequence and upstream of the cPPT/CTS sequence that comprises a portion of the pol integrase polynucleotide comprising the core domain; and a polynucleotide encoding a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0008] In some embodiments, the polynucleotide sequence region is 350-370 nucleotides in length. In some embodiments, the polynucleotide sequence region is about 367 nucleotides in length.

[0009] In some embodiments, the polynucleotide sequence region comprises a nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 8. In some embodiments, the polynucleotide sequence region comprises the nucleotide sequence set forth in SEQ ID NO: 8.

[0010] In some aspects, provided herein is a transfer plasmid comprising a nucleic acid sequence comprising in order: (a) a lentivirus 5’ Long Terminal Repeat (LTR) or a modified 5’ LTR; (b) a polynucleotide encoding a portion of the gag protein, wherein the polynucleotide comprises a y packaging signal; (c) a partial env sequence comprising a Rev responsive element (RRE); (d) a partial pol sequence comprising a central polypurine tract/central termination sequence (cPPT/CTS) but that is devoid of a polynucleotide sequence encoding a portion of the integrase comprising the core domain; and (e) a 3' long terminal repeat (LTR) that is an inactivated 3' LTR or a self-inactivating 3' LTR; wherein the transfer plasmid is devoid of a polynucleotide encoding a Woodchuck hepatitis virus post- transcriptional regulatory element (WPRE).

[0011] In some aspects, provided herein is a transfer plasmid comprising a nucleic acid sequence comprising in order: (a) a lentivirus 5’ Long Terminal Repeat (LTR) or a modified 5’ LTR; (b) a y packaging signal; (c) a Rev response element (RRE); (d) a nucleotide sequence encoding a gp41 peptide sequence; (e) a central polypurine tract/central termination sequence (cPPT/CTS); and (f) a 3' long terminal repeat (LTR) that is an inactivated 3' LTR or a self-inactivating 3' LTR; wherein no more than 300 nucleotides separate the nucleotide sequence encoding the gp41 peptide sequence and the cPPT/CTS and wherein the transfer plasmid is devoid of a polynucleotide encoding a Woodchuck hepatitis virus post- transcriptional regulatory element (WPRE).

[0012] In some embodiments, the transfer plasmid is devoid of at least a portion of the pol integrase polynucleotide comprising the core domain.

[0013] In some embodiments, the transfer plasmid is devoid of a nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 8. In some embodiments, the transfer plasmid is devoid of the nucleotide sequence set forth in SEQ ID NO: 8. In some embodiments, the transfer plasmid is devoid of a WPRE nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 7. In some embodiments, the transfer plasmid is devoid of a WPRE nucleotide sequence set forth in SEQ ID NO: 7.

[0014] In some aspects, provided herein is a transfer plasmid comprising a nucleic acid sequence comprising: (a) a lentivirus 5’ Long Terminal Repeat (LTR) or a modified 5’ LTR; (b) a \|/ packaging signal; (c) a Rev response element (RRE); (d) a central polypurine tract/central termination sequence (cPPT/CTS); and (e) a 3' long terminal repeat (LTR) that is an inactivated 3' LTR or a self-inactivating 3' LTR; wherein the transfer plasmid is devoid of: a polynucleotide comprising a nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 7; and a polynucleotide comprising a nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 8. [0015] In some embodiments, the transfer plasmid is devoid of a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO:7. In some embodiments, the transfer plasmid is devoid of a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 8.

[0016] In some embodiments, the transfer plasmid comprises a nucleotide sequence encoding a gp41 peptide sequence.

[0017] In some embodiments, the 5’ LTR or modified 5’ LTR comprises a U5 and R domain. In some embodiments, the 5’ LTR is a modified 5’ LTR that is truncated to lack a part or all of the U3 region. In some embodiments, the modified 5’ LTR is a modified 5’ LTR that comprises the sequence set forth in SEQ ID NO: 20.

[0018] In some embodiments, the modified 5’ LTR comprises a heterologous regulatory element that is not endogenous to a lentivirus, wherein the heterologous regulatory element is immediately upstream of the modified 5’ LTR. In some embodiments, the heterologous regulatory element is a promoter, enhancer or a promoter/enhancer. In some embodiments, the heterologous regulatory element is a cytomegalovirus enhancer, promoter or enhancer/promoter. In some embodiments, the heterologous regulatory element comprises a nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 36. In some embodiments, the heterologous regulatory element comprises a nucleotide sequence set forth in SEQ ID NO: 36.

[0019] In some embodiments, the 3’ LTR comprises a U5 and R domain. In some embodiments, the 3’ LTR is a truncated 3’ LTR comprising a deleted U3 region in which one or more nucleotide bases of the U3 region of the 3’ LTR are deleted. In some embodiments, the deleted U3 region retains the att sequence and comprises deletions of the enhancer and/or core promoter U3. In some embodiments, the deleted U3 region lacks at least one of an enhancer sequence, a TATA box, an Spl site, an NK-kappa B site, or a polypurine tract (PPT). In some embodiments, the 3’ LTR comprises the sequence set forth in SEQ ID NO: 29.

[0020] In some embodiments, the transfer plasmid comprises a polyadenylation signal within the R region or downstream of the 3’ LTR. In some embodiments, the polyadenylation signal is an SV40 polyadenylation signal.

[0021] In some embodiments, the y packaging signal comprises the nucleotide sequence set forth in SEQ ID NO: 21. In some embodiments, the Rev response element (RRE) comprises the nucleotide sequence set forth in SEQ ID NO: 22. [0022] In some embodiments, the central polypurine tract/central termination sequence (cPPT/CTS) comprises the nucleotide sequence set forth in SEQ ID NO: 10.

[0023] In some embodiments, the nucleotide sequence encoding the gp41 peptide sequence comprises the nucleotide sequence set forth in SEQ ID NO: 23.

[0024] In some embodiments, the transfer plasmid comprises an origin of replication site. In some embodiments, the origin of replication site comprises a pUC origin of replication, a SV40 origin of replication and/or an fl bacteriophage origin of replication.

[0025] In some embodiments, the transfer plasmid comprises a Kozak sequence.

[0026] In some embodiments, the transfer plasmid comprises a multiple cloning site that allows for insertion of a nucleotide sequence of interest (NOI).

[0027] In some embodiments, the transfer plasmid comprises a nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 39.

[0028] In some aspects, provided herein is a transfer plasmid comprising a nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 39.

[0029] In some embodiments, the transfer plasmid comprises the nucleotide sequence set forth in SEQ ID NO: 39. In some embodiments, the transfer plasmid further comprises a nucleotide sequence of interest (NOI) inserted within the multiple cloning site.

[0030] In some embodiments, the nucleotide sequence of interest encodes a protein, an RNA molecule, an enzyme or an antibody or any combination thereof. In some embodiments, the nucleotide sequence of interest (NOI) encodes a chimeric antigen receptor (CAR), a cytokine receptor, an RNA molecule, a synthetic antigen or any combination thereof.

[0031] In some embodiments, the nucleotide sequence of interest is a multicistronic sequence. In some embodiments, the nucleotide sequence of interest is up to 4000 base pairs in length. In some embodiments, the nucleotide sequence of interest is 2000 to 3600 base pairs in length. In some embodiments, the nucleotide sequence of interest is 2800 to 3400 base pairs in length.

[0032] In some embodiments, the transfer plasmid further comprises a non- viral promoter, wherein the non- viral promoter is operably linked to control expression of the nucleotide sequence of interest. In some embodiments, the non-viral promoter comprises an EF- la promoter.

[0033] In some aspects, provided herein is a composition comprising any of the transfer plasmids provided herein, an envelope plasmid and one or more packaging plasmids. [0034] In some embodiments, the one or more packaging plasmid comprises a first packaging plasmid encoding Gag and Pol and a second packaging plasmid encoding Rev. In some embodiments, the one or more envelope plasmid encodes a VSV-G glycoprotein.

[0035] In some aspects, provided herein is a method of producing a lentivirus comprising: (a) contacting a host cell with any of the compositions provided herein; (b) culturing the host cell under conditions that produce the lentivirus; and (c) isolating the lentivirus.

[0036] In some aspects, provided herein is a method of producing a lentivirus comprising: (a) contacting a host cell with any of the transfer plasmids provided herein and one or more packaging plasmids; (b) culturing the host cell under conditions that produce the lentivirus; and (c) isolating the lentivirus.

[0037] In some embodiments, the one or more packaging plasmid comprises a first packaging plasmid encoding Gag and Pol and a second packaging plasmid encoding Rev. In some embodiments, the one or more envelope plasmid encodes a VSV-G glycoprotein.

[0038] In some embodiments, the host cell is an adherent cell. In some embodiments, the host cell is a suspension cell.

[0039] In some embodiments, the host cell is an HEK293 cell, a COS cell, a recombinant Chinese hamster ovary (CHO), a HeLa cell, a NIH3T3 cell, a PC12 cell, a U20S cell, an A549 cell, an HT1080 cell, a CAD cell, a P19 cell, an L929 cell, an N2a cell, an MCF-7, Y79 cell, an SO-Rb50 cell, a Hep G2 cell, a DUKX-X11 cell, a J558L cell, a baby hamster kidney (BHK) cell, or a derivative thereof. In some embodiments, the host cell is an HEK293S cell, an HEK293Ts cell, an HEK293F cell, an HEK293FT cell, an HEK293FTM cell, an HEK293E cell, an LV293 cell, or an HEK293T/17 cell.

[0040] In some aspects, provided herein is a host cell comprising any of the transfer plasmids provided herein, an envelope plasmid, and one or more packaging plasmids.

[0041] In some embodiments, the one or more packaging plasmid comprises a first packaging plasmid encoding Gag and Pol and a second packaging plasmid encoding Rev. In some embodiments, the one or more envelope plasmid encodes a VSV-G glycoprotein.

[0042] In some embodiments, the host cell is an adherent cell. In some embodiments, the host cell is a suspension cell.

[0043] In some embodiments, the host cell is an HEK293 cell, a COS cell, a recombinant Chinese hamster ovary (CHO), a HeLa cell, a NIH3T3 cell, a PC 12 cell, a U20S cell, an A549 cell, an HT1080 cell, a CAD cell, a P19 cell, an L929 cell, an N2a cell, an MCF-7, Y79 cell, an SO-Rb50 cell, a Hep G2 cell, a DUKX-X11 cell, a J558L cell, a baby hamster kidney (BHK) cell, or a derivative thereof. In some embodiments, the host cell is an HEK293S cell, an HEK293Ts cell, an HEK293F cell, an HEK293FT cell, an HEK293FTM cell, an HEK293E cell, an LV293 cell, or an HEK293T/17 cell.

[0044] In some aspects, provided herein is a method of producing a lentivirus comprising: (a) culturing any of the host cells provided herein under conditions that produce the lentivirus; and (b) isolating the lentivirus.

[0045] In some aspects, provided herein is a lentivirus produced by any of the methods provided herein.

[0046] In some aspects, provided herein is a lentivirus comprising a heterologous nucleic acid sequence, comprising (a) a polynucleotide sequence encoding a partial gag, wherein the polynucleotide sequence comprises a lentivirus 5’ Long Terminal Repeat (LTR) or a modified 5’ LTR; a y packaging signal, a Rev response element (RRE), and a nucleotide sequence encoding gp41 peptide sequence; (b) a central polypurine tract/central termination sequence (cPPT/CTS); and (c) a 3' long terminal repeat (LTR) that is an inactivated 3' LTR or a self-inactivating 3' LTR; wherein the lentivirus is devoid of: a polynucleotide encoding a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE); and a polynucleotide sequence region between the polynucleotide sequence encoding the partial gag of (a) and the cPPT/CTS sequence of (b) that comprises a portion of the pol integrase polynucleotide comprising the core domain.

[0047] In some aspects, provided herein is a lentivirus comprising a nucleic acid sequence comprising in order: (a) a lentivirus 5’ Long Terminal Repeat (LTR) or a modified 5’ LTR; (b) a y packaging signal; (c) a Rev response element (RRE); (d) a nucleotide sequence encoding a gp41 peptide sequence; (e) a central polypurine tract/central termination sequence (cPPT/CTS); and (f) a 3' long terminal repeat (LTR) that is an inactivated 3' LTR or a self-inactivating 3' LTR; wherein the lentivirus is devoid of: a polynucleotide sequence region downstream of the gp41 peptide sequence and upstream of the cPPT/CTS sequence that comprises a portion of the pol integrase polynucleotide comprising the core domain; and a polynucleotide encoding a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0048] In some embodiments, the polynucleotide sequence region is 350-370 nucleotides in length. In some embodiments, the polynucleotide sequence region is about 367 nucleotides in length. [0049] In some embodiments, the polynucleotide sequence region comprises a nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 8. In some embodiments, the polynucleotide sequence region comprises the nucleotide sequence set forth in SEQ ID NO: 8.

[0050] In some aspects, provided herein is a lentivirus comprising a nucleic acid sequence comprising in order: (a) a lentivirus 5’ Long Terminal Repeat (LTR) or a modified 5’ LTR; (b) a polynucleotide encoding a portion of the gag protein, wherein the polynucleotide comprises a y packaging signal; (c) a partial env sequence comprising a Rev responsive element (RRE); (d) a partial pol sequence comprising a central polypurine tract/central termination sequence (cPPT/CTS) but that is devoid of a polynucleotide sequence encoding a portion of the integrase comprising the core domain; and (e) a 3' long terminal repeat (LTR) that is an inactivated 3' LTR or a self-inactivating 3' LTR; wherein the lentivirus is devoid of a polynucleotide encoding a Woodchuck hepatitis virus post- transcriptional regulatory element (WPRE).

[0051] In some aspects, provided herein is a lentivirus comprising a nucleic acid sequence comprising in order: (a) a lentivirus 5’ Long Terminal Repeat (LTR) or a modified 5’ LTR; (b) a y packaging signal; (c) a Rev response element (RRE); (d) a nucleotide sequence encoding a gp41 peptide sequence; (e) a central polypurine tract/central termination sequence (cPPT/CTS); and (f) a 3' long terminal repeat (LTR) that is an inactivated 3' LTR or a self-inactivating 3' LTR; wherein no more than 300 nucleotides separate the nucleotide sequence encoding the gp41 peptide sequence and the cPPT/CTS and wherein the lentivirus is devoid of a polynucleotide encoding a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0052] In some embodiments, the lentivirus is devoid of a portion of the pol integrase polynucleotide comprising the core domain. In some embodiments, the lentivirus is devoid of a nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 8. In some embodiments, the lentivirus is devoid of the nucleotide sequence set forth in SEQ ID NO: 8.

[0053] In some embodiments, the lentivirus is devoid of a WPRE nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 7. In some embodiments, the lentivirus is devoid of a WPRE nucleotide sequence set forth in SEQ ID [0054] In some embodiments, the lentivirus further comprises a nucleotide sequence of interest (NOI). In some embodiments, the NOI encodes a chimeric antigen receptor (CAR), a cytokine receptor, an RNA molecule, a synthetic antigen or any combination thereof.

[0055] In some embodiments, the lentivirus results in increased transduction efficiency of an immune cell transduced with the lentivirus compared to an immune cell transduced with a reference lentivirus.

[0056] In some embodiments, the lentivirus results in increased cytotoxicity of an immune cell transduced with the lentivirus compared to an immune cell transduced with a reference lentivirus.

[0057] In some embodiments, the lentivirus results in increased expression of naive phenotype markers of an immune cell transduced with the lentivirus compared to an immune cell transduced with a reference lentivirus.

[0058] In some embodiments, the lentivirus results in reduced expression of exhaustion markers in an immune cell transduced with the lentivirus compared to an immune cell transduced with a reference lentivirus.

[0059] In some embodiments, the reference lentivirus is identical to the lentivirus but further comprises a polynucleotide encoding a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and a polynucleotide encoding a region between a partial Gag sequence and a central polypurine tract/central termination sequence (cPPT/CTS).

[0060] In some aspects, provided herein is a method of transducing a cell comprising contacting a cell with any of the lentiviruses provided herein. In some embodiments, the method comprises incubating the cell contacted with the lentivirus under conditions that promote expression of the NOI. In some embodiments, the cell is an immune cell. In some embodiments, the cell is an effector cell. In some embodiments, the cell is a T cell or an NK cell.

[0061] In some aspects, provided herein is a transduced cell produced according to any of the methods provided herein. In some aspects, provided herein is a composition comprising a population of cells and any of the lentiviruses provided herein.

[0062] In some embodiments, the population of cells comprises a population of immune cells. In some embodiments, the population of cells comprises a population of effector cells. In some embodiments, the population of cells comprises a population of T cells or NK cells.

BRIEF DESCRIPTION OF THE DRAWINGS [0063] FIGS. 1A-1B show representative plasmid maps. FIG. 1A shows a representative pTRPE plasmid map depicting regions targeted for deletion. FIG. IB shows a representative pOpCAS plasmid map.

[0064] FIGS. 2A-2C show NOI expression in primary immune cells and cancer cells transduced with lentiviral vectors lacking cPPT (AcPPT), BTW (ABTW), WPRE (AWPRE), or EF-la-Int (AEF-la-Int). FIG. 2A shows the percentage of primary T cells expressing anti- HER2 4D5 chimeric antigen receptor (CAR). FIG. 2B shows the percentage of primary T cells expressing the anti-HER2 4D5 CAR (left panel) or GFP (right panel). FIG. 2C shows the mean fluorescent intensity (MFI) of GFP in primary T cells (right) and A549 lung carcinoma cells (left) transduced with AcPPT, ABTW, AWPRE, or AEF-la-Int.

[0065] FIGS. 3A-3B show baseline phenotype and exhaustion markers in primary T cells transduced with lentiviral vectors lacking cPPT (AcPPT), BTW (ABTW), WPRE (AWPRE), or EF-la-Int (AEF-la-Int). FIG. 3A shows baseline phenotype markers CD27 and CD45RA. FIG. 3B shows baseline exhaustion markers PD-1, LAG3 and TIM3.

[0066] FIGS. 4A-4C show the normalized cell index of A549 lung carcinoma cells across time in a T cell-mediated killing assay. T cells were transduced with lentiviral vectors lacking cPPT (AcPPT), BTW (ABTW), WPRE (AWPRE), or EF-la-Int (AEF-la-Int). FIG. 4A shows a killing assay with an effector: target ratio of 1:1. FIG. 4B shows a killing assay with an effector:target ratio of 0.5:1. FIG. 4C shows a killing assay with an effector:target ratio of 0.25:1.

[0067] FIGS. 5A-5C show the normalized cell index of SKOV3 ovarian cancer cells across time in a T cell-mediated killing assay. FIG. 5A shows a killing assay with an effector:target ratio of 1: 1. FIG. 5B shows a killing assay with an effector:target ratio of 0.5:1. FIG. 5C shows a killing assay with an effectortarget ratio of 0.25:1.

[0068] FIGS. 6A-6D show phenotype and exhaustion markers in primary T cells after a T cell-mediated killing assay. FIG. 6A shows T cell phenotype for phenotype markers CD27 and CD45RA after SKOV3 cell killing. FIG. 6B shows LAG3 and TIM3 exhaustion markers after SKOV3 cell killing. Effector: target ratios in the killing assay included 1:1, 0.5:1, and 0.25:1. FIG. 6C shows T cell phenotype for phenotype markers CD27 and CD45RA after A549 cell killing. FIG. 6D shows LAG3 and TIM3 exhaustion markers after A549 cell killing for each deletion. Effector: target ratios in the killing assay included 1:1, 0.5:1, and 0.25:1. [0069] FIGS 7A-7B show the CAR expression and T-cell mediated killing in alternative vector systems. FIG. 7A shows CAR expression (4D5) in primary T cells transduced with various lentiviral vectors lacking WPRE. FIG. 7B shows the normalized cell index of SKOV3 ovarian cancer cells across time in a T cell-mediated killing assay where cells are engineered with a different representative lentiviral vector, with an effector: target ratio of 1:1.

[0070] FIGS. 8A-8C show exemplary transfer plasmid maps.

[0071] FIGS. 9A-9C show lentivirus production and nucleotide of interest (NOI)expression in primary T cells transduced with lentiviral vectors encoding scFv-based CAR and LVTPR. FIG. 9A shows the percentage of transduced T cells with different NOI encoding different CARs (4D5 or scFv-based CAR) or GFP normalized to P24 content. FIG. 9B shows the percentage of primary T cells from two different human donors (donor 1 or donor 2) expressing chimeric antigen receptor (CAR) as measured by GFP or anti-HER24D5 CAR. FIG. 9C shows the mean fluorescent intensity (MFI) of GFP in primary T cells transduced with pTRPE or lentiviral vectors LVTPR.

[0072] FIGS. 10A-10C show baseline phenotype and exhaustion markers in T cells from two different human donors transduced with pTRPE or lentiviral vector LVTPR. FIG. 10A shows baseline phenotype markers CD27 and CD45RA in donor 1. FIG. 10B shows baseline phenotype markers CD27 and CD45RA in donor 2. FIG. 10C shows baseline exhaustion markers PD-1, LAG3 and TIM3 of T cells from the two donors.

[0073] FIGS. 11A-11L show the normalized cell index of SKOV3 ovarian cancer cells, PC3 prostate cancer cells, and A549 lung carcinoma cells across time in a T cell-mediated killing assay. In these studies, the T cells were engineered by transduction with LVTPR encoding by T cells from two different donors (donor 1 and donor 2) that had been transduced with LVTPR encoding a anti-HER2 CAR-T2A-GEP bicistronic construct. FIGS. 11A-11B (SKOV3 cells), FIGS. 11E-11F (PC3), and FIGS.11I-11J (A549) show a killing assay with an effector:target ratio of 0.25:1. FIGS. 11C-11D (SKOV3), FIGS.11G-11H (PC3), and FIGS.11K-11L (A549) show a killing assay with an effector: target ratio of 0.5:1.

[0074] FIGS. 12A-12D show CAR expression in primary T cells after T-cell mediated killing of SKOV3 ovarian cancer cells, PC3 prostate cancer cells, and A549 lung carcinoma cells. In these studies, the T cells were engineered by transduction with LVTPR encoding by T cells that had been transduced with LVTPR encoding a anti-HER2 CAR-T2A-GEP bicistronic construct. FIGS. 12A and 12C show GFP expression as a measurement of the percentage of GFP positive T cells in a killing assay with an effector:target ratio of 0.5:1. FIGS. 12B and 12D show CAR expression as a measurement of the percentage of 4D5 positive T cells in a killing assay with an effector: target ratio of 0.5:1.

[0075] FIGS. 13A-13E show the normalized cell index of target cell killing across time in a T cell-mediated killing assay by T cells that had been transduced with LVTPR encoding the larger scFv-based CAR bicistronic construct payload. FIG. 13A shows a killing assay with an effector:target ratio of 1:1. FIG. 13B shows a killing assay with an effector:target ratio of 1:6. FIG. 13C shows a killing assay with an effector: target ratio of 1:12. FIG. 13D shows a killing assay with an effector:target ratio of 1:24. FIG. 13E shows a killing assay with an effector:target ratio of 1:48.

[0076] FIG. 14 shows CAR expression in T cells transduced with 4 different lentiviral vectors: the LVTPR vector with a standard scFv-based CAR NOI with expression from a CMV promoter or an RSV promoter, or the LVTPR vector with a larger bicistronic construct NOI with expression from a CMV promoter or an RSV promoter.

DETAILED DESCRIPTION

[0077] Provided herein is an improved transfer plasmid and uses thereof. In some embodiments, provided herein is an improved transfer plasmid that can be used to produce a functional lentivirus that promotes enhanced expression of a nucleotide sequence of interest (NOI), such as a recombinant receptor (e.g., a chimeric antigen receptor) and/or additional polypeptide, in a target cell.

[0078] A problem with existing viral transfer plasmids is that NOI size (i.e., the size of the polynucleotide encoding the NOI) can impact performance, including downstream viral vector production and NOI expression. The transfer plasmid provided herein is based on the observation that deleting particular viral sequences of an HIV-1 lentiviral transfer plasmid, including a WPRE region and a region between the central polypurine tract (cPPT) and HIV- 1 partial Gag sequence (termed the “BTW” region), improves transduction efficiency and NOI expression, particularly for larger NOIs. The deletions also have the added advantage that they create more space in the viral transfer plasmid and improves viral vector production.

[0079] Embodiments of the transfer plasmid provided herein are also based on the observation that using particular promoters and/or enhancers derived from the Orthoherpesviridae (also known as Herpesviridae) family has utility to produce a lentiviral vector that exhibits improved transduction efficiency and ability to promote an increase in NOI expression from a transduced target cell. In some embodiments, the promoter and/or enhancer derived from the Orthoherpesviridae family, such as a CMV promoter, facilitates production of lentivirus with increased transduction efficiency compared to promoters derived from the Pneumoviridae family. In some embodiments, the promoter derived from the Pneumoviridae family is a respiratory syncytial virus (RSV) promoter. In some embodiments, provided transfer plasmids that include a cytomegalovirus (CMV) promoter and/or enhancer can be used to produce lentivirus that exhibit improved transduction efficiency and NOI expression in a target cell compared to transfer plasmids with alternative promoters and/or enhancers, such as a respiratory syncytial virus (RSV) promoter and/or enhancer. In some embodiments, the CMV promoter and/or enhancer comprises any of the CMV promoters and/or enhancers known in the art. In some embodiments, the CMV promoter and/or enhancer comprises the CMV promoter and/or enhancer provided herein.

[0080] In provided embodiments, the improvement of a provided transfer plasmid is its ability to produce a functional lentivirus that promotes increased transduction efficiency and/or enhanced expression of a NOI in a target cell. In some embodiments, expression is enhanced relative to a reference transfer plasmid. In some embodiments, a reference transfer plasmid is a transfer plasmid that contains a WPRE and/or contains a polynucleotide sequence region that comprises a portion of the pol integrase polynucleotide comprising the core domain, such as set forth in SEQ ID NO: 8. In some embodiments, a reference transfer plasmid is a transfer plasmid in which expression of a NOI is under the control of an RSV promoter. In some embodiments, the reference transfer plasmid comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 1. In some embodiments, the reference transfer plasmid comprises the nucleotide sequence set forth in SEQ ID NO: 1.

[0081] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0082] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. I. Transfer Plasmid

[0083] Provided herein is a lentivirus transfer plasmid that includes polynucleotide sequences that are packaged into a transducing lentivirus. In particular embodiments, the transfer plasmid can include a heterologous nucleic acid encoding a NOI. The transfer plasmids can be used to produce lentiviral vectors, which can be used to transduce target cells for expression of a NOI. For instance, the transfer plasmid comprises a retroviral packaging signal (y), along with the selected NOI sequence and one or more other sequences as described, flanked by long terminal repeat sequences (LTRs). In some embodiments, the elements of the transfer plasmid of the present disclosure are in operable association with one another to enable the transfer plasmid to participate in the formation of a vector, such as a lentivirus, in a transfected cell, together with one or more packaging vectors and envelope vectors.

[0084] In particular, the transfer plasmid provided herein lacks certain lentiviral nucleic acid sequences, including regulatory elements. In some embodiments, the provided transfer plasmid is based on recognition that deletion of certain lentiviral nucleic acid sequences, including regulatory nucleic acid sequences, does not impact lentivirus packaging in host cells or downstream NOI expression in target cells. The provided transfer plasmids are efficient at producing viral vectors when transfected into host cells and also produce functional viral vectors that exhibit improved NOI expression when used to transduce target cells. Surprisingly, as shown herein, the average expression level of the NOI produced using provided transfer plasmids is higher in cells transduced by the lentivirus produced using a provided transfer plasmid lacking one or more lentiviral nucleic acid sequences as compared to a more intact transfer plasmid. Thus, provided herein is an improved version of a transfer plasmid where deletion of certain lentiviral nucleic acid sequences does not compromise the ability of the transfer plasmid to produce functional viral vectors.

[0085] In some embodiments, the sequences for the transfer plasmid include a partial open reading frame from a lentivirus genome that includes cis-acting regulatory elements for the viral life cycle. In some embodiment, transfer plasmid includes a partial gag sequence, a partial env sequence and/or a partial pol sequence from a lentivirus genome that provide for the one or more cis-acting regulatory elements. In some embodiments, a lentiviral transfer vector may include a polynucleotide sequence encoding a partial gag protein that is positioned adjacent to and/or overlapping with the psi ( ) domain that provides a packaging signal to allow assembly into viral particles, a partial env polynucleotide sequence that includes the Rev responsive element (RRE), and a partial pol polynucleotide sequence that includes the central polypurine tract and central termination sequences (cPPT and CTS). In some embodiments, the provided transfer plasmid contains cis-acting regulatory elements that include the psi packaging, the RRE and the cPPT and CTS, as well as 5’ and 3’ LTRs.

[0086] Suitable lentiviral vector genomes include those based on Human Immunodeficiency Virus (HIV-1), HIV-2, feline immunodeficiency virus (FIV), equine infectious anemia virus, Simian Immunodeficiency Virus (SIV) and maedi/visna virus. In some embodiments, the sequences of the transfer plasmid are derived from the HIV-1 genome.

[0087] In some embodiments, the transfer plasmid contains a partial pol nucleotide sequence in which one or more pol gene products are disabled, modified or deleted. In some embodiments, while polynucleotide sequence of the pol gene are included that include the cPPT and CTS, other sequences in the integrase coding sequence are not included. In any of such provided embodiments, it is found that deletion of sequences of the pol gene can be removed without negatively impacting, and in some cases enhancing or improving, transduction efficiency by the generated lentivirus. In some embodiments, the partial pol polynucleotide sequence in the transfer plasmid lacks integrase sequences including the integrase core domain of the pol gene. In some embodiments, the cPPT and CTS are retained but sequences of the pol integrase that include at least the integrase core domain are missing or not present. For purposes herein, such a polynucleotide region sequence upstream of the cPPT/CTS that is not present (i.e. interchangeably referred to as missing, lacking or deleted) is called “the region between” or the “BTW region.” In some embodiments, the polynucleotide sequence region or BTW region that is not present in a provided transfer plasmid is a sequence that includes a least a portion of the pol integrase polynucleotide that contains the core domain. In some such embodiments, the transfer plasmid lacks a sequence between a sequence encoding the partial gag sequence that contains the 5’-LTR, element necessary for genome packaging, RRE and gp41 peptide of the env gene, and the sequence containing the cPPT and CTS of the pol gene. In such embodiments, the transfer plasmid lacks a polynucleotide sequence region upstream of the cPPT and CTS and downstream of the gp41 peptide that includes a portion of the integrase that includes the core domain. In particular embodiments, when producing lentivirus, the integrase gene is supplied by the separate packaging plasmid such that a full length or conserved integrase gene is not required in the transfer plasmid. [0088] In some embodiments, the BTW region that is missing or not present in a provided transfer plasmid is 350-370 nucleotides in length. In some embodiments, the BTW region is about 360 and 370 nucleotides in length. In some embodiments, the BTW region is about 365 and 368 nucleotides in length. In some embodiments, the BTW region is about 367 nucleotides in length. In some embodiments, the BTW region comprises a nucleotide sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 8. In some embodiments, the BTW region comprises a nucleotide sequence at least 85% identical to the sequence set forth in SEQ ID NO: 8. In some embodiments, the BTW region comprises a nucleotide sequence set forth in SEQ ID NO: 8. In some embodiments, the transfer plasmid does not comprise (or is devoid of) a nucleotide sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 8. In some embodiments, the transfer plasmid does not comprise (or is devoid of) a BTW polynucleotide. In some embodiments, the transfer plasmid does not comprise (or is devoid of) the nucleotide sequence set forth in SEQ ID NO: 8. In some embodiments, a transfer plasmid that does not comprise or is devoid of the BTW polynucleotide comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 4. In some embodiments, the transfer plasmid that does not comprise or is devoid of the BTW polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 4.

[0089] In provided embodiments, also not present (such as missing, lacking or deleted) in a provided transfer plasmid are certain cis-acting RNA elements from the Woodchuck Hepatitis Virus (WHV) that are commonly utilized in lentiviral vectors to enhance gene expression, particularly woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). The WPRE can enhance expression from a number of different vector types including lentiviral vectors (U.S. Pat. Nos. 6,136,597; 6,287,814; Zufferey, R., et al. (1999). J. Virol. 73:2886-92). This enhancement is thought to be due to improved RNA processing at the post-transcriptional level, resulting in increased levels of nuclear transcripts. A two-fold increase in mRNA stability also contributes to this enhancement (Zufferey, R., et al. (1999). J. Virol. 73:2886-92). Thus, the present disclosure is based on observations that WPRE is not required to produce functional viral vectors that promote NOI expression in a target cell, and alone or in combination with deletion of the BTW region. In any of such provided embodiments, it is found that sequences of the WPRE, alone or in combination with deletion of the BTW region, can be removed without negatively impacting, and in some cases enhancing or improving, transduction efficiency by the generated lentivirus.

[0090] In some embodiments, the transfer plasmid does not comprise (or is devoid of) a WPRE polynucleotide comprising a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 7. In some embodiments, the transfer plasmid does not comprise (or is devoid of) a WPRE polynucleotide. In some embodiments, the transfer plasmid does not comprise (or is devoid of) a WPRE polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 7. In some embodiments, a transfer plasmid that does not comprise or is devoid of WPRE comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3. In some embodiments, the transfer plasmid that does not comprise or is devoid of WPRE comprises the nucleotide sequence set forth in SEQ ID NO: 3.

[0091] In some embodiments, the transfer plasmid carries more than one deletion. In some embodiments, the transfer plasmid comprises up to a 1000 bp deletion. In some embodiments, the transfer plasmid comprises a 600 bp to 1000 bp deletion, such as a deletion of 956 bp deletion. In some embodiments, the transfer plasmid comprises a 367 bp deletion and a 589 bp deletion. In some embodiments, the transfer plasmid does not comprise (or is devoid of) a polynucleotide sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 8 and a polynucleotide sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 7. In some embodiments, the transfer plasmid does not comprise (or is devoid of) a polynucleotide sequence set forth in SEQ ID NO: 8 and a polynucleotide sequence set forth in SEQ ID NO: 7.

[0092] Provided herein is a transfer plasmid comprising a sequence comprising a 5’ LTR and a 3’ LTR, a retroviral packaging signal (y) and one or more lentiviral regulatory sequences, wherein the transfer plasmid does not comprise (or is devoid of) a polynucleotide comprising a nucleotide sequence that is at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 7 and a polynucleotide comprising a nucleotide sequence that is at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 8. In some embodiments, the transfer plasmid comprises a sequence comprising a 5’ LTR and a 3’ LTR, a retroviral packaging signal (y) and one or more lentiviral regulatory sequences, and does not comprise (or is devoid of) the nucleotide sequence set forth in SEQ ID NO: 7 and the nucleotide sequence set forth in SEQ ID NO: 8.

[0093] In particular embodiments, the 5’ LTR and 3’ LTRs are intact or are modified LTRs thereof. In some embodiments, such 5’ and 3’ LTRs include sequences from a lentivirus. The LTR sequences may include LTR sequences from any lentivirus from any species. Lor example, they may be LTR sequences from HIV, SIV, EIV or BIV. Typically, the LTR sequences include HIV LTR sequences.

[0094] In some embodiments, the 5’ LTR and 3’ LTR elements are modified LTR sequences that comprise an R region and a U5 region but are deleted or truncated of all or a portion of the U3 region. In some embodiments, the 3’ LTR is a sequence from the lentiviral genome that is truncated and devoid of the enhancer of the U3 region. In some embodiments, the 3’ LTR sequence is a modified sequence from the lentiviral genome that is truncated and devoid of the U3 region or partly deleted in the U3 region. In some embodiments, the deletion or truncation of the U3 of the 3’ LTR inactivates the LTR. In some embodiments, the 5’ LTR is a modified sequence in which the U3 region of the 5’ LTR is replaced by a non lentiviral U3 region or by a promoter suitable to drive tat-independent primary transcription. Embodiments of such sequences are described below.

[0095] Typically, the transfer plasmid is replication incompetent and may contain a deletion in the 3’ LTR, rendering the virus “self-inactivating” (SIN) after integration. In a normal lentivirus sequence, the LTR can be divided into three regions including the U3 sequence, the R sequence and the U5 sequence. The U3 sequence comprises the majority of human immunodeficiency virus 1 (HIV-1) LTR. The U3 region contains the enhancer and promoter elements that modulate basal and induced expression of the HIV genome in infected cells and in response to cell activation. Several of the promoter elements are essential for viral replication. Some of the enhancer elements are highly conserved among viral isolates and have been implicated as critical virulence factors in viral pathogenesis. The enhancer elements may act to influence replication rates in the different cellular target of the virus (Marthas et al. J. Virol. (1993) 67:6047-6055). As viral transcription starts at the 3' end of the U3 region of the 5' LTR, those sequences are not part of the viral mRNA and a copy thereof from the 3' LTR acts as template for the generation of both LTR’s in the integrated provirus. If the 3' copy of the U3 region is altered in a retroviral vector construct, the vector RNA still is produced from the intact 5' LTR in producer cells, but cannot be regenerated in target cells. Transduction of such a vector results in the inactivation of both LTR’s in the progeny virus. Thus, the retrovirus is self-inactivating (SIN) and those plasmids are known as SIN transfer plasmids.

[0096] In some embodiments, a SIN lentiviral vector has LTR regions which do not permit replication. In some embodiments, in a SIN retroviral vector, both LTR sequences may be modified to generate the self-inactivating vector. As a result of the self-inactivating 3' LTR, the provirus that is generated following entry and reverse transcription will comprise an inactivated 5' LTR. The rationale is to improve safety by reducing the risk of mobilization of the vector genome and the influence of the LTR on nearby cellular promoters. The skilled artisan is readily familiar with sequences of the modified 3’ and 5’ LTRs of SIN retroviral vectors.

[0097] Any suitable lentiviral 5' LTR can be utilized in accordance with the provided embodiments. A completely intact 5' LTR can be utilized, or a modified copy can be utilized. In some embodiments, the 5’ LTR is a truncated sequence that includes the R and U5 sequences from the 5’ LTR of a lentivirus. In some embodiments, the U3 sequence from the lentiviral 5' LTR may be replaced with a promoter sequence, such as a heterologous promoter sequence. This can increase the titer of virus recovered from the packaging cell line. An enhancer sequence may also be included. Any enhancer/promoter combination that increases expression of the viral RNA genome in the packaging cell line may be used. In some aspects, the U3 region of the 5’-LTR is replaced by heterologous promoter sequences (e.g. CMV or RSV) to achieve a Tat-independent transcription and to increase genomic RNA synthesis, resulting in an increase in viral titer by the resulting viral vector. Because 5’-U3 region drives the expression of primary transcripts, its modifications will not be present in transduced cells (Schambach et al.2009).In some embodiments, the promoter is RSV. In some embodiments, the promoter is CMV. In one example, the CMV enhancer/promoter sequence is used (US Patent No. 5,385,839 and US Patent No. 5,168,062, each of which is incorporated in its entirety).

[0098] In some embodiments, the 3’ LTR is an inactivated or self-inactivating 3’ LTR from a lentivirus. In some embodiments, a self-inactivating vector generally has a deletion of the enhancer and promoter sequences from the 3' long terminal repeat (LTR), which is copied over into the 5' LTR during vector integration. A skilled artisan is familiar with various U3 deletions that can be used. Exemplary deletion of the U3 region of the 3' LTR are described in U.S. Patent No. 7083981. In some embodiments, the 3’ LTR is a deleted U3 (delU3 or AU3) in which a large part of the U3 region is deleted, including portions containing the transcriptional enhancer and promoter. By deleting the transcriptional enhancers and/or the promoter in the U3 region of the LTR, the vector is replication limited so that following reverse transcription a full-length LTR cannot be reconstituted. In some aspects, SIN vectors have a deletion in the 3 ’-LTR covering the promoter/enhancer elements from the U3 region, e.g. about a 50 to about a 400 base pair deletion.

[0099] In provided embodiments, the 5' end of the U3 region is retained because it is involved in vector transfer, being required for integration (terminal dinucleotide + ATT sequence). Thus, the terminal dinucleotide and the ATT sequence may represent the 5' boundary of the U3 sequences which can be deleted. In some embodiments, the delU3 region includes the att sequence, but lacks any sequences having promoter activity, thereby causing the generated viral vector to be self-inactivating (SIN) in that viral transcription cannot go beyond the first round of replication following chromosomal integration. In some embodiments, a delU3 has a sequence in which only the minimal U3 att sequence is retained. In some embodiments, a delU3 has a sequence in which the U3 att sequence and most of the U3 modulatory region is retained with deletions of the enhancer and core promoter U3 regions. In some embodiments, the delU3 region has a deletion that includes a deletion of the TATA box. The deletion may be one that removes the TATA box, preventing transcription initiation and therefore inactivating the virus Miyoshi et al.1998; Zuffrey et al 1998). In some aspects, this 3’-LTR deletion removes the polyadenylation signal distal to the TATA box. In some aspects, the 3’-LTR deletion removes the integrase recognition and processing site. In some aspects, the SIN vector comprises a deletion of the U3 enhancer and/or core regions.

[0100] Modifications include those produce an LTR which retains a minimal amount of functional activity, e.g., transcriptional (promoter-enhancer) functional activity. Such transcriptional activity can be determined routinely, e.g., using a reporter gene. Examples of modifications that produce LTRs with reduced (as compared to the native 3' LTR) and minimal functional activity include, e.g., deletions which are 5' (upstream) to the TATA box in the U3 region. Such deletions can include, e.g., deletions or modifications of one or more of the following transcriptional regulatory sites, such as RBEIII, NF-kB, and/or Spl, as well as the PPT site. An example of a 3' LTR with minimal transcriptional activity includes a modified lentivirus 3 'LTR that comprises TATA box sequence, but is lacking 3' U3 sequences 5' to the said TATA box sequences or in which the 5' sequences are modified (deletion, substitution, addition) such they are not functionally active. [0101] In some embodiments, the U3 element of the 3' LTR contains a deletion of its enhancer sequence, the polypurine tract (PPT), the TATA box, Spl and NF-kappa B sites.

[0102] In some embodiments, the U3 element of the 3' LTR comprises a deletion of at least 400, at least 350, at least 300, at least 250, at least 200, at a least 150, at least 100, or at least 50 base pairs within the U3, wherein said deletion includes a deletion of the TATA box. In some aspects, the U3 element of the 3' LTR comprises a deletion of up to or about 150 base pairs within the U3 core promoter domain, wherein said deletion includes a deletion of the TATA box. In some embodiments, the U3 element of the 3' LTR comprises a deletion of 134 base pairs within the U3 core promoter domain, wherein said deletion includes a deletion of the TATA box. In some embodiments, the U3 element of the 3' LTR comprises a deleted U3 in which TCF-la and TATA sequences are deleted.

[0103] In some aspects, the R region of the viral 3’ LTR of the transfer plasmid also include exogenous elements, such as P-globin or SV40 polyadenylation signals or the upstream sequence element (USE) from simian virus 40 (SV40-USE). In some embodiments, the R region of the viral 3’ LTR includes an SV40 polyadenylation signal. In some embodiments, such one or more additional exogenous elements act to decrease the transcriptional readthrough from the internal promoters or from remnants of the deleted U3 region (Almarza et al. 2011) preventing the potential transcriptional activation of the downstream genes.

[0104] In some embodiments, the 5’ LTR comprises a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 20. In some embodiments, the 5’ LTR comprises SEQ ID NO: 20. In some embodiments, a portion of the 5’ LTR is partially deleted (i.e., truncated) and fused to a heterologous enhancer or promoter. In some embodiments, the one or more promoters comprise a cytomegalovirus (CMV) promoter or an Rous sarcoma virus (RSV) promoter. In some embodiments, the transfer plasmid comprises a Rous sarcoma virus (RSV) promoter. In some embodiments, the RSV promoter comprises a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 19. In some embodiments, the transfer plasmid comprises a cytomegalovirus (CMV) enhancer/promoter. In some embodiments, the CMV enhancer/promoter comprises a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 36. [0105] In some embodiments, the 3’ LTR comprises a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 29. In some embodiments, the 3’ LTR comprises SEQ ID NO: 29.

[0106] In addition to the LTRs (wherein either LTR may comprise one or more modifications, such as one or more deletions as described), the transfer plasmids may further comprise one of more accessory elements to increase transduction efficiency (e.g., a cPPT/CTS), viral packaging (e.g., a Psi packaging signal, RRE), and/or other elements that increase exogenous gene expression (e.g., poly (A) sequences).

[0107] In some embodiments, transfer plasmid includes a nucleic acid whose sequence includes the central polypurine tract and central termination sequences (cPPT and CTS) of a retrovirus, e.g., HIV-1 or HIV-2, in some cases known as a FLAP element. Suitable FLAP elements are described in U.S. Pat. No. 6,682,907 and in Zennou, et ah, 2000, Cell, 101:173, which are herein incorporated by reference in their entireties. During HIV-1 reverse transcription, central initiation of the plus- strand DNA at the central polypurine tract (cPPT) and central termination at the central termination sequence (CTS) can lead to the formation of a three- stranded DNA structure: the HIV-1 central DNA flap. In some embodiments, the FLAP elements (including the cPPT/CTS) upstream or downstream of a NOI sequence. In some embodiments, the cPPT/CTS comprises a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 10. In some embodiments, the cPPT/CTS comprises SEQ ID NO: 10. In some embodiments, the transfer plasmid does not comprise (or is devoid of) a cppT polynucleotide. In some embodiments, the transfer plasmid does not comprise or is devoid of the nucleotide sequence set forth in SEQ ID NO: 10. In some embodiments, a transfer plasmid that does not comprise or is devoid of cPPT comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 5. In some embodiments, the transfer plasmid that does not comprise or is devoid of cppT comprises the nucleotide sequence set forth in SEQ ID NO: 5.

[0108] In some embodiments, the transfer plasmid comprises a y packaging signal. In some embodiments, the y packaging signal is downstream of the 5' LTR. A y packaging sequence downstream of the 5' LTR is recognized by the nucleocapsid (NC) domain of the Gag, which is utilized in cis to facilitate encapsulation of the heterologous sequence of interest into the transducing vector. See, e.g., Lever et al., J. Virol. (1989), 63: 4085-4087; Amarasinghe et al., J. Mol. Bio. (2001), 314(5):961-970. The y packaging sequence is relatively autonomous of neighboring sequences. Its position in the transfer plasmid can be determined routinely. See, e.g., Man and Baltimore, J. Virol., 54(2): 401-407, 1985 which use a reporter gene to optimize positioning of the packaging sequence. In some embodiments, the \|/ packaging sequence comprises a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 21. In some embodiments, the y packaging sequence comprises SEQ ID NO: 21.

[0109] In some embodiments, a transfer plasmid of the present disclosure can include a cis-acting RNA element required for viral replication. In embodiments, the cis-acting RNA element is a lentiviral nucleic acid that comprises one or more export elements, e.g., a cis- acting post-transcriptional regulatory element which regulates the transport of an RNA transcript from the nucleus to the cytoplasm of a cell. Examples of RNA export elements include, but are not limited to, the human immunodeficiency virus (HIV) rev response element (RRE) (see e.g., Cullen et al., 1991. J. Virol. 65: 1053; and Cullen et al., 1991. Cell 58: 423), which are herein incorporated by reference in their entireties. The RRE is necessary for Rev function; it contains a high affinity site for Rev; in all, approximately seven binding sites for Rev exist within the RRE RNA. In some embodiments, RRE encompasses an RNA element encoded within the env region of HIV- 1 of approximately 200 nucleotides (spanning the border of gpl20 and gp41). In some embodiments, the transfer plasmid comprises a Rev response element (RRE). In some embodiments, the RRE comprises a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 22. In some embodiments, the RRE comprises SEQ ID NO: 22.

[0110] In some embodiments, the RRE sequence overlaps with certain env coding sequences such as gp41. In some embodiments, the polynucleotide sequence containing the RRE contains a sequence encoding one or more lentiviral env gene products. In some embodiments, the transfer plasmid includes the RRE and partial env nucleotide sequence encoding a gp41 peptide. In some embodiments, the polynucleotide sequence encoding the gp41 peptide comprises a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 23. In some embodiments, the polynucleotide sequence encoding a gp41 peptide comprises SEQ ID NO: 23. In some embodiments the transfer plasmid comprises a polynucleotide sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 22 and a polynucleotide sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 23. In some embodiments the transfer plasmid comprises a polynucleotide sequence set forth in SEQ ID NO: 22 and a polynucleotide sequence set froth in SEQ ID NO: 23. [0111] In some of any embodiments, the transfer plasmid contains a polynucleotide encoding a partial gag sequence that includes the 5’-LTR, element necessary for genome packaging, RRE and gp41 peptide of the env gene and a sequence encoding a portion of gag. In some embodiments, the 5’ LTR comprises any of the 5’ LTRs described above. In some embodiments, the y comprises any of the y packaging elements described above. In some embodiments, the RRE comprises any of the RREs described above. In some embodiments, gp41 peptide comprises any of the gp41 peptides described above. In some embodiments, the partial Gag sequence comprises a nucleotide sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 35. In some embodiments, such a partial gag sequence is set forth in SEQ ID NO: 35. In some embodiments, the polynucleotide encoding the partial gag sequence incorporates a frameshift that generates a premature stop codon after 21 amino acids (aa) (portion of gag encoded by nucleotides 336-378 of SEQ ID NO: 35).

[0112] Additionally, the transfer plasmid comprises several sequences that are necessary for replication, encapsidation, and expression of any inserted NOIs. These sequences include: a plasmid origin of replication that facilitates replication within bacteria and an SV40 origin (which allows for episomal amplification of plasmids in eukaryotic cells that express SV40 large-T antigen). In some embodiments, one or more additional elements may be included in the transfer plasmid. In some embodiments, the additional element includes a synthetic intron or other sequences utilized to stability mRNA, selectable markers, and transcription termination signals (e.g., polyadenylation site).

[0113] In some embodiments, the transfer plasmid comprises one or more origin of replication site. The origin of replication can be used to increase the copy number of the construct when present in a host cell. Thus, the term “origin of replication” or “ori” is intended to encompass a sequence that is necessary for replication of a plasmid. In some embodiments, the origin of replication is derived from prokaryotic DNA. In some embodiments, the origin of replication is derived from bacteria. In some embodiments, the origin of replication is derived from eukaryotic DNA. In some embodiments, the origin of replication is derived from a mammal. In some embodiments, the origin of replication is derived from a virus. In some embodiments, the transfer plasmid provided herein comprises one or more, two or more, or three or more origins of replication. In some embodiments, the transfer plasmid comprises three origins of replication. In some embodiments, the origin of replication comprises any one of pUC, SV40, fl bacteriophage, ColEl, pMBl, pSClOl, R6K, 15A, pBR322, CloDF13, among others known in the art. In some embodiments, the origin of replication comprises a pUC origin of replication. In some embodiments, the origin of replication comprises an SV40 origin of replication. In some embodiments, the origin of replication is an fl bacteriophage origin of replication. In some embodiments, the transfer plasmid includes a plasmid origin of replication that facilitates replication within bacteria, such as an fl bacteriophage origin and an SV40 origin that allows for episomal amplification of plasmids in eukaryotic cells.

[0114] In some embodiments, the SV40 origin of replication comprises a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 31. In some embodiments, the SV40 origin of replication comprises the sequence set forth in SEQ ID NO: 31.

[0115] In some embodiments, the fl bacteriophage origin of replication comprises a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 34. In some embodiments, the fl bacteriophage origin of replication comprises the sequence set forth in SEQ ID NO: 34.

[0116] In some embodiments, the pUC origin of replication comprises a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 13. In some embodiments, the pUC origin of replication comprises the sequence set forth in SEQ ID NO: 13.

[0117] In some embodiments, the transfer plasmid includes sequences that include: a plasmid origin of replication that facilitates replication within bacteria (e.g., fl bacteriophage origin), a 3' SIN (a self-inactivating 3' HIV LTR), an SV40 origin (which allows for episomal amplification of plasmids in eukaryotic cells that express SV40 large-T antigen), a CMV promoter (a chimeric 5' HIV LTR/CMV promoter hybrid), RU5 (a truncated 5' HIV LTR), Psi packaging element (HIV stem- loops 1-4 that regulate the packaging of the retroviral RNA genome into the viral capsid), a Rev Response Element (RRE; the sequence to which the Rev protein binds for viral replication) and central polypurine tract and central termination sequences (cPPT and CTS), and in which the transfer plasmid is devoid of BTW region sequences as described and a WPRE.

[0118] In some embodiments, the transfer plasmid comprises a sequence that initiates translation of transcribed viral sequences. In some embodiments, the transfer plasmid comprises a Kozak sequence. The Kozak sequence directs the pre-initiation complex and ribosome to the translation initiation site (i.e., start codon) and mediates ribosome assembly ensuring the correct protein sequence is translated. In some embodiments, Kozak sequence comprises a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 25. In some embodiments, the Kozak sequence comprises SEQ ID NO: 25.

[0119] In some embodiments, the transfer plasmid comprises a primer binding site (PBS). A PBS allows for primer binding and subsequent sequencing by polymerase chain reaction (PCR) to confirm insertion of the NOI in the transfer plasmid. In some embodiments, the transfer plasmid comprises a PBS that is 5’ and 3’ to the insert site. The PBS that is 5’ to the insert allows for binding of a reverse primer. The PBS that is 3’ to the insert allows for binding by the forward primer. In some embodiments, the transfer plasmid comprises a forward PBS and a reverse PBS. In some embodiments, the forward PBS is an M13 forward PBS. In some embodiments, the reverse PBS is an M13 reverse PBS. In some embodiments, the transfer plasmid comprises an M13 forward PBS and an M13 reverse PBS. In some embodiments, PBS comprises a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOS: 17 or 33. In some embodiments, the PBS comprises SEQ ID NOS: 17 or 33.

[0120] In some embodiments, the transfer plasmid comprises a protein binding site. In some embodiments, the protein binding site comprises a sequence bound by a catabolite activator protein (CAP). Thus, in some embodiments, the protein binding site comprises a CAP binding site. The CAP is a transcriptional activator that includes a ligand-binding domain at the N-terminus and a DNA-binding domain at the C-terminus. In some embodiments, the CAP binding site comprises a nucleotide sequence that is at least at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 14. In some embodiments, the CAP binding site comprises the nucleotide sequence set forth in SEQ ID NO: 14. In some embodiments, the protein binding site comprises a sequence bound by a lac repressor protein. Thus, in some embodiments, the protein binding site comprises a lac operator binding site. In some embodiments, the transfer plasmid comprises a CAP binding site and/or lac operator binding site. In some embodiments, the lac operator binding site comprises a nucleotide sequence that is at least at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 16. In some embodiments, the lac operator binding site comprises the nucleotide sequence set forth in SEQ ID NO: 16.

[0121] In some embodiments, the transfer plasmids described herein further comprise a transcription termination signal. Elements directing the efficient termination and polyadenylation of the transcripts increases heterologous gene expression. Transcription termination signals are generally found downstream of the polyadenylation signal. In some embodiments, transfer plasmid comprises a polyadenylation sequence 3' of a polynucleotide encoding a polypeptide to be expressed. The term “polyA site” or “polyA sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by addition of a polyA tail to the 3' end of the coding sequence and thus, contribute to increased translational efficiency. Cleavage and poly adenylation is directed by a poly(A) sequence in the RNA. The core poly(A) sequence for mammalian pre-mRNAs has two recognition elements flanking a cleavage-polyadenylation site. Typically, an almost invariant AAUAAA hexamer lies 20-50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements and is coupled to the addition of up to 250 adenosines to the 5’ cleavage product. In some embodiments, the core poly(A) sequence is an ideal polyA sequence (e.g., AATAAA, ATT AAA, AGTAAA). In some embodiments, the poly(A) sequence is an SV40 polyA sequence, a bovine growth hormone polyA sequence (BGHpA), a rabbit P-globin polyA sequence (rPgpA), variants thereof, or another suitable heterologous or endogenous polyA sequence known in the art.

[0122] In some embodiments, the transfer plasmid comprises a polyA sequence. In some embodiments, the polyA sequence comprises an SV40 polyA tail. In some embodiments, the transfer plasmid comprises an SV40 polyA tail. In some embodiments, SV40 polyA tail comprises a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 30. In some embodiments, the SV40 polyA tail comprises SEQ ID NO: 30.

[0123] In some of any embodiments, the functional elements present within a provided transfer plasmid are shown in Table El. In some embodiments, the transfer plasmid may serve as a backbone for introducing a NOI sequence. In some embodiments, an exemplary backbone transfer vector is shown in FIG. 8A (LVTPR) and FIG. 8B (LVTPC).

[0124] In some embodiments, a provided lentiviral transfer plasmid backbone is less than 9000 bp in length, wherein the backbone comprises a multiple cloning site that allows for the insertion of a NOI. In some embodiments, the lentiviral transfer plasmid backbone is 6000 bp to 9000 bp in length, such as 6000 bp to 8500 bp in length, 6000 bp to 8000 bp in length, 6000 bp to 7500 bp in length, 6000 bp to 7000 bp in length or 6000 bp to 6500 bp in length, wherein the backbone comprises a multiple cloning site that allows for the insertion of a NOI. In particular embodiments, the lentiviral transfer plasmid backbone is about 6400 bp in

- l- length, about 6500 bp in length, about 6600 bp in length, about 6700 bp in length, about 6800 bp in length, about 6900 bp in length, about 7000 bp in length, about 7100 bp in length, about 7200 bp in length, about 7300 bp in length or about 7400 bp in length or is a length that has a value between any of the foregoing, wherein the backbone comprises a multiple cloning site that allows for the insertion of a NOI. In some embodiments, the lentiviral transfer plasmid backbone is 6400 bp to 7400 bp in length, wherein the backbone comprises a multiple cloning site that allows for the insertion of a NOI. In some embodiments, the lentiviral transfer plasmid backbone is 6400 bp to 7200 bp in length, wherein the backbone comprises a multiple cloning site that allows for the insertion of a NOI. In some embodiments, the lentiviral transfer plasmid backbone is 6600 bp to 7000 bp in length, wherein the backbone comprises a multiple cloning site that allows for the insertion of a NOI. In some embodiments, the lentiviral transfer plasmid backbone is 6700 bp to 6800 bp in length, wherein the backbone comprises a multiple cloning site that allows for the insertion of a NOI.

[0125] In some embodiments, a multiple cloning site (MCS) is included as a DNA sequence within the transfer plasmid that contains multiple unique restriction enzyme cut sites. The MCS facilitates the insertion of DNA fragments into the lentiviral backbones via standard cloning techniques. In some embodiments, in the lentiviral transfer plasmid backbones described herein, the MCS is located downstream of a promoter that is operably contacted to an inserted NOI from which transcription of any inserted NOI is initiated. In some embodiments, the promoter is an Elongation Factor 1 alpha (EFla) promoter.

[0126] In some embodiments, the transfer plasmid of the present disclosure includes one or more restriction enzyme cut sites to allow for the insertion or deletion of a NOI into the transfer plasmid backbone. In some embodiments, the polynucleotide NOI sequence can contain flanking restriction site sequences that are compatible with cut sites in the transfer plasmid. In some embodiments, insertion of the NOI sequence is accomplished by via two restriction enzymes, blunting the product and circularizing with a Quick Ligase (Quick Ligation Kit; NEB, MA). In some embodiments, the transfer plasmid contains one or more restriction cut sites to facilitate insertion of a NOI. In some embodiments, the restriction cut sites include Nhel, Bmtl, PacI, Acc65I, Acc65I, Kpnl or any combination thereof. In some embodiments, the NOI is inserted between any two restriction cut sites Nhel, Bmtl, PacI, Acc65I, Acc65I, and Kpnl. In particular embodiments, the NOI is interested between Nhel and PacI restriction enzyme cute sites. [0127] In some embodiments, an exemplary transfer plasmid is shown in FIG. 8A in which the 5’ LTR is modified with a heterologous regulatory element that is a promoter and/or enhancer from RSV. In some embodiments, the heterologous regulatory element is an RSV promoter. In some embodiments, the RSV promoter comprises a sequence of nucleotides that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 19. In some embodiments, the RSV promoter comprises the sequence of nucleotides of SEQ ID NO: 19.

[0128] In some embodiments, the disclosure provides a lentiviral transfer plasmid backbone having a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2, wherein the backbone comprises a multiple cloning site that allows for the insertion of a NOI. In some embodiments, the lentiviral transfer plasmid backbone has a sequence that is at least 85% identical to SEQ ID NO: 2, wherein the backbone comprises a multiple cloning site that allows for the insertion of a NOI. In some embodiments, the lentiviral transfer plasmid backbone has a sequence that is at least 90% identical to SEQ ID NO: 2, wherein the backbone comprises a multiple cloning site that allows for the insertion of a NOI. In some embodiments, the lentiviral transfer plasmid backbone has a sequence that is at least 95% identical to SEQ ID NO: 2, wherein the backbone comprises a multiple cloning site that allows for the insertion of a NOI. In some embodiments, the lentiviral transfer plasmid backbone has a sequence that is at least 97% identical to SEQ ID NO: 2, wherein the backbone comprises a multiple cloning site that allows for the insertion of a NOI. In some embodiments, the lentiviral transfer plasmid backbone has the sequence set forth in SEQ ID NO: 2, wherein the backbone comprises a multiple cloning site that allows for the insertion of a NOI. In some aspects, the backbone can be used to generate a population of viral particles with increased viral titer as compared to a conventional lentiviral backbones.

[0129] In some embodiments, an exemplary transfer plasmid is shown in FIG. 8B in which the 5’ LTR is modified with a heterologous regulatory element that is a promoter and/or enhancer from CMV. In some embodiments, the heterologous regulatory element is a CMV promoter. In some embodiments, the CMV promoter comprises a sequence of nucleotides that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 38. In some embodiments, the CMV promoter is set forth in SEQ ID NO: 38. In some embodiments, the CMV enhancer comprises a sequence of nucleotides that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 37. In some embodiments, the CMV enhancer is set forth in SEQ ID NO: 37. In some embodiments, the CMV promoter and enhancer comprises a sequence of nucleotides that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 36. In some embodiments, the CMV promoter and enhancer comprises the sequence of nucleotides of SEQ ID NO: 36.

[0130] In some embodiments, the disclosure provides a lentiviral transfer plasmid backbone having a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 39, wherein the backbone comprises a multiple cloning site that allows for the insertion of a NOI. In some embodiments, the lentiviral transfer plasmid backbone has a sequence that is at least 85% identical to SEQ ID NO: 39, wherein the backbone comprises a multiple cloning site that allows for the insertion of a NOI. In some embodiments, the lentiviral transfer plasmid backbone has a sequence that is at least 90% identical to SEQ ID NO: 39, wherein the backbone comprises a multiple cloning site that allows for the insertion of a NOI. In some embodiments, the lentiviral transfer plasmid backbone has a sequence that is at least 95% identical to SEQ ID NO: 39, wherein the backbone comprises a multiple cloning site that allows for the insertion of a NOI. In some embodiments, the lentiviral transfer plasmid backbone has a sequence that is at least 97% identical to SEQ ID NO: 39, wherein the backbone comprises a multiple cloning site that allows for the insertion of a NOI. In some embodiments, the lentiviral transfer plasmid backbone has the sequence set forth in SEQ ID NO: 39, wherein the backbone comprises a multiple cloning site that allows for the insertion of a NOI. In some aspects, the backbone can be used to generate a population of viral particles with increased viral titer as compared to a conventional lentiviral backbones.

[0131] In some of any of the above embodiments, the transfer plasmid may be used as a backbone for inserting a NOI. In some embodiments, the transfer plasmid comprises a NOI sequence. Techniques for inserting nucleotide sequences are apparent to those skilled in the art. In some embodiments, recombinant DNA techniques are used. In some embodiments, this is accomplished by inserting a polynucleotide comprising the NOI into the lentiviral backbone, typically into the multiple cloning site. In some embodiments, an internal promoter is present upstream of the NOI sequence or expression cassette following the cPPT and CTS.

[0132] In some embodiments, the provided transfer plasmid includes a truncated 5’ LTR that includes the R/U5, a partial gag sequence that includes the psi ( ) packaging signal, a partial env sequence that includes the RRE and gp41, a partial pol gene that includes the cPPT and CTS but lacks integrase sequences including the integrase core domain of the pol gene, an internal promoter to regulate NOI expression, a NOI, and a truncated 3' LTR, and in which the transfer plasmid is devoid of BTW region sequences as described and a WPRE. In some embodiments, the provided transfer plasmid includes a sequence that includes a truncated 5’ LTR that includes the R/U5, the psi ( ) packaging signal, the RRE, gp41 peptide, the cPPT and CTS, an internal promoter to regulate NOI expression, a NOI or expression cassette, and a truncated 3' LTR, in which the transfer plasmid lacks a sequence upstream of the cPPT and CTS and downstream of the gp41 peptide that includes a least a portion of the integrase that includes the core domain, and in which the transfer plasmid is devoid of BTW region sequences as described and a WPRE.

[0133] In some embodiments, the NOI can be any nucleotide sequence of interest. In some embodiments, the NOI is a gene for introduction into the genome of a target cell by a viral vector (e.g., a lentivirus). In the present disclosure, the transfer plasmid comprises a NOI sequence for packaging into a viral vector, such as a lentiviral vector, which can then be used to transduce a target cell for integration of the NOI into the target cell genome for expression. In some embodiments, the transfer plasmid of the present disclosure includes elements suitable for enabling transfer of a heterologous nucleic acid sequence encoding a NOI into a host cell.

[0134] In some embodiments, the NOI is a sequence that encodes a protein or an RNA. In some embodiments, the protein is an enzyme, receptor, cytokine, antibody or other protein molecule. In some embodiments, the protein may be chimeric or synthetic and differ from a native molecule. In some embodiments, the NOI is a polynucleotide encoding a chimeric antigen receptor (CAR). In some embodiments, the NOI is a polynucleotide encoding a cytokine receptor. In some embodiments, the NOI comprises an RNA molecule. In some embodiments, the NOI encodes a detectable marker. In some embodiments, the detectable marker i a fluorescent protein. In some embodiments, the fluorescent protein is GFP, YFP, RFP, dsRed, mCherry, or any derivative thereof. In some embodiments, the transfer plasmid comprises a polynucleotide encoding GFP.

[0135] In some embodiments, the transfer plasmid includes a promoter that is operably connected to an inserted NOI from which transcription of any inserted NOI is initiated. In some embodiments, the promoter comprises a non- viral promoter (e.g., a eukaryotic or mammalian promoter). Non-limiting examples of suitable eukaryotic promoters (promoters functional in a eukaryotic cell) include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, a viral simian virus 40 (SV40) (e.g., early and late SV40), a spleen focus forming virus (SFFV) promoter, long terminal repeats (LTRs) from retrovirus (e.g., a Moloney murine leukemia virus (MoMLV) LTR promoter or a Rous sarcoma virus (RSV) LTR), a herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and Pl l promoters from vaccinia virus, an elongation factor 1-alpha (EFla) promoter, early growth response 1 (EGR1) promoter, a ferritin H (FerH) promoter, a ferritin L (FerL) promoter, a Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, a eukaryotic translation initiation factor 4A1 (EIF4A1) promoter, a heat shock 70kDa protein 5 (HSPA5) promoter, a heat shock protein 90kDa beta, member 1 (HSP90B1) promoter, a heat shock protein 70kDa (HSP70) promoter, a P-kinesin (P-KIN) promoter, the human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), a Ubiquitin C (UBC) promoter, a phosphoglycerate kinase- 1 (PGK) promoter, a cytomegalovirus enhancer/chicken P-actin (CAG) promoter, a P-actin promoter and a myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted (MND) promoter, and mouse metallothionein-1. Selection of the appropriate promoter is well within the level of ordinary skill in the art.

[0136] In some embodiments, the promoter is a non-viral promoter. In some embodiments, the non-viral promoter is operably linked to control expression of a NOI. In some embodiments, the non-viral promoter is operably linked to control expression of a non- viral protein. In some embodiments, the non-viral promoter is operably linked to control expression of a viral protein.

[0137] In some embodiments, the promoter is an EF-la promoter. In some embodiments, the EF-la promoter comprises a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 24 or SEQ ID NO: 41. In some embodiments, the EF-la promoter comprises SEQ ID NO: 24 or SEQ ID NO: 41. In some embodiments, the EF-la promoter comprises an EF-la promoter intron sequence. In some embodiments, the EF-la promoter intron comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 9. In some embodiments, the EF-la promoter intron comprises the nucleotide sequence set forth in SEQ ID NO: 9. In some embodiments, the transfer plasmid does not comprise (or is devoid of) an EF-la promoter intron polynucleotide. In some embodiments, the transfer plasmid does not comprise (or is devoid of) the nucleotide sequence set forth in SEQ ID NO: 9. In some embodiments, a transfer plasmid that does not comprise or is devoid of the EF-la promoter intron polynucleotide comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 6. In some embodiments, the transfer plasmid that does not comprise or is devoid of the EF-la promoter intron polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 6.

[0138] In some embodiments, the transfer plasmid comprises a single NOI. In some embodiments, the transfer plasmid comprises more than one NOI. In some embodiments, the transfer plasmid of the present disclosure includes elements that allow for the production of vectors that can co-express one or multiple NOIs. In some embodiments, the transfer plasmid comprises elements that result in the production of a multicistronic (e.g., bicistronic) viral vector. Multicistronic viral vectors contain elements that allow for simultaneous expression of two or more separate proteins from the same mRNA transcript. Various elements are known that allow for expression of multiple NOIs, including inclusion of Internal Ribosome Entry Site (IRES) to facilitate translation of two open reading frames from a single mRNA or use of self-cleaving peptides, n some embodiments, the self-cleaving peptide comprises T2A, P2A, E2A and/or F2A. In some embodiments, the transfer plasmid comprises T2A. In some cases, the peptide, such as T2A, can cause the ribosome to skip (ribosome skipping) synthesis of a peptide bond at the C-terminus of a 2A element, leading to separation between the end of the 2A sequence and the next peptide downstream (see, for example, de Felipe. Genetic Vaccines and. Ther. 2:13 (2004) and deFelipe et al. Traffic 5:616-626 (2004)). Many 2A elements are known. Examples of 2A sequences that can be used in the methods and nucleic acids disclosed herein, without limitation, 2A sequences from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), and porcine teschovirus- 1 (P2A) as described in U.S. Patent Publication No. 20070116690.

[0139] In some embodiments, the transfer plasmid comprises T2A. In some embodiments, the T2A comprises a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 26. In some embodiments, the T2A comprises SEQ ID NO: 26.

[0140] In some embodiments, the transfer plasmid encodes a multicistronic plasmid comprising a first NOI separated from a marker sequence by a self-cleaving peptide. In some embodiments, the marker sequence is a GFP. In some embodiments, the GFP comprises a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 27. In some embodiments, the GFP comprises SEQ ID NO: 27. In some embodiments, the self-cleaving peptide is a T2A. In some embodiments, the T2A comprises a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 26. In some embodiments, the T2A comprises SEQ ID NO: 26.

[0141] In some embodiments, an exemplary transfer plasmid with a transgene is shown in FIG. 8C (LVTPR with eGFP). In some embodiments, the transfer plasmid with a transgene can have sequence elements as shown in FIG. 8C but in which the RSV promoter is substituted with a CMV promoter and/or enhancer. In some embodiments, the NOI is a bicistronic construct that can contain an additional transgene or other nucleotide sequence separated from the eGFP by a cleavable linker, such as the T2A element as shown in FIG. 8C. In some of any embodiments, the functional elements of LVTPR with eGFP are shown in Table El.

[0142] In some embodiments, the disclosure provides a lentiviral transfer plasmid backbone having a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 40, wherein the backbone comprises a multiple cloning site that allows for the insertion of an NOI as a multicistronic construct with the GFP.

[0143] In some embodiments, the NOI, including a monocistronic NOI sequence or a multicistronic NOI sequence, that is present in or for insertion into the transfer plasmid backbone is any length up to 4000 base pairs (bp). In some embodiments, the NOI is 1000 bp to 4000 bp in length, such as 1000 bp to 3500 bp, 1000 bp to 3000 bp, 1000 bp to 2500 bp or 1000 bp to 2000 bp. In some embodiments, the NOI is 2000 to 4000 bp in length, such as 2000 to 3600 bp, 2000 to 3200 bp, 2000 to 2800 bp or 2000 to 2400 bp. In some embodiments, the NOI is aboutlOOO bp in length, about 1400 bp in length, about 1800 bp in length, about 2200 bp in length, about 2600 bp in length, about 3200 bp in length, about 3600 bp in length or about 4000 bp in length, or any length that is a value between any of the foregoing. In some embodiments, the NOI is 2000 bp to 4000 bp in length. In some embodiments, the NOI is greater than 2500 bp in length. In some embodiments, the NOI is greater than 3000 bp in length. In some embodiments, the NOI is greater than 3200 bp in length. In some embodiments, the NOI is 2600 to 3400 bp in length. In some embodiments, the NOI is 2800 to 3400 bp in length. In some embodiments, the NOI is 3000 to 3400 bp in length. In some embodiments, an exemplary transfer plasmid provided herein comprises ampicillin. In some embodiments, the ampicillin comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 12. In some embodiments, the ampicillin comprises the nucleotide sequence set forth in SEQ ID NO: 12. In some embodiments, expression of the ampicillin is under the control of an ampicillin promoter. In some embodiments, the ampicillin promoter comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 11. In some embodiments, the ampicillin promoter comprises the nucleotide sequence set forth in SEQ ID NO: 11. In some embodiments, an exemplary transfer plasmid provided herein comprises a lac promoter. In some embodiments, the lac promoter comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 15. In some embodiments, the lac promoter comprises the nucleotide sequence set forth in SEQ ID NO: 15. In some embodiments, an exemplary transfer plasmid provided herein comprises a T3 promoter. In some embodiments, the T3 promoter comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 18. In some embodiments, the T3 promoter comprises the nucleotide sequence set forth in SEQ ID NO: 18. In some embodiments, an exemplary transfer plasmid provided herein comprises a Factor Xa site. In some embodiments, the Factor Xa site comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 28. In some embodiments, the Factor Xa site comprises the nucleotide sequence set forth in SEQ ID NO: 28. In some embodiments, an exemplary transfer plasmid provided herein comprises a T7 promoter. In some embodiments, the T7 promoter comprises a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 32. In some embodiments, the T7 promoter comprises the nucleotide sequence set forth in SEQ ID NO: 32.

Payload Sequences

[0144] In some embodiments, the NOI sequence encodes a chimeric antigen receptor (CAR). A CAR comprises an extracellular antigen binding domain fused via hinge and transmembrane domains to a cytoplasmic domain comprising a signaling domain. Generally, the extracellular antigen binding domain comprises a portion of an antibody molecule, e.g., an scFv antibody fragment of a variable heavy (VH) chain region and/or variable light (VL) chain region of the antibody. In some embodiments, the CAR is constructed with specificity for a particular antigen, such as an antigen expressed on a particular cell type to be targeted by the CAR. Antigens targeted by a CAR include those expressed in the context of a disease or condition to be targeted via a CAR cell therapy (e.g., CAR T cell therapy). Diseases and conditions include are proliferative, neoplastic, and malignant diseases and disorders, including cancers and tumors, including hematologic cancers, cancers of the immune system, such as lymphomas, leukemias, and/or myelomas, such as B, T, and myeloid leukemias, lymphomas, and multiple myelomas. CARs specific for a variety of tumor antigens are known in the art, for example CD 171 -specific CARs (Park et al., Mol Ther (2007) 15(4):825- 833), EGFRvIII- specific CARs (Morgan et al., Hum Gene Ther (2012) 23(10): 1043-1053), EGF-R-specific CARs (Kobold et al., J Natl Cancer Inst (2014) 107(l):364), carbonic anhydrase K-specific CARs (Larners et al., Biochem Soc Trans (2016) 44(3):951-959), FR-a- specific CARs (Kershaw et al., Clin Cancer Res (2006) 12(20):6106-6015), HER2-specific CARs (Ahmed et al., J Clin Oncol (2015) 33(15) 1688- 1696;Nakazawa et al., Mol Ther (2011) 19(12):2133-2143; Ahmed et al., Mol Ther (2009) 17(10): 1779-1787; Luo et al., Cell Res (2016) 26(7):850-853; Morgan et al., Mol Ther (2010) 18(4):843-851 ; Grada et al., Mol Ther Nucleic Acids (2013) 9(2):32), CEA-specific CARs (Katz et al., Clin Cancer Res (2015) 21(14):3149-3159), IL13Ra2-specific CARs (Brown et al., Clin Cancer Res (2015) 21(18):4062-4072), GD2-specific CARs (Louis et al., Blood (2011) 118(23):6050-6056; Caruana et al., Nat Med (2015) 21(5):524-529), ErbB 2- specific CARs (Wilkie et al., J Clin Immunol (2012) 32(5): 1059-1070), VEGF-R- specific CARs (Chinnasamy et al., Cancer Res (2016) 22(2):436-447), FAP-specific CARs (Wang et al., Cancer Immunol Res (2014) 2(2): 154-166), MSLN-specific CARs (Moon et al, Clin Cancer Res (2011) 17(14):4719-30), NKG2D-specific CARs (VanSeggelen et al., Mol Ther (2015) 23(10):1600-1610), CD19- specific CARs (Axicabtagene ciloleucel (Yescarta®) and Tisagenlecleucel (Kymriah®). See also_i Li et al., J Hematol and Oncol (2018) 11(22), reviewing clinical trials of tumor- specific CARs.

[0145] In some embodiments, the NOI encodes an engineered T-cell receptor (TCR). Engineered TCRs comprise TCRa and/or TCRP chains that have been isolated and cloned from T cell populations recognizing a particular target antigen. For example, TCRa and/or TCRP genes (i.e., TRAC and TRBC) can be cloned from T cell populations isolated from individuals with particular malignancies or T cell populations that have been isolated from humanized mice immunized with specific tumor antigens or tumor cells. Engineered TCRs recognize antigen through the same mechanisms as their endogenous counterparts (e.g., by recognition of their cognate antigen presented in the context of major histocompatibility complex (MHC) proteins expressed on the surface of a target cell). This antigen engagement stimulates endogenous signal transduction pathways leading to activation and proliferation of the TCR-engineered cells. In some embodiments, the extracellular domain of the TCR binds to an antigen expressed on a cancer cell. In some embodiments, the antigen is selectively expressed or overexpressed on a cancer cell, as compared to normal or non-targeted cells or tissues. In some embodiments, the cancer cell is a blood cancer cell or a solid tumor cancer cell. Engineered TCRs specific for tumor antigens are known in the art, for example WT1- specific TCRs (JTCR016, Juno Therapeutics; WTl-TCRc4, described in US Patent Application Publication No. 20160083449), MART-1 specific TCRs (including the DMF4T clone, described in Morgan et al., Science 314 (2006) 126-129); the DMF5T clone, described in Johnson et al., Blood 114 (2009) 535-546); and the ID3T clone, described in van den Berg et al., Mol. Ther. 23 (2015) 1541-1550), gplOO-specific TCRs (Johnson et al., Blood 114 (2009) 535-546), CEA-specific TCRs (Parkhurst et al., Mol Ther. 19 (2011) 620-626), NY- ESO and LAGE-1 specific TCRs (1G4T clone, described in Robbins et al., J Clin Oncol 26 (2011) 917-924; Robbins et al., Clin Cancer Res 21 (2015) 1019-1027; and Rapoport et al., Nature Medicine 21 (2015) 914-921), and MAGE-A3-specific TCRs (Morgan et al., J Immunother 36 (2013) 133-151) and Linette et al., Blood 122 (2013) 227-242). (See also, Debets et al., Seminars in Immunology 23 (2016) 10-21).

[0146] In some embodiments, the NOI is a synthetic cytokine receptor. In some embodiments, the synthetic cytokine receptor is capable of driving intracellular signaling in the absence of its cognate ligand (i.e., constitutively active). In some embodiments, the synthetic cytokine receptor comprises an extracellular domain, a transmembrane domain, and intracellular domain signaling domain. In some embodiments, the synthetic cytokine receptor can multimerize, typically as a homodimer, to facilitate downstream signaling in the absence of external stimuli of the receptor, such as in the absence of binding of a ligand to the extracellular domain. Thus, in some embodiments, the synthetic cytokine receptor is constitutively active.

[0147] In some embodiments, the at least one engineered receptor is a chimeric switch receptor. Chimeric switch receptors are engineered cell- surface receptors comprising an extracellular domain from an endogenous cell- surface receptor and a heterologous intracellular signaling domain, such that ligand recognition by the extracellular domain results in activation of a different signaling cascade than that activated by the wild type form of the cell-surface receptor. In some embodiments, the chimeric switch receptor comprises the extracellular domain of an inhibitory cell-surface receptor fused to an intracellular domain that leads to the transmission of an activating signal rather than the inhibitory signal normally transduced by the inhibitory cell-surface receptor. In particular embodiments, extracellular domains derived from cell- surface receptors known to inhibit immune effector cell activation can be fused to activating intracellular domains. Engagement of the corresponding ligand will then activate signaling cascades that increase, rather than inhibit, the activation of the immune effector cell. For example, in some embodiments, the transduced immune effector cells described herein comprise a NOI encoding a PD1-CD28 switch receptor, wherein the extracellular domain of PD1 is fused to the intracellular signaling domain of CD28 (See e.g., Liu et al., Cancer Res 76:6 (2016), 1578-1590 and Moon et al., Molecular Therapy 22 (2014), S201). In some embodiments, the transduced immune effector cells described herein comprise a NOI encoding the extracellular domain of CD200R and the intracellular signaling domain of CD28 (See Oda et al., Blood 130:22 (2017), 2410-2419).

[0148] In some embodiments, the engineered molecule comprises an RNA molecule. In some embodiments, the RNA molecule comprises an exogenous RNA molecule, a stimulatory RNA molecule or an immune stimulatory RNA molecule. In some embodiments, the RNA molecule is a viral-like double- stranded RNA molecule. In some embodiments, the RNA molecule is a human RN7SL1 RNA molecule or functional variant thereof. In some embodiments, the RNA molecule increases an immune activity. In some embodiments, the RNA molecule may activate antigen presenting cells, such as dendritic cells, and T cells. Without wishing to be bound by theory, in some embodiments, the activity of the RNA molecule is mediated at least in part by its secondary structure (e.g., a double stranded structure, e.g., a hairpin structure), and a variety of nucleotide sequences would have such activity.

[0149] In some embodiments, the engineered molecule comprises an antigen. In some embodiments, the antigen is a cancer antigen that can be used to tag a cancer cell. In some embodiments, the engineered cancer antigen comprises a target domain and a tumor targeting molecule, wherein the target domain is the extracellular domain or a truncated portion thereof of a target antigen. In some embodiments, the engineered cancer antigen comprises a target domain, wherein the target domain is an extracellular domain or a truncated portion thereof of a target antigen. In some embodiments, the engineered cancer antigen comprises a target domain and a heterologous membrane targeting domain, wherein the target domain is an extracellular domain or a truncated portion thereof of a target antigen.

[0150] In some embodiments, the transduced cell or population of cells further expresses a NOI encoding a safety-switch system. Safety-switch systems (also referred to in the art as suicide gene systems) comprise exogenous NOIs encoding for one or more proteins that enable the elimination of an transduced immune effector cell after the cell has been administered to a subject. Examples of safety-switch systems are known in the art. For example, safety-switch systems include genes encoding for proteins that convert non-toxic pro-drugs into toxic compounds such as the Herpes simplex thymidine kinase (Hsv-tk) and ganciclovir (GCV) system (Hsv-tk/GCV). Hsv-tk converts non-toxic GCV into a cytotoxic compound that leads to cellular apoptosis. As such, administration of GCV to a subject that has been treated with the transduced cells comprising a NOI encoding the Hsv-tk protein can selectively eliminate the transduced cells while sparing endogenous immune effector cells. (See e.g., Bonini et al., Science, 1997, 276(5319): 1719-1724; Ciceri et al., Blood, 2007, 109(11): 1828-1836; Bondanza et al., Blood 2006, 107(5): 1828-1836).

[0151] Additional safety-switch systems include genes encoding for cell-surface markers, enabling elimination of transduced cells by administration of a monoclonal antibody specific for the cell-surface marker via ADCC. In some embodiments, the cell-surface marker is CD20 and the transduced cells can be eliminated by administration of an anti-CD20 monoclonal antibody such as Rituximab (See e.g., Introna et al., Hum Gene Ther, 2000, 11(4):611-620; Serafini et al., Hum Gene Ther, 2004, 14, 63-76; van Meerten et al., Gene Ther, 2006, 13, 789-797). Similar systems using EGF-R and Cetuximab or Panitumumab are described in International PCT Publication No. WO 2018006880. Additional safety-switch systems include NOIs encoding pro-apoptotic molecules comprising one or more binding sites for a chemical inducer of dimerization (CID), enabling elimination of transduced cells by administration of a CID which induces oligomerization of the pro-apoptotic molecules and activation of the apoptosis pathway. In some embodiments, the pro-apoptotic molecule is Fas (also known as CD95) (Thomis et al., Blood, 2001, 97(5), 1249-1257). In some embodiments, the pro-apoptotic molecule is caspase-9 (Straathof et al., Blood, 2005, 105(11), 4247-4254).

[0152] In some embodiments, the transduced cell or population of cells further expresses a NOI encoding a detectable tag. Examples of detectable tags include but are not limited to, FLAG tags, poly-histidine tags (e.g. 6xHis), SNAP tags, Halo tags, cMyc tags, glutathione-S- transferase tags, avidin, enzymes, fluorescent proteins, luminescent proteins, chemiluminescent proteins, bioluminescent proteins, and phosphorescent proteins. In some embodiments the fluorescent protein is selected from the group consisting of blue/UV proteins (such as BFP, TagBFP, mTagBFP2, Azurite, EBFP2, mKalamal, Sirius, Sapphire, and T-Sapphire); cyan proteins (such as CFP, eCFP, Cerulean, SCFP3A, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, and mTFPl); green proteins (such as: GFP, eGFP, meGFP (A208K mutation), Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, Clover, and mNeonGreen); yellow proteins (such as YFP, eYFP, Citrine, Venus, SYFP2, and TagYFP); orange proteins (such as Monomeric Kusabira-Orange, IUKOK, mK02, mOrange, and mOrange2); red proteins (such as RFP, mRaspberry, mCherry, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, and mRuby2); far-red proteins (such as mPlum, HcRed-Tandem, mKate2, mNeptune, and NirFP); near-infrared proteins (such as TagRFP657, IFP1.4, and iRFP); long stokes shift proteins (such as mKeima Red, LSS-mKatel, LSS-mKate2, and mBeRFP); photoactivatable proteins (such as PA-GFP, PamCherryl, and PATagRFP); photoconvertible proteins (such as Kaede (green), Kaede (red), KikGRl (green), KikGRl (red), PS-CFP2, PS- CFP2, mEos2 (green), mEos2 (red), mEos3.2 (green), mEos3.2 (red), PsmOrange, and PsmOrange); and photo switchable proteins (such as Dronpa). In some embodiments, the detectable tag can be selected from AmCyan, AsRed, DsRed2, DsRed Express, E2-Crimson, HcRed, ZsGreen, ZsYellow, mCherry, mStrawberry, mOrange, mBanana, mPlum, mRasberry, tdTomato, DsRed Monomer, and/or AcGFP, all of which are available from Clontech.

II. Methods of Producing Lentiviruses

[0153] Provided herein are methods of manufacturing or producing viral vectors using the provided transfer plasmid. Viral vector production processes include upstream processes for preparing lentiviral vector particles (lentivirus) and downstream processes for isolating and purifying the lentivirus. Provided herein is a method of producing a lentivirus comprising (a) contacting a host cell with any of the transfer plasmids provided herein and one or more helper plasmids including a packaging plasmid and an envelope plasmid; (b) culturing the host cell under conditions that produce the lentivirus; and (c) isolating the lentivirus, thereby producing the lentivirus. In some embodiments, the process further includes methods for purifying the lentivirus.

[0154] The upstream process involves transfecting a particular cell type with a plurality of plasmids including the transfer plasmid described in Section I and one or more other helper plasmids coding for certain viral genes that, when expressed in the particular cell type, ultimately produce the desired viral particles which can then be harvested for use in clinical and/or research settings. That is, in order to generate viral particles, such as lentiviral particles, certain HIV-1 helper packaging proteins have to be introduced concomitantly into the host cell with the transfer plasmid. The helper plasmids provide the helper functions as well as structural and replication proteins in trans required to produce the lentivirus. In some embodiments, the several helper plasmids encode the virus enzymatic and/or structural components, such as Env, Gag, Pol, and/or Rev. Typically, the helper packaging proteins are encoded by two additional plasmids called the packaging plasmid and the envelope plasmid.

[0155] In some embodiments, the packaging plasmid(s) can contain all retroviral, such as HIV-1, proteins other than envelope proteins (Naldini et al., 1998). In some embodiments, the packaging plasmid encodes only the viral proteins essential for viral particle synthesis, which includes Gag, Pol, Rev and, in some cases, Tat. In other embodiments, viral vectors can lack additional viral genes, such as those that are associated with virulence, e.g. vpr, vif, vpu and nef, and/or Tat, a primary transactivator of HIV. In some embodiments, a GagPol packaging plasmid containing the gag and pol genes encoding for structural and enzymatic components and a Rev plasmid containing the rev gene encoding for Rev regulatory protein are separately introduced into a packaging cell line. In some embodiments, a single plasmid vector encoding for each of the Gag, Pol and Rev components can be used.

[0156] In some embodiments, an envelope plasmid encoding an env gene also can be introduced, which, in some cases, can result in viral particles pseudotyped with alternative Env proteins. The envelope plasmid encodes viral capsid proteins, such as glycoproteins, that will be expressed on the viral particle. In some embodiments, it is desirable to engineer lentiviral vectors with different target cell specificities to the native virus, to enable the delivery of genetic material to an expanded or altered range of cell types. One way in which this is achieved is by engineering the virus envelope protein to alter its specificity. Another approach is to introduce a heterologous envelope protein into the lentiviral vector particle to replace or add to the native envelope protein of the virus, which is referred to as pseudotyping. The term pseudotyping includes incorporating at least a part of, or substituting a part of, or replacing all of, an env gene of a viral genome with a heterologous env gene, for example an env gene from another virus. In some embodiments, the retroviral vector particle, such as lentiviral vector particle, is pseudotyped to increase the transduction efficiency of host cells. Various methods of pseudotyping are known, including any as described, for example, in WO 99/61639, WO-A-98105759, WO-A-98/05754, WO-A-97/17457, WO-A- 96/09400, WO-A-91/00047 and Mebatsion et al 1997 Cell 90, 841-847, each of which is incorporated by reference in its entirety. For example, a retroviral vector particle, such as a lentiviral vector particle, is pseudotyped with a VSV-G glycoprotein, which provides a broad cell host range extending the cell types that can be transduced. The envelope plasmid may also be called a pseudotyping plasmid. [0157] The env gene can be derived from any appropriate virus, such as a retrovirus. In some embodiments, the env is an amphotropic envelope protein which allows transduction of cells of human and other species. In some embodiments, env gene can be derived from: human immunodeficiency virus (HIV), Vesicular stomatitis virus, Murine leukemia virus (MLV), Chandipura virus, Gibbon ape leukemia virus (GALV), Feline leukemia virus (RD114), Amphotropic retrovirus (Ampho), 10A1 MLV (10A1), Ecotropic retrovirus (Eco), Baboon ape leukemia virus (BaEV), Moloney murine leukemia virus (MoMuLV or MMLV), Harvey murine sarcoma virus (HaMuSV or HSV), murine mammary tumor virus (MuMTV or MMTV), gibbon ape leukemia virus (GaLV or GALV), or Rous sarcoma virus (RSV). In some embodiments, the env gene comprises Vesicular stomatitis virus (VSV) protein G (VSVG), BaEV glycoprotein, RD114 glycoprotein, H/F glycoprotein, G/F glycoprotein, COCV glycoprotein, glycoprotein 120 (gpl20), glycoprotein 160 (gpl60), or glycoprotein 70 (gp70). In other embodiments, envelope proteins of hepatitis viruses or influenza can be used.

[0158] In some embodiments, methods for producing lentivirus include cotransfecting a provided transfer plasmid carrying a NOI, a packaging plasmids encoding Gag and Pol, a packaging plasmid encoding Rev and an envelope plasmid encoding VSVG envelope protein.

[0159] In some embodiments, a host cell can be transiently transfected with the one or more helper plasmids encoding one or more viral proteins, including at least one packaging plasmid and an envelope plasmid, and the transfer plasmid. The host cells are thus cells or cell-lines that can produce or release viral vector particles carrying the NOI. In some embodiments, the host cell provides components or is made to provide components that are required in trans for the packaging of the viral genomic RNA into lentiviral vector particles, including viral regulatory and structural proteins.

[0160] In some embodiments, the plasmids are introduced by stable transfection, which can result in the generation of packaging and producer cell lines. In some embodiments, packaging and producer cell lines are used that already have elements of the lentiviral packaging system embedded in the cell’s genome. A packaging cell is a host cell modified to express viral structural and/or accessory genes that enable packaging of a viral vector genome into a viral vector. A packaging cell does not contain a packaging signal to package the viral vector genome into a viral vector. A producer cell is a packaging cell that contains a viral vector genome comprising a packaging signal to package the viral vector genome into a viral vector. In some embodiments, the host cells can express or be made to express essential lentiviral (e.g. HIV-1) genes to allow the generation of lentivirus or lentiviral particles. In some embodiments, the transfer plasmid is then transfected into the packaging or producer cell lines for production of lentivirus carrying the NOI.

[0161] In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the viral vector contains a sequence for propagation in a prokaryotic host cell. In some embodiments, the viral vector contains one or more origins of replication for propagation in a prokaryotic cell. In some embodiments, the prokaryotic cell is a bacterial cell. In some embodiments, the host cell is a eukaryotic cell.

[0162] In some embodiments, the host cell can further be anchorage dependent, which means that the cells will grow, survive, or maintain function optimally when attached to a surface such as glass or plastic. In some embodiments, these cells can be suspension-adapted such that these cells do not require attachment to a surface. In some embodiments, the host cells may be neoplastic ally transformed cells.

[0163] In some embodiments, host cells for transfection with the lentiviral vector and packaging plasmids include, for example, mammalian primary cells; established mammalian cell lines, such as COS, CHO, HeLa, NIH3T3, 293T, 293F, LV293, HEK 293, and PC 12 cells; amphibian cells, such as Xenopus embryos and oocytes; other vertebrate cells; insect cells (for example, Drosophila), yeast cells (for example, S. cerevisiae, S. pombe, or Pichia pastoris) and prokaryotic cells (for example, E. coli). In some embodiments, suitable host cells include 293 (ATCC CCL X), 293T, HeLA (ATCC CCL 2), D17 (ATCC CCL 183), MDCK (ATCC CCL 34), BHK (ATCC CCL- 10) and Cf2Th (ATCC CRL 1430) cells. In some embodiments, the host cell includes but is not limited to a recombinant Chinese hamster ovary (CHO), A549, HeLa, U20S, HT1080, CAD, COS, P19, PC12, NIH 3T3, L929, N2a, MCE-7, Y79, SO-Rb50, Hep G2, DUKX-X11, J558L, U20S, L929, and baby hamster kidney (BHK) or a derivative thereof.

[0164] In some embodiments, the host cells are adherent cells and an adherent cell culture is transfected. In some embodiments, the host cell is selected from the group consisting of HEK293, HEK293S, HEK293T adapted for suspension culture (HEK392Ts), HEK293E, HEK293ET, HEK293ETM, HEK293E, HEK 293T/17, or LV293.

[0165] Methods of introducing plasmids into a host cell, such as the transfer plasmid described in Section I and the helper plasmids described above, are known in the art. Suitable methods include e.g., transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE- dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et al., Adv Drug Deliv Rev. 2012 Sep 13. Pii: S0169- 409X(12)00283-9), microfluidics delivery methods (See e.g., International PCT Publication No. WO 2013/059343), and the like. Illustrative examples of polynucleotide delivery systems suitable for use in particular embodiments contemplated include, but are not limited to, those provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, NeonTM Transfection Systems, and Copernicus Therapeutics Inc. Lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient lipofection of polynucleotides have been described in the literature. See e.g., Liu et al. (2003) Gene Therapy. 10:180-187; and Balazs et al. (2011) Journal of Drug Delivery. 2011:1-12.

[0166] When the plasmids are introduced into a host cell, the packaging sequence may permit the RNA transcript of the transfer plasmid to be packaged into viral particles, which then may be secreted into the culture media. The media containing the recombinant retroviruses in some embodiments is then collected. In some embodiments, the collected harvested material is clarified, such as by filtration. In some embodiments, a further downstream process can be used in which the lentiviral vector is concentrated and purified. In some embodiments, harvested materials from an upstream process is subjected to a downstream process for concentration and purification of the lentiviral vector. In some embodiments, the downstream process involves capturing and concentrating the viral vector in the resultant clarified filtrate using chromatography, such as affinity chromatography or cation exchange chromatography; ultrafiltering and diafiltering the viral vector using tangential flow filtration (TFF); and filtering the purified and concentrated material by sterile filtration. In some embodiments, the viral vector can be formulated for fill. In some embodiments, the viral vector can be frozen.

[0167] In some embodiments, the methods produce high titer recombinant virus. In some embodiments, the virus particle preparations can be used to infect target cells using various techniques, such as described in Section IV.

III. Lenti viruses

[0168] Provided herein are viral vectors, such as a lentivirus, produced according to the methods disclosed in Section II. In some embodiments, the lentivirus contains sequences of the transfer plasmid described in Section I. Thus, in some embodiments, the lentivirus does not comprise a polynucleotide encoding a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and a polynucleotide BTW region as described (e.g., a sequence between a partial Gag sequence and a central polypurine tract/central termination sequence (cPPT/CTS)). In some embodiments, the viral vector exhibits improved performance, including transduction efficiency or increased NOI expression.

[0169] A lentivirus of the disclosure may be derived or derivable from any suitable lentivirus. Non-limiting examples of lentivirus include Human Immunodeficiency Virus 1 (HIV-1), HIV-2, an Simian Immunodeficiency Virus (SIV), Human T-lympho tropic virus 1 (HTLV-1), HTLV-2 or equine infection anemia virus (El AV). In some embodiments, the viral transfer plasmid genome is an HIV-1 genome, an SIV genome, mouse mammary tumor virus (MMTV), murine leukemia virus (MLV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A- MLV), Avian myelocytomatosis virus-29 (MC29), or Avian erythroblastosis virus (AEV).

[0170] In some aspects, provided herein is a lentivirus comprising any of the features of any of the transfer plasmids disclosed herein. In some aspects, provided herein is a lentivirus comprising a heterologous nucleic acid sequence. In some embodiments, the heterologous nucleic acid sequence comprises (a) a polynucleotide sequence encoding a partial gag, wherein the polynucleotide sequence comprises a lentivirus 5’ Long Terminal Repeat (LTR) or a modified 5’ LTR; a y packaging signal, a Rev response element (RRE), and a gp41 peptide sequence; (b) a central polypurine tract/central termination sequence (cPPT/CTS); and (c) a 3' long terminal repeat (LTR) that is an inactivated 3' LTR or a self-inactivating 3' LTR. In some aspects, the lentivirus is devoid of: a polynucleotide encoding a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE); and a polynucleotide sequence region between the polynucleotide sequence encoding the partial gag of (a) and the cPPT/CTS sequence of (b) that comprises at least a portion of the pol integrase polynucleotide comprising the core domain.

[0171] A lentivirus provided herein is capable of transducing a target cell with a NOI. In some embodiments, the lentivirus is capable of transducing the target cell with more than one NOI. In some embodiments, the target cell may be transduced in vivo, in vitro or ex vivo. For example, if the cell is a cell from a mammalian subject, the cell may be removed from the subject, transduced, and reimplanted into the subject (i.e., ex vivo transduction). Alternatively the cell may be transduced by direct gene transfer in vivo, using the vector system of the present invention in accordance with standard techniques (such as via injection of vector stocks expressing the NOI). If the cell is part of a cell line which is stable in culture (i.e. survives multiple passages and replicates) then it may be transduced in vitro by standard techniques, for example by exposure of the cell to lentivirus supernatants comprising lentiviruses expressing the NOI.

[0172] In some embodiments, the NOI comprises a recombinant receptor (e.g., a chimeric antigen receptor) and/or additional polypeptide. In some embodiments, the lentivirus comprises a NOI encoding a recombinant protein, such as an antigen receptor such as a chimeric antigen receptor (CAR). In some embodiments, the lentivirus comprises a NOI encoding cytokine receptor. In some embodiments, the lentivirus comprises a NOI encoding an RNA molecule. In some embodiments, the lentivirus comprises a NOI encoding an antigen.

[0173] The target cell may be any cell that is susceptible to transduction. In some embodiments, the transduced cell can be used in a method of treating a disease or condition, e.g., cancer. In some embodiments, the target cell is an immune cell. In some embodiments, the target cell is an immune effector cell. In some embodiments, the immune effector cell is a mammalian cell. In some embodiments, the immune effector cell is a human cell. An immune effector cell refers to a cell involved in mounting innate and adaptive immune responses. In some embodiments, the immune effector cell comprises a lymphocyte, a natural killer cell (NK), a natural killer T (NKT) cell, a macrophage, a monocyte, an eosinophil, a basophil, a neutrophil, a dendritic cell, or a mast cell. In some embodiments, the immune effector cell is a lymphocyte. In some embodiments, the immune effector cell is a cytotoxic T cell, such as a CD4+ T cell, a CD8+ T cell (also referred to as a cytotoxic T cell or CTL), a regulatory T cell (Treg), a Thl cell, a Th2 cell, or a Thl7 cell. In some embodiments, the immune effector cell is a natural killer cell. In some embodiments, the immune effector cell is a myeloid cell. In some embodiments, the immune effector cell is a monocyte. In some embodiments, immune effector cell is a macrophage.

[0174] In some embodiments, the lentivirus promotes enhanced expression of a NOI, such as a recombinant receptor (e.g., a chimeric antigen receptor) and/or additional polypeptide, in a target cell. In some embodiments, the lentivirus results in increased transduction efficiency of an immune cell transduced with the lentivirus compared to an immune cell transduced with a reference lentivirus. In some embodiments, the lentivirus results in increased cytotoxicity of an immune cell transduced with the lentivirus compared to an immune cell transduced with a reference lentivirus. In some embodiments, the lentivirus results in increased expression of naive phenotype markers of an immune cell transduced with the lentivirus compared to an immune cell transduced with a reference lentivirus. In some embodiments, the lentivirus results in reduced expression of exhaustion markers in an immune cell transduced with the lentivirus compared to an immune cell transduced with a reference lentivirus. In some embodiments, the reference lentivirus is identical to the lentivirus but further comprises a polynucleotide encoding a Woodchuck hepatitis virus post- transcriptional regulatory element (WPRE) and a polynucleotide encoding a region between a partial Gag sequence and a central polypurine tract/central termination sequence (cPPT/CTS).

IV. Methods of Transduction and Production of Engineered Cells

[0175] Provided herein is a method of transducing a cell or a population of cells. The recombinant virus can then be isolated and delivered to the engineered mammalian cell in vitro or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In some embodiments, lentivirus vectors are used. In some embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors carrying the immunomodulator (such as immune checkpoint inhibitor) coding sequence and/or selfinactivating lentiviral vectors carrying chimeric antigen receptors can be packaged with protocols known in the art. The resulting lentiviral vectors can be used to transduce a mammalian cell (such as primary human T cells) using methods known in the art. Vectors derived from retroviruses such as lentivirus are suitable tools to achieve long-term gene transfer, because they allow long-term, stable integration of a NOI and its propagation in progeny cells. Lentiviral vectors also have low immunogenicity, and can transduce nonproliferating cells.

[0176] In some embodiments, the method of transducing a cell or a population of cells comprises contacting the cell with any of the lentiviruses disclosed herein. In some embodiments, the method comprises incubating the cell contacted with the lentivirus under conditions that promote expression a NOI. Also provided herein is a transduced cell or a population of transduced cells produced according to any of the methods provided herein.

[0177] In some aspects, the transduced cell or population of transduced cells do not express a polynucleotide encoding the WPRE and a polynucleotide encoding the region between the partial Gag sequence and the central polypurine tract/central termination sequence (cPPT/CTS). [0178] In some embodiments, the transduced cell or population of transduced cells includes an immune cell. In some embodiments, the immune cell is an immune effector cell. In some embodiments, the immune effector cell is a mammalian cell. In some embodiments, the immune effector cell is a human cell. In some embodiments, the immune effector cell comprises a lymphocyte, a natural killer cell (NK), a natural killer T (NKT) cell, a macrophage, a monocyte, an eosinophil, a basophil, a neutrophil, a dendritic cell, or a mast cell. In some embodiments, the immune effector cell is a lymphocyte. In some embodiments, the immune effector cell is a cytotoxic T cell, such as a CD4+ T cell, a CD8+ T cell (also referred to as a cytotoxic T cell or CTL), a regulatory T cell (Treg), a Thl cell, a Th2 cell, or a Thl7 cell. In some embodiments, the immune effector cell is a natural killer cell. In some embodiments, the immune effector cell is a myeloid cell. In some embodiments, the immune effector cell is a monocyte. In some embodiments, immune effector cell is a macrophage.

[0179] In some embodiments, the transduced cell or population of cells expresses at least one engineered receptor. In some embodiments, the at least one engineered receptor is an antigen- specific receptor recognizing a protein target expressed by a target cell. In other embodiments, the at least one engineered receptor is not an antigen- specific receptor and does not recognize a protein target expressed by a cell. In some embodiments, the transduced cell or population of cells expresses at least one engineered molecule.

[0180] In some embodiments, provided herein is a composition comprising a population of cells provided herein and any of the lentiviruses provided herein. In some embodiments, the population of cells comprises any population of cells that can be transduced with a lentivirus. In some embodiments, the population of cells comprises an immune cell as described above. In some embodiments, the population of cells comprises a population of effector cells. In some embodiments, the population of cells comprises a population of T cells or NK cells.

V. Definitions

[0181] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. [0182] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0183] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.”

[0184] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

[0185] The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Polypeptides, including the provided receptors and other polypeptides, e.g., linkers or peptides, may include amino acid residues including natural and/or non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, and phosphorylation. In some aspects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

[0186] As used herein, “percent (%)sequence identity” and “percent identity” when used with respect to a sequence (reference nucleotide sequence or polypeptide sequence) is defined as the percentage of nucleotide or amino acid residues in a candidate sequence (e.g., the sequence of interest such as a transfer plasmid or component thereof) that are identical with the nucleotide or amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various known ways, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For instance, percentage of sequence identity” or “sequence similarity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or amino acid sequence in the comparison window may comprise substitutions, or additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise substitutions, additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Protein and nucleic acid sequence identities are evaluated using the Basic Local Alignment Search Tool (“BLAST”), which is well known in the art (Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268; Altschul et al., 1997, Nucl. Acids Res. 25: 3389-3402). The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as “high-scoring segment pairs,” between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database. Preferably, the statistical significance of a high- scoring segment pair is evaluated using the statistical significance formula (Karlin and Altschul, 1990), the disclosure of which is incorporated by reference in its entirety. The BLAST programs can be used with the default parameters or with modified parameters provided by the user. Percent identity of polynucleotides described herein can be any integer from 85% to 100%, such as at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0187] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof. [0188] The term “vector” is used herein to refer to a nucleic acid molecule, mircroorganism, or virus capable of transferring or transporting another nucleic acid molecule to a cell or genome. Illustrative examples of vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, bacteria, and viral vectors.

[0189] The term “viral vector” refers to a nucleic acid molecule that includes virus- derived nucleic acid elements that typically facilitate transfer of the nucleic acid molecule or integration into a cell and/or genome. The term “viral vector” includes a modified virus or viral particle capable of transferring a nucleic acid into a cell and/or genome. Viral vectors may contain structural and/or functional genetic elements that are primarily derived from a virus. Viral vectors suitable for use in preferred embodiments, include but are not limited to retroviral vectors and lentiviral vectors. In particular embodiments, a viral vector comprises a 5’ LTR, a packaging signal, a cPPT/CTS element, a NOI, and a 3’ LTR. Viral vectors may optionally comprise post-transcriptional regulatory elements and polyadenylation signals/sequences .

[0190] As used herein, the term “retrovirus” or “retroviral vector” refers to a viral vector that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome. Illustrative retroviral vectors suitable for use in particular embodiments, include, but are not limited to those derived from Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)) and lentivirus.

[0191] As used herein, the term “lentivirus” with reference to a lentiviral vector refers to a group (or species) of complex retroviruses. Illustrative lentiviral vectors suitable for use in particular embodiments contemplated herein include, but are not limited to those derived from HIV (human immunodeficiency virus ; including HIV type 1, and HIV type 2); visna- maedi virus (VMV); the caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0192] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0193] As used herein, a “subject” is a mammal, such as a human or other animal, and typically is human. In some embodiments, the subject, e.g., patient, to whom the agent or agents, cells, cell populations, or compositions are administered, is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or an ape. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent.

VI. Embodiments

[0194] Among the provided embodiments are

Embodiment 1. A transfer plasmid comprising a nucleic acid sequence comprising in order:

(a) a polynucleotide sequence encoding a partial gag, wherein the polynucleotide sequence comprises a lentivirus 5’ Long Terminal Repeat (LTR) or a modified 5’ LTR, a y packaging signal, a Rev response element (RRE), and a nucleotide sequence encoding a gp41 peptide sequence;

(b) a central polypurine tract/central termination sequence (cPPT/CTS); and

(c) a 3' long terminal repeat (LTR) that is an inactivated 3' LTR or a self-inactivating 3' LTR; wherein the transfer plasmid is devoid of: a polynucleotide encoding a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE); and a polynucleotide sequence region between the polynucleotide sequence encoding the partial gag of (a) and the cPPT/CTS sequence of (b) that comprises a portion of the pol integrase polynucleotide comprising the core domain.

Embodiment 2. A transfer plasmid comprising a nucleic acid sequence comprising in order: (a) a lentivirus 5’ Long Terminal Repeat (LTR) or a modified 5’ LTR;

(b) a \|/ packaging signal;

(c) a Rev response element (RRE);

(d) a nucleotide sequence encoding a nucleotide sequence encoding a gp41 peptide sequence;

(e) a central polypurine tract/central termination sequence (cPPT/CTS); and

(f) a 3' long terminal repeat (LTR) that is an inactivated 3' LTR or a self-inactivating 3' LTR; wherein the transfer plasmid is devoid of: a polynucleotide sequence region downstream of the gp41 peptide sequence and upstream of the cPPT/CTS sequence that comprises a portion of the pol integrase polynucleotide comprising the core domain; and a polynucleotide encoding a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

Embodiment 3. The transfer plasmid of embodiment 1 or embodiment 2, wherein the polynucleotide sequence region is 350-370 nucleotides in length.

Embodiment 4. The transfer plasmid of any one of embodiments 1 to 3, wherein the polynucleotide sequence region is about 367 nucleotides in length.

Embodiment 5. The transfer plasmid of any one of embodiments 1 to 4, wherein the polynucleotide sequence region comprises a nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 8.

Embodiment 6. The transfer plasmid of any of embodiments 1 to 5, wherein the polynucleotide sequence region comprises the nucleotide sequence set forth in SEQ ID NO: 8.

Embodiment 7. A transfer plasmid comprising a nucleic acid sequence comprising in order:

(a) a lentivirus 5’ Long Terminal Repeat (LTR) or a modified 5’ LTR; (b) a polynucleotide encoding a portion of the gag protein, wherein the polynucleotide comprises a y packaging signal;

(c) a partial env sequence comprising a Rev responsive element (RRE);

(d) a partial pol sequence comprising a central polypurine tract/central termination sequence (cPPT/CTS) but that is devoid of a polynucleotide sequence encoding a portion of the integrase comprising the core domain; and

(e) a 3' long terminal repeat (LTR) that is an inactivated 3' LTR or a self-inactivating 3' LTR; wherein the transfer plasmid is devoid of a polynucleotide encoding a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

Embodiment 8. A transfer plasmid comprising a nucleic acid sequence comprising in order:

(a) a lentivirus 5’ Long Terminal Repeat (LTR) or a modified 5’ LTR;

(b) a \|/ packaging signal;

(c) a Rev response element (RRE);

(e) a central polypurine tract/central termination sequence (cPPT/CTS); and

(f) a 3' long terminal repeat (LTR) that is an inactivated 3' LTR or a self-inactivating 3' LTR; wherein no more than 300 nucleotides separate the nucleotide sequence encoding the gp41 peptide sequence and the cPPT/CTS and wherein the transfer plasmid is devoid of a polynucleotide encoding a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

Embodiment 9. The transfer plasmid of embodiment 8, wherein the transfer plasmid is devoid of a portion of the pol integrase polynucleotide comprising the core domain.

Embodiment 10. The transfer plasmid of any of embodiments 7 to 9, wherein the transfer plasmid is devoid of a nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 8. Embodiment 11. The transfer plasmid of any of embodiments 7 to 10, wherein the transfer plasmid is devoid of the nucleotide sequence set forth in SEQ ID NO: 8.

Embodiment 12. The transfer plasmid of any of embodiments 1 to 11, wherein the transfer plasmid is devoid of a WPRE nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 7.

Embodiment 13. The transfer plasmid of any of embodiments 1 to 12, wherein the transfer plasmid is devoid of a WPRE nucleotide sequence set forth in SEQ ID NO: 7.

Embodiment 14. A transfer plasmid comprising a nucleic acid sequence comprising:

(a) a lentivirus 5’ Long Terminal Repeat (LTR) or a modified 5’ LTR;

(b) a \|/ packaging signal;

(c) a Rev response element (RRE);

(d) a central polypurine tract/central termination sequence (cPPT/CTS); and

(e) a 3' long terminal repeat (LTR) that is an inactivated 3' LTR or a self-inactivating 3' LTR; wherein the transfer plasmid is devoid of: a polynucleotide comprising a nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 7; and a polynucleotide comprising a nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 8.

Embodiment 15. The transfer plasmid of embodiment 14, wherein the transfer plasmid is devoid of a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 7.

Embodiment 16. The transfer plasmid of embodiment 14 or embodiment 15, wherein the transfer plasmid is devoid of a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 8. Embodiment 17. The transfer plasmid of embodiment 15 or embodiment 16, wherein the transfer plasmid comprises a nucleotide sequence encoding a nucleotide sequence encoding a gp41 peptide sequence.

Embodiment 18. The transfer plasmid of any of embodiments 1 to 17, wherein the 5’ LTR or modified 5’ LTR comprises a U5 and R domain.

Embodiment 19. The transfer plasmid of any of embodiments 1 to 18, wherein the 5’ LTR is a modified 5’ LTR that is truncated to lack a part or all of the U3 region.

Embodiment 20. The transfer plasmid of any of embodiments 1 to 19, wherein the 5’ LTR is a modified 5’ LTR that comprises the sequence set forth in SEQ ID NO: 20.

Embodiment 21. The transfer plasmid of embodiment 19 or embodiment 20, wherein the modified 5’ LTR comprises a heterologous regulatory element that is not endogenous to a lentivirus, wherein the heterologous regulatory element is immediately upstream of the modified 5’ LTR.

Embodiment 22. The transfer plasmid of embodiment 21, wherein the heterologous regulatory element is a promoter, enhancer or a promoter/enhancer.

Embodiment 23. The transfer plasmid of embodiment 21 or embodiment 22, wherein the heterologous regulatory element is a cytomegalovirus enhancer, promoter or enhancer/promoter.

Embodiment 24. The transfer plasmid of any of embodiments 21 to 23, wherein the heterologous regulatory element comprises a nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 36.

Embodiment 25. The transfer plasmid of any of embodiments 21 to 24, wherein the heterologous regulatory element comprises a nucleotide sequence set forth in SEQ ID NO: 36. Embodiment 26. The transfer plasmid of any of embodiments 1 to 25, wherein the 3’ LTR comprises a U5 and R domain.

Embodiment 27. The transfer plasmid of any of embodiments 1 to 26, wherein the 3’ LTR is a truncated 3’ LTR comprising a deleted U3 region in which one or more nucleotide bases of the U3 region of the 3’ LTR are deleted.

Embodiment 28. The transfer plasmid of embodiment 27, wherein the deleted U3 region retains the att sequence and comprises deletions of the enhancer and/or core promoter U3.

Embodiment 29. The transfer plasmid of embodiment 27 or embodiment 28, wherein the deleted U3 region lacks at least one of an enhancer sequence, a TATA box, an Spl site, an NK-kappa B site, or a polypurine tract (PPT).

Embodiment 30. The transfer plasmid of any of embodiments 1 to 29, wherein the 3’ LTR comprises the sequence set forth in SEQ ID NO: 29.

Embodiment 31. The transfer plasmid of any of embodiments 1 to 30, wherein the transfer plasmid comprises a polyadenylation signal within the R region or downstream of the 3’ LTR.

Embodiment 32. The transfer plasmid of embodiment 31, wherein the polyadenylation signal is an SV40 polyadenylation signal.

Embodiment 33. The transfer plasmid of any of embodiments 1 to 32, wherein the y packaging signal comprises the nucleotide sequence set forth in SEQ ID NO: 21.

Embodiment 34. The transfer plasmid of any of embodiment 1 to 33, wherein the Rev response element (RRE) comprises the nucleotide sequence set forth in SEQ ID NO: 22. Embodiment 35. The transfer plasmid of any of embodiments 1 to 34, wherein the central polypurine tract/central termination sequence (cPPT/CTS) comprises the nucleotide sequence set forth in SEQ ID NO: 10.

Embodiment 36. The transfer plasmid of any of embodiments 1 to 35, wherein the nucleotide sequence encoding the gp41 peptide sequence comprises the nucleotide sequence set forth in SEQ ID NO: 23.

Embodiment 37. The transfer plasmid of any of embodiments 1 to 36, wherein the transfer plasmid comprises an origin of replication site.

Embodiment 38. The transfer plasmid of embodiment 37, wherein the origin of replication site comprises a pUC origin of replication, a SV40 origin of replication and/or an fl bacteriophage origin of replication.

Embodiment 39. The transfer plasmid of any of embodiments 1 to 38, wherein the transfer plasmid comprises a Kozak sequence.

Embodiment 40. The transfer plasmid of any of embodiments 1 to 39, wherein the transfer plasmid comprises a multiple cloning site that allows for insertion of a nucleotide sequence of interest (NOI).

Embodiment 41. The transfer plasmid of any of embodiments 1 to 40, wherein the transfer plasmid comprises a nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 39.

Embodiment 42. A transfer plasmid comprising a nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 39.

Embodiment 43. The transfer plasmid of embodiment 42, wherein the transfer plasmid comprises a multiple cloning site that allows for insertion of a nucleotide sequence of interest (NOI). Embodiment 44. The transfer plasmid of any of embodiments 1 to 43, wherein the transfer plasmid comprises the nucleotide sequence set forth in SEQ ID NO: 39.

Embodiment 45. The transfer plasmid of any of embodiments 40 to 44, wherein the transfer plasmid further comprises a nucleotide sequence of interest (NOI) inserted within the multiple cloning site.

Embodiment 46. The transfer plasmid of any of embodiment 40 to 45, wherein the nucleotide sequence of interest encodes a protein, an RNA molecule, an enzyme or an antibody or any combination thereof.

Embodiment 47. The transfer plasmid of any of embodiments 40 to 46, wherein the nucleotide sequence of interest (NOI) encodes a chimeric antigen receptor (CAR), a cytokine receptor, an RNA molecule, a synthetic antigen or any combination thereof.

Embodiment 48. The transfer plasmid of any of embodiments 40 to 47, wherein the nucleotide sequence of interest is a multicistronic sequence.

Embodiment 49. The transfer plasmid of any of embodiments 40 to 48, wherein the nucleotide sequence of interest is up to 4000 base pairs in length.

Embodiment 50. The transfer plasmid of any of embodiments 40 to 49, wherein the nucleotide sequence of interest is 2000 to 3600 base pairs in length.

Embodiment 51. The transfer plasmid of any of embodiments 40 to 49, wherein the nucleotide sequence of interest is 2800 to 3400 base pairs in length.

Embodiment 52. The transfer plasmid of any of embodiments 1 to 51, further comprising a non-viral promoter, wherein the non- viral promoter is operably linked to control expression of the nucleotide sequence of interest.

Embodiment 53. The transfer plasmid of embodiment 52, wherein the non-viral promoter comprises an EF- la promoter. Embodiment 54. A composition comprising the transfer plasmid of any of embodiments 1 to 53, an envelope plasmid and one or more packaging plasmids.

Embodiment 55. The composition of embodiment 54, wherein the one or more packaging plasmid comprises a first packaging plasmid encoding Gag and Pol and a second packaging plasmid encoding Rev.

Embodiment 56. The composition of embodiment 54 and embodiment 55, wherein the one or more envelope plasmid encodes a VSV-G glycoprotein.

Embodiment 57. A method of producing a lentivirus comprising:

(a) contacting a host cell with the composition of any one of embodiments 54 to 56;

(b) culturing the host cell under conditions that produce the lentivirus; and

(c) isolating the lentivirus.

Embodiment 58. A method of producing a lentivirus comprising:

(a) contacting a host cell with the transfer plasmid of any of embodiments 1 to 53 and one or more packaging plasmids;

(b) culturing the host cell under conditions that produce the lentivirus; and

(c) isolating the lentivirus.

Embodiment 59. The method of embodiment 57 and embodiment 58, wherein the one or more packaging plasmid comprises a first packaging plasmid encoding Gag and Pol and a second packaging plasmid encoding Rev.

Embodiment 60. The method of any of embodiments 57 to 59, wherein the one or more envelope plasmid encodes a VSV-G glycoprotein.

Embodiment 61. The method of any of embodiments 57 to 60, wherein the host cell is an adherent cell. Embodiment 62. The method of any of embodiments 57 to 60, wherein the host cell is a suspension cell.

Embodiment 63. The method of any of embodiments 57 to 62, wherein the host cell is an HEK293 cell, a COS cell, a recombinant Chinese hamster ovary (CHO), a HeLa cell, a NIH3T3 cell, a PC12 cell, a U20S cell, an A549 cell, an HT1080 cell, a CAD cell, a P19 cell, an L929 cell, an N2a cell, an MCF-7, Y79 cell, an SO-Rb50 cell, a Hep G2 cell, a DUKX- XI 1 cell, a J558L cell, a baby hamster kidney (BHK) cell, or a derivative thereof.

Embodiment 64. The method of any of embodiments 5 to 63, wherein the host cell is an HEK293S cell, an HEK293Ts cell, an HEK293F cell, an HEK293FT cell, an HEK293FTM cell, an HEK293E cell, an LV293 cell, or an HEK293T/17 cell.

Embodiment 65. A host cell comprising the transfer plasmid of any of embodiments 1 to 53, an envelope plasmid, and one or more packaging plasmids.

Embodiment 66. The host cell of embodiment 65, wherein the one or more packaging plasmid comprises a first packaging plasmid encoding Gag and Pol and a second packaging plasmid encoding Rev.

Embodiment 67. The host cell of embodiment 65 or embodiment 66, wherein the one or more envelope plasmid encodes a VSV-G glycoprotein.

Embodiment 68. The host cell of embodiment 66 or embodiment 67, wherein the host cell is an adherent cell.

Embodiment 69. The host cell of embodiment 66 or embodiment 67, wherein the host cell is a suspension cell.

Embodiment 70. The host cell of any of embodiments 67 to 69, wherein the host cell is an HEK293 cell, a COS cell, a recombinant Chinese hamster ovary (CHO), a HeLa cell, a NIH3T3 cell, a PC 12 cell, a U20S cell, an A549 cell, an HT1080 cell, a CAD cell, a P19 cell, an L929 cell, an N2a cell, an MCF-7, Y79 cell, an SO-Rb50 cell, a Hep G2 cell, a DUKX-X1 1 cell, a J558L cell, a baby hamster kidney (BHK) cell, or a derivative thereof.

Embodiment 71. The host cell of any of embodiments 67 to 70, wherein the host cell is an HEK293S cell, an HEK293Ts cell, an HEK293F cell, an HEK293FT cell, an HEK293FTM cell, an HEK293E cell, an LV293 cell, or an HEK293T/17 cell.

Embodiment 72. A method of producing a lentivirus comprising:

(a) culturing the host cell of any of embodiments 65 to 71 under conditions that produce the lentivirus; and

(b) isolating the lentivirus.

Embodiment 73. A lentivirus produced by the method according to any of embodiments 57 to 64 and 72.

Embodiment 74. A lentivirus comprising a heterologous nucleic acid sequence, comprising

(a) a polynucleotide sequence encoding a partial gag, wherein the polynucleotide sequence comprises a lentivirus 5’ Long Terminal Repeat (LTR) or a modified 5’ LTR; a y packaging signal, a Rev response element (RRE), and a nucleotide sequence encoding a gp41 peptide sequence;

(b) a central polypurine tract/central termination sequence (cPPT/CTS); and

(c) a 3' long terminal repeat (LTR) that is an inactivated 3' LTR or a self-inactivating 3' LTR; wherein the lentivirus is devoid of: a polynucleotide encoding a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE); and a polynucleotide sequence region between the polynucleotide sequence encoding the partial gag of (a) and the cPPT/CTS sequence of (b) that comprises a portion of the pol integrase polynucleotide comprising the core domain.

Embodiment 75. A lentivirus comprising a nucleic acid sequence comprising in order: (a) a lentivirus 5’ Long Terminal Repeat (LTR) or a modified 5’ LTR; (b) a \|/ packaging signal;

(c) a Rev response element (RRE);

(e) a central polypurine tract/central termination sequence (cPPT/CTS); and

(f) a 3' long terminal repeat (LTR) that is an inactivated 3' LTR or a self-inactivating 3' LTR; wherein the lentivirus is devoid of: a polynucleotide sequence region downstream of the gp41 peptide sequence and upstream of the cPPT/CTS sequence that comprises a portion of the pol integrase polynucleotide comprising the core domain; and a polynucleotide encoding a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

Embodiment 76. The lentivirus of embodiment 74 or embodiment 75, wherein the polynucleotide sequence region is 350-370 nucleotides in length.

Embodiment 77. The lentivirus of any one of embodiments 74 to 76, wherein the polynucleotide sequence region is about 367 nucleotides in length.

Embodiment 78. The lentivirus of any one of embodiments 74 to 77, wherein the polynucleotide sequence region comprises a nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 8.

Embodiment 79. The lentivirus of any of embodiments 74 to 78, wherein the polynucleotide sequence region comprises the nucleotide sequence set forth in SEQ ID NO: 8.

Embodiment 80. A lentivirus comprising a nucleic acid sequence comprising in order:

(a) a lentivirus 5’ Long Terminal Repeat (LTR) or a modified 5’ LTR;

(b) a polynucleotide encoding a portion of the gag protein, wherein the polynucleotide comprises a y packaging signal;

(c) a partial env sequence comprising a Rev responsive element (RRE); (d) a partial pol sequence comprising a central polypurine tract/central termination sequence (cPPT/CTS) but that is devoid of a polynucleotide sequence encoding a portion of the integrase comprising the core domain; and

(e) a 3' long terminal repeat (LTR) that is an inactivated 3' LTR or a self-inactivating 3' LTR; wherein the lentivirus is devoid of a polynucleotide encoding a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

Embodiment 81. A lentivirus comprising a nucleic acid sequence comprising in order:

(a) a lentivirus 5’ Long Terminal Repeat (LTR) or a modified 5’ LTR;

(b) a \|/ packaging signal;

(c) a Rev response element (RRE);

(e) a central polypurine tract/central termination sequence (cPPT/CTS); and

(f) a 3' long terminal repeat (LTR) that is an inactivated 3' LTR or a self-inactivating 3' LTR; wherein no more than 300 nucleotides separate the nucleotide sequence encoding the gp41 peptide sequence and the cPPT/CTS and wherein the lentivirus is devoid of a polynucleotide encoding a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

Embodiment 82. The lentivirus of embodiment 80 or embodiment 81, wherein the lentivirus is devoid of a portion of the pol integrase polynucleotide comprising the core domain.

Embodiment 83. The lentivirus of any of embodiments 80 to 82, wherein the lentivirus is devoid of a nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 8.

Embodiment 84. The lentivirus of any of embodiments 80 to 83, wherein the lentivirus is devoid of the nucleotide sequence set forth in SEQ ID NO: 8. Embodiment 85. The lentivirus of any of embodiments 74 to 84, wherein the lentivirus is devoid of a WPRE nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 7.

Embodiment 86. The lentivirus of any of embodiments 74 to 85, wherein the lentivirus is devoid of a WPRE nucleotide sequence set forth in SEQ ID NO: 7.

Embodiment 87. The lentivirus of any of embodiments 74 to 86, further comprising a nucleotide sequence of interest (NOI).

Embodiment 88. The lentivirus of embodiment 87, wherein the NOI encodes a chimeric antigen receptor (CAR), a cytokine receptor, an RNA molecule, a synthetic antigen or any combination thereof.

Embodiment 89. The lentivirus of any of embodiments 73 to 88, wherein the lentivirus results in increased transduction efficiency of an immune cell transduced with the lentivirus compared to an immune cell transduced with a reference lentivirus.

Embodiment 90. The lentivirus of any of embodiments 73 to 89, wherein the lentivirus results in increased cytotoxicity of an immune cell transduced with the lentivirus compared to an immune cell transduced with a reference lentivirus.

Embodiment 91. The lentivirus of any of embodiments 73 to 90, wherein the lentivirus results in increased expression of naive phenotype markers of an immune cell transduced with the lentivirus compared to an immune cell transduced with a reference lentivirus.

Embodiment 92. The lentivirus of any of embodiments 73 to 91, wherein the lentivirus results in reduced expression of exhaustion markers in an immune cell transduced with the lentivirus compared to an immune cell transduced with a reference lentivirus.

Embodiment 93. The lentivirus of any of embodiments 73 to 92, wherein the reference lentivirus is identical to the lentivirus but further comprises a polynucleotide encoding a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and a polynucleotide encoding a region between a partial Gag sequence and a central polypurine tract/central termination sequence (cPPT/CTS).

Embodiment 94. A method of transducing a cell comprising contacting a cell with the lentivirus of any of embodiments 73 to 93.

Embodiment 95. The method of embodiment 94, wherein the method comprises incubating the cell contacted with the lentivirus under conditions that promote expression of the NOI.

Embodiment 96. The method of embodiment 94 or embodiment 95, wherein the cell is an immune cell.

Embodiment 97. The method of any of embodiments 94 to 96, wherein the cell is an effector cell.

Embodiment 98. The method of any of embodiments 94 to 97, wherein the cell is a T cell or an NK cell.

Embodiment 99. A transduced cell produced according to the method of any of embodiments 94 to 98.

Embodiment 100. A composition comprising a population of cells and the lentivirus of any of embodiments 73 to 93.

Embodiment 101. The composition of embodiment 100, wherein the population of cells comprises a population of immune cells.

Embodiment 102. The composition of embodiment 100 or embodiment 101, wherein the population of cells comprises a population of effector cells. Embodiment 103. The composition of any of embodiments 100 to 102, wherein the population of cells comprises a population of T cells or NK cells.

VII. Examples

[0195] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1 Generation and Characterization of Modified Lenti viral Vectors

[0196] In order to improve the packaging capacity of HIV-1 based lentiviral vectors, a reference transfer vector pTRPE vector (SEQ ID NO: 1; plasmid map shown in FIG. 1A) encoding a NOI (i.e., pay load) was modified by deleting genomic regions including: EF-la promoter intron (EF-la-Int), which is involved in lentiviral promoter activity; Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), which is involved in mRNA stability and nuclear export; central polypurine tract//central termination sequence (cPPT/CTS), which is involved in recognition for proviral DNA synthesis; and a polynucleotide non-conserved 367 bp region between the a polynucleotide sequence encoding an HIV-1 partial Gag sequence (the polynucleotide sequence encoding the partial gag sequence containing the 5’-LTR, element necessary for genome packaging, RRE, and gp41 peptide) and the cPPT/CTS sequence.

1. Generation of Modified Lentiviral Vectors

[0197] To generate a modified lentiviral vector, the EF-la promoter intron, WPRE, cPPT, or BTW were removed from the lentiviral plasmid pTRPE to individually assess the impact of removal on NOI expression. To delete EF-la promoter intron (EFla-Int), the pTRPE vector was digested with restriction enzymes Miul and Nhel to excise the entire EF- la promoter and then the core of the EF-la promoter was amplified from pTRPE with primers designed to add In-fusion-compatible overlap regions that would hybridize with the digested vector. WPRE was deleted from pTRPE with an EcoRVSall double digest and the ends were blunted and circularized with Quick Ligase (New England Biolabs, MA, Catalog No. M2200L). cPPT and the BTW region were deleted with flanking PCR primers containing a 15 bp overlap at the 5’ end followed by self-ligation of the resulting amplicon in an InFusion reaction (Takara Bio, CA, Catalog No. 638945). The modifications resulted in transfer plasmid lentiviral vectors deleted either for the 939 base pair EF-la promoter intron (lacking EF-la-Int SEQ ID NO: 9; AEF-la-Int transfer plasmid SEQ ID NO: 6), a 589 base pair WPRE region (lacking WPRE SEQ ID NO: 7; AWPRE transfer plasmid SEQ ID NO: 3), a 118 base pair cPPT region (lacking cPPT SEQ ID NO: 10; AcPPT transfer plasmid SEQ ID NO: 5), or the 367 base pair BTW region, corresponding to part of the HIV Pol upstream of cPPT (lacking BTW region SEQ ID NO: 8; ABTW transfer plasmid SEQ ID NO: 4).

[0198] Lentivirus (LV) was produced using the LV-Max system (ThermoFisher Scientific; Catalog # A35684) according to the manufacturer’s instructions. Briefly, a high- density culture of HEK293F cells (LV-Max cells) were cultured, expanded and transfected with total plasmids including a lentiviral packaging plasmid (containing a mix of three packaging plasmids pLPl vector encoding Gag and Pol, pLP2 encoding Rev and pLP/VSVG encoding the VSVG envelope protein) and one of the generated modified lentiviral transfer plasmids that encoded a NOI (e.g., chimeric antigen receptor, CAR). In this experiment, the NOI was anti-HER24D5 CAR. The NOI also encoded GFP separated from the 5’ end of the CAR by a T2A cleavable linker (T2A-GFP). Approximately 48-55 hours post-transfection, lentivirus was harvested and titrated over a 7-point dilution series on primary T cells activated by anti-CD3/anti-CD28 beads for 48 hours. P24 content was measured with Lenti-X GoStix (Takara).

2. Characterization of Modified Lentiviral Vectors on Transduction and NOI Expression

[0199] The impact of deletion of the above regions was assessed by measuring NOI expression in target cells that had been transduced with the above lentivirus preparations. Phenotypic markers, exhaustion markers and function were also assessed.

[0200] Primary T cells from healthy donors (HDs) were isolated and grown in RPMI- 1640 with 10% FBS, 1% pen-strep, 1% NEAA, 1% GlutaMAX, and 1% sodium pyruvate. On day 0, the primary T cells were incubated with 5 ng/mL IL-7 and 5 ng/mL IL- 15 for 6 to 8 hours before overnight activation with anti-CD3/anti-CD28 beads at a ratio of 3:1 (beads:cells). Primary T cells were transduced with CAR-carrying LV generated using one of the transfer plasmids as described above at an MOI of 1 on day 1. The transduced T cells were de-beaded and media containing 5 ng/mL IL-7 and 5 ng/mL IL- 15 was added during culture (e.g. days 3, 6 and 8) for T cell expansion. On about day 9, primary T cells were assessed by flow cytometry for phenotyping and CAR expression. Transduction efficiency and titer was also determined based on expression of GFP.

[0201] Transduction of A549 cells as an alternative target cell also was assessed. A549 lung carcinoma cells were also transduced with the LV preparations at a MOI of 1 for 48 hours. Transduction efficiency of lentivirus preparations was determined by measuring CAR expression using an anti-idiotypic antibody against the anti-HER2 4D5 CAR. The anti- idiotypic antibody was conjugated to AF647 (R&D Systems, AFR1129-020) and used at a dilution of 1:200. The anti-idiotypic antibody :AF647 conjugate was detected by flow cytometry.

[0202] As shown in FIG. 2A, the lentivirus preparations were functional as evidenced by dose-dependent expression of the CAR. CAR expression was highest in T cells transduced with LV deleted for WPRE (AWPRE) or BTW (ABTW), as compared to T cells transduced with LV deleted for cPPT (AcPPT) or EF-la-Int.(AEF-la-Int) or compared to LV produced from the parental pTRPE transfer plasmid. Results in FIG. 2B, depicting expression of either the CAR (4D5) or GFP in primary T cells at the end of CAR production, also showed that LV deleted for either cPPT or EFla-Int had a negative impact on percent of T cells positive for GFP and reduced gene expression of the CAR compared to LV produced using the parental pTRPE vector. In contrast, removing either BTW or WPRE from LV increased percent of cells positive for GFP and percent of cells positive for the CAR. The results thus indicate that LV with either cPPT or EFla-Int deleted produced fewer CAR+ transduced T cells while LV with either BTW or WPRE deletions yielded ~1.5 fold more CAR+ transduced T cells than achieved using LV produced using the pTRPE parental vector.

[0203] As shown in FIG. 2C, deletion of cPPT and EFla-Int in LV had a negative impact on GFP mean fluorescence intensity (MFI) following transduction of LV into either A549 cells or primary human T cells, compared to transduction of LV produced using the pTRPE transfer plasmid. In contrast, increased GFP MFI was observed following transduction of both cell types with LV in which the BTW region was removed. Interestingly, the absence of WPRE had a cell type-specific effect as it reduced GFP expression in A549s but increased GFP MFI in T cells.

3. Characterization of Modified Lentiviral Vectors on T Cell Phenotype After Transduction

[0204] The impact of WPRE, BTW, cPPT and EF-la-Int removal in LV after transduction of LV preparation into T cells and production of NOLexpressing T cells as described above on T cell phenotype and exhaustion was also assessed. T cell phenotype was assessed by monitoring cell surface expression of CD27 and CD45RA by flow cytometry. CD27+/CD45RA- cells represent central memory (CM) T cells; CD27+/CD45RA+ cells represent naive T cells; CD27-/CD45RA- cells represent effector memory (EM) T cells; CD27-/CD45RA+ cells represent effector memory cells re-expressing CD45RA (TEMRA). As shown in FIG. 3A, removal of cPPT, BTW, WPRE and EF-la-Int in LV used to transduce T cells did not significantly impact T cell phenotype relative to T cells transduced with LV produced using pTRPE or untransduced (UTD) cells. The majority of T cells transduced with the LV vectors used for CAR T cell production were CD27+/CD45RA-, which are central memory (CM) T cells. As shown in FIG. 3B, removal of cPPT, BTW, WPRE and EF-la-Int also did not significantly impact T cell exhaustion as measured by PD- 1, LAG3 and TIM3.

4. T-Cell Mediated Killing

[0205] CAR+ T cells produced as described above by transduction with the modified LV preparations followed by a 9 day expansion were assessed for T cell-mediated killing activity. T cell-mediated killing assays were performed against target cells expressing an antigen recognized by the CAR on an xCELLigence microelectronic biosensor system (Agilent). In these experiments, the HER2-expressing target cells were used, including SKOV3 and A549 target cells. Real-Time Cell Analysis 96-well plates were filled with 50 pL media for a blank baseline measurement of impedance. Tumor target cells were then plated in 50 pL at 1 x 104 cells/well and returned to the xCELLigence to track target cell growth overnight. To allow cells to settle, electrical impedance measurements were initiated 30 minutes after plating. On the next day, T cells at desired effector:target ratios were added in 100 pL/well.

Effector: target ratios included 1:1, 0.5:1, and 0.25:1. Measurements were acquired every 15 minutes for the duration of the assay and the cell index was normalized to the time point at which T cells were added to the co-culture. Killing activity was monitored for approximately 168 hours or 7 days. The post-killing phenotype was assessed by flow cytometry immediately following completion of the co-culture.

[0206] As shown in FIGS. 4A-4C, production of CAR T cells by transduction with LV that included removal of either WPRE or EF-la-Int resulted in improved T mediated cell killing of A549 across all effector:target ratios, as depicted at a 1:1 E:T ratio (FIG. 4A), 0.5:1 E:T ratio (FIG. 4B) and a 0.25:1 E:T ratio (FIG. 4C). Similar results were observed in T cell killing assays with SKOV3 target cells as shown in FIGS. 5A-5C. Similar results were also observed by CAR T cells produced from human primary T cells from a second donor The increased T cell killing activity on T cells producing using LV deleted for either WPRE or EF-la-Int was surprising since T cell phenotypes were generally similar at the end of the CAR T cell production protocol even with the different LV preparations as shown by FIGS.

3 A and 3B above.

[0207] To further assess the differences in activity of T cells in the T cell killing assay, T cell phenotype and exhaustion was assessed on T cells at the end of the killing assay. As shown in FIG. 6A (A549 target killing) and FIG. 6C (SKOV3 target killing), T cells transduced with LV deleted for either cPPT or EF-la-Int had a memory phenotype comparable to UTD cells. Whereas T cells transduced with LV deleted for BTW or WPRE had phenotypes comparable to T cells transduced with LV using pTRPE. Consistent with this more naive phenotype, FIG. 6B (A549 target killing) and FIG. 6D (SKOV3 target killing) shows that T cells transduced with LV deleted for either cPPT or EF-la-Int had reduced expression of exhaustion markers LAG3 and TIM3 compared to T cells transduced with LV deleted for BTW or WPRE or compared to T cells transduced with LV using pTRPE.

[0208] The above results are based on deleting WPRE from the exemplary lentiviral plasmid pTRPE. To assess the effect in other LVV systems, the same WPRE region was deleted in the lentiviral plasmid opCasl2. A representative opCAS plasmid is depicted in FIG. IB. As shown in FIG. 7A, CAR expression increased in T cells transduced with lentivirus produced from WPRE deleted opCasl2 (i.e., pOpCAS-dWPRE in FIG. 7A) compared to T cells transduced with lentivirus produced from opCasl2, which retained WPRE. The functional advantage of deleting WPRE on T cell-mediated killing of SKOV3 cells is shown in FIG. 7B. As shown in FIG. 7B, T cell-mediated killing of SKOV3 cells was increased by deleting WPRE in opCasl2.

Example 2 Characterization of Lentiviral Vector with deletion of WPRE and BTW region between the HIV-1 partial Gag sequence and cPPT .

[0209] Based on results described in Example 1, a lentiviral vector was designed with combined deletion of BTW and WPRE, while retaining EF-la-Int and cPPT. Lentivirus was produced and assessed for transduction efficiency and impact on phenotype and function of transduced cells.

A. Design and Generation of LVTPR

[0210] Although removing EF-la-Int and cPPT had clear functional advantages as described in Example 1, a lentiviral vector deleted for EF-la-Int or cPPT failed to express CAR as well as lentiviral vectors deleted for WPRE and BTW. The loss of cPPT had a substantial impact on LV titer, and without out being bound by theory LV characterized by an EFla core promoter without the intron is believed to be too weak to express enough CAR at detectable levels in cells with fewer NOI integrations.

[0211] A transfer plasmid was thus generated to delete BTW (ABTW) and WPRE (AWPRE) and retain EFla-Int and cPPT. Specifically, a transfer plasmid, designated LVTPR, was generated that was deleted for a 589 base pair WPRE region (WPRE; SEQ ID NO: 7) and a 367 base pair BTW region, corresponding to part of the HIV Pol upstream of cPPT (BTW; SEQ ID NO: 8). The LVTPR transfer plasmid retained the 939 base pair EF-la promoter intron (EF-la-Int; SEQ ID NO: 9) and the 118 base pair cPPT region (cPPT; SEQ ID NO: 10).

[0212] Another transfer plasmid was generated to delete BTW, WPRE, and EFla-Int (pTRPEAWPRE/ABTW/AEF-la-Int) and used as a comparator in the experiments described herein.

[0213] The features of LVTPR and LVTPC are set forth in Table El and FIGS. 8A-8C. The nucleotide sequence of LVTPR vector is set forth in SEQ ID NO: 2. The designed LVTPR transfer plasmid is 949 base pairs shorter than pTRPE, which is advantageous for the potential to allow increased NOI capacity. A desired NOI or NOI cassette can be inserted into the backbone plasmid set forth in SEQ ID NO: 2 using a restriction enzyme with a cut site inside the cloning region, i.e. Nhel, Bmtl, PacI, Acc65I, and/or KpnI. [0214] Lentivirus (LV) was produced using the LV-Max system as described in Example 1 using LVTPR as the transfer plasmid. In this example, the LVTPR transfer vector was also engineered to carry a NOI that encoded a CAR by insertion into Nhel and PacI restriction cut sites. In these examples, the NOI encoded either (1) a bicistronic construct of the CAR described in Example 1 (anti-HER2 4D5 CAR) and a GFP separated from the 5’ end of the CAR by a T2A bicistronic element (anti-HER2 CAR-T2A-GFP) or (2) a larger bicistronic construct of an scFv-based monospecific CAR to a target molecule ,a P2A bicistronic element, an armored cytokine receptor and an immunostimulatory non-coding RNA. The resulting size of the NOI coding for anti-HER2 CAR-T2A-GFP bicistronic construct was 2244 base pairs and the NOI encoding the larger bicistronic construct was 3208 basepairs. For comparison, EV was similarly produced using pTRPE transfer plasmid carrying the same NOI.

B. Characterization of LVTPR on Transduction and NOI Expression

[0215] Primary T cells from healthy donors (HDs) were isolated as described in Example 1. CAR expressing T cells were produced as described in Example 1 by activation of the isolated primary T cells, transduction of primary T cells with LV at an MOI of 1 followed by expansion with IL-7 and IL- 15 cytokines. On day 9, primary T cells were assessed by flow cytometry for phenotyping and CAR expression. Transduction efficiency and titer was also determined based on expression of GFP.

[0216] Primary T cells were transduced with LV preparations prepared using the LVTPR transfer plasmid, pTRPE transfer plasmid, or pTRPE transfer plasmid without BTW, WPRE and EF-la-Int (“3xM” in FIG. 9A), and with different CARs. To assess transduction, T cells were then normalized on P24 content, a nonfunctional measure of LV material. As shown in FIG. 9A, there was generally consistent increase in transduction as measured by GFP and CAR (4D5 or scFv-based CAR) expression by LV that had been produced using LVTPR transfer plasmid compared to the other alternative transfer plasmids, particularly of LV carrying the larger NOI. Without wishing to be bound by theory, the results indicate that the smaller size of LVTPR was responsible for the measurable improvement in transduction.

[0217] CAR expression in T cells from two different donors following transduction with the different LV preparations is shown in FIG. 9B as a percent of CAR+ transduced T cells and in FIG. 9C as the mean fluorescence intensity (MFI) of the CAR expression. As shown in FIG. 9B, T cells engineered with LV produced using LVTPR had a higher percent of T cells positive for CAR expression compared to T cells transduced with LV produced using pTRPE across two donors. Although the number of T cells transduced with the CAR was increased, results in FIG. 9C show that the mean fluorescence intensity of CAR expression in the T cells on a per cell basis was similar across the two donors, whether T cells were transduced with LV produced using LVTPR or pTRPE.

C. Characterization of LVTPR on T Cell Phenotype After Transduction

[0218] The impact of WPRE and BTW removal in LV preparations produced from LVTPR on T cell phenotype and exhaustion following transduction was assessed. T cell phenotype was assessed by CD27 and CD45RA and T cell exhaustion was assessed by PD-1, LAG3, and TIM3 substantially as described in Example 1. As shown in FIGS. 10A and 10B, removal of WPRE and BTW in LV produced using LVTPR and used to transduce T cells did not significantly impact T cell phenotype relative to T cells transduced with LV produced using pTRPE or untransduced cells in two different donors. As shown in FIG. 10C, analysis of exhaustion markers as measured by PD-1, LAG3 and TIM3 surface expression by flow cytometry showed that removal of WPRE and BTW in LV produced using LVTPR showed no significant difference relative to T cells transduced with LV produced using pTRPE or untransduced cells, although there was a trend towards a mild reduction in expression of TIM3.

D. T-Cell Mediated Killing

[0219] CAR+ T cells produced as described above by transduction with the modified LV preparations followed by a 9 day expansion were assessed for T cell-mediated killing activity as described in Example 1.

[0220] In an initial set of experiments, T cells transduced with LV expressing the anti- HER2 4D5 CAR were assessed for killing against HER2-expressing target cells, including SKOV3, A549 and the prostate cancer cell line PC3. As shown in FIGS. 11A-11L when challenged in a killing assay co-culture against the HER2 target cells, T cells transduced with LV produced using LVTPR outperformed or matched T cells transduced with LV produced using pTRPE in a donor- specific fashion. T cells at the end of the killing assay were monitored for expression of GEP or for CAR expression in the two donors by flow cytometry. Results from post-killing flow expression analysis revealed that GPP and CAR expression in T cells that had been transduced with LV provided from LVTPR was retained on the cell surface in donor 1 cells with target cell type specificity, especially against PC3 (FIG. 12A and FIG. 12B), whereas CAR expression levels were matched in donor 2 among the different T cells (FIG. 12C and 12D). Without wishing to be bound by theory, since CAR downregulation in response to antigen is known and modifications that improve persistence have demonstrated benefits, it is possible that this feature of LVTPR was at least partially responsible for the enhanced cytotoxicity against PC3.

[0221] In a further set of experiments, T cells transduced with LV expressing the scFv- based CAR were assessed for killing against target cells. As shown in FIG. 13A, donor T cells that were engineered to express the scFv-based CAR by transduction with LV produced using LVTPR had a killing advantage over T cells engineered to express the same scFv-based CAR by transduction with LV produced using pTRPE. A slight killing advantage is maintained at lower E:T ratios as shown in FIGS. 13B-13E.

[0222] Taken together, these data support LVTPR as a unique vector with potential advantages over the standard pTRPE transfer plasmid.

Example 3 Assessment of Promoter in Lentiviral Vectors.

[0223] Experiments were carried out to assess replacement of the RSV promoter in the LVTPR vector described in Example 2 with a CMV promoter.

[0224] For replacement of the RSV promoter in LVTPR with a CMV promoter, CMV promoter was synthesized as two separate fragments and inserted between two unmodified flanking fragments into the vector using restriction enzymes Seal and Notl using the NEBuilder HiFi DNA Assembly (New England Biolabs, MA, Catalog No. E2621X). In this example, the LVTPR transfer plasmid was also engineered to carry a large NOI that was a bicistronic construct of an scFv-based monospecific CAR to a target molecule, a P2A bicistronic element, an armored cytokine receptor and an immunostimulatory non-coding RNA. Experiments also were carried out in which the NOI only encoded the scFv-based CAR. In total, four lentivirus vectors were tested: (1) LVTPR comprising RSV promoter and the scFv-based CAR only; (2) LVTPC comprising the CMV promoter and the scFv-based CAR only; (3) LVTPR comprising RSV promoter and a large bicistronic construct comprising the scFv-based CAR; and (4) LVTPC comprising CMV promoter, and the larger bicistronic construct comprising the scFv-based CAR.

[0225] CAR expression was assessed in primary T cells transduced with the modified LVTPR or LVTPC. As depicted in FIG. 14, replacing the RSV promoter with the CMV promoter increased the population of CAR-expressing cells as shown by detection of the CAR and fluorescent protein reporter in the circuit. These data demonstrate that the vectors provided herein with a CMV promoter support increased or improved transduction efficiency. [0226] The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.

SEQUENCE TABLE

Claims

WHAT IS CLAIMED:

1. A transfer plasmid comprising a nucleic acid sequence comprising in order:

(b) a central polypurine tract/central termination sequence (cPPT/CTS); and

2. A transfer plasmid comprising a nucleic acid sequence comprising in order:

(a) a lentivirus 5’ Long Terminal Repeat (LTR) or a modified 5’ LTR;

(b) a \|/ packaging signal;

(c) a Rev response element (RRE);

(d) a nucleotide sequence encoding a gp41 peptide sequence;(e) a central polypurine tract/central termination sequence (cPPT/CTS); and

(f) a 3' long terminal repeat (LTR) that is an inactivated 3' LTR or a self-inactivating 3' LTR; wherein the transfer plasmid is devoid of: a polynucleotide sequence region downstream of the nucleotide sequence encoding the gp41 peptide sequence and upstream of the cPPT/CTS sequence that comprises a portion of the pol integrase polynucleotide comprising the core domain; and a polynucleotide encoding a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

3. The transfer plasmid of claim 1 or claim 2, wherein the polynucleotide sequence region is 350-370 nucleotides in length.

4. The transfer plasmid of any one of claims 1 to 3, wherein the polynucleotide sequence region is about 367 nucleotides in length.

5. The transfer plasmid of any one of claims 1 to 4, wherein the polynucleotide sequence region comprises a nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 8.

6. The transfer plasmid of any of claims 1 to 5, wherein the polynucleotide sequence region comprises the nucleotide sequence set forth in SEQ ID NO: 8.

7. A transfer plasmid comprising a nucleic acid sequence comprising in order:

(a) a lentivirus 5’ Long Terminal Repeat (LTR) or a modified 5’ LTR;

(c) a partial env sequence comprising a Rev responsive element (RRE);

8. A transfer plasmid comprising a nucleic acid sequence comprising in order:

(a) a lentivirus 5’ Long Terminal Repeat (LTR) or a modified 5’ LTR;

(b) a \|/ packaging signal;

(c) a Rev response element (RRE);

(d) a nucleotide sequence encoding a gp41 peptide sequence;

(e) a central polypurine tract/central termination sequence (cPPT/CTS); and (f) a 3' long terminal repeat (LTR) that is an inactivated 3' LTR or a self-inactivating

3' LTR; wherein no more than 300 nucleotides separate the nucleotide sequence encoding the gp41 peptide sequence and the cPPT/CTS and wherein the transfer plasmid is devoid of a polynucleotide encoding a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

9. The transfer plasmid of claim 8, wherein the transfer plasmid is devoid of a portion of the pol integrase polynucleotide comprising the core domain.

10. The transfer plasmid of any of claims 7 to 9, wherein the transfer plasmid is devoid of a nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 8.

11. The transfer plasmid of any of claims 7 to 10, wherein the transfer plasmid is devoid of the nucleotide sequence set forth in SEQ ID NO: 8.

12. The transfer plasmid of any of claims 1 to 11, wherein the transfer plasmid is devoid of a WPRE nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 7.

13. The transfer plasmid of any of claims 1 to 12, wherein the transfer plasmid is devoid of a WPRE nucleotide sequence set forth in SEQ ID NO: 7.

14. A transfer plasmid comprising a nucleic acid sequence comprising:

(a) a lentivirus 5’ Long Terminal Repeat (LTR) or a modified 5’ LTR;

(b) a \|/ packaging signal;

(c) a Rev response element (RRE);

(d) a central polypurine tract/central termination sequence (cPPT/CTS); and

15. The transfer plasmid of claim 14, wherein the transfer plasmid is devoid of a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 7.

16. The transfer plasmid of claim 14 or claim 15, wherein the transfer plasmid is devoid of a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 8.

17. The transfer plasmid of claim 15 or claim 16, wherein the transfer plasmid comprises a nucleotide sequence encoding a gp41 peptide sequence.

18. The transfer plasmid of any of claims 1 to 17, wherein the 5’ LTR or modified 5’ LTR comprises a U5 and R domain.

19. The transfer plasmid of any of claims 1 to 18, wherein the 5’ LTR is a modified 5’ LTR that is truncated to lack a part or all of the U3 region.

20. The transfer plasmid of any of claims 1 to 19, wherein the 5’ LTR is a modified 5’ LTR that comprises the sequence set forth in SEQ ID NO: 20.

21. The transfer plasmid of claim 19 or claim 20, wherein the modified 5’ LTR comprises a heterologous regulatory element that is not endogenous to a lentivirus, wherein the heterologous regulatory element is immediately upstream of the modified 5’ LTR.

22. The transfer plasmid of claim 21, wherein the heterologous regulatory element is a promoter, enhancer or a promoter/enhancer.

23. The transfer plasmid of claim 21 or claim 22, wherein the heterologous regulatory element is a cytomegalovirus (CMV) enhancer, promoter or enhancer/promoter.

24. The transfer plasmid of any of claims 21 to 23, wherein the heterologous regulatory element comprises a nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 36.

25. The transfer plasmid of any of claims 21 to 24, wherein the heterologous regulatory element comprises a nucleotide sequence set forth in SEQ ID NO: 36.

26. The transfer plasmid of any of claims 1 to 25, wherein the 3’ LTR comprises a U5 and R domain.

27. The transfer plasmid of any of claims 1 to 26, wherein the 3’ LTR is a truncated 3’ LTR comprising a deleted U3 region in which one or more nucleotide bases of the U3 region of the 3’ LTR are deleted.

28. The transfer plasmid of claim 27, wherein the deleted U3 region retains the att sequence and comprises deletions of the enhancer and/or core promoter U3.

29. The transfer plasmid of claim 27 or claim 28, wherein the deleted U3 region lacks at least one of an enhancer sequence, a TATA box, an Spl site, an NK-kappa B site, or a polypurine tract (PPT).

30. The transfer plasmid of any of claims 1 to 29, wherein the 3’ LTR comprises the sequence set forth in SEQ ID NO: 29.

31. The transfer plasmid of any of claims 1 to 30, wherein the transfer plasmid comprises a poly adenylation signal within the R region or downstream of the 3’ LTR.

32. The transfer plasmid of claim 31, wherein the polyadenylation signal is an SV40 polyadenylation signal.

33. The transfer plasmid of any of claims 1 to 32, wherein the y packaging signal comprises the nucleotide sequence set forth in SEQ ID NO: 21.

34. The transfer plasmid of any of claim 1 to 33, wherein the Rev response element (RRE) comprises the nucleotide sequence set forth in SEQ ID NO: 22.

35. The transfer plasmid of any of claims 1 to 34, wherein the central polypurine tract/central termination sequence (cPPT/CTS) comprises the nucleotide sequence set forth in SEQ ID NO: 10.

36. The transfer plasmid of any of claims 1 to 35, wherein the nucleotide sequence encoding the gp41 peptide sequence comprises the nucleotide sequence set forth in SEQ ID NO: 23.

37. The transfer plasmid of any of claims 1 to 36, wherein the transfer plasmid comprises an origin of replication site.

38. The transfer plasmid of claim 37, wherein the origin of replication site comprises a pUC origin of replication, a SV40 origin of replication and/or an fl bacteriophage origin of replication.

39. The transfer plasmid of any of claims 1 to 38, wherein the transfer plasmid comprises a Kozak sequence.

40. The transfer plasmid of any of claims 1 to 39, wherein the transfer plasmid comprises a multiple cloning site that allows for insertion of a nucleotide sequence of interest (NOI).

41. The transfer plasmid of any of claims 1 to 40, wherein the transfer plasmid comprises a nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 39.

42. A transfer plasmid comprising a nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 39.

43. The transfer plasmid of claim 42, wherein the transfer plasmid comprises a multiple cloning site that allows for insertion of a nucleotide sequence of interest (NOI).

44. The transfer plasmid of any of claims 1 to 43, wherein the transfer plasmid comprises the nucleotide sequence set forth in SEQ ID NO: 39.

45. The transfer plasmid of any of claims 40 to 44, wherein the transfer plasmid further comprises a nucleotide sequence of interest (NOI) inserted within the multiple cloning site.

46. The transfer plasmid of any of claim 40 to 45, wherein the nucleotide sequence of interest encodes a protein, an RNA molecule, an enzyme or an antibody or any combination thereof.

47. The transfer plasmid of any of claims 40 to 46, wherein the nucleotide sequence of interest (NOI) encodes a chimeric antigen receptor (CAR), a cytokine receptor, an RNA molecule, a synthetic antigen or any combination thereof.

48. The transfer plasmid of any of claims 40 to 47, wherein the nucleotide sequence of interest is a multicistronic sequence.

49. The transfer plasmid of any of claims 40 to 48, wherein the nucleotide sequence of interest is up to 4000 base pairs in length.

50. The transfer plasmid of any of claims 40 to 49, wherein the nucleotide sequence of interest is 2000 to 3600 base pairs in length.

51. The transfer plasmid of any of claims 40 to 49, wherein the nucleotide sequence of interest is 2800 to 3400 base pairs in length.

52. The transfer plasmid of any of claims 1 to 51, further comprising a non- viral promoter, wherein the non- viral promoter is operably linked to control expression of the nucleotide sequence of interest.

53. The transfer plasmid of claim 52, wherein the non-viral promoter comprises an EF- la promoter.

54. A composition comprising the transfer plasmid of any of claims 1 to 53, an envelope plasmid and one or more packaging plasmids.

55. The composition of claim 54, wherein the one or more packaging plasmid comprises a first packaging plasmid encoding Gag and Pol and a second packaging plasmid encoding Rev.

56. The composition of claim 54 and claim 55, wherein the one or more envelope plasmid encodes a VSV-G glycoprotein.

57. A method of producing a lentivirus comprising:

(a) contacting a host cell with the composition of any one of claims 54 to 56;

(b) culturing the host cell under conditions that produce the lentivirus; and

(c) isolating the lentivirus.

58. A method of producing a lentivirus comprising:

(a) contacting a host cell with the transfer plasmid of any of claims 1 to 53 and one or more packaging plasmids;

(b) culturing the host cell under conditions that produce the lentivirus; and

(c) isolating the lentivirus.

59. The method of claim 57 and claim 58, wherein the one or more packaging plasmid comprises a first packaging plasmid encoding Gag and Pol and a second packaging plasmid encoding Rev.

60. The method of any of claims 57 to 59, wherein the one or more envelope plasmid encodes a VSV-G glycoprotein.

61. The method of any of claims 57 to 60, wherein the host cell is an adherent cell.

62. The method of any of claims 57 to 60, wherein the host cell is a suspension cell.

63. The method of any of claims 57 to 62, wherein the host cell is an HEK293 cell, a COS cell, a recombinant Chinese hamster ovary (CHO), a HeLa cell, a NIH3T3 cell, a PC12 cell, a U20S cell, an A549 cell, an HT1080 cell, a CAD cell, a P19 cell, an L929 cell, an N2a cell, an MCF-7, Y79 cell, an SO-Rb50 cell, a Hep G2 cell, a DUKX-X11 cell, a J558L cell, a baby hamster kidney (BHK) cell, or a derivative thereof.

64. The method of any of claims 5 to 63, wherein the host cell is an HEK293S cell, an HEK293Ts cell, an HEK293F cell, an HEK293FT cell, an HEK293FTM cell, an HEK293E cell, an LV293 cell, or an HEK293T/17 cell.

65. A host cell comprising the transfer plasmid of any of claims 1 to 53, an envelope plasmid, and one or more packaging plasmids.

66. The host cell of claim 65, wherein the one or more packaging plasmid comprises a first packaging plasmid encoding Gag and Pol and a second packaging plasmid encoding Rev.

67. The host cell of claim 65 or claim 66, wherein the one or more envelope plasmid encodes a VSV-G glycoprotein.

68. The host cell of claim 66 or claim 67, wherein the host cell is an adherent cell.

69. The host cell of claim 66 or claim 67, wherein the host cell is a suspension cell.

70. The host cell of any of claims 67 to 69, wherein the host cell is an HEK293 cell, a COS cell, a recombinant Chinese hamster ovary (CHO), a HeLa cell, a NIH3T3 cell, a PC 12 cell, a U20S cell, an A549 cell, an HT1080 cell, a CAD cell, a P19 cell, an L929 cell, an N2a cell, an MCF-7, Y79 cell, an SO-Rb50 cell, a Hep G2 cell, a DUKX-X11 cell, a J558L cell, a baby hamster kidney (BHK) cell, or a derivative thereof.

71. The host cell of any of claims 67 to 70, wherein the host cell is an HEK293S cell, an HEK293Ts cell, an HEK293F cell, an HEK293FT cell, an HEK293FTM cell, an HEK293E cell, an LV293 cell, or an HEK293T/17 cell.

72. A method of producing a lentivirus comprising:

(a) culturing the host cell of any of claims 65 to 71 under conditions that produce the lentivirus; and

(b) isolating the lentivirus.

73. A lentivirus produced by the method according to any of claims 57 to 64 and 72.

74. A lentivirus comprising a heterologous nucleic acid sequence, comprising

(b) a central polypurine tract/central termination sequence (cPPT/CTS); and

(c) a 3' long terminal repeat (LTR) that is an inactivated 3' LTR or a self-inactivating 3' LTR; wherein the lentivirus is devoid of: a polynucleotide encoding a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE); and a polynucleotide sequence region between the polynucleotide sequence encoding the partial gag of (a) and the cPPT/CTS sequence of (b) that comprises at least a portion of the pol integrase polynucleotide comprising the core domain.

75. A lentivirus comprising a nucleic acid sequence comprising in order:

(a) a lentivirus 5’ Long Terminal Repeat (LTR) or a modified 5’ LTR;

(b) a \|/ packaging signal; (c) a Rev response element (RRE);

(d) a nucleotide sequence encoding a gp41 peptide sequence;

(e) a central polypurine tract/central termination sequence (cPPT/CTS); and

76. The lentivirus of claim 74 or claim 75, wherein the polynucleotide sequence region is 350-370 nucleotides in length.

77. The lentivirus of any one of claims 74 to 76, wherein the polynucleotide sequence region is about 367 nucleotides in length.

78. The lentivirus of any one of claims 74 to 77, wherein the polynucleotide sequence region comprises a nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 8.

79. The lentivirus of any of claims 74 to 78, wherein the polynucleotide sequence region comprises the nucleotide sequence set forth in SEQ ID NO: 8.

80. A lentivirus comprising a nucleic acid sequence comprising in order:

(a) a lentivirus 5’ Long Terminal Repeat (LTR) or a modified 5’ LTR;

(c) a partial env sequence comprising a Rev responsive element (RRE);

(d) a partial pol sequence comprising a central polypurine tract/central termination sequence (cPPT/CTS) but that is devoid of a polynucleotide sequence encoding a portion of the integrase comprising the core domain; and (e) a 3' long terminal repeat (LTR) that is an inactivated 3' LTR or a self-inactivating

3' LTR; wherein the lentivirus is devoid of a polynucleotide encoding a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

81. A lentivirus comprising a nucleic acid sequence comprising in order:

(a) a lentivirus 5’ Long Terminal Repeat (LTR) or a modified 5’ LTR;

(b) a \|/ packaging signal;

(c) a Rev response element (RRE);

(d) a nucleotide sequence encoding a gp41 peptide sequence;

(e) a central polypurine tract/central termination sequence (cPPT/CTS); and

82. The lentivirus of claim 80 or claim 81, wherein the lentivirus is devoid of a portion of the pol integrase polynucleotide comprising the core domain.

83. The lentivirus of any of claims 80 to 82, wherein the lentivirus is devoid of a nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 8.

84. The lentivirus of any of claims 80 to 83, wherein the lentivirus is devoid of the nucleotide sequence set forth in SEQ ID NO: 8.

85. The lentivirus of any of claims 74 to 84, wherein the lentivirus is devoid of a WPRE nucleotide sequence that is at least about 85% identical to the sequence set forth in SEQ ID NO: 7.

86. The lentivirus of any of claims 74 to 85, wherein the lentivirus is devoid of a

WPRE nucleotide sequence set forth in SEQ ID NO: 7.

87. The lentivirus of any of claims 74 to 86, further comprising a nucleotide sequence of interest (NOI).

88. The lentivirus of claim 87, wherein the NOI encodes a chimeric antigen receptor (CAR), a cytokine receptor, an RNA molecule, a synthetic antigen or any combination thereof.

89. The lentivirus of any of claims 73 to 88, wherein the lentivirus results in increased transduction efficiency of an immune cell transduced with the lentivirus compared to an immune cell transduced with a reference lentivirus.

90. The lentivirus of any of claims 73 to 89, wherein the lentivirus results in increased cytotoxicity of an immune cell transduced with the lentivirus compared to an immune cell transduced with a reference lentivirus.

91. The lentivirus of any of claims 73 to 90, wherein the lentivirus results in increased expression of naive phenotype markers of an immune cell transduced with the lentivirus compared to an immune cell transduced with a reference lentivirus.

92. The lentivirus of any of claims 73 to 91, wherein the lentivirus results in reduced expression of exhaustion markers in an immune cell transduced with the lentivirus compared to an immune cell transduced with a reference lentivirus.

93. The lentivirus of any of claims 73 to 92, wherein the reference lentivirus is identical to the lentivirus but further comprises a polynucleotide encoding a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and a polynucleotide encoding a region between a partial Gag sequence and a central polypurine tract/central termination sequence (cPPT/CTS).

94. A method of transducing a cell comprising contacting a cell with the lentivirus of any of claims 73 to 93.

95. The method of claim 94, wherein the method comprises incubating the cell contacted with the lentivirus under conditions that promote expression of the NOI.

96. The method of claim 94 or claim 95, wherein the cell is an immune cell.

97. The method of any of claims 94 to 96, wherein the cell is an effector cell.

98. The method of any of claims 94 to 97, wherein the cell is a T cell or an NK cell.

99. A transduced cell produced according to the method of any of claims 94 to 98.

100. A composition comprising a population of cells and the lentivirus of any of claims 73 to 93.

101. The composition of claim 100, wherein the population of cells comprises a population of immune cells.

102. The composition of claim 100 or claim 101, wherein the population of cells comprises a population of effector cells.

103. The composition of any of claims 100 to 102, wherein the population of cells comprises a population of T cells or NK cells.