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WO2024036250A2 - Compositions de virus adéno-associé pour le transfert de gène arsa et procédés pour les utiliser - Google Patents

Compositions de virus adéno-associé pour le transfert de gène arsa et procédés pour les utiliser Download PDF

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WO2024036250A2
WO2024036250A2 PCT/US2023/071985 US2023071985W WO2024036250A2 WO 2024036250 A2 WO2024036250 A2 WO 2024036250A2 US 2023071985 W US2023071985 W US 2023071985W WO 2024036250 A2 WO2024036250 A2 WO 2024036250A2
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amino acid
seq
capsid protein
protein corresponding
nucleotide sequence
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WO2024036250A3 (fr
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Thia Baboval ST. MARTIN
Katherine GALL
Jennifer Newman
Jaime Michelle Prout
Jacinthe GINGRAS
Donald SELBY
Luis Miguel Mendes Soares
Jason Boke WRIGHT
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Homology Medicines Inc
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Homology Medicines Inc
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    • A—HUMAN NECESSITIES
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    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
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    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA

Definitions

  • Metachromatic leukodystrophy is a fatal lysosomal storage disorder with a high unmet medical need.
  • This neurodegenerative disease occurs in three forms (late infantile, juvenile, and adult) and is due to a deficiency in the lysosomal enzyme arylsulfatase- A (ARSA).
  • ARSA is located in cellular structures called lysosomes, where it helps to break down sulfatides. The lack of this enzyme leads to a large accumulation of sulfatides in the brain, spinal cord, and peripheral organs, which results in severe damage of myelin, the main protective layer of the nerve fibers.
  • MLD myelin-producing cells causes progressive destruction of white matter throughout the nervous system, including in the brain, spinal cord, and the nerves connecting the brain and spinal cord to muscles and sensory cells that detect sensations such as touch, pain, heat, and sound. Accordingly, MLD is characterized by progressive axonal demyelination of the central nervous system, and then the peripheral nervous system. This results in loss of acquired functions and/or skills, hypotonia, ataxia, seizures, blindness, hearing loss, and in untimely death.
  • MLD can be managed with several treatments. For example, medications to reduce signs and symptoms of MLD and to relieve associated pain. Hematopoietic stem cell transplants have been shown to delay the progression of MLD by introducing healthy cells to help replace diseased ones. Other treatments include physical, occupational, and speech therapy to promote muscle and joint flexibility and maintain range of motion. However, there is no cure for MLD.
  • MLD arylsulfatase A
  • ARSA arylsulfatase A
  • Carrier mutations have been found in 1 in 100 people, and affect 1 in 40,000 live births in U.S., or 1 in 160,000 worldwide.
  • Retroviral vectors including lentiviral vectors, are capable of integrating nucleic acids into host cell genomes, raising safety concerns due to their non-targeted insertion into the genome. For example, there is a risk of the vector disrupting a tumor suppressor gene or activating an oncogene, thereby causing a malignancy. Indeed, in a clinical trial for treating X-linked severe combined immunodeficiency (SCID) by transducing CD34 + bone marrow precursors with a gammaretroviral vector, four out of ten patients developed leukemia (Hacein-Bey-Abina et al. , J Clin Invest. 2008, 118(9): 3132-42). Non-integrating vectors, on the other hand, often suffer insufficient expression level or inadequate duration of expression in vivo.
  • SCID severe combined immunodeficiency
  • AAV adeno-associated virus
  • the instant disclosure provides a recombinant adeno-associated vims (rAAV) comprising: an AAV capsid comprising an AAV capsid protein; and an rAAV genome comprising a transcriptional regulatory element operably linked to an arylsulfatase A (ARSA) intron-inserted coding sequence comprising an intron.
  • rAAV adeno-associated vims
  • the ARSA intron-inserted coding sequence encodes a human ARSA protein or a variant thereof.
  • the ARSA intron-inserted coding sequence encodes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23.
  • the ARSA intron-inserted coding sequence encodes the amino acid sequence set forth in SEQ ID NO: 23.
  • the intron is a heterologous intron.
  • the intron has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 54.
  • the intron is positioned between nucleotides in the ARSA intron-inserted coding sequence that correspond to positions 369 and 370 of the ARSA coding sequence set forth in SEQ ID NO: 23.
  • the ARSA intron-inserted coding sequence comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 25.
  • the amino acid at amino acid position 204 of SEQ ID NO: 23 is V, and/or the amino acid at amino acid position 446 of SEQ ID NO: 23 is S.
  • the amino acid at amino acid position 204 of SEQ ID NO: 23 is V, and the amino acid at amino acid position 446 of SEQ ID NO: 23 is S.
  • the ARSA intron-inserted coding sequence encodes the amino acid sequence set forth in SEQ ID NO: 81.
  • the intron is a heterologous intron.
  • the intron has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 54.
  • the intron is positioned between nucleotides in the ARSA intron-inserted coding sequence that correspond to positions 369 and 370 of the amino acid sequence set forth in SEQ ID NO: 23.
  • the ARSA intron-inserted coding sequence comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identify to the nucleotide sequence set forth in SEQ ID NO: 27.
  • the instant disclosure provides a recombinant adeno- associated virus (rAAV) comprising: an AAV capsid comprising an AAV capsid protein; and an rAAV genome comprising a transcriptional regulatory element operably linked to an arylsulfatase A (ARSA) coding sequence (e.g, a coding sequence encoding the amino acid sequence set forth in SEQ ID NO: 23), and a micro-RNA (miRNA) binding region.
  • rAAV recombinant adeno- associated virus
  • the ARSA coding sequence is an ARSA intron-inserted coding sequence.
  • the ARSA intron-inserted coding sequence encodes a human ARSA protein or a variant thereof.
  • the ARSA intron-inserted coding sequence encodes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23.
  • the ARSA intron-inserted coding sequence encodes the amino acid sequence set forth in SEQ ID NO: 23.
  • the intron is a heterologous intron.
  • the intron has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 54.
  • the intron is positioned between nucleotides in the ARSA intron-inserted coding sequence that correspond to positions 369 and 370 of the ARSA coding sequence set forth in SEQ ID NO: 23.
  • the ARSA intron-inserted coding sequence comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 25.
  • the amino acid at amino acid position 204 of SEQ ID NO: 23 is V, and/or the amino acid at amino acid position 446 of SEQ ID NO: 23 is S.
  • the amino acid at amino acid position 204 of SEQ ID NO: 23 is V, and the amino acid at amino acid position 446 of SEQ ID NO: 23 is S.
  • the ARSA intron-inserted coding sequence encodes the amino acid sequence set forth in SEQ ID NO: 81.
  • the intron is a heterologous intron.
  • the intron has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 54.
  • the intron is positioned between nucleotides in the ARSA intron-inserted coding sequence that correspond to positions 369 and 370 of the amino acid sequence set forth in SEQ ID NO: 23.
  • the ARSA intron-inserted coding sequence comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 27.
  • the transcriptional regulator)' element comprises one or more of the elements selected from the group consisting of a cytomegalovirus (CMV) enhancer sequence, cytomegalovirus (CMV) promoter, chicken-(3-actin (CBA) promoter, a small chicken-P-actin (SmCBA) promoter, a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, a beta-glucuronidase (GUSB) promoter, a modified human EF-la promoter, a CALM1 promoter, a synthetic promoter, and any combination thereof.
  • CMV cytomegalovirus
  • CBA chicken-(3-actin
  • SmCBA small chicken-P-actin
  • GUSB beta-glucuronidase
  • the transcriptional regulatory element comprises an intron sequence.
  • the intron sequence comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 36.
  • the transcnptional regulatory element comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence set forth in SEQ ID NO: 36, 38, 43, and/or 50.
  • the transcriptional regulatory element comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 53.
  • the rAAV genome further comprises a micro-RNA (miRNA) binding region.
  • the miRNA binding region comprises one or more miRNA binding sites.
  • the miRNA binding region comprises two miRNA binding sites.
  • the miRNA binding region comprises three miRNA binding sites.
  • the one or more miRNA binding sites are each specific for the same miRNA. In certain embodiments, the one or more miRNA binding sites are each specific for different miRNAs.
  • the one or more miRNA binding sites are each specific for a muscle cell-specific miRNA.
  • the muscle cell-specific miRNA is cardiac cell-specific.
  • the miRNA binding site is specific for miR- 208a.
  • the miRNA binding region comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence set forth in SEQ ID NO: 62 or 63.
  • the miRNA binding site is specific for miR-133a.
  • the miRNA binding region compnses a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence set forth in SEQ ID NO: 64 or 65.
  • the rAAV genome further comprises a polyadenylation sequence 3' to the ARSA intron-inserted coding sequence.
  • the polyadenylation sequence is an exogenous polyadenylation sequence.
  • the exogenous polyadenylation sequence is an SV40 polyadenylation sequence.
  • the SV40 polyadenylation sequence comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 34, 35, or 45.
  • the exogenous polyadenylation sequence is a bovine growth hormone (BGH) polyadenylation sequence.
  • BGH polyadenylation sequence comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 52.
  • the rAAV genome comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity' to a nucleotide sequence set forth in SEQ ID NO: 66,
  • the rAAV genome further comprises a 5' inverted terminal repeat (5' ITR) nucleotide sequence, and a 3' inverted terminal repeat (3' ITR) nucleotide sequence.
  • the 5' ITR nucleotide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity' to SEQ ID NO: 18, 20, 49, 72, or 73 and/or the 3' ITR nucleotide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14, 19, 21, 28, 51, or 57.
  • the 5' ITR nucleotide sequence and the 3' ITR nucleotide, respectively comprise the sequence
  • the rAAV genome comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity' to a nucleotide sequence set forth in SEQ ID NO: 67,
  • the AAV capsid protein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of amino acids 203-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17.
  • the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO: 16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I: the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 626
  • the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G:
  • the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H, the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M;
  • the amino acid in the capsid protein corresponding to ammo acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R;
  • the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A
  • the capsid protein comprises the amino acid sequence of amino acids 203-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17.
  • the AAV capsid protein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of amino acids 138-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17.
  • the amino acid in the capsid protein corresponding to amino acid 151 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 160 of SEQ ID NO: 16 is D; the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO: 16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 505 of
  • the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G:
  • the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H, the ammo acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M;
  • the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R;
  • the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A
  • the capsid protein comprises the amino acid sequence of amino acids 138-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17.
  • the AAV capsid protein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of amino acids 1 -736 of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 15, 16, or 17.
  • the amino acid in the capsid protein corresponding to amino acid 2 of SEQ ID NO: 16 is T; the amino acid in the capsid protein corresponding to amino acid 65 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 68 of SEQ ID NO: 16 is V; the amino acid in the capsid protein corresponding to amino acid 77 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 119 of SEQ ID NO: 16 is L; the amino acid in the capsid protein corresponding to amino acid 151 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 160 of SEQ ID NO: 16 is D; the ammo acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO: 16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of
  • the amino acid in the capsid protein corresponding to amino acid 2 of SEQ ID NO: 16 is T, and the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q;
  • the amino acid in the capsid protein corresponding to amino acid 65 of SEQ ID NO: 16 is I, and the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is Y;
  • the amino acid in the capsid protein corresponding to amino acid 77 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K;
  • the amino acid in the capsid protein corresponding to amino acid 119 of SEQ ID NO: 16 is L, and the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S;
  • the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is
  • the instant disclosure provides a method for expressing an arylsulfatase A (ARSA) polypeptide in a cell, the method comprising transducing the cell with a recombinant adeno-associated virus (rAAV) as described herein.
  • ARSA arylsulfatase A
  • rAAV recombinant adeno-associated virus
  • the cell is a cell of the central nervous system.
  • the cell is a cell of the central nervous system region selected from the group consisting of the spinal cord, the motor cortex, the sensory cortex, the hippocampus, the putamen, the cerebellum optionally the cerebellar nuclei, and any combination thereof.
  • the cell is a neuron and/or a glial cell, optionally wherein the cell is a neuron and/or a glial cell of the central nervous system and/or the peripheral nervous system.
  • the cell is a cell selected from the group consisting of a motor neuron, an astrocyte, an oligodendrocyte, a cell of the cerebral cortex in the central nervous system, a sensory neuron of the peripheral nervous system, a Schwann cell, and any combination thereof.
  • the cell is in a mammalian subject and the rAAV is administered to the subject in an amount effective to transduce the cell in the subject.
  • the instant disclosure provides a pharmaceutical composition comprising a rAAV as described herein.
  • the instant disclosure provides a method for treating a subject having metachromatic leukodystrophy (MLD), the method comprising administering to the subject an effective amount of a rAAV as described herein, or a pharmaceutical composition as described herein.
  • MLD metachromatic leukodystrophy
  • the rAAV or pharmaceutical composition is administered intravenously.
  • metachromatic leukodystrophy is associated with an arylsulfatase A (ARSA) gene mutation.
  • ARSA arylsulfatase A
  • the subject is a human subject.
  • the instant disclosure provides a packaging system for preparation of an rAAV, wherein the packaging system comprises: (a) a first nucleotide sequence encoding one or more AAV Rep proteins; (b) a second nucleotide sequence encoding a capsid protein of a rAAV as described herein; and (c) a third nucleotide sequence comprising an rAAV genome sequence of a rAAV as described herein.
  • the packaging system comprises a first vector comprising the first nucleotide sequence and the second nucleotide sequence, and a second vector comprising the third nucleotide sequence.
  • the packaging system further comprises a fourth nucleotide sequence comprising one or more helper virus genes.
  • the fourth nucleotide sequence is comprised within a third vector.
  • the fourth nucleotide sequence comprises one or more genes from a virus selected from the group consisting of adenovirus, herpes virus, vaccinia virus, and cytomegalovirus (CMV).
  • the first vector, second vector, and/or the third vector is a plasmid.
  • the instant disclosure provides a method for recombinant preparation of an rAAV, the method comprising introducing a packaging system as described herein into a cell under conditions whereby the rAAV is produced.
  • the instant disclosure provides a polynucleotide comprising a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 53 or 60, optionally wherein the polynucleotide is comprised within a viral vector or plasmid vector.
  • the instant disclosure provides a polynucleotide comprising a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 25, 27, 61, 66, 67, 68, 69, 70, 74, 75, or 76, optionally wherein the polynucleotide is comprised within a viral vector or plasmid vector.
  • the instant disclosure provides a recombinant cell comprising a polynucleotide as described herein.
  • the instant disclosure provides a rAAV as described herein, a pharmaceutical composition as described herein, a polynucleotide as described herein, or a recombinant cell as described herein, for use as a medicament.
  • the instant disclosure provides a rAAV as described herein, a pharmaceutical composition as described herein, a polynucleotide as described herein, or a recombinant cell as described herein, for use in the treatment of metachromatic leukodystrophy (MLD).
  • MLD metachromatic leukodystrophy
  • the instant disclosure provides a rAAV as described herein, a pharmaceutical composition as described herein, a polynucleotide as descnbed herein, or a recombinant cell as described herein, for use in a method of treating a subject having metachromatic leukodystrophy (MLD), the method comprising administering to the subject an effective amount of the rAAV, the pharmaceutical composition, the polynucleotide, or the cell.
  • MLD metachromatic leukodystrophy
  • FIGs. 1A-1C are graphs showing ARSA protein expression and ARSA enzymatic activity in the brain of ARSA(-/-) mice treated with ARSA rAAV vector ARSA-1 packaged in AAVHSC15 capsid (AAVHSC15-ARSA-1).
  • FIG. 1A is a graph showing integrated pixel intensity determined by quantification of Western blot band obtained from immunoblotting of ARSA from tissue lysates prepared from ARSA(-/-) mice treated with AAVHSC15-ARSA-1 at the indicated doses, using an anti-ARSA antibody. The data is reported as mean integrated pixel intensity from five treated mice.
  • FIG. 1A is a graph showing integrated pixel intensity determined by quantification of Western blot band obtained from immunoblotting of ARSA from tissue lysates prepared from ARSA(-/-) mice treated with AAVHSC15-ARSA-1 at the indicated doses, using an anti-ARSA antibody. The data is reported as mean integrated pixel intensity from five treated mice.
  • FIG. 1A is
  • IB is a graph showing the level of ARSA enzymatic activity detected in the brain, as a percentage of the level of normal human brain ARSA enzymatic activity, in ARSA(-/-) mice treated with AAVHSC15- ARSA-1 at the indicated doses. The data is reported as mean percentage of normal human brain ARSA enzymatic activity from five treated mice. Dotted line indicates the predicted levels of ARSA enzymatic activity required for motor benefit.
  • 1C is a graph showing the level of ARSA enzymatic activity detected in the brain as a function of integrated pixel intensity determined by quantification of Western blot band obtained from immunoblotting of ARSA from tissue lysates prepared from ARSA(-/-) mice treated with AAVHSC15-ARSA-1 at the indicated doses, using an anti-ARSA antibody. Dotted line indicates the predicted levels of ARSA enzymatic activity required for motor benefit.
  • FIGs. 2A-2D are graphs showing the severity of various heart histopathologies observed in ARSA(-/-) mice treated with lel4 vg/kg of AAVHSC15-ARSA-1 or AAVHSC15- ARSA-5, at 4 weeks post-dosing. Control ARSA(-/-) mice were treated with vehicle.
  • FIG. 2A is a graph showing the severity of cardiomyocyte cytoplasmic vacuolation or rarification observed in treated ARSA(-/-) mice.
  • FIG. 2B is a graph showing the severity of histiocytic inflammation observed in treated ARSA(-/-) mice.
  • FIG. 2C is a graph showing the seventy of cardiomyocyte degeneration observed in treated ARSA(-/-) mice.
  • FIG. 2D is a graph showing the severity of myocardial fibrosis observed in treated ARSA(-/-) mice.
  • FIGs. 3A-3D are graphs showing the change in the severity of various heart histopathologies observed in ARSA(-/-) mice treated with lel4 vg/kg of AAVHSC15-ARSA- 1 or AAVHSC15-ARSA-5, at 12 weeks post-dosing. Control ARSA(-/-) mice were treated with vehicle.
  • FIG. 3A is a graph showing the severity of cardiomyocyte cytoplasmic vacuolation or rarification observed in treated ARSA(-/-) mice.
  • FIG. 3B is a graph showing the severity of histiocytic inflammation observed in treated ARSA(-Z-) mice.
  • FIG. 3C is a graph showing the severity of cardiomyocyte degeneration observed in treated ARSA(-/-) mice.
  • FIG. 3D is a graph showing the severity of myocardial fibrosis observed in treated ARSA(-Z-) mice.
  • FIGs. 4A-4C are graphs showing the durability of ARSA protein expression and enzymatic activity in key tissues of ARSA(-Z-) mice treated with lel4 vg/kg of AAVHSC15-ARSA-1, at 12 weeks post-dosing.
  • FIG. 4A is a graph showing ARSA-1 vector genomes detected in the indicated tissues of treated ARSA(-Z-) mice.
  • FIG. 4B is a graph showing ARSA enzymatic activity detected in the indicated tissues of treated ARSA(-Z-) mice. Dotted lines represent ARSA activity levels in corresponding normal human liver, brain, and heart tissue.
  • FIG. 4C is a graph showing ARSA enzymatic activity detected in the serum of treated ARSA(-/-) mice at the indicated doses.
  • FIGs. 5A-5D are graphs showing ARSA enzymatic activity in the brain (FIG. 5A), heart (FIG. 5B), liver (FIG. 5C) and serum (FIG. 5D) of neonatal ARSA(-/-) mice treated with lel3, 3el3, 5el3, or 7el3 vg/kg of ARSA rAAV vector ARSA-1 packaged in AAVHSC15 capsid (AAVHSC15-ARSA-1), at 12 weeks post-dosing.
  • FIGs. 5E-5G are graphs showing ARSA transcript copy number detected in the brain (FIG. 5E), heart (FIG. 5F), and liver (FIG.
  • FIG. 6A-6C are graphs showing ARSA enzymatic activity in the brain (FIG. 6A), liver (FIG. 6B), and heart (FIG. 6C) of neonatal ARSA(-/-) mice treated with vehicle control, or 3el3 or 5el3 vg/kg of ARSA rAAV vector ARSA-1 packaged in AAVHSC15 capsid (AAVHSC15-ARSA-1), at 10 months post-dosing.
  • the graphs also show ARSA enzymatic activity in the brain, liver, and heart of wild-type mice.
  • FIG. 7 is a graph showing the yield, in vector genomes per liter of culture, of ARSA-1, ARSA-2, ARSA-3, ARSA-4, and ARSA-6 packaged in AAVHSC15.
  • AAV adeno-associated virus
  • replication-defective adeno-associated virus refers to an AAV comprising a genome lacking Rep and Cap genes.
  • the term “ARSA gene” refers to the arylsulfatase A gene.
  • the human ARSA gene is identified by National Center for Biotechnology Information (NCBI) Gene ID 410.
  • An exemplary nucleotide sequence of an ARSA coding sequence is provided as SEQ ID NO: 24.
  • An exemplary amino acid sequence of an ARSA polypeptide is provided as SEQ ID NO: 23.
  • the term “recombinant AAV genome” or “rAAV genome” refers to a coding sequence operably linked to an exogenous transcriptional regulatory element that mediates expression of the coding sequence when the rAAV genome is introduced into a cell.
  • the rAAV genome does not integrate in the chromosomal DNA of the cell.
  • the portion of a rAAV genome comprising the transcriptional regulatory element operably linked to an ARSA coding sequence can be in the sense or antisense orientation relative to direction of transcription of the ARSA coding sequence.
  • Clade F capsid protein refers to an AAV VP1, VP2, or VP3 capsid protein that has at least 90% identity with the VP1, VP2, or VP3 amino acid sequences set forth, respectively, in amino acids 1-736, 138-736, and 203-736 of SEQ ID NO: 1 herein.
  • the “percentage identity” between two nucleotide sequences or between two amino acid sequences is calculated by multiplying the number of matches between the pair of aligned sequences by 100, and dividing by the length of the aligned region, including internal gaps. Identity scoring only counts perfect matches, and does not consider the degree of similarity of amino acids to one another. Only internal gaps are included in the length, not gaps at the sequence ends.
  • a disease or disorder associated with an ARSA gene mutation refers to any disease or disorder caused by, exacerbated by, or genetically linked with mutation of an ARSA gene.
  • the disease or disorder associated with an ARSA gene mutation is metachromatic leukodystrophy (MLD).
  • coding sequence refers to the portion of a complementary DNA (cDNA) that encodes a polypeptide, starting at the start codon and ending at the stop codon.
  • a gene may have one or more coding sequences due to alternative splicing, alternative translation initiation, and variation within the population.
  • a coding sequence may either be wild-type, silently-altered, and/or intron-inserted.
  • An exemplary wild-type ARSA coding sequence is set forth in SEQ ID NO: 24.
  • silently altered refers to alteration of a coding sequence or an intron-inserted coding sequence of a gene (e.g., by nucleotide substitution) without changing the amino acid sequence of the polypeptide encoded by the coding sequence or stuffer-inserted coding sequence. Such silent alteration is advantageous in that it may increase the translation efficiency of a coding sequence, and/or prevent recombination with a corresponding sequence of an endogenous gene when a coding sequence is transduced into a cell.
  • the term “intron-ms erted coding sequence” of a gene refers to a nucleotide sequence comprising one or more introns inserted in a coding sequence of the gene.
  • An intron-inserted coding sequence of a gene is also referred to as an intron-inserted coding sequence comprising an intron.
  • at least one of the introns is a normative or heterologous intron, z.e., having a sequence different from a native intron of the gene.
  • all of the introns in the intron-inserted coding sequence are normative introns.
  • a nonnative intron can have the sequence of an intron from a different species or the sequence of an intron in a different gene from the same species or from a different species. Alternatively, or additionally, at least a portion of a normative intron sequence can be synthetic.
  • nonnative intron sequences can be designed to mediate RNA splicing by introducing any consensus splicing motifs known in the art. Exemplary consensus splicing motifs are provided in Sibley et al. Nature Reviews Genetics. 2016, 17:407-21, which is incorporated by reference herein in its entirety.
  • Insertion of a normative mtron may promote the efficiency and robustness of vector packaging, as such sequences may allow for adjustments of the vector to reach an optimal size (e.g., 4.5-4.8 kb).
  • at least one of the introns is a native intron of the gene.
  • all of the introns in the intron-inserted coding sequence are native introns of the gene.
  • the normative or native introns can be inserted at any intemucleotide bonds in the coding sequence.
  • one or more normative or native introns are inserted at intemucleotide bonds predicted to promote efficient splicing (see, e.g., Zhang (1998) Human Molecular Genetics, 7(5):919-32, the disclosure of which is incorporated by reference herein in its entirety).
  • one or more nonnative or native introns are inserted at intemucleotide bonds that link two endogenous exons.
  • an intron-inserted coding sequence of a gene comprises one or more introns designed for efficient splicing.
  • the one or more introns may be inserted into a coding sequence of a gene to enhance expression of the gene (e.g.
  • the ARSA intron-inserted coding sequence comprises an intron positioned between nucleotides in the ARSA intron- inserted coding sequence that correspond to amino acids 369 and 370 of the amino acid sequence set forth in SEQ ID NO: 23.
  • heterologous intron and “normative intron” refer to an intron that is not native to a given gene.
  • nucleotide positions in an ARSA gene are specified relative to the first nucleotide of the start codon.
  • the first nucleotide of a start codon is position 1; the nucleotides 5' to the first nucleotide of the start codon have negative numbers; the nucleotides 3' to the first nucleotide of the start codon have positive numbers.
  • An exemplary nucleotide 1 of the human ARSA gene is nucleotide 374 of the NCBI Reference Sequence: NG_009260.2 (Region: 5028 - 10426), and an exemplary nucleotide 3 of the human ARSA gene is nucleotide 376 of the NCBI Reference Sequence: NG_009260.2 (Region: 5028 - 10426).
  • the nucleotide adjacently 5' to the start codon is nucleotide -1.
  • exons and introns in an ARSA gene are specified relative to the exon encompassing the first nucleotide of the start codon, which is nucleotide 374 of the NCBI Reference Sequence: NG_009260.2 (Region: 5028 - 10426).
  • the exon encompassing the first nucleotide of the start codon is exon 1.
  • Exons 3' to exon 1 are from 5' to 3': exon 2, exon 3, etc.
  • Introns 3' to exon 1 are from 5' to 3': intron 1, intron 2, etc.
  • the ARSA gene comprises from 5' to 3': exon 1, intron 1, exon 2, intron 2, exon 3, etc.
  • An exemplary exon 1 of the human ARSA gene is nucleotides 374-597 of the NCBI Reference Sequence: NG_009260.2 (Region: 5028 - 10426).
  • An exemplary intron 1 of the human ARSA gene is nucleotides 598-746 of the NCBI Reference Sequence: NG_009260.2 (Region: 5028 - 10426).
  • transcriptional regulatory element refers to a cis-acting nucleotide sequence, for example, a DNA sequence, that regulates (e.g. , controls, increases, or reduces) transcription of an operably linked nucleotide sequence by an RNA polymerase to form an RNA molecule.
  • a TRE relies on one or more trans-acting molecules, such as transcription factors, to regulate transcription.
  • one TRE may regulate transcription in different ways when it is in contact with different trans-acting molecules, for example, when it is in different types of cells.
  • a TRE may comprise one or more promoter elements and/or enhancer sequences.
  • promoter and enhancer sequences in a gene may be close in location, and the term “promoter” may refer to a sequence comprising a promoter element and an enhancer sequence. Thus, the term “promoter” does not exclude an enhancer sequence in the sequence.
  • the promoter and enhancer sequences do not need to be derived from the same gene or species, and the sequence of each promoter or enhancer sequence may be either identical or substantially identical to the corresponding endogenous sequence in the genome.
  • operably linked is used to describe the connection between a TRE and a coding sequence to be transcribed.
  • gene expression is placed under the control of a TRE comprising one or more promoter and/or enhancer sequences.
  • the coding sequence is “operably linked” to the TRE if the transcription of the coding sequence is controlled or influenced by the TRE.
  • the promoter and enhancer sequences of the TRE may be in any orientation and/or distance from the coding sequence, as long as the desired transcriptional activity is obtained.
  • the TRE is upstream from the coding sequence.
  • polyadenylation sequence refers to a DNA sequence that when transcribed into RNA constitutes a polyadenylation signal sequence.
  • the polyadenylation sequence can be native (e.g., from the ARSA gene) or exogenous.
  • the exogenous polyadenylation sequence can be a mammalian or a viral polyadenylation sequence (e.g, an SV40 polyadenylation sequence).
  • exogenous polyadenylation sequence refers to a polyadenylation sequence not identical or substantially identical to the endogenous polyadenylation sequence of an ARSA gene e.g , human ARSA gene).
  • an exogenous polyadenylation sequence is a poly adenylation sequence of a non- ARSA gene in the same species (e.g., human).
  • an exogenous polyadenylation sequence is a polyadenylation sequence of a different species (e.g, a vims).
  • the term “effective amount” in the context of the administration of an AAV to a subject refers to the amount of the AAV that achieves a desired prophylactic or therapeutic effect.
  • the term “about,” when in reference to a value or parameter herein, includes a variability of ⁇ 10% of the value or parameter.
  • “about” refers to a range that includes the value 10% below the referenced value, and the value 10% above the referenced value.
  • a dose of about 1E13 vg/kg refers to a dose that encompasses a dose of 0.9E13 vg/kg to 1.1 El 3 vg/kg, inclusive.
  • novel recombinant AAV e.g. , replicationdefective AAV
  • compositions useful for expressing an ARSA polypeptide in cells with reduced or otherwise defective ARSA gene function.
  • the rAAV disclosed herein comprise: an AAV capsid comprising a capsid protein e.g, a Clade F capsid protein); and a rAAV genome comprising a transcriptional regulatory element operably linked to an ARSA coding sequence (e.g., a silently altered ARSA coding sequence), allowing for extrachromosomal expression of ARSA in a cell transduced with the AAV.
  • a capsid protein from any capsid known the art can be used in the rAAV compositions disclosed herein, including, without limitation, a capsid protein from an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 serotype.
  • the capsid protein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of amino acids 203-736 of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17.
  • the capsid protein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity' with the amino acid sequence of amino acids 203-736 of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17, wherein: the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO: 16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N; the amino acid in the capsid
  • the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G.
  • the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H
  • the amino acid in the capsid protein corresponding to ammo acid 464 of SEQ ID NO: 16 is N
  • the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R
  • the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M.
  • the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R.
  • the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A, and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R.
  • the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I
  • the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R
  • the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C.
  • the capsid protein comprises the amino acid sequence of amino acids 203-736 of SEQ ID NO: 2, 3, 4, 6, 7, 10, 11, 12, 13, 15, 16, or 17.
  • the capsid protein comprises an ammo acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of amino acids 138-736 of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17.
  • the capsid protein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of amino acids 138-736 of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17, wherein: the amino acid in the capsid protein corresponding to amino acid 151 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to ammo acid 160 of SEQ ID NO: 16 is D; the ammo acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO: 16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q; the amino acid in the capsid protein
  • the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G.
  • the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H
  • the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N
  • the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R
  • the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M.
  • the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R.
  • the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A, and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R.
  • the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I
  • the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R
  • the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C.
  • the capsid protein comprises the amino acid sequence of amino acids 138-736 of SEQ ID NO: 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 16, or 17.
  • the capsid protein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of amino acids 1-736 of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17.
  • the capsid protein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of amino acids 1-736 of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17, wherein: the amino acid in the capsid protein corresponding to amino acid 2 of SEQ ID NO: 16 is T; the amino acid in the capsid protein corresponding to amino acid 65 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 68 of SEQ ID NO: 16 is V; the amino acid in the capsid protein corresponding to amino acid 77 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 119 of SEQ ID NO: 16 is L; the amino acid in the capsid protein corresponding to
  • the amino acid in the capsid protein corresponding to amino acid 2 of SEQ ID NO: 16 is T, and the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q.
  • the amino acid in the capsid protein corresponding to amino acid 65 of SEQ ID NO: 16 is I, and the ammo acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is Y.
  • the amino acid in the capsid protein corresponding to amino acid 77 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K.
  • the amino acid in the capsid protein corresponding to amino acid 119 of SEQ ID NO: 16 is L, and the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S.
  • the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G.
  • the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H, the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R.
  • the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A, and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R.
  • the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I
  • the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R
  • the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C.
  • the capsid protein comprises the amino acid sequence of amino acids 1-736 of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17.
  • the AAV capsid comprises two or more of: (a) a capsid protein comprising the amino acid sequence of ammo acids 203-736 of SEQ ID NO: 2, 3, 4, 6, 7, 10, 11, 12, 13, 15, 16, or 17; (b) a capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 16, or 17; and (c) a capsid protein comprising the amino acid sequence of amino acids 1-736 of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17.
  • the AAV capsid comprises: (a) a capsid protein having an amino acid sequence consisting of amino acids 203-736 of SEQ ID NO: 2, 3, 4, 6, 7, 10, 11, 12, 13, 15, 16, or 17; (b) a capsid protein having an amino acid sequence consisting of amino acids 138-736 of SEQ ID NO: 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 16, or 17; and (c) a capsid protein having an amino acid sequence consisting of amino acids 1-736 of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17.
  • the AAV capsid comprises one or more of: (a) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of amino acids 203-736 of SEQ ID NO: 8; (b) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of amino acids 138-736 of SEQ ID NO: 8; and (c) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%,
  • the AAV capsid comprises one or more of: (a) a capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 8; (b) a capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 8; and (c) a capsid protein comprising the amino acid sequence of amino acids 1-736 of SEQ ID NO: 8.
  • the AAV capsid comprises two or more of: (a) a capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 8; (b) a capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 8; and (c) a capsid protein comprising the amino acid sequence of amino acids 1-736 of SEQ ID NO: 8.
  • the AAV capsid comprises: (a) a capsid protein having an amino acid sequence consisting of amino acids 203-736 of SEQ ID NO: 8; (b) a capsid protein having an amino acid sequence consisting of amino acids 138-736 of SEQ ID NO: 8; and (c) a capsid protein having an amino acid sequence consisting of amino acids 1-736 of SEQ ID NO: 8.
  • the AAV capsid comprises one or more of: (a) a capsid protein comprising an ammo acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of amino acids 203-736 of SEQ ID NO: 11; (b) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of amino acids 138-736 of SEQ ID NO: 11; and (c) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
  • the AAV capsid comprises one or more of: (a) a capsid protein comprising the ammo acid sequence of amino acids 203-736 of SEQ ID NO: 11; (b) a capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 11; and (c) a capsid protein comprising the amino acid sequence of amino acids 1-736 of SEQ ID NO: 11.
  • the AAV capsid comprises two or more of: (a) a capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 11; (b) a capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 11; and (c) a capsid protein comprising the amino acid sequence of amino acids 1-736 of SEQ ID NO: 11.
  • the AAV capsid comprises: (a) a capsid protein having an amino acid sequence consisting of amino acids 203-736 of SEQ ID NO: 11; (b) a capsid protein having an amino acid sequence consisting of amino acids 138-736 of SEQ ID NO: 11; and (c) a capsid protein having an amino acid sequence consisting of amino acids 1-736 of SEQ ID NO: 11.
  • the AAV capsid comprises one or more of: (a) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of amino acids 203-736 of SEQ ID NO: 13; (b) a capsid protein comprising an amino acid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of amino acids 138-736 of SEQ ID NO: 13; and (c) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
  • the AAV capsid comprises one or more of: (a) a capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 13; (b) a capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 13; and (c) a capsid protein compnsmg the amino acid sequence of amino acids 1-736 of SEQ ID NO: 13.
  • the AAV capsid comprises two or more of: (a) a capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 13; (b) a capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 13; and (c) a capsid protein comprising the amino acid sequence of amino acids 1-736 of SEQ ID NO: 13.
  • the AAV capsid comprises: (a) a capsid protein having an amino acid sequence consisting of amino acids 203-736 of SEQ ID NO: 13; (b) a capsid protein having an amino acid sequence consisting of amino acids 138-736 of SEQ ID NO: 13; and (c) a capsid protein having an amino acid sequence consisting of amino acids 1-736 of SEQ ID NO: 13.
  • the AAV capsid comprises one or more of: (a) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of amino acids 203-736 of SEQ ID NO: 16; (b) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of amino acids 138-736 of SEQ ID NO: 16; and (c) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%,
  • the AAV capsid comprises one or more of: (a) a capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 16; (b) a capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 16; and (c) a capsid protein comprising the amino acid sequence of amino acids 1-736 of SEQ ID NO: 16.
  • the AAV capsid comprises two or more of: (a) a capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 16; (b) a capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 16; and (c) a capsid protein comprising the amino acid sequence of amino acids 1-736 of SEQ ID NO: 16.
  • the AAV capsid comprises: (a) a capsid protein having an amino acid sequence consisting of amino acids 203-736 of SEQ ID NO: 16; (b) a capsid protein having an amino acid sequence consisting of amino acids 138-736 of SEQ ID NO: 16; and (c) a capsid protein having an amino acid sequence consisting of amino acids 1-736 of SEQ ID NO: 16.
  • rAAV genomes useful in the AAV compositions disclosed herein generally comprise a transcriptional regulatory element (TRE) operably linked to an ARSA coding sequence.
  • the rAAV genome comprises a 5' inverted terminal repeat (5' ITR) nucleotide sequence 5' of the TRE and ARSA coding sequence, and a 3' inverted terminal repeat (3' ITR) nucleotide sequence 3' of the TRE and ARSA coding sequence.
  • the ARSA coding sequence comprises all or substantially all of a coding sequence of an ARSA gene. In certain embodiments, the ARSA coding sequence encodes a polypeptide comprising all or substantially all of the amino acids sequence of an ARSA protein. In certain embodiments, the ARSA coding sequence encodes the amino acid sequence of a wild-type ARSA protein (e.g., human ARSA protein).
  • the ARSA coding sequence encodes the ammo acid sequence of a variant ARSA protein (e.g., human ARSA protein), optionally wherein the variant ARSA polypeptide is a functional equivalent of the wild-type ARSA polypeptide, i.e. , can function as a wild-type ARSA polypeptide.
  • the functionally equivalent ARSA polypeptide further comprises at least one characteristic not found in the wild-type ARSA polypeptide, e.g., the ability to resist protein degradation.
  • the ARSA coding sequence encodes the amino acid sequence of a variant ARSA protein (e.g., human ARSA protein), wherein the variant ARSA polypeptide is a hyperfunctional ARSA polypeptide.
  • the ARSA coding sequence encodes an ARSA polypeptide that comprises one or more amino acid substitutions with respect to SEQ ID NO: 23.
  • the amino acid at amino acid position 204 of SEQ ID NO: 23 is V, and/or the amino acid at amino acid position 446 of SEQ ID NO: 23 is S.
  • the amino acid at amino acid position 204 of SEQ ID NO: 23 is V, and the amino acid at amino acid position 446 of SEQ ID NO: 23 is S.
  • the ARSA coding sequence encodes an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23.
  • the ARSA coding sequence encodes an amino acid sequence having at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23.
  • the ARSA coding sequence encodes the amino acid sequence set forth in SEQ ID NO: 23.
  • the ARSA coding sequence encodes the amino acid sequence set forth in SEQ ID NO: 81.
  • the ARSA coding sequence is a contiguous coding sequence (e.g., does not comprise any mtromc sequence).
  • the ARSA coding sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 24 or 61.
  • the ARSA coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 24.
  • the ARSA coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 61.
  • the ARSA coding sequence comprises an intron, and is an ARSA intron-inserted coding sequence.
  • the ARSA mtion- inserted coding sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 25 or 27.
  • the ARSA coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 25.
  • the ARSA coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 27.
  • the rAAV genome can be used to express ARSA in any mammalian cells (e.g. , human cells).
  • the TRE can be active in any mammalian cells (e.g., human cells).
  • the TRE is active in a broad range of human cells.
  • Such TREs may comprise constitutive promoter and/or enhancer sequences including cytomegalovirus (CMV) promoter/ enhancer (e.g.
  • CMV cytomegalovirus
  • SV40 promoter comprising a nucleotide sequence at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 29, 38, 40, 46, or 58
  • CBA chicken beta actin
  • smCBA promoter e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 55
  • human elongation factor 1 alpha (EFla) promoter e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%,
  • a rAAV genome may comprise a CMV enhancer sequence, a CBA promoter, and the splice acceptor from exon 3 of the rabbit beta-globin gene, collectively called a CAG promoter (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 42).
  • a CAG promoter e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 42).
  • a rAAV genome may comprise a hybrid of CMV enhancer sequence and CBA promoter followed by a splice donor and splice acceptor, collectively called a CASI promoter region (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 48).
  • a CASI promoter region e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 48).
  • the rAAV genome comprises a CMV enhancer sequence and a CBA promoter (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 50).
  • a CBA promoter e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 50.
  • the TRE may be a tissue-specific TRE, i. e. , it is active in specific tissue(s) and/or organ(s).
  • a tissue-specific TRE comprises one or more tissue-specific promoter and/or enhancer sequences, and optionally one or more constitutive promoter and/or enhancer sequences.
  • tissue-specific promoter and/or enhancer sequences can be isolated from genes specifically expressed in the tissue by methods well known in the art.
  • the TRE is brain-specific (e.g., neuron-specific, glial cell-specific, astrocyte-specific, oligodendrocyte-specific, microglia-specific, and/or central nervous system-specific).
  • exemplary brain-specific TREs may comprise one or more elements from, without limitation, human glial fibrillary acidic protein (GFAP) promoter, human synapsin 1 (SYN1) promoter, human synapsin 2 (SYN2) promoter, human metallothionein 3 (MT3) promoter, and/or human proteolipid protein 1 (PLP1) promoter. More brain-specific promoter elements are disclosed in WO 2016/100575A1, which is incorporated by reference herein in its entirety.
  • the rAAV genome comprises two or more TREs, optionally comprising at least one of the TREs disclosed above.
  • the transfer vector further comprises an intron 5' to or inserted in the ARSA coding sequence.
  • Such introns can increase transgene expression, for example, by reducing transcriptional silencing and enhancing mRNA export from the nucleus to the cytoplasm.
  • the rAAV genome comprises from 5' to 3': a noncoding exon, an intron, and the ARSA coding sequence.
  • an intron sequence is inserted in the ARSA coding sequence, optionally wherein the intron is inserted at an intemucleotide bond that links two native exons.
  • the intron is inserted at an internucleotide bond that links native exon 1 and exon 2.
  • the intron can comprise a native intron sequence of the ARSA gene, an intron sequence from a different species or a different gene from the same species, and/or a synthetic intron sequence.
  • synthetic intron sequences can be designed to mediate RNA splicing by introducing any consensus splicing motifs known in the art (e.g., in Sibley et al. Nature Reviews Genetics. 2016, 17:407-21, which is incorporated by reference herein in its entirety).
  • Exemplary intron sequences are provided in Lu et al., Molecular Therapy. 2013, 21(5): 954-63, and Lu et al. , Hum. Gene Ther. 2017, 28(1): 125-34, which are incorporated by reference herein in their entirety.
  • the rAAV genome comprises an SV40 intron (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31) or a minute virus of mouse (MVM) intron (e.g, comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 33).
  • SV40 intron e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 33.
  • the rAAV genome comprises a chimeric intron sequence comprising a combination of chicken and rabbit sequences, comprising partially the untranscribed chicken ACTB (cACTB) promoter, all of cACTB exon 1, partially cACTB intron 1, partially rabbit HBB2 (rHBB2) intron 2, and partially rHBB2 exon 3 (e.g, SEQ ID NO: 32).
  • the rAAV genome comprises a chimeric intron sequence (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 32 or 59).
  • the rAAV genome comprises a chimeric intron sequence comprising the nucleotide sequence set forth in SEQ ID NO: 59.
  • the rAAV genome compnses a P-globm intron sequence (e.g. , comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 54).
  • the rAAV genome comprises a P-globin intron sequence comprising the nucleotide sequence set forth in SEQ ID NO: 54.
  • the rAAV genome comprises a TRE comprising a CMV enhancer sequence, a CBA promoter, and a chimeric intron sequence (e.g, comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 60).
  • the rAAV genome comprises a TRE comprising SEQ ID NO: 60.
  • the rAAV genome comprises a TRE comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 36, 38, 43, and/or 50.
  • the rAAV genome comprises a TRE comprising a CMV enhancer sequence, a CBA promoter, and an ARSA intron sequence (e.g, comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 53).
  • the rAAV genome comprises a TRE comprising SEQ ID NO: 53.
  • rAAV genomes that comprise a miRNA binding region.
  • the miRNA binding region can comprise one or more micro-RNA (miRNA) binding sites, e.g, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more miRNA binding sites.
  • the rAAV genome further comprises a micro-RNA (miRNA) binding region that comprises one or more miRNA binding sites.
  • the rAAV genome further comprises a micro-RNA (miRNA) binding region that comprises two miRNA binding sites. In certain embodiments, the rAAV genome further comprises a micro-RNA (miRNA) binding region that comprises three miRNA binding sites. In certain embodiments, the one or more miRNA binding sites are each specific for the same miRNA. In certain embodiments, the one or more miRNA binding sites are each specific for different miRNAs.
  • the one or more miRNA binding sites are incorporated into the 3' UTR of the ARSA coding sequence.
  • the miRNA binding sites are the reverse complement of specific miRNAs expressed in target cells.
  • a cell type-specific miRNA binding site can be engineered into the 3' UTR of the ARSA coding sequence in order to inhibit ARSA gene expression in the specific cell type in which the cognate miRNA is found.
  • the miRNA binding site is specific for a muscle cellspecific miRNA.
  • Muscle cell-specific miRNAs are known in the art as myomiRs and include, without limitation, miR-1, miR-133, miR-206, miR-208b, miR-486, and miR-499.
  • the miRNA binding site is specific for a cardiac cellspecific miRNA.
  • Cardiac cell-specific miRNAs are known in the art and include, without limitation, miR-1, miR-133a, miR-208a/b, and miR-499.
  • the miRNA binding site is specific for miR-208a.
  • the miRNA binding site is specific for miR-133a.
  • the miRNA binding site comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence set forth in SEQ ID NO: 62 or 63.
  • the miRNA binding site comprises the nucleotide sequence set forth in SEQ ID NO: 62.
  • the miRNA binding site comprises the nucleotide sequence set forth in SEQ ID NO: 63.
  • the miRNA binding site comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence set forth in SEQ ID NO: 64 or 65.
  • the miRNA binding site comprises the nucleotide sequence set forth in SEQ ID NO: 64.
  • the miRNA binding site comprises the nucleotide sequence set forth in SEQ ID NO: 65.
  • the rAAV genome further comprises a transcription terminator (e.g , a poly adenylation sequence).
  • the transcription terminator is 3' to the ARSA coding sequence.
  • the transcription terminator may be any sequence that effectively terminates transcription, and a skilled artisan would appreciate that such sequences can be isolated from any genes that are expressed in the cell in which transcription of the ARSA coding sequence is desired.
  • the transcription terminator comprises a polyadenylation sequence.
  • the polyadenylation sequence is identical or substantially identical to the endogenous polyadenylation sequence of the human ARSA gene.
  • the polyadenylation sequence is an exogenous polyadenylation sequence.
  • the polyadenylation sequence is an SV40 polyadenylation sequence (e.g, comprising a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence set forth in SEQ ID NO: 34, 35, or 45, or a nucleotide sequence complementary thereto).
  • the polyadenylation sequence comprises the sequence set forth in SEQ ID NO: 45.
  • the polyadenylation sequence is an bovine growth hormone polyadenylation sequence (e.g., comprising a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence set forth in SEQ ID NO: 52, or a nucleotide sequence complementary thereto).
  • the polyadenylation sequence comprises the sequence set forth in SEQ ID NO: 52.
  • the rAAV genome comprises from 5' to 3': a TRE, an ARSA coding sequence, and a polyadenylation sequence.
  • the TRE comprises a nucleotide sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 43, 58, 59, and/or 60;
  • the ARSA coding sequence comprises a nucleotide sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 61;
  • the polyadenylation sequence comprises a nucleotide sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 34 or 45.
  • the rAAV genome comprises from 5' to 3': a TRE, an ARSA coding sequence, a miRNA binding site, and a polyadenylation sequence.
  • the TRE comprises a nucleotide sequence that has at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 36, 38, 43, and/or 53;
  • the ARSA coding sequence comprises a nucleotide sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 25 or 27;
  • the miRNA binding site comprises a nucleotide sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 63 or 65; and the polyadeny
  • the TRE comprises the nucleotide sequence set forth in SEQ ID NO: 60; the ARSA coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 61 ; and the polyadenylation sequence comprises the nucleotide sequence set forth in SEQ ID NO: 34.
  • the rAAV genome comprises the nucleotide sequences set forth in SEQ ID NOs: 34, 60, and 61.
  • the rAAV genome comprises from 5' to 3': the nucleotide sequence set forth in SEQ ID NO: 60; the nucleotide sequence set forth in SEQ ID NO: 61; and the nucleotide sequence set forth in SEQ ID NO: 34.
  • the TRE comprises the nucleotide sequence set forth in SEQ ID NO: 60; the ARSA coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 61 ; and the polyadenylation sequence comprises the nucleotide sequence set forth in SEQ ID NO: 45.
  • the rAAV genome comprises the nucleotide sequences set forth in SEQ ID NOs: 45, 60, and 61.
  • the rAAV genome comprises from 5' to 3': the nucleotide sequence set forth in SEQ ID NO: 60; the nucleotide sequence set forth in SEQ ID NO: 61; and the nucleotide sequence set forth in SEQ ID NO: 45.
  • the TRE comprises the nucleotide sequence set forth in SEQ ID NO: 53; the ARSA coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 25; the miRNA binding site comprises the nucleotide sequence set forth in SEQ ID NO: 63; and the polyadenylation sequence comprises the nucleotide sequence set forth in SEQ ID NO: 45.
  • the rAAV genome comprises the nucleotide sequences set forth in SEQ ID NOs: 25, 45, 53, and 63.
  • the rAAV genome comprises from 5' to 3': the nucleotide sequence set forth in SEQ ID NO: 53; the nucleotide sequence set forth in SEQ ID NO: 25; the nucleotide sequence set forth in SEQ ID NO: 63; and the nucleotide sequence set forth in SEQ ID NO: 45.
  • the TRE comprises the nucleotide sequence set forth in SEQ ID NO: 53; the ARSA coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 25; the miRNA binding site comprises the nucleotide sequence set forth in SEQ ID NO: 65; and the polyadenylation sequence comprises the nucleotide sequence set forth in SEQ ID NO: 45.
  • the rAAV genome comprises the nucleotide sequences set forth in SEQ ID NOs: 25, 45, 53, and 65.
  • the rAAV genome comprises from 5' to 3': the nucleotide sequence set forth in SEQ ID NO: 53; the nucleotide sequence set forth in SEQ ID NO: 25; the nucleotide sequence set forth in SEQ ID NO: 65; and the nucleotide sequence set forth in SEQ ID NO: 45.
  • the TRE comprises the nucleotide sequence set forth in SEQ ID NO: 53; the ARSA coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 27; the miRNA binding site comprises the nucleotide sequence set forth in SEQ ID NO: 63; and the polyadenylation sequence comprises the nucleotide sequence set forth in SEQ ID NO: 52.
  • the rAAV genome comprises the nucleotide sequences set forth in SEQ ID NOs: 27, 52, 53, and 63.
  • the rAAV genome comprises from 5' to 3': the nucleotide sequence set forth in SEQ ID NO: 53; the nucleotide sequence set forth in SEQ ID NO: 27; the nucleotide sequence set forth in SEQ ID NO: 63; and the nucleotide sequence set forth in SEQ ID NO: 52.
  • the TRE comprises the nucleotide sequence set forth in SEQ ID NO: 53; the ARSA coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 27; the miRNA binding site comprises the nucleotide sequence set forth in SEQ ID NO: 63; and the polyadenylation sequence comprises the nucleotide sequence set forth in SEQ ID NO: 45.
  • the rAAV genome comprises the nucleotide sequences set forth in SEQ ID NOs: 27, 45, 53, and 63.
  • the rAAV genome comprises from 5' to 3': the nucleotide sequence set forth in SEQ ID NO: 53; the nucleotide sequence set forth in SEQ ID NO: 27; the nucleotide sequence set forth in SEQ ID NO: 63; and the nucleotide sequence set forth in SEQ ID NO: 45.
  • the rAAV genome comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 66, 68, 70, 75, 77, or 79.
  • the rAAV genome comprises the nucleotide sequence set forth in SEQ ID NO: 66, 68, 70, 75, 77, or 79.
  • the nucleotide sequence of the rAAV genome consists of the nucleotide sequence set forth in SEQ ID NO: 66, 68, 70, 75, 77, or 79. In certain embodiments, the rAAV genome comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 66. In certain embodiments, the rAAV genome comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 68. In certain embodiments, the rAAV genome comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 70. In certain embodiments, the rAAV genome comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 75.
  • the rAAV genome comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 77. In certain embodiments, the rAAV genome comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 79.
  • the rAAV genomes disclosed herein further comprise a 5' inverted terminal repeat (5' ITR) nucleotide sequence 5' of the TRE, and a 3' inverted terminal repeat (3' ITR) nucleotide sequence 3' of the ARSA coding sequence.
  • ITR sequences from any AAV serotype or variant thereof can be used in the rAAV genomes disclosed herein.
  • the 5' and 3' ITR can be from an AAV of the same serotype or from AAVs of different serotypes.
  • Exemplary ITRs for use in the rAAV genomes disclosed herein are set forth in SEQ ID NO: 14, 18-21, 28, 49, 51, 57, 72, and 73.
  • the 5' ITR or 3' ITR is from AAV2. In certain embodiments, both the 5' ITR and the 3' ITR are from AAV2. In certain embodiments, the 5' ITR nucleotide sequence has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO: 18, or the 3' ITR nucleotide sequence has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO: 19.
  • the 5' ITR nucleotide sequence has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO: 18, and the 3' ITR nucleotide sequence has at least 90% (e.g, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO: 19.
  • the 5' ITR or 3' ITR are from AAV5. In certain embodiments, both the 5' ITR and 3' ITR are from AAV5. In certain embodiments, the 5' ITR nucleotide sequence has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO: 20, or the 3' ITR nucleotide sequence has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO: 21.
  • the 5' ITR nucleotide sequence has at least 90% e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO: 20, and the 3' ITR nucleotide sequence has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO: 21.
  • the 5' ITR nucleotide sequence has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO: 18, 20, 49, or 72.
  • the 3' ITR nucleotide sequence has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO: 14, 19, 21, 28, 51, or 57.
  • the 5' ITR nucleotide sequence and the 3' ITR nucleotide sequence are substantially complementary to each other (e.g., are complementary to each other except for mismatch at 1, 2, 3, 4, or 5 nucleotide positions in the 5' or 3' ITR).
  • the 5' ITR or the 3' ITR is modified to reduce or abolish resolution by Rep protein (“non-resolvable ITR”).
  • the non-resolvable ITR comprises an insertion, deletion, or substitution in the nucleotide sequence of the terminal resolution site. Such modification allows formation of a self-complementary, double-stranded DNA genome of the AAV after the rAAV genome is replicated in an infected cell.
  • Exemplary non-resolvable ITR sequences are known in the art (see, e.g., those provided in U.S. Patent Nos. 7,790,154 and 9,783,824, which are incorporated by reference herein in their entirety).
  • the 3' ITR is flanked by an additional nucleotide sequence derived from a wild-type AAV2 genomic sequence. In certain embodiments, the 3' ITR is flanked by an additional 37 bp sequence derived from a wild-type AAV2 sequence that is adjacent to a wild-type AAV2 ITR. See, e.g., Savy et al., Human Gene Therapy Methods . 2017, 28(5):277-289 (which is hereby incorporated by reference herein in its entirety).
  • the rAAV genome comprises from 5' to 3': a 5' ITR; an internal element comprising from 5' to 3': a TRE, optionally a non-coding exon and an intron, an ARSA coding sequence, and a polyadenylation sequence, as disclosed herein; a non- resolvable ITR; a nucleotide sequence complementary to the internal element; and a 3' ITR.
  • a 5' ITR an internal element comprising from 5' to 3': a TRE, optionally a non-coding exon and an intron, an ARSA coding sequence, and a polyadenylation sequence, as disclosed herein; a non- resolvable ITR; a nucleotide sequence complementary to the internal element; and a 3' ITR.
  • Such rAAV genome can form a self-complementary, double-stranded DNA genome of the AAV after infection and before replication.
  • the rAAV genome comprises from 5' to 3': a 5' ITR, a TRE, an ARSA coding sequence, a poly adenylation sequence; and a 3' ITR.
  • the 5' ITR comprises a nucleotide sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 18, 20, 49, or 72
  • the TRE comprises a nucleotide sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 43, 58, 59, and/or 60
  • the ARSA coding sequence comprises a nucleotide sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:
  • the rAAV genome comprises from 5' to 3': a 5' ITR, a TRE, an ARSA coding sequence, a miRNA binding site, a polyadenylation sequence, and a 3' ITR.
  • the 5' ITR comprises a nucleotide sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 18, 20, 49, or 72;
  • the TRE comprises a nucleotide sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 36, 38, 43, and/or 53;
  • the ARSA coding sequence comprises a nucleotide sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 25 or 27;
  • the miRNA binding site comprises a nucleotide sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO
  • the 5' ITR comprises the nucleotide sequence set forth in SEQ ID NO: 18; the TRE comprises the nucleotide sequence set forth in SEQ ID NO: 60; the ARSA coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 61; the polyadenylation sequence comprises the nucleotide sequence set forth in SEQ ID NO: 34; and the 3' ITR comprises the nucleotide sequence set forth in SEQ ID NO: 14.
  • the rAAV genome comprises the nucleotide sequences set forth in SEQ ID NOs: 14, 18, 34, 60, and 61.
  • the rAAV genome comprises from 5' to 3': the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO: 60; the nucleotide sequence set forth in SEQ ID NO: 61; the nucleotide sequence set forth in SEQ ID NO: 34; and the nucleotide sequence set forth in SEQ ID NO: 14.
  • the 5' ITR comprises the nucleotide sequence set forth in SEQ ID NO: 18; the TRE comprises the nucleotide sequence set forth in SEQ ID NO: 60; the ARSA coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 61 ; the poly adenylation sequence comprises the nucleotide sequence set forth in SEQ ID NO: 45; and the 3' ITR comprises the nucleotide sequence set forth in SEQ ID NO: 14.
  • the rAAV genome comprises the nucleotide sequences set forth in SEQ ID NOs: 14, 18, 45, 60, and 61.
  • the rAAV genome comprises from 5' to 3': the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO: 60; the nucleotide sequence set forth in SEQ ID NO: 61; the nucleotide sequence set forth in SEQ ID NO: 45; and the nucleotide sequence set forth in SEQ ID NO: 14.
  • the 5' ITR comprises the nucleotide sequence set forth in SEQ ID NO: 18; the TRE comprises the nucleotide sequence set forth in SEQ ID NO: 53; the ARSA coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 25; the miRNA binding site comprises the nucleotide sequence set forth in SEQ ID NO: 63; the polyadenylation sequence comprises the nucleotide sequence set forth in SEQ ID NO: 45; and the 3' ITR comprises the nucleotide sequence set forth in SEQ ID NO: 14.
  • the rAAV genome comprises the nucleotide sequences set forth in SEQ ID NOs: 14, 18, 25, 45, 53, and 63.
  • the rAAV genome comprises from 5' to 3': the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO: 53; the nucleotide sequence set forth in SEQ ID NO: 25; the nucleotide sequence set forth in SEQ ID NO: 63; the nucleotide sequence set forth in SEQ ID NO: 45; and the nucleotide sequence set forth in SEQ ID NO: 14.
  • the 5' ITR comprises the nucleotide sequence set forth in SEQ ID NO: 18; the TRE comprises the nucleotide sequence set forth in SEQ ID NO: 53; the ARSA coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 25; the miRNA binding site comprises the nucleotide sequence set forth in SEQ ID NO: 65; the polyadenylation sequence comprises the nucleotide sequence set forth in SEQ ID NO: 45; and the 3' ITR comprises the nucleotide sequence set forth in SEQ ID NO: 14.
  • the rAAV genome comprises the nucleotide sequences set forth in SEQ ID NOs: 14, 18, 25, 45, 53, and 65.
  • the rAAV genome comprises from 5' to 3': the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO: 53; the nucleotide sequence set forth in SEQ ID NO: 25; the nucleotide sequence set forth in SEQ ID NO: 65; the nucleotide sequence set forth in SEQ ID NO: 45; and the nucleotide sequence set forth in SEQ ID NO: 14.
  • the 5' ITR comprises the nucleotide sequence set forth in SEQ ID NO: 18; the TRE comprises the nucleotide sequence set forth in SEQ ID NO: 53; the ARSA coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 27; the miRNA binding site comprises the nucleotide sequence set forth in SEQ ID NO: 63; the polyadenylation sequence comprises the nucleotide sequence set forth in SEQ ID NO: 52; and the 3' ITR comprises the nucleotide sequence set forth in SEQ ID NO: 14.
  • the rAAV genome comprises the nucleotide sequences set forth in SEQ ID NOs: 14, 18, 27, 52, 53, and 63.
  • the rAAV genome comprises from 5' to 3': the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO: 53; the nucleotide sequence set forth in SEQ ID NO: 27; the nucleotide sequence set forth in SEQ ID NO: 63; the nucleotide sequence set forth in SEQ ID NO: 52; and the nucleotide sequence set forth in SEQ ID NO: 14.
  • the 5' ITR comprises the nucleotide sequence set forth in SEQ ID NO: 18; the TRE comprises the nucleotide sequence set forth in SEQ ID NO: 53; the ARSA coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 27; the miRNA binding site comprises the nucleotide sequence set forth in SEQ ID NO: 63; the polyadenylation sequence comprises the nucleotide sequence set forth in SEQ ID NO: 45; and the 3' ITR comprises the nucleotide sequence set forth in SEQ ID NO: 14.
  • the rAAV genome comprises the nucleotide sequences set forth in SEQ ID NOs: 14, 18, 27, 45, 53, and 63.
  • the rAAV genome comprises from 5' to 3': the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO: 53; the nucleotide sequence set forth in SEQ ID NO: 27; the nucleotide sequence set forth in SEQ ID NO: 63; the nucleotide sequence set forth in SEQ ID NO: 45; and the nucleotide sequence set forth in SEQ ID NO: 14.
  • the rAAV genome comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 67, 69, 74, 76, 78, or 80.
  • the rAAV genome comprises the nucleotide sequence set forth in SEQ ID NO: 67, 69, 74, 76, 78, or 80.
  • the nucleotide sequence of the rAAV genome consists of the nucleotide sequence set forth in SEQ ID NO: 67, 69, 74, 76, 78, or 80.
  • the rAAV genome comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 67.
  • the rAAV genome comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 69.
  • the rAAV genome comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 74.
  • the rAAV genome comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 76.
  • the rAAV genome comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 78. In certain embodiments, the rAAV genome comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 80.
  • the present disclosure provides an rAAV comprising: (a) an AAV capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 16, and a rAAV genome comprising 5' to 3' following genetic elements: the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO: 60; the nucleotide sequence set forth in SEQ ID NO: 61; the nucleotide sequence set forth in SEQ ID NO: 34; and the nucleotide sequence set forth in SEQ ID NO: 14; (b) an AAV capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 16, and a rAAV genome comprising 5' to 3' following genetic elements: the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO: 60; the nucleotide sequence set forth in SEQ ID NO:
  • the present disclosure provides an rAAV comprising: (a) an AAV capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 16, and a rAAV genome comprising 5' to 3' following genetic elements: the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO: 60; the nucleotide sequence set forth in SEQ ID NO: 61; the nucleotide sequence set forth in SEQ ID NO: 45; and the nucleotide sequence set forth in SEQ ID NO: 14; (b) an AAV capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 16, and a rAAV genome comprising 5' to 3' following genetic elements: the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO: 60; the nucleotide sequence set forth in SEQ ID NO:
  • the present disclosure provides an rAAV comprising: (a) an AAV capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 16, and a rAAV genome comprising 5' to 3' following genetic elements: the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO: 53; the nucleotide sequence set forth in SEQ ID NO: 25; the nucleotide sequence set forth in SEQ ID NO: 63; the nucleotide sequence set forth in SEQ ID NO: 45; and the nucleotide sequence set forth in SEQ ID NO: 14; (b) an AAV capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 16, and a rAAV genome comprising 5' to 3' following genetic elements: the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO:
  • the present disclosure provides an rAAV comprising: (a) an AAV capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 16, and a rAAV genome comprising 5' to 3' following genetic elements: the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO: 53; the nucleotide sequence set forth in SEQ ID NO: 25; the nucleotide sequence set forth in SEQ ID NO: 65; the nucleotide sequence set forth in SEQ ID NO: 45; and the nucleotide sequence set forth in SEQ ID NO: 14; (b) an AAV capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 16, and a rAAV genome comprising 5' to 3' following genetic elements: the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO: 53
  • the present disclosure provides an rAAV comprising: (a) an AAV capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 16, and a rAAV genome comprising 5' to 3' following genetic elements: the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO: 53; the nucleotide sequence set forth in SEQ ID NO: 27; the nucleotide sequence set forth in SEQ ID NO: 63; the nucleotide sequence set forth in SEQ ID NO: 52; and the nucleotide sequence set forth in SEQ ID NO: 14; (b) an AAV capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 16, and a rAAV genome comprising 5' to 3' following genetic elements: the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO:
  • the present disclosure provides an rAAV comprising: (a) an AAV capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 16, and a rAAV genome comprising 5' to 3' following genetic elements: the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO: 53; the nucleotide sequence set forth in SEQ ID NO: 27; the nucleotide sequence set forth in SEQ ID NO: 63; the nucleotide sequence set forth in SEQ ID NO: 45; and the nucleotide sequence set forth in SEQ ID NO: 14; (b) an AAV capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 1 , and a rAAV genome comprising 5' to 3' following genetic elements: the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO: 53; the nucleo
  • the rAAV comprises: (a) an AAV capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 16, and a rAAV genome comprising the nucleotide sequence set forth in any one of SEQ ID NO: 66, 67, 68, 69, 70, 74, 75, or 76; (b) an AAV capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 16, and a rAAV genome comprising the nucleotide sequence set forth in any one of SEQ ID NO: 66, 67, 68, 69, 70, 74, 75, or 76; and/or (c) an AAV capsid protein comprising the amino acid sequence of SEQ ID NO: 16, and a rAAV genome comprising the nucleotide sequence set forth in any one of SEQ ID NO: 66, 67, 68, 69, 70, 74, 75, or 76.
  • a polynucleotide comprising a nucleotide sequence that is at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the nucleic acid sequence set forth in SEQ ID NO: 66, 67, 68, 69, 70, 74, 75, or 76.
  • a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO: 66, 67, 68, 69, 70, 74, 75, or 76.
  • a polynucleotide that consists of a nucleotide sequence set forth in SEQ ID NO: 66, 67, 68, 69, 70, 74, 75, or 76.
  • the polynucleotide comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 66.
  • the polynucleotide comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 67.
  • the polynucleotide comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 68.
  • the polynucleotide comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 69. In certain embodiments, the polynucleotide comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 70. In certain embodiments, the polynucleotide comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 74. In certain embodiments, the polynucleotide comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 75. In certain embodiments, the polynucleotide comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 76.
  • a polynucleotide comprising a nucleotide sequence that is at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the nucleic acid sequence set forth in SEQ ID NO: 25, 27, or 61.
  • a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO: 25, 27, or 61.
  • a polynucleotide that consists of a nucleotide sequence set forth in SEQ ID NO: 25, 27, or 61.
  • the polynucleotide comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 25.
  • the polynucleotide comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 27.
  • the polynucleotide comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 61.
  • a polynucleotide comprising a nucleotide sequence that is at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the nucleic acid sequence set forth in SEQ ID NO: 53 or 60.
  • a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO: 53 or 60.
  • the polynucleotide compnses or consists of the nucleotide sequence set forth in SEQ ID NO: 53. In certain embodiments, the polynucleotide comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 60.
  • a polynucleotide comprising a nucleotide sequence that is at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the nucleic acid sequence set forth in SEQ ID NO: 63 or 65.
  • a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO: 63 or 65.
  • a polynucleotide that consists of a nucleotide sequence set forth in SEQ ID NO: 63 or 65. In certain embodiments, the polynucleotide comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 63. In certain embodiments, the polynucleotide comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 65.
  • the polynucleotide can comprise DNA, RNA, modified DNA, modified RNA, or a combination thereof.
  • the polynucleotide is an expression vector.
  • the polynucleotide is comprised within a viral vector.
  • the polynucleotide is comprised within a plasmid vector.
  • compositions comprising an AAV as disclosed herein together with a pharmaceutically acceptable excipient, adjuvant, diluent, vehicle or carrier, or a combination thereof.
  • a “pharmaceutically acceptable carrier” includes any material which, when combined with an active ingredient of a composition, allows the ingredient to retain biological activity and without causing disruptive physiological reactions, such as an unintended immune reaction.
  • Pharmaceutically acceptable carriers include water, phosphate buffered saline, emulsions such as oil/water emulsion, and wetting agents. Compositions comprising such carriers are formulated by well-known conventional methods such as those set forth in Remington’s Pharmaceutical Sciences, current Ed., Mack Publishing Co , Easton Pa. 18042, USA; A.
  • the instant disclosure provides methods for expressing an ARSA polypeptide in a cell.
  • the methods generally comprise transducing or transfecting the cell with a rAAV or polynucleotide disclosed herein. Such methods are highly efficient at restoring ARSA expression. Accordingly, in certain embodiments, the methods disclosed herein involve transducing the cell with a rAAV disclosed herein. In certain embodiments, the methods disclosed herein involve transfecting the cell with a polynucleotide disclosed herein.
  • the methods disclosed herein can be applied to any cell harboring a mutation in the ARSA gene.
  • cells that require active endogenous ARSA are of particular interest.
  • the methods are applied to any cell that has lost endogenous ARSA activity.
  • the method is applied to a neuron and/or a glial cell.
  • of particular interest are neurons and/or glial cells that require active endogenous ARSA.
  • the method is applied to cells of the central nervous system, and/or cells of the peripheral nervous system.
  • of particular interest are cells of the central nervous system and/or of the peripheral nervous system that require active endogenous ARSA.
  • of particular interest are cells in the forebrain, midbrain, hindbrain, spinal cord, and any combination thereof.
  • of particular interest are cells of a central nervous system region selected from the group consisting of the spinal cord, the motor cortex, the sensory cortex, the thalamus, the hippocampus, the putamen, the cerebellum (e.g., the cerebellar nuclei), and any combination thereof.
  • of particular interest are cells of the pons and medulla in the brain, ascending fasciculus of the spinal cord, and any combination thereof.
  • of particular interest are cells of a central nervous system region selected from the group consisting of the spinal cord, the motor cortex, the sensory cortex, the thalamus, the hippocampus, the putamen, the cerebellum (e.g., the cerebellar nuclei), and any combination thereof, that require active endogenous ARSA.
  • of particular interest are motor neurons and astrocytic profiles in the central nervous system (CNS), oligodendrocytes (ascending fibers) in the CNS, cellular populations of the cerebral cortex in the CNS, and sensory neurons of the peripheral nervous system (PNS).
  • oligodendrocytes such as those in the dorsal fasciculus of the spinal cord.
  • glial profiles in the central nervous system including but not limited to, astrocytes, oligodendrocytes, Schwann cells, and any combination thereof.
  • of particular interest are motor neurons, astrocytes, oligodendrocytes, cells of the cerebral cortex in the central nervous system, sensory neurons of the peripheral nervous system, glial cells of the peripheral nervous system (e.g, Schwann cells), and any combination thereof.
  • the methods disclosed herein can be performed in vitro for research purposes or can be performed ex vivo or in vivo for therapeutic purposes.
  • the cell to be transduced is in a mammalian subject and the AAV is administered to the subject in an amount effective to transduce the cell in the subject.
  • the instant disclosure provides a method for treating a subject having a disease or disorder associated with an ARSA gene mutation, the method generally comprising administering to the subject an effective amount of a rAAV or polynucleotide disclosed herein.
  • the subject can be a human subject, a non-human primate subject (e.g., a cynomolgus), or a rodent subject (e.g, a mouse) with an ARSA mutation.
  • Any disease or disorder associated with an ARSA gene mutation can be treated using the methods disclosed herein. Suitable diseases or disorders include, without limitation, metachromatic leukodystrophy.
  • the AAV is administered to the subject at a dose of about 0.5E13 vg/kg. In certain embodiments, the AAV is administered at a dose of 0.5E13 vg/kg. In certain embodiments, the AAV is administered at a dose of about 1 El 3 vg/kg. In certain embodiments, the AAV is administered at a dose of 1E13 vg/kg. In certain embodiments, the AAV is administered at a dose of about 3E13 vg/kg. In certain embodiments, the AAV is administered at a dose of 3E13 vg/kg. In certain embodiments, the AAV is administered at a dose of about 5E13 vg/kg.
  • the AAV is administered at a dose of 5E13 vg/kg. In certain embodiments, the AAV is administered at a dose of about 6E13 vg/kg. In certain embodiments, the AAV is administered at a dose of 6E13 vg/kg. In certain embodiments, the AAV is administered at a dose of about 7E13 vg/kg. In certain embodiments, the AAV is administered at a dose of 7E13 vg/kg. In certain embodiments, the AAV is administered at a dose of about 1E14 vg/kg. In certain embodiments, the AAV is administered at a dose of 1E14 vg/kg.
  • the foregoing methods employ a rAAV comprising: (a) an AAV capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 16, and a rAAV genome comprising 5' to 3’ following genetic elements: the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO: 60; the nucleotide sequence set forth in SEQ ID NO: 61; the nucleotide sequence set forth in SEQ ID NO: 34; and the nucleotide sequence set forth in SEQ ID NO: 14; (b) an AAV capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 16, and a rAAV genome comprising 5’ to 3’ following genetic elements: the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO: 60; the nucleotide sequence set forth in SEQ ID NO:
  • the foregoing methods employ a rAAV comprising: (a) an AAV capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 16, and a rAAV genome comprising 5' to 3' following genetic elements: the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO: 60; the nucleotide sequence set forth in SEQ ID NO: 61; the nucleotide sequence set forth in SEQ ID NO: 45; and the nucleotide sequence set forth in SEQ ID NO: 14; (b) an AAV capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 16, and a rAAV genome comprising 5' to 3' following genetic elements: the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO: 60; the nucleotide sequence set forth in SEQ ID NO:
  • the foregoing methods employ a rAAV comprising: (a) an AAV capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 16, and a rAAV genome comprising 5' to 3' following genetic elements: the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO: 53; the nucleotide sequence set forth in SEQ ID NO: 25; the nucleotide sequence set forth in SEQ ID NO: 63; the nucleotide sequence set forth in SEQ ID NO: 45; and the nucleotide sequence set forth in SEQ ID NO: 14; (b) an AAV capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 16, and a rAAV genome comprising 5' to 3' following genetic elements: the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO: 53; the nucle
  • the foregoing methods employ a rAAV comprising: (a) an AAV capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 16, and a rAAV genome comprising 5' to 3' following genetic elements: the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO: 53; the nucleotide sequence set forth in SEQ ID NO: 25; the nucleotide sequence set forth in SEQ ID NO: 65; the nucleotide sequence set forth in SEQ ID NO: 45; and the nucleotide sequence set forth in SEQ ID NO: 14; (b) an AAV capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 1 , and a rAAV genome comprising 5' to 3' following genetic elements: the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO: 53; the nu
  • the foregoing methods employ a rAAV comprising: (a) an AAV capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 16, and a rAAV genome comprising 5' to 3' following genetic elements: the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO: 53; the nucleotide sequence set forth in SEQ ID NO: 27; the nucleotide sequence set forth in SEQ ID NO: 63; the nucleotide sequence set forth in SEQ ID NO: 52; and the nucleotide sequence set forth in SEQ ID NO: 14; (b) an AAV capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 16, and a rAAV genome comprising 5' to 3' following genetic elements: the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO:
  • the foregoing methods employ a rAAV comprising: (a) an AAV capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 16, and a rAAV genome comprising 5' to 3' following genetic elements: the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO: 53; the nucleotide sequence set forth in SEQ ID NO: 27; the nucleotide sequence set forth in SEQ ID NO: 63; the nucleotide sequence set forth in SEQ ID NO: 45; and the nucleotide sequence set forth in SEQ ID NO: 14; (b) an AAV capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 16, and a rAAV genome comprising 5' to 3' following genetic elements: the nucleotide sequence set forth in SEQ ID NO: 18; the nucleotide sequence set forth in SEQ ID NO: 53; the nucle
  • the foregoing methods employ a rAAV comprising: (a) an AAV capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 16, and a rAAV genome comprising the nucleotide sequence set forth in any one of SEQ ID NO: 66, 67, 68, 69, 70, 74, 75, or 76; (b) an AAV capsid protein comprising the amino acid sequence ofamino acids 138-736 of SEQ ID NO: 16, and arAAV genome comprising the nucleotide sequence set forth in any one of SEQ ID NO: 66, 67, 68, 69, 70, 74, 75, or 76; and/or (c) an AAV capsid protein comprising the amino acid sequence of SEQ ID NO: 16, and a rAAV genome comprising the nucleotide sequence set forth in any one of SEQ ID NO: 66, 67, 68, 69, 70, 74, 75, or 76; and
  • the methods disclosed herein are particularly advantageous in that they are capable of expressing an ARSA protein in a cell with high efficiency both in vivo and in vitro.
  • the expression level of the ARSA protein is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the expression level of the endogenous ARSA protein in a cell of the same type that does not have a mutation in the ARSA gene.
  • the expression level of the ARSA protein is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fold higher than the expression level of the endogenous ARSA protein in a cell of the same type that does not have a mutation in the ARSA gene.
  • Any methods of determining the expression level of the ARSA protein can be employed including, without limitation, ELISA, Western blotting, immunostaining, and mass spectrometry.
  • transduction of a cell with an AAV composition disclosed herein can be performed as provided herein or by any method of transduction known to one of ordinary skill in the art.
  • the cell may be contacted with the AAV at a multiplicity of infection (MOI) of 50,000; 100,000; 150,000; 200,000; 250,000; 300,000; 350,000; 400,000; 450,000; or 500,000, or at any MOI that provides for optimal transduction of the cell.
  • MOI multiplicity of infection
  • An AAV composition disclosed herein can be administered to a subject by any appropriate route including, without limitation, intravenous, intrathecal, intraperitoneal, subcutaneous, intramuscular, intranasal, topical, or intradermal routes.
  • the composition is formulated for administration via intravenous injection or subcutaneous injection.
  • the instant disclosure provides packaging systems for recombinant preparation of a recombinant adeno-associated virus (rAAV) disclosed herein.
  • packaging systems generally comprise: first nucleotide encoding one or more AAV Rep proteins; a second nucleotide encoding a capsid protein of any of the AAVs as disclosed herein; and a third nucleotide sequence comprising any of the rAAV genomes as disclosed herein, wherein the packaging system is operative in a cell for enclosing the rAAV genome in the capsid to form the AAV.
  • the packaging system comprises a first vector comprising the first nucleotide sequence encoding the one or more AAV Rep proteins and the second nucleotide sequence encoding the AAV capsid protein, and a second vector comprising the third nucleotide sequence comprising the rAAV genome.
  • a “vector” refers to a nucleic acid molecule that is a vehicle for introducing nucleic acids into a cell (e.g. , a plasmid, a virus, a cosmid, an artificial chromosome, etc.).
  • Any AAV Rep protein can be employed in the packaging systems disclosed herein.
  • the Rep nucleotide sequence encodes an AAV2 Rep protein.
  • Suitable AAV2 Rep proteins include, without limitation, Rep 78/68 or Rep 68/52.
  • the nucleotide sequence encoding the AAV2 Rep protein comprises a nucleotide sequence that encodes a protein having a minimum percent sequence identity to the AAV2 Rep amino acid sequence of SEQ ID NO: 22, wherein the minimum percent sequence identity is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) across the length of the amino acid sequence of the AAV2 Rep protein.
  • the AAV2 Rep protein has the amino acid sequence set forth in SEQ ID NO: 22.
  • the packaging system further comprises a fourth nucleotide sequence comprising one or more helper virus genes.
  • the packaging system further comprises a third vector, e.g., a helper virus vector, comprising the fourth nucleotide sequence comprising the one or more helper virus genes.
  • the third vector may be an independent third vector, integral with the first vector, or integral with the second vector.
  • the helper virus is selected from the group consisting of adenovirus, herpes virus (including herpes simplex virus (HSV)), poxvirus (such as vaccinia virus), cytomegalovirus (CMV), and baculovirus.
  • the adenovirus genome comprises one or more adenovirus RNA genes selected from the group consisting of El, E2, E4, and VA.
  • the HSV genome comprises one or more of HSV genes selected from the group consisting of UL5/8/52, ICPO, ICP4, ICP22, and UL30/UL42.
  • the first, second, and/or third vector are contained within one or more plasmids.
  • the first vector and the third vector are contained within a first plasmid.
  • the second vector and the third vector are contained within a second plasmid.
  • the first, second, and/or third vector are contained within one or more recombinant helper viruses.
  • the first vector and the third vector are contained within a recombinant helper virus.
  • the second vector and the third vector are contained within a recombinant helper virus.
  • the disclosure provides a method for recombinant preparation of an AAV as described herein, wherein the method comprises transfecting or transducing a cell with a packaging system as described herein under conditions operative for enclosing the rAAV genome in the capsid to form the rAAV as described herein.
  • Exemplary methods for recombinant preparation of an rAAV include transient transfection (e.g., with one or more transfection plasmids containing a first, and a second, and optionally a third vector as described herein), viral infection (e.g.
  • helper viruses such as a adenovirus, poxvirus (such as vaccinia virus), herpes virus (including HSV, cytomegalovirus, or baculovirus), containing a first, and a second, and optionally a third vector as described herein), and stable producer cell line transfection or infection (e.g.
  • a stable producer cell such as a mammalian or insect cell, containing a Rep nucleotide sequence encoding one or more AAV Rep proteins and/or a Cap nucleotide sequence encoding one or more capsid proteins as described herein, and with a rAAV genome as described herein being delivered in the form of a plasmid or a recombinant helper virus).
  • the instant disclosure provides a packaging system for preparation of a recombinant AAV (rAAV), wherein the packaging system comprises a first nucleotide sequence encoding one or more AAV Rep proteins; a second nucleotide sequence encoding a capsid protein of any one of the AAVs described herein; a third nucleotide sequence comprising an rAAV genome sequence of any one of the AAVs described herein; and optionally a fourth nucleotide sequence comprising one or more helper virus genes.
  • rAAV recombinant AAV
  • the recombinant AAV vectors disclosed herein mediate highly efficient gene transfer in vitro and in vivo.
  • the following examples demonstrate the efficient restoration of the expression of the ARSA gene (which is mutated in certain human diseases, such as metachromatic leukodystrophy) using an AAV-based vector as disclosed herein. These examples are offered by way of illustration, and not by way of limitation.
  • This example provides human ARSA recombinant adeno-associated virus (rAAV) vectors ARSA-1, ARSA-2, ARSA-3, ARSA-4, ARSA-5, and ARSA-6, for expression of human ARSA (hARSA) in a cell (e.g., a human cell or a mouse cell) to which the vector is transduced.
  • rAAV human ARSA recombinant adeno-associated virus
  • rAAV vector ARSA-1 comprises a rAAV genome comprising from 5' to 3' the following genetic elements: a 5' ITR element, a transcriptional regulatory element comprising a CMV enhancer sequence, a chicken-P-actin promoter, and an ARSA intron sequence; an ARSA intron-inserted coding sequence comprising an intron; a triple miR-208a binding site; an SV40 polyadenylation sequence; and a 3' ITR element.
  • This vector is capable of expressing a human ARSA protein in a cell (e.g. , a human cell or a mouse cell) to which the vector is transduced.
  • ARSA-2 ARSA-2
  • rAAV vector ARSA-2 comprises a rAAV genome comprising from 5' to 3' the following genetic elements: a 5' ITR element, a transcriptional regulatory element comprising a CMV enhancer sequence, a chicken-P-actin promoter, and an ARSA intron sequence; an ARSA intron-inserted coding sequence comprising an intron; a triple miR-133a binding site; an SV40 poly adenylation sequence; and a 3' ITR element.
  • This vector is capable of expressing a human ARSA protein in a cell (e.g. , a human cell or a mouse cell) to which the vector is transduced.
  • ARSA-3 ARSA-3
  • rAAV vector ARSA-3 comprises a rAAV genome comprising from 5' to 3' the following genetic elements: a 5' ITR element, a transcriptional regulatory element comprising a CMV enhancer sequence, a chicken-P-actin promoter, and an ARSA intron sequence; an ARSA intron-inserted coding sequence comprising an intron; a triple miR-208a binding site; a bovine growth hormone (BGH) polyadenylation sequence; and a 3' ITR element.
  • BGH bovine growth hormone
  • rAAV vector ARSA-4 comprises a rAAV genome comprising from 5' to 3' the following genetic elements: a 5' ITR element, a transcriptional regulatory element comprising a CMV enhancer sequence, a chicken-P-actin promoter, and an ARSA intron sequence; an ARSA intron-inserted coding sequence comprising an intron; a triple miR-208a binding site; an SV40 polyadenylation sequence; and a 3' ITR element.
  • This vector is capable of expressing a human ARSA protein in a cell (e.g. , a human cell or a mouse cell) to which the vector is transduced.
  • ARSA-5 ARSA-5
  • rAAV vector ARSA-5 comprises a rAAV genome comprising from 5' to 3' the following genetic elements: a 5' ITR element, a transcriptional regulatory element comprising a CMV enhancer sequence, a chicken-P-actin promoter, and a chimeric intron sequence; an ARSA coding sequence; an SV40 polyadenylation sequence; and a 3' ITR element.
  • This vector is capable of expressing a human ARSA protein in a cell (e.g., a human cell or a mouse cell) to which the vector is transduced.
  • ARSA-6 ARSA-6
  • rAAV vector ARSA-6 comprises a rAAV genome comprising from 5' to 3' the following genetic elements: a 5' ITR element, a transcriptional regulatory element comprising a CMV enhancer sequence, a chicken-P-actin promoter, and a chimeric intron sequence; an ARSA coding sequence; an SV40 polyadenylation sequence; and a 3' ITR element.
  • This vector is capable of expressing a human ARSA protein in a cell (e.g., a human cell or a mouse cell) to which the vector is transduced.
  • the vectors disclosed herein can be packaged in an AAV capsid, such as, without limitation, an AAVHSC15 or AAVHSC17 capsid.
  • the packaged viral particles can be administered to a wild-type animal, or an ARSA-deficient animal.
  • ARSA(-/-) mouse model is an ARSA knock-out mouse produced by insertion of a neomycin cassette into exon 4 of the mouse ARSA gene (see Hess et al. , Proc. Natl. Acad. Sci. U.S.A. 1996, 93(25): 14821-14826, incorporated by reference herein in its entirety).
  • ARSA(-/-) mice develop similar but milder metachromatic leukodystrophy (MLD) compared to humans.
  • ARSA(-/-) mice do not show evidence of widespread demyelination.
  • ARSA rAAV vectors were to ensure safety by reducing overall ARSA protein expression in heart tissue, while maintaining near-normal levels of ARSA activity in brain tissue.
  • ARSA ARSA(-/-) mice 6-8 weeks of age were intravenously administered ARSA-1 or ARSA-5, each packaged in AAVHSC15 capsid (AAVHSC1 -ARSA-1 and AAVHSC15-ARSA-5, respectively), in each case at a dose of lel4 vg/kg.
  • Formulation buffer was used as control.
  • ARSA immunoreactivity was investigated by immunohistochemistry on brain, liver, and heart tissue in ARSA(-/-) mice using an anti -ARSA antibody 4 weeks postdosing.
  • Anti-ARSA immunoreactivity of brain, liver, and heart slices show robust ARSA protein expression in the brain and liver of ARSA(-/-) mice administered AAVHSC 15-ARSA- 1 and AAVHSC 15- ARSA-5 as compared to control ARSA(-/-) mice administered vehicle.
  • ARSA(-/-) mice administered AAVHSC 15-ARS A- 1 maintained robust ARSA protein expression in the brain and liver, while showing lowered expression in the heart, as compared to ARSA(-/-) mice administer AAVHSC15-ARSA-5.
  • Robust ARSA protein expression in the bram and liver, and lowered expression in the heart of ARSA(-/-) mice administered AAVHSC15-ARSA-1 maintained out to 12 weeks post-dosing.
  • the ARSA rAAV vector ARSA-1 comprises an ARSA intron- inserted coding sequence comprising an intron, an improved promoter region comprising a CMV enhancer sequence and chicken (3-actin promoter, and a miR-208a binding site. Transduction of ARSA-1 was shown to exhibit significantly improved expression of ARSA protein in the liver and brain, while maintaining lowered expression of ARSA protein in the heart (see, Example 2).
  • Bram lysates from vector treated ARSA(-/-) mice were used for evaluating ARSA protein expression and enzymatic activity.
  • a colorimetric enzyme assay that measures the cleavage of sulfate from the soluble substrate p-nitrocatechol-sulfate (pNCS) was used to determine ARSA-specific enzyme activity.
  • Non-specific cleavage of sulfate from competing enzymes was eliminated from tissue lysates by use of an anti-ARSA specific immunoprecipitation step.
  • the normal adult human ARSA enzyme activity in brain was determined by analysis of ARSA enzyme activity in adult post-mortem human frontal cortex samples.
  • FIG. 1A a dose responsive relationship was found for the level of ARSA protein expression as presented by integrated pixel intensity determined by quantification of Western blot band obtained from immunoblotting of ARSA from tissue lysates prepared from ARSA(-/-) mice treated with AAVHSC15-ARSA-1 at the indicated doses, using an anti-ARSA antibody.
  • FIG. 1A a dose responsive relationship was found for the level of ARSA protein expression as presented by integrated pixel intensity determined by quantification of Western blot band obtained from immunoblotting of ARSA from tissue lysates prepared from ARSA(-/-) mice treated with AAVHSC15-ARSA-1 at the indicated doses, using an anti-ARSA antibody.
  • IB shows a dose dependent response of ARSA enzymatic activity detected in the brain lysates of vector treated ARSA(-/-) mice.
  • the level of ARSA enzymatic activity detected in brain lysates of AAVHSC15-ARSA-1 treated ARSA(-Z-) mice is predicted to lead to a direct motor benefit in the rotarod assay (z.e. , >50% ARSA enzymatic activity; see, dotted line in FIG. IB).
  • FIG. 1C A direct correlation between ARSA protein expression and ARSA enzymatic activity in vector treated ARSA(-/-) mice is shown in FIG. 1C, demonstrating a favorable dose-range and ARSA protein to activity profile.
  • the ARSA rAAV vector ARSA-5 comprises an ARSA coding sequence (with no intron insertion), and a promoter region comprising a CMV enhancer sequence and chicken -actin promoter.
  • Heart pathology was examined in the vector treated mice 4 weeks post-dosing for any incidence of cardiomyocyte cytoplasmic vacuolation or rarification (FIG. 2A), histiocytic inflammation (FIG. 2B), cardiomyocyte degeneration (FIG.
  • FIG. 2C shows myocardial fibrosis.
  • a clear reduction in the severity of cardiomyocyte cytoplasmic vacuolation or rarification, histiocytic inflammation, and cardiomyocyte degeneration was observed in ARSA-1 treated ARSA(-/-) mice as compared to the ARSA-5 treated ARSA(-/-) mice.
  • the severity of myocardial fibrosis was found to be unchanged in ARSA-1 or ARSA-5 treated ARSA(-/-) mice as compared to untreated ARSA(-/-) mice.
  • the lowered ARSA protein expression levels in the heart observed in ARSA-1 treated ARSA(-/-) mice resulted in a reduction of heart histopathology as compared to ARSA-5 treated ARSA(-/-) mice.
  • the minimal cardiomyocyte cytoplasmic vacuolation or rarification observed at 4 weeks post-dosing did not progress (FIG. 3A).
  • Histiocytic inflammation, cardiomyocyte degeneration, and myocardial fibrosis was also found to be unchanged at 12 weeks post-dosing (FIGs. 3B-3D).
  • ARSA-1 vector genomes and ARSA enzymatic activity were detectable in all tissues out to 12 weeks post-dosing. Further, as shown in FIG.
  • a dose dependent response in ARSA enzymatic activity was detected at 12 weeks post-dosing in the brains of treated mice, reaching and exceeding normal human levels of expression as measured in normal post-mortem human brain tissue samples (FIG. 5A).
  • This range covered the expression levels predicted to lead to motor deficit prevention in the MLD mouse model (50-100% of normal human levels) across multiple doses.
  • a dose dependent response in ARSA enzymatic activity was achieved at 12 weeks post-dosing in tissues of both the heart (FIG. 5B) and the liver (FIG. 5C) of treated mice, reaching supraphysiological levels in the heart and normal levels in actively dividing liver tissue at the highest dose tested.
  • the level of activity in the liver tissue following growth and cell division reached normal human levels, and at all other doses, reached 5-20% of normal human levels, which is the level of ARSA reported in asymptomatic pseudo-deficient ARSA patients.
  • FIGs. 5E-5G show a dose-dependent increase in ARSA transcript copy number per ng of cDNA detected in the brains (FIG. 5E), hearts (FIG. 5F), and livers (FIG. 5G) of the treated mice.
  • MLD biomarkers were assessed in the tissues of wild-type mice, vehicle-treated ARSA(-/-) mice, and ARSA(-/-) mice treated with 5el3 vg/kg AAVHSC15-ARSA-1.
  • ARSA immunoreactivity was investigated by standard immunohistochemistry techniques using an anti-ARSA antibody on brain, liver, and heart tissue of WT and ARSA(-/-) treated with vehicle, as well as AAVHSC15-ARSA-l-treated ARSA(-/-) mice.
  • ARSA(-/-) mice treated with AAVHSC15-ARSA-1 exhibited significantly higher ARSA immunoreactivity compared to wild-type mice and vehicle-treated ARSA(-/-) mice.
  • LAMP-1 lysosomal-associated membrane protein
  • ARSA rAAV vectors ARSA-1, ARSA-2, ARSA-3, ARSA-4, and ARSA-6 packaged in AAVHSC15 was assessed.
  • AAVHSC15-ARSA-1, AAVHSC15-ARSA-2, AAVHSC15-ARSA-3, and AAVHSC15-ARSA-4 performed comparably when produced using a 2L bioreactor, based on productivity, packaging, residuals and impurities.
  • AAVHSC15-ARSA-6 was produced using a 50L bioreactor. As shown in FIG.

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Abstract

L'invention concerne des compositions de virus adéno-associé (AAV) qui peuvent exprimer un polypeptide d'arylsulfatase a (ARSA) dans une cellule, pour ainsi permettre la restauration de la fonction du gène ARSA. L'invention concerne également des procédés d'utilisation des compositions d'AAV, et des systèmes d'encapsidation pour fabriquer les compositions d'AAV.
PCT/US2023/071985 2022-08-10 2023-08-10 Compositions de virus adéno-associé pour le transfert de gène arsa et procédés pour les utiliser Ceased WO2024036250A2 (fr)

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