WO2024130070A2

WO2024130070A2 - Recombinant aav capsids with cardiac- and skeletal muscle- specific targeting motifs and uses thereof

Info

Publication number: WO2024130070A2
Application number: PCT/US2023/084198
Authority: WO
Inventors: Joshua Joyner SIMS; Sharon LIAN; Rosemary MEGGERSEE; Jenny G. SIDRANE
Original assignee: University of Pennsylvania Penn
Current assignee: University of Pennsylvania Penn
Priority date: 2022-12-17
Filing date: 2023-12-15
Publication date: 2024-06-20
Anticipated expiration: 2025-06-17
Also published as: WO2024130070A3

Abstract

Provided herein are compositions including muscle (cardiac and skeletal) cell-targeting peptides linked thereto or inserted in a targeting protein of a recombinant vector having at least one exogenous peptide comprising Xn – non-RGD-n-mer – Xm. Compositions providing such conjugates, targeting peptides, or recombinant vectors having an engineered capsid or envelope protein are provided as are uses thereof.

Description

RECOMBINANT AAV CAPSIDS WITH CARDIAC- AND SKELETAL

MUSCLE- SPECIFIC TARGETING MOTIFS AND USES THEREOF

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (“UPN-22-10106PCT.xnil”; Size: 78,768 bytes; and Date of Creation: December 5, 2023) are herein incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

The adeno-associated virus (AAV) is currently the gene therapy vector of choice. AAVs can deliver a transgene that is stably expressed long-term from a non-integrating genome and are not associated with any human diseases. However, AAV gene therapy is currently limited to a small number of diseases due to challenges in delivery and tropism.

AAV vectors have been approved by the US Food and Drug Administration and other worldwide regulatory authorities for tire treatment of Leber congenital amaurosis, lipoprotein lipase deficiency, and spinal muscular atrophy. A central challenge for gene therapy is the difficulty of modulating and targeting expression of a therapeutic transgene in vivo, more specifically targeting a particular tissue, e.g., heart and skeletal muscle.

There is a high and unmet need in adult- and early- onset forms of muscle cell- related pathologies (cardiac and skeletal). In such instance, lack of effective treatment, results in high rates of organ transplant and/or death. Various cardio- and skeletal -muscle cell-based diseases are amenable to AAV gene replacement therapy, but there is a need for specific and effective targeting to muscles tissue.

There remains a need for vectors that can specifically target selected tissue and cell types.

SUMMARY OF THE INVENTION

In one aspect provided herein is a recombinant adeno-associated particle (rAAV) comprising: (a) an adeno-associated virus (AAV) capsid comprising VP1 proteins, VP2 proteins and VP3 proteins, wherein the capsid proteins have amino acid sequences comprising a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises “Xn - n-mer - Xm”, wherein: (i) optional Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is GQVRVGV (SEQ ID NO: 3), MDAHYVR (SEQ ID NO: 4), TQAVPLK (SEQ ID NO: 5), VVHQTGL (SEQ ID NO: 6), or MAISRER (SEQ ID NO: 7), or an n-mer sequence of at least 6 consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid; and (b) a vector genome packaged in the AAV capsid, wherein the vector genome comprises a nucleic acid sequence encoding a gene product under control of sequences which direct expression thereof. In certain embodiments, tire rAAV comprises an exogenous targeting peptide comprising an n-mer that is GQVRVGV (SEQ ID NO: 3). In certain embodiments, the exogenous targeting peptide is inserted between any two contiguous amino acids in the hypervariable region VIII (HVRVIII) or IV (HVRIV) at a suitable location of a parental AAV capsid. In certain embodiments, the parental AAV capsid is AAV9, AAV8, AAV7, AAV6, AAV5, AAV4, AAV3, AAV1, AAVhu68, AAVhu95, AAVhu96, and AAVrh91. In certain embodiments, the exogenous targeting peptide is inserted in the hypervariable region between amino acids 588 and 589 in an AAV9 parental capsid as determined based on the numbering of VP1 amino acid sequence of SEQ ID NO: 25, or an analogous position in an AAV8. AAV7. AAV6, AAV5, AAV4, AAV3, AAV1, AAVhu68, AAVhu95, AAVhu96, or AAVrh91 capsid. In certain embodiments, the exogenous targeting peptide is immediately preceded by the native AAV residues, e.g., “AQ” In certain embodiments, the rAAV capsid comprises VP1, AAV VP2 and AAV VP3 proteins having a mutant VP3 region of: amino acids 204 to 743 of SEQ ID NO: 80, and wherein each of the VP 1 proteins, VP2 proteins and VP3 proteins have heterogenous populations which further comprise highly deamidated residues in positions N57, N329, N452 and/or N512 (e.g., independently about 50% to about 100% deamidated), wherein the deamidated position numbers are based on the residue positions of SEQ ID NO: 25 or 26. In certain embodiments, the rAAV capsid comprises VP1 proteins, VP2 proteins and VP3 proteins, wherein the VP1 has a mutant sequence of: amino acids 1 to 743 of SEQ ID NO: 80, said VP proteins further comprising highly deamidated residues in positions N57, N329, N452 and/or N512, wherein the deamidated position numbers arc based on the residue positions of SEQ ID NO: 25 or 26 (e.g., independently about 50% to about 100% deamidated). In certain embodiments, the n-mer is encoded by the nucleic acid sequence of SEQ ID NO: 79. or a sequence at least 95% identical thereto.

In another aspect, provided herein is a recombinant muscle cell-targeting peptide, comprising “Xn - n-mer - Xm”, wherein: (a) Xn is 0, 1 , 2, or 3 amino acid residues independently selected from any amino acid; (b) the n-mer is GQVRVGV (SEQ ID NO: 3), MDAHYVR (SEQ ID NO: 4), TQAVPLK (SEQ ID NO: 5), VVHQTGL (SEQ ID NO: 6), or MAISRER (SEQ ID NO: 7), or an n-mer sequence of at least 6 consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid/s independently selected from any amino acid; and optionally wherein the muscle cell-targeting peptide is conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In certain embodiments, the recombinant muscle cell-targeting peptide targets a cardiac muscle cell and/or a skeletal muscle cell, optionally a gastrocnemius muscle cell.

In another aspect provided herein is a nucleic acid molecule comprising a nucleic acid sequence encoding a mutant AAV capsid VP1 comprising n-mer of GQVRVGV (SEQ ID NO: 3). In certain embodiments, the nucleic acid sequence encoding the n-mer comprises SEQ ID NO 78 or a sequence at least 99% identical thereto (encoding GQVRVGV). In certain embodiments, the nucleic acid molecule encoding the AAV capsid comprises SEQ ID NO: 79.

In certain embodiments, a fusion polypeptide or protein comprising a muscle celltargeting peptide and a fusion partner which comprises at least one polypeptide or protein is provided. In certain embodiments, a composition comprising a fusion polypeptide or protein and one or more of a physiologically compatible carrier, excipient, and/or aqueous suspension base is provided.

In another aspect, provided herein are compositions and methods for using an rAAV, a muscle cell targeting peptide, a fusion polypeptide or protein, and/or a composition as described herein of for delivering a therapeutic to a patient in need thereof. In certain embodiments, the therapeutic is targeted to muscle cell, optionally wherein the muscle cell is a cardiac muscle cell or a skeletal muscle cell, optionally a gastrocnemius muscle cell, deltoid muscle cell, a soleus muscle cell, a biceps brachii muscle cell or a diaphragm muscle cell.

In another embodiment, a method is provided for targeting therapy to muscle cell in a patient in need thereof, the method comprising administering to the patient an rAAV as described herein. In certain embodiments, a method is provided for treating a cardiac muscle and/or skeletal muscle disorder and/or disease in a subject in need thereof, the method comprising delivering to the subject a stock of a rAAV as described herein. In certain embodiments, a method is provided for treating a cardiac and/or skeletal (e.g., gastrocnemius, deltoid muscle cell, a soleus muscle cell, a biceps brachii muscle cell or a diaphragm muscle cell) muscle-based disorder and/or a disease in the a subject in need thereof, the method comprising delivering to the subject a stock of an rAAV, wherein the rAAV comprises and sequence that encodes a gene product that is a protein, optionally an antibody.

These and other embodiments and advantages of the invention will be apparent from the specification, including, without limitation, the detailed description of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows plotted heart enrichment score from non-RGD screen round 2 for top performing heart candidates in comparison to top literature heart cap. FIG. IB shows muscle enrichment score from non-RGD screen round 2 for top performing heart and muscle candidates in comparison to the top literature heart candidates.

FIGs. 2A to 2C show RNA, DNA, and protein expression levels in tire collected tissue samples of heart left ventricle following administration of AAV9 and AAV9- GQVRVGV vectors. FIG. 2A shows DNA levels, plotted as Genome Copies (GC)/diploid cell. FIG. 2B shows RNA levels, plotted as copy number (#)/100 nanograms (ng) RNA. FIG. 2C shows protein expression levels, plotted as picograms (pg) GFP/ microgram (pg) protein.

FIGs. 3A to 3C show RNA, DNA, and protein expression levels in the collected tissue samples of heart right ventricle follow ing administration of AAV9 and AAV9- GQVRVGV vectors. FIG. 3 A shows DNA levels, plotted as GC/diploid cell. FIG. 3B shows RNA levels, plotted as copy number (#)/100ng RNA. FIG. 3C shows protein expression levels, plotted as pg GFP/pg protein.

FIGs. 4A to 4C show RNA, DNA, and protein expression levels in the collected tissue samples of gastrocnemius following administration of AAV9 and AAV9- GQVRVGV vectors. FIG. 4A shows DNA levels, plotted as GC/diploid cell. FIG. 4B shows RNA levels, plotted as copy number (#)/100ng RNA. FIG. 4C shows protein expression levels, plotted as pg GFP/pg protein.

FIGs. 5A to 5C show RNA. DNA, and protein expression levels in tire collected tissue samples of liver following administration of AAV9 and AAV9- GQVRVGV vectors. FIG. 5 A shows DNA levels, plotted as GC/diploid cell. FIG. 5B shows RNA levels, plotted as copy number (#)/100ng RNA. FIG. 5C shows protein expression levels, plotted as pg GFP/pg protein.

FIG. 6 shows RNA/DNA ratio (normalized to AAV9) for AAV9 and AAV9- GQVRVGV vector biodistribution (See Table 7, below, for a summary of quantified values).

FIG. 7 shows an alignment of the amino acid sequences of various AAV capsid proteins: AAV9 (amino acids 566 to 615 of AAV9 capsid; SEQ ID NO: 27), AAV8 (amino acids 565 to 614 of AAV8 capsid; SEQ ID NO: 28), AAV7 (amino acids 567 to 616 of AAV7; SEQ ID NO: 29). AAV6 (ammo acids 550 to 599 of AAV6 capsid; SEQ ID NO: 30), AAV5 (ammo acids 556 to 605 of AAV5; SEQ ID NO: 31), AAV4 (amino acids 558 to 607 of AAV4 capsid; SEQ ID NO: 32), AAV3B (ammo acids 564 to 613 of AAV3B capsid; SEQ ID NO: 33), AAV2 (amino acids 566 to 615 of AAV2 capsid; SEQ ID NO: 34), and AAV 1 (amino acids 566 to 615 of AAV 1 capsid; SEQ ID NO: 35). The region shown is the HVRVIII region in which a targeting peptide is inserted (based on structural analysis).

FIG. 8 shows results of an enrichment study in NHPs. plotted as normalized fold change.

FIG. 9A shows RNA/DNA ratio (normalized to AAV9) for AAV9, and various AAV9-X vectors as analyzed in heart tissue. FIG. 9B shows RNA/DNA ratio (normalized to AAV 9) for AAV9, and various AAV9-X vectors as analyzed in liver tissues. FIG. 9C shows RNA levels in heart tissue post-rAAV transduction, plotted as RNA transcript /100ng. FIG. 9D shows DNA levels in heart tissue post-rAAV transduction, plotted as GC per diploid genome. FIG. 9E shows RNA levels in liver tissue post-rAAV transduction, plotted as R A transcript /100ng. FIG. 9F shows DNA levels in liver tissue post-rAAV transduction, plotted as GC per diploid genome.

FIG. 10A shows results of in situ hybridization (ISH) analysis of gastrocnemius tissue, plotted as percent GFP positive as normalized to AAV9 control. FIG. 10B shows results of an ISH analysis of biceps femoris tissue, plotted as percent GFP positive as normalized to AAV9 control.

DETAILED DECR1PT1ON OF THE INVENTION

In certain embodiments, a recombinant muscle cell-targeting peptide and nucleic acid sequences encoding the same are provided herein. Also provided herein are fusion proteins, modified proteins, engineered viral capsids (e.g.. recombinant adeno-associated virus (rAAV) capsids), and other moieties operably linked to an exogenous targeting peptide, wherein the exogenous targeting peptide is “Xn - n-mer - Xm”, wherein the Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the n-mer is GQVRVGV (SEQ ID NO: 3), MDAHYVR (SEQ ID NO: 4), TQAVPLK (SEQ ID NO: 5), VVHQTGL (SEQ ID NO: 6), or MAISRER (SEQ ID NO: 7), or an n-mer sequence of at least 6, at least 7, or the full-length consecutive amino acids of any one of the n-mers, and wherein Xm is 0, 1. 2, or 3 amino acid residues independently selected from any amino acid. Also provided herein are nucleic acid sequences encoding the same. In certain embodiments, the exogenous motif modifies the native tissue specificity of the source (parental) protein, viral vector, viral capsid, or other moiety. In certain embodiments, compositions having one or more of the exogenous targeting peptides have enhanced or altered muscle cell-targeting. In certain embodiments, compositions having one or more of the targeting peptides have enhanced or altered cardiac and/or skeletal muscle cell-targeting, optionally a gastrocnemius muscle cell. In certain embodiments, viral vectors having modified capsids containing the motif exhibit increased transduction of AAV production cells in vitro.

Advantageously, in certain embodiments, provided herein are recombinant muscle cell-targeting peptides (also referred to as “targeting peptides”, “exogenous targeting peptidse”), wherein tire recombinant muscle cell-targeting peptides comprise “Xn - n-mer - Xm”, wherein the Xn is 0, 1, 2, or 3 amino acid residues independently selected from any ammo acid, wherein tire n-mer is GQVRVGV (SEQ ID NO: 3), MDAHYVR (SEQ ID NO: 4), TQAVPLK (SEQ ID NO: 5), VVHQTGL (SEQ ID NO: 6), or MAISRER (SEQ ID NO: 7). or an n-mer sequence of at least 6, at least 7, or the full-length consecutive amino acids of any one of the n-mers, and wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid.

Advantageously, in certain embodiments, provided herein are engineered rAAV capsids comprising an exogenous targeting peptide, wherein the exogenous targeting peptide is “Xn - n-mer - Xm”, wherein Xn is 0. 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the n-mer is: GQVRVGV (SEQ ID NO: 3), MDAHYVR (SEQ ID NO: 4), TQAVPLK (SEQ ID NO: 5), VVHQTGL (SEQ ID NO: 6), or MAISRER (SEQ ID NO: 7), or an n-mer sequence of at least 6, at least 7, or the full-length consecutive amino acids of any one of the n-mers, and wherein Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the exogenous targeting peptides provided herein provide significant transduction advantages in a muscle cell, including a cardiac muscle cell and/or a skeletal muscle cell, optionally a gastrocnemius muscle cell, a deltoid muscle cell, a soleus muscle cell, a biceps brachii muscle cell, or a diaphragm muscle cell, as compared to a parental capsid (e.g., AAV9, or another clade F capsid, or another clade capsid).

In certain embodiments, provided herein are engineered rAAV capsids comprising an exogenous targeting peptide which comprises “Xn - GQVRVGV (SEQ ID NO: 3) - Xm”, wherein Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptides provide significant transduction advantages in a muscle cell, including a cardiac muscle cell and/or a skeletal muscle cell, optionally a gastrocnemius muscle cell, as compared to a parental capsid. In certain embodiments, provided herein are engineered rAAV capsids comprising an exogenous targeting peptide which comprises ‘‘Xn - MDAHYVR (SEQ ID NO: 4) - Xm”, wherein Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein Xm is 0, 1 , 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptides provide significant transduction advantages in a muscle cell, including a cardiac muscle cell and/or a skeletal muscle cell, optionally a gastrocnemius muscle cell, as compared to a parental capsid. In certain embodiments, provided herein are engineered rAAV capsids comprising an exogenous targeting peptide which comprise ‘‘Xn - TQAVPLK (SEQ ID NO: 5) - Xm”. wherein Xn is 0, 1, 2. or 3 amino acid residues independently selected from any amino acid, wherein Xm is 0, 1, 2. or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provides significant transduction advantages in a muscle cell, including a cardiac muscle cell and/or a skeletal muscle cell, optionally a gastrocnemius muscle cell, as compared to a parental capsid. In certain embodiments, provided herein arc engineered rAAV capsids comprising an exogenous targeting peptide which comprises “Xn - VVHQTGL (SEQ ID NO: 6) - Xm”, wherein Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein the optional Xm is 0, 1, 2. or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provides significant transduction advantages in a muscle cell, including a cardiac muscle cell and/or a skeletal muscle cell, optionally a gastrocnemius muscle cell, as compared to a parental capsid. In certain embodiments, provided herein are engineered rAAV capsids comprising an exogenous targeting peptide which comprises "Xn - MAISRER (SEQ ID NO: 7) - Xm”, wherein Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the exogenous targeting peptide provides significant transduction advantages in a muscle cell, including a cardiac muscle cell and/or a skeletal muscle cell, optionally a gastrocnemius muscle cell, as compared to a parental capsid.

In certain embodiments, tire rAAV comprises a mutant AAV capsid having an exogenous targeting peptide as identified herein. In certain embodiments, the mutant AAV capsid comprises an exogenous targeting peptide which is immediately preceded by flanking amino acids that are mutated, as compared to parental AAV capsid. In certain embodiments, the mutated flanking amino acids, together with 1, 2, 3, 4, 5, or 6 inserted amino acids comprise the exogenous targeting peptide. In certain embodiments, the entirety of the exogenous targeting peptide is inserted into the parental AAV capsid. In still other embodiments, the sequence inserted into a capsid may comprise all or a fragment of the exogenous targeting peptide at the carboxy (COO-) or amino terminus (N-) (i.e., via insertion of the 5' or 3' coding sequences therefor) and further comprises 0, 1, 2, or 3 flanking amino acid residues as provided in the above formulae. In certain embodiments, engineered rAAV capsids comprising targeting peptides, as provided herein, demonstrate reduced transduction (i.e., de-targeted/ing) to liver as compared to their parental capsids (e.g., AAV9 or another clade F capsid (e.g., AAVhu68, AAVhu31, AAVhu32, AAVhu95. AAVhu96) or other modification thereof). In certain embodiments, engineered rAAV capsids comprising targeting peptides, as provided herein, demonstrate reduced transduction (i.e., de-targeted/ing) to spleen as compared to their parental capsids (e g., AAV9 or another clade F capsid or other modification thereof.

The targeting peptide may be linked to a recombinant protein (e.g., for enzyme replacement therapy) or a polypeptide (e.g., an immunoglobulin) to form a fusion protein or a conjugate to target a desired tissue (e g., muscle cell, cardiac muscle cell, skeletal muscle cell, or gastrocnemius muscle cell). Additionally, the targeting peptide may be linked to a liposome and/or a nanoparticle (e.g.. a lipid nanoparticle. LNP) forming a peptide-coated liposome and/or LNP to target a desired tissue. Sequences encoding at least one copy of a targeting peptide and optional linking sequences may be fused in frame with the coding sequence for the recombinant protein and co-expressed with the protein or polypeptide to provide fusion proteins or conjugates. Alternatively, other synthetic methods may be used to form a conjugate with a protein, polypeptide or another moiety (e.g., DNA, RNA, or a small molecule). In certain embodiments, multiple copies of a targeting peptide are in the fusion protein/conjugate. Suitable methods for conjugating a targeting peptide to a recombinant protein include modifying the amino (N)-tenninus and one or more residues on a recombinant human protein (e.g., an enzyme) using a first crosslinking agent to give rise to a first crosslinking agent modified recombinant human protein, modifying the N -terminus of a short extension linker region preceding a targeting peptide using a second crosslinking agent to give rise to a second crosslinking agent modified variant target peptide, and then conjugating the first crosslinking agent modified recombinant human protein to the second crosslinking agent modified variant targeting peptide containing a short extension linker. Other suitable methods for conjugating a targeting peptide to a recombinant protein include conjugating a first crosslinking agent modified recombinant human protein to one or more second crosslinking agent modified variant targeting peptides, wherein the first crosslinking agent modified recombinant protein comprises a recombinant protein characterized as having a chemically modified N- terminus and one or more modified lysine residues and the one or more second crosslinking agent modified variant targeting peptides comprise one or more variant targeting peptides comprising a modified N-terminal amino acid of a short extension linker preceding the targeting peptide. Still other suitable methods for conjugating a targeting peptide to a protein, polypeptide, nanoparticle, or another biologically useful chemical moiety may be selected. See. e.g., US Patent No. US 9.545,450 B2 (NHS-phosphine cross-linking agents: NHS-Azide cross-linking agents); US Published Patent Application No. US 2018/0185503 Al (aldehyde-hydrazide crosslinking).

In certain embodiments, the exogenous targeting peptide is engineered (i.e., inserted) at a suitable site within a protein or polypeptide (e.g., a viral capsid protein). In certain embodiments, a targeting peptide is flanked at its amino (N-) (e.g., optional Xn) and/or carboxy (COO-) (e.g., optional Xm) terminus by a short extension linker. Such a linker may be about 1 to about 20 amino acid residues in length, or about 2 to about 20 amino acids residues, or about 1 to about 15 amino acid residues, or about 2 to about 12 amino acid residues, or about 2 to about 7 amino acid residues in length. The short extension linker can also be about 10 amino acids in length. The presence and length of a linker at the N-terminus is independently selected from a linker at the carboxy terminus, and the presence and length of a linker at the carboxy terminus is independently selected from a linker at the N-terminus. Suitable short extension linkers can be provided using an about 5-amino acid flexible GS extension linker (glycine-glycine-glycine-glycine-serine), an about 10-amino acid extension linker comprising 2 flexible GS linkers, an about 15- amino acid extension linker comprising 3 flexible GS linkers, an about 20-amino acid extension linker comprising 4 flexible GS linkers, or any combination thereof.

In certain embodiments, a composition is provided which is useful for targeting a muscle cell. In certain embodiments, the composition comprises an engineered capsid (e.g., rAAV capsid), fusion protein or another conjugate comprising at least one exogenous targeting peptide comprising: a core amino acid sequence (e.g., “n-mer”) of GQVRVGV (SEQ ID NO: 3) flanked at its amino terminus and/or carboxy terminus of the core sequence by 0, 1, 2, or 3 amino acid residues (e.g., Xn and Xm, respectively), and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In certain embodiments, tire composition comprises an engineered capsid, fusion protein or another conjugate comprising at least one exogenous targeting peptide comprising: a core amino acid sequence (e.g., ‘'n-mer”) of MDAHYVR (SEQ ID NO: 4) flanked at its amino terminus and/or carboxy terminus of the core sequence by 0, 1 , 2, or 3 amino acid residues (e.g., Xn and Xm, respectively), and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In certain embodiments, the composition comprises an engineered capsid (e.g., rAAV capsid), fusion protein or another conjugate comprising at least one exogenous targeting peptide comprising: a core amino acid sequence (e.g., “n-mer”) of TQAVPLK (SEQ ID NO: 5) flanked at its amino terminus and/or carboxy terminus of the core sequence by 0, 1. 2. or 3 amino acid residues (e.g., Xn and Xm, respectively), and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In certain embodiments, the composition comprises an engineered capsid (e.g., rAAV capsid), fusion protein or another conjugate comprising at least one exogenous targeting peptide comprising: a core amino acid sequence (e.g., “n- mer”) of VVHQTGL (SEQ ID NO: 6) flanked at its amino terminus and/or carboxy terminus of the core sequence by 0, 1, 2, or 3 amino acid residues (e.g., Xn and Xm, respectively), and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In certain embodiments, the composition comprises an engineered capsid (e.g., rAAV capsid), fusion protein or another conjugate comprising at least one exogenous targeting peptide comprising: a core amino acid sequence (e.g., "n-mer") of MAISRER (SEQ ID NO: 7) flanked at its amino terminus and/or carboxy terminus of the core sequence by 0, 1, 2, or 3 amino acid residues (e.g., Xn and Xm, respectively), and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein.

In certain embodiments, the composition comprises an engineered capsid, fusion protein or another conjugate comprising at least one exogenous targeting peptide comprising: a core amino acid sequence is GQVRVGV (SEQ ID NO: 3), MDAHYVR (SEQ ID NO: 4), TQAVPLK (SEQ ID NO: 5), VVHQTGL (SEQ ID NO: 6), or MAISRER (SEQ ID NO: 7), or an n-mer sequence of at least 6. at least 7. or the full-length consecutive amino acids of any one of the n-mers, flanked at its amino terminus and/or carboxy terminus of the core sequence by 0, 1, 2, or 3 amino acid residues (e.g., Xn and Xm, respectively), and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein.

In certain embodiments, tire targeting peptide core amino acid sequence is encoded by a nucleic acid sequence.

In certain embodiments, tire targeting peptide core (e.g.. “n-mer’^?) is GQVRVGV (SEQ ID NO: 3). In certain embodiments, the targeting peptide core is MDAHYVR (SEQ ID NO: 4). In certain embodiments, the targeting peptide core is TQAVPLK (SEQ ID NO: 5). In certain embodiments, the targeting peptide core is VVHQTGL (SEQ ID NO: 6). In certain embodiments, tire targeting peptide core is MAISRER (SEQ ID NO: 7). In certain embodiments, more than one copy of a targeting peptide within this motif is provided in a conjugate or modified protein (e.g., a parvovirus capsid, rAAV capsid). In certain embodiments, two or more different targeting peptide cores are present.

In certain embodiments, a composition is provided which is useful for targeting a muscle cell. In certain embodiments, a composition is provided which is useful for targeting a cardiac muscle cell (i.e., heart tissue). In certain embodiments, a composition is provided which is useful for targeting skeletal muscle cell. In certain embodiments, a composition is provided which is useful for targeting gastrocnemius muscle cell. In certain embodiments, a composition is provided which is useful for targeting a muscle cell, while also de-targeting cells in liver. In certain embodiment, a composition is provided which is useful for targeting a muscle cell, while also de-targeting cells in spleen.

Provided herein are also compositions comprising an engineered rAAV capsid, fusion protein or another conjugate comprising at least one exogenous targeting peptide comprising: an amino acid core sequence of GQVRVGV (SEQ ID NO: 3), MDAHYVR (SEQ ID NO: 4), TQAVPLK (SEQ ID NO: 5), VVHQTGL (SEQ ID NO: 6), and/or MAISRER (SEQ ID NO: 7), wherein the inserted targeting peptide is flanked at the amino terminus and/or tire carboxy terminus of the motif by 0, 1, 2, 3 amino acids, and optionally further conjugated to a nanoparticle, a second molecule, or a viral capsid protein.

Examples of suitable proteins, including enzymes, immunoglobulins, therapeutic proteins, immunogenic polypeptides, nanoparticles, DNA, RNA, and other moieties (e.g., small molecules, etc.) for targeting are described in more detail below. These and other biologic and chemical moieties are suitable for use with the targeting peptide(s) provided herein.

In certain embodiments, provided herein is a composition comprising a nucleic acid molecule, wherein the nucleic acid molecule is a DNA molecule or RNA molecule, e.g., naked DNA, naked plasmid DNA, messenger RNA (mRNA), linked to a targeting peptide sequence motif described herein. In some embodiments, the nucleic acid molecule is further coupled with various compositions and nano particles, including, e.g., micelles, liposomes, cationic lipid - nucleic acid compositions, poly-glycan compositions and other polymers, lipid and/or cholesterol-based - nucleic acid conjugates, and other constructs such as are described herein. See, e.g., WQ2014/089486, US 2018/0353616A 1 , US2013/0037977A1, WQ2015/074085A1, US9670152B2, and US 8,853,377B2, X. Su, et al., Mol. Pharmaceutics, 2011, 8 (3), pp 774-787; web publication: March 21, 2011; WO2013/182683, WO 2010/053572 and WO 2012/170930, all of which are incorporated herein by reference. In certain embodiments, the targeting peptide motif is chemically linked to a nanoparticle surface, wherein the nanoparticle encapsulates a nucleic acid molecule. In some embodiments, the nanoparticle comprising the targeting peptide linked to the surface is designed for targeted tissue-specific delivery. In some embodiments, two or more different targeting peptides are linked to the surface of the nanoparticle. Suitable chemical linking or cross-linking include those known to one skilled in the art.

Capsids

In certain embodiments, a recombinant parvovirus is provided which has a modified parvovirus capsid, wherein the capsid includes a capsid protein having an amino acid sequence comprising a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1. 2, or 3 amino acid residues independently selected from any amino acid: (ii) the n-mer is GQVRVGV (SEQ ID NO: 3), MDAHYVR (SEQ ID NO: 4), TQAVPLK (SEQ ID NO: 5), VVHQTGL (SEQ ID NO: 6), or MAISRER (SEQ ID NO: 7), or an n- mer sequence of at least 6, at least 7, or the full-length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. Such a recombinant parvovirus may be a hybrid bocavirus/AAV or a recombinant AAV vector (rAAV). In other embodiments, other viral vectors may be generated having one or more exogenous targeting peptides in an exposed capsid protein to modulate and/or alter the targeting specificity of the viral vector as compared to its parental vector, wherein the one or more exogenous targeting peptide comprises '‘Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any ammo acid; (ii) the n-mer is GQVRVGV (SEQ ID NO: 3), MDAHYVR (SEQ ID NO: 4), TQAVPLK (SEQ ID NO: 5), VVHQTGL (SEQ ID NO: 6), or MAISRER (SEQ ID NO: 7), or an n-mer sequence of at least 6, at least 7, or the full-length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2. or 3 amino acid residues independently selected from any amino acid.

The exogenous targeting peptide may be inserted (and/or engineered via mutation of sequences encoding flanking amino acid residue(s)) into a hypervariable loop (HVR) VIII (also referenced as HVR8) at any suitable location. For example, based on the numbering of the AAV9 capsid, the peptide is inserted with linkers of various lengths between amino acids 588 and 589 (Q-A) of the AAV9 capsid protein, based on the numbering of the AAV9 VP1 (also referred to as Vpl or vpl) amino acid sequence (SEQ ID NO: 25). See, also, WO 2019/168961, published September 6, 2019. including Table G providing the deamidation pattern for AAV9 and WO 2020/160582. filed September 7, 2018. The amino acid residue locations (i.e., amino acid numbering reference) are identical in AAVhu68 (SEQ ID NO: 26). However, another site may be selected within HVRVIII. Alternatively, another exposed loop HVR (e.g., HVRIV) may be selected for the insertion. Comparable HVR regions may be selected in other capsids. In certain embodiments, tire location for the HVRVIII and HVRIV is determined using an algorithm and/or alignment technique as described in US Patent No. US 9,737,618 B2 (column 15, lines 3-23), and US Patent No. US 10,308,958 B2 (column 15, line 46 - column 16. line 6), which are incorporated herein by reference in their entireties. In certain embodiments, the targeting peptide may be inserted (and/or engineered) into a hypervariable loop HVRVIII as described in US Provisional Patent Application No. 63/119.863. filed December 1, 2020, and International Patent Application No. PCT/US2021/061312, filed December 1, 2022, which are incorporated herein by reference in their entireties. In certain embodiments, an AAV1 capsid protein is selected as a parental capsid, wherein the targeting peptide with linkers of various lengths is inserted in a suitable location of the HVRVIII region of amino acids 582 to 585, or HVRIV region of amino acids 456 to 459 based on vpl numbering (Gurda, BL., et al., Capsid Antibodies to Different Adeno-Associated Virus Serotypes Bind Common Regions, 2012, Journal of Virology. June 12, 2013, 87(16): 9111-91114). In certain embodiments. AAV 8 is selected as the parental capsid, wherein the targeting peptide with linkers of various lengths is inserted in a suitable location of the HVRVIII region of amino acids 586 to 591, or the HVRIV region of amino acids 456 to 460, based on VP1 numbering (Gurda, BL., et al., Mapping a Neutralizing epitope onto the Capsid of Adeno-Associated Virus Serotype 8, 2012, Journal of Virology , May 16, 2012, 86(15)7739-7751).

In certain embodiments, tire parental AAV capsid is AAV9, AAVhu68, AAVhu31, AAVhu32, AAV8, AAV7, AAV6, AAV5, AAV4, AAV3, AAV1, AAVhu95. AAVhu96, or AAVrh91.

In certain embodiments, the exogenous targeting peptide is engineered and/or inserted in the hypervariable region between amino acids 588 and 589 in an AAV9 parental capsid as determined based on the numbering of VP1 amino acid sequence of SEQ ID NO: 25, or an analogous position in an AAVhu68, AAVhu31, AAVhu32, AAVhu95, AAVhu96, AAV8, AAV7, AAV6, AAV5, AAV4, AAV3, AAV1, or AAVrh91 parental AAV capsid.

In certain embodiments, tire exogenous targeting peptide has an amino acid sequence at its carboxy terminus and its amino terminus that is immediately preceded by “AQ” at position 588 of the parental capsid, which is a Clade F capsid such as an AAV9, AAVhu68, AAVhu31, AAVhu32, AAVhu95, or AAVhu96 capsid.

In certain embodiments, the residues of the parental AAV capsid sequence is preserved (i.e., there are no substitutions and/or deletions) in tire 1, 2, and/or 3 amino acid residues at the N-terminus and/or C-terminus immediately preceding the target peptide insert in the AAV VP3 region, as compared to that of the parental AAV capsid amino acid sequence). In certain embodiments, there are no deletions in the 1, 2 and/or 3 amino acid residues as compared to that of the parental AAV capsid amino acid sequence at the N- terminus and/or C-terminus immediately preceding the target peptide insert. In certain embodiments, one or more amino acid residues, as compared to that of the parental AAV capsid amino acid sequence, are modified at the N-terminus and/or C-terminus immediately preceding the n-mer, and/or to provide the mutant AAV capsid with one or more residues of the n-mer sequence. In certain embodiments, one or more amino acid residues, as compared to that of tire parental AAV capsid amino acid sequence, are modified at the positions at the N-terminus and/or C-terminus immediately flanking the n- mer, and/or to provide the mutant AAV with one or more residues of the n-mer sequence.

In certain embodiments, AAV9 is selected as the parental capsid, wherein the targeting peptide with linkers of various length(s) is inserted (and/or engineered) in a suitable location of the HVRVIII region of amino acids 588 and 589 (Q-A), based on VP1 numbering. In other embodiments, AAVhu68 or another clade F capsid is selected as the parental capsid. In certain embodiments, AAV 8 is selected as the parental capsid, wherein the targeting peptide with linkers of various length(s) is inserted (and/or engineered) in a suitable location of HVRVIII region of amino acid 590 and 591 (N-T), based on VP1 numbering. In certain embodiments, AAV7 is selected as the parental capsid, wherein the targeting peptide with linkers of various length(s) is inserted (and/or engineered) in a suitable location of HVRVIII region of amino acid 589 and 590 (N-T), based on VP 1 numbering. In certain embodiments, AAV6 is selected as the parental capsid, wherein the targeting peptide with linkers of various length(s) is inserted (and/or engineered) in a suitable location of HVRVIII region of amino acid 588 and 589 (S-T), based on VP1 numbering. In certain embodiments, AAV5 is selected as the parental capsid, wherein the targeting peptide with linkers of various length(s) is inserted (and/or engineered) in a suitable location of HVRVIII region of amino acid 577 and 578 (T-T), based on VP1 numbering. In certain embodiments, AAV4 is selected as the parental capsid, wherein the targeting peptide with linkers of various length(s) is inserted (and/or engineered) in a suitable location of HVRVIII region of amino acid 586 and 587 (S-N), based on VP1 numbering. In certain embodiments, AAV3/3B is selected as the parental capsid, wherein the targeting peptide with linkers of various length(s) is inserted (and/or engineered) in a suitable location of HVRVIII region of amino acid 588 and 589 (N-T), based on VP1 numbering. In certain embodiments, AAV2 is selected as the parental capsid, wherein the targeting peptide with linkers of various length(s) is inserted (and/or engineered) in a suitable location of HVRVIII region of amino acid 587 and 589 (N-R), based on VP 1 numbering. In certain embodiments, AAV 1 is selected as the parental capsid, wherein the targeting peptide with linkers of various length(s) is inserted (and/or engineered) in a suitable location of HVRVII1 region of amino acid 588 and 589 (S-T), based on VP1 numbering. See also, FIG. 7 which shows an alignment of tire specified regions of the amino acid sequences of the AAV capsid proteins: AAV9 (amino acids 566 to 615 of AAV9 capsid; SEQ ID NO: 27), AAV8 (amino acids 565 to 614 of AAV8 capsid; SEQ ID NO: 28), AAV7 (amino acids 567 to 616 of AAV7; SEQ ID NO: 29), AAV6 (amino acids 550 to 599 of AAV6 capsid; SEQ ID NO: 30), AAV5 (amino acids 556 to 605 of AAV5; SEQ ID NO: 31). AAV4 (ammo acids 558 to 607 of AAV4 capsid; SEQ ID NO: 32), AAV3B (amino acids 564 to 613 of AAV3B capsid; SEQ ID NO: 33), AAV2 (amino acids 566 to 615 of AAV2 capsid; SEQ ID NO: 34), and AAV1 (amino acids 566 to 615 of AAV1 capsid; SEQ ID NO: 35), which is focused on the HVRVIII region in which the targeting peptide may be inserted (based on structural analysis).

In certain embodiments, tire parental capsid is modified to comprise “Xn - n-mer - Xm”. wherein: (i) Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid; (ii) tire n-mer is GQVRVGV (SEQ ID NO: 3), MDAHYVR (SEQ ID NO: 4), TQAVPLK (SEQ ID NO: 5), VVHQTGL (SEQ ID NO: 6), or MAISRER (SEQ ID NO: 7), or an n-mer sequence of at least 6, at least 7, or the full-length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein the parental capsid is a Clade F AAV (e.g., AAVhu68, AAV9, AAVhu31, AAVhu32, AAVhu95, AAVhu96), Clade E (e.g., AAV8), or Clade A AAV (e.g., AAV1, AAVrh91)) capsid, or a non-parvovirus capsid (e.g., herpes simplex virus, etc.) in order enhance expression and/or otherw ise modulate targeting to muscle cell (e.g.. heart (cardiac cell), or skeletal (gastrocnemius) cell). See, e.g.. WO 2020/223231, published November 5, 2020 (rh91, including table with deamidation pattern), US Provisional Patent Application No. 63/065,616, filed August 14, 2020, US Provisional Patent Application No. 63/109,734, filed November 4, 2020, and International Patent Application No. PCT/US21/45945, filed August 13, 2021, which is now published as WO 2022/036220, all of which are incorporated herein by reference in their entireties. In certain embodiments. AAV capsids having reduced capsid deamidation may be selected. See, e.g.. PCT/US 19/19804 and PCT/US 18/19861, both filed Feb 'll, 2019, and incorporated by reference in their entireties. In certain embodiments, the mutant capsids described herein are characterized by having a deamidation pattern similar to their parental AAV capsid, e.g., such as described in US 2020/0056159, published Feb 20, 2020 (AAVhu68; highly deamidated in N57, N329, N452 and N512), with minor optional amounts of deamidation); US 2020/0407750, published Dec 31, 2020 (AAV9, highly deamidated in N57, N329, N452 and N512), each of which is incorporated herein by reference in its entirety.

In certain embodiments, a recombinant adeno-associated virus particle (rAAV) is provided comprising: (a) an adeno-associated virus (AAV) capsid comprising VP1 proteins, VP2 proteins and VP3 proteins, wherein the capsid proteins have an amino acid sequence comprising a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises ”Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) n-mer selected from GQVRVGV (SEQ ID NO: 3), MDAHYVR (SEQ ID NO: 4), TQAVPLK (SEQ ID NO: 5), VVHQTGL (SEQ ID NO: 6), MAISRER (SEQ ID NO: 7), or a n-mer sequence of at least 6, at least 7, or the full-length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid; and (b) a vector genome packaged in the AAV capsid, wherein the vector genome comprises a nucleic acid sequence encoding a gene product under control of sequences which direct expression thereof. In certain embodiments, an rAAV comprises capsid proteins having an amino acid sequence comprising a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises: GQVRVGV (SEQ ID NO: 3), MDAHYVR (SEQ ID NO: 4). TQAVPLK (SEQ ID NO: 5), VVHQTGL (SEQ ID NO: 6). MAISRER (SEQ ID NO: 7), and/or any combination thereof. In certain embodiment, the rAAV comprising capsid proteins comprising exogenous targeting peptide as described herein (i.e., an engineered rAAV capsid) has a greater muscle specificity, targeting, and/or efficacy as compared to a parental rAAV capsid.

In certain embodiments, a recombinant adeno-associated viral particle (rAAV) is provided comprising an AAV capsid, wherein the AAV capsid is not an AAV2 capsid. In certain embodiments, the rAAV comprises an AAV capsid, wherein the AAV capsid is not a mutant AAV2 capsid comprising a NDVRAVS (SEQ ID NO: 36) sequence. In certain embodiments, the rAAV comprises an AAV2 capsid having AAV2 capsid proteins comprising at least one or more of the exogenous targeting peptides comprising '‘Xn - n- mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any ammo acid; (n) the n-mer is GQVRVGV (SEQ ID NO: 3), MDAHYVR (SEQ ID NO: 4), TQAVPLK (SEQ ID NO: 5), VVHQTGL (SEQ ID NO: 6), MAISRER (SEQ ID NO: 7), or an n-mer sequence of at least 6, at least 7, or the full-length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid.

In certain embodiments, tire rAAV comprises a capsid having AAV9 capsid proteins comprising one or more exogenous targeting peptides comprising "Xn - n-mer - Xm”. wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any ammo acid; (n) the n-mer is GQVRVGV (SEQ ID NO: 3), MDAHYVR (SEQ ID NO: 4), TQAVPLK (SEQ ID NO: 5), VVHQTGL (SEQ ID NO: 6), MAISRER (SEQ ID NO: 7), or an n-mer sequence of at least 6, at least 7, or the full-length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises: GQVRVGV (SEQ ID NO: 3), MDAHYVR (SEQ ID NO: 4), TQAVPLK (SEQ ID NO: 5), VVHQTGL (SEQ ID NO: 6), MAISRER (SEQ ID NO: 7), and/or any combination thereof. In certain embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises GQVRVGV (SEQ ID NO: 3). In certain embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises MDAHYVR (SEQ ID NO: 4). In certain embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises TQAVPLK (SEQ ID NO: 5). In certain embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises VVHQTGL (SEQ ID NO: 6). In certain embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises MAISRER (SEQ ID NO: 7). In certain embodiments, the rAAV comprises AAVhu68 capsid proteins, wherein the AAVhu68 capsid proteins comprising one or more exogenous targeting peptides comprising “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is GQVRVGV (SEQ ID NO: 3), MDAHYVR (SEQ ID NO: 4), TQAVPLK (SEQ ID NO: 5), VVHQTGL (SEQ ID NO: 6), MAISRER (SEQ ID NO: 7), or an n-mer sequence of at least 6, at least 7, or tire full- length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the rAAV comprises an AAVhu68 capsid having an AAVhu68 capsid protein comprising a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises: GQVRVGV (SEQ ID NO: 3), MDAHYVR (SEQ ID NO: 4), TQAVPLK (SEQ ID NO: 5), VVHQTGL (SEQ ID NO: 6), MAISRER (SEQ ID NO: 7), and/or any combination thereof.

In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising one or more exogenous peptides comprising “Xn - GQVRVGV (SEQ ID NO: 3) - Xm”, wherein Xn is 0. 1, 2 or 3 amino acid residues independently selected from any amino acid, and wherein Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid proteins comprise one or more exogenous peptides comprising GQVRVGV (SEQ ID NO: 3).

In certain embodiments, the rAAV comprises an AAV9 capsid protein having AAV9 capsid proteins comprising one or more exogenous peptides comprising “Xn - MDAHYVR (SEQ ID NO: 4)- Xm”, wherein Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, and wherein Xm is 0, 1, 2. or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid proteins comprise one or more exogenous peptides comprising MDAHYVR (SEQ ID NO: 4).

In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising one or more exogenous peptides comprising “Xn - TQAVPLK (SEQ ID NO: 5)- Xm”. wherein Xn is 0, 1, 2. or 3 amino acid residues independently selected from any amino acid, and wherein Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid proteins comprise one or more exogenous peptides comprising TQAVPLK (SEQ ID NO: 5).

In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising one or more exogenous peptides comprising “Xn - VVHQTGL (SEQ ID NO: 6) - Xm”, wherein Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and wherein Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid proteins comprise one or more exogenous peptides comprising VVHQTGL (SEQ ID NO: 6).

In certain embodiments, the rAAV comprises an AAV9 capsid having AAV9 capsid proteins comprising one or more of exogenous peptides comprising “Xn - MAISRER (SEQ ID NO: 7) - Xm”, wherein Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and wherein Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid. In certain embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid proteins comprise one or more exogenous peptides comprising MAISRER (SEQ ID NO: 7).

In certain embodiments, tire rAAV comprises a mutant AAV9- capsid or a mutant AAVhu68-GQVRVGV capsid, which comprises mutant VP1 , mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the GQVRVGV (SEQ ID NO: 3) peptide insert. In certain embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and/or N512. and optional deamidation in other positions within the parental capsid sequence. In certain embodiments, the rAAV has a capsid comprising AAV9- GQVRVGV (or AAVhu68- GQVRVGV) - in which each the AAV VP1, AAV VP2 AAV VP3 proteins are a heterogenous population and comprise the AAV VP3 region of SEQ ID NO: 80 (about amino acid 203 to about amino acid 736 based on the residue positions in SEQ ID NO: 25 (AAV9)). The proteins further comprising a heterogenous population of each of the capsid proteins having deamidation in about 50% to about 100% of positions N57 (VP1 only), N329. N452, and/or N512, based on the parental capsid residue positions. In certain embodiments, the percentage of deamidation at one or more of these positions is over 60%, over 70%, over 80%. over 90%, over 95%, or about 70% to about 100%, or values therebetween. The percentage of deamidation in each of the highly deamidated positions may differ from each other within an rAAV capsid. For example, the percent of deamidation at position N57 in an AAV capsid, may differ from the percent of deamidation at each of the other highly deamidated residues, which may have higher (or lower) deamidation percentages. Similarly, the percent of deamidation at position N329, N452, and/or N512, may each vary’ from one another. In certain embodiments, tire percentage of deamidation for each of the highly deamidated positions is determined for the total VP proteins in a single rAAV capsid or a stock of rAAV capsids. In certain embodiments, a parental clade F capsid comprises VP proteins which are highly deamidated in all four of these positions. In certain embodiments, the percentage of deamidation in one or more of these highly deamidated positions is over 50%, over 55%, over 60%, over 65%, over 70%, over 75%, over 80%, over 85%, over 90%, over 95%, or about 70% to about 100%, or values therebetween.

In certain embodiments, the rAAV comprises an AAV capsid having AAV capsid proteins comprising exogenous peptides, wherein the peptides are inserted with linkers of various lengths between amino acids 588 and 589 (Q-A) of the AAV capsid protein, based on the numbering of the SEQ ID NO: 25, optionally further wherein flanking amino acids at positions 587, 588, 589, and/or 590 are mutated. In certain embodiments, the rAAV comprises an AAV capsid having AAV capsid proteins comprising an exogenous peptide which is immediately preceded by “AQ”. In certain embodiments, tire rAAV comprises an AAV capsid having AAV capsid proteins comprising an exogenous peptide wherein the exogenous peptide is flanked by "AQ" (e.g., "AQ- GQVRVGV (SEQ ID NO: 3)’", "AQ- GQVRVGV (SEQ ID NO: 3)-AQ”, or “GQVRVGV (SEQ ID NO: 3)-AQ”). In certain embodiments, the rAAV comprises an AAV capsid having AAV capsid proteins comprising an exogenous peptide that is listed in Table A, below.

In certain embodiments, the rAAV comprises an AAV capsid having AAV capsid proteins comprising an exogenous peptide insert that is immediately preceded by the native residues of the parental AAV capsid which may be unmodified at tire amino (N-) terminus, and/or at the carboxy (COO-) terminus. In certain embodiments, tire rAAV comprises an AAV capsid having AAV capsid proteins comprisin an exogenous peptide that is immediately preceded by the native residues of the parental AAV capsid which may be mutated at the amino (N-) terminus, and/or at the carboxy (COO-) terminus. In certain embodiments, wherein the parental capsid is an AAV9 capsid or other Clade F capsid, tire AAV9 parental capsid or other Clade F parental capsid is unmodified at the residues flanking the inserted exogenous targeting peptide. In certain embodiments, the rAAV comprises an AAV capsid having capsid proteins comprising an exogenous peptide insert that is flanked by "SAQ" at the amino (N-) terminus of the exogenous peptide. In certain embodiments, the rAAV comprises an AAV capsid having AAV capsid proteins comprising an exogenous peptide insert wherein the exogenous peptide insert is flanked by ”AQA" at its carboxy (COO-) terminus. In certain embodiments, wherein the parental capsid is an AAV9 capsid or other Clade F capsid, the AAV9 parental capsid or other Clade F parental capsid is modified (i.e., mutated) at the residues flanking the inserted exogenous targeting peptide. In certain embodiments, the rAAV comprises an AAV capsid having AAV capsid proteins comprising an exogenous peptide insert that is flanked by the mutated trimer “ENT” at the amino (N-) terminus of the exogenous peptide. In certain embodiments, the rAAV comprises an AAV capsid having AAV capsid proteins comprising an exogenous peptide insert that is flanked by the mutated trimer “SSQ”, “SQQ”, “ENR”, “SHQ”, SWQ”, SAI”, “GAQ”, “FAQ”, “QAQ”, “AAQ”, “SGQ”, or “SGM” at its amino (N-) terminus of the exogenous peptide insert. In certain embodiments, the rAAV comprises an AAV capsid having AAV capsid proteins comprising an exogenous peptide insert that is flanked by the mutated trimer “QQA”, “NQA”, “EQA”, “AMA”, “AQC”. “GQA”, “ARA”, or “GRA” at its carboxy (COO-) terminus. In other embodiments, the flanking residues may be modified, e.g., where a non-Clade F parental AAV is selected and/or to reduce the number of AAV residues inserted.

In certain embodiments, the rAAV comprises an AAV capsid having AAV capsid proteins comprising an exogenous peptide insert that is flanked by modified amino acid residues, wherein the capsid proteins comprise the amino acid sequence of “SAQMDAHYSRQQA” (SEQ ID NO 70). In certain embodiments, the rAAV comprises an AAV capsid having AAV capsid proteins comprising an exogenous peptide insert that is flanked by modified amino acid residues, wherein the capsid proteins comprise an amino acid sequence at least 99% identical to “SAQMDAHYSRQQA” (SEQ ID NO 70). In certain embodiments, the rAAV comprises an AAV capsid having AAV capsid proteins comprising an exogenous peptide insert that is flanked by modified amino acid residues, wherein the capsid proteins comprises the amino acid sequence of “SSQMDAHYVRNQA” (SEQ ID NO 71). Tn certain embodiments, the rAAV comprises an AAV capsid having AAV capsid proteins comprising an exogenous peptide insert that is flanked by modified amino acid residues, wherein the capsid proteins comprise an amino acid sequence at least 99% identical to “SSQMDAHYVRNQA” (SEQ ID NO 71). In certain embodiments, the rAAV comprises an AAV capsid having AAV capsid proteins comprising an exogenous peptide insert that is flanked by modified amino acid residues, wherein the capsid proteins comprise the amino acid sequence of “SAQMDAHYRREQA” (SEQ ID NO 72). In certain embodiments, the rAAV comprises an AAV capsid having AAV capsid proteins comprising an exogenous peptide insert that is flanked by modified amino acid residues, wherein the capsid proteins comprise an amino acid sequence at least 99% identical to “SAQMDAHYRREQA” (SEQ ID NO 72). In certain embodiments, the rAAV comprises an AAV capsid having AAV capsid proteins comprising an exogenous peptide insert that is flanked by modified amino acid residues, wherein the capsid proteins comprise the amino acid sequence of “SQQGQVRVGVAMA” (SEQ ID NO 73). In certain embodiments, the rAAV comprises an AAV capsid having AAV capsid proteins comprising an exogenous peptide insert that is flanked by modified amino acid residues, wherein the capsid proteins comprise an amino acid sequence at least 99% identical to “SQQGQVRVGVAMA” (SEQ ID NO 73). In certain embodiments, the rAAV comprises an AAV capsid having AAV capsid proteins comprising an exogenous peptide insert that is flanked by modified amino acid residues, wherein the capsid proteins comprise the amino acid sequence of “SAQLDAHYVRNQA” (SEQ ID NO 74). In certain embodiments, the rAAV comprises an AAV capsid having AAV capsid proteins comprising an exogenous peptide insert that is flanked by modified amino acid residues, wherein the capsid proteins comprise an amino acid sequence at least 99% identical to “SAQLDAHYVRNQA” (SEQ ID NO 74). In certain embodiments, tire rAAV comprises an AAV capsid having AAV capsid proteins comprising an exogenous peptide insert that is flanked by modified amino acid residues, wherein the capsid proteins comprise the amino acid sequence of “ENRRGDFNNTAQA” (SEQ ID NO 75). In certain embodiments, the rAAV comprises an AAV capsid having AAV capsid proteins comprising an exogenous peptide insert that is flanked by modified amino acid residues, wherein the capsid proteins comprise an amino acid sequence at least 99% identical to “ENRRGDFNNTAQA” (SEQ ID NO 75). In certain embodiments, a capsid from among Clade F AAVs. such as AAVhu68 or AAV9 is selected for the parental capsids. Methods of generating vectors having the AAV9 capsid or AAVhu68 capsid, and/or chimeric capsids derived from AAV9 have been described. See, e.g., US 7,906,111, which is incorporated by reference herein. See also, US Provisional Patent Application No. 63/093,275, filed October 18, 2020, which is incorporated herein by reference. Other AAV serotypes which transduce nasal cells or another suitable target (e.g., muscle or lung) may be selected as sources for capsids of AAV viral vectors including, e.g., AAV1. AAV2. AAV3. AAV4, AAV5, AAV6, AAV6.2, AAV7. AAV8. AAV9. rhlO, AAVrh64Rl, AAVrh64R2, rh8. AAVrh32.33 (See, e.g.. US Published Patent Application No. 2007-0036760-Al; US Published Patent Application No. 2009-0197338-Al; and EP 1310571). See also, WO 2003/042397 (AAV7 and other simian AAV), US Patent 7790449 and US Patent 7282199 (AAV8), WO 2005/033321 (AAV9), and WO 2006/110689, or yet to be discovered, or a recombinant AAV based thereon, may be used as a source for the AAV capsid. See, e.g., WO 2020/223232 Al (AAV rh90), WO 2020/223231 Al International Application No. PCT/US21/45945. filed August 13, 2021 (AAV rh91), and WO 2020/223236 Al (AAV rh92, AAV rh93. AAV rh91.93), which are incorporated herein by reference in its entirety. These documents also describe other AAV which may be selected for generating AAV and are incorporated by reference. In some embodiments, an AAV capsid (cap) for use in the viral vector can be generated by mutagenesis (i.e., by insertions, deletions, or substitutions) of one of tire aforementioned AAV caps or its encoding nucleic acid. In some embodiments, the AAV capsid is chimeric, comprising domains from two or three or four or more of the aforementioned AAV capsid proteins. In some embodiments, tire AAV capsid is a mosaic of Vpl. Vp2, and Vp3 (also referred to as vpl, vp2. vp3, or VP1, VP2, VP3) monomers from two or three different AAVs or recombinant AAVs. In some embodiments, an rAAV composition comprises more than one of the aforementioned caps.

In certain embodiments, mutant AAV capsids are produced by engineering a nucleic acid sequence encoding an exogenous targeting peptide insert into a AAV VP 1 coding sequence. In certain embodiments, the coding sequence for the exogenous targeting peptide insert is SEQ ID NO: 78, or a sequence 95% to 100%, or at least 99% identical thereto encoding SEQ ID NO: 3 (GQVRVGV).

In certain embodiments, peptide inserts are engineered between amino acids 588 and 589 of the AAVhu68 capsid (as based on amino acid sequence of SEQ ID NO: 26). In other embodiments, one or more peptides are inserted between amino acids 588 and 89 in the AAV9 capsid (as based on amino acid sequence of SEQ ID NO: 25). Still other suitable locations for these inserts may be determined. In still other embodiments, the peptides may be used in other vectors or compositions for improving targeting of muscle cells.

In certain embodiments, the coding sequence of a mutant AAV9 capsid having the exogenous targeting peptide inserted in the hypervariable region between amino acids 588 and 589 in the AAV9 parental capsid is: SEQ ID NO: 79 (GQVRVGV). In other embodiments, a mutant AAV9 is encoded by any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 80 (GQVRVGV mutant VP 1). In certain embodiments, the coding sequence of a mutant AAV9 capsid is SEQ ID NO: 79, or a sequence 95% to 100% identical, or at least 99% identical thereto encoding SEQ ID NO: 79 (GQVRVGV mutant VP1).

As used herein, the term "clade" as it relates to groups of AAV refers to a group of AAV which are phylogenetically related to one another as determined using a Neighbor- Joining algorithm by a bootstrap value of at least 75% (of at least 1000 replicates) and a Poisson correction distance measurement of no more than 0.05, based on alignment of the AAV vpl amino acid sequence. The Neighbor-Joining algorithm has been described in the literature. See, e.g., M. Nei and S. Kumar, Molecular Evolution and Phylogenetics (Oxford University Press, New York (2000). Computer programs are available that can be used to implement this algorithm. For example, tire MEGA v2. 1 program implements the modified Nei-Gojobori method. Using these techniques and computer programs, and the sequence of an AAV vpl capsid protein, one of skill in the art can readily determine whether a selected AAV is contained in one of the clades identified herein, in another clade, or is outside these clades. See, e.g., G Gao, et al, J Virol, 2004 Jun: 78(12): 6381-6388, which identifies Clades A, B, C, D, E and F, and provides nucleic acid sequences of novel AAV, GenBank Accession Numbers AY530553 to AY530629. See, also, WO 2005/033321.

As used herein, an “AAV9 capsid” is a self-assembled AAV capsid composed of multiple AAV9 vp proteins. The AAV9 vp proteins are typically expressed as alternative splice variants encoded by a nucleic acid sequence which encodes the vp 1 amino acid sequence of GenBank accession: AAS99264. These splice variants result in proteins of different length. In certain embodiments, “AAV9 capsid” includes an AAV having an amino acid sequence which is 99% identical to AAS99264 or 99% identical thereto. See, also, WO 2019/168961, published September 6, 2019, including Table G providing the deamidation pattern for AAV9. See, also US7906111 and WO 2005/033321. When specified, “AAV9 variants” may include those described in, e.g., WO2016/049230, US 8,927,514, US 2015/0344911, and US 8,734,809.

A rAAVhu68 is composed of an AAVhu68 capsid and a vector genome. An AAVhu68 capsid is an assembly of a heterogenous population of vpl, a heterogenous population of vp2, and a heterogenous population of vp3 proteins. As used herein when used to refer to vp capsid proteins, the term “heterogenous” or any grammatical variation thereof, refers to a population consisting of elements that are not the same, for example, having vpl, vp2 or vp3 monomers (proteins) with different modified amino acid sequences. See, also, PC T/US2018/019992, WO 2018/160582, entitled “Adeno-Associated Virus (AAV) Clade F Vector and Uses Therefor”, and which are incorporated herein by reference in its entirety.

For other recombinant viral vectors, suitable exposed portions of the viral capsid or envelope protein which is responsible for targeting specificity are selected for insertion of the targeting peptide. For example, in an adenovirus, it may be desirable to modify tire hexon protein. In a lentivirus, an envelope fusion protein may modified comprise one or more copies of the targeting motif. For vaccinia virus, the major glycoprotein may be modified to comprise one or more copies of the targeting motif. Suitably, these recombinant viral vectors are replication-defective for safety purposes.

Expression Cassette and Vectors

Vector genome sequences that are packaged into an AAV capsid and delivered to a host cell are typically composed of, at a minimum, a transgene and its regulatory sequences, and AAV inverted terminal repeats (ITRs). Both single-stranded AAV and self- complementary (sc) AAV are can be used. The transgene is a nucleic acid coding sequence, heterologous to tire vector sequences, that encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product of interest. The nucleic acid coding sequence is operatively linked to regulatory components in a manner that permits transgene transcription, translation, and/or expression in a cell of a target tissue.

The AAV sequences of the vector genome typically comprise cis-acting AAV 5’ and AAV 3’ inverted terminal repeat (ITR) sequences (See, e.g., B. J. Carter, in “Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 168 (1990)). The ITR sequences are about 145 base pairs (bp) in length. Preferably, substantially the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of tire art. (See, e.g., texts such as Sambrook et al, ’‘Molecular Cloning. A Laboratory' Manual”, 2d ed., Cold Spring Harbor Laboratory', New York (1989); and K. Fisher et al., J. Virol., 70:520 532 (1996)). An example of such a molecule employed in the present invention is a “cis-acting” plasmid containing the transgene, in which the selected transgene sequence and associated regulatory elements are flanked by the 5’ and 3' AAV ITR sequences (also referred to as “AAV 5’ ITR”, “5’ ITR”, “AAV 5' ITR”, or “5' ITR”, “AAV 3’ ITR”, “3’ ITR”, “AAV 3' ITR”, or “3' ITR”). In one embodiment, the ITRs are from an AAV different than that supplying a capsid. In one embodiment, the ITR sequences are from AAV2. However, ITRs from other AAV sources may be selected. A shortened version of the 5’ ITR, termed AITR, has been described in which the D-sequence and terminal resolution site (trs) are deleted. In certain embodiments, the vector genome includes a shortened AAV2 ITR of 130 base pairs, wherein the external A elements is deleted. Without wishing to be bound by theory, it is believed that the shortened ITR reverts back to the wild-type (WT) length of 145 base pairs during vector DNA amplification using the internal (A’) element as a template. In other embodiments, full- length AAV 5’ and 3’ ITRs are used. Where the source of the ITRs is from AAV2 and tire AAV capsid is from another AAV source, the resulting vector may be termed pseudotyped. However, other configurations of these elements may be suitable.

In certain embodiments, the provided herein is an rAAV comprising a nucleic acid molecule comprising a vector genome comprising at least one AAV ITR at the extreme 5’ and/or extreme 3' end of the nucleic acid molecule which is the vector genome and an expression cassette. In certain embodiments, the vector genome is a nucleic acid molecule which comprises a 5' - AAV ITR, the expression cassette and a 3' - AAV ITR.

In certain embodiments, the rAAV comprises vector genome comprising a nucleic acid molecule comprising, 5' to 3', AAV- 5' ITR - an optional enhancer - a promoter - an optional nitron - coding sequence (e.g., test transgene) - polyadenylation (polyA) signal sequence - AAV3' - ITR. In other embodiments, the orientation of the ITRs may change from the orientation presented in the vector genome of the nucleic acid used in production (e.g., a plasmid). Thus, in certain embodiments, the rAAV may comprise a vector genome flanked by 3' and 5' AAV ITRs, respectively. In certain embodiments, the rAAV may comprise a vector genome flanked by two 5' AAV ITRs. In certain embodiments, the rAAV may comprise a vector genome flanked by two 3' AAV ITRs. In other embodiments, an rAAV as provided herein may be partially truncated such that the 5' AAV ITR and/or the 3' AAV ITR is not detectable in the vector genome packaged in a final rAAV product.

In addition to tire major elements identified above for the recombinant AAV vector, the vector also includes conventional control elements necessary which are operably linked to tire transgene in a manner which permits its transcription, translation and/or expression in a cell transfected with the plasmid vector or infected with the virus produced by the invention. As used herein, “operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

The regulatory control elements ty pically contain a promoter sequence as part of the expression control sequences, e.g., located between tire selected 5' ITR sequence and the coding sequence. Constitutive promoters, regulatable promoters [see, e.g., WO 2011/126808 and WO 2013/04943], tissue specific promoters, or a promoter responsive to physiologic cues may be used may be utilized in the vectors described herein.

Examples of constitutive promoters suitable for controlling expression of the therapeutic products include, but are not limited to chicken beta (0)-actin (CB) promoter, CB7 promoter (promoter comprising a cytomegalovirus immediate-early (CMV IE) enhancer and tire chicken 0-actin promoter, optionally with spacer sequence, optionally with a chimeric intron comprising chicken beta actin intron and further comprising a chicken beta-actin splicing donor (including tire exon sequence, chicken beta actin intron) and rabbit beta-globin splicing acceptor), human cytomegalovirus (CMV) promoter, ubiquitin C promoter (UbC), the early and late promoters of simian virus 40 (SV40), U6 promoter, metallothionein promoters, EFla promoter, ubiquitin promoter, hypoxanthine phosphoribosyl transferase (HPRT) promoter, dihydrofolate reductase (DHFR) promoter (Scharfmann ct al., Proc. Natl. Acad. Sci. USA 88:4626-4630 (1991), adenosine deaminase promoter, phosphoglycerol kinase (PGK) promoter, pyruvate kinase promoter phosphoglycerol mutase promoter, the 0-actin promoter (Lai et al., Proc. Natl. Acad. Sci. USA 86: 10006-10010 (1989)). the long terminal repeats (LTR) of Moloney Leukemia Virus and other retroviruses, the thymidine kinase promoter of Herpes Simplex Vims and other constitutive promoters known to those of skill in the art. Examples of tissue- or cell- specific promoters suitable for use in the present invention include, but are not limited to, endothelin-I (ET -I) and Flt-I, which are specific for endothelial cells, FoxJl (that targets ciliated cells). In other embodiments, selection of cardiac-specific promoters may be desired. See, e.g., R. M. Deviatiirov, et al, “Human library of cardiac promoters and enhancers”, bioRxiv, pp. 1-27, bioRxiv preprint; posted June 15, 2020. Preferably, such promoters are of human origin. In other embodiments, selection of muscle-specific promoters may be desired, e.g., muscle creatine kinase promoter, human skeletal a-actin promoter, desmin gene promoter. See, e.g.. Skopenkova, V.V., Muscle Specific Promoters for Gene Therapy, Acta Naturae, 2021, Jan-Mar; 13(1): 47-58, which is incorporated herein by reference in its entirety.

In certain embodiments, the regulatory sequences comprise one or more of a promoter, an enhancer, an intron, a transcription factor, a transcription terminator, an efficient RNA processing signals such as splicing and polyadenylation signals (poly A), a sequences that stabilize cytoplasmic mRNA, for example Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE), and sequences that enhance translation efficiency (i.e., Kozak consensus sequence). In certain embodiments the selected promoter is a constitutive promoter. In certain embodiments, the promoter is a ubiquitous promoter. For example such promoters may include chicken beta-actin (CB) promoter, hybrid of a cytomegalovirus immediate-early enhancer and the chicken -actin promoter (a CB7 promoter), human cytomegalovirus (CMV) promoter, ubiquitin C promoter (UbC), the early and late promoters of simian virus 40 (SV40), U6 promoter, metallothionein promoters, EFla promoter, ubiquitin promoter, hypoxanthine phosphoribosyl transferase (HPRT) promoter, dihydrofolate reductase (DHFR) promoter (Scharfmann et al.. Proc. Natl. Acad. Sci. USA 88:4626-4630 (1991). adenosine deaminase promoter, phosphoglycerol kinase (PGK) promoter, pyruvate kinase promoter phosphoglycerol mutase promoter, the -actin promoter (Lai et al.. Proc. Natl. Acad. Sci. USA 86: 10006-10010 (1989)), the long terminal repeats (LTR) of Moloney Leukemia Virus and other retroviruses, the thymidine kinase promoter of Herpes Simplex Virus and other constitutive promoters known to those of skill in the art.

In certain embodiments, tire promoter is a tissue- or cell specific-promoter. In certain embodiments, the promoter is cardiac specific promoter, e.g., cardiac troponin T (cTNT), desmin (DES), alpha-myosin heavy chain (a-MHC), myosin light chain 2 (MLC- 2) promoters. See also, Pacak, C.A., et al.. Tissue specific promoters improve specificity of AAV9 mediated transgene expression following intra-vascular gene delivery in neonatal mice. Genetic Vaccines and Therapy 2008, 6: 13. In certain embodiments, the expression cassette comprises a promoter which is a chicken cardiac Troponin T promoter (also referred to as chicken TnT or chTnT). In certain embodiments, the promoter is a hybrid cardiac promoter comprising a cytomegalovirus immediate early (CMV IE) enhancer and a chicken cardiac troponin T (chicken cTnT or chTnT) promoter. See also, International Patent Application No. PCT/US2022/082384. filed December 24, 2022, now published WO 2023/122804, which is incorporated herein by reference in its entirety. In certain embodiments, and enhancer is a a-myosin heavy-chain enhancer.

Inducible promoters suitable for controlling expression of the therapeutic product include promoters responsive to exogenous agents (e.g., pharmacological agents) or to physiological cues. These response elements include, but are not limited to, a hypoxia response element (HRE) that binds HIF-Ia and p, a metal-ion response element such as described by Mayo et al. (1982, Cell 29:99-108); Brinster et al. (1982, Nature 296:39-42) and Searle et al. (1985, Mol. Cell. Biol. 5: 1480-1489); or a heat shock response element such as described by Nouer et al. (in: Heat Shock Response, ed. Nouer, L., CRC, Boca Raton, Fla., ppI67-220, 1991).

In one embodiment, expression of the gene product is controlled by a regulatable promoter that provides tight control over the transcription of the sequence encoding the gene product, e.g., a pharmacological agent, or transcription factors activated by a pharmacological agent or in alternative embodiments, physiological cues. Promoter systems that are non-leaky and that can be tightly controlled are preferred. Examples of regulatable promoters which are ligand-dependent transcription factor complexes that maybe used in the invention include, without limitation, members of the nuclear receptor superfamily activated by their respective ligands (e.g., glucocorticoid, estrogen, progestin, retinoid, ecdysone, and analogs and mimetics thereof) and rTTA activated by tetracycline. In one aspect of the invention, the gene switch is an EcR-based gene switch. Examples of such systems include, without limitation, the systems described in US Patent Nos. 6,258,603, 7,045,315, U.S. Published Patent Application Nos. 2006/0014711, 2007/0161086, and International Published Application No. WO 01/70816. Examples of chimeric ecdysone receptor systems are described in U.S. Pat. No. 7,091,038, U.S. Published Patent Application Nos. 2002/0110861, 2004/0033600, 2004/0096942, 2005/0266457, and 2006/0100416, and International Published Application Nos. WO 01/70816, WO 02/066612, WO 02/066613. WO 02/066614, WO 02/066615, WO 02/29075, and WO 2005/108617, each of which is incorporated by reference in its entirety. An example of a non-steroidal ecdysone agonist-regulated system is the RheoSwitch® Mammalian Inducible Expression System (New England Biolabs, Ipswich, MA).

Still other promoter systems may include response elements including but not limited to a tetracycline (tet) response element (such as described by Gossen & Bujard (1992, Proc. Natl. Acad. Sci. USA 89:5547-551); or a hormone response element such as described by Lee et al. (1981. Nature 294:228-232); Hynes et al. (1981, Proc. Natl. Acad. Sci. USA 78:2038-2042); Klock et al. (1987, Nature 329:734-736); and Israel & Kaufman (1989, Nucl. Acids Res. 17:2589-2604) and other inducible promoters known in the art. Using such promoters, expression of the soluble hACE2 construct can be controlled, for example, by the Tet-on/off system (Gossen et al., 1995, Science 268: 1766-9; Gossen et al., 1992, Proc. Natl. Acad. Sci. USA., 89( 12):5547-51); the TetR-KRAB system (Urrutia R„ 2003, Genome Biol., 4(10):231; Deuschle U et al., 1995, Mol Cell Biol. (4): 1907-14); the mifepristone (RU486) regulatable system (Geneswitch; Wang Y et al., 1994, Proc. Natl. Acad. Sci. USA.. 91( 17): 8180-4; Schillinger et al., 2005, Proc. Natl. Acad. Sci. U S A. 102(39): 13789-94); and the humanized tamoxifen-dep regulatable system (Roscilli et al., 2002, Mol. Ther. 6(5):653-63).

In another aspect, the gene switch is based on heterodimerization of FK506 binding protein (FKBP) with FKBP rapamycin associated protein (FRAP) and is regulated through rapamycin or its non-immunosuppressive analogs. Examples of such systems, include, without limitation, the ARGENT™ Transcriptional Technology (ARIAD Pharmaceuticals, Cambridge, Mass.) and the systems described in U.S. Pat. Nos. 6,015.709, 6,117,680, 6,479,653, 6.187.757, and 6,649,595. U.S. Publication No. 2002/0173474, U.S. Publication No. 200910100535, U.S. Patent No. 5,834,266, U.S. Patent No. 7,109,317, U.S. PatentNo. 7,485,441, U.S. Patent No. 5,830,462, U.S. Patent No. 5,869,337, U.S. Patent No. 5,871,753, U.S. Patent No. 6,011,018, U.S. Patent No. 6,043,082, U.S. Patent No. 6,046,047, U.S. Patent No. 6,063,625, U.S. Patent No. 6,140,120, U.S. Patent No. 6,165,787, U.S. Patent No. 6,972.193, U.S. Patent No. 6,326,166, U.S. Patent No. 7,008,780, U.S. Patent No. 6,133.456, U.S. Patent No. 6,150,527, U.S. Patent No. 6,506,379, U.S. Patent No. 6.258.823, U.S. Patent No. 6,693,189, U.S. Patent No. 6,127,521, U.S. Patent No. 6,150,137, U.S. Patent No. 6,464,974, U.S. Patent No. 6,509,152, U.S. Patent No. 6,015,709, U.S. Patent No. 6,1 17,680, U.S. Patent No. 6,479,653, U.S. Patent No. 6.187.757, U.S. Patent No. 6,649,595, U.S. Patent No. 6,984,635, U.S. Patent No. 7,067,526, U.S. Patent No. 7,196,192, U.S. Patent No.

6,476,200, U.S. Patent No. 6,492,106, WO 94/18347, WO 96/20951, WO 96/06097, WO 97/31898, WO 96/41865, WO 98/02441, WO 95/33052, WO 99110508, WO 99110510, WO 99/36553, WO 99/41258, WO 01114387, ARGENT™ Regulated Transcription Retrovirus Kit, Version 2.0 (9109102), and ARGENT™ Regulated Transcription Plasmid Kit, Version 2.0 (9109/02), each of which is incorporated herein by reference in its entirety . The Ariad system is designed to be induced by rapamycin and analogs thereof referred to as ‘‘rapalogs”. Examples of suitable rapamycins are provided in the documents listed above in connection with the description of the ARGENT™ system. In one embodiment, the molecule is rapamycin [e.g., marketed as Rapamune™ by Pfizer], In another embodiment, a rapalog known as AP21967 [ARIAD] is used. Examples of these dimerizer molecules that can be used in the present invention include, but are not limited to rapamycin, FK506, FK1012 (a homodimer of FK506), rapamycin analogs (“rapalogs”) which are readily prepared by chemical modifications of the natural product to add a "bump" that reduces or eliminates affinity for endogenous FKBP and/or FRAP. Examples of rapalogs include, but are not limited to, such as AP26113 (Ariad), AP1510 (Amara, J.F., et al., 1997, Proc Natl Acad Sci USA, 94(20): 10618-23) AP22660, AP22594, AP21370, AP22594, AP23054, AP1855, AP1856, AP1701, AP1861, AP1692 and AP1889, with designed ’bumps' that minimize interactions with endogenous FKBP. Still other rapalogs may be selected, e.g., AP23573 [Merck], In certain embodiments, rapamycin or a suitable analog may be delivered locally to the AAV-transfected cells of the nasopharynx. This local delivery may be by intranasal injection, topically to the cells via bolus, cream, or gel. See. US Patent Application US 2019/0216841 Al, which is incorporated herein by reference.

Other suitable enhancers include those that are appropriate for a desired target tissue indication. In one embodiment, the expression cassette comprises one or more expression enhancers. In one embodiment, the expression cassette contains two or more expression enhancers. These enhancers may be the same or may differ from one another. For example, an enhancer may include a CMV immediate early enhancer. This enhancer may be present in two copies which are located adjacent to one another. Alternatively, the dual copies of tire enhancer may be separated by one or more sequences. In still another embodiment, the expression cassette further contains an intron, e.g., the chicken beta-actin intron. Other suitable introns include those known in the art, e.g., such as are described in WO 2011/126808. Examples of suitable polyadenylation (polyA) sequences include, e.g.. rabbit beta globin (rBG), SV40, SV50, bovine growth hormone (bGH), human growth hormone, and synthetic polyAs. Optionally, one or more sequences may be selected to stabilize mRNA. An example of such a sequence is a modified WPRE sequence, which may be engineered upstream of the polyA sequence and downstream of the coding sequence (see, e.g., MA Zanta-Boussif, et al, Gene Therapy (2009) 16: 605-619).

In certain embodiments, the expression cassette includes one or more expression enhancers such as post-transcriptional regulatory element from hepatitis viruses of woodchuck (WPRE). human (HPRE), ground squirrel (GPRE) or arctic ground squirrel (AGSPRE), or a synthetic post-transcriptional regulatory element. These expressionenhancing elements are particularly advantageous when placed in a 3' UTR and can significantly increase mRNA stability and/or protein yield. In certain embodiments, the expressions cassettes provided include a regulator sequence that is a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) or a variant thereof. Suitable WPRE sequences are provided in the vector genomes described herein and are known in the art (e.g., such as those are described in US Patent Nos. 6.136,597, 6,287,814. and 7,419,829, which are incorporated by reference). In certain embodiments, the WPRE is a variant that has been mutated to eliminate expression of the woodchuck hepatitis B virus X (WHX) protein, including, for example, mutations in the start codon of the WHX gene. In certain embodiments, modified WPRE element is engineered to eliminate expression of the WHX protein, wherein the modified WPRE is a mutated version that contains five-point mutations in the putative promoter region of the WHX gene, along with an additional mutation in the start codon of the WHX gene (ATG mutated to TTG). This mutant WPRE is considered sufficient to eliminate expression of truncated WHX protein based on sensitive flow cytometry analyses of various human cell lines transduced with lentivirus containing a WPRE-GFP fusion construct (Zanta-Boussif et al., 2009). See also, Kingsman S.M., Mitrophanous K., & Olsen J.C. (2005), Potential Oncogene Activity⁷ of the Woodchuck Hepatitis Post-Transcriptional Regulator}’ Element (WPRE). Gene Thcr. 12(1): 3-4; and Zanta-Boussif M. A., Charrier S.. Brice-Ouzet A., Martin S., Opolon P., Thrasher A. J., Hope T.J., & Galy A. (2009), Validation of a Mutated Pre-Sequence Allowing High and Sustained Transgene Expression While Abrogating Whv-X Protein Synthesis: Application to the Gene Therapy of Was, Gene Ther. 16(5):605- 19, both of which are incorporated herein by reference in its entirety. In other embodiments, enhancers are selected from a non-viral source. In certain embodiments, no WPRE sequence is present.

An AAV viral vector may include multiple transgenes. In certain embodiments, the transgene may be used to correct or ameliorate gene deficiencies, which may include deficiencies in which normal genes are expressed at less than normal levels or deficiencies in which the functional gene product is not expressed. Alternatively, the transgene may provide a product to a cell which is not natively expressed in the cell type or in tire host. A preferred type of transgene sequence encodes a therapeutic protein or polypeptide which is expressed in a host cell. The invention further includes using multiple transgenes. In certain situations, a different transgene may be used to encode each subunit of a protein, or to encode different peptides or proteins. This is desirable when the size of the DNA encoding tire protein subunit is large, e.g., for an immunoglobulin, the platelet-derived growth factor, or a dystrophin protein. In certain situations, a different transgene may be used to encode each subunit of a protein (e.g., an immunoglobulin domain, an immunoglobulin heavy chain, an immunoglobulin light chain). In one embodiment, a cell produces the multisubunit protein following infected/transfection with the virus containing each of the different subunits. In another embodiment, different subunits of a protein may be encoded by the same transgene. An IRES is desirable when the size of the DNA encoding each of tire submits is small, e.g., tire total size of the DNA encoding the subunits and tire IRES is less than five kilobases. As an alternative to an IRES, the DNA may be separated bysequences encoding a 2A peptide, which self-cleaves in a post-translational event. See, e.g., ML Donnelly, et al, (Jan 1997) J. Gen. Virol., 78(Pt 1): 13-21; S. Furler, S et al, (June 2001) Gene Ther., 8(11):864-873; H. Klump, et al., (May 2001) Gene Ther.. 8(10):811-817. This 2A peptide is significantly smaller than IRES, making it well suited for use when space is a limiting factor. More often, w hen the transgene is large, consists of multi-subunits, or tw o transgenes are co-delivered, rAAV carrying the desired transgene(s) or subunits are coadministered to allow them to concatamerize in vivo to form a single vector genome. In such an embodiment, a first AAV may carry- an expression cassette which expresses a single transgene and a second AAV may carry an expression cassette which expresses a different transgene for co-expression in the host cell. However, the selected transgene may encode any biologically active product or other product, e.g., a product desirable for study.

In addition to the elements identified above for the expression cassette, the vector also includes conventional control elements w hich are operably linked to the coding sequence in a maimer which permits transcription, translation and/or expression of tire encoded product in a cell transfected with the plasmid vector or infected with the virus produced by the invention. Examples of other suitable transgenes are provided herein. As used herein, “operably linked” sequences include both expression control sequences that are contiguous w ith the gene of interest and expression control sequences that act in trans or at a distance to control tire gene of interest.

Expression control sequences include appropriate enhancer; transcription factor; transcription terminator; promoter; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA. for example Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE); sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability: and when desired, sequences that enhance secretion of the encoded product.

In one embodiment, the regulatory sequences are selected such that tire total rAAV vector genome is about 2.0 to about 5.5 kilobases in size. In one embodiment, it is desirable that the rAAV vector genome approximate the size of the native AAV genome. Thus, in one embodiment, the regulatory sequences are selected such that the total rAAV vector genome is about 4.7 kb in size. In another embodiment, the total rAAV vector genome is less about 5.2kb in size. The size of the vector genome may be manipulated based on the size of the regulator ’ sequences including tire promoter, enhancer, intron, poly A, etc. See, Wu et al, Mol Ther, Jan 2010 18( 1): 80-6, which is incorporated herein by reference.

Thus, in one embodiment, an intron is included in the vector. Suitable introns include chicken beta-actin intron, the human beta globin IV S2 (Kelly et al, Nucleic Acids Research, 43(9):4721-32 (2015)); the Promega chimeric intron (Almond, B. and Schenbom, E. T. A Comparison of pCI-neo Vector and pcDNA4/HisMax Vector): and the hFIX intron. Various introns suitable herein are known in the art and include, without limitation, those found at bpg.utoledo.edu/~afedorov/lab/eid.html, which is incorporated herein by reference. Sec also, Shepelev V., Fedorov A. Advances in tire Exon-Intron Database. Briefings in Bioinfonnatics 2006, 7: 178-185, which is incorporated herein by reference.

In certain embodiments, the mutant rAAV comprises an expression cassette which further comprising at least one miRNA target sequences operably linked to a selected transgene, optionally in its 3' UTR and/or its 5' UTR. In certain embodiments, the mutant rAAV comprises a vector genome that further comprises at least one miRNA seed, binding site or full sequence. MicroRNAs (or miRNA or miR) are 19-25 nucleotide noncoding RNAs that bind to the sites of nucleic acid targets and down-regulate gene expression either by reducing nucleic acid molecule stability or by inhibiting translation. In some embodiments, a microRNA sequence comprises a seed region, e.g., a sequence in the region of positions 2-8 of tire mature microRNA, which has Watson-Crick sequence fully or partially complementarity to the miRNA target sequence of the nucleic acid. Such miRNA may be used in combinations, including in a vector genome also comprising a coding sequence for therapeutic protein, enzyme, or other moiety.

In some embodiments, the miR binding site is complementary to a miR expressed in a DRG (dorsal root ganglion) neuron, e.g., a miR183, and/or a miR182, binding site. In some embodiments, the miR binding site complementary to a miR expressed in expressed in a DRG neuron comprises a nucleotide sequence disclosed, e.g., in WO2020/132455, and in WO 2023/087019, the contents of which are incorporated by reference herein in its entirety.

As another non-limiting example, a miR- 122 miRNA may be encoded in the viral genome to modulate, e.g., reduce, the expression of the viral genome in the liver. In some embodiments, a miRNA, e.g., a miR- 142-3p, may be encoded in the viral genome to modulate, e.g., reduce, the expression, of the viral genome in a cell or tissue of the hematopoietic lineage, including for example immune cells (e.g., antigen presenting cells or APC, including dendritic cells (DCs), macrophages, and B-lymphocytes). Other suitable miR may include, e.g., miR-206 (skeletal muscle), miR-018b, miR-431.See. e g., Kim., H.K.. Muscle- specifi c microRNA miR-206 promotes muscle differentiation, The Journal of Cell Biology, 2006, 174(5):677-687: WO 2019/035690A1; WO 2019/035690A1; WO 2022/147181 A 1 , and Brazilian Patent Publication No. BR102018067702A2, which are all incorporate herein by reference.

Several different viral genomes were generated in the studies described herein. However, it will be understood by the skilled artisan that other genomic configurations, including other regulatory sequences may be substituted for the promoter, enhancer and other coding sequences may be selected. rAAV Vector Production For use in producing an AAV viral vector (e.g., a recombinant (r) AAV), the expression cassettes can be carried on any suitable vector, e.g., a plasmid, which is delivered to a production (packaging) host cell. The plasmids useful in this invention may be engineered such that they are suitable for replication and packaging in vitro in prokaryotic cells, insect cells, mammalian cells, among others. Suitable transfection techniques and packaging host cells are known and/or can be readily designed by one of skill in the art. In certain embodiments, the production host cell is a human cell or insect cell. In certain embodiments, the production host cell in is HEK293 cell, HuH-7 cell, BHK cell, or Vero cell. In certain embodiments, production host cell is in a suspension cell culture.

In certain embodiments, provided herein is a production host cell comprising a recombinant nucleic acid molecule as described herein, a nucleic acid sequence encoding an AAV capsid protein, and sufficient AAV rep functions and helper functions to pennit packaging of the vector genome into tire AAV capsid.

In certain embodiments, tire insertion of the exogenous targeting peptide, wherein the exogenous targeting peptide comprises ‘‘Xn - n-mer - Xm”. wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) n-mer selected from GQVRVGV (SEQ ID NO: 3), MDAHYVR (SEQ ID NO: 4), TQAVPLK (SEQ ID NO: 5), VVHQTGL (SEQ ID NO: 6), MAISRER (SEQ ID NO: 7), or a n-mer sequence of at least 6, at least 7 or full length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, wherein tire inclusion of exogenous targeting peptide into an AAV capsid provides advantages in production as compared to the method without inclusion of at least one copy of motif in AAV capsid, and wherein the production cells are 293 cells.

In certain embodiments, a host cell is stably or transiently transfected with a genetic element (e.g., a plasmid or other nucleic acid molecule) which expresses a mutant AAV capsid as provided herein. In certain embodiments, such a genetic element comprises a nucleic acid sequence encoding a mutant AAV VP 1 coding sequence comprising the mutant peptide(s) inserted therein, operably linked to expression control sequences which enable expression of the AAV capsid proteins in the packaging host cell. In certain embodiments, the coding sequence for the peptide insert is a sequence encoding SEQ ID NO: 80 (AAV-GQVRVGV), which is optionally a nucleic acid sequence of SEQ ID NO: 79 or a sequence 95% to 100% identical, or at least 99% identical, thereto. In certain embodiments, a host cell is stably or transiently transfected with a genetic element (e.g., a plasmid or other nucleic acid molecule) which expresses a mutant AAV capsid as provided herein. In certain embodiments, such a genetic element comprises a nucleic acid sequence encoding a mutant AAV VP 1 coding sequence comprising the mutant peptide(s) (i.e., the exogenous targeting peptide(s)) inserted therein, operably linked to expression control sequences which enable expression of the AAV capsid proteins in the packaging host cell. In certain embodiments, the mutant peptides are engineered between amino acids 588 and 589 in the AAVhu68 capsid. In other embodiments, these peptides are inserted between amino acids 588 and 589 in the AAV9 capsid. Still other suitable locations for these inserts may be determined. In still other embodiments, these peptides may be used in other vectors or compositions for targeting. In certain embodiments, the coding sequence of a mutant AAV9 capsid having the exogenous targeting peptide inserted in the hypervariable region betw een amino acids 588 and 589 in the AAV9 parental capsid is SEQ ID NO: 79 (GQVRVGV). In other embodiments, a mutant AAV9 is encoded by any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 80 (GQVRVGV mutant VP 1 ).

Methods of preparing AAV-based vectors (e.g., having an AAV9 or another AAV capsid) are known. See, e.g., US Published Patent Application No. 2007/0036760 (February' 15, 2007), which is incorporated by reference herein. The invention is not limited to tire use of AAV9 or other clade F AAV amino acid sequences, but encompasses peptides and/or proteins containing the terminal 0-gaIactose binding generated by other methods known in tire art, including, e.g., by chemical synthesis, by other synthetic techniques, or by other methods. The sequences of any of the AAV capsids provided herein can be readily generated using a variety of techniques. Suitable production techniques are well known to those of skill in the art. See, e g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY). Alternatively, peptides can also be synthesized by the well-known solid phase peptide synthesis methods (Merrifield, (1962) J. Am. Chcm. Soc., 85:2149; Stewart and Young, Solid Phase Peptide Synthesis (Freeman, San Francisco, 1969) pp. 27-62). These methods may involve, e.g., culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid; a functional rep gene; a minigene composed of. at a minimum, AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the minigene into the AAV capsid protein. These and other suitable production methods are within the knowledge of those of skill in the art and are not a limitation of the present invention.

The components required to be cultured in the host cell to package an AAV minigene in an AAV capsid may be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., minigene, rep sequences, cap sequences, and/or helper functions) may be provided by a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art. Most suitably, such a stable host cell will contain the required component(s) under the control of an inducible promoter. However, the required component(s) may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein, in the discussion of regulatory elements suitable for use with the transgene. In still another alternative, a selected stable host cell may contain selected component(s) under tire control of a constitutive promoter and other selected component(s) under tire control of one or more inducible promoters. For example, a stable host cell may be generated which is derived from 293 cells (which contain E 1 helper functions under the control of a constitutive promoter), but which contains the rep and/or cap proteins under the control of inducible promoters. Still other stable host cells may be generated by one of skill in the art.

These rAAVs are particularly well suited to gene delivery for therapeutic purposes and for preventing infection. Further, the compositions of the invention may also be used for production of a desired gene product in vitro. For in vitro production, a desired product (e.g., a protein) may be obtained from a desired culture following transfection of host cells with a rAAV containing the molecule encoding the desired product and culturing the cell culture under conditions which permit expression. The expressed product may then be purified and isolated, as desired. Suitable techniques for transfection, cell culturing, purification, and isolation are known to those of skill in the art. Methods for generating and isolating AAVs suitable for use as vectors are known in the art. See generally, e.g., Grieger & Samulski, 2005, "Adcno-associatcd virus as a gene therapy vector: Vector development, production and clinical applications,’' Adv. Biochem. Engin/Biotechnol. 99: 119-145; Buning et al.. 2008. “Recent developments in adeno-associated virus vector technology,” J. Gene Med. 10:717-733; and the references cited below, each of which is incorporated herein by reference in its entirety. For packaging a transgene into virions, the ITRs are the only AAV components required in cis in the same construct as tire nucleic acid molecule containing the expression cassettes. The cap and rep genes can be supplied in trans.

In one embodiment, the expression cassettes described herein are engineered into a genetic element (e.g., a shuttle plasmid) which transfers the immunoglobulin construct sequences carried thereon into a packaging host cell for production a viral vector. In one embodiment, the selected genetic element may be delivered to an AAV packaging cell by any suitable method, including transfection, electroporation, liposome delivery , membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion. Stable AAV packaging cells can also be made. Alternatively, the expression cassettes may be used to generate a viral vector other than AAV, or for production of mixtures of antibodies in vitro. The methods used to make such constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Molecular Cloning: A Laboratory Manual, ed. Green and Sambrook, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

The term “AAV intermediate'’ or “AAV vector intermediate” refers to an assembled rAAV capsid which lacks the desired genomic sequences packaged therein. These may also be termed an “empty” capsid. Such a capsid may contain no detectable genomic sequences of an expression cassette, or only partially packaged genomic sequences which are insufficient to achieve expression of the gene product. These empty capsids are non-functional to transfer the gene of interest to a host cell.

The recombinant AAV described herein may be generated using techniques which are known. See, e.g., WO 2003/042397; WO 2005/033321, WO 2006/110689; US 7588772 B2. Such a method involves culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid; a functional rep gene; an expression cassette composed of, at a minimum, AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the expression cassette into the AAV capsid protein. Methods of generating the capsid, coding sequences therefore, and methods for production of rAAV viral vectors have been described. Sec, e.g., Gao, ct al, Proc. Natl. Acad. Sci. U.S.A. 100 (10), 6081-6086 (2003) and US 2013/0045186A1.

In certain embodiment, the rAAV are generated (manufactured) using triple transfection techniques. In certain embodiments the rAAV are generated using a stable mammalian cell line. In certain embodiments, the stable cell line comprises one or more of: (a) a first plurality of polynucleotide molecules which comprise a coding sequence for at least one adeno-associated virus (AAV) replicase (Rep) protein necessary for production of a replication-defective rAAV vector (Rep52 and Rep78), wherein said rep proteins coding sequences are operably linked to a doxycycline-inducible promoter which directs expression of the rep proteins in the cell line; (b) at least a second plurality of polynucleotide molecules each encoding adenovirus (Ad) helper proteins necessary for production of a replication-defective rAAV vector comprising at least an Ad E2A DNA Binding Protein (DBP) coding sequence, and Ad E4ORF6 coding sequence, wherein the Ad E2A DBP coding sequences and the Ad E4ORF6 coding sequences are operably linked to a doxycycline-inducible promoter which direct expression of the Ad helper proteins in the cell line; (c) a nucleic acid molecule comprising an Ad El coding sequence operably linked to a constitutive promoter which directs expression of the Ad El in the cell line; (d) at least a third plurality' of nucleic acid molecules each of which comprises an AAV VP 1 coding sequence which encodes AAV VP1 proteins, AAV VP2 proteins and AAV VP3 proteins which self-assemble to form an AAV capsid following expression in tire cell, said AAV VP 1 coding sequence being operably linked to a promoter which directs expression of the VP1 coding sequences in the cell line. See also. US Provisional Patent Application No. 63/490,222, filed March 14, 2023, which is incorporated herein by reference.

In one embodiment, cells are manufactured in a suitable cell culture (e.g., HEK 293 cells). Methods for manufacturing the gene therapy vectors described herein include methods well known in tire art such as generation of plasmid DNA used for production of tire gene therapy vectors, generation of the vectors, and purification of the vectors. In some embodiments, the gene therapy vector is an AAV vector and the plasmids generated are an AAV cis-plasmid encoding the AAV genome and the gene of interest for packaging into the capsid, an AAV trans-plasmid containing AAV rep and cap genes, and an adenovirus helper plasmid. The vector generation process can include method steps such as initiation of cell culture, passage of cells, seeding of cells, transfection of cells with the plasmid DNA, post-transfection medium exchange to serum free medium, and the harvest of vectorcontaining cells and culture media. The harvested vector-containing cells and culture media are referred to herein as crude cell harvest. In yet another system, the gene therapy vectors are introduced into insect cells by infection with baculovirus-based vectors. For reviews on these production systems, see generally, e.g., Zhang et al.. 2009, "Adenovirus-adeno- associated virus hybrid for large-scale recombinant adeno-associated virus production," Human Gene Therapy 20:922-929, which is incorporated herein by reference in its entirety. Methods of making and using these and other AAV production systems are also described in the following U.S. patents, the contents of each of which is incorporated herein by reference in its entirety: 5,139,941 ; 5,741 ,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898; 7,229,823; and 7,439,065.

The crude cell harvest may thereafter be subject method steps such as concentration of the vector harvest, diafiltration of the vector harvest, microfluidization of the vector harvest, nuclease digestion of the vector harvest, filtration of microfluidized intermediate, crude purification by chromatography, crude purification by ultracentrifugation, buffer exchange by tangential flow filtration, and/or formulation and filtration to prepare bulk vector.

A two-step affinity chromatography purification at high salt concentration followed anion exchange resin chromatography are used to purify the vector drug product and to remove empty capsids. These methods are described in more detail in International Patent Application No. PCT/US2016/065970, filed December 9, 2016, and US 11,098,286 B2, entitled “Scalable Purification Method for AAV9”, which are incorporated by reference. Purification methods for AAV8, International Patent Application No.

PCT/US2016/065976, filed December 9, 2016, and US 11,015,174 B2, entitled “Scalable Purification Method for AAV8”, which are incorporated herein by reference. Purification methods for rhlO, International Patent Application No. PCT/US16/066013, filed December 9, 2016, and US 11,028,372 B2, entitled “Scalable Purification Method for AAVrhlO”, which are incorporated herein by reference. Purification methods for AAV1, International Patent Application No. PCT/US2016/065974. filed December 9, 2016, and US 11,015,173 B2. entitled “Scalable Purification Method for AAV1”, which are incorporated herein by reference.

To calculate empty and full particle content, vp3 band volumes for a selected sample (e.g., in examples herein an iodixanol gradient-purified preparation where number of GC = number of particles) are plotted against GC particles loaded. The resulting linear equation (y = mx+c) is used to calculate the number of particles in the band volumes of the test article peaks. The number of particles (pt) per 20 LLL loaded is then multiplied by 50 to give particles (pt) /mL. Pt/mL divided by GC/mL gives the ratio of particles to genome copies (pt/GC). Pt/mL-GC/mL gives empty pt/mL. Empty pt/mL divided by pt/mL and x 100 gives the percentage of empty particles. Generally, methods for assaying for empty capsids and AAV vector particles with packaged genomes have been known in the art. See, e.g., Grimm et al., Gene Therapy (1999) 6: 1322-1330; and Sommer et al., Molec. Ther. (2003) 7: 122-128. To test for denatured capsid, the methods include subjecting the treated AAV stock to SDS- polyacrylamide gel electrophoresis, consisting of any gel capable of separating the three capsid proteins, for example, a gradient gel containing 3-8% Tris-acetate in the buffer, then running the gel until sample material is separated, and blotting tire gel onto nylon or nitrocellulose membranes, preferably nylon. Anti-AAV capsid antibodies are then used as the primary antibodies that bind to denatured capsid proteins, preferably an anti- AAV capsid monoclonal antibody, most preferably the Bl anti-AAV-2 monoclonal antibody (Wobus et al., J. Virol. (2000) 74:9281-9293). A secondary antibody is then used, one that binds to the primary antibody and contains a means for detecting binding with tire primary antibody, more preferably an anti-IgG antibody containing a detection molecule covalently bound to it, most preferably a sheep anti-mouse IgG antibody covalently linked to horseradish peroxidase. A method for detecting binding is used to semi-quantitatively determine binding between the primary and secondary’ antibodies, preferably a detection method capable of detecting radioactive isotope emissions, electromagnetic radiation, or colorimetric changes, most preferably a chemiluminescence detection kit. For example, for SDS-PAGE, samples from column fractions can be taken and heated in SDS-PAGE loading buffer containing reducing agent (e.g., DTT), and capsid proteins were resolved on pre-cast gradient polyacrylamide gels (e.g., Novex). Silver staining may be performed using SilverXpress (Invitrogen, CA) according to the manufacturer's instructions or other suitable staining method, i.e., SYPRO ruby or coomassie stains. In one embodiment, tire concentration of AAV vector genomes (vg) in column fractions can be measured by quantitative real time PCR (Q-PCR). Samples are diluted and digested with DNase I (or another suitable nuclease) to remove exogenous DNA. After inactivation of the nuclease, tire samples are further diluted and amplified using primers and a TaqMan™ fluorogenic probe specific for tire DNA sequence betw een tire primers. The number of cycles required to reach a defined level of fluorescence (threshold cycle, Ct) is measured for each sample on an Applied Biosystems Prism 7700 Sequence Detection System. Plasmid DNA containing identical sequences to that contained in tire AAV vector is employed to generate a standard curve in the Q-PCR reaction. The cycle threshold (Ct) values obtained from the samples are used to determine vector genome titer by normalizing it to tire Ct value of the plasmid standard curve. End-point assays based on the digital PCR can also be used.

Additionally, another example of measuring empty to full particle ratio is also known in the art. Sedimentation velocity, as measured in an analytical ultracentrifuge (AUC) can detect aggregates, other minor components as well as providing good quantitation of relative amounts of different particle species based upon their different sedimentation coefficients. This is an absolute method based on fundamental units of length and time, requiring no standard molecules as references. Vector samples are loaded into cells with 2-channel charcoal-epon centerpieces with 12mm optical path length. The supplied dilution buffer is loaded into the reference channel of each cell. The loaded cells are then placed into an AN-60Ti analytical rotor and loaded into a Beckman-Coulter ProteomeLab XL-I analytical ultracentrifuge equipped with both absorbance and RI detectors. After full temperature equilibration at 20 °C the rotor is brought to the final run speed of 12,000 rpm. A280 scans are recorded approximately every 3 minutes for ~5.5 hours (110 total scans for each sample). The raw data is analyzed using the c(s) method and implemented in the analysis program SEDFIT. The resultant size distributions are graphed and the peaks integrated. The percentage values associated with each peak represent the peak area fraction of the total area under all peaks and are based upon the raw data generated at 280nm; many labs use these values to calculate empty: full particle ratios. How ever, because empty and full particles have different extinction coefficients at this wavelength, the raw data can be adjusted accordingly. The ratio of the empty particle and full monomer peak values both before and after extinction coefficient- adj ustment is used to determine tire empty-full particle ratio.

In one aspect, an optimized q-PCR method is used which utilizes a broad-spectrum serine protease, e.g., proteinase K (such as is commercially available from Qiagen). More particularly, the optimized qPCR genome titer assay is similar to a standard assay, except that after the DNase I digestion, samples are diluted with proteinase K buffer and treated with proteinase K followed by heat inactivation. Suitably samples arc diluted with proteinase K buffer in an amount equal to the sample size. The proteinase K buffer may be concentrated to 2- fold or higher. Typically, proteinase K treatment is about 0.2 mg/mL, but may be varied from 0. 1 mg/mL to about 1 mg/mL. The treatment step is generally conducted at about 55 °C for about 15 minutes, but may be performed at a lower temperature (e.g., about 37 °C to about 50 °C) over a longer time period (e.g., about 20 minutes to about 30 minutes), or a higher temperature (e.g., up to about 60 °C) for a shorter time period (e.g., about 5 to 10 minutes). Similarly, heat inactivation is generally at about 95 °C for about 15 minutes, but the temperature may be lowered (e g., about 70 to about 90 °C) and the time extended (e.g., about 20 minutes to about 30 minutes). Samples are then diluted (e.g., 1000-fold) and subjected to TaqMan analysis as described in tire standard assay. Quantification also can be done using ViroCyt or flow cytometry.

Additionally, or alternatively, droplet digital PCR (ddPCR) may be used. For example, methods for determining single-stranded and self-complementary AAV vector genome titers by ddPCR have been described. See, e.g.. M. Lock et al, Hu Gene Therapy Methods, Hum Gene Ther Methods. 2014 Apr;25(2): 115-25. doi: 10.1089/hgtb.2013. 131. Epub 2014 Feb 14.

In certain embodiments, the manufacturing process for rAAV as described herein (e.g., comprising engineered rAAV) involves a method as described in US Provisional Patent Application No. 63/371,597, filed August 16, 2022, and US Provisional Patent Application No. 63/371,592, filed August 16, 2022, which are incorporated herein by reference in their entireties.

Therapeutic Proteins and Delivery Systems

In certain embodiments, provided here are fusion partners, conjugate partners and recombinant vectors containing a muscle targeting peptide comprising “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is GQVRVGV (SEQ ID NO: 3). MDAHYVR (SEQ ID NO: 4), TQAVPLK (SEQ ID NO: 5), VVHQTGL (SEQ ID NO: 6), or MAISRER (SEQ ID NO: 7), or an n-mer sequence of at least 6, at least 7, or the full-length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, which are useful w ith a variety of different therapeutic proteins, polypeptides, nanoparticles, and delivery systems. Examples of proteins and compounds useful in compositions provided herein and targeted delivery are described below. It will be understood that the viral vectors, nanoparticles and other delivery systems contain sequences encoding the selected proteins (or conjugates) for expression in vivo.

In some embodiments, provided herein is an rAAV having a modified capsid with one or more exogenous targeting peptides, wherein the exogenous targeting peptides comprises ‘ Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is GQVRVGV (SEQ ID NO: 3), MDAHYVR (SEQ ID NO: 4), TQAVPLK (SEQ ID NO: 5), VVHQTGL (SEQ ID NO: 6), or MAISRER (SEQ ID NO: 7), or an n-mer sequence of at least 6, at least 7, or the full- length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and the rAAV comprises a vector genome comprising the desired transgene and promoter for use in the target cells as detailed above is optionally assessed for contamination by conventional methods and then formulated into a pharmaceutical composition intended for administration to a subject in need thereof. In some embodiments, provided herein is a rAAV having a modified capsid with one or more exogenous targeting peptides, wherein the exogenous targeting peptides comprise “Xn - GQVRVGV (SEQ ID NO: 3)- Xm”, wherein Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, and Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and the rAAV comprises a vector genome comprising the desired transgene and promoter for use in the target cells as detailed above is optionally assessed for contamination by conventional methods and then formulated into a pharmaceutical composition intended for administration to a subject in need thereof. In some embodiments, provided herein is a rAAV having a modified capsid with at one or more exogenous targeting peptides, wherein the exogenous targeting peptide comprises GQVRVGV (SEQ ID NO: 3), and the rAAV comprises a vector genome comprising tire desired transgene and promoter for use in the target cells as detailed above is optionally assessed for contamination by conventional methods and then formulated into a pharmaceutical composition intended for administration to a subject in need thereof. In some embodiments, provided herein is a rAAV having a modified capsid with at least one core one or more exogenous targeting peptides, wherein the exogenous targeting peptide comprises AQGQVRVGVAQ (SEQ ID NO: 39), and the rAAV comprises a vector genome comprising tire desired transgene and promoter for use in the target cells as detailed above is optionally assessed for contamination by conventional methods and then formulated into a pharmaceutical composition intended for administration to a subject in need thereof. Such formulations involve the use of a pharmaceutically and/or physiologically acceptable vehicle or carrier, such as buffered saline or other buffers, e.g., HEPES, to maintain pH at appropriate physiological levels, and, optionally, other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc. For injection, the carrier will typically be a liquid. In certain embodiments, proteins, polypeptides, nanoparticles, and/or delivery systems including viral vectors (rAAV) and nanoparticles, comprising the exogenous targeting peptide provided herein, are useful in treatment of one or more of cardiac and/or skeletal (e.g., gastrocnemius) muscle-based disorders. Such disease and/or disorders may include, without limitation, auto immune disease, cancer, muscular dystrophy, a neuromuscular disease, a sugar or glycogen storage disease, cardiomyopathy, infectious disease affecting the muscle cell. More specifically, such disease and/or disorders may include, without limitation, Huntington’s disease, a Myotonic Dystrophy (Type 1 or Type 2), Facioscapulohumeral muscular dystrophy (FSHD), Duchene muscular dystrophy. Becker Muscular dystrophy, Limb-Girdle muscular dystrophy, Emery Dreifuss muscular dystrophy, Oculopharyngeal muscular dystrophy, Barth syndrome, MPS type III disease, Pompe disease, Charcot-Marie-Tooth disease, Friedreich’s Ataxia, dilated cardiomyopathy, hypertrophic cardiomyopathy, DMD-associated cardiomyopathy, Myotubular myopathy, Primary merosin deficiency, Dannon disease, idiopathic dilated cardiomyopathy (DCM) or a disease associated with a mutation in the LMNA gene. Examples of genes and proteins those associated with disease and/or disorders, e.g., spinal muscular atrophy (SMA, SMN1), Duchenne Type Muscular dystrophy, Friedrichs Ataxia (e.g., frataxin), cardiomyopathy (LMNA), Charcot-Marie-Tooth disease (MFN2). See also, US Provisional Patent Application No. 63/293,680, filed December 24, 2021, International Patent Application No. PCT/US2021/041406, filed July 13, 2021, now Publication No. WO 2022/015715A1, International Patent Application No. PCT/US2022/076939, filed September 23, 2022, International Patent Application No. PCT/US2020/066167, filed December 18, 2020, now Publication No. WO 2021/127533A1. International Patent Application No. PCT/US2022/025879, filed April 22. 2022. now Publication No. WO 2022/226263A1, which are all incorporated herein by reference in their entireties.

In certain embodiments, the protein useful in compositions provided herein is encoded by a transgene sequence including hormones and grow th and differentiation factors including, without limitation, insulin, glucagon, glucagon-like peptide 1 (GLP-1), growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH). luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietins. angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor a (TGFa), platelet-derived growth factor (PDGF), insulin growth factors I and II (IGF-I and IGF-II), any one of the transforming growth factor f> superfamily, including TGF p, activins, inhibins, or any of the bone morphogenic proteins (BMP) BMPs 1-15, any one of the heregluin/neuregulin/ARIA/neu differentiation factor (NDF) family of growth factors, nen e growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT-4/5, ciliary neurotrophic factor (CNTF), glial cell line derived neurotrophic factor (GDNF), lysosomal acid lipase (LIPA or LAL), neurturin. agrin, anyone of the family of semaphorins/collapsins, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrins, noggin, sonic hedgehog and tyrosine hydroxylase. Other useful transgene encode lysosomal enzymes that cause mucopolysaccharidoses (MPS), including a-L-iduronidase (MPSI), iduronate sulfatase (MPSII), heparan N-sulfatase (sulfaminidase) (MPS IIIA, Sanfilippo A), a-N-acetyl-glucosaminidase (MPS IIIB, Sanfdippo B), acetyl- CoA:a-glucosaminide acetyltransferase (MPS IIIC, Sanfdippo C). N-acetylglucosamine 6- sulfatase (MPS IIID, Sanfdippo D). galactose 6-sulfatase (MPS IVA, Morquio A), P- Galactosidase (MPS IVB, Morquio B), N-acetyl-galactosamine 4-sulfatase (MPS VI, Maroteaux-Lamy), P-Glucuronidase (MPS VIL Sly), and hyaluronidase (MPS IX).

In certain embodiments, the protein useful in compositions provided herein is encoded by a transgene sequence including a reporter sequence, which upon expression produces a detectable signal. Such reporter sequences include, without limitation, DNA sequences encoding -lactamase, -galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), enhanced GFP (EGFP), chloramphenicol acetyltransferase (CAT), luciferase, membrane bound proteins including, for example. CD2. CD4. CD8. the influenza hemagglutinin protein, and others well known in the art. to which high affinity antibodies directed thereto exist or can be produced by conventional means, and fusion proteins comprising a membrane bound protein appropriately- fused to an antigen tag domain from, among others, hemagglutinin or Myc.

An rAAV having a mutant rAAV capsid as provided herein has a vector genome which comprises nucleic acid sequence encoding a protein, for example that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for example but are not limited to RNAi, shRNAi. siRNA, micro RNAi (mRNAi or miRNA), antisense oligonucleotides etc. These may be in additional to or in alternative to a protein to be delivered. Compositions and Uses

Provided herein are compositions containing at least one rAAV stock (e.g., an rAAV9 engineered stock or rAAVhu68 engineered stock, wherein the engineered capsid comprises an exogenous targeting motif as described herein) and an optional carrier, excipient and/or preservative.

In one aspect, provided is a pharmaceutical composition comprising na rAAV as described herein in a formulation buffer. In one embodiment, the rAAV is formulated at about 1 x 10⁹ genome copies (GC)/mL to about 1 x 10¹⁴ GC/mL. In a further embodiment, the rAAV is formulated at about 3 x 10⁹ GC/mL to about 3 x 10¹³ GC/mL. In yet a further embodiment, the rAAV is formulated at about 1 x 10⁹ GC/mL to about 1 x 10¹³ GC/mL. In one embodiment, tire rAAV is formulated at least about 1 x 10¹¹ GC/mL.

Provided herein, also, are compositions containing at least one therapeutic protein, polypeptide, nanoparticles and/or delivery system comprising the targeting motif as provided herein, and an optional carrier, excipient and/or preservative.

Provided herein are methods for use of compositions as described herein. In certain embodiments, a method for targeted therapy to muscle cells is provided that includes administering to a patient in need thereof a stock of an rAAV as described herein, wherein a therapeutic is targeted for delivery to muscle cells (e.g., cardiac cells (heart), skeletal muscle cells (e.g., gastrocnemius)), and is de-targeted for cells in liver.

Additionally, provided herein is a method of delivering of a transgene to one or more muscle cells of a subject comprising administering to the subject a recombinant adeno-associated vims (rAAV) vector comprising engineered capsid proteins comprising one or more exogenous targeting peptides, wherein the exogenous targeting peptides comprise “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is GQVRVGV (SEQ ID NO: 3), MDAHYVR (SEQ ID NO: 4), TQAVPLK (SEQ ID NO: 5), VVHQTGL (SEQ ID NO: 6), or MAISRER (SEQ ID NO: 7), or an n-mer sequence of at least 6, at least 7, or the full- length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, and the rAAV further comprises a vector genome comprising the transgene operably linked to regulatory sequences that direct expression of the transgene in muscle cells. In certain embodiments, the target muscle cell is a cardiac, smooth, and/or skeletal muscle cell. In certain embodiments, the transgene encodes a secreted gene product. In certain embodiments, the AAV vector is delivered intravenously.

Provided herein are also uses of an rAAV having a engineered capsid with one or more exogenous targeting peptides, to target muscle cells at higher levels of transduction than achieved using an AAV9 vector, wherein the exogenous targeting peptides comprise “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1. 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is GQVRVGV (SEQ ID NO: 3), MDAHYVR (SEQ ID NO: 4). TQAVPLK (SEQ ID NO: 5), VVHQTGL (SEQ ID NO: 6), MAISRER (SEQ ID NO: 7), or a n-mer sequence of at least 6, at least 7, or the full-length consecutive amino acids of any one of tire n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid.

In certain embodiments, a composition may contain at least a second, different rAAV stock. This second vector stock may vary from the first by having a different AAV capsid and/or a different vector genome. In certain embodiments, a composition as described herein may contain a different vector expressing an expression cassette as described herein, or another active component (e.g., an antibody construct, another biologic, and/or a small molecule drug).

As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host. Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present invention into suitable host cells. In particular, the rAAV vector delivered transgcncs maybe formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.

In one embodiment, a composition includes a final formulation suitable for delivery to a subject, e.g., is an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration. Suitably, the final formulation is adjusted to a physiologically acceptable pH, e.g.. the pH may be in the range of pH 6 to 9, or pH 6.5 to 7.5, pH 7.0 to 7.7, or pH 7.2 to 7.8. For intravenous delivery, a pH of 6.8 to about 7.2 may be desired. However, other pHs within the broadest ranges and these subranges may be selected for other routes of delivery. Optionally, one or more surfactants are present in tire formulation. In another embodiment, the composition may be transported as a concentrate which is diluted for administration to a subject. In other embodiments, the composition may be lyophilized and reconstituted at the time of administration.

A suitable surfactant, or combination of surfactants, may be selected from among non-ionic surfactants that are nontoxic. In one embodiment, a difunctional block copolymer surfactant terminating in primary hydrox l groups is selected, e.g., such as Pluronic® F68 [BASF], also known as Poloxamer 188, which has a neutral pH, has an average molecular weight of 8400. Other surfactants and other Poloxamers may be selected, i.e., nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly (propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly (ethylene oxide)), SOLUTOL HS 15 (Macrogol (polyethylene glycol) -15 Hydroxy stearate), LABRAS OL® (Polyoxy capryllic glyceride), poly oxy 10 oleyl ether. TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are commonly named with tire letter "P" (for poloxamer) followed by three digits: the first two digits x 100 give the approximate molecular mass of the poly oxypropylene core, and the last digit x 10 gives the percentage polyoxyethylene content. In one embodiment Poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005 % to about 0.001% of the suspension.

In another embodiment, the composition includes a carrier, diluent, excipient and/or adjuvant. Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the transfer virus is directed. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil. sesame oil, and water. The buffer/carrier should include a component that prevents the rAAV, from sticking to the infusion tubing but does not interfere with tire rAAV binding activity in vivo. A suitable surfactant, or combination of surfactants, may be selected from among non-ionic surfactants that are nontoxic. In one embodiment, a difunctional block copolymer surfactant terminating in primary hydroxyl groups is selected, e.g., such as Poloxamer 188 (also known under the commercial names Pluronic® F68 [BASF], Lutrol® F68, Synperonic® F68, Kolliphor® Pl 88) which has a neutral pH, has an average molecular weight of 8400. Other surfactants and other Poloxamers may be selected, i.e., nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly (propylene oxide)) flanked by tw o hydrophilic chains of polyoxyethylene

(poly (ethylene oxide)), SOLUTOL HS 15 (Macrogol-15 Hydroxystearate), LABRASOL (Polyoxy capryllic glyceride), polyoxy -oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are commonly named with the letter "P" (for poloxamer) followed by three digits: the first two digits x 100 give the approximate molecular mass of the poly oxypropylene core, and the last digit x 10 gives the percentage polyoxyethylene content. In one embodiment Poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005 % to about 0.001% of the suspension.

In certain embodiments, tire formulation may contain a buffered saline aqueous solution not comprising sodium bicarbonate. Such a formulation may contain a buffered saline aqueous solution comprising one or more of sodium phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride and mixtures thereof, in water, such as a Harvard’s buffer. In one embodiment, the buffer is phosphate-buffered saline (PBS). In one embodiment, the formulation buffer PBS with total salt concentration of 200 mM, 0.001% (w/v) pluronic F68 (Final Formulation Buffer, FFB).

Optionally, the compositions of the invention may contain, in addition to tire rAAV and carrier(s), other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

The compositions according to the present invention may comprise a pharmaceutically acceptable carrier, such as defined above. Suitably, the compositions described herein comprise an effective amount of one or more AAV suspended in a pharmaceutically suitable carrier and/or admixed with suitable excipients designed for delivery to the subject via injection, or for delivery by another route and/or device.

In certain embodiments, the composition comprises a viral vector (i.e., rAAV vector). The vectors are administered in sufficient amounts to transfect the cells and to provide sufficient levels of gene transfer and expression to provide a therapeutic benefit without undue adverse effects, or with medically acceptable physiological effects, which can be determined by those skilled in the medical arts. In certain embodiments, the vectors are formulated for delivery via systemic or direct delivery to a desired organ (e.g., lung), oral inhalation, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral routes of administration.

As used herein, the term ‘"dosage” or “amount” can refer to the total dosage or amount delivered to the subject in the course of treatment, or the dosage or amount delivered in a single unit (or multiple unit or split dosage) administration. Dosages of the viral vector will depend primarily on factors such as tire condition being treated, the age, weight and health of the patient, and may thus vary among patients. For example, a therapeutically effective human dosage of tire viral vector is generally in the range of from about 25 to about 1000 microliters to about 5 mL of aqueous suspending liquid containing doses of from about 10⁹ to 4xl0¹⁴ GC of AAV vector. The dosage will be adjusted to balance the therapeutic benefit against any side effects and such dosages may vary depending upon the therapeutic application for which the recombinant vector is employed. The levels of expression of the transgene can be monitored to determine the frequency of dosage resulting in viral vectors, preferably AAV vectors containing the minigene. Optionally, dosage regimens similar to those described for therapeutic purposes may be utilized for immunization using tire compositions of the invention.

The replication-defective virus compositions can be formulated in dosage units to contain an amount of replication-defective virus that is in the range of about 1.0 x 10⁹ GC to about 1.0 x 10¹⁶ GC (to treat an average subject of 70 kg in body weight) including all integers or fractional amounts within the range, and preferably 1.0 x 10¹² GC to 1.0 x 10¹⁴ GC for a human patient. In one embodiment, the compositions are formulated to contain at least IxlO⁹, 2xl0⁹, 3xl0⁹, 4xl0⁹, 5xl0⁹, 6xl0⁹, 7xl0⁹, 8xl0⁹, or 9xl0⁹ GC per dose including all integers or fractional amounts w ithin the range. In another embodiment, the compositions arc formulated to contain at least IxlO¹⁰, 2xlO¹⁰, 3xl0¹⁰, 4xlO¹⁰, 5xl0¹⁰, 6xlO¹⁰, 7xlO¹⁰, 8xl0¹⁰, or 9xlO¹⁰ GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO¹¹, 2x10", 3x10", 4x10", 5x 10". OxlO¹¹. 7x10". 8x10", or 9x10" GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO¹², 2xl0¹², 3xl0¹², 4xl0¹², 5xl0¹², 6xl0¹². 7xl0¹², 8xl0¹², or 9xl0¹² GC per dose including all integers or fractional amounts within the range. In another embodiment, tire compositions are formulated to contain at least Ixl O¹³, 2xl0¹³, 3x l0¹³, 4xl0¹³, 5xl0¹³, 6xl0¹³, 7xl0¹³, 8xl0¹³, or 9xl0¹³ GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO¹⁴, 2xl0¹⁴, 3xl0¹⁴, 4x1014, 5xl0¹⁴, 6xl0¹⁴, 7xl0¹⁴, 8xl0¹⁴, or 9xl0¹⁴ GC per dose including all integers or fractional amounts within the range. In another embodiment, tire compositions are formulated to contain at least IxlO¹⁵, 2xl0¹⁵, 3xl0¹⁵, 4xl0¹⁵, 5xl0¹⁵, 6xl0¹⁵. 7xl0¹⁵. 8xl0¹⁵, or 9xl0¹⁵ GC per dose including all integers or fractional amounts within the range. In one embodiment, for human application the dose can range from IxlO¹⁰ to about IxlO¹² GC per dose including all integers or fractional amounts within tire range. In certain embodiments, the rAAV compositions is formulated in dosage units to contain about 1 x 10¹³ GC/kg. In certain embodiments, the rAAV compositions is formulated in dosage units to contain about 2.5 x 10¹³ GC/kg. In certain embodiments, the rAAV compositions is formulated in dosage units to contain about 5 x 10¹³ GC/kg.

In one embodiment, for human application the dose can range from 10⁹ to about 7x10¹³ GC per dose including all integers or fractional amounts within the range.

These above doses may be administered in a variety of volumes of carrier, excipient or buffer formulation, ranging from about 25 to about 1000 microliters, or higher volumes, including all numbers within the range, depending on the size of the area to be treated, tire viral titer used, the route of administration, and the desired effect of the method. In one embodiment, the volume of carrier, excipient or buffer is at least about 25 pL. In one embodiment, the volume is about 50 pL. In another embodiment, the volume is about 75 pL. In another embodiment, the volume is about 100 pL. In another embodiment, the volume is about 125 pL. In another embodiment, the volume is about 150 pL. In another embodiment, the volume is about 175 pL. In yet another embodiment, tire volume is about 200 pL. In another embodiment, the volume is about 225 pL. In yet another embodiment, tire volume is about 250 pL. In yet another embodiment, the volume is about 275 pL. In yet another embodiment, the volume is about 300 pL. In yet another embodiment, the volume is about 325 pL. In another embodiment, the volume is about 350 pL. In another embodiment, the volume is about 375 pL. In another embodiment, the volume is about 400 pL. In another embodiment, the volume is about 450 pL. In another embodiment, the volume is about 500 pL. In another embodiment, the volume is about 550 pL. In another embodiment, the volume is about 600 pL. In another embodiment, the volume is about 650 pL. In another embodiment, the volume is about 700 pL. In another embodiment, the volume is between about 700 and 1000 pL.

In one embodiment, tire viral constructs may be delivered in doses of from at least about least IxlO⁹ GCs to about 1 x 10¹⁵, or about 1 x 10¹¹ to 5 x 10¹³ GC. Suitable volumes for delivery of these doses and concentrations may be determined by one of skill in the art. For example, volumes of about 1 pL to 150 mL may be selected, with the higher volumes being selected for adults. Typically, for newborn infants a suitable volume is about 0.5 mL to about 10 mL, for older infants, about 0.5 mL to about 15 mL may be selected. For toddlers, a volume of about 0.5 mL to about 20 mL may be selected. For children, volumes of up to about 30 mL may be selected. For pre-teens and teens, volumes up to about 50 mL may be selected. Other suitable volumes and dosages may be determined. The dosage will be adjusted to balance the therapeutic benefit against any side effects and such dosages may vary depending upon the therapeutic application for which the recombinant vector is employed.

The compositions according to the present invention may comprise a pharmaceutically acceptable carrier, such as defined above. Suitably, the compositions described herein comprise an effective amount of one or more AAV suspended in a pharmaceutically suitable carrier and/or admixed with suitable excipients designed for delivery to the subject via injection.

The composition, the suspension or the pharmaceutical compositions described herein are designed for delivery to subjects in need thereof by any suitable route or a combination of different routes. In certain embodiments, the rAAV or the pharmaceutical composition comprises a formulation buffer suitable for intravenous administration to a patient in the need thereof.

In certain embodiments, provided herein is a composition comprising one or more exogenous muscle cell-targeting peptide(s) comprising “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is GQVRVGV (SEQ ID NO: 3), MDAHYVR (SEQ ID NO: 4), TQAVPLK (SEQ ID NO: 5), VVHQTGL (SEQ ID NO: 6), or MAISRER (SEQ ID NO: 7), or a n-mer sequence of at least 6, at least 7, or the full-length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, together with one or more of a physiologically compatible carrier. excipient, and/or aqueous suspension base. Further provided are compositions comprising nucleic acid sequences encoding same.

In certain embodiments, a composition comprising a fusion polypeptide or protein, or a nucleic acid sequence encoding the fusion polypeptide or protein, or a nanoparticle containing same are provided. The composition may further comprise one or more of a physiologically compatible carrier, excipient, and/or aqueous suspension base.

In certain embodiments, a nucleic acid sequence encoding the fusion polypeptide protein is encapsulated in a lipid nanoparticle (LNP). As used herein, the phrase “lipid nanoparticle” or “nanoparticle” refers to a transfer vehicle comprising one or more lipids (e.g., cationic lipids, non- cationic lipids, and PEG-modified lipids). Preferably, the lipid nanoparticles are formulated to deliver one or more nucleic acid sequences to one or more target cells (e.g., muscle cell (cardiac, skeletal, smooth)). Examples of suitable lipids include, for example, the phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides). Also contemplated is the use of polymers as transfer vehicles, whether alone or in combination with other transfer vehicles. Suitable polymers may include, for example, polyacrylates, polyalkycyanoacrylates, polylactide, polylactidepolyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrins, dendrimers and polyethylenimine. In one embodiment, the transfer vehicle is selected based upon its ability to facilitate the transfection of a nucleic acid sequence encapsulated therein to a target cell. Useful lipid nanoparticles for nucleic acid sequence comprise a cationic lipid to encapsulate and/or enhance the delivery of such nucleic acid sequence into the target cell that will act as a depot for protein production. As used herein, the phrase “cationic lipid” refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH. The contemplated lipid nanoparticles may be prepared by including multi-component lipid mixtures of varying ratios employing one or more cationic lipids, non-cationic lipids and PEG- modified lipids. Several cationic lipids have been described in the literature, many of which arc commercially available. See, e.g., WO2014/089486, US 2018/0353616A1, and US 8,853,377B2. which are incorporated by reference. In certain embodiments, LNP formulation is performed using routine procedures comprising cholesterol, ionizable lipid, helper lipid, PEG-lipid and polymer forming a lipid bilayer around encapsulated nucleic acid sequence (Kowalski et al., 2019, Mol. Ther. 27(4):710-728). In some embodiments. LNP comprises a cationic lipids (i.e. N-[l-(2,3-dioleoyloxy)propyl]-N,N,N- trimethyl ammonium chloride (DOTMA), or l,2-dioleoyl-3-trimethylammonium-propane (DOTAP)) with helper lipid DOPE. In some embodiments, LNP comprises an ionizable lipid Dlin-MC3-DMA ionizable lipids, or diketopiperazine-based ionizable lipids (cKK- E12). In some embodiments, polymer comprises a polyethyleneimine (PEI), or a poly( - amino)esters (PBAEs). See, e.g., WO2014/089486, US 2018/0353616A1, US2013/0037977A1, W02015/074085A1, US9670152B2, and US 8,853,377B2, which are incorporated by reference. In certain embodiments, a lipid nanoparticle (LNP) comprises at least one exogenous targeting peptide comprising "Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is GQVRVGV (SEQ ID NO: 3), MDAHYVR (SEQ ID NO: 4), TQAVPLK (SEQ ID NO: 5), VVHQTGL (SEQ ID NO: 6), or MAISRER (SEQ ID NO: 7), or a n-mer sequence of at least 6, at least 7, or the full-length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, i.e., decorated surface with targeting peptide. In certain embodiments, a lipid nanoparticle (LNP) comprises at least one GQVRVGV (SEQ ID NO: 3) peptide.

In certain embodiments, provided herein is a composition comprising, e.g., an rAAV having an engineered capsid with at least one exogenous targeting peptide comprising “Xn - n-mer - Xm”, wherein: (i) Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid; (ii) the n-mer is GQVRVGV (SEQ ID NO: 3), MDAHYVR (SEQ ID NO: 4), TQAVPLK (SEQ ID NO: 5), VVHQTGL (SEQ ID NO: 6), or MAISRER (SEQ ID NO: 7), or an n-mer sequence of at least 6, at least 7, or the full- length consecutive amino acids of any one of the n-mers; and (iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid, that is useful for delivering a therapeutic to a patient in need thereof. In certain embodiments, a composition comprising an rAAV having a modified capsid with at least one GQVRVGV (SEQ ID NO: 3) peptide is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for deliver}’ to muscle cell. In certain embodiments, a composition comprising an rAAV having a modified capsid with at least one GQVRVGV (SEQ ID NO: 3) peptide is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery to muscle cell, and de-targeted for liver.

In certain embodiments, the methods and compositions provided are useful for treatment of mitochondrial cardiomyopathy associated with Barth Syndrome. Barth Syndrome is a rare. X-linked recessive disorder characterized by a loss of function mutation in TAZ gene (i.e., amenable gene therapy). Bart Syndrome is associated with pediatric onset cardiomyopathy (i.e., by age 5) with neutropenia, mild mitochondrial myopathy (skeletal muscle weakness), and mild intellectual impairment. See also, Sabbah, H.N., Barth syndrome cardiomyopathy: targeting the mitochondria with elamipretide, Heart Failure Reviews (2021) 26:237-253, which is incorporated herein by reference in its entirety.

In certain embodiments, tire methods and compositions are useful for treatment of autosomal dominant form of long-QT syndrome caused by a loss-of-function and partial dominant negative mutations in KCNQ1 gene (i.e., amenable to gene replacement or knockdown/replace approach). The autosomal dominant form of long-QT syndrome is associated with syncope and sudden cardiac death usually occurring during exercise or emotional stress, and many patients remain at-risk despite standard of care (beta blockers, cardiac sympathetic denervation) and require Implantable Cardioverter Defibrillator (ICD). See also, Huang H., et al., Mechanisms of KCNQ1 channel dysfunction in long QT syndrome involving voltage sensor domain mutations, Sci. Adv. 2018, 4: 1-12. epub March 7, 2018, which is incorporated herein by reference in its entirety.

In certain embodiments, the methods and compositions are useful for treatment of hypertrophic cardiomyopathy. In certain embodiments, the methods and compositions may be used in treatment of hypertrophic cardiomyopathy caused by loss-of-function mutations in the MYBPC3 gene (i.e., amenable to gene therapy). See also, Mearini G., et al., Mybpc3 gene therapy for neonatal cardiomyopathy enables long-term disease prevention in mice, Nature Communication, 2014, 5:5515, epub December 2, 2014, which is incorporated herein by reference in its entirety.

In certain embodiments, the methods and compositions may be used in treatment of transthyretin amyloid cardiomyopathy (ATTR-CM). In certain embodiments, the methods and compositions may be used in treatment of ATTR-CM caused by mutations in the transthyretin (TTR) gene (i.e., amenable to gene therapy). Sec also, Yamamoto H, Yokochi T. Transthyretin cardiac amyloidosis: an update on diagnosis and treatment. ESC Heart Fail. 2019 De c;6(6): 1128-1139; and Jain A, Zahra F. Transthyretin Amyloid Cardiomyopathy (ATTR-CM) [Updated 2023 Apr 27], In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan, ncbi.nlm.nih.gov/books/NBK574531/, which are incorporated herein by reference in their entirety. In certain embodiments, the methods and compositions are useful for treatment of long WT syndrome type 2 caused by a loss-of-function mutation in hERG (Kvll.l; also, Kv 11 . 1 voltage-gated potassium channel) gene. See also, Curran ME., et al., A Molecular Basis for Cardiac Arrhythmia: HERG Mutations Cause Long QT Syndrome, Cell, Voi. 80, 795-803, March 10, 1995, and Hylten-Cavallius, L., et al., Patients With Long-QT Syndrome Caused by Impaired hERG-Encoded Kvl 1. 1 Potassium Channel Have Exaggerated Endocrine Pancreatic and Incretin Function Associated With Reactive Hypoglycemia. Circulation. 2017;135: 1705-1719, which are incorporated herein by reference in their entirety.

In certain embodiments, the methods and compositions are useful for treatment of LMNA cardiomyopathy, or a disease caused by loss-of-function mutation in the LMNA gene. See also, Kang, S., et al., Laminopathies; Mutations on single gene and various human genetic diseases, BMB Rep. 2018; 51(7): 327-337, and US Provisional Patent application No. 63/293,680, filed December 24, 2021. International Patent Application No. PCT/US2022/082383, filed December 24, 2022, now Publication No. WO 2023/122803 Al, published June 29, 2023, which are incorporated herein by reference in their entirety.

In certain embodiments, tire methods and compositions are useful for treatment of heart failure, ischemia-reperfusion injury, myocardial infarction, ventricular remodeling, or a disease associated with extracellular superoxide dismutase 3 (SOD3 or EcSOD). See also, US Patent Application Publication No. US20130136729A1, which is incorporated herein by reference in its entirety’.

In certain embodiments, the methods and compositions are useful for treatment of myocardial infarction, reduced ejection fraction of the heart or a disease associated with myc transcription factor, cyclin T 1 and cyclin-dependent kinase 9 (CDK9). See also, International Patent Application Publication No. W02020/165603A1, which is incorporated herein by reference in its entirety.

In certain embodiments, the methods and compositions are useful for treatment of heart failure, or heart tissue damage, or degeneration, or a disease associated with cy clin A2 protein. See also, International Patent Application Publication No. W02020/051296A1, which is incorporated herein by reference in its entirety.

In certain embodiments, tire methods and compositions are useful for treatment of dilated cardiomyopathy (DCM), a heart failure, a cardiac fibrosis, a heart inflammation, an ischemic heart disease, a myocardial infarction, an ischemic/reperfusion (I/R) related injuries, a transverse aortic constriction, or a disease associated with YY 1 or BMP7 protein. See also. International Patent Application Publication No. WO2021/021021 Al, which is incorporated herein by reference in its entirety.

In certain embodiments, the methods and compositions are useful for treatment of dilated cardiomyopathy, or a disease associated with cardiac Apoptosis Repressor with Caspase Recruitment Domain (cARC). See also, International Patent Application Publication No. W02021/016126A1, which is incorporated herein by reference in its entirety.

Symptoms of cardiomyopathy or a disease associated with a mutation in a LMNA gene, KCNQ1 gene, MYBPC3 gene, TAZ gene, or hERG gene include atrioventricular (AV) conduction block, atrial fibrillation, arrhythmia including atrial arrhythmia such as atrial flutter and atrial tachycardia, and ventricular arrhythmias including sustained ventricular tachycardias, and ventricular fibrillation (VF) and/or heart failure.

In certain embodiments, the methods and compositions described herein are useful to ameliorate one or more symptoms of cardiomyopathy including increased average life span, and/or reduction in progression towards heart failure.

In certain embodiments, tire methods and compositions are used for treatment, or to ameliorate one or more symptoms of muscular dystrophy.

In certain embodiments, the methods and compositions are useful for treatment, or to ameliorate one or more symptoms of Duchenne muscular dystrophy (Dystrophin, DMD), Becker muscular dystrophy (Dystrophin, DMD), Danon disease (LAMP2), Myotubular myopathy (Myotubularin, MRM1), Primary merosin deficiency (Merosin, LAMA2), Pompe disease (a-l,4-Glucosidase. GAA), Limb-girdle muscular dystrophy (Calpain 3. CAPN3). Oculopharyngeal muscular dystrophy (PABPN1). or muscular dystrophies associated with Dysferlin (DYSF), a-Sarcoglycan (LGMD 2D), -Sarcoglycan (SGCB) or Fukutinrelated protein (FKRP) proteins.

In certain embodiments, an rAAV having a modified capsid as described herein may be delivered in a co-thcrapcutic regimen which further comprises one or more other active components. In certain embodiments, the regimen may involve co-administration of an immunomodulatory component. Such an immunomodulatory regimen may include, e.g., but are not limited to immunosuppressants such as. a glucocorticoid, steroids, antimetabolites, T-cell inhibitors, a macrolide (e.g., a rapamycin or rapalog), and cytostatic agents including an alkylating agent, an anti-metabolite, a cytotoxic antibiotic, an antibody, or an agent active on immunophilin. The immune suppressant may include a nitrogen mustard, nitrosourea, platinum compound, methotrexate, azathioprine, mercaptopurine, fluorouracil, dactinomycin, an anthracycline, mitomycin C, bleomycin, mithramycin, IL-2 receptor- (CD25-) or CD3-directed antibodies, anti-IL-2 antibodies, cyclosporin, tacrolimus, sirolimus, IFN-0, IFN-y, an opioid, or TNF-a (tumor necrosis factor-alpha) binding agent. In certain embodiments, the immunosuppressive therapy may be started prior to the gene therapy administration. Such therapy may involve co-administration of two or more drugs, the (e.g., prednelisone, micophenolate mofetil (MMF) and/or sirolimus (i.e. , rapamycin)) on the same day. One or more of these drugs may be continued after gene therapy administration, at the same dose or an adjusted dose. Such therapy may be for about 1 week, about 15 days, about 30 days, about 45 days, 60 days, or longer, as needed. Still other co-therapeutics may include, e.g., anti-IgG enzymes, which have been described as being useful for depleting anti-AAV antibodies (and thus may permit administration to patients testing above a threshold level of antibody for the selected AAV capsid), and/or delivery of anti-FcRN antibodies which is described, e.g., in WO 2021/257668, filed 23 December 2021, (claiming priority to US Provisional Patent Application No. 63/040,381, filed June 17, 2020, US Provisional Patent Application No. 62/135,998, filed January 1 1, 2021, and US Provisional Patent Application No. 63/152,085, filed February 22, 2021) entitled "Compositions and Methods for Treatment of Gene Therapy Patients”, and/or one or more of a) a steroid or combination of steroids and/or (b) an IgG-cleaving enzyme, (c) an inhibitor of Fc-IgE binding; (d) an inhibitor of Fc-IgM binding; (e) an inhibitor of Fc-IgA binding; and/or (f) gamma interferon.

Kit

In certain embodiments, a kit is provided which includes a concentrated vector suspended in a formulation (optionally frozen), optional dilution buffer, and devices and components required for intravenous administration. In another embodiment, the kit may additional or alternatively include components for intravenous delivery. In one embodiment, the kit provides sufficient buffer to allow for injection. Such buffer may allow for about a 1 : 1 to a 1 :5 dilution of the concentrated vector, or more. In other embodiments, higher or lower amounts of buffer or sterile water are included to allow for dose titration and other adjustments by the treating clinician. In still other embodiments. one or more components of the device are included in the kit. Suitable dilution buffer is available, such as, a saline, a phosphate buffered saline (PBS) or a glycerol/PBS.

It should be understood that the compositions in kit described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.

An “immunoglobulin molecule” is a protein containing the immunologically-active portions of an immunoglobulin heavy chain and immunoglobulin light chain covalently coupled together and capable of specifically combining with antigen. Immunoglobulin molecules are of any type (e.g.. IgG, IgE. IgM, IgD. IgA and IgY), class (e.g.. IgGl. lgG2, IgG3, IgG4, IgAl and IgA2) or subclass. The terms “antibody” and “immunoglobulin” may be used interchangeably herein.

“Neutralizing antibody titer” (NAb titer) a measurement of how much neutralizing antibody (e.g., anti-AAV NAb) is produced which neutralizes tire physiologic effect of its targeted epitope (e.g.. an AAV). Anti-AAV NAb titers may be measured as described in, e.g., Calcedo. R., et al., Worldwide Epidemiology of Neutralizing Antibodies to Adeno- Associated Viruses. Journal of Infectious Diseases, 2009, 199 (3): p. 381-390. which is incorporated by reference herein.

As used herein when used to refer to vp capsid proteins, the term “heterogenous” or any grammatical variation thereof, refers to a population consisting of elements that are not tire same, for example, having vpl, vp2 or vp3 monomers (proteins) with different modified amino acid sequences. SEQ ID NO: 26 provides tire encoded amino acid sequence of the AAVhu68 vpl protein. The term “heterogenous” as used in connection with vpl. vp2 and vp3 proteins (alternatively termed isoforms), refers to differences in the amino acid sequence of the vpl, vp2 and vp3 proteins within a capsid. The AAV capsid contains subpopulations within the vp 1 proteins, within the vp2 proteins and within the vp3 proteins which have modifications from tire predicted amino acid residues. These subpopulations include, at a minimum, certain deamidated asparagine (N or Asn) residues. For example, certain subpopulations comprise at least one, two, three or four highly deamidated asparagines (N) positions in asparagine - glycine (N - G) pairs and optionally further comprising other deamidated amino acids, wherein the deamidation results in an amino acid change and other optional modifications.

As used herein, a “subpopulation” of vp proteins refers to a group of vp proteins which has at least one defined characteristic in common and which consists of at least one group member to less than all members of tire reference group, unless otherwise specified. For example, a “subpopulation” of vpl proteins is at least one (1) vpl protein and less than all vpl proteins in an assembled AAV capsid, unless otherwise specified. A “subpopulation” of vp3 proteins may be one (1) vp3 protein to less than all vp3 proteins in an assembled AAV capsid, unless otherwise specified. For example, vpl proteins may be a subpopulation of vp proteins; vp2 proteins may be a separate subpopulation of vp proteins, and vp3 are yet a further subpopulation of vp proteins in an assembled AAV capsid. In another example, vpl, vp2 and vp3 proteins may contain subpopulations having different modifications, e.g.. at least one. two, three or four highly deamidated asparagines, e.g., at asparagine - glycine pairs. Unless otherwise specified, highly deamidated refers to at least 45% deamidated, at least 50% deamidated, at least 60% deamidated, at least 65% deamidated, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 97%, 99%, up to about 100% deamidated, 50% to 100% deamidated, 70% to 100% deamidated, 75% to 100% deamidated, or 70% to 90% deamidated at a referenced amino acid position, as compared to the predicted amino acid sequence at tlie reference amino acid position. Such percentages may be determined using 2D-gel. mass spectrometry techniques, or other suitable techniques.

As used herein, a “stock” of rAAV refers to a population of rAAV. Despite heterogeneity in their capsid proteins due to deamidation, rAAV in a stock are expected to share an identical vector genome. A stock can include rAAV having capsids with, for example, heterogeneous deamidation patterns characteristic of the selected AAV capsid proteins and a selected production system. The stock may be produced from a single production system or pooled from multiple runs of the production system. A variety of production systems, including but not limited to those described herein, may be selected. See, e g., WO 2019/168961, published September 6, 2019, including Table G providing the deamidation pattern for AAV9 and WO 2020/160582, filed September 7, 2018. See, also, e.g., WO 2020/223231, published November 5, 2020 (rh91, including table with deamidation pattern), US Provisional Patent Application No. 63/065,616, filed August 14, 2020, and US Provisional Patent Application No. 63/109,734, filed November 4, 2020 and International Patent Application No. PCT/US21/45945, filed August 13, 2021, which are all incorporated herein by reference in its entirety.

The compositions described herein may be used in a regimen involving coadministration of other active agents. Any suitable method or route can be used to administer such other agents. Routes of administration include, for example, systemic, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration. Optionally, the AAV compositions described herein may also be administered by one of these routes.

The abbreviation “sc” refers to self-complementary. “Self-complementary AAV” refers a construct in which a coding region carried by a recombinant AAV nucleic acid sequence has been designed to form an intra-molecular double-stranded DNA template. Upon infection, rather than waiting for cell mediated synthesis of the second strand, the two complementary halves of scAAV will associate to form one double stranded DNA (dsDNA) unit drat is ready for immediate replication and transcription. See. e.g., D M McCarty et al, “Self-complementary recombinant adeno-associated vims (scAAV) vectors promote efficient transduction independently of DNA synthesis”, Gene Therapy, (August 2001), Vol 8, Number 16, Pages 1248-1254. Self-complementary AAVs are described in, e.g., U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety.

The term “heterologous” when used with reference to a protein or a nucleic acid indicates that the protein or the nucleic acid comprises two or more sequences or subsequences which are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid. For example, in one embodiment, tire nucleic acid has a promoter from one gene arranged to direct the expression of a coding sequence from a different gene. Thus, with reference to the coding sequence, the promoter is heterologous.

A “replication-defective vims” or “viral vector” refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, where any viral genomic sequences also packaged within the viral capsid or envelope are replication-deficient; i.e., they cannot generate progeny virions but retain the ability to infect target cells. In one embodiment, the genome of the viral vector docs not include genes encoding the enzymes required to replicate (the genome can be engineered to be "gutless" - containing only the transgene of interest flanked by the signals required for amplification and packaging of the artificial genome), but these genes may be supplied during production. Therefore, it is deemed safe for use in gene therapy since replication and infection by progeny virions cannot occur except in the presence of the viral enzyme required for replication. A ‘‘recombinant AAV” or “rAAV" is a DNAse-resistant viral particle containing two elements, an AAV capsid and a vector genome containing at least non-AAV coding sequences packaged within the AAV capsid. In certain embodiments, the capsid contains about 60 proteins composed of vp 1 proteins, vp2 proteins, and vp3 proteins, which selfassemble to form the capsid. Unless otherwise specified, “recombinant AAV” or “rAAV” may be used interchangeably with the phrase “rAAV vector”. The rAAV is a “replicationdefective virus" or "viral vector", as it lacks any functional AAV rep gene or functional AAV cap gene and cannot generate progeny. In certain embodiments, tire only AAV sequences are die AAV inverted terminal repeat sequences (ITRs), typically located at the extreme 5 ’ and 3 ’ ends of the vector genome in order to allow the gene and regulatory sequences located between the ITRs to be packaged within the AAV capsid.

The term “nuclease-resistant” indicates that the AAV capsid has assembled around die expression cassete which is designed to deliver a transgene to a host cell and protects these packaged genomic sequences from degradation (digestion) during nuclease incubation steps designed to remove contaminating nucleic acids which may be present from the production process.

As used herein, the term “host cell” may refer to die packaging cell line in which the rAAV is produced from the plasmid. In the alternative, the term “host cell” may refer to die target cell in which expression of the transgene is desired.

As used herein, a “vector genome” refers to the nucleic acid sequence packaged inside the rAAV capsid which forms a viral particle. Such a nucleic acid sequence contains AAV inverted tenninal repeat sequences (ITRs). In the examples herein, a vector genome contains, at a minimum, from 5’ to 3'. an AAV 5’ ITR, expression cassette comprising coding sequence(s) (i.e., transgene(s)), and an AAV 3^? ITR. In certain embodiments, the ITRs are from AAV2, a different source AAV than the capsid, or other than full-length ITRs may be selected. In certain embodiments, the ITRs are from the same AAV source as die AAV which provides the rep function during production or a trans-complementing AAV. Furtiicr, other ITRs, c.g., sclf-complcmcntary (scAAV) ITRs, may be used. Both single-stranded AAV and self-complementary (sc) AAV are encompassed witii the rAAV. The transgene is a nucleic acid coding sequence, heterologous to the vector sequences, which encodes a polypeptide, protein, functional RNA molecule (e.g.. miRNA, miRNA inhibitor) or other gene product, of interest. The nucleic acid coding sequence is operatively linked to regulatory components in a manner which permits transgene transcription, translation, and/or expression in a cell of a target tissue. Suitable components of a vector genome are discussed in more detail herein. In one example, a ' vector genome⁷’ contains, at a minimum, from 5’ to 3’, a vector-specific sequence, a nucleic acid sequence encoding protein of interest operably linked to regulatory control sequences (which direct their expression in a target cell), where the vector-specific sequence may be a terminal repeat sequence which specifically packages the vector genome into a viral vector capsid or envelope protein. For example, AAV inverted terminal repeats are utilized for packaging into AAV and certain other parvovirus capsids.

As used herein, "operably linked” sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

In certain embodiments, non- viral genetic elements used in manufacture of a rAAV, will be referred to as vectors (e.g., production vectors). In certain embodiments, these vectors are plasmids, but the use of other suitable genetic elements is contemplated. Such production plasmids may encode sequences expressed during rAAV production, e.g., AAV capsid or rep proteins required for production of a rAAV, which are not packaged into the rAAV. Alternatively, such a production plasmid may carry the vector genome which is packaged into the rAAV.

As used herein, a "parental capsid” refers to a non-mutated, non-engineered or a non-modified capsid selected from parvovirus or other viruses (e.g., AAV, adenovirus, HSV. RSV, etc ). In certain embodiments, the parental capsid includes any naturally occurring AAV capsids comprising a wild-type genome encoding for capsid proteins (i.e., vp proteins), wherein the capsid proteins direct the AAV transduction and/or tissue-specific tropism. In some embodiments, the parent capsid is selected from AAV which natively targets muscle cell. In other embodiments, the parental capsid is selected from AAV which do not natively target muscle cell.

As used herein, the terms “target cell” and “target tissue” can refer to any cell or tissue which is intended to be transduced by the subject AAV vector. The term may refer to any one or more of muscle, liver, lung, airway epithelium, central nervous system, neurons, eye (ocular cells), or heart. In one embodiment, the target tissue is muscle tissue. In certain embodiments, the target cell is one or more muscle cell type (e.g.. cardio muscle cell or gastrocnemius muscle cell). As used herein, a “cardiac cell” refers to general cardiac tissue cells including but not limited to heart cells, cardiac muscle cells (cardiomyocyte), conduction cells, fibroblasts, endothelial cells, smooth muscle cells and peri-vascular cells.

As used herein, a “variant capsid” or a “variant AAV” or “variant AAV capsid” refers to a modified capsid, engineered capsid or a mutated capsid, wherein the capsid protein comprises an insertion of a tissue-specific targeting peptide, wherein modified insert is not a naturally occurring mutation.

As used herein, an “expression cassette” refers to a nucleic acid molecule which comprises a biologically useful nucleic acid sequence (e.g., a gene cDNA encoding a protein, enzyme or other useful gene product, mRNA, etc.) and regulatory sequences operably linked thereto which direct or modulate transcription, translation, and/or expression of the nucleic acid sequence and its gene product. As used herein, “operably linked” sequences include both regulatory sequences that are contiguous or non-contiguous with the nucleic acid sequence and regulatory sequences that act in trans or cis nucleic acid sequence. Such regulatory sequences typically include, e.g., one or more of a promoter, an enhancer, an intron, a Kozak sequence, a polyadenylation sequence, and a TATA signal. The expression cassette may contain regulatory sequences upstream (5’ (5') to) of the gene sequence, e.g., one or more of a promoter, an enhancer, an intron, etc., and one or more of an enhancer, or regulatory sequences downstream (3’ (3') to) a gene sequence, e.g., 3’ untranslated region (3’ UTR) comprising a polyadenylation site, among other elements. In certain embodiments, tire regulatory sequences are operably linked to the nucleic acid sequence of a gene product, wherein the regulatory sequences are separated from nucleic acid sequence of a gene product by an intervening nucleic acid sequences, i.e., 5'- untranslated regions (5 ’UTR). In certain embodiments, the expression cassette comprises nucleic acid sequence of one or more of gene products. In some embodiments, the expression cassette can be a monocistronic or a bicistronic expression cassette. In other embodiments, the term “transgene” refers to one or more DNA sequences from an exogenous source which arc inserted into a target cell. Typically, such an expression cassette can be used for generating a viral vector and contains the coding sequence for the gene product described herein flanked by packaging signals of the viral genome and other expression control sequences such as those described herein. In certain embodiments, a vector genome may contain two or more expression cassettes. The term “translation’" in the context of the present invention relates to a process at the ribosome, wherein an mRNA strand controls the assembly of an amino acid sequence to generate a protein or a peptide.

The term “expression” is used herein in its broadest meaning and comprises tire production of RNA or of RNA and protein. Expression may be transient or may be stable. The term “substantial homolog} ” or “substantial similarity ,” when referring to a nucleic acid, or fragment thereof, indicates that, when optimally aligned w ith appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95 to 99% of tire aligned sequences. Preferably, the homology is over full-length sequence, or an open reading frame thereof, or another suitable fragment which is at least 15 nucleotides in length. Examples of suitable fragments are described herein.

The term “percent (%) identity", “sequence identity ”, “percent sequence identity”, or “percent identical” in the context of nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for correspondence. The length of sequence identity comparison may be over the full-length of the genome, the full-length of a gene coding sequence, or a fragment of at least about 500 to 5000 nucleotides, is desired. However, identity^ among smaller fragments, e g., of at least about nine nucleotides, usually at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides, may also be desired.

Percent identity may be readily determined for amino acid sequences over tire full- length of a protein, polypeptide, about 32 amino acids, about 330 amino acids, or a peptide fragment thereof or the corresponding nucleic acid sequence coding sequences. A suitable amino acid fragment may be at least about 7 amino acids in length, and may be up to about 700 amino acids.

Examples of suitable fragments are described herein. By the term “highly? conserved” is meant at least 80% identity , preferably at least 90% identity, and more preferably, over 97% identity. Identity’ is readily determined by one of skill in the art byresort to algorithms and computer programs known by those of skill in the art.

Generally, when referring to “identity”, “homology”, or “similarity” between two different sequences, “identity”, “homology” or “similarity” is determined in reference to “aligned” sequences. “Aligned” sequences or “alignments” refer to multiple nucleic acid sequences or protein (amino acids) sequences, often containing corrections for missing or additional bases or amino acids as compared to a reference sequence.

Identity may be determined by preparing an alignment of the sequences and through the use of a variety of algorithms and/or computer programs known in the art or commercially available (e.g., BLAST, ExPASy; Clustal Omega; FASTA; using, e.g., Needleman-Wunsch algorithm, Smith-Watennan algorithm). Alignments are performed using any of a variety of publicly or commercially available Multiple Sequence Alignment Programs. Multiple sequence alignment programs are available for nucleic acid sequences. Examples of such programs include, ‘‘Clustal Omega’; “Clustal W”, “MUSCLE”. “CAP Sequence Assembly”, “BLAST”, “MAP”, and “MEME”, which are accessible through Web Servers on the internet. Other sources for such programs are known to those of skill in tire art. Alternatively, Vector NTI utilities are also used. There are also a number of algorithms known in the art that can be used to measure nucleotide sequence identity, including those contained in tire programs described above. As another example, polynucleotide sequences can be compared using Fasta™, a program in GCG Version 10. 1. Fasta™ provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. For instance, percent sequence identity between nucleic acid sequences can be determined using Fasta™ with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) as provided in GCG Version 10.1, herein incorporated by reference. Sequence alignment programs are also available for amino acid sequences, e.g., the “Clustal Omega”, “Clustal X”, “MUSCLE”, “MAP”, “PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box” programs. Generally, any of these programs are used at default settings, although one of skill in the art can alter these settings as needed. Alternatively, one of skill in the art can utilize another algorithm or computer program which provides at least the level of identity or alignment as that provided by the referenced algorithms and programs. See, e.g., J. D. Thomson et al, Nucl. Acids. Res., “A comprehensive comparison of multiple sequence alignments”, 27(13):2682-2690 (1999).

In certain embodiments, an effective amount may be determined based on an animal model, rather than a human patient.

As described above, tire term “about” when used to modify a numerical value means a variation of ±10%, (±10%, e.g., ±1, ±2, ±3, ±4, ±5, ±6, ±7, ±8, ±9, ±10, or values therebetween) from the reference given, unless otherwise specified. In certain instances, the term “E+#” or the term “e+#” is used to reference an exponent. For example, “5E10” or “5el0” is 5 x 1O^1CI. These terms may be used interchangeably.

As used throughout this specification and the claims, the terms “comprise” and “contain” and its variants including, “comprises”, “comprising”, “contains” and “containing”, among other variants, is inclusive of other components, elements, integers, steps and the like. The term “consists of’ or “consisting of’ are exclusive of other components, elements, integers, steps and the like.

It is to be noted that the term “a” or “an”, refers to one or more, for example, “an enhancer”, is understood to represent one or more enhancer(s). As such, the terms “a” (or “an”), “one or more,” and “at least one” is used interchangeably herein.

With regard to the description of these inventions, it is intended that each of the compositions herein described, is useful, in another embodiment, in the methods of the invention. In addition, it is also intended that each of the compositions herein described as useful in the methods, is, in another embodiment, itself an embodiment of the invention.

Unless defined otherwise in this specification, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in tire art to which this invention belongs and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application.

EXAMPLES

The following examples are illustrative only and are not a limitation on the invention described herein.

There are currently no approved AAV gene therapies for cardiac disorders. One reason for this is the inherent difficulty of transducing cardiac myocytes via systemic AAV injection. Though appreciable cardiac transduction is achievable via the naturally occurring AAV variant AAV9, the specificity of this transduction is lacking. Indeed, the majority of injected vector ends up transducing the liver, and thus the higher doses of vector required for therapeutically relevant cardiac transduction risks liver damage, adverse immune responses, and potentially death. One solution to this problem is localized injection of gene therapy agents: however this technology is invasive and difficult to control, thus posing another set of safety risks that will require further innovation to overcome. A primary goal in the gene therapy field then is to create gene transfer agents that can specifically and non- invasively transduce cells of the myocardium. Thus, there is an unmet clinical need for AAV vectors that can more efficiently transduce cells of the myocardium.

Similarly, AAV9 is poor at transducing skeletal muscle, a great hindrance to the current efforts aimed at treating numerous genetic disorders, incuding Duchenne’s Muscular Dystrophy. AAV vectors with enhanced skeletal muscle tropism are thus another strong target for viral vector engineering.

It has been shown that small peptide insertions into flexible loops on the surface of the AAV capsid can mediate interactions with new cellular receptors. In one case discovered at CalTech (AAV9-PHP.B). a seven amino acid peptide inserted into the HVR8 loop on AAV9 mediated interaction with Ly6a, a GPI-anchored receptor on the brain vasculature of some mouse strains. This interaction drives transport of AAV9-PHP.B across the blood brain barrier, resulting in ~50-fold higher transduction of brain cells than AAV9.

Studies were conducted to identify peptide inserts that can improve AAV9 capsid transduction of both skeletal and cardiac muscle. To accomplish this, we leveraged previous work in AAV capsid engineering that indicated the well-studied RGD peptide motif (which is a targeting motif for numerous integrins) was able to target AAV vectors to skeletal muscle (and to a lesser extent cardiac muscle) in the context of the AAV9 HVR8 loop.

We generated a library of AAV9 insertion mutants containing hundreds of thousands of RGD and non-RGD containing peptides, all inserted individually at the HVR8 locus (between position 588 and 589). Each variant was barcoded such that the transgene it carried was the capsid protein it was encased in, allowing for analysis of relative abundance for each variant after RNA extraction from transduced tissue. This library was injected at a high does intravenously into Rhesus macaques, and at two weeks animals were sacrificed for RNA extraction and analysis.

We then collected total RNA, enriched mRNA from it and converted that mRNA into cDNA, and performed next generation sequencing of the resultant cDNA library. Thus, by sequencing all expressed genomes in both cardiac and skeletal muscle, we were able to identify hits for validation in a second, less noisy and more targeted library experiment. Variants were selected for high levels of enrichment (tissue rpm/injected vector library rpm) across a range of vector library representations. Additionally, some vectors from a separate screen in NHPs (driven by the syna sin promoter but still expressing in heart) were added to tire second round screen as well. The second round library also included a number of important controls, AAV9 - PHP.B (insert TLAVPFK (SEQ ID NO: 37)) and a related insert (TLAGPFK (SEQ ID NO: 38)), both of which perform similarly to AAV9 in tire heart despite having an insert at site 588. We also included a number of previously reported vectors with known skeletal muscle tropism as positive controls. Each variant was coded for with three different synonymous codons, to give better confidence in performance metrics based on clustering of synonymous variants. From this secondary screen we were able to identify a large number of variants with 2 to 22x increases in both cardiac and skeletal muscle transduction.

EXAMPLE 1. Production of rAAV comprising a gene (protein) of interest

In the studies herein, engineered rAAVs comprising engineered AAV capsids comprising exogenous targeting peptides are generated and comparative studies are performed. In some cases, a rAAV comprising a GFP gene is generated and used to evaluate rAAV transduction and transgene expression. In some cases, a rAAV comprising a test gene X (TGX, wherein X is 1, 2, 3, etc.) is generated and used to evaluate rAAV transduction and gene expression.

The rAAV are generated using triple transfection techniques, utilizing (1) a cis plasmid encoding AAV2 rep proteins and tire AAV9 VP1 cap gene, (2) a cis plasmid comprising adenovirus helper genes not provided by the packaging cell line which expresses adenovirus El a, and (3) a trans plasmid containing the vector genome for packaging in the AAV capsid. See, e.g.. US 2020/0056159. The trans plasmid is designed to contain the vector genome comprising transgene of interest (e.g., GFP). The vector genome contains an AAV 5’ inverted terminal repeat (ITR) and an AAV 3’ ITR at the extreme 5 ’ and 3 ’ end, respectively. The ITRs flank the sequences of the expression cassette packaged into the AAV capsid which have sequences encoding protein of interest. The expression cassette further comprises regulator}’ sequences operably linked to tire protein coding sequences, the regulatory control sequence of which include at least one or more of promoter, enhancer, polyA sequence.

EXAMPLE 2: Improving AAV9 heart transduction

In this study, we designed and generated an AAV9 library to include a comprehensive collection of all possible 7-mer peptides (unbiased library (~ 2 billion) neuron-specific promoter (hSyn)) inserted in HVR8 (HVRVIII) region of AAV9 capsid), which was used for selection by 2-rounds of selection in NHP (dose 5xl0¹³ GC/kg, with heart tissue collected on day 14) with focus on the heart. In Round 2 of the NHP heart selection, a mini library design with embedded control sequences, AAV9-like negative control vectors, and best reported heart variants from literature as positive controls were used. FIG. 1A shows plotted heart enrichment score from non-RGD screen round 2 for top performing heart candidates in comparison to the top literature capsids. FIG. IB shows muscle enrichment score from non-RGD screen round 2 for top performing heart and muscle candidates in comparison to the top heart candidates.

Next, we used vector genome barcoding to evaluate over 30 engineered capsids, in comparison to controls in a single animal. First, we looked at individual vector production at pre-clinical scale, then we evaluated top heart and muscle capsid variants individually at full dose (5E12 GC/kg).

Overall, a comprehensive screen of AAV9-non-RGD variants in NHP yielded hits with enhanced heart and skeletal muscle transduction vs AAV9 capsids. Top non-RGD variants are summarized in Table 1 below.

Next, we performed a barcoded cardiac vector study using MyoAAV (literature control), AAV9 (negative control), Gal vectors (modified amino acids in the Gal-binding pocket of AAV capsid), and W503A. Vectors were administered to NHP (n=l) at a dose of IxlO¹³ GC/kg, with time points observed at D14. Table 2 shows results of the barcode study with normalized RNA values. Table 3 shows results of the barcode DNA values.

All values were normalized to AAV9. The individually tested vectors were AAV9 and GQVRVGV. MY0AAV4E (literature control, Manini, A., Adeno-Associated Virus (AAV)-Mediated Gene Therapy for Duchenne Muscular Dystrophy: The Issue of Transgene Persistence, Front. Neurol., 2021, 12:814174) is a previously published vector with good performance in heart and muscle. The non-RGD hit (GQVRVGV, SEQ ID NO: 3) was performed the best among the heart capsids identified.

Next, we performed testing of individual heart capsids for cardiac transduction, in which AAV9 and AAV9-GQVRVGV vectors were administered to NHPs (n=8) at a dose of IxlO¹³ GC/kg. On D13-15 tissues samples were collected and histology analysis was performed, including staining, imaging, and quantification (i.e., computer analyzed). Representative IHC microscopy images of heart left longitudinal tissue samples were taken and signal (dark brown in IHC, positive for GFP) was quantified (data not shown). Representative IHC microscopy images of heart left transverse tissue samples were taken and signal (dark brown in IHC, positive for GFP) was quantified (data not shown). Table 4, below, shows a summary of the values calculated from IHC analysis (% positive cells for each slide per NHP).

Next, we examined RNA (expression PCR), DNA (biodistribution PCR), and protein expression (ELISA) biodistribution in the collected tissue samples following administration with AAV9 and AAV9-GQVRVGV vectors. DNA and RNA analyses were performed on 3-4 pieces of tissue per NHP. There were 2 NHPs per vector. Left and right ventricles of tire heart were examined separately.

FIGs. 2A to 2C show RNA. DNA, and protein expression levels in tire collected tissue samples of heart left ventricle following administration of AAV9 and AAV9- GQVRVGV vectors. FIG. 2A shows DNA levels, plotted as GC/diploid cell. FIG. 2B shows RNA levels, plotted as copy number (#)/100ng RNA. FIG. 2C shows protein expression levels, plotted as pg GFP/pg protein. Table 5 show s a summary of quantified values.

FIGs. 3 A to 3C show RNA, DNA, and protein expression levels in the collected tissue samples of heart right ventricle following administration of AAV9 and AAV9- GQVRVGV vectors. FIG. 3 A shows DNA levels, plotted as GC/diploid cell. FIG. 3B shows RNA levels, plotted as copy number (#)/100ng RNA. FIG. 3C shows protein expression levels, plotted as pg GFP/pg protein. Table shows a summary of quantified values.

FIGs. 4A to 4C show RNA. DNA, and protein expression levels in tire collected tissue samples of gastrocnemius following administration of AAV9. and AAV9- GQVRVGV vectors. FIG. 4A shows DNA levels, plotted as GC/diploid cell. FIG. 4B shows RNA levels, plotted as copy number (#)/100ng RNA. FIG. 4C shows protein expression levels, plotted as pg GFP/pg protein. Table 7 shows a summary of quantified values.

FIGs. 5A to 5C show RNA, DNA, and protein expression levels in the collected tissue samples of liver following administration of AAV9, and AAV9-GQVRVGV vectors. FIG. 5A shows DNA levels, plotted as GC/diploid cell. FIG. 5B shows RNA levels, plotted as copy number (#)/100ng RNA. FIG. 5C shows protein expression levels, plotted as pg GFP/pg protein. Table 8 shows a summary of quantified values.

FIG. 6 shows RNA/DNA ratios (normalized to AAV9) for AAV9 and AAV9-

GQVRVGV vector biodistribution. Table 9 shows a summary of quantified values.

Tables 10A to 10D show molecular summaries of individual hits. Table 10A. Left Ventricle

Table 10B. Right Ventricle

Table IOC. Gastrocnemius

Table 10D. Liver

Next, we evaluated more of the hSyn-heart hits (e g., GQVRVGV (SEQ ID NO: 3), MDAHYVR (SEQ ID NO: 4), TQAVPLK (SEQ ID NO: 5), VVHQTGL (SEQ ID NO: 6), MAISRER (SEQ ID NO: 7). Briefly, '‘2SEL” (site evaluation library including 2 amino acid modifications) libraries of each peptide were made (library size total 285,696 variants), including mutations flanking amino acids at position 587/588 and 589/590 (based on WT numbering of SEQ ID NO: 25). Table 11, below, shows summary of tire results.

Table 12 shows a summary of tire enrichment of original sequences and PHP.B.

Fold change values normalized to PHP.B (AAV9 like control). Many sequences across all 5 2SEL peptides outperformed their original sequences.

Table 13 shows enrichment results of tire follow up study in NHP using a mini library (286 hits) with and without a flanking “AQ”. FIG. 8 shows results of the enrichment study in NHPs, plotted as normalized FC.

These results confirmed the heart targeting capabilities of the muscle-targeting peptides. Overall, these results show that unbiased library (non-RGD) screen yielded AAV9 insert capsids with enhanced heart transduction and greater heart specificity' versus skeletal muscle. EXAMPLE 3: Evaluation of engineered rAAV9 for transduction.

In this study, we performed testing of individual heart capsids (i.e., engineered rAAV comprising exogenous targeting peptides) for cardiac, gastrocnemius, liver cell transduction, in which AAV9 and AAV9-X vectors (including modified flanking regions comprising “SAQMDAH YSRQQA (SEQ ID NO 70), “SSQMDAHYVRNQA^?? (SEQ ID NO 71), ‘-SAQMDAHYRREQA’' (SEQ ID NO 72), “SQQGQVRVGVAMA’⁷ (SEQ ID NO 73), ‘-SAQLDAHYVRNQA” (SEQ ID NO 74), “ENRRGDFNNT AQA” (SEQ ID NO 75)) were administered to mice at a dose of 5 x 10¹² GC/kg. Mice were observed for 14 days, after which mice were necropsied. Heart, liver, and gastrocnemius tissues were collected and analyzed for vector biodistribution and histological analysis was performed. Table 14 shows AAV9-X variants evaluated (FIGs. 9).

FIG. 9A shows RNA/DNA ratios (normalized to AAV 9) for AAV9 and AAV9-X various vectors as analyzed in heart tissue. FIG. 9B shows RNA/DNA ratios (normalized to AAV9) for AAV9 and AAV9-X various vectors as analyzed in liver tissues. FIG. 9C shows RNA levels in heart tissue post rAAV transduction, plotted as RNA transcript /lOOng. FIG. 9D shows DNA levels in heart tissue post-rAAV transduction, plotted as GC per diploid genome. FIG. 9E shows RNA levels in liver tissue post-rAAV transduction, plotted as RNA transcript / lOOng. FIG. 9F shows DNA levels in liver tissue post-rAAV transduction, plotted as GC per diploid genome FIG. 10A shows results of an in situ hybridization (ISH) analysis of gastrocnemius tissue, plotted as percent GFP positive (normalized to AAV9 control). FIG. 10B shows results of an ISH analysis of bicep femoris tissue, plotted as percent GFP positive (normalized to AAV9 control). All documents cited in this specification are incorporated herein by reference. US Provisional Patent Application No. 63/387, 943, filed December 17, 2022, and US Provisional Patent Application No. 63/ 14,401 , filed July 19, 2023, are incorporated herein by reference in their entireties. While the invention has been described with reference to particular embodiments, it will be appreciated that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of tire appended claims.

Claims

WHAT IS CLAIMED IS:

1. A recombinant adeno-associated virus particle (rAAV) comprising:

(a) an adeno-associated virus (AAV) capsid comprising VP 1 proteins, VP2 proteins, and VP3 proteins, wherein the capsid proteins have amino acid sequences comprising a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises “Xn - n-mer - Xm”, wherein:

(i) Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid;

(ii) the n-mer is GQVRVGV (SEQ ID NO: 3), MDAHYVR (SEQ ID NO: 4), TQAVPLK (SEQ ID NO: 5), VVHQTGL (SEQ ID NO: 6), or MAISRER (SEQ ID NO: 7), or an n-mer sequence of at least 6. at least 7. or the full-length consecutive amino acids of any one of the n-mers; and

(iii) Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid; and

(b) a vector genome packaged in the AAV capsid, wherein the vector genome comprises a nucleic acid sequence encoding a gene product under control of sequences which direct expression thereof.

2. The rAAV of claim 1. wherein the exogenous targeting peptide comprises n-mer GQVRVGV

(SEQ ID NO: 3).

4. The rAAV of any one of claims 1 to 3, wherein the exogenous targeting peptide is inserted between any two contiguous amino acids in the hypervariable region VIII (HVRVIII) or IV (HVRIV) at a suitable location of a parental AAV capsid.

5. The rAAV of claim 4. wherein tire parental AAV capsid is selected from AAV9. AAV8. AAV7, AAV6, AAV5, AAV4, AAV3, AAV1, AAVhu68, AAVhu95. AAVhu96, and AAVrh91.

6. The rAAV of any one of claims 1 to 4, wherein the exogenous targeting peptide is inserted in the hypervariable region between amino acids 588 and 589 in an AAV9 capsid, as determined based on the numbering of VP1 amino acid sequence of SEQ ID NO: 25. or is inserted in an analogous position in an AAV8, AAV7, AAV6, AAV5, AAV4, AAV3, AAV1, AAVhu68, AAVhu95, AAVhu96, or AAVrh91 capsid.

7. The rAAV of any one of claims 1 to 4 or 6, wherein the exogenous targeting peptide is immediately preceded by “AQ”.

8. The rAAV of any one of claims 1 to 6, wherein the rAAV capsid comprises VP3 proteins having a mutant VP3 region of: amino acids 204 to 743 of SEQ ID NO: 80, further comprising highly deamidated residues at positions N57, N329, N452 and/or N512, wherein the deamidated position numbers are based on the residue positions of SEQ ID NO: 25 or 26.

9. The rAAV of any one of claims 1 to 6, wherein the rAAV capsid comprises VP1 proteins, VP2 proteins, and VP3 proteins having a mutant VP1 of: amino acids 1 to 743 of SEQ ID NO: 80. further comprising highly deamidated residues at positions N57, N329, N452 and N512. wherein tire deamidated position numbers are based on the residue positions of SEQ ID NO: 25 or 26.

10. The rAAV of any one of claims 1 to 5, wherein the n-mer is encoded by the nucleic acid sequence of SEQ ID NOs: 78, or a sequence at least 95% identical thereto.

11. A composition comprising a stock of the rAAV of any one of claims 1 to 10, and one or more of a physiologically compatible carrier, excipient, and/or aqueous suspension base.

12. A recombinant muscle cell-targeting peptide comprising “Xn - n-mer - Xm”, wherein:

(a) Xn is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid;

(b) the n-mer is GQVRVGV (SEQ ID NO: 3). MDAHYVR (SEQ ID NO: 4), TQAVPLK (SEQ ID NO: 5), VVHQTGL (SEQ ID NO: 6), or MAISRER (SEQ ID NO: 7), or an n-mer sequence of at least 6, at least 7, of a the full-length consecutive amino acids of any one of the n-mers; and (c) Xm is 0. 1, 2, or 3 amino acid/s independently selected from any amino acid: optionally wherein the muscle cell-targeting peptide is conjugated to a nanoparticle, a second molecule, or a viral capsid protein.

13. The recombinant muscle cell-targeting peptide of claim 12, wherein the n- mer is GQVRVGV (SEQ ID NO: 3).

14. The recombinant muscle targeting peptide of claim 12, wherein the recombinant muscle cell-targeting peptide targets cardiac muscle cell.

15. The recombinant muscle cell-targeting peptide of claim 12, wherein the recombinant muscle cell-targeting peptide targets skeletal a muscle cell, optionally a gastrocnemius muscle cell.

16. A composition comprising tire recombinant muscle cell-targeting peptide of any one of claims 12 to 15, and one or more of a physiologically compatible carrier, excipient, and/or aqueous suspension base.

17. A nucleic acid molecule encoding tire recombinant muscle cell-targeting peptide of any one of claims 12 to 15.

18. A nucleic acid molecule comprising a nucleic acid sequence encoding a mutant AAV capsid VP1 comprising n-mer GQVRVGV (SEQ ID NO: 3).

19. The nucleic acid molecule of claim 18, wherein the sequence encoding tire n-mer is SEQ ID NO 78 or a sequence at least 99% identical thereto (encoding GQVRVGV).

20. The nucleic acid molecule of claim 18 or 19, wherein the nucleic acid sequence encoding the AAV capsid VP1 is SEQ ID NO 79.

21. A fusion polypeptide or protein comprising the recombinant muscle celltargeting peptide according to any one of claims 12 to 15 and a fusion partner which comprises at least one polypeptide or protein.

22. A composition comprising the fusion polypeptide or protein of claim 21 and one or more of a physiologically compatible carrier, excipient, and/or aqueous suspension base.

23. Use of a stock of the rAAV of any one of claims 1 to 10, the recombinant muscle cell-targeting peptide of any one of claims 12 to 15, or the fusion polypeptide or protein of claim 16, or the composition of any one of claims 11, 16, or 22 for delivering a gene product to a patient in need thereof.

24. A method for targeted therapy to muscle cells in a subject in need thereof, the method comprising administering to the subject a stock of the rAAV of claim 1.

25. A method for targeted therapy to muscle cells in a subject in need thereof, the method comprising administering to the subject a stock of the rAAV of claim 1, wherein the gene product is targeted for delivery to muscle cell, optionally wherein the muscle cells are cardiac muscle cells and/or skeletal muscle cells, optionally gastrocnemius muscle cells.

26. A method for treating a muscle cell disorder and/or a disease in a subject in need thereof, the method comprising delivering to the subject the rAAV of claim 1, wherein the encoded gene product is a protein, optionally an antibody.