Compositions and methods for treating dystrophin mediated diseases

Abstract

Provided herein are compositions and methods for treating dystrophin mediated diseases, such as, Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD).

Description

[0001] Attorney Docket No. GMA-068WO

[0002] COMPOSITIONS AND METHODS FOR TREATING DYSTROPHIN MEDIATED DISEASES CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims the benefit of and priority to U. S. Provisional Patent Application No. 63 / 748,906 filed on January 23, 2025, the entire disclosure of which is hereby incorporated by reference in its entirety for all purposes.

[0004] BACKGROUND OF THE INVENTION

[0005] The dystrophin -deficient muscle diseases include Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). They are among the most common genetic diseases and affect more than one male infant in every 3,000 newborns. The progressive muscle degeneration and weakness usually confine the patients to wheelchairs by their early teens, and lead to death by their early twenties. Women carriers are also affected in their 40s to 50s. The above mentioned muscle diseases are caused by mutations in the dystrophin gene, specifically, the X-linked recessive mutations. The dystrophin gene is the largest gene known to date, which spans nearly 2.4 million base-pairs on the X-chromosome with 79 exons, a coding sequence of about 11.5 kb, and a high rate of de novo mutations.

[0006] Currently, there is no effective treatment for dystrophin mediated diseases. The large size of the gene make it difficult to simply replace the gene via gene therapy treatments. Gene therapy vectors encoding for mini-dystrophins have been tested, but have not been ideal drug candidates because of expression and immunogenicity caused by the encoded protein. Accordingly, there is still a need for treatments for such diseases. The present embodiments fulfills these needs as well as others.

[0007] There remains a need in the art for vectors that can specifically target selected tissue and cell types.

[0008] SUMMARY OF THE INVENTION

[0009] Provided for herein are mini -dystrophin polypeptides and methods of using the same. In some embodiments, polypeptides are provided comprising an amino acid sequence comprising: i) an N-terminal portion of the ABDI domain from the dystrophin gene; a hinge region of exon 8-11 from the utrophin gene, or a fragment

[0010] 1

[0011] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0012] thereof; a Spectrin 1 domain of the dystrophin or utrophin protein; and a spectrin 16-CT udys5 domain.

[0013] In some embodiments, nucleic acid molecules are provided encoding the same. In some embodiments, vectors comprising nucleic acid molecules provided for herein are provided.

[0014] In some embodiments, lipid nanoparticles comprising polypeptides provided herein are provided or comprising nucleic acid molecules encoding the same.

[0015] In some embodiments, recombinant adeno-associated virus (rAAV) are provided comprising an adeno-associated virus (AAV) capsid and packaged therein a vector genome, wherein the vector genome comprises: (a) an AAV 5' inverted terminal repeat (ITR), (b) an expression cassette comprising a coding sequence for polypeptide provided for herein, or a sequence that is at least 80% identical to the polypeptides provided for herein, wherein the coding sequence is operably linked to expression control sequences which direct expression of the polypeptide, and (c) an AAV 3' ITR.

[0016] In some embodiments, methods of using such polypeptides, nucleic acid molecules, vectors, and compositions are provided. In some embodiments, isolated cells comprising a nucleic acid molecule provided for herein are provided

[0017] These and other embodiments and advantages of the embodiments provided herein will be apparent from the specification, including, without limitation, the detailed description of the invention.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 illustrates various embodiments provided for herein.

[0020] Figure 2 illustrates various embodiments provided for herein.

[0021] Figure 3 illustrates various embodiments provided for herein.

[0022] Figure 4 illustrates various embodiments provided for herein.

[0023] Figure 5 illustrates various embodiments provided for herein.

[0024] Figure 6 illustrates various embodiments provided for herein.

[0025] Figure 7 illustrates various embodiments provided for herein.

[0026] 2

[0027] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0028] DETAILED DESCRIPTION

[0029] Embodiments provided for herein provide a dystrophin variant polypeptide that can be used to treat dystrophin mediated diseases, including, but not limited to Duchenne muscular dystrophy (DMD) and / or Becker Muscular Dystrophy (BMD). Without being bound to any particular theory, both DMD and BMD are caused by the misexpression of wild-type dystrophin protein. To overcome this defect, therapies have been tested to increase the expression of dystrophin, most notably through gene therapy vectors expressing version of dystrophin. Dystrophin is difficult to replace by gene therapy due to the size of the gene and the encoded protein. Dystrophin is believed to be the largest human gene, encompassing about 2.4 megabases. To circumvent this problem, minidystrophin gene products have been produced and tested, but none have been sufficient. Expressing the gene in therapy can lead to an immunogenic protein due to factors that are not well understood. A hotspot for such immunogenic epitopes is located in the region encoded by exons 8 to 11, including the hinge 1 domain which, without being bound to any particular theory, is believed to be driving most of the immunogenicity due to poor identity with utrophin. As provided for herein, the variant mini-dystrophin gene and gene product can be utilized that has reduced immunogenicity, thereby facilitating the expression and function of what would be a hybrid mini utrophin-dystrophin protein if produced in sufficient amounts to avoid the muscle issues seen in patients with DMD and BMD.

[0030] Accordingly, provided for herein, is a variant mini-dystrophin polypeptide or mini-dystrophin-utrophin hybrid that supplements the dystrophin function without the immunogenicity seen with other dystrophin gene therapies. Accordingly, in some embodiments, polypeptides are provided that comprise an amino acids sequence comprising, i) an N-terminal portion of the ABDI domain from the dystrophin gene, ii) the amino acid fragment encoded by exons 8-11 from the utrophin gene, or a fragment thereof; iii) a Spectrin 1 domain of the dystrophin protein or utrophin, and iv) and the amino acids of the spectrinl6-CT dystrophin domains. In some embodiments, the polypeptide comprises a functional udys that prevents myofiber degeneration.

[0031] In some embodiments, the fragment of the exon 8-11 from the utrophin gene comprises the hinge 1 and Spectrin 1 region of the utrophin gene. In some embodiments, the polypeptide comprises the Spectrin 1 region of the dystrophin protein. The hinge region can be present in the polypeptide in multiple place and still function without disrupting the function of the dystrophin gene product. In some embodiments, the hinge

[0032] 3

[0033] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0034] region fragment is positioned at the C-terminus of the N-terminal portion of the of the ABDI domain from the dystrophin gene. In some embodiments, the hinge region is inserted between the C-terminus of the N-terminal portion of the of the ABDI domain from the dystrophin gene and the N-terminus of the spectrin 16-CT udys5 domain.

[0035] In some embodiments, the polypeptide has a formula of Z1-Z2-Z3-Z4, wherein: Z1 comprises an amino acid sequence is an N-terminal portion of the ABDI domain from the dystrophin gene, Z2 comprises an amino acid sequence comprising an amino acid hinge fragment encoded by exons 8-11 from the utrophin gene, or a fragment thereof; Z3, which can be present or absent, wherein when present is a a Spectrin 1 domain of the dystrophin protein, and Z4 comprises an the amino acid of the spectrin 16-CT udys5 domain. The difference domains (i.e., Zi, Z2, Z3, and Z4) can be directly linked to one other or have peptide linkers, such as, but not limited to, glycine / serine or glycine / alanine linkers.

[0036] However, in some embodiments, there are no exogenous peptide linkers linking the different domains.

[0037] In some embodiments, wherein the N-terminal portion of the ABDI domain (actin-binding domain 1) from the dystrophin gene comprises the amino acid sequence of:

[0038] MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLT GQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNI IL HWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQVNVINFTTSWSDGLALNALIH SHRPDLFDWNSWCQQSATQRLEHAFNIARYQLGIEKLLDPE (SEQ ID NO: 1 ).

[0039] In some embodiments, wherein the N-terminal portion of the ABDI domain from the dystrophin gene comprises the amino acid sequence of:

[0040] MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLT GQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNI IL HWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQVNVINFTTSWSDGLALNALIH SHRPDLFDWNSWCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYI TSLFQVLP (SEQ ID NO: 2 ).

[0041] In some embodiments, the N-terminal methionine is not present when the protein is expressed in the cell, but it can be encoded by the expression cassette to provide the polypeptide to the subject. In some embodiments, the expression cassette comprises a nucleic acid molecule encoding such sequence without the N-terminal methionine.

[0042] In some embodiments, the Spectrin 1 domain from the dystrophin gene comprises the amino acid sequence of:

[0043] VNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEWKDQFHTHEGYMMDLTAHQGRV GNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLH (SEQ ID NO: 3 )

[0044] 4

[0045] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0046] In some embodiments, the polypeptide of any one of claims 1-6, wherein the exon 8-11 from the utrophin gene, or a fragment thereof, comprises the amino acid sequence of, or a fragment thereof of:

[0047] DVAVQLPDKKSIIMYLTSLFEVLPQQVTIDAIREVETLPRKYKKECEEEAINIQSTAP EEEHESPRAETPSTVTEVDMDLDSYQIALEEVLTWLLSAEDTFQEQDDISDDVEEVKD QFATHEAFMMELTAHQSSVGSVLQAGNQLITQGTLSDEEEFEIQEQMTLLNARWEALR VESMDRQSRLH (SEQ ID NO: 4 ).

[0048] In some embodiments, the polypeptide of any one of claims 1-6, wherein the exon 8-11 from the utrophin gene, or a fragment thereof, comprises the amino acid sequence of, or a fragment thereof of:

[0049] QQVTIDAIREVETLPRKYKKECEEEAINIQSTAPEEEHESPRAETPSTVTEVD (SEQ ID NO: 5 ).

[0050] In some embodiments, the spectrin 16-CT domain comprises the amino acid sequence of SYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQEESLKNIKDSLQQSSGR IDI IHSKKTAALQSATPVERVKLQEALSQLDFQWEKVNKMYKDRQGRFDRSVEKWRRF HYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTWRTLNATGE EIIQQSSKTDASILQEKLGSLNLRWQEVCKQLSDRKKRLEEQSDQWKRLHLSLQELLV WLQLKDDELSRQAPIGGDFPAVQKQNDVHRAFKRELKTKEPVIMSTLETVRIFLTEQP LEGLEKLYQEPRELPPEERAQNVTRLLRKQAEEVNTEWEKLNLHSADWQRKIDETLER LQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVS HVNDLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQH FLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTA MKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNN LVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASS TGFCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLD WMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPI IGFRYRSLKHFNYDICQSCF FSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQT VLEGDNMETDTM (SEQ ID NO: 6),

[0051] or a fragment thereof.

[0052] In some embodiments, the polypeptides, which can be referred to herein as a dystrophin gene product, or variant thereof, comprises the amino acid sequence of:

[0053] MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLT GQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNI IL HWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQVNVINFTTSWSDGLALNALIH SHRPDLFDWNSWCQQSATQRLEHAFNIARYQLGIEKLLDPEDVAVQLPDKKSIIMYL TSLFEVLPQQVTIDAIREVETLPRKYKKECEEEAINIQSTAPEEEHESPRAETPSTVT EVDMDLDSYQIALEEVLTWLLSAEDTFQEQDDISDDVEEVKDQFATHEAFMMELTAHQ SSVGSVLQAGNQLITQGTLSDEEEFEIQEQMTLLNARWEALRVESMDRQSRLHSYVPS TYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQEESLKNIKDSLQQSSGRIDIIH SKKTAALQSATPVERVKLQEALSQLDFQWEKVNKMYKDRQGRFDRSVEKWRRFHYDIK IFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTWRTLNATGEEIIQQ SSKTDASILQEKLGSLNLRWQEVCKQLSDRKKRLEEQSDQWKRLHLSLQELLVWLQLK DDELSRQAPIGGDFPAVQKQNDVHRAFKRELKTKEPVIMSTLETVRIFLTEQPLEGLE KLYQEPRELPPEERAQNVTRLLRKQAEEVNTEWEKLNLHSADWQRKIDETLERLQELQ EATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVSHVNDL ARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHFLSTS VQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRR

[0054] 5

[0055] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0056] LQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQI INCLTTI YDRLEQEHNNLVNVP LCVDMCLNWLLNVYDTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCD QRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLE PQSMVWLPVLHRVAAAETAKHQAKCNICKECPI IGFRYRSLKHFNYDICQSCFFSGRV AKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGD NMETDTM (SEQ ID NO: 7).

[0057] In some embodiments, the polypeptides, which can be referred to herein as a dystrophin gene product, or variant thereof, comprises the amino acid sequence of:

[0058] MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLT GQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNI IL HWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQVNVINFTTSWSDGLALNALIH SHRPDLFDWNSWCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYI TSLFQVLPQQVTIDAIREVETLPRKYKKECEEEAINIQSTAPEEEHESPRAETPSTVT EVDVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEWKDQFHTHEGYMMDLTAHQ GRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHSYVPS TYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQEESLKNIKDSLQQSSGRIDIIH SKKTAALQSATPVERVKLQEALSQLDFQWEKVNKMYKDRQGRFDRSVEKWRRFHYDIK IFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTWRTLNATGEEIIQQ SSKTDASILQEKLGSLNLRWQEVCKQLSDRKKRLEEQSDQWKRLHLSLQELLVWLQLK DDELSRQAPIGGDFPAVQKQNDVHRAFKRELKTKEPVIMSTLETVRIFLTEQPLEGLE KLYQEPRELPPEERAQNVTRLLRKQAEEVNTEWEKLNLHSADWQRKIDETLERLQELQ EATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVSHVNDL ARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHFLSTS VQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRR LQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQI INCLTTI YDRLEQEHNNLVNVP LCVDMCLNWLLNVYDTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCD QRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLE PQSMVWLPVLHRVAAAETAKHQAKCNICKECPI IGFRYRSLKHFNYDICQSCFFSGRV AKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGD NMETDTM (SEQ ID NO: 8 ).

[0059] In addition to the polypeptides provided for herein, variants of such polypeptides area also provided. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8. In some embodiments, the variant retains the actin binding function of the ABDI domain.

[0060] The polypeptides can be delivered or administered to subjects (patients) as provided herein. For example, an expression cassette comprising a nucleic acid molecule encoding the polypeptides provided for herein can be administered to the subject to a dystrophin mediated disease, such as, but not limited to, DMD and BMD. The expression cassette can be part of a viral vector, such as an recombinant AAV vector (rAAV), which are utilized in gene therapy treatments. Non-limiting examples of such (rAAV) vectors are provided herein and any such vectors can be combined with nucleic acid molecules encoding the polypeptides provided for herein. As provided for herein, the rAAV can comprise a capsid that has a targeting peptide. The targeting peptide can be used to target

[0061] 6

[0062] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0063] the rAAV to a specific tissue or cell type, such as muscle, which includes skeletal muscle. The targeting peptides can be an RGD based targeting peptide. Non-limiting examples of RGD peptides that can be incorporated into the rAAV are also provided for herein.

[0064] Accordingly, in some embodiments, provided herein are nucleic acid molecules encoding the polypeptides provided for herein. The nucleic acid molecule can be DNA or RNA, such as mRNA. In some embodiments, the nucleic acid molecule comprises a is a codon optimized nucleotide sequence. In some embodiments, the codon optimized nucleotide sequence is optimized for expression in human cells, such as muscle cells, including but not limited to skeletal muscle cells. In some embodiments, the nucleic acid molecule encoding the polypeptides provided for herein comprise the nucleic acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12. However, because the genetic code has built in degeneracy, any nucleic acid molecule can be used to encode for the polypeptides provided for herein. Thus, the nucleic acid molecules of SEQ ID NO: 11 and SEQ ID NO: 12 are non-limiting examples that can be utilized. As provided for herein, the nucleic acid molecules can be incorporated into an expression cassette and then delivered via a vector, which can be either non-viral or viral vector, such as, but not limited to an rAAV. Nonlimiting examples of which are provided for herein. In some embodiments, the codon optimized genome comprises the nucleic acid sequence of SEQ ID NO: 9 (NTDIudys5co) or SEQ ID NO: 10 (HIDIudys5co) or a sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the same.

[0065] 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' 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' to) a gene sequence, e.g., 3' untranslated region (3″ UTR) comprising a polyadenylation site, among other elements. In

[0066] 7

[0067] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0068] some embodiments, the 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 some 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 are inserted into a target cell.

[0069] 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 some embodiments, a vector genome may contain two or more expression cassettes. In some embodiments, the expression cassette encoding the dystrophin constructs provided for herein is delivered in a non- viral vector, such as a lipid nanoparticle. The expression cassette can be a DNA or mRNA molecule when encompassed by a lipid nanoparticle.

[0070] In some embodiments, the transgene provided for herein 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 or a mutant gene is expressed, which leads to dysfunction. Alternatively, the transgene may provide a product to a cell which is not natively expressed in the cell type or in the host. In some embodiments, the transgene sequence encodes a therapeutic protein, such as the dystrophin polypeptides provided for herein, or polypeptide which is expressed in a host cell.

[0071] As used herein, a “vector genome” refers to the nucleic acid sequence packaged inside a parvovirus (e.g., rAAV) capsid which forms a viral particle. Such a nucleic acid sequence contains AAV inverted terminal repeat sequences (ITRs). In the examples herein, a vector genome contains, at a minimum, from 5' to 3', an AAV 5' ITR, coding sequence(s) (i.e., transgene(s)), and an AAV 3' ITR. ITRs from AAV2, a different source AAV than the capsid, or other than full-length ITRs may be selected. In some embodiments, the ITRs are from the same AAV source as the AAV which provides the rep function during production or a transcomplementing AAV. Further, other ITRs, e.g., self-complementary (scAAV) ITRs, may be used. Both single-stranded AAV and self-complementary (sc) AAV are

[0072] 8

[0073] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0074] encompassed with 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 genome” contains, at a minimum, from 5' to 3', a vector-specific sequence, a nucleic acid sequence encoding the mini-dystrophin gene 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. The vector genome can also be packaged in other viruses that can be used to deliver the transgene to the cell in the subject.

[0075] In some embodiments, the AAV sequences of the vector typically comprise the cisacting 5' and 3' inverted terminal repeat 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 bp in length. In some embodiments, 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 the 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., 70:520532 (1996)). An example of such a molecule employed provided for herein 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. In some embodiments, the ITRs are from an AAV different than that supplying a capsid. In some embodiments, the ITR sequences from AAV2. However, ITRs from other AAV sources may be selected. A shortened version of the 5' ITR, termed MITR, has been described in which the D-sequence and terminal resolution site (trs) are deleted. In some 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 length of 145 base pairs during vector DNA amplification using the internal (A') element as a template. In other

[0076] 9

[0077] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0078] embodiments, full-length AAV 5' and 3' ITRs are used. Where the source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be termed pseudotyped. However, other configurations of these elements may be suitable.

[0079] In some embodiments, provided herein is a recombinant adeno-associated virus (rAAV) comprising an adeno-associated virus (AAV) capsid and packaged therein a vector genome, wherein the vector genome comprises: (a) an AAV 5' inverted terminal repeat (ITR), (b) an expression cassette comprising at least one open reading frame (ORF) comprising a coding sequence for dystrophin product provided for herein, which coding sequence is operably linked to expression control sequences which direct expression of the dystrophin product, and (c) an AAV 3' ITR. In some embodiments, the coding sequence comprises a nucleic acid molecule as provided for herein or one that is it at least 70, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the same.

[0080] In addition to the major elements identified for the recombinant AAV vector provided for herein, the vector also includes conventional control elements (i.e., regulatory sequences) necessary which are operably linked to the 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. As described herein, regulatory elements comprise but not limited to: promoter: enhancer: transcription factor: transcription terminator: efficient RNA processing signals such as splicing and polyadenylation signals (poly A): 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).

[0081] The regulatory control elements, in some embodiments, contain a promoter sequence as part of the expression control sequences, e.g., located between the 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.

[0082] In some embodiments, the promoter is a muscle specific promoter. In some embodiments, the muscle specific promoter is the spc5-12 promoter. In some embodiments, the spc5-12 promoter comprises the sequence of:

[0083] 10

[0084] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0085] C C GC C T T C GGC AC C AT C C T C AC GAC AC C CAAAT AT GGC GAC GGGT G AGGAAT GGT GGGGAGT AT T T T T AGAGC GGT GAGGAAGGT GGGC AG GC AGC AGGT GTTGGCGCTC TAAAAATAAC T C C C GGGAGT T AT T T T T AGAGC GGAGGAAT GGT GGAC AC C CAAAT AT GGC GAC GGT T C C T C AC CCGTCGCCATATTTGGGTGTCCGCCCTCGGCCGGGGCCGCATTCCT GGGGGCCGGGCGGTGCTCCCGCCCGCCTC GAT AAAAGGC T C C GGGG CCGGCGGCGGCCCAC GAGC T AC C C GGAGGAGC GGGAGGC GC C AAGC TCTAGA (SEQ ID NO: 13 )

[0086] In some embodiments, the spc5-12 promoter comprises the sequence of:

[0087] CGGCCGTCCGCCTTCGGCACCATCCTCACGACACCCAAATATGGCG AC GGGT GAGGAAT GGT GGGGAGT T AT T T T T AGAGC GGT GAGGAAGG T GGGC AGGC AGC AGGT GTTGGCGCTC TAAAAATAAC T C C C GGGAGT T AT T T T T AGAGC GGAGGAAT GGT GGAC AC C CAAAT AT GGC GAC GGT TCCTCACCCGTCGCCATATTTGGGTGTCCGCCCTCGGCCGGGGCCG CATTCCTGGGGGCCGGGCGGTGCTCCCGCCCGCCTCGATAAAAGGC TCCGGGGCCGGCGGCGGCC C AC GAGC T AC C C GGAGGAGC GGGAGGC GCCAAGCTCTAGA (SEQ ID NO: 14 )

[0088] In some embodiments, the spc5-12 promoter comprises the sequence of:

[0089] TGGCCACCGCCTTCGGCAC CAT C C T C AC GAC AC C CAAAT AT GGC GA C GGGT GAGGAAT GGT GGGGAGT T AT T T T T AGAGC GGT GAGGAAGGT GGGC AGGC AGC AGGT GTTGGCGCTC TAAAAATAAC T C C C GGGAGT T AT T T T T AGAGC GGAGGAAT GGT GGAC AC C CAAAT AT GGC GAC GGT T CCTCACCCGTCGCCATATTTGGGTGTCCGCCCTCGGCCGGGGCCGC ATTCCTGGGGGCCGGGCGGTGCTCCCGCCCGCCTCGATAAAAGGCT CCGGGGCCGGCGGCGGCCCAC GAGC T AC C C GGAGGAGC GGGAGGC G CCAAGCTCTAGA (SEQ ID NO: 15 )

[0090] Other muscle specific promoters are suitable and can be interchanged with the promoters provided for herein. In some embodiments, the promoters are as provided for in U. S. Patent No. 10,731,177, which is hereby incorporated by reference in its entirety.

[0091] Examples of constitutive promoters suitable for controlling expression of the therapeutic products (e.g., the dystrophin product) include, but are not limited to chicken beta-actin (CB) promoter, CB7 promoter (also referred to as CB7 hybrid promoter comprising a cytomegalovirus immediate early (CMV IE) enhancer, optionally a linker sequence, and a chicken beta-actin 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 P-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 some embodiments, the

[0092] 11

[0093] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0094] CB promoter comprises nucleic acid sequence of SEQ ID NO: 16 as provided for in U. S. Patent Application Publication No. 20240269328, which is hereby incorporated by reference in its entirety. In some embodiments, the CB7 hybrid promoter comprises a CMV IE enhancer comprising nucleic acid sequence of SEQ ID NO: 15 as provided for in U. S. Patent Application Publication No. 20240269328, which is hereby incorporated by reference in its entirety, optionally a linker sequence, and a CB promoter comprising nucleic acid sequence of SEQ ID NO: 16 as provided for in U. S. Patent Application Publication No. 20240269328, which is hereby incorporated by reference in its entirety. In some embodiments, the CB7 hybrid promoter comprises nucleic acid sequence of SEQ ID NO: 10 as provided for in U. S. Patent Application Publication No. 20240269328, which is hereby incorporated by reference in its entirety.

[0095] In some embodiments, 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 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 U. S. Pat. 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.

[0096] 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 (R Mammalian Inducible Expression System (New England Biolabs, Ipswich, MA).

[0097] Still other promoter systems may include response elements including but not limited to a tetracycline (tet) response element (such as described by Gossen & Bujard

[0098] 12

[0099] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0100] (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. These response elements may include, a hypoxia response element (HRE) that binds HIF-la and B, 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).

[0101] Using such promoters, expression of the transgene 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.

[0102] USA.102(39): 13789-94); and the humanized tamoxifen-dep regulatable system (Roscilli et al., 2002, Mol. Then 6(5):653-63).

[0103] In some embodiments, 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.

[0104] 6,015,709, 6,117,680, 6,479,653, 6,187,757, and 6,649,595, U. S. Publication No.

[0105] 2002 / 0173474, U. S. Publication No. 200910100535, U. S. Pat. Nos. 5,834,266, 7,109,317, 7,485,441, 5,830,462, 5,869,337, 5,871,753, 6,011,018, 6,043,082, 6,046,047, 6,063,625, 6,140,120, 6,165,787, 6,972,193, 6,326,166, 7,008,780, 6,133,456, 6,150,527, 6,506,379, 6,258,823, 6,693,189, 6,127,521, 6,150,137, 6,464,974, 6,509,152, 6,015,709, 6,117,680, 6,479,653, 6,187,757, 6,649,595, 6,984,635, 7,067,526, 7,196,192, 6,476,200, 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

[0106] 13

[0107] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0108] designed to be induced by rapamycin and analogs thereof referred to as “rapalogs”.

[0109] Examples of suitable rapamycins are provided in the documents listed above in connection with the description of the ARGENT™ system. In some embodiments, 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 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 API 889, with designed ‘bumps’ that minimize interactions with endogenous FKBP. Still other rapalogs may be selected, e.g., AP23573 [Merck], In some embodiments, rapamycin or a suitable analog may be delivered locally or systemically to the AAV-transfected cells.

[0110] In some embodiments, the regulatory elements comprise an enhancer. In some embodiments, suitable enhancers include those that are appropriate for a desired target tissue indication. In some embodiments, the expression cassette comprises one or more expression enhancers. In some embodiments, 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 cytomegalovirus immediate early (CMV IE) enhancer. This enhancer may be present in two copies which are located adjacent to one another. Alternatively, the dual copies of the enhancer may be separated by one or more sequences. In a further embodiment, the enhancer(s) is selected from one or more of an APB enhancer, an ABPS enhancer, an alpha mic / bik enhancer, a TTR enhancer, an en34 enhancer, an ApoE enhancer, a CMV enhancer, or an RSV enhancer. In yet another embodiment, the regulatory elements comprise an intron. In a further embodiment, the intron is selected from chicken beta actin intron (CBA), human beta globin, IVS2, SV40, bGH, alpha-globulin, beta-globulin, collagen, ovalbumin, or p53. In some embodiments, the chicken beta actin intron comprises nucleic acid sequence as provided for in WO 2011 / 126808, which is hereby incorporated by reference in its entirety. In some embodiments, the regulatory elements comprise a polyA. In a further embodiment, the polyA is a synthetic polyA or from bovine growth hormone (bGH), human growth

[0111] 14

[0112] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0113] hormone (hGH), SV40, rabbit B-globin (also referenced as rabbit beta-globin or RBG), or modified RGB (mRBG). In another embodiment, the regulatory elements may comprise a WPRE sequence, which may be engineered upstream of the polyA sequence and downstream of the coding sequence. 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 U. S. Pat. Nos. 6,136,597, 6,287,814, and 7,419,829, which are incorporated by reference). In some 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. See also, Kingsman S. M., Mitrophanous K., & Olsen J. C. (2005), Potential Oncogene Activity of the Woodchuck Hepatitis Post-Transcriptional Regulatory Element (Wpre).” Gene Ther. 12(l):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 Why-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. A WPRE mutated element can also be used. In other embodiments, enhancers are selected from a non-viral source. In some embodiments, no WPRE sequence is present. In yet another embodiment, the regulatory elements comprise a Kozak sequence.

[0114] In some embodiments, the expression cassette comprises regulatory elements which direct expression of a sequence encoding one or more elements of a gene replacement system for delivering the dystrophin gene products provided for herein. In some embodiments, the regulatory elements comprise one or more promoters. In some embodiments, the expression cassette includes a constitutive or a regulatable promoter. In some embodiments, the promoter is a tissue-specific (e.g., muscle specific) promoter.

[0115] In some embodiments, the expression cassette may include miRNA target sequences in the untranslated region(s). The miRNA target sequences are designed to be specifically recognized by miRNA present in cells in which transgene expression is undesirable and / or reduced levels of transgene expression are desired. In some embodiments, the expression cassette includes miRNA target sequences that specifically reduce expression of the dystrophin gene products provided for herein in non-desired tissues. In some embodiments, the miRNA target sequences are located in the 3' UTR, 5' UTR, and / or in both 3' and 5' UTR. In some embodiments, the miRNA target sequences are operably linked to the regulatory sequences in the expression cassette. In some

[0116] 15

[0117] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0118] embodiments, the expression cassette comprises at least two tandem repeats of non-desired tissue specific miRNA target sequences, wherein the at least two tandem repeats comprise at least a first miRNA target sequence and at least a second miRNA target sequence which may be the same or different. In some embodiments, the tandem miRNA target sequences are continuous or are separated by a spacer of 1 to 10 nucleic acids, wherein said spacer is not a miRNA target sequence.

[0119] In some embodiments, the miRNA binding site is complementary to a miRNA 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.

[0120] As another non-limiting example, a vector genome (expression cassette) may comprise miR- 122 miRNA to modulate, e.g., reduce, the expression of a gene product in the liver. In some embodiments, the vector genome (expression cassette) may comprise a miRNA, e.g., a miR-142-3p, to modulate, e.g., reduce, the expression, of the gene product 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 miRNA may include, e.g., miR-206 (skeletal muscle), miR-018b, miR-431. See. e.g., Kim., H. K., Muscle-specific microRNA miR-206 promotes muscle differentiation, The Journal of Cell Biology, 2006, 174(5):677-687; WO 2019 / 035690A1; WO 2019 / 035690A1; WO 2022 / 147181A1, and Brazilian Patent Publication No. BR102018067702A2, which are all incorporate herein by reference.

[0121] It should be understood that the compositions in the expression cassettes described herein are intended to be applied to the compositions and methods described across the specification.

[0122] In some embodiments, the rAAV. dystrophin, which refers to an recombinant AAV vector encoding for the dystrophin products or polypeptides provided for herein, comprises a capsid which targets muscle cells, such as in the quadriceps, heart, or other skeletal muscles. This may be done via direct delivery to the muscle. Alternatively, other routes of delivery may be utilized. In some embodiments, the rAAV comprises the vector genome which comprises muscle specific promoter, such as those provided for herein promoter for use in targeting skeletal muscles.

[0123] 16

[0124] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0125] In some embodiments, the AAV capsid is a natural Clade F AAV, e.g., AAV9, hu31, hu32, hu95, or AAVhu68. Alternatively, engineered or mutant Clade F capsids may be selected. Methods of generating vectors having the AAV9 capsid or AAVhu68 capsid, and / or chimeric capsids derived from AAV9 have been described. See, e.g., U. S. Pat. No.

[0126] 7,906,111, which is incorporated by reference herein. See also, WO 2022 / 082109, published Apr. 21, 2022, which is incorporated herein by reference. In one embodiment, the rAAV comprises an AAVhu68 capsid. SEQ ID NO: 16 provides the encoded amino acid sequence of the AAVhu68 vpl protein.

[0127] In some embodiments, the AAV capsid may selected from another clade, e.g., Clade A. In some embodiments, the AAV capsid is selected from capsids which target the CNS (e.g., Clade F AAV (e.g., AAVhu68 or AAV9), Clade E (e.g., AAV8, AAVrhlO, and AAV10, rh74), or certain Clade A AAV (e.g., AAV1, AAVrh91)) capsids. See, e.g., WO 2020 / 223231, published Nov. 5, 2020 (rh91, including table with deamidation pattern), U. S. Provisional Patent Application No. 63 / 065,616, filed Aug. 14, 2020 and U. S.

[0128] Provisional Patent Application No. 63 / 109,734, fded Nov. 4, 2020, and International Patent Application No. PCT / US21 / 45945, filed Aug. 13, 2021 which is now published as WO 2022 / 036220A 1 (published Feb. 17, 2022). In some embodiments, the AAV capsid having reduced capsid deamidation may be selected. See, e.g., PCT / US19 / 19804 (which is now published as WO 2019 / 168961A1, published Sep. 6, 2019) filed Feb. 27, 2019 and PCT / US18 / 19861 (which is now published as WO 2018 / 160533 Al, published Sep. 7, 2018) filed Feb. 27, 2018 and incorporated by reference in their entireties. In some embodiments, AAV capsid is selected from Clade F, E or A as a parental capsid, wherein the selected parental capsid is further modified to include the targeting peptide inserted into a hypervariable region loop of as described in U. S. Provisional Patent Application No. 63 / 178,881, filed Apr. 23, 2021, and International Patent Application No.

[0129] PCT / US22 / 25879, filed Apr. 22, 2022 which are incorporated herein by reference in their entireties. In some embodiments, the targeting peptides are RGD peptides that can be used to target muscle cells, such as present in skeletal muscles. Examples of such peptides are known and also provided for herein.

[0130] The term “AAV” as used herein refers to naturally occurring adeno-associated viruses, adeno-associated viruses available to one of skill in the art and / or in light of the composition(s) and method(s) described herein, as well as artificial AAVs. An adeno-associated virus (AAV) viral vector is an AAV DNase-resistant particle having an AAV

[0131] 17

[0132] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0133] protein capsid into which is packaged expression cassette flanked by AAV inverted terminal repeat sequences (ITRs) for delivery to target cells. An AAV capsid is composed of 60 capsid (cap) protein subunits, VP1, VP2, and VP3, that are arranged in an icosahedral symmetry typically in a ratio of approximately 1: 1: 10 to 1:1:20, depending upon the selected AAV. Various AAVs may be selected as sources for capsids of AAV viral vectors as identified above. See, e.g., US Published Patent Application No. 2007-0036760-Al: US Published Patent Application No. 2009-0197338-Al: EP 1310571. See also, PCT / US19 / 19861, filed Feb. 27, 2019, and PCT / US19 / 19804, filed Feb. 27, 2019. See also, WO 2003 / 042397 (AAV7 and other simian AAV), U. S. Pat. Nos. 7,790,449 and 7,282,199 (AAV8), WO 2005 / 033321 and U. S. Pat. No. 7,906,111 (AAV9), and WO 2006 / 110689, and WO 2003 / 042397 (rh. 10). These documents also describe other AAV which may be selected for generating AAV and are incorporated by reference. Among the AAVs isolated or engineered from human or non-human primates (NHP) and well characterized, human AAV2 is the first AAV that was developed as a gene transfer vector: it has been widely used for efficient gene transfer experiments in different target tissues and animal models. Unless otherwise specified, the AAV capsid, ITRs, and other selected AAV components described herein, may be readily selected from among any AAV, including, without limitation, the AAVs commonly identified as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV8, AAVrh 10, AAVhu37, AAV7M8 and AAVAnc80, AAVrh90 (PCTUS20 / 30273, filed Apr. 28, 2020), AAVrh91 (PCTUS20 / 30266, filed Apr. 28, 2020), and AAVrh92, rh93, and rh91.93 (PCTUS20 / 30281, filed Apr. 28, 2020). See, e.g., WO 2005 / 033321, which is incorporated herein by reference. In one embodiment, the AAV capsid is an AAV9 variant. See, U. S. Provisional Patent Application No. 63 / 119,863, filed Dec. 1, 2020, and International Patent Application No. PCT / US21 / 61312, filed Dec. 1, 2021: US Provisional Patent Application No. 63 / 178,881, filed Apr. 23, 2021, and International Patent Application No.

[0134] PCT / US22 / 25879, filed Apr. 22, 2022: U. S. Provisional Patent Application No.

[0135] 63 / 107,030, filed Oct. 29, 2020, U. S. Provisional Patent Application No. 63 / 214,530, filed Jun. 24, 2021, and International Patent Application No. PCT / US2021 / 057201, filed Oct.

[0136] 29, 2021, which is now published as WO 2022 / 094180 (published May 5, 2022), all of which are incorporated herein by reference. In some embodiments, the capsid protein is designated by a number or a combination of numbers and letters following the term “AAV” in the name of the rAAV vector.

[0137] 18

[0138] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0139] An AAVhu68 capsid is described in WO 2018 / 160582, which incorporated by reference in its entirety herein, and in this detailed description. In some embodiments, an AAVhu68 capsid is further characterized by one or more of the following: AAVhu68 vpl proteins produced by expression from a nucleic acid sequence which encodes the predicted amino acid sequence of 1 to 736 of the following amino acid sequence:

[0140] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLV LPGYKYLGPGNGLDKGEPVNEADAAALEHDKAYDQQLKAGDNPYLK YNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKT APGKKRPVEQSPQEPDSSVGIGKSGAQPAKKRLNFGQTGDTESVPD P Q P I GE P PAAP S GVGS LTMAS GGGAPVADNNE GADGVGS S S GNWHC DSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFG YSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQV KEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFP ADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFS YEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQT LKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGA SSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLI FGKQGTGRDN VDADKVMI TNEEE I KTTNPVATES YGQVATNHQSAQAQAQTGWVQN QGI LPGMVWQDRDVYLQGP IWAKI PHTDGNFHP S PLMGGFGMKHP P PQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKE NSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

[0141] (SEQ ID NO: 16),

[0142] vpl proteins which can be produced from the following nucleic acid sequence (SEQ ID NO: 17),

[0143] AT GGC T GC C GAT GGTTATCTTC C AGAT T GGC T C GAGGAC AAC C T C A GT GAAGGC AT T C GC GAGT GGT GGGC T T T GAAAC C T GGAGC C C C T C A AC C CAAGGCAAAT C AAC AAC AT C AAGAC AAC GCTCGGGGTCTTGTG CTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACAAGG GGGAGC C GGT C AAC GAAGC AGAC GC GGC GGC C C T C GAGC AC GAC AA GGC C T AC GAC C AGC AGC T C AAGGC C GGAGAC AAC C C GT AC C T C AAG T AC AAC C AC GC C GAC GC C GAGT T C C AGGAGC GGC T C AAAGAAGAT A CGTCTTTT GGGGGC AAC C T C GGGC GAGC AGT CTTCCAGGC CAAAAA GAGGC T T C T T GAAC C T C T T GGT C T GGT T GAGGAAGC GGC T AAGAC G GC T C C T GGAAAGAAGAGGC C T GT AGAGC AGT C T C C T C AGGAAC C GG ACTCCTCCGTGGGTATT GGCAAAT C GGGT GC AC AGC C C GC TAAAAA GAGAC T C AAT T T C GGT C AGAC T GGC GAC AC AGAGT C AGT C C C C GAC C C T CAAC C AAT C GGAGAAC C T C C C GC AGC C C C C T C AGGT GT GGGAT CTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAA C GAAGGT GC C GAT GGAGT GGGT AGT T C C T C GGGAAAT T GGC AT TGC GAT T C C C AAT GGC T GGGGGAC AGAGT CAT C AC C AC C AGC AC C C GAA CCTGGGCCCTGCCCACC T ACAAC AAT C AC C T C T ACAAGC AAAT C T C CAAC AGC AC AT C T GGAGGAT C T T CAAAT GAC AAC GCCTACTTCGGC TACAGCACCCCCTGGGGGTATTTT GAC T T CAAC AGAT TCCACTGCC AC T T C T C AC C AC GT GAC T GGC AAAGAC T C AT C AACAAC AAC T GGGG AT T C C GGC C TAAGC GAC T CAAC T T CAAGC T C T T CAAC AT T C AGGT C AAAGAGGT T AC GGACAAC AAT GGAGT C AAGAC C AT C GC T AAT AAC C T T AC C AGC AC GGT C C AGGT C T T C AC GGAC T C AGAC TAT C AGC T C C C GTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCA GC GGAC GT T T T C AT GAT T C C T C AGT AC GGGT AT C TAAC GC T T AAT G AT GGAAGC CAAGC CGTGGGTCGTTCGTCCTTTTACTGCCT GGAAT A TTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAGC T AC GAGT T T GAGAAC GT AC C T T T C CAT AGC AGC T AT GC T C AC AGC C AAAGC C T GGAC C GAC T C AT GAAT C C AC T C AT C GAC C AAT AC T T GT A

[0144] 19

[0145] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0146] C AT CT C CAAAGAC TAT T AAC GGT T C T GGAC AGAAT C AACAAAC G C T AAAAT T C AGT GT GGC C GGAC C C AGC AAC AT GGC T GT C C AGGGAA GAAAC T AC AT AC C T GGAC C C AGC T AC C GACAAC AAC GT GT C T C AAC C AC T GT GAC T C AAAAC AAC AAC AGC GAAT TTGCTTGGCCT GGAGC T TCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGGAC C T GC T AT GGC C AGC C AC AAAGAAGGAGAGGAC CGTTTCTTTCCTTT GT C T GGAT C T T T AAT T T T T GGC AAACAAGGAAC T GGAAGAGAC AAC GTGGATGCGGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAA CTACCAACCCAGTAGCAACGGAGTCCTATGGACAAGTGGCCACAAA C C AC C AGAGT GC C C AAGC AC AGGC GC AGAC CGGCTGGGTT CAAAAC C AAGGAAT AC TTCCGGGTATGGTTT GGC AGGAC AGAGAT GT GT AC C T GC AAGGAC CCATTTGGGC CAAAAT T C C T C AC AC GGAC GGCAAC T T TCACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCT C C T C AGAT C C T C AT CAAAAAC AC AC C T GT AC C T GC GGAT C C T C C AA C GGC T T T C AAC AAGGAC AAGC T GAAC TCTTTCATCACC C AGT AT T C T AC T GGC C AAGT C AGC GT GGAGAT T GAGT GGGAGC T GC AGAAGGAA AAC AGC AAGC GC T GGAAC C C GGAGAT C C AGT AC AC T T C C AAC T AT T AC AAGT C T AAT AAT GT T GAAT T T GC T GT T AAT AC T GAAGGT GT T T A TTCTGAACCCCGCCCCATTGGCACCAGATACCTGACTCGTAATCTG TAA

[0147] or vpl proteins produced from a nucleic acid sequence at least 70% identical to SEQ ID NO: 17 which encodes the predicted amino acid sequence of 1 to 736 of SEQ ID NO: 16; AAVhu68 vp2 proteins produced by expression from a nucleic acid sequence which encodes the predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO: 16, vp2 proteins produced from a sequence comprising at least nucleotides 412 to 2211 of SEQ ID NO: 17, or vp2 proteins produced from a nucleic acid sequence at least 70% identical to at least nucleotides 412 to 2211 of SEQ ID NO: 17 which encodes the predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO: 16; and / or AAVhu68 vp3 proteins produced by expression from a nucleic acid sequence which encodes the predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO: 16, vp3 proteins produced from a sequence comprising at least nucleotides 607 to 2211 of SEQ ID NO: 17, or vp3 proteins produced from a nucleic acid sequence at least 70% identical to at least nucleotides 607 to 2211 of SEQ ID NO: 17, which encodes the predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO: 17.

[0148] The AAVhu68 vpl, vp2 and vp3 proteins are typically expressed as alternative splice variants encoded by the same nucleic acid sequence which encodes the full-length vpl amino acid sequence (amino acid (aa) 1 to 736). Optionally the vpl-encoding sequence is used alone to express the vpl, vp2 and vp3 proteins. Alternatively, this sequence may be co-expressed with one or more of a nucleic acid sequence which encodes the AAVhu68 vp3 amino acid sequence (about aa 203 to 736) without the vpl -unique region (about aa 1 to about aa 137) and / or vp2 -unique regions (about aa 1 to about aa 202), or a strand complementary thereto, the corresponding mRNA or tRNA (for example, the mRNA

[0149] 20

[0150] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0151] transcribed from about nucleotide (nt) 607 to about nt 2211 of SEQ ID NO: 17, or a sequence at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99%) identical to SEQ ID NO: 17 which encodes aa 203 to 736 of SEQ ID NO: 16.

[0152] Additionally, or alternatively, the vpl -encoding and / or the vp2-encoding sequence may be co-expressed with the nucleic acid sequence which encodes the AAVhu68 vp2 amino acid sequence of SEQ ID NO: 16 (about aa 138 to 736) without the vpl -unique region (about aa 1 to about 137 of SEQ ID NO: 16, or a strand complementary thereto, the corresponding mRNA or tRNA (for example, the mRNA transcribed from nt 412 to 2211 of SEQ ID NO: 17, or a sequence at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99%) identical to SEQ ID NO: 17 which encodes about aa 138 to 736 of SEQ ID NO: 16.

[0153] In some embodiments, the AAVhu68 vpl nucleic acid sequence has the sequence of SEQ ID NO: 17, or a strand complementary thereto, e.g., the corresponding mRNA or tRNA. In some embodiments, the vp2 and / or vp3 proteins may be expressed additionally or alternatively from different nucleic acid sequences than the vpl, e.g., to alter the ratio of the vp proteins in a selected expression system. In some embodiments, also provided is a nucleic acid sequence which encodes the AAVhu68 vp3 amino acid sequence of SEQ ID NO: 16 (about aa 203 to 736 without the vpl -unique region (about aa 1 to about aa 137 of SEQ ID NO: 16 and / or vp2-unique regions (about aa 1 to about aa 202 of SEQ SEQ ID NO: 16, or a strand complementary thereto, the corresponding mRNA or tRNA (about nt 607 to about nt 2211 of SEQ ID NO: 17. In some embodiments, also provided is a nucleic acid sequence which encodes the AAVhu68 vp2 amino acid sequence of SEQ ID NO: 16 (about aa 138 to 736 of SEQ ID NO: 16 without the vpl -unique region (about aa 1 to about 137 of SEQ ID NO: SEQ ID NO: 16, or a strand complementary thereto, the corresponding mRNA or tRNA (nt 412 to 2211 of SEQ ID NO: 17.

[0154] However, other nucleic acid sequences which encode the amino acid sequence of SEQ ID NO: 8 may be selected for use in producing rAAVhu68 capsids. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO: 17 or a sequence at least 70% to 99% identical, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, identical to SEQ ID NO: 17 which encodes SEQ ID NO: 16. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO: 17 or a sequence at least 70% to 99%, at least 75%, at least

[0155] 21

[0156] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0157] 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, identical to about nt 412 to about nt 2211 of SEQ ID NO: 17 which encodes the vp2 capsid protein (about aa 138 to 736) of SEQ ID NO: 16. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of about nt 607 to about nt 2211 of SEQ ID NO: 17 or a sequence at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, identical to nt 607 to about nt 2211 of SEQ ID NO: 17 which encodes the vp3 capsid protein (about aa 203 to 736) of SEQ ID NO: 16.

[0158] Alternatively, the AAV clade that can be used can be AAV9 or a variant thereof as provided for herein. As provided for herein any of the AAV vectors that are used can comprise capsids that comprise targeting peptides, such as targeting peptides that target the AAV virus to muscle cells. Thus, in some embodiments, the AAV vector comprises a muscle specific capsid and the expression of the transgene (i.e., the dystrophin product / polypeptide) is under the control of a muscle specific promoter. This combination can be utilized to increase the dose of the vector while still limiting or avoiding expression of the dystrophin product in cells that are not muscle cells.

[0159] As used herein, the terms “rAAV” and “recombinant AAV vector” are used interchangeably, mean, without limitation, an AAV comprising a capsid protein and a vector genome packaged therein, wherein the vector genome comprising a nucleic acid heterologous to the AAV. rAAV includes “pseudotyped rAAV”, wherein the viral vector contains a vector genome containing the inverted terminal repeat of one AAV (e.g., AAV2) packaged into the capsid of a different AAV capsid protein. In one embodiment, the capsid protein is a non-naturally occurring capsid. Such an artificial capsid may be generated by any suitable technique, using a selected AAV sequence (e.g., a fragment of a vpl capsid protein) in combination with heterologous sequences which may be obtained from a different selected AAV, non-contiguous portions of the same AAV, from a non-AAV viral source, or from a non-viral source. The selected genetic element may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion. 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., Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0160] 22

[0161] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0162] 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. SEQ ID NO: 16 provides the 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 vpl proteins, within the vp2 proteins and within the vp3 proteins which have modifications from the 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.

[0163] 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 the 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.

[0164] 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

[0165] 23

[0166] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0167] implement this algorithm. For example, the 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.

[0168] rAAV production

[0169] The rAAV described herein may be generated using techniques which are known. See, e.g., WO 2003 / 042397: WO 2005 / 033321, WO 2006 / 110689; U. S. Pat. No. 7,588,772 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 as described herein flanked by AAV inverted terminal repeats (ITRs); and sufficient helper functions to permit packaging of the expression cassette into the AAV capsid protein. Also provided herein is the host cell which contains a nucleic acid sequence encoding an AAV capsid: a functional rep gene: a vector genome as described; and sufficient helper functions to permit packaging of the vector genome into the AAV capsid protein. In one embodiment, the host cell is a cell culture. In another embodiment, the host cell is a suspension. In some embodiments, the host cell is a HEK 293 cell. These methods are described in more detail in W02017 / 160360 A2, which is incorporated by reference herein. See also, 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.

[0170] Other methods of producing rAAV available to one of skill in the art may be utilized. Suitable methods may include without limitation, baculovirus expression system or production via yeast. See, e.g., Robert M. Kotin, Large-scale recombinant adeno-associated virus production. Hum Mol Genet. 2011 Apr 15: 2O(R1): R2-R6. Published online 2011 Apr 29. doi: 10.1093 / hmg / ddrl41: Aucoin MG et al., Production of adeno-associated viral vectors in insect cells using triple infection: optimization of baculovirus concentration ratios. Biotechnol Bioeng. 2006 Dec 20:95(6): 1081-92; SAMI S. THAKUR, Production of Recombinant Adeno-associated viral vectors in yeast. Thesis presented to the Graduate School of the University of Florida, 2012: Kondratov O et al. Direct Head-to-Head Evaluation of Recombinant Adeno-associated Viral Vectors Manufactured in Human versus Insect Cells, Mol Ther. 2017 Aug 10. pii: S1525-0016(17)30362-3. doi:

[0171] 24

[0172] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0173] 10.1016 / j.ymthe.2017.08.003. [Epub ahead of print]: Mietzsch M et al, OneBac 2.0: Sf9 Cell Lines for Production of AAV1, AAV2, and AAV8 Vectors with Minimal Encapsidation of Foreign DNA. Hum Gene Ther Methods. 2017 Feb:28(l):15-22. doi: 10.1089 / hgtb.2016.164.; Li L et al. Production and characterization of novel recombinant adeno-associated virus replicative-form genomes: a eukaryotic source of DNA for gene transfer. PLOS One. 2013 Aug 1:8(8): e69879. doi: 10.1371 / joumal.pone.0069879. Print 2013; Galibert L et al, Latest developments in the large-scale production of adeno-associated virus vectors in insect cells toward the treatment of neuromuscular diseases. J Invertebr Pathol. 2011 Jul: 107 Suppl: S80-93. doi: 10.1016 / j.jip.2011.05.008; and Kotin RM, Large-scale recombinant adeno-associated virus production. Hum Mol Genet. 2011 Apr 15:20(Rl): R2-6, doi: 10.1093 / hmg / ddrl41. Epub 2011 Apr 29.

[0174] A two-step affinity chromatography purification at high salt concentration followed by anion exchange resin chromatography is used to purify the rAAV product and to remove empty capsids. These methods are described in more detail in WO 2017 / 160360 entitled “Scalable Purification Method for AAV9”, and WO 2017 / 100674 entitled “Scalable Purification Method for AAV1”, which are incorporated by reference herein. In brief, the method for separating rAAV particles having packaged genomic sequences from genomedeficient AAV intermediates involves subjecting a suspension comprising recombinant AAV9 or AAV viral particles and AAV capsid intermediates to fast performance liquid chromatography, wherein the AAV9 viral particles and AAV intermediates are bound to a strong anion exchange resin equilibrated at a pH of about 10.2 for rAAV9 or about 9.8 for AAV1, and subjected to a salt gradient while monitoring eluate for ultraviolet absorbance at about 260 and about 280. In this method, the AAV full capsids are collected from a fraction which is eluted when the ratio of A260 / A280 reaches an inflection point. In one example, for the affinity chromatography step, the diafiltered product may be applied to an AAV-specific resin that efficiently captures the selected AAV serotype. Under these ionic conditions, a significant percentage of residual cellular DNA and proteins flow through the column, while AAV particles are efficiently captured.

[0175] Conventional methods for characterization or quantification of rAAV are available to one of skill in the art. 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 #of GC=#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

[0176] 25

[0177] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0178] test article peaks. The number of particles (pt) per 20 pL 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: 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% Trisacetate in the buffer, then running the gel until sample material is separated, and blotting the 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 the 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., Dithiothreitol (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, the concentration of AAV vector genomes (vg) in column fractions can be measured by quantitative real time PCR (Q-PCR, q-PCR or qPCR). Samples are diluted and digested with DNase I (or another suitable nuclease) to remove exogenous DNA. After inactivation of the nuclease, the samples are further diluted and amplified using primers and a TaqMan™ fluorogenic probe specific for the DNA sequence between the primers. The number of cycles required to reach a defined level of fluorescence (threshold cycle, Ct) is measured for each sample on

[0179] 26

[0180] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0181] an Applied Biosystems Prism 7700 Sequence Detection System. Plasmid DNA containing identical sequences to that contained in the 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 the Ct value of the plasmid standard curve. End-point assays based on the digital PCR can also be used.

[0182] 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 (oqPCR) 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 are 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 the standard assay.

[0183] 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 Then Methods. 2014 Apr:25(2): 115-25. doi: 10.1089 / hgtb.2013.131. Epub 2014 Feb 14.

[0184] Methods for determining the ratio among vpl, vp2, and vp3 of capsid protein are also available. See, e.g., Vamseedhar Rayaprolu et al., Comparative Analysis of Adeno-Associated Virus Capsid Stability and Dynamics, J Virol. 2013 Dec: 87(24): 13150-13160; Buller RM, Rose JA. 1978. Characterization of adenovirus-associated virus-induced polypeptides in KB cells. J. Virol. 25:331-338; and Rose JA, Maizel JV, Inman JK, Shatkin AJ. 1971. Structural proteins of adenovirus-associated viruses. J. Virol. 8:766-770. For use in producing an AAV viral vector (e.g., a recombinant (r) AAV), the expression cassettes

[0185] 27

[0186] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0187] can be carried on any suitable vector, e.g., a plasmid, which is delivered to a 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.

[0188] In some embodiments, the rAAV comprises an AAV hu68 or AAV9 capsid with a vector genome comprising an AAV 5' ITR, a muscle specific promoter, a dystrophin product coding sequence as provided for herein, and an AAV 3' ITR. In certain embodiments, a plasmid has a vector genome comprising ITRs with a shortened 130 nucleotide sequence, optionally derived from AAV2, which reverts to the full-length 145 nucleotide ITR when it replicates and is packaged into the AAV capsid. In certain embodiments, the vector genome comprises stuffer sequences between the AAV 5' ITR and the promoter and / or the polyA and the AAV 3' ITR. In certain embodiments, the vector genome comprises an AAV 5' ITR, spacer sequences, spacer sequences, and an AAV 3' ITR. In certain embodiments, the vector genome comprises the sequence of SEQ ID NO: 11 or SEQ ID NO: 12.

[0189] As described herein, a rAAVhu68 has a rAAVhu68 capsid produced in a production system expressing capsids from an AAVhu68 nucleic acid sequence which encodes the vpl amino acid sequence of SEQ ID NO: 16, and optionally additional nucleic acid sequences, e.g., encoding a vp3 protein free of the vpl and / or vp2-unique regions. The rAAVhu68 resulting from production using a single nucleic acid sequence vpl produces the heterogeneous populations of vpl proteins, vp2 proteins and vp3 proteins. More particularly, the AAVhu68 capsid contains subpopulations within the vpl proteins, within the vp2 proteins and within the vp3 proteins which have modifications from the predicted amino acid residues in SEQ ID NO: 16. These subpopulations include, at a minimum, deamidated asparagine (N or Asn) residues. For example, asparagines in asparagine — glycine pairs are highly deamidated. In certain embodiments, the rAAVhu68 has a rAAVhu68 capsid produced in a production system expressing capsids from an AAVhu68 nucleic acid sequence of SEQ ID NO: 17.

[0190] Accordingly, in some embodiments, provided herein are recombinant adeno-associated virus (rAAV) comprising an adeno-associated virus (AAV) capsid and packaged therein a vector genome, wherein the vector genome comprises: (a) an AAV 5' inverted terminal repeat (ITR); (b) an expression cassette comprising a coding sequence for

[0191] 28

[0192] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0193] polypeptide provided for herein, or any variant thereof, wherein the coding sequence is operably linked to expression control sequences which direct expression of the polypeptide; and (c) an AAV 3' ITR.

[0194] In some embodiments, the AAV capsid is suitable for targeting to muscles, such as skeletal muscles. In some embodiments, the AAV capsid is a Clade F AAV. In some embodiments, the AAV capsid is an AAVhu68 capsid or an AAV9 capsid, or a variant thereof. In some embodiments, the capsid proteins have an amino acid sequence comprising a hypervariable region comprising an exogenous targeting peptide. In some embodiments, the exogenous targeting peptide comprises a targeting peptide that targets the AAV to muscle, such as skeletal muscle. Non-limiting examples of such targeting peptides are provided for herein and any one can be used in combination with the AAV vectors.

[0195] It should be understood that the compositions in the vectors described herein are intended to be applied to other compositions and methods described across the specification.

[0196] As provided for herein, the vector delivering the dystrophin products provided herein or the capsids of the AAV vector can comprise targeting peptides incorporated into the capsid. Non-limiting examples of such peptides are provided in PCT Publication No. WO2024 / 130067, PCT Publication No. WO2024 / 130070, PCT Application No.

[0197] PCT / US2024 / 55910, filed November 14, 2024, U. S. Provisional Application No.

[0198] 63 / 598,718, filed November 14, 2023, U. S. Provisional Application No. 63 / 612,676, filed December 20, 2023, U. S. Provisional Application No. 63 / 661,133, filed June 18, 2024, PCT Publication No. WO 2024 / 173802, and U. S. Patent No. 11,920,150, each of which is hereby incorporated by reference in its entirety.

[0199] In some embodiments, the muscle cell-targeting peptide is a peptide having the formula of “Xn - n-mer - Xm”, wherein Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein n-mer is RGDYREV, RGDYREI, RGDYREL, RGDYHQV, VYTRGDV, RGDYSQI, RGDYASV, QNRGDPH, RGDYHYQ, VHRGDLN, RGDFSGY, RGDYVYQ, RGDYSYT, QVRGDIK, PQYTRGD, VRGDIRL, AVIRGDV, IIRGDPA, RGDLHGY, PYQRGDH, RGDYAQV, or RGDYSTM, or a sequence comprising at least 6 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, or a targeting peptide comprising an amino acid sequence as provided

[0200] 29

[0201] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0202] for in PCT Publication No. WO2024 / 130067, PCT Publication No. WO2024 / 130070, PCT Application No. PCT / US2024 / 55910, filed November 14, 2024, U. S. Provisional Application No. 63 / 598,718, filed November 14, 2023, U. S. Provisional Application No.

[0203] 63 / 612,676, filed December 20, 2023, U. S. Provisional Application No. 63 / 661,133, filed June 18, 2024, PCT Publication No. WO 2024 / 173802, and U. S. Patent No. 11,920,150, each of which is hereby incorporated by reference in its entirety.

[0204] . Also provided herein are nucleic acid sequences encoding the same. In some embodiments, this exogenous motif modifies the native tissue specificity of the source (parental) protein, viral vector, viral capsid, or another moiety. In some embodiments, compositions having one or more of these exogenous targeting peptides have enhanced or altered muscle cell-targeting. In some embodiments, compositions having one or more of these targeting peptides have enhanced or altered cardiac and / or skeletal muscle celltargeting, optionally improved targeting of gastrocnemius muscle cells. In some embodiments, viral vectors having modified capsids containing this motif exhibit increased transduction of AAV production cells in vitro.

[0205] In some embodiments provided herein is an engineered rAAV capsid comprising the exogenous targeting peptide, wherein the exogenous targeting peptide has the formula of “Xn - n-mer - Xm”, wherein Xn is 0, 1, 2 or 3 amino acid residues independently selected from any amino acid, wherein n-mer is RGDYREV, RGDYREI, RGDYREL, RGDYHQV, VYTRGDV, RGDYSQI, RGDYASV, QNRGDPH, RGDYHYQ, VHRGDLN, RGDFSGY, RGDYVYQ, RGDYSYT, QVRGDIK, PQYTRGD, VRGDIRL, AVIRGDV, IIRGDPA, RGDLHGY, PYQRGDH, RGDYAQV, or RGDYSTM or a sequence comprising at least 6 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, or a targeting peptide comprising an amino acid sequence as provided for in PCT Publication No. WO2024 / 130067, PCT Publication No. WO2024 / 130070, PCT Application No. PCT / US2024 / 55910, filed November 14, 2024, U. S. Provisional Application No. 63 / 598,718, filed November 14, 2023, U. S. Provisional Application No. 63 / 612,676, filed December 20, 2023, U. S. Provisional Application No. 63 / 661,133, filed June 18, 2024, PCT Publication No. WO 2024 / 173802, and U. S. Patent No. 11,920,150, each of which is hereby incorporated by reference in its entirety. In some embodiments, the exogenous targeting peptide provided herein provide significant transduction advantages in muscle cells, including cardiac muscle cells and / or skeletal muscle cells, optionally improved

[0206] 30

[0207] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0208] targeting of gastrocnemius muscle cells, deltoid muscle cells, soleus muscle cells, bicep brachii muscle cells, or a diaphragm muscle cells, as compared to a parental capsid (e.g., AAV9, AAVhu68, or another clade F capsid, or another clade capsid).

[0209] In some embodiments, the engineered rAAV capsids comprise an exogenous targeting peptide that comprises “Xn - n-mer - 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 provide significant transduction advantages in muscle cells, including cardiac muscle cells and / or skeletal muscle cells, optionally improved targeting of gastrocnemius muscle cells, deltoid muscle cells, soleus muscle cells, bicep brachii muscle cells, or diaphragm muscle cells, as compared to a parental capsid. In some embodiments, the engineered rAAV capsids comprise an exogenous targeting peptide that comprises “Xn -RGDYREV- 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 muscle cell, including cardiac muscle cells and / or skeletal muscle cells, optionally improved targeting of gastrocnemius muscle cells, deltoid muscle cells, soleus muscle cells, bicep brachii muscle cells, or diaphragm muscle cells, as compared to a parental capsid. In some embodiments, the engineered rAAV capsid comprises an exogenous targeting peptide that comprises “Xn - RGDYHQV- 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 muscle cells, including cardiac muscle cells and / or skeletal muscle cells, optionally improved targeting of gastrocnemius muscle cells, deltoid muscle cells, soleus muscle cells, biceps brachii muscle cells, or a diaphragm muscle cells, as compared to a parental capsid. In some embodiments, the engineered rAAV capsid comprises an exogenous targeting peptide that comprises “Xn - VYTRGDV - 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 muscle cells, including cardiac muscle cells and / or skeletal muscle cells, optionally improved targeting of gastrocnemius muscle cells, deltoid muscle cells, soleus muscle cells, bicep brachii muscle

[0210] 31

[0211] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0212] cells, or a diaphragm muscle cells, as compared to a parental capsid. In some embodiments, the engineered rAAV capsid comprises an exogenous targeting peptide that comprises “Xn - RGDYSQI- 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 muscle cells, including cardiac muscle cells and / or skeletal muscle cells, optionally improved targeting of gastrocnemius muscle cells, deltoid muscle cells, soleus muscle cells, biceps brachii muscle cells or diaphragm muscle cells, as compared to a parental capsid. In some embodiments, the engineered rAAV capsid comprises an exogenous targeting peptide that comprises “Xn - RGDYASV- 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 muscle cell, including cardiac muscle cells and / or skeletal muscle cells, optionally improved targeting of gastrocnemius muscle cells, deltoid muscle cells, soleus muscle cells, biceps brachii muscle cells, or diaphragm muscle cells, as compared to a parental capsid. In some embodiments, the engineered rAAV capsid comprises an exogenous targeting peptide that comprises “Xn - QNRGDPH - 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 muscle cell, including cardiac muscle cells and / or skeletal muscle cells, optionally gastrocnemius muscle cells, deltoid muscle cells, soleus muscle cells, bicep brachii muscle cells, or diaphragm muscle cells, as compared to a parental capsid. In some embodiments, the engineered rAAV capsid comprises an exogenous targeting peptide that comprises “Xn - RGDYHYQ- 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 muscle cells, including cardiac muscle cells and / or skeletal muscle cells, optionally improved targeting of a gastrocnemius muscle cells, deltoid muscle cells, soleus muscle cells, bicep brachii muscle cell or, diaphragm muscle cells, as compared to a parental capsid. In some embodiments, the engineered rAAV capsid comprises an exogenous targeting peptide that comprises “Xn - VHRGDLN - Xm”, wherein Xn is 0, 1, 2 or 3

[0213] 32

[0214] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0215] 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 the muscle cell, including cardiac muscle cells and / or skeletal muscle cells, optionally improved targeting of gastrocnemius muscle cells, deltoid muscle cells, soleus muscle cells, bicep brachii muscle cells, or diaphragm muscle cells, as compared to a parental capsid. In some embodiments, the engineered rAAV capsid comprises an exogenous targeting peptide that comprises “Xn - RGDFSGY - 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 muscle cells, including cardiac muscle cells and / or skeletal muscle cells, optionally improved targeting of gastrocnemius muscle cells, deltoid muscle cells, soleus muscle cells, bicep brachii muscle cells or diaphragm muscle cells, as compared to a parental capsid. In some embodiments, the engineered rAAV capsid comprises an exogenous targeting peptide that comprises “Xn - RGDYVYQ- 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 muscle cells, including cardiac muscle cells and / or skeletal muscle cells, optionally improved targeting of gastrocnemius muscle cells, deltoid muscle cells, soleus muscle cells, biceps brachii muscle cells, or diaphragm muscle cells, as compared to a parental capsid. In some embodiments, the engineered rAAV capsid comprises an exogenous targeting peptide that comprises “Xn - RGDYSYT - 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 muscle cells, including cardiac muscle cells and / or skeletal muscle cells, optionally improved targeting of gastrocnemius muscle cells, deltoid muscle cells, soleus muscle cells, biceps brachii muscle cells, or diaphragm muscle cells, as compared to a parental capsid. In some embodiments, the engineered rAAV capsid comprises an exogenous targeting peptide comprises “Xn -QVRGDIK- 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

[0216] 33

[0217] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0218] transduction advantages in muscle cells, including cardiac muscle cells and / or skeletal muscle cells, optionally improved targeting of gastrocnemius muscle cells, deltoid muscle cells, soleus muscle cells, bicep brachii muscle cells, or diaphragm muscle cells, as compared to a parental capsid. In some embodiments, the engineered rAAV capsid comprises an exogenous targeting peptide that comprises “Xn - PQYTRGD - 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 muscle cells, including cardiac muscle cells and / or skeletal muscle cells, optionally improved targeting of gastrocnemius muscle cells, deltoid muscle cells, soleus muscle cells, bicep brachii muscle cells, or diaphragm muscle cells, as compared to a parental capsid. In some embodiments, the engineered rAAV capsid comprises an exogenous targeting peptide that comprises “Xn - VRGDIRL - 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 muscle cells, including cardiac muscle cells and / or skeletal muscle cells, optionally improved targeting of gastrocnemius muscle cells, deltoid muscle cells, soleus muscle cells, bicep brachii muscle cells, or diaphragm muscle cells, as compared to a parental capsid.

[0219] In some embodiments, the rAAV comprises a mutant AAV capsid having an exogenous targeting peptide as identified herein. In some embodiments, the mutant AAV capsid comprises an exogenous targeting peptide which is immediately preceded by flanking amino acids which are mutated, as compared to parental AAV capsid. In some embodiments, the mutated flanking amino acids, together with 1, 2, 3, 4, 5, or 6 inserted amino acids comprise the exogenous targeting peptide. In some 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 to 3 flanking amino acid residues as provided in the above formulae. In some embodiments, engineered rAAV capsids comprising the targeting peptides, as provided herein, demonstrate reduced transduction (i.e., de-targeted / de-targeting) of liver as compared to its parental capsid (e.g., AAV9 or another clade F capsid (e.g., AAVhu68, AAVhu31, AAVhu32, AAVhu95,

[0220] 34

[0221] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0222] AAVhu96) or other modification thereof). In some embodiments, engineered rAAV capsids comprising the targeting peptides, as provided herein, demonstrate reduced transduction (i.e., de-targeted / ing) to spleen as compared to its parental capsid (e.g., AAV9 or another clade F capsid or other modification thereof.

[0223] 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, gastrocnemius muscle cell). Additionally, the targeting peptide may be linked to a liposome and / or a nanoparticle (a lipid nanoparticle, LNP) forming a peptide-coated liposome and / or LNP to target the 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 some 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)-terminus 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 amino (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

[0224] 35

[0225] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0226] Patent No. US 9,545,450 B2 (NHS-phosphine cross-linking agents; NHS-Azide crosslinking agents); US Published Patent Application No. US 2018 / 0185503 A1 (aldehydehydrazide crosslinking), which are incorporated herein by reference.

[0227] In some embodiments, the exogenous targeting peptide may be engineered (e.g., inserted) at a suitable site within a protein or polypeptide (e.g., a viral capsid protein). In some embodiments, a targeting peptide may be 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.

[0228] In some embodiments, a composition is provided which is useful for targeting a muscle cell. In some embodiments, the composition comprises an engineered capsid (e.g., an 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 RGDYREV 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 some 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 (e.g., “n-mer”) of RGDYHQV 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 some 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 (e.g., “n-mer”) of VYTRGDV flanked at

[0229] 36

[0230] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0231] 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 some 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 (e.g., “n-mer”) of RGDYSQI, 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 some 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 (e.g., “n-mer”) of RGDYASV 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 some 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 (e.g., “n-mer”) of QNRGDPH 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 some 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 (e.g., “n-mer”) of RGDYHYQ 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 some 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 (e.g., “n-mer”) of VHRGDLN 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 some 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 (e.g., “n-mer”) of RGDFSGY flanked at

[0232] 37

[0233] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0234] 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 some 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 (e.g., “n-mer”) of RGDYVYQ 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 some 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 (e.g., “n-mer”) as provided for herein.

[0235] In some embodiments, a recombinant parvovirus encoding the transgene is provided that has a modified parvovirus capsid, wherein the capsid has an amino acid sequence comprising a hypervariable region comprising an exogenous targeting peptide, such as those provided for herein and incorporated by reference above, 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 is RGDYREV, RGDYREI, RGDYREL, RGDYHQV, VYTRGDV, RGDYSQI, RGDYASV, QNRGDPH, RGDYHYQ, VHRGDLN, RGDFSGY, RGDYVYQ, RGDYSYT, QVRGDIK, PQYTRGD, VRGDIRL, AVIRGDV, IIRGDPA, RGDLHGY, PYQRGDH, RGDYAQV, or RGDYSTM, or a sequence comprising 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, or a targeting peptide comprising an amino acid sequence as provided for in PCT Publication No. WO2024 / 130067, PCT Publication No. WO2024 / 130070, PCT Application No. PCT / US2024 / 55910, filed November 14, 2024, U. S. Provisional Application No. 63 / 598,718, filed November 14, 2023, U. S. Provisional Application No. 63 / 612,676, filed December 20, 2023, U. S. Provisional Application No.

[0236] 63 / 661,133, filed June 18, 2024, PCT Publication No. WO 2024 / 173802, and U. S. Patent No. 11,920,150, each of which is hereby incorporated by reference in its entirety. 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 the parental vector, wherein the one

[0237] 38

[0238] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0239] 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 amino acid; (ii) the n-mer RGDYREV, RGDYREI, RGDYREL, RGDYHQV, VYTRGDV, RGDYSQI, RGDYASV, QNRGDPH, RGDYHYQ, VHRGDLN, RGDFSGY, RGDYVYQ, RGDYSYT, QVRGDIK, PQYTRGD, VRGDIRL, AVIRGDV, IIRGDPA, RGDLHGY, PYQRGDH, RGDYAQV, or RGDYSTM, or an n-mer sequence comprising 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, or a targeting peptide comprising an amino acid sequence as provided for in PCT Publication No. WO2024 / 130067, PCT Publication No. WO2024 / 130070, PCT Application No. PCT / US2024 / 55910, filed November 14, 2024, U. S. Provisional Application No. 63 / 598,718, filed November 14, 2023, U. S. Provisional Application No. 63 / 612,676, filed December 20, 2023, U. S. Provisional Application No.

[0240] 63 / 661,133, filed June 18, 2024, PCT Publication No. WO 2024 / 173802, and U. S. Patent No. 11,920,150, each of which is hereby incorporated by reference in its entirety.

[0241] 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 referenced as Vpl or vpl) amino acid sequence, which is provided for in WO 2019 / 168961, published September 6, 2019, including Table G providing the deamidation pattern for AAV9 and WO 2020 / 160582, filed September 7, 2018, each of which is incorporated by reference in its entirety. The amino acid residue locations (i.e., amino acid numbering reference) are identical in AAVhu68. However, another site may be selected within HVRVIII. Alternatively, another exposed loop HVR (e.g., HVRIV) may be selected for the site of insertion. Comparable HVR regions may be selected in other capsids. In some embodiments, the 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 entirety. In some embodiments, the targeting peptide may be inserted (and / or engineered) into a hypervariable loop HVRVIII as described in International Patent Application No. PCT / US2021 / 061312, filed December 1, 2022, which is now published as WO

[0242] 39

[0243] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0244] 2022 / 119871A2 (published June 9, 2022) which is incorporated herein by reference in its entirety. In some embodiments, an AAV 1 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 acid 582 to 585, or HVRIV region of amino acid 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 some embodiments, an AAV8 capsid is selected as a parental capsid, wherein the targeting peptide with linkers of various length is inserted in a suitable location of HVRVIII region of amino acid 586 to 591, or HVRIV region of amino acid 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).

[0245] In some embodiments, the parental AAV capsid is an AAV9, AAVhu68, AAVhu31, AAVhu32, AAV8, AAV7, AAV6, AAV5, AAV4, AAV3, AAV1, AAVhu95, AAVhu96, or AAVrh91 capsid.

[0246] In some 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: 18, or an analogous position in an AAVhu68, AAVhu31, AAVhu32, AAVhu95, AAVhu96, AAV8, AAV7, AAV6, AAV5, AAV4, AAV3, AAV1, or AAVrh91 parental AAV capsid.

[0247] In some embodiments, the exogenous targeting peptide has an amino acid sequence at its carboxy terminus and its amino terminus which is immediately preceded by “AQ” at position 588 of the parental capsid, which is a Clade F capsid, optionally an AAV9, AAVhu68, AAVhu31, AAVhu32, AAVhu95, or AAVhu96 capsid.

[0248] In some embodiments, the residues of the parental AAV capsid sequence protein are preserved (i.e., there are no substitutions and / or deletions in the 1, 2, and / or 3 amino acid residues at the N-terminus and / or C-terminus immediately preceding the target peptide insert, as compared to that of the parental AAV capsid amino acid sequence). In some embodiments, there are no deletions in the 1, 2 and / or 3 amino acid residues as compared to that of the parental AAV capsid protein amino acid sequence at the N-terminus and / or C-terminus immediately preceding the target peptide insert. In some embodiments, one or more amino acid residues, as compared to that of the parental AAV capsid protein amino

[0249] 40

[0250] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0251] 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 some embodiments, one or more amino acid residues, as compared to that of the parental AAV capsid protein amino acid sequence, are modified at positions at the N-terminus and / or the C-terminus immediately flanking the n-mer, and / or to provide the mutant AAV capsid protein with one or more residues of the n-mer sequence.

[0252] In some embodiments, AAV9 is selected as a 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 (Q-A), based on VP1 numbering. In other embodiments, AAVhu68 or another clade F capsid is selected as the parental capsid. In some embodiments, AAV8 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 some 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 VP1 numbering. In some 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 some 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 the HVRVIII region of amino acid 577 and 578 (T-T), based on VP1 numbering. In some 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 some 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 some 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 VP1 numbering. In some embodiments, AAV1 is selected as the parental capsid, wherein the targeting peptide with linkers of various length(s) is inserted (and / or engineered) in a

[0253] 41

[0254] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0255] suitable location of HVRVIII region of amino acid 588 and 589 (S-T), based on VP1 numbering. In other embodiments, inserts may be additionally or alternatively located in: AAV9 (amino acids 566 to 615 of AAV9 capsid), AAV8 (amino acids 565 to 614 of AAV8 capsid;), AAV7 (amino acids 567 to 616 of AAV7 ), AAV6 (amino acids 550 to 599 of AAV6 capsid), AAV5 (amino acids 556 to 605 of AAV5), AAV4 (amino acids 558 to 607 of AAV4 capsid), AAV3B (amino acids 564 to 613 of AAV3B capsid), AAV2 (amino acids 566 to 615 of AAV2 capsid), and AAV1 (amino acids 566 to 615 of AAV1 capsid), which is focused on the region HVRVIII in which the targeting peptide may be inserted (based on structural analysis).

[0256] In some embodiments, there are no substitutions in the 1, 2, or 3 amino acid residues of the parental AAV capsid protein at the N-terminus and / or C-terminus of the target peptide inserted. In some embodiments, there are no deletions in the 1, 2 or 3 amino acid residues of the parental AAV capsid protein at the N-terminus and / or C-terminus of the target peptide inserted. In some embodiments, one or more amino acid residues of the parental AAV capsid protein are modified at the N-terminus and / or the C-terminus of the n-mer, and / or to provide the mutant AAV capsid protein with one or more residues of the n-mer sequence.

[0257] In some embodiments, the parental capsid protein 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) the n-mer RGDYREV, RGDYREI, RGDYREL, RGDYHQV, VYTRGDV, RGDYSQI, RGDYASV, QNRGDPH, RGDYHYQ, VHRGDLN, RGDFSGY, RGDYVYQ, RGDYSYT, QVRGDIK, PQYTRGD, VRGDIRL, AVIRGDV, IIRGDPA, RGDLHGY, PYQRGDH, RGDYAQV, or RGDYSTM, or an n-mer sequence comprising 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, or a targeting peptide comprising an amino acid sequence as provided for in PCT Publication No.

[0258] WO2024 / 130067, PCT Publication No. WO2024 / 130070, PCT Application No.

[0259] PCT / US2024 / 55910, filed November 14, 2024, U. S. Provisional Application No.

[0260] 63 / 598,718, filed November 14, 2023, U. S. Provisional Application No. 63 / 612,676, filed December 20, 2023, U. S. Provisional Application No. 63 / 661,133, filed June 18, 2024, PCT Publication No. WO 2024 / 173802, and U. S. Patent No. 11,920,150, each of which is hereby incorporated by reference in its entirety, wherein the parental capsid is selected from parvoviruses of Clade F AAV (e.g., AAVhu68, AAV9, AAVhu31, AAVhu32,

[0261] 42

[0262] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0263] AAVhu95, AAVhu96), Clade E (e.g., AAV8), or Clade A AAV (e.g., AAV1, AAVrh91)) capsids, or non-parvovirus capsids (e.g., herpes simplex virus, etc.) in order enhance expression and / or otherwise 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), 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 some embodiments, AAV capsids having reduced capsid deamidation may be selected. See, e.g., PCT / US19 / 19804 and PCT / US18 / 19861, both filed Feb 27, 2019, and incorporated by reference in their entireties. See also, International Patent Application No.

[0264] PCT / US2021 / 055436, filed October 18, 2021, now publication No. WO 2022 / 082109, and International Patent Application No. PCT / US2022 / 077315, filed September 30, 2022, now publication No. WO 2023 / 056399 are incorporated herein, and incorporated by reference in their entireties.

[0265] In some embodiments, the mutant capsids described herein are characterized by having a deamidation pattern similar to their parental AAV, 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.

[0266] In some embodiments, provided herein is a recombinant adeno-associated virus (rAAV) 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 is RGDYREV, RGDYREI, RGDYREL, RGDYHQV, VYTRGDV, RGDYSQI, RGDYASV, QNRGDPH, RGDYHYQ, VHRGDLN, RGDFSGY, RGDYVYQ, RGDYSYT, QVRGDIK, PQYTRGD, VRGDIRL, AVIRGDV, IIRGDPA, RGDLHGY, PYQRGDH, RGDYAQV, or RGDYSTM, or an n-mer sequence comprising 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, or a targeting peptide comprising an amino acid sequence as provided for in PCT Publication No. WO2024 / 130067, PCT Publication No. WO2024 / 130070, PCT

[0267] 43

[0268] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0269] Application No. PCT / US2024 / 55910, filed November 14, 2024, U. S. Provisional Application No. 63 / 598,718, filed November 14, 2023, U. S. Provisional Application No.

[0270] 63 / 612,676, filed December 20, 2023, U. S. Provisional Application No. 63 / 661,133, filed June 18, 2024, PCT Publication No. WO 2024 / 173802, and U. S. Patent No. 11,920,150, each of which is hereby incorporated by reference in its entirety; and (b) a vector genome packaged in the AAV capsid, wherein the vector genome comprises a nucleic acid sequence encoding a gene product operably linked to regulatory sequences. In some 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: RGDYREV, RGDYREI, RGDYREL, RGDYHQV, VYTRGDV, RGDYSQI, RGDYASV, QNRGDPH, RGDYHYQ, VHRGDLN, RGDFSGY, RGDYVYQ, RGDYSYT, QVRGDIK, PQYTRGD, VRGDIRL, AVIRGDV, IIRGDPA, RGDLHGY, PYQRGDH, RGDYAQV, or RGDYSTM, or a targeting peptide comprising an amino acid sequence as provided for in PCT Publication No. WO2024 / 130067, PCT Publication No. WO2024 / 130070, PCT Application No.

[0271] PCT / US2024 / 55910, filed November 14, 2024, U. S. Provisional Application No.

[0272] 63 / 598,718, filed November 14, 2023, U. S. Provisional Application No. 63 / 612,676, filed December 20, 2023, U. S. Provisional Application No. 63 / 661,133, filed June 18, 2024, PCT Publication No. WO 2024 / 173802, and U. S. Patent No. 11,920,150, each of which is hereby incorporated by reference in its entirety, and / or combination of any thereof. In certain embodiment, the rAAV comprising capsid proteins comprising exogenous targeting peptides as described herein (i.e., engineered rAAV capsid) has a greater muscle specificity, targeting, and / or efficacy as compared to a parental AAV capsid.

[0273] In some embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid proteins comprise 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 amino acid; (ii) n-mer is RGDYREV, RGDYREI, RGDYREL, RGDYHQV, VYTRGDV, RGDYSQI, RGDYASV, QNRGDPH, RGDYHYQ, VHRGDLN, RGDFSGY, RGDYVYQ, RGDYSYT, QVRGDIK, PQYTRGD, VRGDIRL, AVIRGDV, IIRGDPA, RGDLHGY, PYQRGDH, RGDYAQV, or RGDYSTM, or an n-mer sequence comprising 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, or a targeting peptide comprising an amino acid sequence as provided for in PCT Publication No.

[0274] 44

[0275] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0276] WO2024 / 130067, PCT Publication No. WO2024 / 130070, PCT Application No.

[0277] PCT / US2024 / 55910, filed November 14, 2024, U. S. Provisional Application No.

[0278] 63 / 598,718, filed November 14, 2023, U. S. Provisional Application No. 63 / 612,676, filed December 20, 2023, U. S. Provisional Application No. 63 / 661,133, filed June 18, 2024, PCT Publication No. WO 2024 / 173802, and U. S. Patent No. 11,920,150, each of which is hereby incorporated by reference in its entirety. In some 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: RGDYREV, RGDYREI, RGDYREL, RGDYHQV, VYTRGDV, RGDYSQI, RGDYASV, QNRGDPH, RGDYHYQ, VHRGDLN, RGDFSGY, RGDYVYQ, RGDYSYT, QVRGDIK, PQYTRGD, VRGDIRL, AVIRGDV, IIRGDPA, RGDLHGY, PYQRGDH, RGDYAQV, or RGDYSTM, or a targeting peptide comprising an amino acid sequence as provided for in PCT Publication No. WO2024 / 130067, PCT Publication No. WO2024 / 130070, PCT Application No. PCT / US2024 / 55910, filed November 14, 2024, U. S. Provisional Application No. 63 / 598,718, filed November 14, 2023, U. S. Provisional Application No. 63 / 612,676, filed December 20, 2023, U. S. Provisional Application No. 63 / 661,133, filed June 18, 2024, PCT Publication No. WO 2024 / 173802, and U. S. Patent No. 11,920,150, each of which is hereby incorporated by reference in its entirety, and / or combination of any thereof. In some 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: RGDYREV, RGDYREI, RGDYREL, RGDYHQV, VYTRGDV, RGDYSQI, RGDYASV, QNRGDPH, RGDYHYQ, VHRGDLN, RGDFSGY, RGDYVYQ, RGDYSYT, QVRGDIK, PQYTRGD, VRGDIRL, AVIRGDV, IIRGDPA, RGDLHGY, PYQRGDH, RGDYAQV, or RGDYSTM, or a targeting peptide comprising an amino acid sequence as provided for in PCT Publication No.

[0279] WO2024 / 130067, PCT Publication No. WO2024 / 130070, PCT Application No.

[0280] PCT / US2024 / 55910, filed November 14, 2024, U. S. Provisional Application No.

[0281] 63 / 598,718, filed November 14, 2023, U. S. Provisional Application No. 63 / 612,676, filed December 20, 2023, U. S. Provisional Application No. 63 / 661,133, filed June 18, 2024, PCT Publication No. WO 2024 / 173802, and U. S. Patent No. 11,920,150, each of which is hereby incorporated by reference in its entirety.

[0282] 45

[0283] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0284] In some embodiments, the rAAV comprises an AAVhu68 capsid wherein the AAVhu68 capsid protein comprises 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 amino acid; (ii) the n-mer is RGDYREV, RGDYREI, RGDYREL, RGDYHQV, VYTRGDV, RGDYSQI, RGDYASV, QNRGDPH, RGDYHYQ, VHRGDLN, RGDFSGY, RGDYVYQ, RGDYSYT, QVRGDIK, PQYTRGD, VRGDIRL, AVIRGDV, IIRGDPA, RGDLHGY, PYQRGDH, RGDYAQV, or RGDYSTM, or an n-mer sequence comprising 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, or a targeting peptide comprising an amino acid sequence as provided for in PCT Publication No. WO2024 / 130067, PCT Publication No. WO2024 / 130070, PCT Application No. PCT / US2024 / 55910, filed November 14, 2024, U. S. Provisional Application No. 63 / 598,718, filed November 14, 2023, U. S. Provisional Application No.

[0285] 63 / 612,676, filed December 20, 2023, U. S. Provisional Application No. 63 / 661,133, filed June 18, 2024, PCT Publication No. WO 2024 / 173802, and U. S. Patent No. 11,920,150, each of which is hereby incorporated by reference in its entirety. In some embodiments, the rAAV comprises an AAVhu68 capsid wherein the AAVhu68 capsid protein comprises a hypervariable region comprising an exogenous targeting peptide, wherein the exogenous targeting peptide comprises: RGDYREV, RGDYREI, RGDYREL, RGDYHQV, VYTRGDV, RGDYSQI, RGDYASV, QNRGDPH, RGDYHYQ, VHRGDLN, RGDFSGY, RGDYVYQ, RGDYSYT, QVRGDIK, PQYTRGD, VRGDIRL, AVIRGDV, IIRGDPA, RGDLHGY, PYQRGDH, RGDYAQV, or RGDYSTM, or a targeting peptide comprising an amino acid sequence as provided for in PCT Publication No.

[0286] WO2024 / 130067, PCT Publication No. WO2024 / 130070, PCT Application No.

[0287] PCT / US2024 / 55910, filed November 14, 2024, U. S. Provisional Application No.

[0288] 63 / 598,718, filed November 14, 2023, U. S. Provisional Application No. 63 / 612,676, filed December 20, 2023, U. S. Provisional Application No. 63 / 661,133, filed June 18, 2024, PCT Publication No. WO 2024 / 173802, and U. S. Patent No. 11,920,150, each of which is hereby incorporated by reference in its entirety, and / or combination of any thereof.

[0289] In some embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises one or more of the exogenous peptides comprising “Xn -RGDYREV- Xm”, wherein Xn is 0, 1, 2 or 3 amino acid residues independently selected

[0290] 46

[0291] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0292] from any amino acid, and wherein Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid.

[0293] In some embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises one or more of the exogenous peptides comprising “Xn -RGDYHQV - 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.

[0294] In some embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises one or more of the exogenous peptides comprising “Xn -VYTRGDV - 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.

[0295] In some embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises one or more of the exogenous peptides comprising “Xn -RGDYSQI - 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.

[0296] In some embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises one or more of the exogenous peptides comprising “Xn -RGDYASV - 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.

[0297] In some embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises one or more of the exogenous peptides comprising “Xn -QNRGDPH- 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.

[0298] In some embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises one or more of the exogenous peptides comprising “Xn -RGDYHYQ - 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.

[0299] 47

[0300] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0301] In some embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises one or more of the exogenous peptides comprising “Xn -VHRGDLN - 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.

[0302] In some embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises one or more of the exogenous peptides comprising “Xn -RGDFSGY - 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.

[0303] In some embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises one or more of the exogenous peptides comprising “Xn -RGDYVYQ - 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.

[0304] In some embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises one or more of the exogenous peptides comprising “Xn -RGDYSYT- 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.

[0305] In some embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises one or more of the exogenous peptides comprising “Xn -QVRGDIK - 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.

[0306] In some embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises one or more of the exogenous peptides comprising “Xn -PQYTRGD- 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.

[0307] In some embodiments, the rAAV comprises an AAV9 capsid wherein the AAV9 capsid protein comprises one or more of the exogenous peptides comprising “Xn -VRGDIRL - Xm”, wherein Xn is 0, 1, 2 or 3 amino acid residues independently selected

[0308] 48

[0309] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0310] from any amino acid, and wherein Xm is 0, 1, 2, or 3 amino acid residues independently selected from any amino acid.

[0311] In some embodiments, the rAAV comprises a mutant AAV9 capsid comprising one or more of the exogenous peptides comprising RGDYREV, RGDYREI, RGDYREL, RGDYHQV, VYTRGDV, RGDYSQI, RGDYASV, QNRGDPH, RGDYHYQ, VHRGDLN, RGDFSGY, RGDYVYQ, RGDYSYT, QVRGDIK, PQYTRGD, VRGDIRL, AVIRGDV, IIRGDPA, RGDLHGY, PYQRGDH, RGDYAQV, or RGDYSTM, or a targeting peptide comprising an amino acid sequence as provided for in PCT Publication No. WO2024 / 130067, PCT Publication No. WO2024 / 130070, PCT Application No.

[0312] PCT / US2024 / 55910, filed November 14, 2024, U. S. Provisional Application No.

[0313] 63 / 598,718, filed November 14, 2023, U. S. Provisional Application No. 63 / 612,676, filed December 20, 2023, U. S. Provisional Application No. 63 / 661,133, filed June 18, 2024, PCT Publication No. WO 2024 / 173802, and U. S. Patent No. 11,920,150, each of which is hereby incorporated by reference in its entirety.

[0314] In some embodiments, the rAAV comprises a mutant AAV9-RGDYREV capsid or a mutant AAVhu68 -RGDYREV capsid, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the RGDYREV peptide insert. In some embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In some embodiments, a rAAV has a capsid comprising AAV9-RGDYREV (or AAVhu68-RGDYREV) - VP proteins in which each the VP1, VP2 VP3 proteins are a heterogenous population and comprise the VP3 region (about amino acid 203 to about amino acid 736). The proteins further comprising deamidation in about 50% to about 100% of positions N57 (VP1 only), N329, N452, or N512, based on the parental capsid residue positions. In some embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In some 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.

[0315] In some embodiments, the rAAV comprises a mutant AAV9-RGDYHQV capsid or a mutant AAVhu68 -RGDYHQV capsid, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the

[0316] 49

[0317] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0318] RGDYHQV peptide insert. In some embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In some embodiments, a rAAV has a mutant capsid comprising AAV9-RGDYHQV (or AAVhu68 -RGDYHQV) - VP proteins in which each the VP1, VP2 VP3 proteins are a heterogenous population and comprise the VP3 region (about amino acid 203 to about amino acid 736). The proteins further comprising deamidation in about 50% to about 100% of positions N57 (VP1 only), N329, N452, or N512, based on the parental capsid residue positions. In some embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In some 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.

[0319] In some embodiments, the rAAV comprises a mutant AAV9-VYTRGDV capsid or a mutant AAVhu68-VYTRGDV capsid, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the VYTRGDV peptide insert. In some embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In some embodiments, a rAAV has a mutant capsid comprising AAV9-VYTRGDV (or AAVhu68 -VYTRGDV) VP proteins in which each the VP1, VP2 VP3 proteins are a heterogenous population and comprise the VP3 region (about amino acid 203 to about amino acid 736). The proteins further comprising deamidation in about 50% to about 100% of positions N57 (VP1 only), N329, N452, or N512, based on the parental capsid residue positions. In some embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In some 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.

[0320] In some embodiments, the rAAV comprises a mutant AAV9-RGDYSQI capsid or a mutant AAVhu68-RGDYSQI capsid, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the RGDYSQI peptide insert. In some embodiments, the proteins are further characterized by

[0321] 50

[0322] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0323] having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In some embodiments, a mutant rAAV has a mutant capsid comprising AAV9-RGDYSQI VP proteins, or AAVhu68-RGDYSQI VP proteins, in which each the VP1, VP2 VP3 proteins are a heterogenous population and comprise the VP3 region (about amino acid 203 to about amino acid 736 based on the residue positions in SEQ ID 26). The proteins further comprising deamidation in about 50% to about 100% of positions N57 (VP1 only), N329, N452, or N512, based on the parental capsid residue positions. In some embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In some 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.

[0324] In some embodiments, the rAAV comprises a mutant AAV9-RGDYASV capsid or a mutant AAVhu68-RGDYASV capsid, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the RGDYASV peptide insert. In some embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In some embodiments, a mutant rAAV9 has a mutant capsid comprising AAV9-RGDYASV -VP proteins (and a mutant rAAVhu68 has a mutant capsid comprising AAVhu68-RGDYASV) in which each the VP1, VP2 VP3 proteins are a heterogenous population and comprise the VP3 region, about amino acid 203 to about amino acid 736 based on the residue positions in SEQ ID 26. The proteins further comprising deamidation in about 50% to about 100% of positions N57 (VP1 only), N329, N452, or N512, based on the parental capsid residue positions. In some embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In some 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.

[0325] In some embodiments, the rAAV comprises a mutant AAV9-QNRGDPH capsid or a mutant AAVhu68-QNRGDPH capsid, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the

[0326] 51

[0327] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0328] QNRGDPH peptide insert. In some embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In some embodiments, a mutant rAAV9 or a mutant AAVhu68 has a mutant capsid comprising AAV-QNRGDPH - VP proteins in which each the VP1, VP2 VP3 proteins are a heterogenous population and comprise the VP3 region, about amino acid 203 to about amino acid 736 based on the residue positions in SEQ ID 26. The mutant AAV proteins further comprising deamidated residues n in about 50% to about 100% of positions N57 (VP1 only), N329, N452, or N512, based on the parental capsid residue positions. In some embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In some 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.

[0329] In some embodiments, the rAAV comprises a mutant AAV9-RGDYHYQ capsid or a mutant AAVhu68-RGDYHYQ capsid, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the RGDYHYQ peptide insert. In some embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In some embodiments, a mutant rAAV9 or a mutant rAAVhu68 has a mutant capsid comprising AAV-RGDYHYQ - VP proteins in which each the VP1, VP2 VP3 proteins are a heterogenous population having deamidation in about 50% to about 100% of positions N57 (VPl only), N329, N452, or N512, based on the parental capsid residue positions. In some embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In some 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.

[0330] In some embodiments, the rAAV comprises a mutant AAV9-VHRGDLN capsid, which comprises mutant VPl, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the VHRGDLN peptide insert. In some embodiments, the proteins are further characterized by having three or four highly

[0331] 52

[0332] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0333] deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In some embodiments, a mutant rAAV9 or a mutant rAAVhu68 has a mutant capsid comprising AAV-VHRGDLN - VP proteins in which each the VP1, VP2 VP3 proteins are a heterogenous population and comprise deamidated residues in about 50% to about 100% of positions N57 (VP1 only), N329, N452, or N512, based on the parental capsid residue positions. In some embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In some 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.

[0334] In some embodiments, the rAAV comprises a mutant AAV9-RGDFSGY capsid or a mutant AAVhu68-RGDFSGY capsid, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the RGDFSGY peptide insert. In some embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In some embodiments, a mutant rAAV9 or a mutant AAVhu68 has a mutant capsid comprising AAV-RGDFSGY - VP proteins in which each the VP1, VP2 VP3 proteins are a heterogenous population and comprise deamidated residues in about 50% to about 100% of positions N57 (VP1 only), N329, N452, or N512, based on the parental capsid residue positions. In some embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In some 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.

[0335] In some embodiments, the rAAV comprises a mutant AAV9-RGDYVYQ capsid or a mutant AAVhu68-RGDYVYQ capsid, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the RGDYVYQ peptide insert. In some embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In some embodiments, a mutant rAAV9 or a mutant AAVhu68 has a mutant capsid

[0336] 53

[0337] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0338] comprising AAV-RGDYVYQ - VP proteins in which each the VP1, VP2 VP3 proteins are a heterogenous population and comprise deamidated residues in about 50% to about 100% of positions N57 (VP1 only), N329, N452, or N512, based on the parental capsid residue positions. In some embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In some 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.

[0339] In some embodiments, the rAAV comprises a mutant AAV9-RGDYSYT capsid or a mutant AAVhu68-RGDYSYT, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the RGDYSYT peptide insert. In some embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In some embodiments, a rAAV9 or AAVhu68 has a mutant capsid comprising AAV-RGDYSYT - VP proteins in which each the VP1, VP2 VP3 proteins are a heterogenous population and comprise deamidate residues in about 50% to about 100% of positions N57 (VP1 only), N329, N452, or N512, based on the parental capsid residue positions. In some embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In some 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.

[0340] In some embodiments, the rAAV comprises a mutant AAV9-QVRGDIK capsid or a mutant AAVhu68-QVRGDIK, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the QVRGDIK peptide insert. In some embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In some embodiments, a rAAV9 or AAVhu68 has a mutant capsid comprising AAV-QVRGDIK - VP proteins in which each the VP1, VP2 VP3 proteins are a heterogenous population and comprise deamidate residues in about 50% to about 100% of positions N57 (VP1 only), N329, N452, or N512, based on the parental capsid residue positions. In some

[0341] 54

[0342] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0343] embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In some 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.

[0344] In some embodiments, the rAAV comprises a mutant AAV9-PQYTRGD capsid or a mutant AAVhu68-PQYTRGD, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the PQYTRGD peptide insert. In some embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In some embodiments, a rAAV9 or AAVhu68 has a mutant capsid comprising AAV-PQYTRGD - VP proteins in which each the VP1, VP2 VP3 proteins are a heterogenous population and comprise deamidate residues in about 50% to about 100% of positions N57 (VP1 only), N329, N452, or N512, based on the parental capsid residue positions. In some embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In some 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.

[0345] In some embodiments, the rAAV comprises a mutant AAV9-VRGDIRL capsid or a mutant AAVhu68-VRGDIRL, which comprises mutant VP1, mutant VP2, and mutant VP3 proteins, each having a heterogenous population of proteins comprising the VRGDIRL peptide insert. In some embodiments, the proteins are further characterized by having three or four highly deamidated asparagines in positions: N57, N329, N452 and N512, and optional deamidation in other positions within the parental capsid sequence. In some embodiments, a rAAV9 or AAVhu68 has a mutant capsid comprising AAV- VRGDIRL -VP proteins in which each the VP1, VP2 VP3 proteins are a heterogenous population and comprise deamidate residues in about 50% to about 100% of positions N57 (VP1 only), N329, N452, or N512, based on the parental capsid residue positions. In some embodiments, the capsid comprises VP proteins which are highly deamidated in all of these positions. In some 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

[0346] 55

[0347] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0348] 75%, over 80%, over 85%, over 90%, over 95%, or about 70% to about 100%, or values therebetween.

[0349] In some embodiments, the rAAV comprises an AAV capsid wherein the AAV capsid protein comprise an exogenous peptide that is immediately preceded by “AQ”. In some embodiments, the rAAV comprises an AAV capsid wherein the AAV capsid protein comprises an exogenous peptide that is flanked by “AQ” (e.g., “AQ- RGDYREV”. In some embodiments, the rAAV comprises an AAV capsid wherein the AAV capsid protein comprises exogenous peptide which is immediately preceded by the native residues of the parent AAV which may be unmodified at the amino (N-) terminus, and / or at the carboxy (COO-) terminus. In some embodiments, the rAAV comprises an AAV capsid wherein the AAV capsid protein comprises exogenous peptide that is immediately preceded by the native residues of the parent AAV which may be mutated at the amino (N-) terminus, and / or at the carboxy (COO-) terminus. In some embodiments, wherein the parent capsid is an AAV9 capsid or other Clade F capsid, the AAV9 parent capsid or other Clade F parent capsid is unmodified at the residues flanking the inserted exogenous targeting peptide. In some embodiments, the rAAV comprises an AAV capsid wherein the AAV capsid protein comprises exogenous peptide that is flanked by “SAQ” at amino (N-) terminus of the exogenous peptide. In some embodiments, the rAAV comprises an AAV capsid wherein the AAV capsid protein comprises exogenous peptide that is flanked by “AQA” at carboxy (COO-) terminus. In some embodiments, wherein the parent capsid is an AAV9 capsid or other Clade F capsid, the AAV9 parent capsid or other Clade F parent capsid is modified (i.e., mutated) at the residues flanking the inserted exogenous targeting peptide. In some embodiments, the rAAV comprises an AAV capsid wherein the AAV capsid protein comprises exogenous peptide that is flanked by mutated trimer “ENT” at amino (N-) terminus of the exogenous peptide. In some embodiments, the rAAV comprises an AAV capsid wherein the AAV capsid protein comprises exogenous peptide that is flanked by a mutated trimer “SHQ”, SWQ”, SAI”, “GAQ”, “FAQ”, “QAQ”, “AAQ”, “SGQ”, or “SGM” at the amino (N-) terminus of the exogenous peptide. In some embodiments, the rAAV comprises an AAV capsid wherein the AAV capsid protein comprises exogenous peptide that is flanked by a mutated trimer “QQA”, “NQA”, “AMA”, “AQC”, “GQA”, “ARA”, or “GRA” at the 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.

[0350] 56

[0351] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0352] In some embodiments, capsids from Clade F AAV such as AAVhu68 or AAV9 are selected for 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 International Patent Application No. PCT / US2021 / 055436, filed October 18, 2021, now publication No. WO 2022 / 082109, 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 the 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, the 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 capsids.

[0353] In some embodiments, the mutant AAV capsid may be produced by engineering a nucleic acid sequence encoding a mutant peptide insert into the AAV VP1 coding sequence.

[0354] In some embodiments, peptide inserts 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

[0355] 57

[0356] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0357] may be determined. In still other embodiments, these peptides may be used in other vectors or compositions for targeting. In some 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 comprises a sequence encoding for RGDYREV, RGDYREI, RGDYREL, RGDYHQV, VYTRGDV, RGDYSQI, RGDYASV, QNRGDPH, RGDYHYQ, VHRGDLN, RGDFSGY, RGDYVYQ, RGDYSYT, QVRGDIK, PQYTRGD, VRGDIRL, AVIRGDV, IIRGDPA, RGDLHGY, PYQRGDH, RGDYAQV, or RGDYSTM, or a targeting peptide comprising an amino acid sequence as provided for in PCT Publication No. WO2024 / 130067, PCT Publication No. WO2024 / 130070, PCT Application No. PCT / US2024 / 55910, filed November 14, 2024, U. S. Provisional Application No. 63 / 598,718, filed November 14, 2023, U. S. Provisional Application No.

[0358] 63 / 612,676, filed December 20, 2023, U. S. Provisional Application No. 63 / 661,133, filed June 18, 2024, PCT Publication No. WO 2024 / 173802, and U. S. Patent No. 11,920,150, each of which is hereby incorporated by reference in its entirety.

[0359] 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, the 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.

[0360] 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 vpl amino acid sequence of GenBank accession: AAS99264. These splice variants result in proteins of different length. In some embodiments, “AAV9 capsid” includes an AAV having an amino

[0361] 58

[0362] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0363] 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., W02016 / 049230, US 8,927,514, US 2015 / 0344911, and US 8,734,809.

[0364] 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, PCT / 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.

[0365] 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 the 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.

[0366] Methods of preparing AAV-based vectors (e.g., having an AAV9 or another AAV parental 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 the use of AAV9 or other clade F AAV amino acid sequences, but encompasses peptides and / or proteins containing the terminal β-galactose binding generated by other methods known in the 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. Chem. Soc., 85:2149; Stewart and Young, Solid Phase Peptide

[0367] 59

[0368] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0369] 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 nucleic acid molecule comprising a functional rep gene; at least one nucleic acid molecule comprising sufficient helper functions to permit packaging of the minigene into the AAV capsid protein, and a nucleic acid molecule comprising an engineered AAV vector genome to be packaged into the mutant AAV capsid. The AAV vector genome is described herein, and may be composed of, at a minimum, AAV inverted terminal repeats (ITRs) flanking (at the extreme 5' and 3' ends of a nucleic acid molecule comprising an expression cassette comprising nucleic acid sequences, typically exogenous to the AAV, which provide a physiologic useful effect (one or more of a protein or peptide coding sequence, one or more other DNA sequences, one or more miR targeting sequences, and / or combinations thereof optionally, with other useful coding sequences). Typically, the AAV ITRs of the vector genome are selected to be transcomplemented by the rep. 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.

[0370] The components required to be cultured in the host cell to package an AAV expression cassette, comprising transgene, in an AAV capsid may be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., expression cassette comprising transgene, 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 the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, a stable host cell may be generated which is derived from 293 cells (which contain El 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.

[0371] 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

[0372] 60

[0373] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0374] 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, “Adeno-associated 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 the nucleic acid molecule containing the expression cassettes. The cap and rep genes can be supplied in trans.

[0375] In some embodiments, 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 some embodiments, 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).

[0376] 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.

[0377] 61

[0378] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0379] 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. See, e.g., Gao, et al, Proc. Natl. Acad. Sci. U. S. A. 100 (10), 6081-6086 (2003) and US 2013 / 0045186A1.

[0380] 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 some 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 VP1 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 the cell, said AAV VP1 coding sequence being operably linked to a promoter which directs expression of the VP1 coding sequences in the cell line.

[0381] In some embodiments, 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 the art such as generation of plasmid DNA used for production of the gene therapy vectors, generation of the vectors, and purification of the vectors. In some

[0382] 62

[0383] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0384] 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.

[0385] 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.

[0386] 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.

[0387] 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

[0388] 63

[0389] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0390] 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. See also, International Patent Application No. PCT / US2018 / 019992, filed February 27, 2018, now published WO 2018 / 160582, and International Patent Application No. PCT / US2021 / 055436, filed October 18, 2021, now published WO 2022 / 082109, which are incorporated herein by reference in their entireties. Other suitable methods may be selected.

[0391] 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 pL 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.

[0392] 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. Then (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 the 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 the 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

[0393] 64

[0394] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0395] 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 some embodiments, the 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, the samples are further diluted and amplified using primers and a TaqMan™ fluorogenic probe specific for the DNA sequence between the 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 the 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 the Ct value of the plasmid standard curve. End-point assays based on the digital PCR can also be used.

[0396] 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.

[0397] 65

[0398] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0399] However, 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-adjustment is used to determine the empty-full particle ratio.

[0400] 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 are 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 the standard assay. Quantification also can be done using ViroCyt or flow cytometry.

[0401] 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.

[0402] In some embodiments, the manufacturing process for rAAV as described herein (e.g., comprising engineered rAAV) involves 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 its entirety.

[0403] 66

[0404] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0405] Compositions and Uses

[0406] Provided herein are compositions containing at least one rAAV stock (e.g.,a rAAV encoding for a dystrophin product as provided for herein, including, but not limited to, a mutant rAAV9 engineered stock or mutant rAAVhu68 engineered stock, wherein engineered capsid comprises an exogenous targeting motif as described herein) and an optional carrier, excipient and / or preservative.

[0407] In one aspect, provided is a pharmaceutical composition comprising a rAAV as described herein in a formulation buffer. In some embodiments, the rAAV is formulated at about 1 x 109genome copies (GC) / mL to about 1 x 1014GC / mL. In a further embodiment, the rAAV is formulated at about 3 x 109GC / mL to about 3 x 1013GC / mL. In yet a further embodiment, the rAAV is formulated at about 1 x 109GC / mL to about 1 x 1013GC / mL. In some embodiments, the rAAV is formulated at least about 1 x 1011GC / mL.

[0408] 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.

[0409] Provided herein, also, are methods of use of compositions as described herein. In some embodiments, a method for targeted therapy to muscle cells comprising administering to a patient in need thereof a stock of the rAAV as described herein, wherein a therapeutic is targeted for delivery to muscle cell (e.g., cardiac cell (heart), skeletal muscle cell (e.g., gastrocnemius)), and is de-targeted for cells in liver.

[0410] 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 virus (rAAV) vector comprising engineered capsid as provided herein, which can have a muscle targeting motif. In some embodiments, the targeting motif, which can be referred to as an exogenous targeting peptide has a formula of “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 RGDYREV, RGDYREI, RGDYREL, RGDYHQV, VYTRGDV, RGDYSQI, RGDYASV, QNRGDPH, RGDYHYQ, VHRGDLN, RGDFSGY, RGDYVYQ, RGDYSYT, QVRGDIK, PQYTRGD, VRGDIRL, AVIRGDV, IIRGDPA, RGDLHGY, PYQRGDH, RGDYAQV, or RGDYSTM, or an n-mer sequence comprising 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, or a targeting peptide comprising an amino acid sequence as provided for in PCT Publication No.

[0411] 67

[0412] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0413] WO2024 / 130067, PCT Publication No. WO2024 / 130070, PCT Application No.

[0414] PCT / US2024 / 55910, filed November 14, 2024, U. S. Provisional Application No.

[0415] 63 / 598,718, filed November 14, 2023, U. S. Provisional Application No. 63 / 612,676, filed December 20, 2023, U. S. Provisional Application No. 63 / 661,133, filed June 18, 2024, PCT Publication No. WO 2024 / 173802, and U. S. Patent No. 11,920,150, each of which is hereby incorporated by reference in its entirety, and rAAV further comprising a vector genome comprising the transgene operably linked to regulatory sequences that direct expression of the transgene in muscle cell.

[0416] In some embodiments, the target muscle cells include cardiac, smooth, and / or skeletal muscle cells. In some embodiments, the transgene encodes a secreted gene product. In some embodiments, the AAV vector is delivered intravenously.

[0417] Provided herein are also uses of an rAAV comprising a transgene as provided for herein having a engineered capsid with at least one core one or more of exogenous targeting peptide, to target muscle cells at higher levels of transduction than achieved using an AAV9 vector, 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 RGDYREV, RGDYREI, RGDYREL, RGDYHQV, VYTRGDV, RGDYSQI, RGDYASV, QNRGDPH, RGDYHYQ, VHRGDLN, RGDFSGY, RGDYVYQ, RGDYSYT, QVRGDIK, PQYTRGD, VRGDIRL, AVIRGDV, IIRGDPA, RGDLHGY, PYQRGDH, RGDYAQV, or RGDYSTM, or an n-mer sequence comprising 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, or a targeting peptide comprising an amino acid sequence as provided for in PCT Publication No.

[0418] WO2024 / 130067, PCT Publication No. WO2024 / 130070, PCT Application No.

[0419] PCT / US2024 / 55910, filed November 14, 2024, U. S. Provisional Application No.

[0420] 63 / 598,718, filed November 14, 2023, U. S. Provisional Application No. 63 / 612,676, filed December 20, 2023, U. S. Provisional Application No. 63 / 661,133, filed June 18, 2024, PCT Publication No. WO 2024 / 173802, and U. S. Patent No. 11,920,150, each of which is hereby incorporated by reference in its entirety.

[0421] In some 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 some embodiments, a composition as described herein may contain a different vector expressing an expression cassette as described herein, or

[0422] 68

[0423] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0424] another active component (e.g., an antibody construct, another biologic, and / or a small molecule drug).

[0425] 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 transgenes may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.

[0426] In some embodiments, 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 the 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.

[0427] A suitable surfactant, or combination of surfactants, may be selected from among non-ionic surfactants that are nontoxic. In some embodiments, a difunctional block copolymer surfactant terminating in primary hydroxyl 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 (polyethylene oxide)), SOLUTOL HS 15 (Macrogol (polyethylene glycol) -15 Hydroxystearate), LABRASOL® (Polyoxy capryllic glyceride), polyoxy 10

[0428] 69

[0429] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0430] oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol. In some embodiments, 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 polyoxypropylene 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.

[0431] 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 the rAAV binding activity in vivo. A suitable surfactant, or combination of surfactants, may be selected from among non-ionic surfactants that are nontoxic. In some embodiments, 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 (polypropylene oxide)) flanked by two 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 some embodiments, 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 polyoxypropylene 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.

[0432] In some embodiments, the formulation may contain a buffered saline aqueous solution not comprising sodium bicarbonate. Such a formulation may contain a buffered

[0433] 70

[0434] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0435] 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 some embodiments, the buffer is phosphate-buffered saline (PBS). In some embodiments, the formulation buffer PBS with total salt concentration of 200 mM, 0.001% (w / v) pluronic F68 (Final Formulation Buffer, FFB).

[0436] Optionally, the compositions of the invention may contain, in addition to the 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, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0437] 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.

[0438] In some embodiments, the composition comprises a 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 some 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.

[0439] 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 recombinant vector (e.g., rAAV) will depend primarily on factors such as the 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 the recombinant 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 109to 4x1014GC 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

[0440] 71

[0441] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0442] vector is employed. The levels of expression of the transgene can be monitored to determine the frequency of dosage resulting in recombinant vectors, preferably AAV vectors comprising transgene. Optionally, dosage regimens similar to those described for therapeutic purposes may be utilized for immunization using the compositions of the invention.

[0443] 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 109GC to about 1.0 x 1016GC (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 1012GC to 1.0 x 1014GC for a human patient. In some embodiments, the compositions are formulated to contain at least IxlO9, 2xl09, 3xl09, 4xl09, 5xl09, 6xl09, 7xl09, 8xl09, or 9xl09GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO10, 2xlO10, 3xlO10, 4xlO10, 5xl010, 6xlO10, 7xlO10, 8xl010, or 9xlO10GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO11, 2xlO11, 3xlO11, 4xlO11, 5xl011, 6xlO11, 7xlO11, 8xl011, or 9xlO11GC 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 9xl012GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO13, 2xl013, 3xl013, 4xl013, 5xl013, 6xl013, 7xl013, 8xl013, or 9xl013GC 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, 6x1014, 7x1014, 8x1014, or 9x1014GC per dose including all integers or fractional amounts within the range. In another embodiment, the compositions are formulated to contain at least IxlO15, 2xl015, 3xl015, 4xl015, 5xl015, 6xl015, 7xl015, 8xl015, or 9xl015GC per dose including all integers or fractional amounts within the range. In some embodiments, for human application the dose can range from IxlO10to about IxlO12GC per dose including all integers or fractional amounts within the range. In some embodiments, the rAAV compositions is formulated in dosage units to contain about 1 x 1013GC / kg. In some embodiments, the rAAV compositions is formulated in dosage units to contain about 2.5 x 1013GC / kg. In some embodiments, the rAAV compositions is formulated in dosage units to contain about 5 x 1013GC / kg.

[0444] 72

[0445] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0446] In some embodiments, for human application the dose can range from 109to about 7xl013GC per dose including all integers or fractional amounts within the range.

[0447] 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, the viral titer used, the route of administration, and the desired effect of the method. In some embodiments, the volume of carrier, excipient or buffer is at least about 25 μL. In some embodiments, the volume is about 50 μL. In another embodiment, the volume is about 75 μL. In another embodiment, the volume is about 100 μL. In another embodiment, the volume is about 125 μL. In another embodiment, the volume is about 150 μL. In another embodiment, the volume is about 175 μL. In yet another embodiment, the volume is about 200 μL. In another embodiment, the volume is about 225 μL. In yet another embodiment, the volume is about 250 μL. In yet another embodiment, the volume is about 275 μL. In yet another embodiment, the volume is about 300 μL. In yet another embodiment, the volume is about 325 μL. In another embodiment, the volume is about 350 μL. In another embodiment, the volume is about 375 μL. In another embodiment, the volume is about 400 μL. In another embodiment, the volume is about 450 μL. In another embodiment, the volume is about 500 μL. In another embodiment, the volume is about 550 μL. In another embodiment, the volume is about 600 μL. In another embodiment, the volume is about 650 μL. In another embodiment, the volume is about 700 μL. In another embodiment, the volume is between about 700 and 1000 μL.

[0448] In some embodiments, the viral constructs may be delivered in doses of from at least about least IxlO9GCs to about 1 x 1015, or about 1 x 1011to 5 x 1013GC. 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 μL 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.

[0449] 73

[0450] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0451] 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.

[0452] 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 some embodiments, the rAAV or the pharmaceutical composition comprises a formulation buffer suitable for intravenous administration to a patient in the need thereof.

[0453] In some embodiments, provided herein is a composition, such as a pharmaceutical 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 RGDYREV, RGDYREI, RGDYREL, RGDYHQV, VYTRGDV, RGDYSQI, RGDYASV, QNRGDPH, RGDYHYQ, VHRGDLN, RGDFSGY, RGDYVYQ, RGDYSYT, QVRGDIK, PQYTRGD, VRGDIRL, AVIRGDV, IIRGDPA, RGDLHGY, PYQRGDH, RGDYAQV, or RGDYSTM, or an n-mer sequence comprising 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, or a targeting peptide comprising an amino acid sequence as provided for in PCT Publication No. WO2024 / 130067, PCT Publication No. WO2024 / 130070, PCT Application No. PCT / US2024 / 55910, filed November 14, 2024, U. S. Provisional Application No. 63 / 598,718, filed November 14, 2023, U. S. Provisional Application No.

[0454] 63 / 612,676, filed December 20, 2023, U. S. Provisional Application No. 63 / 661,133, filed June 18, 2024, PCT Publication No. WO 2024 / 173802, and U. S. Patent No. 11,920,150, each of which is hereby incorporated by reference in its entirety, 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.

[0455] In some embodiments, provided herein is 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.

[0456] 74

[0457] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0458] In some embodiments, provided herein is a nucleic acid sequence encoding the fusion polypeptide protein that 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, polylactide- polyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrins, dendrimers and polyethylenimine. In some embodiments, 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 are commercially available. See, e.g., WO2014 / 089486, US 2018 / 0353616A1, and US 8,853,377B2, which are incorporated by reference. In some 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. Then 27(4):710-728). In some embodiments, LNP comprises a cationic lipid (i.e. N-[l-(2,3-dioleoyloxy)propyl]-N, N, N-trim ethylammonium 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(P-amino)esters (PBAEs). See, e.g., WO2014 / 089486,

[0459] 75

[0460] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0461] US 2018 / 0353616A1, US2013 / 0037977A1, W02015 / 074085A1, US9670152B2, and US 8,853,377B2, which are incorporated by reference. In some 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) n-mer is RGDYREV, RGDYREI, RGDYREL, RGDYHQV, VYTRGDV, RGDYSQI, RGDYASV, QNRGDPH, RGDYHYQ, VHRGDLN, RGDFSGY, RGDYVYQ, RGDYSYT, QVRGDIK, PQYTRGD, VRGDIRL, AVIRGDV, IIRGDPA, RGDLHGY, PYQRGDH, RGDYAQV, or RGDYSTM, or an n-mer sequence comprising 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, or a targeting peptide comprising an amino acid sequence as provided for in PCT Publication No. WO2024 / 130067, PCT Publication No. WO2024 / 130070, PCT Application No.

[0462] PCT / US2024 / 55910, filed November 14, 2024, U. S. Provisional Application No.

[0463] 63 / 598,718, filed November 14, 2023, U. S. Provisional Application No. 63 / 612,676, filed December 20, 2023, U. S. Provisional Application No. 63 / 661,133, filed June 18, 2024, PCT Publication No. WO 2024 / 173802, and U. S. Patent No. 11,920,150, each of which is hereby incorporated by reference in its entirety, i.e., decorated surface with targeting peptide. In some embodiments, a lipid nanoparticle (LNP) comprises at least one IIRGDPA peptide. In some embodiments, a lipid nanoparticle (LNP) comprises at least one AVIRGDV peptide.

[0464] In some embodiments, provided herein is a composition, e.g., an rAAV having a 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) n-mer is RGDYREV, RGDYREI, RGDYREL, RGDYHQV, VYTRGDV, RGDYSQI, RGDYASV, QNRGDPH, RGDYHYQ, VHRGDLN, RGDFSGY, RGDYVYQ, RGDYSYT, QVRGDIK, PQYTRGD, VRGDIRL, AVIRGDV, IIRGDPA, RGDLHGY, PYQRGDH, RGDYAQV, or RGDYSTM, or an n-mer sequence comprising 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, or a targeting peptide comprising an amino acid sequence as provided for in PCT Publication No.

[0465] WO2024 / 130067, PCT Publication No. WO2024 / 130070, PCT Application No.

[0466] PCT / US2024 / 55910, filed November 14, 2024, U. S. Provisional Application No.

[0467] 63 / 598,718, filed November 14, 2023, U. S. Provisional Application No. 63 / 612,676, filed

[0468] 76

[0469] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0470] December 20, 2023, U. S. Provisional Application No. 63 / 661,133, filed June 18, 2024, PCT Publication No. WO 2024 / 173802, and U. S. Patent No. 11,920,150, each of which is hereby incorporated by reference in its entirety, is useful for delivering a therapeutic to a patient in need thereof.

[0471] In some embodiments, provided herein is a composition comprising an rAAV having a modified capsid which is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery to muscle cell. In some embodiments, provided herein is a composition comprising an rAAV having a modified capsid which is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery to in muscle cell, and de-targeted for liver.

[0472] In some embodiments, a composition comprises an rAAV having a modified capsid with at least one RGDYREV peptide is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery to muscle cell. In some embodiments, a composition comprises an rAAV having a modified capsid with at least one RGDYHQV peptide is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery to muscle cell. In some embodiments, a composition comprises an rAAV having a modified capsid with at least one VYTRGDV peptide is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery to muscle cell. In some embodiments, a composition comprises an rAAV having a modified capsid with at least one RGDYSQI peptide is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery to muscle cell. In some embodiments, a composition comprises an rAAV having a modified capsid with at least one RGDYASV peptide is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery to muscle cell. In some embodiments, a composition comprises an rAAV having a modified capsid with at least one QNRGDPH peptide is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery to muscle cell. In some embodiments, a composition comprises an rAAV having a modified capsid with at least one RGDYHY Q peptide is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery to muscle cell. In some embodiments, a composition comprises an rAAV having a modified capsid with at least one VHRGDLN peptide is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery to muscle cell. In some embodiments, a

[0473] 77

[0474] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0475] composition comprises an rAAV having a modified capsid with at least one RGDFSGY peptide is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery to muscle cell. In some embodiments, a composition comprises an rAAV having a modified capsid with at least one RGDYVYQ peptide is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery to muscle cell. In some embodiments, a composition comprises an rAAV having a modified capsid with at least one RGDYSYT peptide is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery to muscle cell. In some embodiments, a composition comprises an rAAV having a modified capsid with at least one QVRGDIK peptide is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery to muscle cell. In some embodiments, a composition comprises an rAAV having a modified capsid with at least one PQYTRGD peptide is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery to muscle cell. In some embodiments, a composition comprises an rAAV having a modified capsid with at least one VRGDIRL peptide is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery to muscle cell. In some embodiments, a composition comprises an rAAV having a modified capsid with at least one core RGDYHQV or VYTRGDV is useful for delivering a therapeutic to a patient in need thereof, wherein the therapeutic is targeted for delivery to in muscle cell, and de-targeted for liver.

[0476] In some embodiments, the methods and compositions may be used for treatment of Duchenne muscular dystrophy (DMD) and related conditions that are associated with misexpression, dysregulation, or mutations of the dystrophin gene.

[0477] In some embodiments, the methods and compositions described herein may be used to ameliorate one or more symptoms of DMD, including increased average life span.

[0478] In some embodiments, the methods and compositions may be used for treatment, or to ameliorate one or more symptoms of muscular dystrophy.

[0479] In some embodiments, the methods and compositions may be used for treatment, or to ameliorate one or more symptoms of Duchenne muscular dystrophy (Dystrophin, DMD) or Becker muscular dystrophy (Dystrophin, DMD).

[0480] In some embodiments, methods for treating one or more of muscle cell disorders, and / or a disease in a patient in need thereof are provided. In some embodiments, the

[0481] 78

[0482] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0483] methods comprise delivering a polypeptide as provided for herein, a nucleic acid molecule as provided for herein, a vector as provided for herein, a lipid nanoparticle as provided for herein, a stock of rAAV as provided for herein, or a combination of any of the foregoing. In some embodiments, the composition that is administered is referred to as a pharmaceutical composition.

[0484] For clarity, the term “subject” and “patient” can be used interchangeably.

[0485] In some embodiments, methods of treating Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy in a patient in need thereof are provided. In some embodiments, the methods comprise delivering a polypeptide as provided for herein, a nucleic acid molecule as provided for herein, a vector as provided for herein, a lipid nanoparticle as provided for herein, a stock of rAAV as provided for herein, or a combination of any of the foregoing. In some embodiments, the composition that is administered is referred to as a pharmaceutical composition.

[0486] In some embodiments, methods for inhibiting the progression of Duchenne muscular dystrophy or Becker muscular dystrophy in a patient in need thereof are provided. In some embodiments, the methods comprise delivering a polypeptide as provided for herein, a nucleic acid molecule as provided for herein, a vector as provided for herein, a lipid nanoparticle as provided for herein, a stock of rAAV as provided for herein, or a combination of any of the foregoing. In some embodiments, the composition that is administered is referred to as a pharmaceutical composition.

[0487] In some embodiments, methods of treating skeletal muscle mass deficiency in a subject having a mutation in the dystrophin gene are provided. In some embodiments, the methods comprise delivering a polypeptide as provided for herein, a nucleic acid molecule as provided for herein, a vector as provided for herein, a lipid nanoparticle as provided for herein, a stock of rAAV as provided for herein, or a combination of any of the foregoing. In some embodiments, the composition that is administered is referred to as a pharmaceutical composition.

[0488] In some embodiments, methods of increasing skeletal muscle mass in a subject with DMD or Becker muscular dystrophy are provided. In some embodiments, the methods comprise delivering a polypeptide as provided for herein, a nucleic acid molecule as provided for herein, a vector as provided for herein, a lipid nanoparticle as provided for herein, a stock of rAAV as provided for herein, or a combination of any of the foregoing.

[0489] 79

[0490] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0491] In some embodiments, the composition that is administered is referred to as a pharmaceutical composition.

[0492] In some embodiments, methods of preventing or inhibiting the decrease of muscle mass in a subject with DMD or Becker muscular dystrophy are provided. In some embodiments, the methods comprise delivering a polypeptide as provided for herein, a nucleic acid molecule as provided for herein, a vector as provided for herein, a lipid nanoparticle as provided for herein, a stock of rAAV as provided for herein, or a combination of any of the foregoing. In some embodiments, the composition that is administered is referred to as a pharmaceutical composition.

[0493] In some embodiments, medicaments for treating Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy are provided. In some embodiments, the medicament comprises a polypeptide as provided for herein, a nucleic acid molecule as provided for herein, a vector as provided for herein, a lipid nanoparticle as provided for herein, a stock of rAAV as provided for herein, or a combination of any of the foregoing.

[0494] In some embodiments, medicaments for treating skeletal muscle mass deficiency in a subject having a mutation in the dystrophin gene are provided. In some embodiments, the medicament comprises a polypeptide as provided for herein, a nucleic acid molecule as provided for herein, a vector as provided for herein, a lipid nanoparticle as provided for herein, a stock of rAAV as provided for herein, or a combination of any of the foregoing.

[0495] In some embodiments, medicaments for preventing or inhibiting the decrease of muscle mass in a subject with DMD or Becker muscular dystrophy are provided. In some embodiments, the medicament comprises a polypeptide as provided for herein, a nucleic acid molecule as provided for herein, a vector as provided for herein, a lipid nanoparticle as provided for herein, a stock of rAAV as provided for herein, or a combination of any of the foregoing.

[0496] In some embodiments, a rAAV having a modified capsid as described herein may be delivered in a co-therapeutic regimen which further comprises one or more other active components. In some embodiments, the regimen may involve co-administration of an immunomodulatory component. In some embodiments, provided herein is a rAAV having a modified capsid as described herein for use in delivery with immunosuppressive co-therapeutic regimen, and methods thereof. Without wishing to be bound by theory, immune suppression co-therapy does one or more of the following: induces anergy or immunologic

[0497] 80

[0498] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0499] tolerance to the rAAV and / or transgene; blocks an immune response to optimize efficacy; minimize de novo immune response against transgene; minimize impact of pre-existing immune response to transgene; minimize impact of pre-existing immune response to AAV; prevent immune medicated toxicity; minimize destruction of TG expressing cells.

[0500] In some embodiments, provided herein is rAAV having modified capsid as described herein for use in a method further comprising a combination therapy, such as transient co-treatment with an immunosuppressant before and / or during treatment with rAAV. Optionally, immunosuppressive co-therapy may be used as a precautionary measure without prior assessment of neutralizing antibodies to the AAV vector capsid and / or other components of the formulation. Prior immunosuppression therapy may be desirable to prevent potential adverse immune reaction to the transgene product (gene of interest) i.e., where the transgene product may be seen as “foreign.”

[0501] Immunosuppressants for such co-therapy include, but are not limited to, a glucocorticoid, steroids or corticosteroids, antimetabolites, T-cell inhibitors, a macrolide (e.g., arapamycin or rapalog), and cytostatic agents including an alkylating agent, an antimetabolite, a cytotoxic antibiotic, an antibody, or an agent active on immunophilin. The immune suppressant may include a nitrogen mustard, nitrosourea, platinum compound, methotrexate, azathioprine, mycophenolate mofetil, methotrexate, leflunomide (Arava), cyclophosphamide, chlorambucil (Leukeran), a chloroquine (e.g., hydroxychloroquine), quinine sulfate, mefloquine, a combination of atovaquone and proguanil, sulfasalazine, mercaptopurine, fluorouracil, dactinomycin, an anthracycline, mitomycin C, bleomycin, mithramycin, IL-2 receptor- (CD25-) or CD3 -directed antibodies, anti-IL-2 antibodies, ciclosporinabatacept (Orencia), adalimumab (Humira), anakinra (Kineret), certolizumab (Cimzia), etanercept (Enbrel), golimumab (Simponi), infliximab (Remicade), rituximab (Rituxan), tocilizumab (Actemra) and tofacitinib (Xeljanz), cyclosporine, tacrolimus, sirolimus, IFN-β, IFN-γ, an opioid, or TNF-a (tumor necrosis factor-alpha) binding agent, and combinations of these drugs.

[0502] In some embodiments, the immunosuppressive therapy may be started 0, 1, 2, 7, or more days prior to the gene therapy administration. Such therapy may involve coadministration 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 (7 days), about 60 days, or longer, as needed. In some

[0503] 81

[0504] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0505] embodiments, a tacrolimus-free regimen is selected. In some embodiments, the two or more drugs may be, e.g., one or more corticosteroids (e.g., a prednelisone or prednisone) and optionally, MMF and / or a calcinuerin inhibitor (e.g., tacrolimus or sirolimus (i.e., rapamycin)). In some embodiments, the two or more drugs are micophenolate mofetil (MMF) and / or sirolimus. In another embodiment, the two or more drugs may be, e.g., methylprednisolone, prednisone, tacrolimus, and / or sirolimus. In some embodiments, the drugs are MMF and tacrolimus for 0 to 15 days pre-vector delivery and maintaining for about 8 weeks with MMF and / or throughout follow-up appointments with tacrolimus. One or more of these drugs may be continued after gene therapy administration, at the same dose or an adjusted dose. In some embodiments, patients are dosed initially with an IV steroid (e.g., methylprednisolone) to load the dose, followed by with an oral steroid (e.g., prednisolone) that is gradually tapered down so that the patient is off steroids by week 12. The corticosteroid treatment is supplemented by tacrolimus (for 24 weeks) and / or sirolimus (for 12 weeks), and can be further supplemented with MMF. When using both tacrolimus and sirolimus, the dose of each should be a low dose adjusted to maintain a blood trough level of about 4 ng / mL to about 8 ng / ml, or a total of about 8 ng / mL to about 16 ng / mL. In some embodiments, when only one of these agents is used, the total dose for tacrolimus and / or sirolimus may be in the range of about 16 ng / mL to about 24 ng / mL. If only one of the agents is used, the label dose (higher dose) should be employed; e.g., tacrolimus at 0.15-0.20 mg / kg / day given as two divided doses every 12 hours; and sirolimus at 1 mg / m2 / day; the loading dose should be 3 mg / m2. If MMF is added to the regimen, the dose for tacrolimus and / or sirolimus can be maintained since the mechanisms of action differ. These and other therapies may be started at about day - 14 to day -1 (e.g., day -2, day 0, etc.), and continue to about to up to about 1 week (7 days), or up to about 60 days, or up to about 12 weeks, or up to about 16 weeks, or up to about 24 weeks, or up to about 48 weeks, or longer, as needed. In some embodiments, a tacrolimus-free regimen is selected.

[0506] In some embodiments, patients will receive immune suppression (IS) as follows: corticosteroids: methylprednisolone 10 mg / kg IV once on Day 1 pre-dose and oral prednisone starting at 0.5 mg / kg / day on Day 2 with gradual tapering and discontinuation by Week 12; Tacrolimus: 1 mg BID by mouth Day 2 to Week 24 with tapering over 8 weeks between Week 24 and 32; Sirolimus: (a loading dose on Day -2 and then sirolimus 0.5 mg / m2 / day divided in BID dosing until Week 48. In some embodiments, IS therapy is discontinued at Week 48 post dosing with the rAAV.

[0507] 82

[0508] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0509] In some embodiments, the method further comprises administering subject with intramuscular steroid or corticosteroid prior to and / or post administration of rAAV. In some embodiments, the method further comprises administering subject with oral steroid or corticosteroid prior to and / or post administration with rAAV.

[0510] In some embodiments, the immunosuppressive therapy regimen is as follows:

[0511] Corticosteroids:

[0512] In the morning of vector administration (Day 1 pre-dose), patients receive methylprednisolone 10mg / kg IV (maximum of 500 mg) over at least 30 minutes. The methylprednisolone is administered before the lumbar puncture and intrathecal (IC) injection of rAAV. Premedication with acetaminophen and an antihistamine is optional.

[0513] On Day 2, oral prednisone is started with the goal to discontinue prednisone by Week 12. The dose of prednisone is as follows: Day 2 to the end of Week 2: 0.5 mg / kg / day. Week 3 and 4: 0.35 mg / kg / day. Week 5-8: 0.2 mg / kg / day. Week 9-12: 0.1 mg / kg.

[0514] Prednisone is discontinued after Week 12. The exact dose of prednisone can be adjusted to the next higher clinically practical dose.

[0515] Sirolimus: 2 days prior to vector administration (Day -2): a loading dose of sirolimus 1 mg / m2 every 4 hours x 3 doses is administered. From Day -1: sirolimus 0.5 mg / m2 / day divided in twice a day dosing with target blood level of 4-8 ng / ml. Sirolimus is discontinued after the Week 48 visit.

[0516] Tacrolimus: Tacrolimus is started on Day 2 (the day following rAAV) at a dose of 1 mg twice daily and adjusted to achieve a blood level 4-8 ng / mL for 24 Weeks. Starting at Week 24 visit, tacrolimus is tapered off over 8 weeks. At week 24 the dose is decreased by approximately 50%. At Week 28 the dose is further decreased by approximately 50%. Tacrolimus is discontinued at Week 32.

[0517] In some embodiments, the method further comprises administering to a subject anti-AAV neutralizing antibodies (NAb) to reduce peripheral transduction, and mitigate the potential risk of transgene-induced toxicity. In some embodiments, the method further comprises detect the presence of systemic AAV NAb prior to treating with anti-AAV NAb, wherein patients with levels of anti-AAV NAb in excess of a predetermined level against the rAAV capsid (or a sero-crossreactive capsid) do not require pretreatment. Such levels may be, e.g., in excess of about 1:10, about 1:20, about 1:50, about 1:100, about 1:250, or higher or lower levels. In some embodiments, the method further comprises

[0518] 83

[0519] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0520] intravenously administering human anti-AAV polyclonal antibodies (e.g., plasma-derived, pooled human immunoglobulin (IVIG)), an anti-AAV monoclonal antibody, or a cocktail of anti-AAV antibodies, to a patient about 1 day to about 2 hours before treatment with a rAAV as described herein.

[0521] In some embodiments, a combination regimen is provided for preventing off-target delivery rAAV, the regimen comprising (a) pretreating the patient by systemically administering a composition comprising anti-AAV capsid neutralizing antibodies directed against an AAV capsid in a recombinant AAV vector, and (b) administering to the central nervous system (CNS) rAAV as described herein (e.g., rAAV). See also, US Provisional Patent Application No. 63 / 328,227, filed April 6, 2022, and International Patent Application No. PCT / US2023 / 065422, filed April 6, 2023, now Publication No.

[0522] WO2023 / 196892A1 which are incorporated herein by reference in their entirety.

[0523] As used herein, the term “NAb titer” a measurement of how much neutralizing antibody (e.g., anti-AAV Nab) is produced which neutralizes the 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.

[0524] In some embodiments, a combination regimen is provided including an 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 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. anti-FcRN antibodies include, e.g., rozanolixizumab (UCB7665) (UCB SA); IMVT-1401, RVT-1401 (HL161), HBM9161 (all form HanAll BioPhrma Co. Ltd), Nipocalimab (M281) (Momenta Pharmaceuticals Inc), ARGX-113 (efgartigimod) (Argenx S. E.), orilanolimab (ALXN 1830, SYNT001, Alexion Pharmaceuticals Inc), SYNT002, ABY-039 (Affibody AB), or DX-2507 (Takeda Pharmaceutical Co. Ltd). In some embodiments, a combinations of anti-FcRN antibodies is administered. In some embodiments, an anti-FcRN antibody is administered in combination with a suitable anti- FcRn ligand (i.e., a peptide or protein construct binding human FcRn so as to inhibit IgG binding).

[0525] 84

[0526] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0527] In some embodiments, a combination regimen for treating a patient with MPSIIIB is provided, wherein the regiment includes administering a vector describe herein in combination with a ligand which inhibits binding of human FcRn and pre-existing patient neutralizing antibodies (e.g., IgG). In some embodiments, the patient may be naive to any therapeutic treatment with a vector and may have pre-existing immunity due to prior infections with a wild-type virus. In other embodiments, the patient may have neutralizing antibodies as a result of a prior treatment or vaccination. In some embodiments, the patient may have neutralizing antibodies 1: 1 to 1:20, or in excess of 1:2, in excess of 1:5, in excess of 1:10, in excess of 1:20, in excess of 1:50, in excess of 1:100, in excess of 1:200, in excess of 1:300 or higher. In some embodiments, a patient has neutralizing antibodies in the range of 1:1 to 1:200, or 1:5 to 1:100, or 1:2 to 1: 20, or 1:5 to 1: 50, or 1:5 to 1:20. In some embodiments, a patient receives a single anti-FcRn ligand (e.g., anti-FcRn antibody) as the sole agent to modulate FcRn-IgG binding and to permit effective vector delivery. In other embodiments, a patient may receive a combination of one or more anti-FcRn ligands and a second component (e.g., an Fc receptor down-regulator (e.g., interferon gamma), an IgG enzyme, or another suitable component). Such combinations may be particularly desirable for patients having particularly high neutralizing antibody levels (e.g., in excess of 1:200).

[0528] In some embodiments, an anti-FcRn ligand(s) (e.g., antibodies) is administered to a patient having neutralizing antibodies prior to and, optionally, concurrently with a selected viral vector. In some embodiments, continued expression of an anti-FcRn ligand post administration of the gene therapy vector may desired on a short-term (transient basis), e.g., until such time as the viral vector clears from the patient. In some embodiments, persistent expression of an anti-FcRn ligand may be desired. Optionally, in this embodiment, the ligand may be delivered via a viral vector, including, e.g., in the viral vector expressing the therapeutic transgene. However, this embodiment is not desirable where the therapeutic gene being delivered is an antibody or antibody construct or another construct comprising an IgG chain. In such embodiments, where an antibody construct having an IgG chain is being delivered via a viral vector to a patient having pre-existing immunity, the anti-FcRn ligand is delivered or dosed transiently so that the amount of anti-FcRn ligand in the circulation is cleared from the sera before effective levels of vector-mediated transgene product are expressed.

[0529] 85

[0530] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0531] In some embodiments, the FcRn ligand is delivered one to seven days prior to administration of the vector (e.g., rAAV). In some embodiments, the FcRn ligand is delivered daily. In some embodiments, the FcRn ligand (e.g., immunoglobulin construct(s)) is delivered on the same day as the vector is administered. In some embodiments, the FcRn ligand (e.g., immunoglobulin construct(s)) is delivered at least one day to four weeks post-rAAV administration. In some embodiments, the ligand is delivered for four weeks to six months post-rAAV administration. In some embodiments, the ligand is dosed via a different route of administration than the rAAV. In some embodiments, the ligand is dosed orally, intravenously, or intraperitoneally. See also, International Patent Application No. PCT / US2021 / 037575, filed June 16, 2021, and now published WO 2021 / 257668 Al, which is incorporated herein by reference in its entirety.

[0532] In some embodiments, the rAAV is for use in a co-therapeutic regimen which further comprises administering at least one or more of immunosuppressive agents comprising corticosteroid, an antimetabolite, a T-cell inhibitor, a macrolide, or a cytostatic agent. In some embodiments, the rAAV is for use in a co-therapeutic regimen which further comprises administering one or more of: (a) a corticosteroid or combination of corticosteroids 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. In some embodiments, the one or more of immunosuppressive agents is administered (i) prior to rAAV administration, (ii) post-rAAV administration, or (iii) prior to and post-rAAV administration. In some embodiments, the rAAV is for use in a co-therapeutic regimen which further comprises co-administering a ligand which specifically prevents binding between human neonatal Fc receptor (FcRn) and the neutralizing antibodies without interfering with albumin binding to FcRn. In some embodiments, the rAAV is for use in a co-therapeutic regimen which further comprises pretreating the patient or the subject by systemically administering a composition comprising anti-AAV capsid neutralizing antibodies directed against an AAV capsid of the recombinant AAV vector.

[0533] It should be understood that the compositions in the method described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.

[0534] Kit

[0535] 86

[0536] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0537] In some 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 some embodiments, 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.

[0538] 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.

[0539] 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, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass. The terms “antibody” and “immunoglobulin” may be used interchangeably herein.

[0540] “Neutralizing antibody titer” (NAb titer) a measurement of how much neutralizing antibody (e.g., anti-AAV NAb) is produced which neutralizes the 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.

[0541] As used herein when used to refer to vp capsid proteins, the term “heterogeneous” 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. SEQ ID NO: 20 provides the encoded amino acid sequence of AAVhu68. SEQ ID NO: 18 provides the encoded amino acid sequence of the AAV9 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

[0542] 87

[0543] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0544] the vpl, vp2 and vp3 proteins within a capsid. The AAV capsid contains subpopulations within the vpl proteins, within the vp2 proteins and within the vp3 proteins which have modifications from the 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.

[0545] 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 the 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 the reference amino acid position. Such percentages may be determined using 2D-gel, mass spectrometry techniques, or other suitable techniques.

[0546] 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

[0547] 88

[0548] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0549] 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.

[0550] 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.

[0551] 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 that is ready for immediate replication and transcription. See, e.g., D M McCarty et al, “Self-complementary recombinant adeno-associated virus (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.

[0552] 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 some embodiments, the 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.

[0553] 89

[0554] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0555] A “replication-defective virus” 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 some embodiments, the genome of the viral vector does 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.

[0556] 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 some embodiments, the capsid contains about 60 proteins composed of vpl 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 some embodiments, the only AAV sequences are the 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.

[0557] The term “nuclease-resistant” indicates that the AAV capsid has assembled around the expression cassette 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.

[0558] As used herein, the term “host cell” may refer to the packaging cell line in which the rAAV is produced from the plasmid. Such a cell may be transiently transfected for production, with one, two, three, or more genetic elements (e.g., plasmids). Alternatively or additionally, a host cell may stably transformed with one or more of the required sequences.

[0559] 90

[0560] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0561] In the alternative, the term “host cell” may refer to the target cell in which expression of the transgene is desired.

[0562] The rAAV provided herein are not limited by the type of nucleic acid molecule (e.g, vector genome) which is packaged in the mutant capsids. As used herein, a “vector genome” refers to the nucleic acid molecule packaged inside the rAAV capsid which forms a viral particle. Such a nucleic acid sequence contains AAV inverted terminal repeat sequences (ITRs). In the examples herein, a vector genome in a nucleic acid molecule useful in production 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 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 some embodiments, the rAAV may comprise a vector genome flanked by 3' and 5' AAV ITRs, respectively. In some embodiments, the rAAV may comprise a vector genome flanked by two 5' AAV ITRs. In some 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 final rAAV product. In some embodiments, the ITRs are from AAV2, a different source AAV than the capsid, or other than full-length ITRs may be selected. In some embodiments, the ITRs are from the same AAV source as the AAV which provides the rep function during production or a trans-complementing AAV. Further, other ITRs, e.g., self-complementary (scAAV) ITRs, may be used. Both single-stranded AAV and self-complementary (sc) AAV are encompassed with 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 genome” 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

[0563] 91

[0564] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0565] envelope protein. For example, AAV inverted terminal repeats are utilized for packaging into AAV and certain other parvovirus capsids.

[0566] 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.

[0567] In some embodiments, non-viral genetic elements used in manufacture of a rAAV, will be referred to as vectors (e.g., production vectors). In some 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.

[0568] 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 some 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.

[0569] 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 some embodiments, the target tissue is muscle tissue. In some embodiments, the target cell is one or more muscle cell type (e.g., cardio muscle cell or gastrocnemius muscle cell).

[0570] 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.

[0571] 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 mutant.

[0572] 92

[0573] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0574] 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.

[0575] The term “expression” is used herein in its broadest meaning and comprises the production of RNA or of RNA and protein. Expression may be transient or may be stable.

[0576] The term “substantial homology” or “substantial similarity,” when referring to a nucleic acid, or fragment thereof, indicates that, when optimally aligned with 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 the 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.

[0577] 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.

[0578] Percent identity may be readily determined for amino acid sequences over the 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.

[0579] 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 by resort to algorithms and computer programs known by those of skill in the art.

[0580] 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

[0581] 93

[0582] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0583] sequences or protein (amino acids) sequences, often containing corrections for missing or additional bases or amino acids as compared to a reference sequence.

[0584] 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-Waterman 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 the 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 the 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).

[0585] In some embodiments, an effective amount may be determined based on an animal model, rather than a human patient.

[0586] As described above, the 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

[0587] 94

[0588] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0589] term “E+#” or the term “e+#” is used to reference an exponent. For example, “5E10” or “5el0” is 5 x 1010. These terms may be used interchangeably.

[0590] 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.

[0591] 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.

[0592] Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Herein, “up to” a number (for example, up to 50) includes the number (for example, 50). The term “in the range” or “within a range” (and similar statements) includes the endpoints of the stated range.

[0593] With regard to the description of these embodiments, it is intended that each of the compositions herein described, is useful, in another embodiment, in the methods provided for herein. In addition, it is also intended that each of the compositions herein described as useful in the methods, is, in another embodiment.

[0594] 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 the 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.

[0595] EXAMPLES

[0596] The following examples are illustrative only and are not a limitation on the invention described herein.

[0597] Example 1

[0598] Recent findings from the ENDEAVOR trial (NCT04626674) phase one clinical trial of DMD patients with delandistrogene moxeparvovec micro-dystrophin (μDys) has uncovered the increased risk of eliciting an adverse immune response in individuals who have a portion of the DMD gene sequence deleted.

[0001] HLA epitope mapping investigation of immune-mediated myositis (IMM) in one of the treated patients in this clinical trial indicated that exons 8 and / or 9 (corresponding to Hinge 1 domain of dystrophin), and

[0599] 95

[0600] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0601] perhaps 10 and 11 (corresponding to repeat 1 domain of dystrophin) appear to be highly immunogenic. These results suggest that patients with deletions in regions of the DMD gene overlapping those expressed in a given μDys may be at an increased risk of an immune-mediated myositis event following gene therapy, and are most likely be excluded from receiving the treatment.

[0602] As a novel strategy to circumvent immune response to dystrophin, we have replaced some of its highly immunogenic regions with those of utrophin, a dystrophin homolog (65% nucleic-acid identity) that is expressed in a wide range of tissues. We have developed 3 next-generation AAV vectors expressing a codon-optimized micro dystrophin (μDys) 5 (μDys5co) (SEQ ID NO: 36, encoded by SEQ ID NO: 37), or one of the two codon-optimized μDys-utrophin hybrid peptides, H1DIμDys5co (hinge 1 de-immunized) and NTDIμDys5co (Nter de-immunized). In H1DIμDys5co transgene, Hinge 1 (H1) region of Dyst is swapped with the corresponding H1 region of human utrophin, and in NTDIμDys5co transgene, both Hinge 1 and Repeat 1 domains (abbreviated N-terminal Domaine; NTD) are swapped with the corresponding Hl and R1 domains of utrophin. All three transgenes are expressed from a muscle-tropic synthetic promoter, which can be referred to as SPC5-12. In an in vitro study, we confirmed high level and comparable expression levels of the μDys5co and hybrid proteins from these vectors in transfected cell lines by Western blot analysis.

[0603] Subsequently, in an in vivo study we aimed to evaluate the expression and efficacy of these next-generation hybrid μDys-utrophin transgenes following IV administration of adult mdx (Dystrophin model) mice with AAV vectors. In a short-duration (4 week-long) study we evaluated expression levels and localization of the hybrid peptides in comparison to μDys5co. A total of 20 mice (n=5 / vector group and KO and WT control groups; dosed at 5-6 weeks of age via tail IV; 1e14 GC / kg; RGDYREV modified capsid) were enrolled in this study. Blood was collected prior to necropsy to measure serum Creatine Kinase (CK). At necropsy, liver, heart and muscle tissue including diaphragm, quadriceps, tibialis anterior, biceps and triceps were collected for histology, DNA and RNA biodistribution, and Western blot analysis.

[0604] While the in-life stage of this study is now completed, data analysis is still ongoing. Our findings from analysis of quadriceps muscle fibers and heart cardiomyocytes indicated that i) all three μDys5co, H1DIμDys5co and NTDIμDys5co transgenes are highly and similarly expressed in AAV-dosed mdx mice muscle fibers and heart cardiomyocytes, and at levels

[0605] 96

[0606] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0607] indistinguishable from that of dystrophin expression in WT mice; ii) expression of all three μDys5co, H1DIμDys5co and NTDIμDys5co transgenes led to a similar and near complete prevention of embryonic myosin heavy chain (eMHC)* expression in quadriceps muscle fibers of treated mdx mice; iii) all three μDys5co, H1DIμDys5co and NTDIμDys5co transgenes mediated high and similar levels of nNOS** translocation to quadriceps muscle fiber sarcolemma of treated mdx mice at levels indistinguishable from that achieved in dystrophin expressing WT mice; iv) expression of all three μDys5co, H1DIμDys5co and NTDIμDys5co transgenes led to reduction in central nuclei (centronucleation)*** of quadriceps muscle fiber in treated mdx mice at similar levels.

[0608] Overall, this data demonstrates that at the lel4 GC / kg (2el2 GC per mouse) dose, our engineered hybrid μDys5-utrophin transgenes performed similarly to that of unmodified μDys5co transgene without any loss of expression or activity, and were expressed at a level sufficient for suppression of the tested histological signs of muscular dystrophy, including myofiber centronucleation and embryonic myosin heavy chain (eMHC) expression, while mediating efficient sarcolemma localization of nNOS. The trend is quite convincing, which could not have been predicted and expected in such small groups (N=5 only) and for such a short-duration study (4 weeks).

[0609] *eMHCs serve as a useful regeneration markers in post-development skeletal muscle. These myosins are re-expressed during muscle regeneration and provide a specific marker of regenerating fibers in the pathologic skeletal muscle. [2]

[0610] ** nNOS is component of the dystrophin-associated glycoprotein complex (DGC). In the absence of dystrophin, delocalization of nNOS impairs compensatory vasodilation during muscle contraction and subsequently leads to focal ischemia and necrosis. [3] All three of our μDys5co vectors contain the nNOS binding domains (repeats 16 and 17) to allow nNOS binding and localization to sarcolemma where it releases nitrous oxide (NO) for mediation of vasodilation and proper oxygenation of muscle fibers during activity and preventing ischemia and cell death.

[0611] *** Centronucleation is quantitatively the most sensitive indicator of myoprotection in mdx mice because it reflects previous cycles of degeneration and regeneration. [4] Materials and Methods

[0612] Animal Experiments. Male wildtype (C57BL / 6) and dystrophic mdx mice were used in this study. For this pilot study, dystrophic mdx mice (5-6 weeks old) were administered

[0613] 97

[0614] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0615] 1E14 GC / kg recombinant AAV vector via tail iv injection (N = 5). Four weeks post-dosing, mice were humanely euthanized, and target tissue were collected.

[0616] Vector Cloning and Virus Production. The hybrid codon-optimized (co) pDys5co and the hybrid μDys5-utrophin (HIDIμDys5co and NTDIμDys5co) transgenes were constructed using standard DNA synthesis and cloning techniques. All three transgenes were expressed from the synthetic muscle-tropic SPC5-12 promoter and were followed by a short version (75 bp) poly A. The vectors were produced by triple transfection of adherent HEK293 cells and purified by iohexol gradient.

[0617] Table 1. Test Articles

[0618] Designation Lot ddPCR Number Titer AAV9-INS588(RGDYREV).SPC512.μDys5co.pA(p0540; WL4783S 1.52E+13p7515)

[0619] AAV9- WL4782S 1.52E+13 INS588(RGDYREV). SPC512. HlDIpDys5co.pA(p0540;

[0620] p7516)

[0621] AAV9-INS588(RGDYREV).SPC5-12.NTDIμDys5co.pA(p0540; p7517)

[0622]

[0623] Histological Processing and Evaluation. Muscle tissues were harvested and immediately frozen without prior fixation. The muscle tissues were placed on a small piece of cork, secured and oriented with tragacanth paste, and dropped into isopentane cooled with liquid nitrogen. Cryosections were prepared from the frozen tissues with a cryostat.

[0624] IF staining for transgene and other biomarkers. The following IF staining were performed:

[0625] Immunostaining for dystrophin, embryonic myosin heavy chain, and nNOS Briefly, cryosections were fixed with 4% paraformaldehyde, blocked, and incubated with primary antibodies against these proteins. After washing the corresponding secondary antibodies labeled with fluorochromes were applied. Sections were counterstained with wheat germ agglutinin (W GA) to outline muscle fibers and mounted with Fluoromount G containing DAPI to stain nuclei.

[0626] Central nuclei in muscle fibers

[0627] 98

[0628] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0629] The sections stained for dystrophin, WGA and DAPI were used to assess the number of muscle fibers containing central nuclei.

[0630] VisioPharm analysis

[0631] Transgene, eMHC, and nNOS expression levels were quantified as percentage positive area per field using VisioPharm analysis software.

[0632] Statistical Analysis

[0633] Analyses were performed using Prism (GraphPad Software). When applicable, statistical analyses were performed to analyze treatment effect using a 1-way ANOVA followed by post-hoc multiple comparison test using Prism.

[0634] Figure Legends

[0635] Figure 1. (A) Schematics of full-length dystrophin, dystrophin from Becker’s phenotype, utrophin, and the Sarepta Therapeutics’ Delandistrogene moxeparvovec micro dystrophin (pDys) construct. The immunogenic Hing 1 and spectrin-like Repeat 1 domains of Delandistrogene moxeparvovec pDys are shown in red box. (B) Schematics of the pDys5co (based on published pDys5 peptide sequence that were backtranslated and codon-optimized), and HlDIpDys5co and NTDIpDys5co (hybrid pDys5co-utrophin) constructs engineered by us and evaluated in this study. The Hinge 1 and Hing 1 -Repeat 1 domains of pDys were swapped with the corresponding regions from human utrophin in HlDIpDys5co (exons 8 and 9) and NTIDμμDys5co (exons 8-11), respectively. ACBD, Actin Binding Domain; H, hinge; R, spectrin-like repeat; nNOS, syntrophin-binding domain (R16 and R17) that enables localization of neuronal nitric oxide synthase; CR, cysteine-rich domain; CT, carboxyl terminal domain.

[0636] Figure 2. (A) Western blot analysis of protein lysates extracted from in vitro transfected HEK293T cells with GFP-, μDys5, μDys5co, H1DIμDys5co, and NTDIμDys5co-expressing AAV vectors. Endogenously expressed vinculin (124 kD) was used as internal loading control (B) Quantification of relative expression from μDys5-, μDys5co-, H1DIμDys5co-, and NTDIμDys5co-expressing AAV vectors in transfected 293T cells. μDys band intensities were measured and normalized to that of vinculin loading control band intensity.

[0637] Figure 3. (A) Expression levels of dystrophin in quadriceps muscle fibers of PBS-dosed WT and mdx mice, and in mdx mice dosed with μDys5co-, H1DIμDys5co-, and NTDIμDys5co-expressing AAV vectors. Six-week-old dystrophic mdx mice were injected by tail vein with PBS or the indicated μDys vectors. PBS-dosed WT C57Bl6 mice served

[0638] 99

[0639] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0640] as the positive control group. Skeletal muscles were harvested 4 weeks post-treatment and immunostained for dystrophin, for cell membrane by WGA, and for nuclei with DAPI. Shown are representative quadriceps cryosections. Top row, dystrophin (green), WGA (red), and DAPI (blue); Bottom row, dystrophin (green). Magnification, 200X. (B) Myofibers from quadriceps were quantified for dystrophin expression using the VisioPharm software at 4 weeks post-treatment (N = 5 per group; mean ± SEM).

[0641] Significance is compared between each group and that of PBS-dosed mdx mice. *p<0.05, **p<0.01.

[0642] Figure 4. (A) Expression levels of dystrophin in heart cardiomyocytes of PBS-dosed WT and mdx mice, and in mdx mice dosed with μDys5co-, H1DIμDys5co-, and NTDIμDys5co-expressing AAV vectors. Six-week-old dystrophic mdx mice were injected by tail vein with PBS or the indicated μDys vectors. PBS-dosed WT C57Bl6 mice served as the positive control group. Hearts were harvested 4 weeks post-treatment and immunostained for dystrophin, for cell membrane by WGA, and for nuclei with DAPI. Shown are representative heart cryosections. Top row, dystrophin (green), WGA (red), and DAPI (blue); Bottom row, dystrophin (green). Magnification, 200X. (B) Cardiomyocytes from hearts were quantified for dystrophin expression using the VisioPharm software at 4 weeks post-treatment (N = 5 per group; mean ± SEM). Significance is compared between each group and that of PBS-dosed mdx mice. *p<0.05, **p<0.01.

[0643] Figure 5. (A) Expression levels of mouse embryonic myocyte heavy chain (eMHC) in quadriceps muscle fibers of PBS-dosed WT and mdx mice, and in mdx mice dosed with μDys5co-, H1DIμDys5co-, and NTDIμDys5co-expressing AAV vectors. Six-week-old dystrophic mdx mice were injected by tail vein with PBS or the indicated μDys vectors. PBS-dosed WT C57Bl6 mice served as the negative control group. Skeletal muscles were harvested 4 weeks post-treatment and immunostained for eMHC, for cell membrane by WGA, and for nuclei with DAPI. Shown are representative quadriceps cryosections. Top row, eMHC (green), WGA (red), and DAPI (blue); Bottom row, eMHC (green).

[0644] Magnification, 200X. (B) Myofibers from quadriceps were quantified for eMHC expression using the VisioPharm software at 4 weeks post-treatment (N = 5 per group; mean ± SEM). Significance is compared between each group and that of PBS-dosed mdx mice. *p<0.05, **p<0.01.

[0645] Figure 6. (A) Expression levels of neuronal nitrous oxide synthetase (nNOS) in quadriceps muscle fibers of PBS-dosed WT and mdx mice, and in mdx mice dosed with μDys5co-,

[0646] 100

[0647] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0648] HlDIpDys5co-, and NTDIpDys5co-expressing AAV vectors. Six-week-old dystrophic mdx mice were injected by tail vein with PBS or the indicated pDys vectors. PBS-dosed WT C57B16 mice served as the positive control group. Skeletal muscles were harvested 4 weeks post-treatment and immunostained for nNOS, for cell membrane by WGA, and for nuclei with DAPI. Shown are representative quadriceps cryosections. Top row, nNOS (green), WGA (red), and DAPI (blue); Bottom row, nNOS (green). Magnification, 200X.

[0649] (B) Myofibers from quadriceps were quantified for dystrophin expression using the VisioPharm software at 4 weeks post-treatment (N = 5 per group; mean ± SEM).

[0650] Significance is compared between each group and that of PBS-dosed mdx mice. *p<0.05, **p<0.01.

[0651] Figure 7. (A) Quantification of central nuclei in quadriceps muscle fibers of PBS-dosed WT and mdx mice, and in mdx mice dosed with pDys5co-, HlDIpDys5co-, and NTDIpDys5co-expressing AAV vectors. Six-week-old dystrophic mdx mice were injected by tail vein with PBS or the indicated pDys vectors. PBS-dosed WT C57B16 and mdx mice served as the control groups. Skeletal muscles were harvested 4 weeks post-treatment and immunostained for cell membrane by WGA, and for nuclei with DAPI. Shown are representative quadriceps cryosections. WGA (red) and DAPI (blue). Magnification, 200X.

[0652] (B) Myofibers from quadriceps were quantified for central nuclei using the VisioPharm software at 4 weeks post-treatment (N = 5 per group; mean ± SEM). Significance is compared between each group and that of PBS-dosed mdx mice. *p<0.05, **p<0.01.

[0653] Sequences:

[0654] NtDIμdys5

[0655] Amino Acid Sequence of NtDIμdys5 (Nterminus from ABD1 from dystrophin-exon8-11 entirely from utrophin;and spectrin16-CT udys5):

[0656] MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTR VHALNNVNKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNI ILHWQVKNVMKNIMAGLQQTNSEKIL LSWVRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFDWNSWCQQSATQRLEHAFNIARYQLGIE KLLDPEDVAVQLPDKKSIIMYLTSLFEVLPQQVTIDAIREVETLPRKYKKECEEEAINIQSTAPEEEHES PRAETPSTVTEVDMDLDSYQIALEEVLTWLLSAEDTFQEQDDISDDVEEVKDQFATHEAFMMELTAHQSS VGSVLQAGNQLITQGTLSDEEEFEIQEQMTLLNARWEALRVESMDRQSRLHSYVPSTYLTEITHVSQALL EVEQLLNAPDLCAKDFEDLFKQEESLKNIKDSLQQSSGRIDIIHSKKTAALQSATPVERVKLQEALSQLD FQWEKVNKMYKDRQGRFDRSVEKWRRFHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGI GQRQTWRTLNATGEEIIQQSSKTDASILQEKLGSLNLRWQEVCKQLSDRKKRLEEQSDQWKRLHLSLQE LLVWLQLKDDELSRQAPIGGDFPAVQKQNDVHRAFKRELKTKEPVIMSTLETVRIFLTEQPLEGLEKLYQ EPRELPPEERAQNVTRLLRKQAEEVNTEWEKLNLHSADWQRKIDETLERLQELQEATDELDLKLRQAEVI KGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVSHVNDLARQLTTLGIQLSPYNLSTLEDLNTRWKL LQVAVEDRVRQLHEAHRDFGPASQHFLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLA DLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQEHN NLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGL

[0657] 101

[0658] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0659] LLHDS IQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEI EAALFLDWMRLEPQSMVWLPVLHRVAAAET AKHQAKCNICKECPI IGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVL KNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETDTM ( SEQ ID NO: 7 )

[0660] Nucleic Acid Sequence Encoding for NtDIμdys5

[0661] AT GC T T T GGT GGGAAGAAGT T GAGGAC T GC T AC GAGAGAGAGGAC GT GC AGAAGAAAAC C T T C AC C AAGT GGGTCAACGCCCAGTTCAGCAAGTTTGGCAAGCAGCACATTGAGAACCTGTTCAGTGACCTGCAGGACGG C AGAAGGC T GC T GGAT C T GC T GGAAGGC C T GAC AGGAC AGAAGC T GC C C AAAGAGAAGGGC AGC AC AAGA GTGCACGCCCTGAACAACGTGAACAAGGCCCTGAGAGTGCTGCAGAACAACAACGTGGACCTGGTCAACA TTGGCAGCACTGACATTGTGGACGGCAACCACAAACTGACCCTGGGCCTGATCTGGAACATCATCCTGCA C T GGC AAGT GAAGAAC GT GAT GAAGAAC AT C AT GGC T GGC C T GC AGC AGAC CAAC AGT GAGAAGAT T C T G CTGAGCTGGGTCAGGCAGAGCACCAGGAATTACCCTCAAGTGAACGTGATCAACTTCACCACCTCTTGGT CTGACGGACTGGCCCTGAATGCCCTGATCCACAGCCACAGACCTGACCTGTTTGACTGGAACAGCGTGGT GT GT C AGC AGAGT GC C AC AC AGAGGC T GGAAC AC GC C T T C AAT AT TGC C AGAT AC C AGC T GGGC AT T GAG AAACTGCTGGACCCTGAGGACGTGGCTGTGCAGCTGCCTGACAAGAAATCCATCATCATGTACCTGACCA GCCTGTTTGAGGTGCTGCCCCAGCAAGTGACCATTGACGCCATCAGAGAGGTGGAAACCCTGCCTAGGAA GT ACAAGAAAGAGT GT GAGGAAGAGGC CAT CAAC AT C C AGAGC AC AGC C C C T GAGGAAGAAC AT GAGAGC CCTAGAGCTGAGACACCTAGCACAGTGACAGAGGTGGACATGGACCTGGACAGCTATCAGATTGCCCTGG AAGAGGTGCTGACCTGGCTGCTGTCTGCTGAGGATACCTTCCAAGAGCAGGATGACATCTCTGATGATGT GGAAGAAGTGAAGGACCAGTTTGCCACACATGAGGCCTTCATGATGGAACTGACAGCCCACCAGAGCTCT GTGGGATCTGTTCTGCAAGCTGGCAACCAGCTGATCACCCAGGGCACCCTGTCTGATGAGGAAGAGTTTG AGATTCAAGAGCAGATGACCCTGCTGAACGCCAGGTGGGAAGCCCTGAGAGTGGAATCCATGGATAGGCA GAGCAGACTGCATAGCTACGTGCCCAGCACATACCTGACAGAGATCACCCACGTGTCTCAGGCCCTGCTG GAGGTGGAACAGCTGCTGAACGCTCCTGATCTGTGTGCCAAGGACTTTGAGGATCTGTTCAAGCAAGAGG AAAGC C T GAAGAAT AT C AAGGAC T C T C T GC AGC AGT CCAGTGGCC GGAT AGAT AT CAT C C AC AGC AAGAA AACTGCTGCACTGCAGTCTGCCACACCTGTGGAAAGAGTGAAGCTGCAAGAGGCCCTGTCTCAGCTGGAC T T C C AGT GGGAGAAAGT GAAC AAGAT GT ACAAGGAC AGGC AGGGC AGAT T T GAC C GC T C T GT T GAAAAGT GGAGAAGGTTCCACTACGACATCAAGATCTTCAACCAGTGGCTGACTGAGGCTGAGCAGTTCCTGAGAAA GACACAGATCCCTGAGAACTGGGAGCACGCCAAGTACAAGTGGTATCTGAAAGAGCTGCAGGACGGCATT GGC C AGAGGC AGAC AGT GGT C AGAAC AC T GAAC GC C AC AGGAGAGGAAAT C AT C C AGC AGAGC AGC AAGA C AGAC GC C AGC AT C C T GC AAGAGAAGC T GGGC AGC C T GAAC C T T AGGT GGC AAGAAGT GT GC AAGC AGC T GTCTGACAGGAAGAAGAGACTGGAAGAACAGAGTGACCAGTGGAAGAGACTGCACCTGTCTCTGCAAGAA CTGCTTGTGTGGCTGCAGCTGAAGGACGACGAGCTGAGTAGACAGGCCCCTATTGGAGGAGATTTTCCTG C T GT GCAGAAAC AGAAC GAC GT GC AC AGAGC C T T CAAGAGGGAGC T GAAAACAAAAGAAC C C GT GAT CAT GAGCACCCTGGAAACAGTGCGGATCTTTCTGACTGAGCAGCCCCTGGAAGGACTGGAAAAGCTGTACCAA GAGCCTAGAGAGCTGCCTCCTGAAGAAAGGGCCCAGAACGTGACCAGACTGCTGAGAAAGCAGGCTGAGG AAGTGAACACTGAGTGGGAGAAGCTGAATCTGCACTCTGCTGACTGGCAGAGGAAGATTGACGAGACACT GGAAAGACTCCAAGAGCTTCAAGAAGCCACAGACGAACTGGATCTGAAGCTGAGGCAGGCTGAAGTGATC AAAGGC AGC T GGC AGC C AGT T GGGGAC C T GC T GAT T GAT T C T C T GC AGGAC C AC C T GGAAAAAGT GAAAG CCCTGAGAGGAGAGATTGCCCCTCTGAAAGAAAACGTGTCCCACGTGAACGACCTGGCCAGACAGCTGAC AAC AC T GGGC AT C C AGC T GT C C C C AT AC AAC C T GT C C AC AC T GGAAGAT C T GAAC AC C AGGT GGAAGC T G CTCCAGGTGGCCGTGGAAGATAGAGTGAGGCAGCTGCACGAAGCCCACAGAGATTTTGGACCAGCCAGCC AGCACTTCCTGAGCACCTCAGTTCAAGGCCCCTGGGAGAGAGCTATCAGCCCTAACAAGGTGCCCTACTA CAT CAAC C AC GAGAC AC AGAC C AC C T GT T GGGAT C AC C C C AAGAT GAC T GAGC T GT AT C AGAGC C T GGC T GACCTGAACAACGTGAGGTTTAGTGCCTACAGGACAGCTATGAAGCTGCGGAGACTGCAGAAAGCTCTGT GCCTGGACCTGCTGTCTCTGAGTGCTGCTTGTGATGCCCTGGACCAGCACAATCTGAAGCAGAACGACCA GC C TAT GGAT AT C C T GC AGAT CAT CAAC T GC C T GAC C AC C AT C T AC GAC AGGC T GGAAC AAGAGCACAAC AACCTGGTGAACGTGCCCCTGTGTGTGGACATGTGCCTGAATTGGCTGCTGAACGTGTACGACACAGGCA GAAC AGGC AGGAT C AGAGT GC T GAGC T T T AAGAC T GGC AT CAT C T C C C T GT GC AAGGC C C AT C T GGAAGA TAAGTACCGCTACCTGTTCAAACAGGTGGCCAGCTCCACAGGCTTTTGTGACCAAAGAAGGCTGGGCCTG CTGCTGCACGACAGCATCCAGATTCCTAGACAGCTGGGAGAAGTGGCCTCTTTCGGAGGCTCTAATATTG AGCCTTCTGTGAGGAGCTGCTTCCAGTTTGCCAACAACAAGCCTGAGATTGAGGCTGCCCTGTTCCTGGA CTGGATGAGACTGGAACCCCAGAGCATGGTCTGGCTGCCTGTGCTGCATAGAGTTGCTGCTGCTGAAACA GCCAAGCACCAGGCCAAGTGCAACATCTGCAAAGAGTGCCCCATCATTGGCTTCCGGTACAGATCCCTGA AGCACTTCAACTACGATATCTGCCAGAGCTGTTTCTTCTCTGGCAGGGTGGCCAAGGGCCACAAAATGCA CTACCCCATGGTGGAATACTGCACCCCTACCACATCTGGAGAGGATGTGAGGGATTTTGCCAAGGTGCTG

[0662] 102

[0663] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0664] AAGAAC AAGT T C AGGAC C AAGAGGT AC T T T GC T AAGC AC C C C AGAAT GGGC T AC C T GC C T GT GC AGAC AG TGCTGGAGGGAGACAACATGGAAACTGATACCATGTGA ( SEQ ID NO: 11 )

[0665] H1DIμdys5 (ABD from dystrophin; hinge 1 from utrophin; spectrin 1 from dystrophin; and spectrin16-CT udys5):

[0666] MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTR VHALNNVNKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNI ILHWQVKNVMKNIMAGLQQTNSEKIL LSWVRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFDWNSWCQQSATQRLEHAFNIARYQLGIE KLLDPEDVDTTYPDKKS ILMYITSLFQVLPQQVTIDAIREVETLPRKYKKECEEEAINIQSTAPEEEHES PRAETPSTVTEVDVNLDRYQTALEEVLSWLLSAEDTLQAQGEI SNDVEWKDQFHTHEGYMMDLTAHQGR VGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHSYVPSTYLTEITHVSQALL EVEQLLNAPDLCAKDFEDLFKQEESLKNIKDSLQQSSGRI DI IHSKKTAALQSATPVERVKLQEALSQLD FQWEKVNKMYKDRQGRFDRSVEKWRRFHYDIKI FNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGI GQRQTWRTLNATGEEI IQQSSKTDAS ILQEKLGSLNLRWQEVCKQLSDRKKRLEEQSDQWKRLHLSLQE LLVWLQLKDDELSRQAPIGGDFPAVQKQNDVHRAFKRELKTKEPVIMSTLETVRI FLTEQPLEGLEKLYQ EPRELPPEERAQNVTRLLRKQAEEVNTEWEKLNLHSADWQRKIDETLERLQELQEATDELDLKLRQAEVI KGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVSHVNDLARQLTTLGI QLSPYNLSTLEDLNTRWKL LQVAVEDRVRQLHEAHRDFGPASQHFLSTSVQGPWERAIS PNKVPYYINHETQTTCWDHPKMTELYQSLA DLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDI LQI INCLTTIYDRLEQEHN NLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTGI I SLCKAHLEDKYRYLFKQVASSTGFCDQRRLGL LLHDS IQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEI EAALFLDWMRLEPQSMVWLPVLHRVAAAET AKHQAKCNICKECPI IGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVL KNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETDTM ( SEQ ID NO: 8 )

[0667] Nucleic Acid Sequence Encoding for H1DIμdys5

[0668] AT GC T T T GGT GGGAAGAAGT T GAGGAC T GC T AC GAGAGAGAGGAC GT GC AGAAGAAAAC C T T C AC C AAGT GGGTCAACGCCCAGTTCAGCAAGTTTGGCAAGCAGCACATTGAGAACCTGTTCAGTGACCTGCAGGACGG C AGAAGGC T GC T GGAT C T GC T GGAAGGC C T GAC AGGAC AGAAGC T GC C C AAAGAGAAGGGC AGC AC AAGA GTGCACGCCCTGAACAACGTGAACAAGGCCCTGAGAGTGCTGCAGAACAACAACGTGGACCTGGTCAACA TTGGCAGCACTGACATTGTGGACGGCAACCACAAACTGACCCTGGGCCTGATCTGGAACATCATCCTGCA C T GGC AAGT GAAGAAC GT GAT GAAGAAC AT C AT GGC T GGC C T GC AGC AGAC CAAC AGT GAGAAGAT T C T G CTGAGCTGGGTCAGGCAGAGCACCAGGAATTACCCTCAAGTGAACGTGATCAACTTCACCACCTCTTGGT CTGACGGACTGGCCCTGAATGCCCTGATCCACAGCCACAGACCTGACCTGTTTGACTGGAACAGCGTGGT GT GT C AGC AGAGT GC C AC AC AGAGGC T GGAAC AC GC C T T C AAT AT TGC C AGAT AC C AGC T GGGC AT T GAG AAACTGCTGGACCCTGAGGACGTGGACACCACCTATCCTGACAAGAAATCCATCCTCATGTACATCACCA GCCTGTTCCAGGTGCTGCCCCAGCAAGTGACCATTGACGCCATCAGAGAGGTGGAAACCCTGCCTAGGAA GT ACAAGAAAGAGT GT GAGGAAGAGGC CAT CAAC AT C C AGAGC AC AGC C C C T GAGGAAGAAC AT GAGAGC CCTAGAGCTGAGACACCTAGCACAGTGACAGAGGTGGACGTGAACCTGGACCGCTACCAGACAGCCCTGG AAGAGGTTCTGAGCTGGCTGCTGTCTGCAGAGGATACACTGCAGGCTCAGGGAGAGATCAGCAACGACGT GGAAGT GGT C AAGGAC C AGT T T C AC AC C C AC GAGGGC T AC AT GAT GGAC C T GAC AGC C C AC C AGGGC AGA GTGGGCAATATTCTGCAGCTGGGCTCCAAGCTGATTGGCACAGGCAAGCTGAGTGAGGACGAAGAGACAG AGGTGCAAGAGCAGATGAACCTGCTGAACAGCAGGTGGGAGTGTCTGAGAGTGGCCAGCATGGAAAAGCA GAGCAACCTGCACAGCTACGTGCCCAGCACATACCTGACAGAGATCACCCACGTGTCTCAGGCCCTGCTG GAGGTGGAACAGCTGCTGAACGCTCCTGATCTGTGTGCCAAGGACTTTGAGGATCTGTTCAAGCAAGAGG AAAGC C T GAAGAAT AT C AAGGAC T C T C T GC AGC AGT CCAGTGGCC GGAT AGAT AT CAT C C AC AGC AAGAA AACTGCTGCACTGCAGTCTGCCACACCTGTGGAAAGAGTGAAGCTGCAAGAGGCCCTGTCTCAGCTGGAC T T C C AGT GGGAGAAAGT GAAC AAGAT GT ACAAGGACAGGC AGGGC AGAT T T GAC C GC T C T GT T GAAAAGT GGAGAAGGTTCCACTACGACATCAAGATCTTCAACCAGTGGCTGACTGAGGCTGAGCAGTTCCTGAGAAA GACACAGATCCCTGAGAACTGGGAGCACGCCAAGTACAAGTGGTATCTGAAAGAGCTGCAGGACGGCATT GGC C AGAGGC AGAC AGT GGT C AGAAC AC T GAAC GC C AC AGGAGAGGAAAT C AT C C AGC AGAGC AGC AAGA C AGAC GC C AGC AT C C T GC AAGAGAAGC T GGGC AGC C T GAAC C T T AGGT GGC AAGAAGT GT GC AAGC AGC T GTCTGACAGGAAGAAGAGACTGGAAGAACAGAGTGACCAGTGGAAGAGACTGCACCTGTCTCTGCAAGAA CTGCTTGTGTGGCTGCAGCTGAAGGACGACGAGCTGAGTAGACAGGCCCCTATTGGAGGAGATTTTCCTG C T GT GCAGAAAC AGAAC GAC GT GC AC AGAGC C T T CAAGAGGGAGC T GAAAACAAAAGAAC C C GT GAT CAT GAGCACCCTGGAAACAGTGCGGATCTTTCTGACTGAGCAGCCCCTGGAAGGACTGGAAAAGCTGTACCAA GAGCCTAGAGAGCTGCCTCCTGAAGAAAGGGCCCAGAACGTGACCAGACTGCTGAGAAAGCAGGCTGAGG

[0669] 103

[0670] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0671] AAGTGAACACTGAGTGGGAGAAGCTGAATCTGCACTCTGCTGACTGGCAGAGGAAGATTGACGAGACACT GGAAAGACTCCAAGAGCTTCAAGAAGCCACAGACGAACTGGATCTGAAGCTGAGGCAGGCTGAAGTGATC AAAGGC AGC T GGC AGC C AGT T GGGGAC C T GC T GAT T GAT T C T C T GC AGGAC C AC C T GGAAAAAGT GAAAG CCCTGAGAGGAGAGATTGCCCCTCTGAAAGAAAACGTGTCCCACGTGAACGACCTGGCCAGACAGCTGAC AAC AC T GGGC AT C C AGC T GT C C C C AT AC AAC C T GT C C AC AC T GGAAGAT C T GAAC AC C AGGT GGAAGC T G CTCCAGGTGGCCGTGGAAGATAGAGTGAGGCAGCTGCACGAAGCCCACAGAGATTTTGGACCAGCCAGCC AGCACTTCCTGAGCACCTCAGTTCAAGGCCCCTGGGAGAGAGCTATCAGCCCTAACAAGGTGCCCTACTA CAT CAAC C AC GAGAC AC AGAC C AC C T GT T GGGAT C AC C C C AAGAT GAC T GAGC T GT AT C AGAGC C T GGC T GACCTGAACAACGTGAGGTTTAGTGCCTACAGGACAGCTATGAAGCTGCGGAGACTGCAGAAAGCTCTGT GCCTGGACCTGCTGTCTCTGAGTGCTGCTTGTGATGCCCTGGACCAGCACAATCTGAAGCAGAACGACCA GC C TAT GGAT AT C C T GC AGAT CAT CAAC T GC C T GAC C AC C AT C T AC GAC AGGC T GGAAC AAGAGC ACAAC AACCTGGTGAACGTGCCCCTGTGTGTGGACATGTGCCTGAATTGGCTGCTGAACGTGTACGACACAGGCA GAAC AGGC AGGAT C AGAGT GC T GAGC T T T AAGAC T GGC AT CAT C T C C C T GT GC AAGGC C C AT C T GGAAGA TAAGTACCGCTACCTGTTCAAACAGGTGGCCAGCTCCACAGGCTTTTGTGACCAAAGAAGGCTGGGCCTG CTGCTGCACGACAGCATCCAGATTCCTAGACAGCTGGGAGAAGTGGCCTCTTTCGGAGGCTCTAATATTG AGCCTTCTGTGAGGAGCTGCTTCCAGTTTGCCAACAACAAGCCTGAGATTGAGGCTGCCCTGTTCCTGGA CTGGATGAGACTGGAACCCCAGAGCATGGTCTGGCTGCCTGTGCTGCATAGAGTTGCTGCTGCTGAAACA GCCAAGCACCAGGCCAAGTGCAACATCTGCAAAGAGTGCCCCATCATTGGCTTCCGGTACAGATCCCTGA AGCACTTCAACTACGATATCTGCCAGAGCTGTTTCTTCTCTGGCAGGGTGGCCAAGGGCCACAAAATGCA CTACCCCATGGTGGAATACTGCACCCCTACCACATCTGGAGAGGATGTGAGGGATTTTGCCAAGGTGCTG AAGAAC AAGT T C AGGAC C AAGAGGT AC T T T GC T AAGC AC C C C AGAAT GGGC T AC C T GC C T GT GC AGAC AG TGCTGGAGGGAGACAACATGGAAACTGATACCATGTGA ( SEQ ID NO: 12 )

[0672] Spc512 promoter used in the present Examples.

[0673] CGGCCGTCCGCCTTCGGCACCATCCTCACGACACCCAAATATGGCGACGGGTGAGGAATGGTGGGGAGTT ATTTTTAGAGCGGTGAGGAAGGTGGGCAGGCAGCAGGTGTTGGCGCTCTAAAAATAACTCCCGGGAGTTA TTTTTAGAGCGGAGGAATGGTGGACACCCAAATATGGCGACGGTTCCTCACCCGTCGCCATATTTGGGTG TCCGCCCTCGGCCGGGGCCGCATTCCTGGGGGCCGGGCGGTGCTCCCGCCCGCCTCGATAAAAGGCTCCG GGGCCGGCGGCGGCCCACGAGCTACCCGGAGGAGCGGGAGGCGCCAAGCTCTAGA ( SEQ ID NO: 14 )

[0674] Bibliography

[0675] [1] Kan, S. et al. Poster presented at the 28th International Annual Congress of the World Muscle Society (WMS), Charleston, USA; 3-7, 2023, which is incorporated by reference in its entirety.

[0676] [2] Schiaffino S. et al. Developmental myosins: expression patterns and functional significance. Skeletal Muscle. 5:22, 2015, which is incorporated by reference in its entirety.

[0677] [3] Yi, Lai. Et al. Dystrophins carrying spectrin-like repeats 16 and 17 anchor nNOS to the sarcolemma and enhance exercise performance in a mouse model of muscular dystrophy. J Clin Invest. 119(3):624-35, 2009, which is incorporated by reference in its entirety.

[0678] [4] Massopust, R. T. et al. Lifetime analysis of mdx skeletal muscle reveals a progressive pathology that leads to myofiber loss. Sci Rep 14; 10(1 ): 17248, 2020, which is incorporated by reference in its entirety.

[0679] Other Sequences Referenced herein:

[0680] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNEADA AALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAP GKKRPVEQSPQEPDSSVGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAP

[0681] 104

[0682] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0683] VADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDY QLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENV PFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVS TTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFH PSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQ YTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 20 ) MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADA AALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAP GKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAP VADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDY QLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENV PFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVS TTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFH PSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQ YTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 18 )

[0684] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADA AALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAP GKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAP VADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDY QLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENV PFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVS TTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAQRGDYREVAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIP HTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSK RWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 28 ) MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADA AALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAP GKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAP VADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDY QLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENV PFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVS TTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAQRGDYHQVAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIP HTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSK RWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 21 ) MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADA AALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAP GKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAP VADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDY QLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENV PFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVS TTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAQVYTRGDVAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIP

[0685] 105

[0686] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0687] HTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSK RWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 32 ) MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADA AALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAP GKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAP VADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDY QLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENV PFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVS TTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAQQNRGDPHAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIP HTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSK RWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 22 ) MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADA AALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAP GKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAP VADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDY QLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENV PFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVS TTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAQRGDYASVAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIP HTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSK RWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 24 ) MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADA AALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAP GKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAP VADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDY QLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENV PFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVS TTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAQRGDYSQIAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIP HTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSK RWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 35 ) uDys5co MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTR VHALNNVNKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNI ILHWQVKNVMKNIMAGLQQTNSEKIL LSWVRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFDWNSWCQQSATQRLEHAFNIARYQLGIE KLLDPEDVDTTYPDKKSILMYITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITV SLAQGYERTSSPKPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEE VLSWLLSAEDTLQAQGEISNDVEWKDQFHTHEGYMMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEV QEQMNLLNSRWECLRVASMEKQSNLHSYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQEES LKNIKDSLQQSSGRIDI IHSKKTAALQSATPVERVKLQEALSQLDFQWEKVNKMYKDRQGRFDRSVEKWR RFHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTWRTLNATGEEIIQQSSKTD ASILQEKLGSLNLRWQEVCKQLSDRKKRLEEQSDQWKRLHLSLQELLVWLQLKDDELSRQAPIGGDFPAV QKQNDVHRAFKRELKTKEPVIMSTLETVRIFLTEQPLEGLEKLYQEPRELPPEERAQNVTRLLRKQAEEV NTEWEKLNLHSADWQRKIDETLERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKAL RGEIAPLKENVSHVNDLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQH FLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCL DLLSLSAACDALDQHNLKQNDQPMDILQI INCLTTI YDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRT

[0688] 106

[0689] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0690] GRIRVLSFKTGI I SLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSI QI PRQLGEVASFGGSNIEP SVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPI IGFRYRSLKH FNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVL EGDNMETDTM ( SEQ ID NO: 36 )

[0691] Vector Genome for uDys5co (5' ITR to 3' ITR) CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGG CCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAAT GATTAACCCGCCATGCTACTTATCTACCAGGGTAATGGCGGCCGTCCGCCTTCGGCACCATCCTCACGAC AC C CAAAT AT GGC GAC GGGT GAGGAAT GGT GGGGAGT T AT T T T T AGAGC GGT GAGGAAGGT GGGC AGGC A GCAGGTGTTGGCGCTCTAAAAATAACTCCCGGGAGTTATTTTTAGAGCGGAGGAATGGTGGACACCCAAA TATGGCGACGGTTCCTCACCCGTCGCCATATTTGGGTGTCCGCCCTCGGCCGGGGCCGCATTCCTGGGGG CCGGGCGGTGCTCCCGCCCGCCTCGATAAAAGGCTCCGGGGCCGGCGGCGGCCCACGAGCTACCCGGAGG AGCGGGAGGCGCCAAGCTCTAGACTTTAAGCTGCAGTAACCGGAATTCGCCACCATGCTTTGGTGGGAAG AAGT T GAGGAC T GC T AC GAGAGAGAGGAC GT GC AGAAGAAAAC C T T C AC CAAGT GGGT C AAC GC C C AGT T C AGCAAGT T T GGC AAGC AGC AC AT T GAGAAC C T GT T C AGT GAC C T GC AGGAC GGC AGAAGGC T GC T GGAT C T GC T GGAAGGC C T GAC AGGAC AGAAGC T GC C C AAAGAGAAGGGC AGC ACAAGAGT GC AC GC C C T GAAC A ACGTGAACAAGGCCCTGAGAGTGCTGCAGAACAACAACGTGGACCTGGTCAACATTGGCAGCACTGACAT T GT GGAC GGC AAC C AC AAAC T GAC CCTGGGCCT GAT C T GGAAC AT CAT C C T GC AC T GGC AAGT GAAGAAC GTGATGAAGAACATCATGGCTGGCCTGCAGCAGACCAACAGTGAGAAGATTCTGCTGAGCTGGGTCAGGC AGAGCACCAGGAATTACCCTCAAGTGAACGTGATCAACTTCACCACCTCTTGGTCTGACGGACTGGCCCT GAATGCCCTGATCCACAGCCACAGACCTGACCTGTTTGACTGGAACAGCGTGGTGTGTCAGCAGAGTGCC ACACAGAGGCTGGAACACGCCTTCAATATTGCCAGATACCAGCTGGGCATTGAGAAACTGCTGGACCCTG AGGACGTGGACACCACCTATCCTGACAAGAAATCCATCCTCATGTACATCACCAGCCTGTTCCAGGTGCT GCCCCAGCAGGTTTCCATTGAGGCCATTCAAGAGGTGGAGATGCTGCCCAGACCTCCTAAAGTGACCAAA GAGGAAC AC T T C C AGC T GC AC C AC C AGAT GC AC T AC T C T C AGC AGAT C AC AGT GTCTCTGGCCCAGGGCT ACGAGAGAACAAGCAGCCCCAAGCCTAGATTCAAGAGCTACGCCTATACACAGGCTGCCTACGTGACCAC CAGTGATCCTACAAGAAGCCCATTTCCTAGCCAGCACCTGGAGGCCCCTGAGGATAAGAGCTTTGGCAGC AGCCTGATGGAAAGTGAAGTGAACCTGGACCGCTACCAGACAGCCCTGGAAGAGGTTCTGAGCTGGCTGC T GT C T GC AGAGGAT AC AC T GC AGGC T C AGGGAGAGAT C AGCAAC GAC GT GGAAGT GGT C AAGGAC C AGT T T C AC AC C C AC GAGGGC T AC AT GAT GGAC C T GAC AGC C C AC C AGGGC AGAGT GGGC AAT AT T C T GC AGC T G GGC T C C AAGC T GAT T GGC AC AGGCAAGC T GAGT GAGGAC GAAGAGAC AGAGGT GC AAGAGC AGAT GAAC C T GC T GAAC AGC AGGT GGGAGT GT C T GAGAGT GGC C AGC AT GGAAAAGC AGAGC AAC C T GC AC AGC T AC GT GC C C AGC AC AT AC C T GAC AGAGAT C AC C C AC GT GT C T C AGGC C C T GC T GGAGGT GGAAC AGC T GC T GAAC GC T C C T GAT C T GT GT GC C AAGGAC T T T GAGGAT C T GT T CAAGC AAGAGGAAAGC C T GAAGAAT AT C AAGG ACTCTCTGCAGCAGTCCAGTGGCCGGATAGATATCATCCACAGCAAGAAAACTGCTGCACTGCAGTCTGC CACACCTGTGGAAAGAGTGAAGCTGCAAGAGGCCCTGTCTCAGCTGGACTTCCAGTGGGAGAAAGTGAAC AAGAT GT AC AAGGAC AGGC AGGGC AGAT T T GAC CGCTCTGTT GAAAAGT GGAGAAGGT T C C AC T AC GAC A TCAAGATCTTCAACCAGTGGCTGACTGAGGCTGAGCAGTTCCTGAGAAAGACACAGATCCCTGAGAACTG GGAGC AC GC CAAGT AC AAGT GGT AT C T GAAAGAGC T GC AGGAC GGC AT T GGC C AGAGGC AGAC AGT GGT C AGAAC AC T GAAC GC C AC AGGAGAGGAAAT CAT C C AGC AGAGC AGC AAGAC AGAC GCCAGCATCCT GCAAG AGAAGC T GGGC AGC C T GAAC C T T AGGT GGCAAGAAGT GT GCAAGC AGC T GT C T GAC AGGAAGAAGAGAC T GGAAGAACAGAGTGACCAGTGGAAGAGACTGCACCTGTCTCTGCAAGAACTGCTTGTGTGGCTGCAGCTG AAGGAC GAC GAGC T GAGT AGAC AGGC C C C TAT T GGAGGAGAT TTTCCTGCTGT GCAGAAAC AGAAC GAC G TGCACAGAGCCTTCAAGAGGGAGCTGAAAACAAAAGAACCCGTGATCATGAGCACCCTGGAAACAGTGCG GATCTTTCTGACTGAGCAGCCCCTGGAAGGACTGGAAAAGCTGTACCAAGAGCCTAGAGAGCTGCCTCCT GAAGAAAGGGC C C AGAAC GT GAC CAGAC T GC T GAGAAAGC AGGC T GAGGAAGT GAAC AC T GAGT GGGAGA AGCTGAATCTGCACTCTGCTGACTGGCAGAGGAAGATTGACGAGACACTGGAAAGACTCCAAGAGCTTCA AGAAGC C AC AGAC GAAC T GGAT C T GAAGC T GAGGC AGGC T GAAGT GAT C AAAGGC AGC T GGC AGC C AGT T GGGGACCTGCTGATTGATTCTCTGCAGGACCACCTGGAAAAAGTGAAAGCCCTGAGAGGAGAGATTGCCC CTCTGAAAGAAAACGTGTCCCACGTGAACGACCTGGCCAGACAGCTGACAACACTGGGCATCCAGCTGTC CCCATACAACCTGTCCACACTGGAAGATCTGAACACCAGGTGGAAGCTGCTCCAGGTGGCCGTGGAAGAT AGAGTGAGGCAGCTGCACGAAGCCCACAGAGATTTTGGACCAGCCAGCCAGCACTTCCTGAGCACCTCAG T T C AAGGC C C C T GGGAGAGAGC T AT C AGC C C T AACAAGGT GC C C T AC T AC AT C AAC C AC GAGAC AC AGAC

[0692] 107

[0693] IPTS / 200297809.2Attorney Docket No. GMA-068WO

[0694] CACCTGTTGGGATCACCCCAAGATGACTGAGCTGTATCAGAGCCTGGCTGACCTGAACAACGTGAGGTTT AGTGCCTACAGGACAGCTATGAAGCTGCGGAGACTGCAGAAAGCTCTGTGCCTGGACCTGCTGTCTCTGA GTGCTGCTTGTGATGCCCTGGACCAGCACAATCTGAAGCAGAACGACCAGCCTATGGATATCCTGCAGAT CAT CAAC T GC C T GAC C AC CAT C T AC GAC AGGC T GGAAC AA...

Claims

Attorney Docket No. GMA-068WOWHAT IS CLAIMED IS:

1. A polypeptide comprising an amino acid sequence comprising:i) an N-terminal portion of the ABDI domain from the dystrophin gene,ii) a hinge region of exon 8-11 from the utrophin gene, or a fragment thereof;iii) a Spectrin 1 domain of the dystrophin or utrophin protein; and iv) a spectrinl6-CT udys5 domain.

2. The polypeptide of claim 1, wherein the polypeptide comprisesi) an N-terminal portion of the ABDI domain from the dystrophin gene,ii) a hinge region of exon 8-11 from the utrophin gene, or a fragment thereof;iii) a Spectrin 1 domain of the dystrophin protein; and iv) a spectrinl6-CT udys5 domain.

3. The polypeptide of claim 1, wherein the fragment of the exon 8-11 from the utrophin gene comprises the hinge 1 region of the utrophin gene.

4. The polypeptide of claim 1, wherein the N-terminal portion of the ABDI domain from the dystrophin gene comprises the amino acid sequence of:MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLT GQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNI IL HWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQVNVINFTTSWSDGLALNALIH SHRPDLFDWNSWCQQSATQRLEHAFNIARYQLGIEKLLDPE (SEQ ID NO: 1 ).

5. The polypeptide of any one of claims 1-4, wherein the N-terminal portion of the ABDI domain from the dystrophin gene comprises the amino acid sequence of:MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLT GQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNI IL HWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQVNVINFTTSWSDGLALNALIH SHRPDLFDWNSWCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYI TSLFQVLP (SEQ ID NO: 2 ).

6. The polypeptide of any one of claims 1-5, wherein the Spectrin 1 domain from the dystrophin gene comprises the amino acid sequence of:112IPTS / 200297809.2Attorney Docket No. GMA-068WOVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEWKDQFHTHEGYMMDLTAHQGRV GNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLH (SEQ ID NO: 3 ).

7. The polypeptide of any one of claims 1-6, wherein the hinge region of exon 8-11 from the utrophin gene, or a fragment thereof, comprises the amino acid sequence of, or a fragment thereof of:DVAVQLPDKKSIIMYLTSLFEVLPQQVTIDAIREVETLPRKYKKECEEEAINIQSTAP EEEHESPRAETPSTVTEVDMDLDSYQIALEEVLTWLLSAEDTFQEQDDISDDVEEVKD QFATHEAFMMELTAHQSSVGSVLQAGNQLITQGTLSDEEEFEIQEQMTLLNARWEALR VESMDRQSRLH (SEQ ID NO: 4 ),or a fragment thereof; or QQVTIDAIREVETLPRKYKKECEEEAINIQSTAPEEEHESPRAETPSTVTEVD (SEQ ID NO: 5 ),or a fragment thereof.

8. The polypeptide of any one of claims 1-7, wherein the spectrinl6-CT udys5 domain comprises the amino acid sequence of SYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQEESLKNIKDSLQQSSGR IDI IHSKKTAALQSATPVERVKLQEALSQLDFQWEKVNKMYKDRQGRFDRSVEKWRRF HYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTWRTLNATGE EIIQQSSKTDASILQEKLGSLNLRWQEVCKQLSDRKKRLEEQSDQWKRLHLSLQELLV WLQLKDDELSRQAPIGGDFPAVQKQNDVHRAFKRELKTKEPVIMSTLETVRIFLTEQP LEGLEKLYQEPRELPPEERAQNVTRLLRKQAEEVNTEWEKLNLHSADWQRKIDETLER LQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVS HVNDLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQH FLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTA MKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNN LVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASS TGFCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLD WMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPI IGFRYRSLKHFNYDICQSCF FSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQT VLEGDNMETDTM (SEQ ID NO: 6),or a fragment thereof9. The polypeptide of any one of claims 1-8, wherein the polypeptide comprises the amino acid sequence of:MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLT GQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNI IL HWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQVNVINFTTSWSDGLALNALIH SHRPDLFDWNSWCQQSATQRLEHAFNIARYQLGIEKLLDPEDVAVQLPDKKSIIMYL TSLFEVLPQQVTIDAIREVETLPRKYKKECEEEAINIQSTAPEEEHESPRAETPSTVT EVDMDLDSYQIALEEVLTWLLSAEDTFQEQDDISDDVEEVKDQFATHEAFMMELTAHQ SSVGSVLQAGNQLITQGTLSDEEEFEIQEQMTLLNARWEALRVESMDRQSRLHSYVPS TYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQEESLKNIKDSLQQSSGRIDIIH SKKTAALQSATPVERVKLQEALSQLDFQWEKVNKMYKDRQGRFDRSVEKWRRFHYDIK IFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTWRTLNATGEEIIQQ SSKTDASILQEKLGSLNLRWQEVCKQLSDRKKRLEEQSDQWKRLHLSLQELLVWLQLK DDELSRQAPIGGDFPAVQKQNDVHRAFKRELKTKEPVIMSTLETVRIFLTEQPLEGLE113IPTS / 200297809.2Attorney Docket No. GMA-068WOKLYQEPRELPPEERAQNVTRLLRKQAEEVNTEWEKLNLHSADWQRKIDETLERLQELQ EATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVSHVNDL ARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHFLSTS VQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRR LQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQI INCLTTI YDRLEQEHNNLVNVP LCVDMCLNWLLNVYDTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCD QRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLE PQSMVWLPVLHRVAAAETAKHQAKCNICKECPI IGFRYRSLKHFNYDICQSCFFSGRV AKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGD NMETDTM (SEQ ID NO: 7).

10. The polypeptide of any one of claims 1-8, wherein the polypeptide comprises the amino acid sequence of:MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLT GQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNI IL HWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQVNVINFTTSWSDGLALNALIH SHRPDLFDWNSWCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYI TSLFQVLPQQVTIDAIREVETLPRKYKKECEEEAINIQSTAPEEEHESPRAETPSTVT EVDVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEWKDQFHTHEGYMMDLTAHQ GRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHSYVPS TYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQEESLKNIKDSLQQSSGRIDIIH SKKTAALQSATPVERVKLQEALSQLDFQWEKVNKMYKDRQGRFDRSVEKWRRFHYDIK IFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTWRTLNATGEEIIQQ SSKTDASILQEKLGSLNLRWQEVCKQLSDRKKRLEEQSDQWKRLHLSLQELLVWLQLK DDELSRQAPIGGDFPAVQKQNDVHRAFKRELKTKEPVIMSTLETVRIFLTEQPLEGLE KLYQEPRELPPEERAQNVTRLLRKQAEEVNTEWEKLNLHSADWQRKIDETLERLQELQ EATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVSHVNDL ARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHFLSTS VQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRR LQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQI INCLTTI YDRLEQEHNNLVNVP LCVDMCLNWLLNVYDTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCD QRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLE PQSMVWLPVLHRVAAAETAKHQAKCNICKECPI IGFRYRSLKHFNYDICQSCFFSGRV AKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGD NMETDTM (SEQ ID NO: 8 ).

11. A nucleic acid molecule encoding the polypeptide of any one of claims 1-10, wherein the nucleic acid molecule is DNA or mRNA.

12. The nucleic acid molecule of claim 11, wherein the nucleic acid molecule comprises a codon optimized nucleotide sequence.

13. The nucleic acid molecule of claim 12, wherein the codon optimized nucleotide sequence is optimized for expression in human cells, such as muscle cells, including but not limited to skeletal muscle cells.

14. The nucleic acid molecule of any one of claims 1-11, wherein the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12.114IPTS / 200297809.2Attorney Docket No. GMA-068WO15. A vector comprising an expression cassette comprising a nucleic acid molecule encoding the polypeptide of any one of claims 1-10.

16. The vector of claim 15, wherein the nucleic acid molecule encoding the polypeptide of any one of claims 1-10 is operatively connected to a promoter.

17. The vector of claim 15, wherein the promoter is a muscle specific promoter, such as a skeletal muscle specific promoter.

18. The vector of claim 17, wherein the muscle specific promoter is Spc512, or a variant thereof.

19. The vector of any one of claims 16-18, wherein the promoter comprises a nucleotide sequence of SEQ ID NO: 14, SEQ ID NO: 13, or SEQ ID NO: 15.

20. The vector of any one of claims 16-18, wherein the promoter comprises a nucleotide sequence of SEQ ID NO: 14.

21. A lipid nanoparticle comprising the nucleic acid molecule or vector of any one of claims 11-20.

22. A recombinant adeno-associated virus (rAAV) comprising an adeno-associated virus (AAV) capsid and packaged therein a vector genome, wherein the vector genome comprises:(a) an AAV 5' inverted terminal repeat (ITR),(b) an expression cassette comprising a coding sequence for polypeptide of any one of claims 1-11, or a sequence that is at least 80% identical to the polypeptide of any one of claims 1-11, wherein the coding sequence is operably linked to expression control sequences which direct expression of the polypeptide, and(c) an AAV 3' ITR.115IPTS / 200297809.2Attorney Docket No. GMA-068WO23. The rAAV of claim 22, wherein the AAV capsid is suitable for targeting to muscles, such as skeletal muscles.

24. The rAAV of claims 22 or 23, wherein the AAV capsid is a Clade F AAV.

25. The rAAV of any one of claims 22-24, wherein the AAV capsid is an AAVhu68 capsid or AAV9 capsid, or a variant thereof.

26. The rAAV of claim 25, the capsid proteins have an amino acid sequence comprising a hypervariable region comprising an exogenous targeting peptide.

27. The rAAV of claim 26, wherein the exogenous targeting peptide comprises a targeting peptide that targets the AAV to muscle, such as skeletal muscle.

28. The rAAV of claim 27, wherein the targeting peptide has a formula of “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 is RGDYREV, RGDYREI, RGDYREL, RGDYHQV, VYTRGDV, RGDYSQI, RGDYASV, QNRGDPH, RGDYHYQ, VHRGDLN, RGDFSGY, RGDYVYQ, RGDYSYT, QVRGDIK, PQYTRGD, VRGDIRL, AVIRGDV, IIRGDPA, RGDLHGY, PYQRGDH, RGDYAQV, or RGDYSTM, or a targeting peptide as provided for in targeting peptide comprising an amino acid sequence as provided for in PCT Publication No. WO2024 / 130067, PCT Publication No. WO2024 / 130070, PCT Application No. PCT / US2024 / 55910, filed November 14, 2024, U. S. Provisional Application No. 63 / 598,718, filed November 14, 2023, U. S. Provisional Application No.63 / 612,676, filed December 20, 2023, U. S. Provisional Application No. 63 / 661,133, filed June 18, 2024, PCT Publication No. WO 2024 / 173802, and U. S. Patent No. 11,920,150, or a sequence comprising 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.116IPTS / 200297809.2Attorney Docket No. GMA-068WO29. The rAAV of any one of claims 26-28, 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.

30. The rAAV of claim 29, wherein the parental AAV capsid is an AAV9, AAVhu68, AAVhu95, AAVhu96, or AAVrh91 capsid.

31. The rAAV of any one of claims 26-30, wherein the exogenous targeting peptide is inserted in the hypervariable region between amino acids 588 and 589 in an AAV9 or an AAVhu68 parental capsid as determined based on the numbering of VP1 amino acids in the sequence of SEQ ID NO: 18, or an analogous position in an AAV8, AAV7, AAV6, AAV5, AAV4, AAV3, AAV1, AAVhu95, AAVhu96, or AAVrh91 parental AAV capsid.

32. The rAAV of any one of claims 26-31, wherein the exogenous targeting peptide is immediately preceded by the dipeptide of AQ.

33. The rAAV of any one of claims 22 to 32, wherein the rAAV capsid proteins comprise VP1, VP2 and VP3 proteins having a mutant VP3 region of: amino acids 204 to 743 of SEQ ID NO: 28, SEQ ID NO: 21, SEQ ID NO: 32, SEQ ID NO: 22, SEQ ID NO: 24, or SEQ ID NO: 35, further comprising highly deamidated residues at positions N57, N329, N452 and N512, wherein the deamidated position numbers are based on the residue positions of SEQ ID NO: 20 or 26.

34. The rAAV of any one of claims 22 to 33, wherein the mutant rAAV capsid VP1 proteins comprising a mutant VP1 protein having amino acids 1 to 743 of SEQ ID NO: 28, SEQ ID NO: 21, SEQ ID NO: 32, SEQ ID NO: 22, SEQ ID NO: 24, or SEQ ID NO: 35, further comprising highly deamidated residues at positions N57, N329, N452, and N512, wherein the deamidated position numbers are based on the residue positions of SEQ ID NO: 20 or 26.

35. A composition comprising a stock of the rAAV according to any one of claims 22 to 34, and one or more of a physiologically compatible carrier, excipient, and / or aqueous suspension base.117IPTS / 200297809.2Attorney Docket No. GMA-068WO36. A method for treating one or more of muscle cell disorders, and / or a disease in a patient in need thereof, the method comprising delivering a peptide of any one of claims 1-10, a nucleic acid molecule of any one of claims 11-14, a vector of any one of claims 15-20, a lipid nanoparticle of claim 21, a stock of rAAV according to any one of claims 22 to 34, or a combination of any of the foregoing, or a pharmaceutical composition of any of the foregoing, to the patient.

37. A method of treating Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy in a patient in need thereof, the method comprising delivering a peptide of any one of claims 1-10, a nucleic acid molecule of any one of claims 11-14, a vector of any one of claims 15-20, a lipid nanoparticle of claim 21, a stock of rAAV according to any one of claims 22 to 34, or a combination of any of the foregoing, or a pharmaceutical composition of any of the foregoing, to the patient.

38. A method for inhibiting the progression of Duchenne muscular dystrophy or Becker muscular dystrophy in a patient in need thereof, the method comprising delivering a peptide of any one of claims 1-10, a nucleic acid molecule of any one of claims 11-14, a vector of any one of claims 15-20, a lipid nanoparticle of claim 21, a stock of rAAV according to any one of claims 22 to 34, or a combination of any of the foregoing, or a pharmaceutical composition of any of the foregoing, to the patient.

39. A method of treating skeletal muscle mass deficiency in a subject having a mutation in the dystrophin gene, the method comprising delivering a peptide of any one of claims 1-10, a nucleic acid molecule of any one of claims 11-14, a vector of any one of claims 15-20, a lipid nanoparticle of claim 21, a stock of rAAV according to any one of claims 22 to 34, or a combination of any of the foregoing, or a pharmaceutical composition of any of the foregoing, to the patient.

40. A method of increasing skeletal muscle mass in a subject with DMD or Becker muscular dystrophy, the method comprising delivering a peptide of any one of claims 1-10, a nucleic acid molecule of any one of claims 11-14, a vector of any one of claims 15-20, a lipid nanoparticle of claim 21, a stock of rAAV according to any one of claims 22 to 34, orAttorney Docket No. GMA-068WOa combination of any of the foregoing, or a pharmaceutical composition of any of the foregoing, to the patient.

41. A method of preventing or inhibiting the decrease of muscle mass in a subject with DMD or Becker muscular dystrophy, the method comprising delivering a peptide of any one of claims 1-10, a nucleic acid molecule of any one of claims 11-14, a vector of any one of claims 15-20, a lipid nanoparticle of claim 21, a stock of rAAV according to any one of claims 22 to 34, or a combination of any of the foregoing, or a pharmaceutical composition of any of the foregoing, to the patient.

42. A medicament for treating Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy, the medicament comprising a peptide of any one of claims 1-10, a nucleic acid molecule of any one of claims 11-14, a vector of any one of claims 15-20, a lipid nanoparticle of claim 21, a stock of rAAV according to any one of claims 22 to 34, or a combination of any of the foregoing.

43. A medicament for treating skeletal muscle mass deficiency in a subject having a mutation in the dystrophin gene, the medicament comprising a peptide of any one of claims 1-10, a nucleic acid molecule of any one of claims 11-14, a vector of any one of claims 15-20, a lipid nanoparticle of claim 21, a stock of rAAV according to any one of claims 22 to 34, or a combination of any of the foregoing.

44. A medicament for preventing or inhibiting the decrease of muscle mass in a subject with DMD or Becker muscular dystrophy, the medicament comprising a peptide of any one of claims 1-10, a nucleic acid molecule of any one of claims 11-14, a vector of any one of claims 15-20, a lipid nanoparticle of claim 21, a stock of rAAV according to any one of claims 22 to 34, or a combination of any of the foregoing.

Images