WO2023240236A1 - Compositions and methods for the treatment of spinal muscular atrophy related disorders - Google Patents

Compositions and methods for the treatment of spinal muscular atrophy related disorders Download PDF

Info

Publication number
WO2023240236A1
WO2023240236A1 PCT/US2023/068202 US2023068202W WO2023240236A1 WO 2023240236 A1 WO2023240236 A1 WO 2023240236A1 US 2023068202 W US2023068202 W US 2023068202W WO 2023240236 A1 WO2023240236 A1 WO 2023240236A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
amino acid
nucleotide sequence
acid sequence
sequence
Prior art date
Application number
PCT/US2023/068202
Other languages
French (fr)
Inventor
Mathieu Emmanuel NONNENMACHER
Amy Zhen REN
Damien MAURA
Wei Wang
Heather YONUTAS
Elisabeth KNOLL
Original Assignee
Voyager Therapeutics, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Voyager Therapeutics, Inc. filed Critical Voyager Therapeutics, Inc.
Publication of WO2023240236A1 publication Critical patent/WO2023240236A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14145—Special targeting system for viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

  • compositions and methods relating to polynucleotides e.g., polynucleotides encoding survival motor neuron (SMN) proteins and peptides for use in the treatment of Spinal Muscular Atrophy and related disorders, including Werdnig-Hoffmann disease and Kugelberg- Welander disease (collectively, “SMA-related disorders”).
  • SMA-related disorders Spinal Muscular Atrophy and related disorders, including Werdnig-Hoffmann disease and Kugelberg- Welander disease
  • compositions may be delivered in an adeno-associated viral (AAV) vector.
  • compositions described herein may be used to treat a subject in need thereof, such as a human subject diagnosed with SMA-related disorders or other condition resulting from a deficiency in the quantity and/or function of SMN protein.
  • SMA Spinal Muscular Atrophy
  • CNS central nervous system
  • SMA is the second most common autosomal recessive disorder with an incidence of approximately 1 in 10,000 live births. Childhood SMA is classically subdivided into three clinical groups on the basis of age of onset and clinical course.
  • the acute form of Werdnig-Hoffmann disease (Type I) is characterized by severe generalized muscle weakness and hypotonia at birth or in the 3 months following birth. Death, from respiratory failure, usually occurs within the first two years. This disease may be distinguished from the intermediate (Type II) and juvenile (Type III, Kugelberg- Welander disease) forms. Type II children were able to sit but unable to stand or walk unaided, and they live beyond 4 years. Type III patients had proximal muscle weakness, starting after the age of two.
  • SMA IV slow evolving adult form of SMA
  • SMA is linked to a genetic defect in the SMN1 gene that encodes the Survival of Motor Neuron (SMN) protein.
  • the SMN protein is ubiquitously expressed and required by all cells and tissue types plays important roles in multiple fundamental cellular homeostatic pathways, including a well- characterized role in the assembly of the spliceosome and biogenesis of ribonucleoproteins. More recent studies have shown that SMN is also involved in other housekeeping processes, including mRNA trafficking and local translation, cytoskeletal dynamics, endocytosis and autophagy. Moreover, SMN has been shown to influence mitochondria and bioenergetic pathways as well as regulate function of the ubiquitin-proteasome system.
  • SMN human isoform of SMN
  • SMN1 contains nine exons, 1, 2a, 2b, 3, 4, 5, 6, 7 and 8, with exon 8 remaining untranslated. Chaytow et al., (Cell Mol Life Sci. 2018: 75(21): 3877-3894).
  • SMN2 differs from SMN1 at 5 bases, and a C-to-T transition in exon 7 of SMN2 favors skipping of exon 7 during splicing, resulting in the majority of SMN2 products being a truncated isoform referred as SMNA7.
  • SMNA7 is highly unstable and quickly subjected to the ubiquitin-tin-proteasome pathway for degradation.
  • SMA characterized by a homozygous loss of function mutation in the survival motor neuron gene SMN1 on 5ql3, while retaining the modifying SMN2 gene.
  • the SMN2 gene has a similar structure to SMN1, but only a small amount (10%) of the SMN protein it produces is fully functional. This low level of SMN protein is not effective enough to sustain the survival of motor neurons in the CNS.
  • compositions and methods directed to AAV-based gene delivery of SMN to ameliorate loss-of-function and to increase SMN protein levels.
  • the compositions and methods are useful to improve motor neuron function, and to slow, halt, or reverse neurodegenerative and other symptoms of SMA-related disorders (e.g., Werdni-Hoffman disease, Dubowitz disease, Kugelberg-Welander disease), in a subject (e.g., a subject having a mutation in an SMN1 gene).
  • SMA-related disorders e.g., Werdni-Hoffman disease, Dubowitz disease, Kugelberg-Welander disease
  • the present disclosure provides an AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant, e.g., an AAV5 capsid variant, comprises an amino acid other than T at position 577 (e.g., Y, N, or C), numbered relative to SEQ ID NO: 138.
  • the AAV capsid variant comprises a Y at position 577, numbered relative to SEQ ID NO: 138.
  • the AAV capsid variant comprises a N at position 577, numbered relative to SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises a C at position 577, numbered relative to SEQ ID NO: 138.
  • the present disclosure provides an AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant, e.g., a variant of the wild-type AAV5 capsid, comprises more than one amino acid that replaces the threonine (T) at position 577, numbered relative to SEQ ID NO: 138.
  • T threonine
  • an insert of two, three, four, five, six, seven, eight, nine, or ten amino acids replaces the T at position 577, numbered relative to SEQ ID NO: 138.
  • an insert of eight amino acids replaces the T at position 577, numbered relative to SEQ ID NO: 138.
  • the present disclosure provides an AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant comprises an amino acid sequence having the following formula: [N2]-[N3], wherein (i) [N2] comprises positions XI, X2, X3, X4, and X5, wherein: (a) position XI is Y, N, or C; (b) position X2 is P, K, T, or Q; (c) position X3 is A or P; (d) position X4 is E, S, or A; and (e) position X5 is V, L, or E; and (ii) [N3] comprises the amino acid sequence of VQK, EQK, VKK, VHK, VQQ, or LQK; ; and optionally wherein the AAV capsid variant comprises an
  • [N2] comprises Y at position XI. In some embodiments, [N2] comprises P at position X2. In some embodiments, [N2] comprises A at position X3. In some embodiments, [N2] comprises E at position X4. In some embodiments, [N2] comprises V at position X5. In some embodiments, the amino acid sequence of [N3] is VQK.
  • XI of [N2] is present at position 577
  • X2 of [N2] is present at position 578
  • X3 of [N2] is present at position 579
  • X4 of [N2] is present at position 580
  • X5 of [N2] is present at position 581, numbered according to SEQ ID NO: 982.
  • [N2] is present at positions 577-581, numbered according to SEQ ID NO: 982.
  • [N3] is present at positions 582-584, numbered according to SEQ ID NO: 982.
  • [N2]-[N3] is present at positions 577-584, numbered according to SEQ ID NO: 982.
  • the amino acid sequence of [N2] consists of YPAEV (SEQ ID NO: 39). In some embodiments, the amino acid sequence of [N3] consists of VQK. In some embodiments, [N2]-[N3] replaces the threonine (T) at position 577 of wild-type AAV5, e.g., SEQ ID NO: 138.
  • [N2]-[N3] replaces the threonine (T) at position 577 of wild-type AAV5, e.g., SEQ ID NO: 138, and the amino acid sequence of [N2] consists of YPAEV (SEQ ID NO: 39), and the amino acid sequence of [N3] consists of VQK.
  • [N2] is present at positions 577-581, numbered according to SEQ ID NO: 982.
  • [N3] is present at positions 582-584, numbered according to SEQ ID NO: 982.
  • [N2]-[N3] is present at positions 577- 584, numbered according to SEQ ID NO: 982.
  • the present disclosure AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant comprises one, two, three, or all of: (i) an [NO], wherein the amino acid sequence of [NO] comprises TNN, TNT, INN, TNS, NNN, or TNK; (ii) an [Nl], wherein the amino acid sequence of [Nl] comprises QSS, QSK, TSL, SSS, QSR, AGA, IGS, QAS, ASS, LGS, QST, HSS, LSS, or QRS; (iii) an [N2], wherein the amino acid sequence of [N2] comprises YPAEV (SEQ ID NO: 39), YPPSL (SEQ ID NO: 40), NKAEV (SEQ ID NO: 41), YTAEV (SEQ ID NO: 39),
  • the amino acid sequence of [NO] is TNN. In some embodiments, the amino acid sequence of [Nl] is QSS. In some embodiments, the amino acid sequence of [N2] is YPAEV (SEQ ID NO: 39). In some embodiments, the amino acid sequence of [N3] is VQK. In some embodiments, the amino acid sequence of [N4] is TA. In some embodiments, the amino acid sequence of [NO] is TNN, the amino acid sequence of [Nl] is QSS, the amino acid sequence of [N2] is YPAEV (SEQ ID NO: 39), the amino acid sequence of [N3] is VQK, and/or the amino acid sequence of [N4] is TA.
  • the amino acid sequence of [NO] is TNN
  • the amino acid sequence of [Nl] is QSS
  • the amino acid sequence of [N2] is YPAEV (SEQ ID NO: 39)
  • the amino acid sequence of [N3] is VQK
  • the amino acid sequence of [N4] is TA.
  • [NO] is present immediately subsequent to position 570, numbered relative to SEQ ID NO: 138.
  • [NO] replaces positions 571- 573 (e.g., T571, N572, and N573), numbered relative to SEQ ID NO: 138.
  • [NO] is present immediately subsequent to position 570, and [NO] positions 571-573 (e.g., T571, N572, and N573), numbered relative to SEQ ID NO: 138.
  • [Nl] is present immediately subsequent to position 573, numbered relative to SEQ ID NO: 138.
  • [Nl] replaces positions 574-576 (e.g., Q574, S575, and S576), numbered relative to SEQ ID NO: 138.
  • [Nl] is present immediately subsequent to position 573, and [Nl] replaces positions 574-576 (e.g., Q574, S575, and S576), numbered relative to SEQ ID NO: 138.
  • [N2] is present immediately subsequent to position 576, numbered relative to SEQ ID NO: 138.
  • [N2] replaces position 577 (e.g., T577), numbered relative to SEQ ID NO: 138.
  • [N2] is present immediately subsequent to position 576, and [N2] replaces position 577 (e.g., T577), numbered relative to SEQ ID NO: 138.
  • [N2]-[N3] is present immediately subsequent to position 576, numbered relative to SEQ ID NO: 138. In any of these embodiments, [N2]-[N3] replaces position 577 (e.g., T577), numbered relative to SEQ ID NO: 138. In any of these embodiments, [N2]-[N3] is present immediately subsequent to position 576, and [N2]-[N3] replaces position 577 (e.g., T577), numbered relative to SEQ ID NO: 138.
  • [N2]-[N3]-[N4] replaces positions 577-579 (e.g., T577, T578, and A579), numbered relative to SEQ ID NO: 138.
  • [N2]-[N3]-[N4] is present immediately subsequent to position 576, and [N2J-[N3J- [N4J replaces positions 577-579 (e.g., T577, T578, and A579), numbered relative to SEQ ID NO: 138.
  • - [N1]-[N2]-[N3]-[N4] is present immediately subsequent to position 570, numbered relative to SEQ ID NO: 138.
  • [N0]-[Nl]-[N2]-[N3]-[N4] replaces positions 571-579 (e.g., T571, N572, N573, Q574, S575, S576, T577, T578, and A579), numbered relative to SEQ ID NO: 138.
  • [N0]-[N1 ]-[N2]-[N3]-[N4] is present immediately subsequent to position 570, and [NO]-[N1]-[N2]-[N3]-[N4] replaces positions 571-579 (e.g., T571, N572, N573, Q574, S575, S576, T577, T578, and A579), numbered relative to SEQ ID NO: 138.
  • [NO]-[N1]-[N2]-[N3]-[N4] is TNNQSSYPAEVVQKTA (SEQ ID NO: 1533) and is present immediately subsequent to position 570, numbered relative to SEQ ID NO: 138, wherein [N2]-[N3] (YPAEVVQK; SEQ ID NO: 943) replaces position 577 (e.g., replaces T577), numbered relative to SEQ ID NO: 138.
  • [NO] is present at positions 571-573, numbered according to SEQ ID NO: 982.
  • [Nl] is present at positions 574-576, numbered according to SEQ ID NO: 982.
  • [N2] is present at positions 577-581, numbered according to SEQ ID NO: 982.
  • [N3] is present at positions 582-584, numbered according to SEQ ID NO: 982.
  • [N4] is present at positions 585-586, numbered according to SEQ ID NO: 982.
  • [N2]-[N3] is present at positions 577-584, numbered according to SEQ ID NO: 982.
  • [NO]-[N1]-[N2]-[N3]-[N4] is present at positions 571-586, numbered according to SEQ ID NO: 982.
  • the present disclosure provides an AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant comprises the formula [B]-[C], wherein: (i) [B] comprises positions XI, X2, and X3, wherein: (a) position XI is Q, T, S, A, I, L, or H; (b) position X2 is S, G, or A; and (c) position X3 is S, K, L, R, or A: and (ii) [C] comprises the amino acid sequence of YPAEVVQK (SEQ ID NO: 943); and optionally wherein the AAV capsid variant comprises an amino acid modification, e.g., a conservative substitution, of any of the aforesaid amino acids in (i) and/or (
  • [B] comprises Q at position XI. In some embodiments, [B] comprises S at position X2. In some embodiments, [B] comprises S at position X3. In some embodiments, the amino acid sequence of [B] is QSS. In some embodiments, [B] is present immediately subsequent to position 573, relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, [B] replaces positions 574-576 (e.g., Q574, S575, and S576), relative to a reference sequence numbered according to SEQ ID NO: 138.
  • positions 574-576 e.g., Q574, S575, and S576
  • [BJ is present immediately subsequent to position 573, and [B] replaces positions 574-576 (e.g., Q574, S575, and S576), relative to a reference sequence numbered according to SEQ ID NO: 138.
  • [C] is present immediately subsequent to position 576, relative to a reference sequence numbered according to SEQ ID NO: 138.
  • [C] replaces position 577 (e.g., T577), relative to a reference sequence numbered according to SEQ ID NO: 138.
  • [C] is present immediately subsequent to position 576, and [C] replaces position 577 (e.g., T577), relative to a reference sequence numbered according to SEQ ID NO: 138.
  • [B]-[C] is present immediately subsequent to position 573, relative to a reference sequence numbered according to SEQ ID NO: 138.
  • [B]-[C] replaces positions 574-577 (e.g., Q574, S575, S576, and T577), relative to a reference sequence numbered according to SEQ ID NO: 138.
  • [B]-[C] is present immediately subsequent to position 573, and [B]-[C] replaces positions 574-577 (e.g., Q574, S575, S576, and T577), relative to a reference sequence numbered according to SEQ ID NO: 138.
  • XI of [B] is present at position 574
  • X2 of [B] is present at position 575
  • X3 of [B] is present at position 576, numbered according to SEQ ID NO: 982.
  • [B] is present at positions 574-576, numbered according to SEQ ID NO: 982.
  • [C] is present at positions 577-584, numbered according to SEQ ID NO: 982.
  • [B]-[C] is present at positions 574-584, numbered according to SEQ ID NO: 982.
  • the present disclosure provides an AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant comprises one, two, three, or all of (i) an [A], wherein the amino acid sequence of [A] comprises TNN, TNT, INN, NNN, TNS, or TNK; (ii) a [B], wherein the amino acid sequence of [B] comprises QSS, TSL, SSS, QSR, QSK, AGA, IGS, QAS, ASS, LGS, or HSS; (iii) a [C], wherein [C] comprises the amino acid sequence of YPAEVVQK (SEQ ID NO: 943); and (iv) a [D], wherein [D] comprises the amino acid sequence of TA or PA; and optionally wherein the AAV capsid variant comprises one, two
  • the amino acid sequence of [A] is TNN. In some embodiments, the amino acid sequence of [B] is QSS. In some embodiments, the amino acid sequence of [A] is TNN and the amino acid sequence of [B] is QSS. In some embodiments, the amino acid sequence of [A] is TNN, the amino acid sequence of [B] is QSS, and the amino acid sequence of [C] is YPAEVVQK (SEQ ID NO: 943). In some embodiments, the amino acid sequence of [A] is TNN, the amino acid sequence of [B] is QSS, the amino acid sequence of [C] is YPAEVVQK (SEQ ID NO: 943), and the amino acid sequence of [D] is TA.
  • [A] is present immediately subsequent to position 570, numbered relative to SEQ ID NO: 138. In any of these embodiments, [A] replaces positions 571-573 (e.g., T571, N572, and N573) numbered relative to SEQ ID NO: 138. In any of these embodiments, [A] is present immediately subsequent to position 570, and [A] replaces positions 571-573 (e.g., T571, N572, and N573) numbered relative to SEQ ID NO: 138. In any of these embodiments, [B J is present immediately subsequent to position 573, relative to a reference sequence numbered according to SEQ ID NO: 138.
  • replaces positions 574-576 (e.g., Q574, S575, and S576), numbered relative to SEQ ID NO: 138.
  • [B] is present immediately subsequent to position 573, and [B] replaces positions 574-576 (e.g., Q574, S575, and S576), numbered relative to SEQ ID NO: 138.
  • [C] is present immediately subsequent to position 576, numbered relative to SEQ ID NO: 138.
  • [C] replaces position 577 (e.g., T577), numbered relative to SEQ ID NO: 138.
  • [C] is present immediately subsequent to position 576, and [C] replaces position 577 (e.g., T577), numbered relative to SEQ ID NO: 138.
  • [B]-[C] is present immediately subsequent to position 573, numbered relative to SEQ ID NO: 138.
  • [B]-[C] replaces positions 574-577 (e.g., Q574, S575, S576, and T577), numbered relative to SEQ ID NO: 138.
  • [B]-[C] is present immediately subsequent to position 573, and [B]-[C] replaces positions 574-577 (e.g., Q574, S575, S576, and T577), numbered relative to SEQ ID NO: 138.
  • [C]-[D] is present immediately subsequent to position 576, numbered relative to SEQ ID NO: 138.
  • [C]-[D] replaces positions 577-579 (e.g., T577, T578, and A579), numbered relative to SEQ ID NO: 138.
  • [A]-[B]-[C]-[D] is present immediately subsequent to position 570, numbered relative to SEQ ID NO: 138.
  • [A]-[B]-[C]-[D] replaces positions 571-579 (e.g., T571, N572, N573, Q574, S575, S576, T577, T578, and A579), numbered relative to SEQ ID NO: 138.
  • [A]-[B]-[C]-[D] is present immediately subsequent to position 570, and [A]-[B]- [C]-[D] replaces positions 571-579 (e.g., T571, N572, N573, Q574, S575, S576, T577, T578, and A579), numbered relative to SEQ ID NO: 138.
  • positions 571-579 e.g., T571, N572, N573, Q574, S575, S576, T577, T578, and A579
  • [A]- [B] - [C] - [D] is TNNQSSYPAEVVQKTA (SEQ ID NO: 1533) and is present immediately subsequent to position 570, numbered relative to SEQ ID NO: 138, wherein [C] (YPAEVVQK; SEQ ID NO: 943) replaces position 577 (e.g., replaces T577) numbered relative to SEQ ID NO: 138.
  • [A] is present at positions 571-573, numbered according to SEQ ID NO: 982.
  • [B] is present at positions 574-576, numbered according to SEQ ID NO: 982.
  • [C] is present at positions 577-584, numbered according to SEQ ID NO: 982.
  • [D] is present at positions 585-586, numbered according to SEQ ID NO: 982.
  • [A]-[B]-[C]-[D] is present at positions 571-586, numbered according to SEQ ID NO: 982.
  • the present disclosure provides an AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant comprises the amino acid Y at position 577, and comprises the amino acid sequence of PAEVVQK (SEQ ID NO: 20), which is present immediately subsequent to position 577, numbered relative to SEQ ID NO: 982.
  • SSN survival motor neuron
  • the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 739, or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto. In some embodiments, the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 738, or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto. In some embodiments, the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 982, or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.
  • the present disclosure provides an AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant comprises the amino acid Y at position 577 and the amino acid sequence of PAEVVQK (SEQ ID NO: 20) at positions 578-584, numbered relative to SEQ ID NO: 982.
  • the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 739, or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.
  • the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 738, or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto. In some embodiments, the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 982, or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.
  • the present disclosure provides a method of making an isolated, e.g., recombinant AAV particle.
  • the method comprising providing a host cell comprising an AAV viral genome described herein and incubating the host cell under conditions suitable to enclose the viral genome in the AAV particle, e.g., an AAV capsid variant described herein, thereby making The AAV particle.
  • the present disclosure provides a method of delivering an exogenous SMN protein, e.g., an SMN1 protein, to a cell or tissue (e.g., a CNS cell or a CNS tissue).
  • the method comprising administering an effective amount of an AAV particle comprising an AAV capsid or an AAV capsid variant, e.g., an AAV capsid variant described herein.
  • the present disclosure provides method of delivering an exogenous SMN protein, e.g., an SMN1 protein, to a subject.
  • the method comprising administering an effective amount of an AAV particle or a plurality of AAV particles, described herein, said AAV particle comprising an AAV viral genome described herein, e.g., a viral genome comprising a nucleic acid comprising a transgene encoding an SMN protein, e.g., an SMN1 protein, described herein.
  • AAV particle comprising an AAV viral genome described herein, e.g., a viral genome comprising a nucleic acid comprising a transgene encoding an SMN protein, e.g., an SMN1 protein, described herein.
  • the present disclosure provides a method of treating a subject having or being diagnosed as having disease and/or a disorder associated with decreased SMN protein expression, e.g., a mutation in an SMN1 gene.
  • the method comprising administering to the subject an effective amount of an AAV particle or a plurality of AAV particles, described herein, comprising an AAV viral genome described herein.
  • the disease and/or disorder associated with decreased expression of the SMN protein is Spinal Muscular Atrophy (SMA), an SMA-related disorder, Werdnig- Hoffman disease, Dubowitz disease, or Kugelberg-Welander disease.
  • An AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant comprises an amino acid sequence having the following formula: [N2]-[N3], wherein:
  • SNS survival motor neuron
  • [N2] comprises positions XI, X2, X3, X4, and X5, wherein:
  • position X2 is P, K, T, or Q;
  • (ii) [N3] comprises the amino acid sequence of VQK, EQK, VKK, VHK, VQQ, or LQK; or wherein the AAV capsid variant comprises an amino acid modification, e.g., a conservative substitution, of any of the aforesaid amino acids in (i) and/or (ii).
  • An AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant comprises one, two, three, four, or all of:
  • SNS survival motor neuron
  • an [NO] comprising TNN, TNT, INN, TNS, NNN, or TNK;
  • an [Nl] comprising QSS, QSK, TSL, SSS, QSR, AGA, IGS, QAS, ASS, LGS, QST, HSS, LSS, or QRS;
  • an [N2] comprising YPAEV (SEQ ID NO: 39), YPPSL (SEQ ID NO: 40), NKAEV (SEQ ID NO: 41), YTAEV (SEQ ID NO: 42), YPAEE (SEQ ID NO: 43), YQAEV (SEQ ID NO: 44), YTPSL (SEQ ID NO: 45), YPAAV (SEQ ID NO: 46), NPAEV (SEQ ID NO: 47), CPAEV (SEQ ID NO: 48), or YQAEE (SEQ ID NO: 49);
  • an [N4] comprising TA, PA, or NA; and optionally wherein the AAV capsid variant comprises an amino acid modification, e.g., a conservative substitution, of any of the aforesaid amino acids in (i)-(v).
  • AAV particle of any one of embodiments 1, or 3, wherein [N2] comprises YP, NK, YT, YQ, NP, CP, TH, AE, PS, AA, AS, PA, PP, KA, TA, QA, TP, HA, EV, SL, EE, AV, or SH.
  • AAV particle of any one of embodiments 1, or 3-5, wherein [N2] comprises YPAE (SEQ ID NO: 286), YPPS (SEQ ID NO: 287), NKAE (SEQ ID NO: 288), YTAE (SEQ ID NO: 289), YQAE (SEQ ID NO: 290), YTPS (SEQ ID NO: 291), YPAA (SEQ ID NO: 292), NPAE (SEQ ID NO: 293), CPAE (SEQ ID NO: 294), THAS(SEQ ID NO: 295) , PAEV (SEQ ID NO: 296), PPSL (SEQ ID NO: 297), KAEV (SEQ ID NO: 298), TAEV (SEQ ID NO: 299), PAEE (SEQ ID NO: 300), QAEV (SEQ ID NO: 301), TPSL (SEQ ID NO: 302), PAAV (SEQ ID NO: 303), or QAEE (SEQ ID NO: 304).
  • AAV particle of any one of embodiments 1-6, wherein [N2] is or comprises YPAEV (SEQ ID NO: 39), YPPSL (SEQ ID NO: 40), NKAEV (SEQ ID NO: 41), YTAEV (SEQ ID NO: 42), YPAEE (SEQ ID NO: 43), YQAEV (SEQ ID NO: 44), YTPSL (SEQ ID NO: 45), YPAAV (SEQ ID NO: 46), NPAEV (SEQ ID NO: 47), CPAEV (SEQ ID NO: 48), or YQAEE(SEQ ID NO: 49).
  • [N3] comprises the amino acid sequence of VQK, EQK, or VKK.
  • [N2] is or comprises the amino acid sequence of YTPSL (SEQ ID NO: 45) and [N3] is or comprises the amino acid sequence of VQK;
  • [N2] is or comprises the amino acid sequence of YPPSL (SEQ ID NO: 40) and [N3] is or comprises the amino acid sequence of VQK;
  • [N2] is or comprises the amino acid sequence of YPPSL (SEQ ID NO: 40) and [N3] is or comprises the amino acid sequence of EQK; or
  • [N2] is or comprises the amino acid sequence of YPPSL (SEQ ID NO: 40) and [N3] is or comprises the amino acid sequence of VKK.
  • AEVVQK (SEQ ID NO: 50), PSLVQK (SEQ ID NO: 51), AEVEQK (SEQ ID NO: 52), AEEVQK (SEQ ID NO: 53), PSLEQK (SEQ ID NO: 54), PSLVKK (SEQ ID NO: 55), AEVVKK (SEQ ID NO: 56), AEVVHK (SEQ ID NO: 57), AAVVQK (SEQ ID NO: 58), AEVVQQ (SEQ ID NO: 59), or AEVLQK (SEQ ID NO: 60);
  • amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, or 5 amino acids, e.g., consecutive amino acids, thereof;
  • amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or
  • AAV particle of any one of embodiments 1 or 3-14, wherein [N2]-[N3] comprises: (i) PAEVVQK (SEQ ID NO: 61) , PPSLVQK (SEQ ID NO: 62), KAEVVQK (SEQ ID NO: 63), TAEVVQK (SEQ ID NO: 64), PAEVEQK (SEQ ID NO: 65), PAEEVQK (SEQ ID NO: 66), QAEVVQK (SEQ ID NO: 67), TPSLVQK (SEQ ID NO: 68), PPSLEQK (SEQ ID NO: 69), PPSLVKK (SEQ ID NO: 70), PAEVVKK (SEQ ID NO: 71), PAEVVHK (SEQ ID NO: 72), PAAVVQK (SEQ ID NO; 73), PAEVVQQ (SEQ ID NO: 74), TAEVVKK (SEQ ID NO: 75), PAEVLQK (SEQ ID NO: 76), or QAE
  • amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, or 6 amino acids, e.g., consecutive amino acids, thereof;
  • amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or
  • amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
  • YPAEVVQK (SEQ ID NO: 943), YPPSLVQK (SEQ ID NO: 946), NKAEVVQK, YTAEVVQK (SEQ ID NO: 948), YPAEVEQK (SEQ ID NO: 949), YPAEEVQK(SEQ ID NO: 950), YQAEVVQK (SEQ ID NO: 951), YTPSLVQK (SEQ ID NO: 952), YPPSLEQK (SEQ ID NO: 953), YPPSLVKK (SEQ ID NO: 954), YPAEVVKK (SEQ ID NO: 955), YPAEVVHK (SEQ ID NO: 956), YPAAVVQK (SEQ ID NO: 957), NPAEVVQK (SEQ ID NO: 958), YPAEVVQQ (SEQ ID NO: 959), CPAEVVQK (SEQ ID NO: 960), YTAEVVKK (SEQ ID NO:
  • amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, 6, or 7 amino acids, e.g., consecutive amino acids, thereof;
  • amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or
  • amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
  • the AAV capsid variant further comprises:
  • position XE is S, G, A, or R
  • position XF is S, K, L, R, A, or T
  • amino acid modification e.g., a conservative substitution, of any of the aforesaid amino acids in (a)-(c).
  • AAV particle of any one of embodiments 2, 19, or 20, wherein [Nl] is or comprises QSS, QSK, TSL, SSS, QSR, AGA, IGS, QAS, ASS, LGS, QST, HSS, LSS, or QRS.
  • SSYPA SEQ ID NO: 78
  • SKYPA SEQ ID NO: 79
  • SLYPA SEQ ID NO: 80
  • SRYPA SEQ ID NO: 81
  • SSYPP SEQ ID NO: 82
  • GAYPA SEQ ID NO: 83
  • GSYPA SEQ ID NO: 84
  • ASYPA SEQ ID NO: 85
  • STNKA SEQ ID NO: 86
  • SSYTA SEQ ID NO: 87
  • SSYQA SEQ ID NO: 88
  • SSYTP SEQ ID NO: 89
  • SSNPA SEQ ID NO: 90
  • SLCPA SEQ ID NO: 91
  • RSYTA SEQ ID NO: 92
  • SSTHA SEQ ID NO: 93
  • amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, or 4 amino acids, e.g., consecutive amino acids, thereof;
  • amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or
  • amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
  • amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, or 5 amino acids, e.g., consecutive amino acids, thereof;
  • amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or
  • amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
  • amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, 6, or 7 amino acids, e.g., consecutive amino acids, thereof;
  • amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or
  • amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
  • AAV particle of any one of embodiments 19-24, wherein [N1]-[N2]-[N3] comprises:
  • amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, 6, 7, or 8 amino acids, e.g., consecutive amino acids, thereof;
  • amino acid sequences in (i) comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
  • QSSYPAEVVQK (SEQ ID NO: 176), QSKYPAEVVQK (SEQ ID NO: 177), TSLYPAEVVQK (SEQ ID NO: 178), SSSYPAEVVQK (SEQ ID NO: 179), QSRYPAEVVQK (SEQ ID NO: 180), QSSYPPSLVQK (SEQ ID NO: 181), AGAYPAEVVQK (SEQ ID NO: 182), IGSYPAEVVQK (SEQ ID NO: 183), QASYPAEVVQK (SEQ ID NO: 184), ASSYPAEVVQK (SEQ ID NO: 186), LGSYPAEVVQK (SEQ ID NO: 187), QSTNKAEVVQK (SEQ ID NO: 188), HSSYPAEVVQK (SEQ ID NO: 189), SSSYTAEVVQK (SEQ ID NO: 190), QSKYPAEVEQK (SEQ ID NO: 191),
  • amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, e.g., consecutive amino acids, thereof;
  • amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or
  • amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
  • (c) position XC is N, T, S, or K; and optionally, wherein the AAV capsid variant comprises an amino acid modification, e.g., a conservative substitution, of any of the aforesaid amino acids in (a)-(c).
  • AAV particle of embodiment 27, wherein [NO] comprises TN, IN, NN, NT, NS, or NK. 29.
  • TNNQSS (SEQ ID NO; 210), TNNQSK (SEQ ID NO: 211), TNNTSL (SEQ ID NO: 212), TNNSSS (SEQ ID NO: 213), TNNQSR (SEQ ID NO: 214), TNNAGA (SEQ ID NO: 215), TNNIGS (SEQ ID NO: 216), TNNQAS (SEQ ID NO: 217), TNTASS (SEQ ID NO: 218), TNNLGS (SEQ ID NO: 219), TNNQST (SEQ ID NO: 220), TNNHSS (SEQ ID NO: 221), TNNLSS (SEQ ID NO: 223), INNQSS (SEQ ID NO: 224), TNSQSS (SEQ ID NO: 225), NNNQSR (SEQ ID NO: 226), TNSTSL (SEQ ID NO: 227), TNNQRS (SEQ ID NO: 228), or TNKQAS (SEQ ID NO: 229);
  • amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, or 5 amino acids, e.g., consecutive amino acids, thereof;
  • amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or
  • amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
  • TNNQSSYPAEVVQK (SEQ ID NO: 230), TNNQSKYPAEVVQK (SEQ ID NO: 231), TNNTSL YPAEVVQK (SEQ ID NO: 232), TNNSSSYPAEVVQK (SEQ ID NO: 233), TNNQSRYPAEVVQK (SEQ ID NO: 234), TNNQSSYPPSLVQK (SEQ ID NO: 235), TNNAGA YPAEVVQK (SEQ ID NO: 236), TNNIGSYPAEVVQK (SEQ ID NO: 237), TNNQAS YPAEVVQK (SEQ ID NO: 238), TNTASS YPAEVVQK (SEQ ID NO: 239), TNNLGSYPAEVVQK (SEQ ID NO: 240), TNNQSTNKAEVVQK (SEQ ID NO: 241), TNNHSSYPAEVVQK (SEQ ID NO: 242),
  • amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acids, e.g., consecutive amino acids, thereof;
  • amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or
  • amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
  • AAV particle of embodiment 2 or 27-31, wherein [NO]-[N1]-[N2]-[N3] is or comprises TNNAGAYPAEVVQK (SEQ ID NO: 236), TNNTSLYPAEVVQK (SEQ ID NO: 232), TNNQSKYPAEVVQK (SEQ ID NO: 2 1), TNNQSSYTPSLVQK (SEQ ID NO: 247), TNNQSSYPPSLVQK (SEQ ID NO: 235), TNNQSRYPAEVVQK (SEQ ID NO: 234), TNNQSSYPPSLEQK (SEQ ID NO: 249), TNNQSSYPPSLVKK (SEQ ID NO: 250), or INNQSSYPAEVVQK (SEQ ID NO: 254).
  • (b) position XH is A; and optionally wherein the AAV capsid variant comprises an amino acid modification, e.g., a conservative substitution, of any of the aforesaid amino acids in (a) or (b).
  • an amino acid modification e.g., a conservative substitution, of any of the aforesaid amino acids in (a) or (b).
  • VQKTA (SEQ ID NO: 268), EQKTA (SEQ ID NO: 269), VKKTA (SEQ ID NO: 270), VQKPA (SEQ ID NO: 271), VHKTA (SEQ ID NO: 272), VQQTA (SEQ ID NO: 273), VQKNA (SEQ ID NO: 274), or LQKTA (SEQ ID NO: 275);
  • amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, or 4 amino acids, e.g., consecutive amino acids, thereof; (iii) an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or
  • amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
  • TNNQSSYPAEVVQKTA (SEQ ID NO: 1533), TNNQSKYPAEVVQKTA (SEQ ID NO: 1538), TNNTSLYPAEVVQKTA (SEQ ID NO: 1232), TNNSSSYPAEVVQKTA (SEQ ID NO: 1539), TNNQSRYPAEVVQKTA(SEQ ID NO: 1327), TNNQSSYPPSLVQKTA (SEQ ID NO: 1300), TNNAGAYPAEVVQKTA (SEQ ID NO: 1021), TNNIGSYPAEVVQKTA (SEQ ID NO: 1112), TNNQASYPAEVVQKTA (SEQ ID NO: 1586), TNTASSYPAEVVQKTA (SEQ ID NO: 1575), TNNLGSYPAEVVQKTA (SEQ ID NO: 1027), TNNQSTNKAEVVQKTA (SEQ ID NO: 1578), TNNHSSYPAEVVQKTA (SEQ ID NO
  • amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids, e.g., consecutive amino acids, thereof;
  • amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or
  • an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i). 38.
  • AAV particle of any one of embodiments 2 or 34-37, wherein [NO]-[N1]-[N2]-[N3]-[N4] is or comprises TNNAGAYPAEVVQKTA (SEQ ID NO: 1021), TNNTSLYPAEVVQKTA (SEQ ID NO: 1232), TNNQSKYPAEVVQKTA (SEQ ID NO: 1538), TNNQSSYTPSLVQKTA (SEQ ID NO: 1585), TNNQSSYPPSLVQKTA (SEQ ID NO: 1300), TNNQSRYPAEVVQKTA (SEQ ID NO: 1327), TNNQSSYPPSLEQKTA (SEQ ID NO: 1590), TNNQSSYPPSLVKKTA (SEQ ID NO: 1591), or INNQSSYPAEVVQKTA(SEQ ID NO: 1024) .
  • An AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant comprises an amino sequence comprising the following formula: [B]-[C], wherein
  • SNS survival motor neuron
  • [A] comprises the amino acid sequence of TNN, TNT, INN, NNN, TNS, or TNK;
  • [B] comprises the amino acid sequence of QSS, TSL, SSS, QSR, QSK, AGA, IGS, QAS, ASS, LGS, or HSS;
  • SSYPAEVVQK (SEQ ID NO: 276), SKYPAEVVQK (SEQ ID NO: 277), SLYPAEVVQK (SEQ ID NO: 278), SRYPAEVVQK (SEQ ID NO: 279), GAYPAEVVQK (SEQ ID NO: 280), GSYPAEVVQK (SEQ ID NO: 281), or ASYPAEVVQK (SEQ ID NO: 282);
  • amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, 6, 7, 8, or 9 amino acids, e.g., consecutive amino acids, thereof;
  • amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
  • AAV particle of any one of embodiments embodiment 40-46, wherein [B]-[C] is or comprises AGAYPAEVVQK (SEQ ID NO: 182), TSLYPAEVVQK (SEQ ID NO: 178), QSKYPAEVVQK (SEQ ID NO: 177), or QSRYPAEVVQK (SEQ ID NO: 180).
  • an amino acid other than T at position 571 e.g., I or N
  • an amino acid other than N at position 573 e.g., T, S, or K
  • amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or
  • an amino acid other than T at position 578 e.g., P or N
  • an amino acid other than T at position 578 e.g., P or N
  • an amino acid other than T at position 585 e.g., P or N
  • an amino acid other than T at position 585 e.g., P or N
  • amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
  • TNNQSSYPAEVVQKTA (SEQ ID NO: 1533), TNNQSKYPAEVVQKTA (SEQ ID NO: 1538), TNNTSLYPAEVVQKTA (SEQ ID NO: 1232), TNNSSS YPAEVVQKTA (SEQ ID NO: 1539), TNNQSRYPAEVVQKTA (SEQ ID NO: 1327), TNNAGAYPAEVVQKTA (SEQ ID NO: 1021), TNNIGSYPAEVVQKTA (SEQ ID NO: 1112), TNNQ AS YPAEVVQKTA (SEQ ID NO: 1586), TNTASSYPAEVVQKTA (SEQ ID NO: 1575), TNNLGSYPAEVVQKTA (SEQ ID NO: 1027), TNNHSS YPAEVVQKTA (SEQ ID NO: 1310), TNNSSS YPAEVVQKPA (SEQ ID NO: 1142), INNQSSYPAEVVQK
  • amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids, e.g., consecutive amino acids, thereof;
  • amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or
  • TNNAGAYPAEVVQKTA SEQ ID NO: 1021
  • TNNTSLYPAEVVQKTA SEQ ID NO: 1232
  • TNNQSKYPAEVVQKTA SEQ ID NO: 1538
  • TNNQSRYPAEVVQKTA SEQ ID NO: 1327
  • INNQSSYPAEVVQKTA SEQ ID NO: 1024
  • loop VIII comprises positions 571-592 (e.g., amino acids TNNQSSTTAPATGTYNLQEIVP (SEQ ID NO: 285) numbered according to SEQ ID NO: 138, or positions 571-599, numbered according to SEQ ID NO: 982.
  • loop VIH comprises positions 571-592 (e.g., amino acids TNNQSSTTAPATGTYNLQEIVP (SEQ ID NO: 285) numbered according to SEQ ID NO: 138, or positions 571-599, numbered according to SEQ ID NO: 982.
  • loop VIII comprises positions 571-592 (e.g., amino acids TNNQSSTTAPATGTYNLQEIVP (SEQ ID NO: 285) numbered according to SEQ ID NO: 138, or positions 571-599, numbered according to SEQ ID NO: 982.
  • AAV particle of any one of embodiments 1-73 which comprises a Y at position 577, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or 982.
  • 75 The AAV particle of any one of embodiments 1-39, 58, 59, or 66-74, wherein XI of [N2] is present at position 577 (e.g., T577), and positions X2-X5 of [N2] are present immediately subsequent to position 577, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • AAV particle of any one of embodiments 1-39, 58, 59, or 66-75 wherein XI of [N2] corresponds to position 577 (e.g., Y577), positions X2 corresponds to position 578 (e.g., P588), X3 of [N2] corresponds to position 579 (e.g., A579), X4 of [N2] corresponds to position 580 (e.g., E580), and X5 of [N2] corresponds to position 581 (e.g., V581) of SEQ ID NO: 982.
  • XI of [N2] corresponds to position 577 (e.g., Y577)
  • positions X2 corresponds to position 578 (e.g., P588)
  • X3 of [N2] corresponds to position 579 (e.g., A579)
  • X4 of [N2] corresponds to position 580 (e.g., E580)
  • [N2]-[N3]-[N4] corresponds to positions 577-586 (e.g., Y577, P578, A579, E580, V581, V582, Q583, K584, T585, A586) of SEQ ID NO: 982.
  • [N1]-[N2]-[N3]- [N4] replaces positions 574-579 (e.g., Q574, S575, S576, T577, T578, and A579), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • positions 574-579 e.g., Q574, S575, S576, T577, T578, and A579
  • [N1]-[N2]-[N3]- [N4] corresponds to positions 574-586 (e.g., Q574, S575, S576, Y577, P578, A579, E58O, V581, V582, Q583, K584, T585, A586) of SEQ ID NO: 982.
  • positions 571-579 e.g., T571, N572, N573, Q574, S575, S576, T577, T578, and A579
  • AAV particle of any one of embodiments 2, 27-39, 58, 59, or 66-114 which comprises from N- terminus to C-terminus [NO]-[N1]-[N2]-[N3].
  • 116. The AAV particle of any one of embodiments 2, 34-39, 58, 59, or 66-115, which comprises from N- terminus to C-terminus [N1]-[N2]-[N3]-[N4].
  • AAV particle of any one of embodiments 2, 34-39, 58, 59, or 66-116 which comprises from N- terminus to C-terminus [NO]-[N1]-[N2]-[N3]-[N4],
  • loop VUI comprises positions 571-592 (e.g., amino acids TNNQSSTTAPATGTYNLQEIVP (SEQ ID NO: 285) numbered according to SEQ ID NO: 138, or positions 571-599, numbered according to SEQ ID NO: 982.
  • loop VIII comprises positions 571-592 (e.g., amino acids TNNQSSTTAPATGTYNLQEIVP (SEQ ID NO: 285)) numbered according to SEQ ID NO: 138, or positions 571-599, numbered according to SEQ ID NO: 982.
  • loop VUI comprises positions 571-592 (e.g., amino acids TNNQSSTTAPATGTYNLQEIVP (SEQ ID NO: 285) numbered according to SEQ ID NO: 138, or positions 571-599, numbered according to SEQ ID NO: 982.
  • 125 The AAV particle of any one of embodiments 40-65 or 118-124, wherein [B] replaces positions 574-576 (e.g., Q574, S575, and S576), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 982.
  • [B]-[C] corresponds to positions 574-584 (e.g., Q574, S575, S576, Y577, P578, A579, E580, V581, V582, Q583, K584) of SEQ ID NO: 982.
  • [B]-[C]-[D] replaces positions 574-579 (e.g., Q574, S575, S576, T577, T578, and A579), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. 134.
  • [B]-[C]-[D] replaces positions 574-579 (e.g., Q574, S575, S576, T577, T578, and A579), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • [B]-[C]-[D] corresponds to positions 574-586 (e.g., Q574, S575, S576, Y577, P578, A579, E580, V581, V582, Q583, K584, T585, A586) of SEQ ID NO: 982.
  • positions 571-579 e.g., T571, N572, N573, Q574, S575, S576, T577, T578, and A579
  • positions 571-586 e.g., T571, N572, N573, Q574, S575, S576, Y577, P578, A579, E58O, V581, V582, Q583, K584, T585, A586
  • [A] is present at positions 571-573, numbered according to SEQ ID NO: 982;
  • AAV particle of any one of embodiments 41, 51-65, or 118-158 which comprises from N- terminus to C-terminus [A]-[B]-[C].
  • AAV particle of any one of embodiments 41, 51-65, or 118-159 which comprises from N- terminus to C-terminus [B]-[C]-[D].
  • An AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant comprises:
  • an amino sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of any one of SEQ ID NOs: 943, 1021, 1024, 1027, 1112, 1142, 1214, 1232, 1254, 1300, 1310, 1327, 1331, 1342, 1419, 1453, 1533, 1538, 1539, 1575, 1578, 1583-1587, 1590, 1591-1593, 1598-1608, or 1610-1624.
  • An AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant comprises:
  • amino acid sequence comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive amino acids from any one of SEQ ID NOs: 1021 , 1024, 1232, 1300, 1327, 1533, 1538, 1585, 1590, or 1591;
  • amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to the amino acid sequence of any one of SEQ ID NOs: 1021, 1024, 1232, 1300, 1327, 1533, 1538, 1585, 1590, or 1591; or
  • AAV particle of any one of embodiments 162-164 which comprises at least 3, 4, 5, 6, or 7 consecutive amino acids from of any one of SEQ ID NOs: 943 or 946-966.
  • AAV particle of embodiment 162-169, wherein the 7 consecutive amino acids comprise YPAEVVQ (SEQ ID NO: 152).
  • AAV particle of embodiment 162-170, wherein the amino acid sequence comprises YPAEVVQK (SEQ ID NO: 943).
  • the 5 consecutive amino acids comprise YTPSL (SEQ ID NO: 145) ;
  • the 7 consecutive amino acids comprise YTPSLVQ (SEQ ID NO: 154); and/or
  • the 6 consecutive amino acids comprise YPPSLV (SEQ ID NO: 155);
  • the 7 consecutive amino acids comprise YPPSLEQ (SEQ ID NO: 158); and/or
  • amino acid sequence comprises YPPSLVKK (SEQ ID NO: 954).
  • substitutions e.g., conservative substitutions
  • substitutions e.g., conservative substitutions
  • insertions e.g., or deletions
  • nucleotide sequence comprising at least one, two, three, four, five, six, or seven, but no more than ten different nucleotides relative to the nucleotide sequence of SEQ ID NO: 944.
  • AAV particle of any one of embodiments 162-171, 176, 179 or 182, wherein the nucleotide sequence encoding the amino acid sequence comprises:
  • nucleotide sequence comprising at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to the nucleotide sequence of SEQ ID NO: 944; or
  • nucleotide sequence comprising at least one, two, three, four, five, six, or seven, but no more than ten different nucleotides relative to the nucleotide sequence of SEQ ID NO: 944.
  • amino acid sequence replaces one, two, three, four, five or all of positions 571, 572, 573, 574, 575, and/or 576 (e.g., positions T571, N572, N573, Q574, S575, S576, T577, T578, and/or A579), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • AAV particle of any one of embodiments 162-187 which comprises an amino acid residue other than T at position 577, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • AAV particle of any one of embodiments 162-188, wherein the AAV capsid variant comprises the amino acid Y at position 577, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • an amino acid other than Q at position 574 (e.g., A or T)
  • S at position 575 e.g., G
  • S e.g., A, L, K, or R
  • AAV capsid variant comprises Y at position 577 and the amino acid sequence PPSLEQK (SEQ ID NO: 69), which is present immediately subsequent to position 577, all numbered according to SEQ ID NO: 138 or 982.
  • a modification e.g., an insertion, substitution (e.g., conservative substitution), and/or deletion, in loop I, II, IV, and/or VI.
  • the AAV particle of any one of the preceding embodiments, wherein the nucleotide sequence encoding the AAV capsid variant comprises the nucleotide sequence of SEQ ID NO: 137, or a sequence with at least 80% (e.g., at least about 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto. 218.
  • the AAV particle of any one of the preceding embodiments, wherein the AAV capsid variant comprises a VP1 protein, a VP2 protein, a VP3 protein, or a combination thereof.
  • AAV capsid variant of any one of embodiments 162-171, 176, 179, 182, 183, 184-196 or 211- 218, comprising an amino acid sequence comprising at least 3, 4, 5, or 6 consecutive amino acids from the amino acid sequence of YPAEVVQK (SEQ ID NO: 943), wherein:
  • the 6 consecutive amino acids comprise YPAEVV (SEQ ID NO: 151);
  • the 7 consecutive amino acids comprise YPAEVVQ (SEQ ID NO: 152); wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 739, or an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 739.
  • the 6 consecutive amino acids comprise YPAEVV (SEQ ID NO: 151);
  • a VP1 protein comprising the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 982;
  • a VP2 protein comprising the amino acid sequence of positions 137-724 of SEQ ID NO: 138 or positions 137-731 of SEQ ID NO: 982;
  • the AAV particle of any one of embodiments 1-171, 176, 179, 182, 183, 184-196, 211-219, or 224-
  • the AAV capsid variant comprises an amino acid sequence comprising at least one, two or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but not more than 30, 20 or 10 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SEQ ID NO: 982.
  • An AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 739, and wherein the AAV capsid variant comprises the amino acid sequence of YPAEVVQK (SEQ ID NO: 943).
  • SEQ ID NO: 739 e.g., human SMN
  • a brain region e.g., a temporal cortex, perirhinal cortex,
  • a non-human primate and rodent e.g., rat and/or mouse
  • the AAV particle of any one of embodiments 1-243, wherein the AAV capsid variant is enriched at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, 30, 35, 40, 45-fold, in the brain of at least two to three species, e.g., a non-human primate and rodent (e.g., rat and/or mouse), compared to a reference sequence of SEQ ID NO: 138, e.g., when measured by an assay as described in Examples 4-7.
  • a non-human primate and rodent e.g., rat and/or mouse
  • the AAV particle of embodiment 243 or 244, wherein the at least two to three species are Macaca fascicularis , Chlorocebus sabaeus, Callithrixjacchus, rat, and/or mouse (e.g., BALB/c mice).
  • the AAV particle of any one of embodiments 1-245, wherein the AAV capsid variant is enriched at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, or 225-fold, in the brain compared to a reference sequence of SEQ ID NO: 982, e.g., when measured by an assay as described in Example 6.
  • an assay e.g., a qRT-PCR or a qPCR assay (e.g., as described in Example 5).
  • an assay e.g., a qRT- PCR or a qPCR assay (e.g., as described in Example 5).
  • AAV particle of any one of embodiments 1-258, wherein the encoded SMN protein comprises an amino acid sequence having at least one, two, three or four, but no more than 30, 20 or 10 different amino acids relative to SEQ ID NO: 2000.
  • the AAV particle of any one of embodiments 1-261, wherein the nucleotide sequence encoding the SMN protein comprises:
  • nucleotide sequence comprising at least one, two, three, or four but no more than 30, 20, or 10 different nucleotides relative to any one of SEQ ID NOs: 6-8;
  • nucleotide sequence encoding the SMN protein comprises SEQ ID NO: 8, or a nucleotide sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto.
  • the AAV particle of any one of embodiments 1-265, wherein the nucleotide sequence encoding the SMN protein comprises SEQ ID NO: 8.
  • the AAV particle of any one of embodiments 1-262, wherein the nucleotide sequence encoding the SMN protein comprises the nucleotide sequence of any one of SEQ ID NOs: 2001-2004, or a nucleotide sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto. 269.
  • nucleotide sequence encoding the SMN protein comprises the nucleotide sequence of SEQ ID NO: 9, or a nucleotide sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto.
  • the encoded SMN protein comprises the amino acid sequence of SEQ ID NO: 2005 or an amino acid sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto; and/or
  • the nucleotide sequence encoding the SMN protein comprises SEQ ID NO: 2006, or a nucleotide sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto.
  • nucleic acid encoding the SMN protein further comprises a nucleotide sequence encoding a splicing modulator element.
  • the AAV particle of embodiment 278 or 279, wherein the UlsnRNA comprises one or more of SEQ ID NOs: 2014-2019.
  • the AAV particle of any one of the preceding embodiments which comprises a viral genome comprising a promoter operably linked to the nucleic acid sequence encoding the SMN protein.
  • the promoter comprises the nucleotide sequence of any one of SEQ ID NOs: 26-35, a nucleotide sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions, relative to the nucleotide sequence of any one of SEQ ID NOs: 26-35, or a nucleotide sequence with at least 80% (e
  • the AAV particle of embodiment 295, wherein the intron is an SV40 intron or a variant thereof.
  • the ITR positioned 3’ relative to the nucleic acid comprising the transgene encoding the SMN protein comprises the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical thereto.
  • the AAV particle of embodiment 313 or 314, wherein the encoded miR binding site modulates, e.g., reduces, expression of the encoded antibody molecule in a cell or tissue of the DRG, liver, heart, hematopoietic lineage, or a combination thereof.
  • the AAV particle of embodiment 317 wherein the at least 3 copies of the encoded miR binding sites are continuous (e.g., not separated by a spacer), or are separated by a spacer, optionally wherein the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions, but no more than four modifications, e.g., substitutions, relative to GATAGTTA.
  • the encoded miR122 binding site comprises the nucleotide sequence of SEQ ID NO: 1865, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions relative to SEQ ID NO: 1865;
  • the encoded miR183 binding site comprises the nucleotide sequence of SEQ ID NO: 1847, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 1847;
  • the encoded miR-1 binding site comprises the nucleotide sequence of SEQ ID NO: 4679, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 4679; and/or
  • the encoded miR-142-3p binding site comprises the nucleotide sequence of SEQ ID NO: 1869, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 1869.
  • AAV particle of any one of embodiments 282-324, wherein the viral genome comprises:
  • (A) (i) a first encoded miR122 binding site comprising the nucleotide sequence of SEQ ID NO: 1865, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 1865;
  • a first spacer comprising the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions, but no more than four modifications, e.g., substitutions, relative to GATAGTTA;
  • a second encoded miR122 binding site comprising the nucleotide sequence of SEQ ID NO: 1865, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 1865; or
  • a first encoded miR122 binding site comprising the nucleotide sequence of SEQ ID NO: 1865, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 1865;
  • a first spacer comprising the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions, but no more than four modifications, e.g., substitutions, relative to GATAGTTA;
  • a second encoded miR122 binding site comprising the nucleotide sequence of SEQ ID NO: 1865, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 1865;
  • a second spacer comprising the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions, but no more than four modifications, e.g., substitutions, relative to GATAGTTA;
  • (A) (i) a first encoded miR183 binding site comprising the nucleotide sequence of SEQ ID NO: 1847, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, live, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 1847;
  • a first spacer comprising the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions, but no more than four modifications, e.g., substitutions, relative to GATAGTTA;
  • a second encoded miR183 binding site comprising the nucleotide sequence of SEQ ID NO: 1847, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 1847; or
  • a first encoded miR183 binding site comprising the nucleotide sequence of SEQ ID NO: 1847, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 1847;
  • a second spacer comprising the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions, but no more than four modifications, e.g., substitutions, relative to GATAGTTA;
  • a third encoded miR183 binding site comprising the nucleotide sequence of SEQ ID NO: 1847, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 1847.
  • a 5’ ITR optionally wherein the 5’ ITR comprises the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical thereto;
  • a promoter optionally wherein the promoter comprises the nucleotide sequence of any one of SEQ ID NOs: 26-35, or a nucleotide sequence at least 80%' (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical to any one of SEQ ID NOs: 26-35;
  • an intron optionally wherein the intron comprises the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical thereto;
  • nucleotide sequence encoding an SMN protein optionally comprising the nucleotide sequence of any one of SEQ ID NOs: 6-9, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical to any one of SEQ ID NOs: 6-8;
  • a polyA sequence optionally wherein the polyA sequence comprises the nucleotide sequence of SEQ ID NO: 4, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical thereto;
  • nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical thereto.
  • a 5’ ITR optionally wherein the 5’ ITR comprises the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical thereto;
  • a promoter optionally wherein the promoter comprises the nucleotide sequence of any one of SEQ ID NOs: 26, 27, 29, or 31, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical to any one of SEQ ID NOs: 26, 27, 29, or 31;
  • nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical thereto;
  • nucleotide sequence encoding an SMN protein optionally comprising the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical thereto;
  • polyA sequence optionally wherein the polyA sequence comprises the nucleotide sequence of SEQ ID NO: 4, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical thereto; and
  • a 3’ ITR optionally wherein the 3’ ITR comprises the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical thereto.
  • AAV particle of any one of embodiments 282-337 which comprises the nucleotide sequence of any one of SEQ ID NOs: 10-25 or 36-38, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical to any one of SEQ ID NOs: 10-25 or 36-38.
  • AAV particle of any one of embodiments 282-338 which comprises the nucleotide sequence of any one of SEQ ID NOs: 20-22 or 24, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical to any one of SEQ ID NOs: 20-22 or 24.
  • a cell e.g., a host cell, comprising the AAV particle of any one of the preceding embodiments.
  • the cell of embodiment 340 wherein the cell is a mammalian cell (e.g., an HEK293 cell) or an insect cell (e.g., an Sf9 cell).
  • the cell is a cell of the temporal cortex, perirhinal cortex, globus pallidus, putamen, caudate, thalamus, hippocampus, geniculate nucleus, Purkinje Layer, deep cerebellar nuclei, cerebellum, cervical spinal cord, thoracic spinal cord, lumbar spinal cord, or a combination thereof.
  • a method of making the AAV particle of any one of embodiments 1-339 comprising:
  • a pharmaceutical composition comprising the AAV particle of any one of embodiments 1-339, and a pharmaceutically acceptable excipient.
  • a method of delivering a payload to a cell or tissue comprising administering an effective amount of the pharmaceutical composition of embodiment 334, or the AAV particle of any one of embodiments 1-339.
  • a method of treating a subject having or diagnosed with having a neurological disorder comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 348, or the AAV particle of any one of embodiments 1-339.
  • a method of treating a subject having or diagnosed with having a disease related to decreased SMN protein expression, e.g., a mutation in an SMN1 gene, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 348, or the AAV particle of any one of embodiments 1-339.
  • a method of treating a subject having or diagnosed with Spinal Muscular Atrophy comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 348, or the AAV particle of any one of embodiments 1-339.
  • SMA Spinal Muscular Atrophy
  • 365 The method of any one of embodiments 352-362, wherein the subject comprises 1 to 2 copies, e.g., 1 copy or 2 copies, of an SMN2 gene (e.g., a type I SMA).
  • an SMN2 gene e.g., a type I SMA
  • compositions and methods described herein provides improved features compared to prior SMN gene replacement approaches, including (i) increased SMN activity in a cell, tissue, (e.g., a cell or tissue of the CNS, e.g., the cortex, striatum, thalamus, cerebellum, and/or brainstem), and/or fluid (e.g., CSF and/or serum), of the subject; (ii) increased biodistribution throughout the CNS (e.g., the cortex, striatum, thalamus, cerebellum, brainstem, and/or spinal cord), and the periphery (e.g., the liver), and/or (iii) elevated payload expression, e.g., SMN mRNA expression, in multiple brain regions (e.g., cortex, thalamus, and brain stem) and the periphery (e.g., the liver).
  • SMN activity in a cell, tissue e.g., a cell or tissue of the
  • an AAV viral genome encoding an SMN protein described herein which comprise an optimized nucleotide encoding the SMN protein result in high biodistribution in the CNS; increased SMN activity in the CNS, peripheral tissues, and/or fluid; and successful transgene transcription and expression.
  • the compositions and methods described herein can be used in the treatment of disorders associated with a lack of an SMN protein and/or SMN activity (e.g., SMA, Werdnig-Hoffman disease, Dubowitz disease, Kugelberg-Welander disease), such as SMA-related disorders associated with a mutation in an SMN gene.
  • certain AAV capsid variants described herein show multiple advantages over wild-type AAV5 and/or wild-type AAV9, including (i) increased penetrance through the blood brain barrier following intravenous administration, (ii) wider distribution throughout the multiple brain regions, e.g., frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate, and/or hippocampus, (iii) elevated payload expression in multiple brain regions, (iv) wider distribution in one or more peripheral tissues, e.g., the heart, muscle, and/or liver, and/or (v) elevated payload expression in one or more peripheral tissues.
  • AAV capsid variants described herein show multiple advantages over wild-type AAV5 and/or wild-type AAV9, including (i) increased penetrance through the blood brain barrier following intravenous administration, (ii) wider distribution throughout the multiple brain regions, e.g., frontal cortex, sensory cortex, motor cortex, putamen,
  • the AAV capsids described herein enhance the delivery of a payload to multiple regions of the brain including, for example, a temporal cortex, perirhinal cortex, globus pallidus, putamen, caudate, thalamus, hippocampus, geniculate nucleus, Purkinje Layer, deep cerebellar nuclei, cerebellum, or a combination thereof.
  • AAV capsid variants disclosed herein comprise a modification in loop VIII of AAV5, e.g., at positions between 571-579, e.g., at position 577, numbered relative to SEQ ID NO: 138.
  • the aforesaid region (e.g., positions between 571-579, e.g., at position 577) of the AAV5 capsid protrudes above the 3- fold axis of symmetry, e.g., is a surface-exposed location in the AAV5 capsid , e.g., as described in Govindasamy et al.
  • loop VIII (e.g., VR-VIII) comprises positions 571-599 (e.g., amino acids TNNQSSYPAEVVQKTAPATGTYNLQEIVP (SEQ ID NO: 756)), numbered according to SEQ ID NO: 982 .
  • loop VIII or variable region VIII (VR- VIII) is as described in Govindasamy et al. (supra) (the contents of which are hereby incorporated by reference in their entirety).
  • SMA is caused by a reduction in the expression of the survival motor neuron (SMN) protein caused by mutations in the survival motor neuron 1 (SMN1) gene and loss of encoded SMN protein (Lefebvre et al., Cell (1995) 80:155-165).
  • SMN survival motor neuron
  • SMN1 survival motor neuron 1
  • SMN is a ubiquitously expressed protein that functions in the assembly of the spliceosome and may also mediate mRNA trafficking in the axon and nerve terminus of neurons.
  • the lack of SMN results in motor neuron degeneration in the ventral (anterior) horn of the spinal cord, which leads to weakness of the proximal muscles responsible for crawling, walking, neck control and swallowing, and the involuntary muscles that control breathing and coughing (Sumner C. J., NeuroRx (2006) 3:235-245). Consequently, SMA patients present with increased tendencies for pneumonia and other pulmonary problems such as restrictive lung disease.
  • SMN1 and SMN2 In humans there are two very similar copies of the SMN gene termed SMN1 and SMN2. The amino acid sequence encoded by the two genes is identical.
  • the SMN1 and SMN2 genes lie within the telomeric and centromeric halves, respectively, of a large, inverted duplication on chromosome 5ql3. These genes share more than 99% nucleotide identity, and both are capable of encoding SMN (a 294- amino acid RNA-binding protein). However, there is a single, silent nucleotide change in SMN 2 in exon 7 that results in exon 7 being excluded in 80-90% of transcripts from SMN2.
  • SMNA7 The resulting truncated protein, called SMNA7, is less stable and rapidly degraded.
  • the remaining 10-20% of transcript from SMN2 encodes the full length SMN protein.
  • SMN2 acts as a phenotypic modifier in SMA in that patients with a higher SMN2 copy number generally exhibit later onset and less severe disease.
  • patients with a high SMN2 copy number (3-4 copies) exhibit a less severe form of the disease (referred to as Types II or III), whereas 1-2 copies of SMN2 typically result in the more severe Type I disease (Campbell et al., Am. J. Hum. Genet. (1997) 61:40-50; Lefebvre et al., Nat. Genet. (1997) 16:265-269).
  • the SMN1 and SMN2 genes differ by live nucleotides; one of these differences - a translationally silent C to T substitution in an exonic splicing region - results in frequent exon 7 skipping during transcription of SMN2.
  • SMNAEx7 exon 7
  • the majority of transcripts produced from SMN2 lack exon 7 (SMNAEx7), and encode a truncated protein which is rapidly degraded (about 10% of the SMN2 transcripts are full length and encode a functional SMN protein).
  • AAV Adeno-associated viral
  • AAV are used as a biological tool due to a relatively simple structure, their ability to infect a wide range of cells (including quiescent and dividing cells) without integration into the host genome and without replicating, and their relatively benign immunogenic profile.
  • the genome, e.g., viral genome, of the virus may be manipulated to contain a minimum of components for the assembly of a functional recombinant virus, or viral particle, which is loaded with or engineered to target a particular tissue and express or deliver a desired payload, e.g., a polypeptide encoding polynucleotide, e.g., an SMN protein described herein, e.g., an SMN1 and/or SMN2 protein described herein.
  • a polypeptide encoding polynucleotide e.g., an SMN protein described herein, e.g., an SMN1 and/or SMN2 protein described herein.
  • the AAV particle is a naturally occurring (e.g., wild-type) AAV or a recombinant AAV.
  • the wild-type AAV viral genome is a linear, single- stranded DNA (ssDNA) molecule approximately 5,000 nucleotides (nt) in length.
  • ssDNA single- stranded DNA
  • nt nucleotides
  • inverted terminal repeats (ITRs) cap the viral genome at both the 5’ and the 3’ end, providing origins of replication for the viral genome.
  • an AAV viral genome typically comprises two ITR sequences.
  • ITRs have a characteristic T-shaped hairpin structure defined by a self- complementary region (145nt in wild-type AAV) at the 5’ and 3’ ends of the ssDNA which form an energetically stable double stranded region.
  • the double stranded hairpin structures comprise multiple functions including, but not limited to, acting as an origin for DNA replication by functioning as primers for the endogenous DNA polymerase complex of the host viral replication cell.
  • the wild-type AAV viral genome further comprises nucleotide sequences for two open reading frames, one for the four non-structural Rep proteins (Rep78, Rep68, Rep52, Rep40, encoded by Rep genes) and one for the three capsid, or structural, proteins (VP1, VP2, VP3, encoded by capsid genes or Cap genes).
  • the Rep proteins are used for replication and packaging, while the capsid proteins are assembled to create the protein shell of the AAV, or AAV capsid polypeptide, e.g., an AAV capsid variant.
  • VP1 refers to amino acids 1-724
  • VP2 refers to amino acids 137- 724
  • VP3 refers to amino acids 193-724.
  • amino acid sequence of SEQ ID NO: 982 VP1 comprises amino acids 1-731
  • VP2 comprises amino acids 137-731
  • VP3 comprises amino acids 193-731.
  • VP1 is the full-length capsid sequence
  • VP2 and VP3 are shorter components of the whole.
  • changes in the sequence in the VP3 region are also changes to VP1 and VP2, however, the percent difference as compared to the parent sequence will be greatest for VP3 since it is the shortest sequence of the three.
  • the nucleic acid sequence encoding these proteins can be similarly described.
  • the three capsid proteins assemble to create the AAV capsid protein. While not wishing to be bound by theory, the AAV capsid protein typically comprises a molar ratio of 1:1:10 of VP1:VP2:VP3.
  • a viral genome of a wild-type, e.g., naturally occurring, AAV can be modified to replace the rep/cap sequences with a nucleic acid comprising a transgene encoding a payload, e.g., an antibody molecule, wherein the viral genome comprises at least one ITR region.
  • the viral genome of a recombinant AAV comprises two ITR regions, e.g., a 5TTR or a 3TTR.
  • the rep/cap sequences can be provided in trans during production to generate AAV particles.
  • an AAV particle in addition to the viral genome comprising a nucleic acid encoding a transgene encoding a payload (e.g., a therapeutic protein, e.g., an SMN protein), an AAV particle, e.g., an AAV particle described herein, may comprise the viral genome, in whole or in part, of any naturally occurring and/or recombinant AAV serotype nucleotide sequence or variant.
  • AAV variants may have sequences of significant homology at the nucleic acid (viral genome or capsid) and amino acid levels (capsids), to produce constructs which are generally physical and functional equivalents, replicate by similar mechanisms, and assemble by similar mechanisms.
  • the viral genome or the AAV vector of the AAV particles described herein comprise at least one control element which provides for the replication, transcription, and translation of a coding sequence encoded therein.
  • a sufficient number of control elements are present such that the coding sequence of the transgene encoded by the viral genome is capable of being replicated, transcribed, and/or translated in a host cell.
  • the AAV particles described herein may be modified to enhance the efficiency of delivery, e.g., delivery of a transgene encoding a payload, e.g., an antibody molecule.
  • a modified, e.g., recombinant, AAV particle can be packaged efficiently and successfully infect target cells at high frequency and with minimal toxicity.
  • the capsid protein of the AAV particles is engineered according to the methods described in US Publication Number US20130195801, the contents of which are incorporated herein by reference in their entirety.
  • the nucleotide sequence encoding the AAV capsid polypeptide or the AAV capsid variant comprises the nucleotide sequence of SEQ ID NO: 137 or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto.
  • the AAV capsid variant comprises an amino acid other than the wild-type, e.g., native, amino acid, at position T577, numbered according to SEQ ID NO: 138 or corresponding to equivalent positions in any other AAV serotype (e.g., AAV1, AAV2, AAV3, AAV3b, AAV4, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrhlO, AAVrh32.33, AAVrh74, SEQ ID NO: 139, PHP.N, PHP.B, or an AAV serotype as provided in Table 6 of WO 2021/230987 (the contents of which are hereby incorporated by reference in their entirety)).
  • AAV serotype e.g., AAV1, AAV2, AAV3, AAV3b, AAV4, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrhlO, AAVrh32.33, AAVrh74, SEQ ID NO: 139
  • the AAV capsid variant comprises a modification, e.g., substitution, at position T577, numbered according to SEQ ID NO: 138 or corresponding to equivalent positions in any other AAV serotype (e.g., AAV1, AAV2, AAV3, AAV3b, AAV4, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrhlO, AAVrh32.33, AAVrh74, SEQ ID NO: 139, PHP.N, PHP.B, or an AAV serotype as provided in Table 6 of WO 2021/230987 (the contents of which are hereby incorporated by reference in their entirety).
  • AAV serotype e.g., substitution, at position T577, numbered according to SEQ ID NO: 138 or corresponding to equivalent positions in any other AAV serotype (e.g., AAV1, AAV2, AAV3, AAV3b, AAV4, AAV6, AAV7, AAV8, AAV9, AAVrh
  • an AAV capsid described herein does not comprise a T at position 581, an A at position 582, an A at position 584, a V at position 585, a T at position 585, a V at position 569, an A at position 652, an M at position 362, a Q at position 359, a Q at position 350, an S at position 533, or a combination thereof, all numbered relative to SEQ ID NO: 138.
  • an AAV capsid described herein does not comprise a modification, e.g., substitution, at positions 581-589 (numbered according to SEQ ID NO: 138), wherein the modification has the amino acid sequence of any of the sequences provided in Tables 2, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, or 71-86 of WO 2021/242909.
  • an AAV particle described herein comprises an AAV capsid variant, e.g., an AAV capsid variant described herein (e.g., an AAV capsid variant comprising a peptide or an amino acid sequence described herein).
  • an AAV capsid variant comprises a peptide as set forth in any of Tables 2A, 2B, 2C, 15 or 21.
  • [N2] comprises A at position X3. In some embodiments, [N2] comprises E at position X4. In some embodiments, [N2] comprises V at position X5. In some embodiments, [N2] comprises YPA, YPP, NKA, YTA, YQA, YTP, NPA, CPA, THA, PAE, PPS, KAE, TAE, QAE, TPS, PAA, HAS, AEV, PSL, AEE, or AAV.
  • [N2] comprises YPAE (SEQ ID NO: 286), YPPS (SEQ ID NO: 287), NKAE (SEQ ID NO: 288), YTAE (SEQ ID NO: 289), YQAE (SEQ ID NO: 290), YTPS (SEQ ID NO: 291), YPAA (SEQ ID NO: 292), NPAE (SEQ ID NO: 293), CPAE (SEQ ID NO: 294), THAS(SEQ ID NO: 295) , PAEV (SEQ ID NO: 296), PPSL (SEQ ID NO: 297), KAEV (SEQ ID NO: 298), TAEV (SEQ ID NO: 299), PAEE (SEQ ID NO: 300), QAEV (SEQ ID NO: 301), TPSL (SEQ ID NO: 302), PAAV (SEQ ID NO: 303), or QAEE (SEQ ID NO: 304).
  • [N2] is or comprises YPAEV (SEQ ID NO: 39), YPPSL (SEQ ID NO: 40), NKAEV (SEQ ID NO: 41), YTAEV (SEQ ID NO: 42), YPAEE (SEQ ID NO: 43), YQAEV (SEQ ID NO: 44), YTPSL (SEQ ID NO: 45) , YPAAV (SEQ ID NO: 46), NPAEV (SEQ ID NO: 47), CPAEV (SEQ ID NO: 48), or YQAEE (SEQ ID NO: 49).
  • [N2] is YPAEV (SEQ ID NO: 39).
  • [Nl] comprises SK, SL, SS, SR, GA, GS, AS, ST, RS, QS, TS, AG, IG, QA, LG, HS, LS, or QR.
  • [Nl] is or comprises QSS, QSK, TSL, SSS, QSR, AGA, IGS, QAS, ASS, LGS, QST, HSS, LSS, or QRS.
  • [N1]-[N2] is QSSYPAEV (SEQ ID NO: 113).
  • [N1]-[N2]-[N3] comprises SSYPAEVVQ (SEQ ID NO: 142), SKYPAEVVQ (SEQ ID NO: 143), SLYPAEVVQ (SEQ ID NO: 101), SRYPAEVVQ (SEQ ID NO: 144), SSYPPSLVQ (SEQ ID NO: 145), GAYPAEVVQ (SEQ ID NO: 146), GSYPAEVVQ (SEQ ID NO: 147), ASYPAEVVQ (SEQ ID NO: 148), STNKAEVVQ (SEQ ID NO: 149), SSYTAEVVQ (SEQ ID NO: 150), SKYPAEVEQ (SEQ ID NO: 160), SLYPAEEVQ (SEQ ID NO: 161), SSYQAEVVQ (SEQ ID NO: 162), SSYTPSLVQ (SEQ ID NO: 143
  • [N0]-[Nl]-[N2]-[N3] is or comprises TNNQSSYPAEVVQK (SEQ ID NO: 230), TNNQSKYPAEVVQK (SEQ ID NO: 231), TNNTSLYPAEVVQK (SEQ ID NO: 232), TNNSSSYPAEVVQK (SEQ ID NO: 233), TNNQSRYPAEVVQK (SEQ ID NO: 234), TNNQSSYPPSLVQK (SEQ ID NO: 235), TNNAGAYPAEVVQK (SEQ ID NO: 236), TNNIGSYPAEVVQK (SEQ ID NO: 237), TNNQASYPAEVVQK (SEQ ID NO: 238), TNTASSYPAEVVQK (SEQ ID NO: 239), TNNLGSYPAEVVQK (SEQ ID NO: 240), TNNQSTNKAEVVQK (SEQ ID NO: 24
  • [N0]-[Nl]-[N2]-[N3]-[N4] is or comprises TNNQSSYPAEVVQKTA (SEQ ID NO: 1533), TNNQSKYPAEVVQKTA (SEQ ID NO: 1538), TNNTSLYPAEV VQKTA (SEQ ID NO: 1232), TNNSSSYPAEV VQKTA (SEQ ID NO: 1539), TNNQSRYPAEVVQKTA (SEQ ID NO: 1327), TNNQSSYPPSLVQKTA (SEQ ID NO: 1300), TNNAGAYPAEVVQKTA (SEQ ID NO: 1021), TNNIGSYPAEVVQKTA (SEQ ID NO: 1112) , TNNQ AS YPAEV VQKTA (SEQ ID NO: 1586), TNTASSYPAEVVQKTA (SEQ ID NO: 1575), TNNLGSYPAEVVQKTA (SEQ ID NO: 10
  • [NO] is present immediately subsequent to position 570, numbered relative to SEQ ID NO: 138.
  • [NO] replaces positions 571-573 (e.g., T571, N572, and N573), numbered relative to SEQ ID NO: 138.
  • [NO] is present immediately subsequent to position 570, and [NO] replaces positions 571-573 (e.g., amino acids T571, N572, and N573), numbered relative to SEQ ID NO: 138.
  • [Nl] is present immediately subsequent to position 573, numbered relative to SEQ ID NO: 138.
  • [Nl] replaces positions 574-576 (e.g., Q574, S575, and S576), numbered relative to SEQ ID NO: 138. In some embodiments, [Nl] is present immediately subsequent to position 573, and [Nl] replaces positions 574- 576 (e.g., Q574, S575, and S576), numbered relative to SEQ ID NO: 138. In some embodiments, [N2] is present immediately subsequent to position 576, numbered relative to SEQ ID NO: 138. In some embodiments, [N2] replaces position 577 (e.g., T577), numbered relative to SEQ ID NO: 138.
  • position 577 e.g., T577
  • [N2] is present immediately subsequent to position 576, and [N2] replaces position 577 (e.g., T577), numbered relative to SEQ ID NO: 138.
  • [N2]-[N3] is present immediately subsequent to position 576, numbered relative to SEQ ID NO: 138.
  • [N2]-[N3] replaces position 577 (e.g., T577), numbered relative to SEQ ID NO: 138.
  • [N2]-[N3] is present immediately subsequent to position 576, and [N2]-[N3] replaces position 577 (e.g., T577), numbered relative to SEQ ID NO: 138.
  • [N1]-[N2]-[N3]-[N4] is present immediately subsequent to position 573, numbered relative to SEQ ID NO: 138.
  • [N1]-[N2]- [N3]-[N4] replaces positions 574-579 (e.g., Q574, S575, S576, T577, T578, and A579), numbered relative to SEQ ID NO: 138.
  • [N1]-[N2]-[N3]-[N4] is present immediately subsequent to position 573, and [N1]-[N2]-[N3]-[N4] replaces positions 574-579 (e.g., Q574, S575, S576, T577, T578, and A579), numbered relative to SEQ ID NO: 138.
  • [NO]- [N1]-[N2]-[N3]-[N4] is present immediately subsequent to position 570.
  • [N0]- [N 1 J-LN2J- [N3]-[N4] replaces positions 571-579 (e.g., T571, N572, N573, Q574, S575, S576, T577, T578, and A579), numbered relative to SEQ ID NO: 138.
  • - [N4] is present immediately subsequent to position 570, and [N0]-[Nl]-[N2]-[N3]-[N4] replaces positions 571-579 (e.g., T571, N572, N573, Q574, S575, S576, T577, T578, and A579), numbered relative to SEQ ID NO: 138.
  • the AAV capsid variant comprises an amino acid other than T at position 577, relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises Y at position 577, relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, XI of [N2] is present at position 577 (e.g., T577), and positions X2 and X3 of [N2] arc present immediately subsequent to position 577, relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, [N3] is present immediately subsequent to [N2].
  • XA of [NO] is present at position 571
  • XB of [NO] is present at position 572
  • Xc of [NO] is present at position 573, numbered according to SEQ ID NO: 982.
  • XD of [Nl] is present at position 574
  • XE of [Nl] is present at position 575
  • XF of [Nl] is present at position 576, numbered according to SEQ ID NO: 982.
  • XI of [N2] is present at position 577
  • X2 of [N2] is present at position 578
  • X3 of [N2] is present at position 579
  • X4 of [N2] is present at position 580
  • X5 of [N2] is present at position 581, numbered according to SEQ ID NO: 982.
  • [N3] is present at positions 582-584, numbered according to SEQ ID NO: 982.
  • XQ of [N4] is present at position 585 and Xu of [N4] is present at position 586, numbered according to SEQ ID NO: 982.
  • [NO] is present at positions 571 -573, numbered according to SEQ ID NO: 982.
  • [Nl] is present at positions 574-576, numbered according to SEQ ID NO: 982.
  • [N2] is present at positions 577-581, numbered according to SEQ ID NO:
  • [N3] is present at positions 582-584, numbered according to SEQ ID NO:
  • [N4] is present at positions 585-586, numbered according to SEQ ID NO:
  • [N2]-[N3] is present at positions 577-584, numbered according to SEQ ID NO:
  • [NO]-[N1]-[N2]-[N3]-[N4] is present at positions 571-586, numbered according to SEQ ID NO: 982.
  • [Nl] is present immediately subsequent to [NO]. In some embodiments, [N2] is present immediately subsequent to [Nl]. In some embodiments, [N3] is present immediately subsequent to [N2J. In some embodiments, [N4J is present immediately subsequent to [N3J.
  • the AAV capsid variant comprises from N-terminus to C-terminus, [N2]- [N3]. In some embodiments, the AAV capsid variant comprises from N-terminus to C-terminus, [Nl]- [N2]-[N3].
  • the AAV capsid variant comprises from N-terminus to C-terminus, [N1]-[N2]-[N3]-[N4], In some embodiments, the AAV capsid variant comprises from N-terminus to C- terminus, [NO]-[N1]-[N2]-[N3], In some embodiments, the AAV capsid variant comprises from N- terminus to C-terminus, [N0]-[Nl]-[N2]-[N3]-[N4],
  • [N2]-[N3] is YPAEVVQK (SEQ ID NO: 943), wherein YPAEVVQK (SEQ ID NO: 943) replaces position 577, numbered relative to SEQ ID NO: 138.
  • [NO]-[N1]-[N2]-[N3]-[N4] is TNNQSSYPAEVVQKTA (SEQ ID NO: 1533) and is present immediately subsequent to position 570, numbered relative to SEQ ID NO: 138, wherein [N2]-[N3] (YPAEVVQK; SEQ ID NO: 943) replaces position 577 (e.g., replaces T577) numbered relative to SEQ ID NO: 138.
  • [N2]-[N3] is YPAEVVQK, wherein [N2]-[N3] is present at positions 577-584, numbered according to SEQ ID NO: 982.
  • [NO]-[N1]-[N2]-[N3]-[N4] is TNNQSSYPAEVVQKTA (SEQ ID NO: 1533) and is present at positions 571-586, numbered according to SEQ ID NO: 982.
  • an AAV capsid variant described herein comprises an amino acid sequence having the formula [B]-[C], wherein [B] comprises positions XI, X2, and X3, and [C] comprises the amino acid sequence YPAEVVQK (SEQ ID NO: 943).
  • position XI of [B] is Q, T, S, A, I, L, or H.
  • position XI of [B] is Q, T, S, A, or H.
  • position X2 of [B] is S, G, or A.
  • position X2 of [B] is S or G.
  • [B] is QSS.
  • [B]-[C] comprises SSYPAEVVQK (SEQ ID NO: 276), SKYPAEVVQK (SEQ ID NO: 277), SLYPAEVVQK (SEQ ID NO: 278), SRYPAEVVQK (SEQ ID NO: 279), GAYPAEVVQK (SEQ ID NO: 280), GSYPAEVVQK (SEQ ID NO: 281), or ASYPAEVVQK (SEQ ID NO: 282).
  • [B]-[C] is or comprises QSSYPAEVVQK (SEQ ID NO: 176), QSKYPAEVVQK (SEQ ID NO: 177), TSLYPAEVVQK (SEQ ID NO: 178), SSSYPAEVVQK (SEQ ID NO: 179), QSRYPAEVVQK (SEQ ID NO: 180), AGAYPAEVVQK (SEQ ID NO: 182), IGSYPAEVVQK (SEQ ID NO: 183), QASYPAEVVQK (SEQ ID NO: 184), ASSYPAEVVQK (SEQ ID NO: 186), LGSYPAEVVQK (SEQ ID NO: 187), or HSSYPAEVVQK (SEQ ID NO: 189); an amino acid sequence comprising any portion of any of the aforesaid amino acid sequences (e.g., any 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, e.g., consecutive amino acids) thereof;
  • an AAV capsid variant comprising the formula [B]-[C], further comprises [A], which comprises positions XA, XB, and Xc-
  • position XA of [A] is T, I, or N.
  • position XB of [A] is N.
  • position Xc of [A] is N, T, S, or K.
  • [A] comprises TN, IN, NN, NT, NS, or NK.
  • [A] is or comprises TNN, TNT, INN, NNN, TNS, or TNK.
  • [A] is TNN.
  • [A]-[B] is or comprises TNNQSS (SEQ ID NO: 210), TNNQSK (SEQ ID NO: 211), TNNTSL (SEQ ID NO: 212), TNNSSS (SEQ ID NO: 213), TNNQSR (SEQ ID NO: 214), TNNAGA (SEQ ID NO: 215), TNNIGS (SEQ ID NO: 216), TNNQAS (SEQ ID NO: 217), TNTASS (SEQ ID NO: 218), TNNLGS (SEQ ID NO: 219), TNNHSS (SEQ ID NO: 221), INNQSS (SEQ ID NO: 224), NNNQSR (SEQ ID NO: 226), TNSTSL (SEQ ID NO: 227), or TNKQAS (SEQ ID NO: 229); an amino acid sequence comprising any portion of any of the aforesaid amino acid sequences (e.g., any 2, 3, 4, or 5 amino acids, e.g., consecutive
  • [A]-[B] is TNNQSS (SEQ ID NO: 210).
  • [A]-[B]-[C] is or comprises TNNQSSYPAEVVQK (SEQ ID NO: 230), TNNQSKYPAEVVQK (SEQ ID NO: 231), TNNTSLYPAEVVQK (SEQ ID NO: 232), TNNS SSYPAEVVQK (SEQ ID NO: 233), TNNQSRYPAEVVQK (SEQ ID NO: 234), TNNAGAYPAEVVQK (SEQ ID NO: 236), TNNIGSYPAEVVQK (SEQ ID NO: 237), TNNQASYPAEVVQK (SEQ ID NO: 238), TNTASSYPAEVVQK (SEQ ID NO: 239), TNNLGSYPAEVVQK (SEQ ID NO: 240), TNNHSSYPAEVVQK (SEQ ID NO: 242), INNQSSYPAEVVQ
  • an AAV capsid variant comprising the formula [B]- [C] , further comprises [D], wherein [D] comprises position X4 and X5.
  • position X4 of [D] is T or N.
  • position X5 of [D] is A.
  • [D] is or comprises TA or PA.
  • [D] is TA.
  • [C]-[D] is or comprises YPAEVVQKTA (SEQ ID NO: 283) or YPAEVVQKPA (SEQ ID NO: 284); an amino acid sequence comprising any portion of any of the aforesaid amino acid sequences (e.g., any 2, 3, 4, 5, 6, 7, 8, or 9 amino acids, e.g., consecutive amino acids) thereof; an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforesaid amino acid sequences; or an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the aforesaid amino acid sequences.
  • [C]-[D] is YPAEVVQKTA (SEQ ID NO: 283).
  • [A]-[B]-[C]-[D] is or comprises TNNQSSYPAEVVQKTA (SEQ ID NO: 1533), TNNQSKYPAEVVQKTA (SEQ ID NO: 1538), TNNTSLYPAEVVQKTA (SEQ ID NO: 1232), TNNSSSYPAEVVQKTA (SEQ ID NO: 1539), TNNQSRYPAEVVQKTA (SEQ ID NO: 1327), TNNAGAYPAEVVQKTA(SEQ ID NO: 1021), TNNIGSYPAEVVQKTA(SEQ ID NO: 1112), TNNQ AS YPAEVVQKTA (SEQ ID NO: 1586), TNTASSYPAEVVQKTA (SEQ ID NO: 1575), TNNLGSYPAEVVQKTA (SEQ ID NO: 1027), TNNHSSYPAEVVQKTA (SEQ ID NO: 1310), TNNSSSYPAEVVQKTA (SEQ ID NO:
  • [A]-[B]-[C]-[D] is TNNQSSYPAEVVQKTA (SEQ ID NO: 1533). [0081] In some embodiments, [B]-[C] is present in loop VIII of the AAV capsid variant. In some embodiments, [A] and/or [D] is present in loop VUI of the AAV capsid variant. In some embodiments,
  • loop VIH comprises positions 571-592 numbered according to SEQ ID NO: 138.
  • loop VIII comprises positions 571-599, numbered according to SEQ ID NO: 982.
  • [A] is present immediately subsequent to position 570, numbered relative to SEQ ID NO: 138. In any of these embodiments, [A] replaces positions 571-573 (e.g., T571, N572, and N573) numbered relative to SEQ ID NO: 138. In some embodiments, [A] is present immediately subsequent to position 570, and [A] replaces positions 571-573 (e.g., T571, N572, and N573) numbered relative to SEQ ID NO: 138. In some embodiments, [B] is present immediately subsequent to position 573, relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments,
  • [B] replaces positions 574-576 (e.g., Q574, S575, and S576), relative to a reference sequence numbered according to SEQ ID NO: 138.
  • [B] is present immediately subsequent to position 573, and [B] replaces positions 574-576 (e.g., Q574, S575, and S576), relative to a reference sequence numbered according to SEQ ID NO: 138.
  • [C] is present immediately subsequent to position 576, relative to a reference sequence numbered according to SEQ ID NO: 138.
  • [C] replaces position 577 (e.g., T577), relative to a reference sequence numbered according to SEQ ID NO: 138.
  • [C] is present immediately subsequent to position 576, wherein [C] replaces position 577 (e.g., T577), relative to a reference sequence numbered according to SEQ ID NO: 138.
  • [B]-[C] is present immediately subsequent to position 573, relative to a reference sequence numbered according to SEQ ID NO: 138.
  • [B]-[C] replaces positions 574-577 (e.g., Q574, S575, S576, and T577), relative to a reference sequence numbered according to SEQ ID NO: 138.
  • [B] is present at position 574, X2 of [B] is present at position 575, and X3 of [B] is present at position 576, numbered according to SEQ ID NO: 982.
  • [B] is present at positions 574-576, numbered according to SEQ ID NO: 982.
  • [C] is present at positions 577-584, numbered according to SEQ ID NO: 982.
  • [B]-[C] is present at positions 574-584, numbered according to SEQ ID NO: 982.
  • [C]-[D] replaces positions 577-579 (e.g., T577, T578, and A579), relative to a reference sequence numbered according to SEQ ID NO: 138.
  • [A] is present at positions 571-573, numbered according to SEQ ID NO: 982.
  • [B] is present at positions 574-576, numbered according to SEQ ID NO: 982.
  • [C] is present at positions 577-584, numbered according to SEQ ID NO: 982.
  • [D] is present at positions 585-586, numbered according to SEQ ID NO: 982.
  • [A]-[B]-[C]-[D] is present at positions 571-586, numbered according to SEQ ID NO: 982.
  • [A]-[B]-[C]-[D] is present immediately subsequent to position 570, numbered relative to SEQ ID NO: 138.
  • [A]-[B]-[C]-[D] replaces positions 571-579 (e.g., T571, N572, N573, Q574, S575, S576, T577, T578, and A579), numbered relative to SEQ ID NO: 138.
  • [A] -[B] - [C] - [D] is present immediately subsequent to position 570, and [A]-[B]-[C]-[D] replaces positions 571 -579 (e.g., T571 , N572, N573, Q574, S575, S576, T577, T578, and A579), numbered relative to SEQ ID NO: 138.
  • positions 571 -579 e.g., T571 , N572, N573, Q574, S575, S576, T577, T578, and A57
  • XA of [A] is present at position 571
  • XB of [A] is present at position 572
  • Xc of [A] is present at position 573, numbered according to SEQ ID NO: 982.
  • XI of [B] is present at position 574
  • X2 of [B] is present at position 575
  • X3 of [B] is present at position 576, numbered according to SEQ ID NO: 982.
  • [C] is present at positions 577-584, numbered according to SEQ ID NO: 982.
  • X4 of [D] is present at position 585 and position X5 of [D] is present at position 586, numbered according to SEQ ID NO: 982.
  • the AAV capsid variant comprises from N-terminus to C-terminus, [B]- [C]. In some embodiments, the AAV capsid variant comprises from N-terminus to C-terminus, [B]-[C], In some embodiments, the AAV capsid variant comprises from N-terminus to C-terminus, [A]-[B]-[C], In some embodiments, the AAV capsid variant comprises from N-terminus to C-terminus, [B]-[C]-[D]. In some embodiments, the AAV capsid variant comprises from N-terminus to C-terminus, [A]-[B]-[C]- [D],
  • [C] is YPAEVVQK (SEQ ID NO: 943), wherein YPAEVVQK (SEQ ID NO: 943) replaces position 577 (e.g., T577), numbered relative to SEQ ID NO: 138.
  • [A]-[B]-[C]-[D] is TNNQSSYPAEVVQKTA (SEQ ID NO: 1533)and is present immediately subsequent to position 570, numbered relative to SEQ ID NO: 138, wherein [C] (YPAEVVQK) replaces position 577 (e.g., replaces T577) numbered relative to SEQ ID NO: 138.
  • [C] is YPAEVVQK (SEQ ID NO: 943), wherein [C] is present at positions 577-584, numbered according to SEQ ID NO: 982.
  • an AAV capsid variant described herein comprises an amino acid sequence comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive amino acids from any one of the sequences provided in Tables 2A, 2B, 2C, 15 and 21 .
  • the AAV capsid variant comprises an amino acid sequence comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive amino acids from any one of SEQ ID NOs: 943, 1021, 1024, 1027, 1112, 1142, 1214, 1232, 1254, 1300, 1310, 1327, 1331, 1342, 1419, 1453, 1533, 1538, 1539, 1575, 1578, 1583- 1587, 1590, 1591-1593, 1598-1608, or 1610-1624.
  • the AAV capsid variant comprises at least 3, 4, 5, 6, or 7 consecutive amino acids from any one of SEQ ID NOs: 943 or 946-966.
  • the amino acid sequence is present in loop VIII. In some embodiments, the amino acid sequence is present immediately subsequent to position 570, 571, 572, 573, 574, 575, or 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • the 3 consecutive amino acids comprise YPA.
  • the 4 consecutive amino acids comprise YPAE.
  • the 5 consecutive amino acids comprise YPAEV (SEQ ID NO: 39).
  • the 6 consecutive amino acids comprise YPAEVV (SEQ ID NO: 151).
  • the 7 consecutive amino acids comprise YPAEVVQ (SEQ ID NO: 152).
  • the amino acid sequence comprises YPAEVVQK (SEQ ID NO: 943).
  • the amino acid sequence consists of YPAEVVQK (SEQ ID NO: 943).
  • an AAV capsid variant described herein comprises an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of any one of the sequences provided in Tables 2A, 2B, 2C, 15 and 21.
  • the AAV capsid variant comprises an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to the amino acid sequence of any one of the sequences provided in Tables 2A, 2B, 2C, 15 and 21.
  • the AAV capsid variant comprises an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of any one of SEQ ID NOs: 943, 1021, 1024, 1027, 1112, 1142, 1214, 1232, 1254, 1300, 1310, 1327, 1331, 1342, 1419, 1453, 1533, 1538, 1539, 1575, 1578, 1583- 1587, 1590, 1591-1593, 1598-1608, or 1610-1624.
  • substitutions e.g., conservative substitutions
  • the AAV capsid variant comprises an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to the amino acid sequence of any one of SEQ ID NOs: 943, 1021, 1024, 1027, 1112, 1142, 1214, 1232, 1254, 1300, 1310, 1327, 1331, 1342, 1419, 1453, 1533, 1538, 1539, 1575, 1578, 1583- 1587, 1590, 1591-1593, 1598-1608, or 1610-1624.
  • the AAV capsid variant comprises an amino acid sequence comprising one, two, or three but no more than four different amino acids relative to the amino acid sequence of any one of SEQ ID NOs: 943 or 2064-2080.
  • the amino acid sequence is present in loop VIII.
  • loop VIII comprises positions 571-592 numbered according to SEQ ID NO: 138.
  • loop VIII comprises positions 571-599, numbered according to SEQ ID NO: 982.
  • the amino acid sequence is present immediately subsequent to position 570, 571, 572, 573, 574, 575, or 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • the amino acid sequence replaces position 577 (e.g., T577), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • the AAV capsid variant comprises an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of YPAEVVQK (SEQ ID NO: 943).
  • the AAV capsid variant comprises an amino acid sequence comprising one, two, or three, but no more than four different amino acids that relative to the amino acid sequence of YPAEVVQK (SEQ ID NO: 943).
  • the AAV capsid variant comprises the amino acid sequence of any of the sequences provided in Tables 2A, 2B, 2C, 15 and 21.
  • the AAV capsid variant comprises the amino acid sequence of any of SEQ ID NOs: 943, 1021, 1024, 1027, 1112, 1142, 1214, 1232, 1254, 1300, 1310, 1327, 1331, 1342, 1419, 1453, 1533, 1538, 1539, 1575, 1578, 1583- 1587, 1590, 1591-1593, 1598-1608, or 1610-1624.
  • the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 943.
  • the amino acid sequence is present in loop VIII. In some embodiments, the amino acid sequence is present immediately subsequent to position 570, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces positions 571-579 (e.g., T571, N572, N573, Q574, S575, S576, T577, T578, and T579), relative to a reference sequence numbered according to SEQ ID NO 138.
  • positions 571-579 e.g., T571, N572, N573, Q574, S575, S576, T577, T578, and T579
  • the amino acid sequence is present immediately subsequent to position 570, and the amino acid sequence replaces positions 571-579 (e.g., T571, N572, N573, Q574, S575, S576, T577, T578, and T579), relative to a reference sequence numbered according to SEQ ID NO 138.
  • the amino acid sequence is present immediately subsequent to position 571, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • the amino acid sequence replaces positions 572-579 (e.g., N572, N573, Q574, S575, S576, T577, T578, and T579), relative to a reference sequence numbered according to SEQ ID NO 138.
  • the amino acid sequence is present immediately subsequent to position 572, and the amino acid sequence replaces positions 573-579 (e.g., N573, Q574, S575, S576, T577, T578, and T579), relative to a reference sequence numbered according to SEQ ID NO 138.
  • the amino acid sequence is present immediately subsequent to position 573, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • the amino acid sequence replaces positions 574-579 (e.g., Q574, S575, S576, T577, T578, and T579), relative to a reference sequence numbered according to SEQ ID NO 138.
  • the amino acid sequence is present immediately subsequent to position 573, and the amino acid sequence replaces positions 574-579 (e.g., Q574, S575, S576, T577, T578, and T579), relative to a reference sequence numbered according to SEQ ID NO 138.
  • the amino acid sequence is present immediately subsequent to position 574, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • the amino acid sequence replaces positions 575-579 (e.g., S575, S576, T577, T578, and T579), relative to a reference sequence numbered according to SEQ ID NO 138.
  • the amino acid sequence is present immediately subsequent to position 574, and the amino acid sequence replaces positions 575-579 (e.g., S575, S576, T577, T578, and T579), relative to a reference sequence numbered according to SEQ ID NO 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 575, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces positions 576-579 (e.g., S576, T577, T578, and T579), relative to a reference sequence numbered according to SEQ ID NO 138.
  • positions 575-579 e.g., S575, S576, T577, T578, and T579
  • the amino acid sequence is present immediately subsequent to position 575, and the amino acid sequence replaces positions 576-579 (e.g., S575, S576, T577, T578, and T579), relative to a reference sequence numbered according to SEQ ID NO 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces position 577 (e.g., T577), numbered relative to SEQ ID NO: 138.
  • the amino acid sequence replaces positions 577-579 (e.g., T577, T578, and T579), relative to a reference sequence numbered according to SEQ ID NO 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 576, and the amino acid sequence replaces position 577 (e.g., T577), relative to a reference sequence numbered according to SEQ ID NO 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 576, and the amino acid sequence replaces positions 577-579 (e.g., T577, T578, and T579), relative to a reference sequence numbered according to SEQ ID NO 138.
  • positions 577-579 e.g., T577, T578, and T579
  • the AAV capsid variant comprises the amino acid sequence of any of SEQ ID NOs: 943 or 946-966, wherein the amino acid sequence is present immediately subsequent to position 576, and wherein the amino acid sequence replaces position 577 (e.g., T577), numbered relative to SEQ ID NO: 138.
  • the AAV capsid variant (e.g., an AAV capsid variant described herein), comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 944, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto.
  • the AAV capsid variant described herein comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 944, or a nucleotide sequence comprising at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, insertions, or deletions, but no more than ten modifications, e.g., substitutions, insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 944.
  • the AAV capsid variant comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one, two, three, four, five, six, or seven, but no more than ten different nucleotides relative to the nucleotide sequence of SEQ ID NO: 944.
  • the nucleotide sequence encoding the AAV capsid variant comprises the nucleotide sequence of SEQ ID NO: 944, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto.
  • the nucleic acid sequence encoding the AAV capsid variant comprises a nucleotide sequence comprising at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, insertions, or deletions, but no more than ten modifications, e.g., substitutions, insertions, or deletions, relative to the nucleotide sequences of SEQ ID NO: 944.
  • the nucleotide sequence encoding an AAV capsid variant described herein comprises a nucleotide sequence comprising at least one, two, three, four, five, six, or seven, but no more than ten different nucleotides relative to the nucleotide sequence of SEQ ID NO: 944.
  • an AAV capsid variant described herein comprises the amino acid sequence of YPAEVVQK (SEQ ID NO: 943), wherein the amino acid sequence is present in loop VIII.
  • an AAV capsid variant described herein comprises the amino acid sequence of YPAEVVQK (SEQ ID NO: 943), wherein the amino acid sequence is present immediately subsequent to position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • an AAV capsid variant described herein comprises the amino acid sequence of YPAEVVQK (SEQ ID NO: 943), wherein the amino acid sequence of YPAEVVQK (SEQ ID NO: 943) replaces position 577 (e.g., T577), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • an AAV capsid variant described herein comprises the amino acid sequence of YPAEVVQK (SEQ ID NO: 943), wherein the amino acid sequence of YPAEVVQK (SEQ ID NO: 943) is present immediately subsequent to position 576, and wherein the amino acid sequence of YPAEVVQK (SEQ ID NO: 943) replaces position 577 (e.g., T577), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • an AAV capsid variant described herein comprises the amino acid Y at position 577, and further comprises the amino acid sequence of PAEVVQK (SEQ ID NO: 61), which is present immediately subsequent to position 577, numbered relative to SEQ ID NO: 138.
  • an AAV capsid variant described herein comprises the amino acid Y at position 577 and the amino acid sequence of PAEVVQK (SEQ ID NO: 20) at positions 578-584, numbered relative to SEQ ID NO: 982.
  • an AAV capsid variant described herein comprises the amino acid Y al position 577, and comprises the amino acid sequence of PAEVVQK (SEQ ID NO: 20), which is present immediately subsequent to position 577, numbered relative to SEQ ID NO: 982.
  • the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 739, or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.
  • the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 738, or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto. In some embodiments, the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 982, or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.
  • an AAV capsid variant described herein comprises the amino acid Y at position 577 and the amino acid sequence of PAEVVQK (SEQ ID NO: 20) at positions 578-584, numbered relative to SEQ ID NO: 982.
  • the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 739, or an amino acid sequence at least 95% (e.g., at least 96,
  • the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 738, or an amino acid sequence at least 95% (e.g., at least 96, 97,
  • the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 982, or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.
  • an AAV capsid variant described herein comprises the amino acid sequence of TNNQSSYPAEVVQKTA (SEQ ID NO: 1533), wherein the amino acid sequence is present in loop VIII.
  • the AAV capsid variant comprises TNNQSSYPAEVVQKTA (SEQ ID NO: 1533) and is present immediately subsequent to position 570, numbered relative to SEQ ID NO: 138 or 982, wherein YPAEVVQK (SEQ ID NO: 943) replaces position 577 (e.g., replaces T577) numbered relative to SEQ ID NO: 138.
  • an AAV capsid variant described herein comprises the amino acid Y at position 577 and the amino acid sequence of PAEVVQK (SEQ ID NO: 61) at positions 578-584, numbered relative to SEQ ID NO: 982.
  • the AAV capsid variant further one, two, three or all of an amino acid other than Q at position 574 (e.g., T, S, A, I, L, or H), an amino acid other than S at position 575 (e.g., G, A, L, T, or R), and/or an amino acid other than S at position 576 (e.g., K, L, R, A, Y, or T), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • an amino acid other than Q at position 574 e.g., T, S, A, I, L, or H
  • an amino acid other than S at position 575 e.g., G, A, L, T, or R
  • an amino acid other than S at position 576 e.g., K, L, R, A, Y, or T
  • the AAV capsid variant further comprises a Q at position 574, an S at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a T at position 574, an S at position 575, and/or a L at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an S at position 574, an S at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • the AAV capsid variant further comprises a Q at position 574, an S at position 575, and/or an R at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises Q at position 574, an S at position 575, and/or a K at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an A at position 574, a G at position 575, and/or an A at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • the AAV capsid variant further comprises an I at position 574, a G at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a Q at position 574, an A at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an A at position 574, an S at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • the AAV capsid variant further comprises an L at position 574, a G at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a Q at position 574, an S at position 575, and/or a T at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an H at position 574, an S at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • the AAV capsid variant further comprises an L at position 574, an S at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a Q at position 574, an R at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an S at position 574, an L at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • the AAV capsid variant further comprises an S at position 574, an L at position 575, and/or a Y at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an S at position 574, an A at position 575, and/or a T at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a Q at position 574, a T at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • the AAV capsid variant comprises amino acid other than Q at position 574 (c.g., S), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises S at position 574, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • the AAV capsid variant further comprises one or both of an amino acid other than T at position 571 (e.g., I or N), and/or an amino acid other than N at position 573 (e.g., T, S, or K), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • the AAV capsid variant further comprises an R at position 456, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • the AAV capsid variant further comprises a T at position 571, an N at position 572, and/or an N at position 573, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a T at position 571, an N at position 572, and/or a T at position 573, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an I at position 571, an N at position 572, and/or an N at position 573, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • the AAV capsid variant further comprises a T at position 571, an N at position 572, and/or an S at position 573, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an N at position 571, an N at position 572, and/or an N at position 573, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a T at position 571, an N at position 572, and/or a K at position 573, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • the AAV capsid variant further comprises an amino acid other than T at position 578 (e.g., P or N), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • the AAV capsid variant further comprises one or both of an amino acid other than T at position 578 (e.g., P or N), and/or an amino acid other than A at position 589 (e.g., D), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • the AAV capsid variant further comprises a T at position 578 and/or an A at position 588, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a P at position 578 and/or an A at position 588, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an N at position 578 and/or an A at position 588, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a T at position 578 and/or a D at position 579, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
  • the AAV capsid variant further comprises an amino acid other than T (e.g., Y) at position 577, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises Y at position 577, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. [00109] In some embodiments, the AAV capsid variant further comprises a modification, e.g., an insertion, substitution, and/or deletion in loop I, H, IV, and/or VI. In some embodiments, loop I, II, IV, VI, and VIII can be identified as described in Govindasamy et al.
  • additional modifications e.g., substitutions (e.g., conservative substitutions), insertions, and/or deletions can be introduced into an AAV capsid variant described herein at positions determined using a structural map of wild-type AAV5, e.g., a structural map described and generated by Govindasamy et al. et al. Structural Insights into Adeno- Associated Virus Serotype 5. Journal of Virology. 2013 Oct. 87(20): 11187-11199 (the contents of which are hereby incorporated herein by reference in their entirety) or Walters et al. “Structure of Adeno- Associated Virus Serotype 5,” Journal of Virology, 2004, 78(7):3361-3371 (the contents of which are hereby incorporated by reference in their entirety).
  • substitutions e.g., conservative substitutions
  • insertions, and/or deletions can be introduced into an AAV capsid variant described herein at positions determined using a structural map of wild-type
  • an AAV capsid variant described herein comprises a modification as described in Jose et al. “High-Resolution Structural Characterization of a New Adenoassociated Virus Serotype 5 Antibody Epitope toward Engineering Antibody-Resistant Recombinant Gene Delivery Vectors,” Journal of Virology, 2020, 93(1): e01394-18; Qian et al. “Directed Evolution of AAV Serotype 5 for Increased Hepatocyte Transduction and Retained Low Humoral Seroreactivity,” Molecular Therapy: Methods and Clinical Development, 2021, 20: 122-132; Afione et al.
  • the AAV capsid variant further comprises an amino acid sequence comprising at least one, two or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but not more than 30, 20 or 10 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, of the amino acid sequence of SEQ ID NO: 138.
  • the AAV capsid variant further comprises an amino acid sequence comprising at least one, two or three, but not more than 30, 20 or 10 different amino acids relative to the amino acid sequence of SEQ ID NO: 138.
  • the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 138, or an amino acid sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.
  • the AAV capsid variant further comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 137, or a sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.
  • the AAV capsid variant further comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one, two or three modifications, e.g., substitutions, insertions, or deletions, but not more than 30, 20 or 10 modifications, e.g., substitutions, insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 137.
  • the AAV capsid variant further comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one, two or three, but not more than 30, 20 or 10 different nucleotides, relative to the amino acid sequence of SEQ ID NO: 137.
  • the nucleotide sequence encoding the AAV capsid variant further comprises the nucleotide sequence of SEQ ID NO: 137, or a sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.
  • the nucleotide sequence encoding the AAV capsid variant further comprises a nucleotide sequence comprising at least one, two or three modifications, e.g., substitutions, insertions, or deletions, but not more than 30, 20 or 10 modifications, e.g., substitutions, insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 137.
  • the nucleotide sequence encoding the AAV capsid variant further comprises a nucleotide sequence comprising at least one, two or three, but not more than 30, 20 or 10 different nucleotides, relative to the amino acid sequence of SEQ ID NO: 137.
  • an AAV capsid variant of the present disclosure comprises an amino acid sequence as described herein, e.g., an amino acid sequence of an AAV capsid variant of TTN-002, TTN-003, TTN-004, TTN-005, or TTN-006, e.g., as described in Tables 3 and 4.
  • an AAV capsid variant of the present disclosure comprises an amino acid sequence as described herein, e.g., an amino acid sequence of an AAV capsid variant of TTN-002, e.g., as described in Tables 3 and 4.
  • an AAV capsid variant described herein comprises a VP1, VP2, and/or VP3 protein comprising an amino acid sequence described herein, e.g., an amino acid sequence of an AAV capsid variant of TTN-002, TTN-003, TTN-004, TTN-005, or TTN-006, e.g., as described in Tables 3 and 4.
  • an AAV capsid variant described herein comprises a VP1, VP2, and/or VP3 protein comprising an amino acid sequence described herein, e.g., an amino acid sequence of an AAV capsid variant of TTN-002, e.g., as described in Tables 3 and 4.
  • an AAV capsid variant described herein comprises an amino acid sequence encoded by a nucleotide sequence as described herein, e.g., a nucleotide sequence of an AAV capsid variant of TTN-002, e.g., as described in Tables 3 and 5.
  • a polynucleotide or nucleic acid encoding an AAV capsid variant, of the present disclosure comprises a nucleotide sequence described herein, e.g., a nucleotide sequence of an AAV capsid variant of TTN-002, e.g., as described in Tables 3 and 5.
  • an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 982, or an amino acid sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.
  • an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 982 or an amino acid sequence with at least 90% sequence identity thereto.
  • an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 982 or an amino acid sequence with at least 95% sequence identity thereto.
  • an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 982 or an amino acid sequence with at least 96% sequence identity thereto. In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 982 or an amino acid sequence with at least 97% sequence identity thereto. In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 982 or an amino acid sequence with at least 98% sequence identity thereto. In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 982 or an amino acid sequence with at least 99% sequence identity thereto.
  • an AAV capsid variant described herein comprises an amino acid sequence comprising at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but not more than 30, 20 or 10 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SEQ ID NO: 982.
  • the AAV capsid variant comprises an amino acid sequence comprising at least one, two or three, but not more than 30, 20 or 10 different amino acids, relative to the amino acid sequence of SEQ ID NO: 982.
  • an AAV capsid variant described herein comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 984, or a nucleotide sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.
  • an AAV capsid variant described herein comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one, two or three, but not more than 30, 20 or 10 different nucleotides, relative to the amino acid sequence of SEQ ID NO: 984.
  • an AAV capsid variant described herein comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one, two or three modifications, e.g., substitutions, insertions, or deletions, but not more than 30, 20 or 10 modifications, e.g., substitutions, insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 984.
  • the nucleotide sequence encoding an AAV capsid variant, described herein comprises the nucleotide sequence of SEQ ID NO: 984, or a nucleotide sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.
  • the nucleotide sequence encoding an AAV capsid variant described herein comprises the nucleotide sequence of SEQ ID NO: 984, or a nucleotide sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.
  • the nucleotide sequence encoding an AAV capsid variant described herein comprises a nucleotide sequence comprising at least one, two or three modifications, e.g., substitutions, insertions, or deletions, but not more than 30, 20 or 10 modifications, e.g., substitutions, insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 984.
  • the nucleotide sequence encoding an AAV capsid variant described herein comprises a nucleotide sequence comprising at least one, two or three, but not more than 30, 20 or 10 different nucleotides, relative to the amino acid sequence of SEQ ID NO: 984.
  • the nucleic acid sequence encoding an AAV capsid variant described herein is codon optimized.
  • an AAV capsid variant described herein comprises a VP1, VP2, VP3 protein, or a combination thereof.
  • an AAV capsid variant comprises the amino acid sequence corresponding to positions 137-731, e.g., a VP2, of SEQ ID NO: 982, or a sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.
  • the AAV capsid protein comprises the amino acid sequence corresponding to positions 193-731, e.g., a VP3, of SEQ ID NO: 982, or a sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.
  • the AAV capsid variant comprises the amino acid sequence corresponding to positions 1-731, e.g., a VP1, of SEQ ID NO: 982, or an amino acid sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.
  • an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 739, or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.
  • the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 738, or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.
  • the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 982, or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.
  • an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 739 (e.g., VP3). In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 738 (e.g., VP2). In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 982 (e.g., VP1).
  • an AAV capsid variant, described herein has an increased tropism for a CNS cell or tissue, e.g., a brain cell, brain tissue, spinal cord cell, or spinal cord tissue, relative to the tropism of a reference sequence comprising the amino acid sequence of SEQ ID NO: 138.
  • an AAV capsid variant described herein has an increased tropism for a CNS cell or tissue, e.g., a brain cell, brain tissue, spinal cord cell, or spinal cord tissue, relative to the tropism of a reference sequence comprising the amino acid sequence of SEQ ID NO: 982.
  • an AAV capsid variant described herein has an increased tropism for a CNS cell or tissue, e.g., a brain cell, brain tissue, spinal cord cell, or spinal cord tissue, relative to the tropism of a reference sequence comprising the amino acid sequence of SEQ ID NO: 139.
  • an AAV capsid variant described herein transduces a brain region, e.g., a temporal cortex, perirhinal cortex, globus pallidus, putamen, caudate, thalamus, hippocampus, geniculate nucleus, Purkinje Layer, deep cerebellar nuclei, and/or cerebellum.
  • the level of transduction is at least .5, 2.2, 2.4, 2.5, 2.6, 2.7, 3.0, 3.2, 3.5, 3.7, 4.0, 4.2, 4.5, 4.7, 4.9, 5, 10, 15, 20, 25, 30, or 35-fold greater as compared to a reference sequence of SEQ ID NO: 139.
  • an AAV capsid variant described herein is enriched at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 65-fold in the brain compared to a reference sequence of SEQ ID NO: 138. In some embodiments, an AAV capsid variant described herein is enriched at least about 10, 12, 15, 17, 20, 25, 30, 35, 40, 45, 50, 55, 60, 61, 62, 63, 64, or 65-fold in the brain compared to a reference sequence of SEQ ID NO: 138.
  • an AAV capsid variant described herein is enriched in the brain of at least two to three species, e.g., a non-human primate and rodent (e.g., mouse and/or rat) species, compared to a reference sequence of SEQ ID NO: 138.
  • a non-human primate and rodent e.g., mouse and/or rat
  • an AAV capsid variant described herein is enriched at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100-fold in the brain of at least two to three species, e.g., a non-human primate and rodent (e.g., mouse and/or rat) species, compared to a reference sequence of SEQ ID NO: 138 or 982.
  • the at least two to three species are Macaca fascicularis, Chlorocebus sabaeus, Callithrix jacchus, rat and/or mouse (e.g., BALB/c mice).
  • an AAV capsid variant described herein is enriched about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, or 225-fold in the brain compared to a reference sequence of SEQ ID NO: 982.
  • an AAV capsid variant described herein delivers an increased level of viral genomes to a brain region.
  • the level of viral genomes is increased by at least 1.5, 2.2, 2.4, 2.5, 2.6, 2.7, 3.0, 3.2, 3.5, 3.7, 4.0, 4.2, 4.5, 4.7, 4.9, or 5-fold, as compared to a reference sequence of SEQ ID NO: 139.
  • the brain region comprises a midbrain region (e.g., the hippocampus or thalamus) and/or the brainstem.
  • an AAV capsid variant described herein delivers an increased level of a payload to a brain region.
  • the level of the payload is increased by at least 20, 25, 30, 35-fold, as compared to a reference sequence of SEQ ID NO: 139.
  • the brain region comprises a temporal cortex, perirhinal cortex, globus pallidus, putamen, caudate, thalamus, hippocampus, geniculate nucleus, Purkinje Layer, deep cerebellar nuclei, cerebellum, or a combination thereof.
  • an AAV capsid variant described herein is enriched at least about 3, 3.5, 4.0, 4.5, 5, 5.0, 6.0, or 6.5-fold, in a spinal cord region compared to a reference sequence of SEQ ID NO: 139.
  • the spinal cord region comprises a cervical spinal cord region, a lumbar spinal cord region, a thoracic spinal cord region, or a combination thereof
  • an AAV capsid variant described herein shows preferential transduction in a brain region relative to the transduction in the dorsal root ganglia (DRG). In some embodiments an AAV capsid variant described herein shows preferential transduction in a brain region relative to the transduction in the liver.
  • DDG dorsal root ganglia
  • an AAV capsid variant described herein is capable of transducing neuronal cells.
  • an AAV capsid variant described herein is capable of transducing nonneuronal cells, e.g., glial cells (e.g., oligodendrocytes or astrocytes).
  • the AAV capsid variant is capable of transducing neuronal cells and non-neuronal cells, e.g., glial cells (e.g., oligodendrocytes or astrocytes).
  • the non-neuronal cells are glial cells (e.g., oligodendrocytes or astrocytes).
  • an AAV capsid variant described herein has decreased tropism for the liver.
  • an AAV capsid variant comprises a modification, e.g., substitution, insertion, or deletion, that results in reduced tropism (e.g., de-targeting) and/or activity in the liver.
  • the reduced tropism in the liver is compared to an otherwise similar capsid that does not comprise the modification, e.g., a wild-type capsid polypeptide.
  • an AAV capsid variant described comprises a modification, e.g., substitution, insertion, or deletion, that results in one or more of the following properties: (1) reduced tropism in the liver; (2) de -targeted expression in the liver; (3) reduced activity in the liver; and/or (4) reduced binding to galactose.
  • the reduction in any one, or all of properties (1 )-(3) is compared to an otherwise similar AAV capsid variant that does not comprise the modification.
  • the AAV capsid variant e.g., the AAV capsid variant having reduced tropism in the liver, comprises one or more of: an amino acid other than A, G, K, M, N, Q, R, S, and/or T at position 581; an amino acid other than A, C, H, I, K, S, T, and/or V at position 582; an amino acid other than A, G, H, K, M, N, Q, R, S, T, and/or V at position 583; an amino acid other than L, M, P, Q, R.
  • T and/or W at position 584; an amino acid other than F, H, I, K, M, T and/or Y at position 585; an amino acid other than E, G, H, L, M, N, Q, T, and/or W at position 586; an amino acid other than A, C, G, H, L, M, R, and/or S at position 587; an amino acid other than A, C, D, F, G, H, M, Q, S, V, W, and/or Y at position 588; and/or an amino acid other than A, C, E, G, H, M, N, P, Q, S, V, and/or W at position 589, all numbered relative to SEQ ID NO: 138.
  • an AAV capsid variant of the present disclosure is isolated, e.g., recombinant.
  • a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, of the present disclosure is isolated, e.g., recombinant.
  • polynucleotide sequences encoding any of the AAV capsid variants described above and AAV particles, vectors, and cells comprising the same.
  • an, AAV capsid variant of the present disclosure is isolated, e.g., recombinant.
  • a polynucleotide encoding an AAV capsid variant of the present disclosure is isolated, e.g., recombinant.
  • the present disclosure refers to structural capsid proteins (including VP1, VP2 and VP3) which are encoded by capsid (Cap) genes. These capsid proteins form an outer protein structural shell (i.e., capsid) of a viral vector such as AAV.
  • VP capsid proteins synthesized from Cap polynucleotides generally include a methionine as the first amino acid in the peptide sequence (Metl), which is associated with the start codon (AUG or ATG) in the corresponding Cap nucleotide sequence.
  • first-methionine (Metl) residue or generally any first amino acid (AA1) to be cleaved off after or during polypeptide synthesis by protein processing enzymes such as Met-aminopeptidases.
  • Met/AA-clipping often correlates with a corresponding acetylation of the second amino acid in the polypeptide sequence (e.g., alanine, valine, serine, threonine, etc.). Met-clipping commonly occurs with VP1 and VP3 capsid proteins but can also occur with VP2 capsid proteins.
  • Met/AA-clipping is incomplete, a mixture of one or more (one, two or three) VP capsid proteins comprising the viral capsid may be produced, some of which may include a Metl/AAl amino acid (Met+/AA+) and some of which may lack a Metl/AAl amino acid as a result of Met/AA- clipping (Met-/AA-).
  • Met/AA-clipping in capsid proteins see Jin, et al. Direct Liquid Chromatography/Mass Spectrometry Analysis for Complete Characterization of Recombinant Adeno- Associated Virus Capsid Proteins. Hum Gene Ther Methods. 2017 Oct. 28(5):255- 267; Hwang, et al. N-Terminal Acetylation of Cellular Proteins Creates Specific Degradation Signals. Science. 2010 February 19. 327(5968): 973-977; the contents of which are each incorporated herein by reference in their entirety.
  • references to capsid proteins is not limited to either clipped (Met-/AA-) or unclipped (Met+/AA+) and may, in context, refer to independent capsid proteins, viral capsids comprised of a mixture of capsid proteins, and/or polynucleotide sequences (or fragments thereof) which encode, describe, produce or result in capsid proteins of the present disclosure.
  • a direct reference to a “capsid protein” or “capsid polypeptide” may also comprise VP capsid proteins which include a Metl/AAl amino acid (Met+/AA+) as well as corresponding VP capsid proteins which lack the Metl/AAl amino acid as a result of Met/AA-clipping (Met-/AA-).
  • a reference to a specific “SEQ ID NO:” (whether a protein or nucleic acid) which comprises or encodes, respectively, one or more capsid proteins which include a Metl/AAl amino acid (Met+/AA+) should be understood to teach the VP capsid proteins which lack the Metl/AAl amino acid as upon review of the sequence, it is readily apparent any sequence which merely lacks the first listed amino acid (whether or not Metl/AAl).
  • VP1 polypeptide sequence which is 736 amino acids in length and which includes a “Metl” amino acid (Met+) encoded by the AUG/ATG start codon may also be understood to teach a VP1 polypeptide sequence which is 735 amino acids in length and which does not include the “Metl” amino acid (Met-) of the 736 amino acid Met+ sequence.
  • VP1 polypeptide sequence which is 736 amino acids in length and which includes an “AA1 ” amino acid (AA1 +) encoded by any NNN initiator codon may also be understood to teach a VP1 polypeptide sequence which is 735 amino acids in length and which does not include the “AA1” amino acid (AA1-) of the 736 amino acid AA1+ sequence.
  • references to viral capsids formed from VP capsid proteins can incorporate VP capsid proteins which include a Metl/AAl amino acid (Met+/AA1+), corresponding VP capsid proteins which lack the Metl/AAl amino acid as a result of Mct/AAl -clipping (Mct-/AA1-), and combinations thereof (Mct+/AA1+ and Mct-/AA1-).
  • an AAV capsid serotype can include VP1 (Met+/AA1+), VP1 (Met- /AA1-), or a combination of VP1 (Met+/AA1+) and VP1 (Met-/AA1-).
  • An AAV capsid serotype can also include VP3 (Met+/AA1+), VP3 (Met-/AA1-), or a combination of VP3 (Met+/AA1+) and VP3 (Met-/AA1-); and can also include similar optional combinations of VP2 (Met+/AA1) and VP2 (Met- /AA1-).
  • polynucleotide sequences encoding any of the AAV capsid variants described above and AAV particles, vectors, and cells comprising the same.
  • the AAV particle of the present disclosure serves as an expression vector comprising a viral genome which encodes an SMN protein.
  • expression vectors are not limited to AAV and may be adenovirus, retrovirus, lentivirus, plasmid, vector, or any variant thereof.
  • an AAV particle e.g., an AAV particle for the vectorized delivery of an SMN protein described herein, comprises a viral genome, e.g., an AAV viral genome (e.g., a vector genome or AAV vector genome).
  • the viral genome e.g., the AAV viral genome, further comprises an inverted terminal repeat (ITR) region, an enhancer, a promoter, an intron region, a Kozak sequence, an exon region, a nucleic acid encoding a transgene encoding a payload (e.g., an SMN protein described herein) with or without an enhancement element, a nucleotide sequence encoding a miR binding site (e.g., a miR183 binding site), a poly A signal region, or a combination thereof.
  • ITR Inverted Terminal Repeat
  • the viral genome may comprise at least one inverted terminal repeat (ITR) region.
  • ITR inverted terminal repeat
  • the AAV particles of the present disclosure comprise a viral genome with at least one ITR region and a payload region.
  • the viral genome has two ITRs. These two ITRs flank the payload region at the 5’ and 3’ ends.
  • the ITR functions as an origin of replication comprising a recognition site for replication.
  • the ITR comprises a sequence region which can be complementary and symmetrically arranged.
  • the ITR incorporated into a viral genome described herein may be comprised of a naturally occurring polynucleotide sequence or a recombinantly derived polynucleotide sequence.
  • the ITRs may be derived from the same serotype as the capsid, selected from any of the serotypes listed in Table 1, or a derivative thereof.
  • the ITR may be of a different serotype than the capsid.
  • the AAV particle has more than one ITR.
  • the AAV particle has a viral genome comprising two ITRs.
  • the ITRs are of the same serotype as one another.
  • the ITRs are of different serotypes.
  • Non-limiting examples include zero, one or both of the ITRs having the same serotype as the capsid.
  • both ITRs of the viral genome of the AAV particle are AAV2 ITRs.
  • each ITR may be 141 nucleotides in length.
  • each ITR may be 130 nucleotides in length.
  • the ITR comprises the nucleotide sequence of SEQ ID NO: 1 or 2, or a nucleotide sequence substantially identical (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical) to any of the aforesaid sequences.
  • the ITR comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a nucleotide sequence having one, two, or three but no more than four modifications, e.g., substitutions, relative to SEQ ID NO: 1 or SEQ ID NO: 2.
  • a viral genome encoding an SMN protein described herein comprises an ITR comprising the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence substantially identical (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical), which is present 5’ relative to the nucleotide sequence encoding the SMN protein.
  • a viral genome encoding an SMN protein described herein comprises an ITR comprising the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence substantially identical (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical), thereto which is present 3’ relative to the nucleotide sequence encoding the SMN protein.
  • the payload region of the viral genome comprises at least one element to enhance the transgene target specificity and expression.
  • elements to enhance the transgene target specificity and expression include promoters, endogenous miRNAs, post- transcriptional regulatory elements (PREs), polyadenylation (Poly A) signal sequences, upstream enhancers (USEs), CMV enhancers, and introns.
  • expression of the polypeptides in a target cell may be driven by a promoter, including but not limited to, a promoter that is species specific, inducible, tissue-specific, ubiquitous, or cell cycle-specific (e.g., as described in Parr et al., Nat. Med. '.1145-9 (1997); the contents of which are herein incorporated by reference in their entirety).
  • a promoter including but not limited to, a promoter that is species specific, inducible, tissue-specific, ubiquitous, or cell cycle-specific (e.g., as described in Parr et al., Nat. Med. '.1145-9 (1997); the contents of which are herein incorporated by reference in their entirety).
  • the viral genome comprises a that is sufficient for expression, e.g., in a target cell, of a payload (e.g., an SMN protein) encoded by a transgene.
  • a payload e.g., an SMN protein
  • the promoter is deemed to be efficient when it drives expression of the polypeptide(s) encoded in the payload region of the viral genome of the AAV particle.
  • the promoter is a promoter deemed to be efficient when it drives expression in the cell or tissue being targeted.
  • Promoters may be naturally occurring or non-naturally occurring.
  • Non-limiting examples of promoters include viral promoters, plant promoters and mammalian promoters.
  • the promoters may be human promoters.
  • the promoter may be truncated.
  • the viral genome comprises a promoter that results in expression in one or more, e.g., multiple, cells and/or tissues, e.g., a ubiquitous promoter.
  • a promoter which drives or promotes expression in most mammalian tissues includes, but is not limited to, human elongation factor la-subunit (EFla), cytomegalovirus (CMV) immediate-early enhancer and/or promoter, chicken 0-actin (CBA) and its derivative CAG, glucuronidase (GUSB), and ubiquitin C (UBC).
  • Tissue-specific expression elements can be used to restrict expression to certain cell types such as, but not limited to, CNS-specific promoters, B cell promoters, monocyte promoters, leukocyte promoters, macrophage promoters, pancreatic acinar cell promoters, endothelial cell promoters, lung tissue promoters, astrocyte promoters, or various specific nervous system cell- or tissue-type promoters which can be used to restrict expression to neurons, astrocytes, or oligodendrocytes, for example.
  • the viral genome comprises a nervous system specific promoter, e.g., a promoter that results in expression of a payload in a neuron, an astrocyte, and/or an oligodendrocyte.
  • tissue-specific expression elements for neurons include neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B-chain (PDGF-0), synapsin (Syn), synapsin 1 (Synl), methyl-CpG binding protein 2 (MeCP2), Ca 2+ /calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), neurofilament light (NFL) or heavy (NFH), -globin minigene n [ 2, preproenkephalin (PPE), enkephalin (Enk) and excitatory amino acid transporter 2 (EAAT2) promoters.
  • NSE neuron-specific enolase
  • tissue-specific expression elements for astrocytes include glial fibrillary acidic protein (GFAP) and EAAT2 promoters.
  • GFAP glial fibrillary acidic protein
  • EAAT2 promoters glial fibrillary acidic protein
  • a non-limiting example of a tissue-specific expression element for oligodendrocytes includes the myelin basic protein (MBP) promoter.
  • MBP myelin basic protein
  • Prion promoter represents an additional tissue specific promoter useful for driving protein expression in CNS tissue (see Loftus, Stacie K., et al. Human molecular genetics 11.24 (2002): 3107- 3114, the disclosure of which is incorporated by reference in its entirety).
  • the viral genome comprises a ubiquitous promoter.
  • ubiquitous promoters include CMV, CBA (including derivatives CAG, CB6, CBh, etc.), EFla, PGK, UBC, GUSB (hGBp), and UCOE (promoter of HNRPA2B1-CBX3).
  • the viral genome comprises an EF-lapromoter or EF-la promoter variant, e.g., as provided in Table 7.
  • the EF-lapromoter comprises the nucleotide sequence of any one of SEQ ID NOs: 26-35 or 987, 988, 990, 991, 995, 996, 998-1007, or any one of the nucleotide sequences in Table 7, a nucleotide sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions, relative to the nucleotide sequence of SEQ ID NOs: 26-35 or 987-1007, or a nucleotide sequence with at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 26-35 or 995, 996, 998-1007, or any one of the nucleotide sequences in Table 7.
  • the promoter is a ubiquitous promoter.
  • the ubiquitous promoter is as described in Yu et al. (Molecular Pain 2011, 7:63), Soderblom et al. (E. Neuro 2015), Gill et al., (Gene Therapy 2001, Vol. 8, 1539-1546), and Husain et al. (Gene Therapy 2009), each of which are incorporated by reference in their entirety.
  • a viral genome encoding an SMN protein described herein comprises a ubiquitous promoter, e.g., an EF- la promoter, an insulin promoter, a PGK promoter, a CBA promoter, or a functional variant, e.g., truncation, thereof.
  • the promoter is a tissue specific promoter. In some embodiments, the promoter is a cell specific promoter. In some embodiments, a viral genome encoding an SMN protein described herein comprises a neuron-specific promoter, e.g., a synapsin promoter, an Hb9 promoter, an MeCP2 promoter, or a functional variant thereof. In some embodiments the promoter is a motor neuron specific promoter, e.g., an Hb9 promoter or a functional variant thereof.
  • the promoter is not cell specific.
  • the promoter is a ubiquitin c (UBC) promoter. In some embodiments, the promoter is a 0-glucuronidase (GUSB) promoter. In some embodiments, the promoter is a neurofilament light (NFL) promoter. In some embodiments, the promoter is a neurofilament heavy (NFH) promoter. In some embodiments, the promoter is a scn8a promoter.
  • the promoter is a phosphoglycerate kinase 1 (PGK) promoter, or a functional variant thereof.
  • PGK phosphoglycerate kinase 1
  • the promoter is a synapsin promoter or a functional variant thereof.
  • the promoter is a MeCP2 promoter or a functional variant thereof.
  • the promoter is a chicken P-actin (CBA) promoter, or a functional variant thereof.
  • the promoter is a CB6 promoter, or a functional variant thereof.
  • the promoter is a CB promoter, or a functional variant thereof . In some embodiments, the promoter is a minimal CB promoter, or a functional variant thereof.
  • the promoter is a CBA promoter, or functional variant thereof. In some embodiments, the promoter is a minimal CBA promoter, or functional variant thereof.
  • the promoter is a cytomegalovirus (CMV) promoter, or a functional variant thereof.
  • CMV cytomegalovirus
  • the promoter is a CAG promoter, or a functional variant thereof.
  • the promoter is an EFla promoter or functional variant thereof, e.g., a truncated EFla promoter.
  • the promoter is a GFAP promoter (e.g., as described, for example, in Zhang, Min, et al. Journal of neuroscience research 86.13 (2008): 2848-2856, the disclosure of which is incorporated by reference in its entirety) to drive expression of an SMN polypeptide, or an SMN polypeptide and a splicing modulator (e.g., SMN and a UlsnRNA, SMN and an antisense molecule)) in astrocytes.
  • a GFAP promoter e.g., as described, for example, in Zhang, Min, et al. Journal of neuroscience research 86.13 (2008): 2848-2856, the disclosure of which is incorporated by reference in its entirety
  • a splicing modulator e.g., SMN and a UlsnRNA, SMN and an antisense molecule
  • the promoter is an endogenous promoter, e.g., endogenous SMN1 (e.g., human SMN1) promoter.
  • the promoter is an SMN promoter, e.g., an SMN1 promoter.
  • the promoter is an SMN1 promoter.
  • the promoter is an RNA pol III promoter.
  • the RNA pol III promoter is U6.
  • the RNA pol HI promoter is Hl.
  • the viral genome comprises two promoters.
  • the promoters are an EFl a promoter and a CMV promoter.
  • the viral genome comprises an enhancer element, a promoter and/or a 5’UTR intron.
  • the enhancer element may be, but is not limited to, a CMV enhancer
  • the promoter may be, but is not limited to, a CMV, CBA, UBC, GUSB, NSE, Synapsin, MeCP2, and GFAP promoter
  • the 5’UTR/intron may be, but is not limited to, SV40, and CBA-MVM.
  • the enhancer, promoter and/or intron used in combination may be: (1) CMV enhancer, CMV promoter, SV40 5’UTR intron; (2) CMV enhancer, CBA promoter, SV 40 5’UTR intron; (3) CMV enhancer, CBA promoter, CBA-MVM 5’UTR intron; (4) UBC promoter; (5) GUSB promoter; (6) NSE promoter; (7) Synapsin promoter; (8) MeCP2 promoter; and (9) GFAP promoter.
  • the viral genome comprises an enhancer.
  • the enhancer comprises a CMVie enhancer.
  • the viral genome comprises a CMVie enhancer and a CB promoter.
  • the viral genome comprises a CMVie enhancer and a CMV promoter (e.g., a CMV promoter region).
  • the viral genome comprises a CMVie enhancer, a CBA promoter or functional variant thereof, and an intron (e.g., a CAG promoter).
  • the viral genome comprises an engineered promoter. In another embodiments, the viral genome comprises a promoter from a naturally expressed protein.
  • the vector genome comprises at least one intron or a fragment or derivative thereof.
  • the at least one intron may enhance expression of an SMN protein and/or an splicing modulator or variant thereof
  • introns include, MVM (67-97 bps), F.IX truncated intron 1 (300 bps), P-globin SD/immunoglobulin heavy chain splice acceptor (250 bps), adenovirus splice donor/immunoglobin splice acceptor (500 bps), SV40 late splice donor/splice acceptor (19S/16S) (180 bps), and hybrid adenovirus splice donor/IgG splice acceptor (230 bps).
  • the viral genome e.g., a viral genome encoding an SMN protein described herein, may comprise an SV40 intron or fragment or variant thereof.
  • the viral genome e.g., a viral genome encoding an SMN protein described herein, may comprise a beta-globin intron or a fragment or variant thereof.
  • the intron comprises one or more human beta-globin sequences (e.g., including fragments/variants thereof).
  • a viral genome e.g., a viral genome encoding an SMN protein described herein, comprises a chimeric intron or functional variant thereof.
  • the viral genome e.g., a viral genome encoding an SMN protein described herein comprises an intron comprising the nucleotide sequence of SEQ ID NO: 3, a nucleotide sequence substantially identical (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical) to SEQ ID NO: 3, a nucleotide sequence comprising one, two, or three but no more than four modifications, e.g., substitutions, relative to the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence comprising one, two, or three but no more than four different nucleotides relative to the nucleotide sequence of SEQ ID NO: 3.
  • UTRs Untranslated Regions
  • a wild type untranslated region (UTR) of a gene is transcribed but not translated.
  • the 5’ UTR starts at the transcription start site and ends at the start codon and the 3’ UTR starts immediately following the stop codon and continues until the termination signal for transcription.
  • UTRs features typically found in abundantly expressed genes of specific target organs may be engineered into UTRs to enhance the stability and protein production.
  • a 5’ UTR from mRNA normally expressed in the liver e.g. , albumin, serum amyloid A, Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, or Factor VIII
  • albumin e.g. , albumin, serum amyloid A, Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, or Factor VIII
  • the viral genome encoding a transgene described herein comprises a Kozak sequence.
  • wild- type 5' untranslated regions include features that play roles in translation initiation.
  • Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes, are usually included in 5’ UTRs.
  • Kozak sequences have the consensus CCR(A/G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (ATG), which is followed by another ’G’.
  • the 5’ UTR in the viral genome includes a Kozak sequence.
  • the 5’ UTR in the viral genome does not include a Kozak sequence.
  • wild-type 3' UTRs are known to have stretches of adenosines and uridines embedded therein. These AU rich signatures are particularly prevalent in genes with high rates of turnover.
  • the AU rich elements (AREs) can be separated into three classes (Chen et al, 1995, the contents of which are herein incorporated by reference in their entirety): Class I AREs, such as, but not limited to, c-Myc and MyoD, contain several dispersed copies of an AUUUA motif within U-rich regions.
  • Class II AREs such as, but not limited to, GM-CSF and TNF-a, possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers.
  • Class 111 ARES such as, but not limited to, c-Jun and Myogenin, are less well defined. These U rich regions do not contain an AUUUA motif.
  • Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA.
  • HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo.
  • AREs 3' UTR AU rich elements
  • AREs 3' UTR AU rich elements
  • polynucleotides When engineering specific polynucleotides, e.g., payload regions of viral genomes, one or more copies of an ARE can be introduced to make polynucleotides less stable and thereby curtail translation and decrease production of the resultant protein.
  • AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein.
  • the 3’ UTR of the viral genome may include an oligo(dT) sequence for templated addition of a poly-A tail.
  • any UTR from any gene known in the art may be incorporated into the viral genome of the AAV particle. These UTRs, or portions thereof, may be placed in the same orientation as in the gene from which they were selected or they may be altered in orientation or location.
  • the UTR used in the viral genome of the AAV particle may be inverted, shortened, lengthened, or made with one or more other 5' UTRs or 3' UTRs known in the art.
  • the term “altered,” as it relates to a UTR means that the UTR has been changed in some way in relation to a reference sequence.
  • a 3' or 5' UTR may be altered relative to a wild type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides.
  • the viral genome of the AAV particle comprises at least one artificial UTR, which is not a variant of a wild type UTR.
  • the viral genome of the AAV particle comprises UTRs which have been selected from a family of transcripts whose proteins share a common function, structure, feature, or property.
  • Tissue- or cell-specific expression of the AAV viral particles of the invention can be enhanced by introducing tissue- or cell-specific regulatory sequences, e.g., promoters, enhancers, microRNA binding sites, e.g., a detargeting site.
  • tissue- or cell-specific regulatory sequences e.g., promoters, enhancers, microRNA binding sites, e.g., a detargeting site.
  • an encoded miR binding site can modulate, e.g., prevent, suppress, or otherwise inhibit, the expression of a gene of interest on the viral genome of the invention, based on the expression of the corresponding endogenous microRNA (miRNA) or a corresponding controlled exogenous miRNA in a tissue or cell, e.g., a non-targeting cell or tissue.
  • a miR binding site modulates, e.g., reduces, expression of the payload encoded by a viral genome of an AAV particle described herein in a cell or tissue where the corresponding mRNA is expressed.
  • the miR binding site modulates, e.g., reduces, expression of the encoded SMN protein in a cell or tissue of the DRG, liver, hematopoietic lineage, or a combination thereof.
  • the viral genome of an AAV particle described herein comprises a nucleotide sequence encoding a microRNA binding site, e.g., a detargeting site.
  • the viral genome of an AAV particle described herein comprises a nucleotide sequence encoding a miR binding site, a microRNA binding site series (miR BSs), or a reverse complement thereof.
  • the nucleotide sequence encoding the miR binding site series or the miR binding site is located in the 3’-UTR region of the viral genome (e.g., 3’ relative to the nucleic acid sequence encoding a payload), e.g., before the polyA sequence, 5’ -UTR region of the viral genome (e.g., 5’ relative to the nucleic acid sequence encoding a payload), or both.
  • the encoded miR binding site series comprise at least 1-5 copies, e.g., at least 1-3, 2-4, 3-5, 1, 2, 3, 4, 5 or more copies of a miR binding site (miR BS).
  • the encoded miR binding site series comprises 4 copies of a miR binding site. In some embodiments, all copies are identical, e.g., comprise the same miR binding site. In some embodiments, the miR binding sites within the encoded miR binding site series are continuous and not separated by a spacer. In some embodiments, the miR binding sites within an encoded miR binding site series are separated by a spacer, e.g. , a non-coding sequence. In some embodiments, the spacer is about 1 to 6 nucleotides or about 5 to 10 nucleotides, e.g., about 7-8 nucleotides, nucleotides in length. In some embodiments, the spacer is about 8 nucleotides in length.
  • the spacer sequence comprises one or more of (i) GGAT; (ii) CACGTG; (iii) GCATGC, or a repeat of one or more of (i)-(iii).
  • the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, but no more than four modifications of GATAGTTA.
  • the encoded miR binding site series comprise at least 1 -5 copies, e.g., at least 1-3, 2-4, 3-5, 1, 2, 3, 4, 5 or more copies of a miR binding site (miR BS). In some embodiments, at least 1, 2, 3, 4, 5, or all of the copies are different, e.g., comprise a different miR binding site.
  • the miR binding sites within the encoded miR binding site series are continuous and not separated by a spacer. In some embodiments, the miR binding sites within an encoded miR binding site series are separated by a spacer, e.g., a non-coding sequence.
  • the spacer is about 1 to 6 nucleotides or about 5 to 10 nucleotides, e.g., about 7-8 nucleotides or about 8 nucleotides, in length.
  • the spacer sequence comprises one or more of (i) GGAT; (ii) CACGTG; (iii) GCATGC, or a repeat of one or more of (i)-(iii).
  • the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, but no more than four modifications of GATAGTTA.
  • the encoded miR binding site is substantially identical (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical), to the miR in the host cell.
  • the encoded miR binding site comprises at least 1, 2, 3, 4, or 5 mismatches or no more than 6, 7, 8, 9, or 10 mismatches to a miR in the host cell.
  • the mismatched nucleotides are contiguous. In some embodiments, the mismatched nucleotides are non-contiguous. In some embodiments, the mismatched nucleotides occur outside the seed region- binding sequence of the miR binding site, such as at one or both ends of the miR binding site. In some embodiments, the encoded miR binding site is 100% identical to the miR in the host cell.
  • the nucleotide sequence encoding the miR binding site is substantially complimentary (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% complementary), to the miR in the host cell.
  • the sequence complementary to the nucleotide sequence encoding the miR binding site comprises at least 1, 2, 3, 4, or 5 mismatches or no more than 6, 7, 8, 9, or 10 mismatches relative to the corresponding miR in the host cell.
  • the mismatched nucleotides are contiguous. In some embodiments, the mismatched nucleotides are non-contiguous.
  • the mismatched nucleotides occur outside the seed region-binding sequence of the miR binding site, such as at one or both ends of the miR binding site.
  • the encoded miR binding site is 100% complementary to the miR in the host cell.
  • the encoded miR binding site or the encoded miR binding site series is about 10 to about 125 nucleotides in length, e.g., about 10 to 50 nucleotides, 10 to 100 nucleotides, 50 to 100 nucleotides, 50 to 125 nucleotides, or 100 to 125 nucleotides in length.
  • an encoded miR binding site or the encoded miR binding site series is about 7 to about 28 nucleotides in length, e.g., about 8-28 nucleotides, 7-28 nucleotides, 8-18 nucleotides, 12-28 nucleotides, 20-26 nucleotides, 22 nucleotides, 24 nucleotides, or 26 nucleotides in length, and optionally comprises at least one consecutive region (e.g., 7 or 8 nucleotides) complementary (e.g., full complementary or partially complementary) to the seed sequence of a miRNA (e.g., a miR122, a miR142, a miR-1, a miR183).
  • a miRNA e.g., a miR122, a miR142, a miR-1, a miR183.
  • the encoded miR binding site is complementary (e.g., fully complementary or partially complementary) to a miR expressed in liver or hepatocytes, such as miR122.
  • the encoded miR binding site or encoded miR binding site series comprises a miR122 binding site sequence.
  • the encoded miR122 binding site comprises the nucleotide sequence of ACAAACACCATTGTCACACTCCA (SEQ ID NO: 1865), or a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95%, at least 99%, or 100% sequence identity, or having at least one, two, three, four, five, six, or seven modifications but no more than ten modifications to SEQ ID NO: 1865, e.g., wherein the modification can result in a mismatch between the encoded miR binding site and the corresponding miRNA.
  • the viral genome comprises at least 3, 4, or 5 copies of the encoded miR122 binding site, e.g., an encoded miR122 binding site series, optionally wherein the encoded miR122 binding site series comprises the nucleotide sequence of:
  • At least two of the encoded miR122 binding sites are connected directly, e.g., without a spacer.
  • at least two of the encoded miR122 binding sites are separated by a spacer, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length, which is located between two or more consecutive encoded miR122 binding site sequences.
  • the spacer is about 1 to 6 nucleotides or about 5 to 10 nucleotides, e.g., about 7-8 nucleotides or about 8 nucleotides, in length.
  • the spacer sequence comprises one or more of (i) GGAT; (ii) CACGTG; (iii) GCATGC, or a repeat of one or more of (i)-(iii).
  • the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, but no more than four modifications of GATAGTTA.
  • the encoded niiR binding site is complementary (e.g., fully or partially complementary) to a miR expressed in the heart.
  • the encoded miR binding site or encoded miR binding site series comprises a miR-1 binding site.
  • the encoded miR- 1 binding site comprises the nucleotide sequence of ATACATACTTCTTTACATTCCA (SEQ ID NO: 4679), a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95%, at least 99%, or 100% sequence identity, or having at least one, two, three, four, five, six, or seven modifications but no more than ten modifications to SEQ ID NO: 4679, e.g., wherein the modification can result in a mismatch between the encoded miR binding site and the corresponding miRNA.
  • the viral genome comprises at least 2, 3, 4, or 5 copies of the encoded miR-1 binding site, e.g., an encoded miR-1 binding site series.
  • the at least 2, 3, 4, or 5 copies (e.g., 2 or 3 copies) of the encoded miR-1 binding site are continuous (e.g., not separated by a spacer) or separated by a spacer.
  • the spacer is about 1 to 6 nucleotides or about 5 to 10 nucleotides, e.g., about 7-8 nucleotides or about 8 nucleotides, in length.
  • the spacer sequence comprises one or more of (i) GGAT; (ii) CACGTG; (iii) GCATGC, or a repeat of one or more of (i)-(iii).
  • the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, but no more than four modifications of GATAGTTA.
  • the encoded miR binding site is complementary (e.g., fully complementary or partially complementary) to a miR expressed in hematopoietic lineage, including immune cells (e.g., antigen presenting cells or APC, including dendritic cells (DCs), macrophages, and B-lymphocytes).
  • the encoded miR binding site is complementary (e.g., fully complementary or partially complementary) to a miR expressed in hematopoietic lineage comprises a nucleotide sequence disclosed, e.g., in US 2018/0066279, the contents of which are incorporated by reference herein in its entirety.
  • the encoded miR binding site or encoded miR binding site series comprises a miR-142-3p binding site sequence.
  • the encoded miR-142-3p binding site comprises the nucleotide sequence of TCCATAAAGTAGGAAACACTACA (SEQ ID NO: 1869), a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95%, at least 99%, or 100% sequence identity, or having at least one, two, three, four, five, six, or seven modifications but no more than ten modifications to SEQ ID NO: 1842, e.g., wherein the modification can result in a mismatch between the encoded miR binding site and the corresponding miRNA.
  • the viral genome comprises at least 3, 4, or 5 copies of an encoded miR-142-3p binding site, e.g., an encoded miR-142-3p binding site series.
  • the at least 3, 4, or 5 copies (e.g., 4 copies) of the encoded miR-142-3p binding site are continuous (e.g., not separated by a spacer) or separated by a spacer.
  • the spacer is about 1 to 6 nucleotides or about 5 to 10 nucleotides, e.g., about 7-8 nucleotides or about 8 nucleotides, in length.
  • the spacer sequence comprises one or more of (i) GGAT; (ii) CACGTG; (iii) GCATGC, or a repeat of one or more of (i)-(iii).
  • the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, but no more than four modifications of GATAGTTA.
  • the encoded miR binding site is complementary (e.g., fully complementary or partially complementary) to a miR expressed in a DRG (dorsal root ganglion) neuron, e.g., a miR183, a miR182, and/or miR96 binding site.
  • the encoded miR binding site is complementary (e.g., fully complementary or partially complementary) to a miR expressed in expressed in a DRG neuron.
  • the encoded miR binding site comprises a nucleotide sequence disclosed, e.g., in W02020/132455, the contents of which are incorporated by reference herein in its entirety.
  • the encoded miR binding site or encoded miR binding site series comprises a miR183 binding site sequence.
  • the encoded miR183 binding site comprises the nucleotide sequence of AG T GAAT T C T AC C AG T G C CAT A (SEQ ID NO: 1847), or a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95%, at least 99%, or 100% sequence identity, or having at least one, two, three, four, five, six, or seven modifications but no more than ten modifications to SEQ ID NO: 1847, e.g., wherein the modification can result in a mismatch between the encoded miR binding site and the corresponding miRNA.
  • the sequence complementary (e.g., fully complementary or partially complementary) to the seed sequence corresponds to the double underlined of the encoded miR- 183 binding site sequence.
  • the viral genome comprises at least comprises at least 3, 4, or 5 copies (e.g., 4 copies) of the encoded miR183 binding site, e.g., an encoded miR183 binding site.
  • the viral genome comprises at least comprises 4 copies of the encoded miR183 binding site, e.g., an encoded miR183 binding site comprising 4 copies of a miR183 binding site.
  • the at least 3, 4, or 5 copies (e.g., 4 copies) of the encoded miR183 binding site are continuous (e.g., not separated by a spacer) or separated by a spacer.
  • the spacer is about 1 to 6 nucleotides or about 5 to 10 nucleotides, e.g., about 7-8 nucleotides or about 8 nucleotides, in length.
  • the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, but no more than four modifications of GATAGTTA.

Abstract

The disclosure provides compositions and methods for the preparation, manufacture and use of an adeno-associated virus (AAV) particle for the vectorized delivery of an SMN protein, e.g., an SMN1 protein.

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT OF SPINAL MUSCULAR
ATROPHY RELATED DISORDERS
RELATED APPLICATIONS
[001] This application claims priority to U.S. Provisional Application No. 63/351,058 filed on June 10, 2022, the entire contents of which are incorporated by reference in its entirety.
FIELD OF THE DISCLOSURE
[002] Described herein arc compositions and methods relating to polynucleotides, e.g., polynucleotides encoding survival motor neuron (SMN) proteins and peptides for use in the treatment of Spinal Muscular Atrophy and related disorders, including Werdnig-Hoffmann disease and Kugelberg- Welander disease (collectively, “SMA-related disorders”). In some embodiments, compositions may be delivered in an adeno-associated viral (AAV) vector. In other embodiments, compositions described herein, may be used to treat a subject in need thereof, such as a human subject diagnosed with SMA-related disorders or other condition resulting from a deficiency in the quantity and/or function of SMN protein.
BACKGROUND
[003] Spinal Muscular Atrophy (SMA) is a collection of inherited and acquired central nervous system (CNS) diseases characterized by motor neuron degeneration and loss along the length of the entire spinal cord cells of the spinal cord leading to progressive symmetrical limb and trunk paralysis associated with muscular atrophy. The cell body of these neurons is located in the spinal cord of the central nervous system (CNS) and their nerve extensions, the axons, leave the CNS to innervate muscle fibers in order to generate movement.
[004] SMA is the second most common autosomal recessive disorder with an incidence of approximately 1 in 10,000 live births. Childhood SMA is classically subdivided into three clinical groups on the basis of age of onset and clinical course. The acute form of Werdnig-Hoffmann disease (Type I) is characterized by severe generalized muscle weakness and hypotonia at birth or in the 3 months following birth. Death, from respiratory failure, usually occurs within the first two years. This disease may be distinguished from the intermediate (Type II) and juvenile (Type III, Kugelberg- Welander disease) forms. Type II children were able to sit but unable to stand or walk unaided, and they live beyond 4 years. Type III patients had proximal muscle weakness, starting after the age of two. In addition, there is known to exist a slowly evolving adult form of SMA, sometimes referred to as SMA IV.
[005] SMA is linked to a genetic defect in the SMN1 gene that encodes the Survival of Motor Neuron (SMN) protein. The SMN protein is ubiquitously expressed and required by all cells and tissue types plays important roles in multiple fundamental cellular homeostatic pathways, including a well- characterized role in the assembly of the spliceosome and biogenesis of ribonucleoproteins. More recent studies have shown that SMN is also involved in other housekeeping processes, including mRNA trafficking and local translation, cytoskeletal dynamics, endocytosis and autophagy. Moreover, SMN has been shown to influence mitochondria and bioenergetic pathways as well as regulate function of the ubiquitin-proteasome system. The full-length, 294 amino acids, 38 kDa, human isoform of SMN (also known as FL-SMN, referred to as SMN hereafter) is mainly transcribed from the telomeric SMN1 gene, located on chromosome 5ql3. SMN1 contains nine exons, 1, 2a, 2b, 3, 4, 5, 6, 7 and 8, with exon 8 remaining untranslated. Chaytow et al., (Cell Mol Life Sci. 2018: 75(21): 3877-3894).
[006] SMN2 differs from SMN1 at 5 bases, and a C-to-T transition in exon 7 of SMN2 favors skipping of exon 7 during splicing, resulting in the majority of SMN2 products being a truncated isoform referred as SMNA7. SMNA7 is highly unstable and quickly subjected to the ubiquitin-tin-proteasome pathway for degradation.
[007] SMA characterized by a homozygous loss of function mutation in the survival motor neuron gene SMN1 on 5ql3, while retaining the modifying SMN2 gene. The SMN2 gene has a similar structure to SMN1, but only a small amount (10%) of the SMN protein it produces is fully functional. This low level of SMN protein is not effective enough to sustain the survival of motor neurons in the CNS.
[008] The correlation between loss of SMN function and disease severity makes SMA a potential target for gene therapy. Previous studies involving administration of an adeno-associated virus, AAV8-hSMN, to the CNS (central nervous system) in SMA-mousc models demonstrated expression of SMN in the spinal cord and that the SMA phenotype could be rescued; however, only modest preservation in the number of motor neurons was produced and long-term survival was not achieved. (Passini et al., 2010, J Clin Invest 120: 1253-1264).
[009] Although several therapies for SMA have been developed, there remains a need for treatments that increase intracellular SMN activity in motor neurons involved in spinal muscular atrophy for patients having different levels of disease severity.
SUMMARY OF THE DISCLOSURE
[0010] The present disclosure addresses these challenges by providing AAV-based compositions and methods for treating SMN deficiency in patients. Disclosed herein are compositions and methods directed to AAV-based gene delivery of SMN to ameliorate loss-of-function and to increase SMN protein levels. The compositions and methods are useful to improve motor neuron function, and to slow, halt, or reverse neurodegenerative and other symptoms of SMA-related disorders (e.g., Werdni-Hoffman disease, Dubowitz disease, Kugelberg-Welander disease), in a subject (e.g., a subject having a mutation in an SMN1 gene).
[0011] Accordingly, in one aspect, the present disclosure provides an AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant, e.g., an AAV5 capsid variant, comprises an amino acid other than T at position 577 (e.g., Y, N, or C), numbered relative to SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises a Y at position 577, numbered relative to SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises a N at position 577, numbered relative to SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises a C at position 577, numbered relative to SEQ ID NO: 138.
[0012] In another aspect, the present disclosure provides an AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant, e.g., a variant of the wild-type AAV5 capsid, comprises more than one amino acid that replaces the threonine (T) at position 577, numbered relative to SEQ ID NO: 138. In some embodiments, an insert of two, three, four, five, six, seven, eight, nine, or ten amino acids replaces the T at position 577, numbered relative to SEQ ID NO: 138. In some embodiments an insert of eight amino acids replaces the T at position 577, numbered relative to SEQ ID NO: 138.
[0013] In yet another aspect, the present disclosure provides an AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant comprises an amino acid sequence having the following formula: [N2]-[N3], wherein (i) [N2] comprises positions XI, X2, X3, X4, and X5, wherein: (a) position XI is Y, N, or C; (b) position X2 is P, K, T, or Q; (c) position X3 is A or P; (d) position X4 is E, S, or A; and (e) position X5 is V, L, or E; and (ii) [N3] comprises the amino acid sequence of VQK, EQK, VKK, VHK, VQQ, or LQK; ; and optionally wherein the AAV capsid variant comprises an amino acid modification, e.g., a conservative substitution, of any of the aforesaid amino acids in (i) and/or (ii); optionally wherein [N2]-[N3] is present immediately subsequent to position 576, numbered relative to SEQ ID NO: 138 and replaces position 577, numbered relative to SEQ ID NO: 138. In some embodiments, [N2] comprises Y at position XI. In some embodiments, [N2] comprises P at position X2. In some embodiments, [N2] comprises A at position X3. In some embodiments, [N2] comprises E at position X4. In some embodiments, [N2] comprises V at position X5. In some embodiments, the amino acid sequence of [N3] is VQK. In some embodiments, XI of [N2] is present at position 577, X2 of [N2] is present at position 578, X3 of [N2] is present at position 579, X4 of [N2] is present at position 580, and of X5 of [N2] is present at position 581, numbered according to SEQ ID NO: 982. In some embodiments, [N2] is present at positions 577-581, numbered according to SEQ ID NO: 982. In some embodiments, [N3] is present at positions 582-584, numbered according to SEQ ID NO: 982. In some embodiments, [N2]-[N3] is present at positions 577-584, numbered according to SEQ ID NO: 982.
[0014] In some embodiments, the amino acid sequence of [N2] consists of YPAEV (SEQ ID NO: 39). In some embodiments, the amino acid sequence of [N3] consists of VQK. In some embodiments, [N2]-[N3] replaces the threonine (T) at position 577 of wild-type AAV5, e.g., SEQ ID NO: 138. In some embodiments, [N2]-[N3] replaces the threonine (T) at position 577 of wild-type AAV5, e.g., SEQ ID NO: 138, and the amino acid sequence of [N2] consists of YPAEV (SEQ ID NO: 39), and the amino acid sequence of [N3] consists of VQK. In some embodiments, [N2] is present at positions 577-581, numbered according to SEQ ID NO: 982. In some embodiments, [N3] is present at positions 582-584, numbered according to SEQ ID NO: 982. In some embodiments, [N2]-[N3] is present at positions 577- 584, numbered according to SEQ ID NO: 982.
[0015] In another aspect, the present disclosure AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant comprises one, two, three, or all of: (i) an [NO], wherein the amino acid sequence of [NO] comprises TNN, TNT, INN, TNS, NNN, or TNK; (ii) an [Nl], wherein the amino acid sequence of [Nl] comprises QSS, QSK, TSL, SSS, QSR, AGA, IGS, QAS, ASS, LGS, QST, HSS, LSS, or QRS; (iii) an [N2], wherein the amino acid sequence of [N2] comprises YPAEV (SEQ ID NO: 39), YPPSL (SEQ ID NO: 40), NKAEV (SEQ ID NO: 41), YTAEV (SEQ ID NO: 42), YPAEE (SEQ ID NO: 43), YQAEV (SEQ ID NO: 44), YTPSL (SEQ ID NO: 45), YPAAV (SEQ ID NO: 46), NPAEV (SEQ ID NO: 47), CPAEV (SEQ ID NO: 48, or YQAEE (SEQ ID NO: 49); (iv) an [N3], wherein the amino acid sequence of [N3] comprises VQK, EQK, VKK, VHK, VQQ, or LQK; and/or (v) an [N4], wherein the amino acid sequence of [N4] comprises TA, PA, or NA; and optionally wherein the AAV capsid variant comprises an amino acid modification, e.g., a conservative substitution, of any of the aforesaid amino acids in (i)-(v); optionally wherein [NO] is present immediately subsequent to position 570, numbered relative to SEQ ID NO: 138. In some embodiments, the amino acid sequence of [NO] is TNN. In some embodiments, the amino acid sequence of [Nl] is QSS. In some embodiments, the amino acid sequence of [N2] is YPAEV (SEQ ID NO: 39). In some embodiments, the amino acid sequence of [N3] is VQK. In some embodiments, the amino acid sequence of [N4] is TA. In some embodiments, the amino acid sequence of [NO] is TNN, the amino acid sequence of [Nl] is QSS, the amino acid sequence of [N2] is YPAEV (SEQ ID NO: 39), the amino acid sequence of [N3] is VQK, and/or the amino acid sequence of [N4] is TA. In some embodiments, the amino acid sequence of [NO] is TNN, the amino acid sequence of [Nl] is QSS, the amino acid sequence of [N2] is YPAEV (SEQ ID NO: 39), the amino acid sequence of [N3] is VQK, and the amino acid sequence of [N4] is TA. In any of these embodiments, [NO] is present immediately subsequent to position 570, numbered relative to SEQ ID NO: 138. In any of these embodiments, [NO] replaces positions 571- 573 (e.g., T571, N572, and N573), numbered relative to SEQ ID NO: 138. In any of these embodiments, [NO] is present immediately subsequent to position 570, and [NO] positions 571-573 (e.g., T571, N572, and N573), numbered relative to SEQ ID NO: 138. In any of these embodiments, [Nl] is present immediately subsequent to position 573, numbered relative to SEQ ID NO: 138. In any of these embodiments, [Nl] replaces positions 574-576 (e.g., Q574, S575, and S576), numbered relative to SEQ ID NO: 138. In any of these embodiments, [Nl] is present immediately subsequent to position 573, and [Nl] replaces positions 574-576 (e.g., Q574, S575, and S576), numbered relative to SEQ ID NO: 138. In any of these embodiments, [N2] is present immediately subsequent to position 576, numbered relative to SEQ ID NO: 138. In any of these embodiments, [N2] replaces position 577 (e.g., T577), numbered relative to SEQ ID NO: 138. In any of these embodiments, [N2] is present immediately subsequent to position 576, and [N2] replaces position 577 (e.g., T577), numbered relative to SEQ ID NO: 138. In any of these embodiments, [N2]-[N3] is present immediately subsequent to position 576, numbered relative to SEQ ID NO: 138. In any of these embodiments, [N2]-[N3] replaces position 577 (e.g., T577), numbered relative to SEQ ID NO: 138. In any of these embodiments, [N2]-[N3] is present immediately subsequent to position 576, and [N2]-[N3] replaces position 577 (e.g., T577), numbered relative to SEQ ID NO: 138. In any of these embodiments, [N2]-[N3]-[N4] replaces positions 577-579 (e.g., T577, T578, and A579), numbered relative to SEQ ID NO: 138. In any of these embodiments, [N2]-[N3]-[N4] is present immediately subsequent to position 576, and [N2J-[N3J- [N4J replaces positions 577-579 (e.g., T577, T578, and A579), numbered relative to SEQ ID NO: 138. In any of these embodiments, [N0|- [N1]-[N2]-[N3]-[N4] is present immediately subsequent to position 570, numbered relative to SEQ ID NO: 138. In some embodiments, [N0]-[Nl]-[N2]-[N3]-[N4] replaces positions 571-579 (e.g., T571, N572, N573, Q574, S575, S576, T577, T578, and A579), numbered relative to SEQ ID NO: 138. In some embodiments, [N0]-[N1 ]-[N2]-[N3]-[N4] is present immediately subsequent to position 570, and [NO]-[N1]-[N2]-[N3]-[N4] replaces positions 571-579 (e.g., T571, N572, N573, Q574, S575, S576, T577, T578, and A579), numbered relative to SEQ ID NO: 138. For example, in some embodiments, [NO]-[N1]-[N2]-[N3]-[N4] is TNNQSSYPAEVVQKTA (SEQ ID NO: 1533) and is present immediately subsequent to position 570, numbered relative to SEQ ID NO: 138, wherein [N2]-[N3] (YPAEVVQK; SEQ ID NO: 943) replaces position 577 (e.g., replaces T577), numbered relative to SEQ ID NO: 138. In some embodiments, [NO] is present at positions 571-573, numbered according to SEQ ID NO: 982. In some embodiments, [Nl] is present at positions 574-576, numbered according to SEQ ID NO: 982. In some embodiments, [N2] is present at positions 577-581, numbered according to SEQ ID NO: 982. In some embodiments, [N3] is present at positions 582-584, numbered according to SEQ ID NO: 982. In some embodiments, [N4] is present at positions 585-586, numbered according to SEQ ID NO: 982. In some embodiments, [N2]-[N3] is present at positions 577-584, numbered according to SEQ ID NO: 982. In some embodiments, [NO]-[N1]-[N2]-[N3]-[N4] is present at positions 571-586, numbered according to SEQ ID NO: 982.
[0016] In yet another aspect, the present disclosure provides an AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant comprises the formula [B]-[C], wherein: (i) [B] comprises positions XI, X2, and X3, wherein: (a) position XI is Q, T, S, A, I, L, or H; (b) position X2 is S, G, or A; and (c) position X3 is S, K, L, R, or A: and (ii) [C] comprises the amino acid sequence of YPAEVVQK (SEQ ID NO: 943); and optionally wherein the AAV capsid variant comprises an amino acid modification, e.g., a conservative substitution, of any of the aforesaid amino acids in (i) and/or (ii); and further optionally wherein [B] is present immediately subsequent to position 573, numbered relative to SEQ ID NO: 138.
[0017] In some embodiments, [B] comprises Q at position XI. In some embodiments, [B] comprises S at position X2. In some embodiments, [B] comprises S at position X3. In some embodiments, the amino acid sequence of [B] is QSS. In some embodiments, [B] is present immediately subsequent to position 573, relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, [B] replaces positions 574-576 (e.g., Q574, S575, and S576), relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, [BJ is present immediately subsequent to position 573, and [B] replaces positions 574-576 (e.g., Q574, S575, and S576), relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, [C] is present immediately subsequent to position 576, relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, [C] replaces position 577 (e.g., T577), relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, [C] is present immediately subsequent to position 576, and [C] replaces position 577 (e.g., T577), relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, [B]-[C] is present immediately subsequent to position 573, relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, [B]-[C] replaces positions 574-577 (e.g., Q574, S575, S576, and T577), relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, [B]-[C] is present immediately subsequent to position 573, and [B]-[C] replaces positions 574-577 (e.g., Q574, S575, S576, and T577), relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, XI of [B] is present at position 574, X2 of [B] is present at position 575, and X3 of [B] is present at position 576, numbered according to SEQ ID NO: 982. In some embodiments, [B] is present at positions 574-576, numbered according to SEQ ID NO: 982. In some embodiments, [C] is present at positions 577-584, numbered according to SEQ ID NO: 982. In some embodiments, [B]-[C] is present at positions 574-584, numbered according to SEQ ID NO: 982.
[0018] In another aspect, the present disclosure provides an AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant comprises one, two, three, or all of (i) an [A], wherein the amino acid sequence of [A] comprises TNN, TNT, INN, NNN, TNS, or TNK; (ii) a [B], wherein the amino acid sequence of [B] comprises QSS, TSL, SSS, QSR, QSK, AGA, IGS, QAS, ASS, LGS, or HSS; (iii) a [C], wherein [C] comprises the amino acid sequence of YPAEVVQK (SEQ ID NO: 943); and (iv) a [D], wherein [D] comprises the amino acid sequence of TA or PA; and optionally wherein the AAV capsid variant comprises an amino acid modification, e.g., a conservative substitution, of any of the aforesaid amino acids in (i)-(v); optionally wherein [A] is present immediately subsequent to position 570, numbered relative to SEQ ID NO: 138 and [C] replaces position 577, numbered relative to SEQ ID NO: 138. In some embodiments, the amino acid sequence of [A] is TNN. In some embodiments, the amino acid sequence of [B] is QSS. In some embodiments, the amino acid sequence of [A] is TNN and the amino acid sequence of [B] is QSS. In some embodiments, the amino acid sequence of [A] is TNN, the amino acid sequence of [B] is QSS, and the amino acid sequence of [C] is YPAEVVQK (SEQ ID NO: 943). In some embodiments, the amino acid sequence of [A] is TNN, the amino acid sequence of [B] is QSS, the amino acid sequence of [C] is YPAEVVQK (SEQ ID NO: 943), and the amino acid sequence of [D] is TA. In any of these embodiments, [A] is present immediately subsequent to position 570, numbered relative to SEQ ID NO: 138. In any of these embodiments, [A] replaces positions 571-573 (e.g., T571, N572, and N573) numbered relative to SEQ ID NO: 138. In any of these embodiments, [A] is present immediately subsequent to position 570, and [A] replaces positions 571-573 (e.g., T571, N572, and N573) numbered relative to SEQ ID NO: 138. In any of these embodiments, [B J is present immediately subsequent to position 573, relative to a reference sequence numbered according to SEQ ID NO: 138. In any of these embodiments, [B| replaces positions 574-576 (e.g., Q574, S575, and S576), numbered relative to SEQ ID NO: 138. In any of these embodiments, [B] is present immediately subsequent to position 573, and [B] replaces positions 574-576 (e.g., Q574, S575, and S576), numbered relative to SEQ ID NO: 138. In any of these embodiments, [C] is present immediately subsequent to position 576, numbered relative to SEQ ID NO: 138. In any of these embodiments, [C] replaces position 577 (e.g., T577), numbered relative to SEQ ID NO: 138. In any of these embodiments, [C] is present immediately subsequent to position 576, and [C] replaces position 577 (e.g., T577), numbered relative to SEQ ID NO: 138. In any of these embodiments, [B]-[C] is present immediately subsequent to position 573, numbered relative to SEQ ID NO: 138. In any of these embodiments, [B]-[C] replaces positions 574-577 (e.g., Q574, S575, S576, and T577), numbered relative to SEQ ID NO: 138. In any of these embodiments, [B]-[C] is present immediately subsequent to position 573, and [B]-[C] replaces positions 574-577 (e.g., Q574, S575, S576, and T577), numbered relative to SEQ ID NO: 138. In any of these embodiments, [C]-[D] is present immediately subsequent to position 576, numbered relative to SEQ ID NO: 138. In any of these embodiments, [C]-[D] replaces positions 577-579 (e.g., T577, T578, and A579), numbered relative to SEQ ID NO: 138. In any of these embodiments, [A]-[B]-[C]-[D] is present immediately subsequent to position 570, numbered relative to SEQ ID NO: 138. In any of these embodiments, [A]-[B]-[C]-[D] replaces positions 571-579 (e.g., T571, N572, N573, Q574, S575, S576, T577, T578, and A579), numbered relative to SEQ ID NO: 138. In any of these embodiments, [A]-[B]-[C]-[D] is present immediately subsequent to position 570, and [A]-[B]- [C]-[D] replaces positions 571-579 (e.g., T571, N572, N573, Q574, S575, S576, T577, T578, and A579), numbered relative to SEQ ID NO: 138. For example, in some embodiments, [A]- [B] - [C] - [D] is TNNQSSYPAEVVQKTA (SEQ ID NO: 1533) and is present immediately subsequent to position 570, numbered relative to SEQ ID NO: 138, wherein [C] (YPAEVVQK; SEQ ID NO: 943) replaces position 577 (e.g., replaces T577) numbered relative to SEQ ID NO: 138. In some embodiments, [A] is present at positions 571-573, numbered according to SEQ ID NO: 982. In some embodiments, [B] is present at positions 574-576, numbered according to SEQ ID NO: 982. In some embodiments, [C] is present at positions 577-584, numbered according to SEQ ID NO: 982. In some embodiments, [D] is present at positions 585-586, numbered according to SEQ ID NO: 982. In some embodiments, [A]-[B]-[C]-[D] is present at positions 571-586, numbered according to SEQ ID NO: 982. In yet another aspect, the present disclosure provides an AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant comprises the amino acid Y at position 577, and comprises the amino acid sequence of PAEVVQK (SEQ ID NO: 20), which is present immediately subsequent to position 577, numbered relative to SEQ ID NO: 982. In some embodiments, the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 739, or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto. In some embodiments, the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 738, or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto. In some embodiments, the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 982, or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.
[0019] In yet another aspect, the present disclosure provides an AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant comprises the amino acid Y at position 577 and the amino acid sequence of PAEVVQK (SEQ ID NO: 20) at positions 578-584, numbered relative to SEQ ID NO: 982. In some embodiments, the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 739, or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto. In some embodiments, the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 738, or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto. In some embodiments, the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 982, or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.
[0020] In yet another aspect, the present disclosure provides a method of making an isolated, e.g., recombinant AAV particle. The method comprising providing a host cell comprising an AAV viral genome described herein and incubating the host cell under conditions suitable to enclose the viral genome in the AAV particle, e.g., an AAV capsid variant described herein, thereby making The AAV particle.
[0021] In yet another aspect, the present disclosure provides a method of delivering an exogenous SMN protein, e.g., an SMN1 protein, to a cell or tissue (e.g., a CNS cell or a CNS tissue). The method comprising administering an effective amount of an AAV particle comprising an AAV capsid or an AAV capsid variant, e.g., an AAV capsid variant described herein. [0022] In yet another aspect, the present disclosure provides method of delivering an exogenous SMN protein, e.g., an SMN1 protein, to a subject. The method comprising administering an effective amount of an AAV particle or a plurality of AAV particles, described herein, said AAV particle comprising an AAV viral genome described herein, e.g., a viral genome comprising a nucleic acid comprising a transgene encoding an SMN protein, e.g., an SMN1 protein, described herein.
[0023] In another aspect, the present disclosure provides a method of treating a subject having or being diagnosed as having disease and/or a disorder associated with decreased SMN protein expression, e.g., a mutation in an SMN1 gene. The method comprising administering to the subject an effective amount of an AAV particle or a plurality of AAV particles, described herein, comprising an AAV viral genome described herein. In some embodiments, the disease and/or disorder associated with decreased expression of the SMN protein is Spinal Muscular Atrophy (SMA), an SMA-related disorder, Werdnig- Hoffman disease, Dubowitz disease, or Kugelberg-Welander disease.
[0024] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following enumerated embodiments.
Enumerated Embodiments
1. An AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant comprises an amino acid sequence having the following formula: [N2]-[N3], wherein:
(i) [N2] comprises positions XI, X2, X3, X4, and X5, wherein:
(a) position XI is Y, N, or C;
(b) position X2 is P, K, T, or Q;
(c) position X3 is A or P;
(d) position X4 is E, S, or A; and
(e) position X5 is V, L, or E; and
(ii) [N3] comprises the amino acid sequence of VQK, EQK, VKK, VHK, VQQ, or LQK; or wherein the AAV capsid variant comprises an amino acid modification, e.g., a conservative substitution, of any of the aforesaid amino acids in (i) and/or (ii).
2. An AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant comprises one, two, three, four, or all of:
(i) an [NO] comprising TNN, TNT, INN, TNS, NNN, or TNK;
(ii) an [Nl] comprising QSS, QSK, TSL, SSS, QSR, AGA, IGS, QAS, ASS, LGS, QST, HSS, LSS, or QRS;
(iii) an [N2] comprising YPAEV (SEQ ID NO: 39), YPPSL (SEQ ID NO: 40), NKAEV (SEQ ID NO: 41), YTAEV (SEQ ID NO: 42), YPAEE (SEQ ID NO: 43), YQAEV (SEQ ID NO: 44), YTPSL (SEQ ID NO: 45), YPAAV (SEQ ID NO: 46), NPAEV (SEQ ID NO: 47), CPAEV (SEQ ID NO: 48), or YQAEE (SEQ ID NO: 49);
(iv) an [N3] comprising VQK, EQK, VKK, VHK, VQQ, or LQK; and
(v) an [N4] comprising TA, PA, or NA; and optionally wherein the AAV capsid variant comprises an amino acid modification, e.g., a conservative substitution, of any of the aforesaid amino acids in (i)-(v).
3. The AAV capsid particle of embodiment 1, wherein:
(a) position XI is Y or N;
(b) position X2 is P, T or Q;
(c) position X3 is A;
(d) position X4 is E or S; and/or
(e) position X5 is V or L.
4. The AAV particle of any one of embodiments 1, or 3, wherein [N2] comprises YP, NK, YT, YQ, NP, CP, TH, AE, PS, AA, AS, PA, PP, KA, TA, QA, TP, HA, EV, SL, EE, AV, or SH.
5. The AAV particle of any one of embodiments 1, 3, or 4, wherein [N2] comprises YPA, YPP, NKA, YTA, YQA, YTP, NPA, CPA, THA, PAE, PPS, KAE, TAE, QAE, TPS, PAA, HAS, AEV, PSL, AEE, or AAV.
6. The AAV particle of any one of embodiments 1, or 3-5, wherein [N2] comprises YPAE (SEQ ID NO: 286), YPPS (SEQ ID NO: 287), NKAE (SEQ ID NO: 288), YTAE (SEQ ID NO: 289), YQAE (SEQ ID NO: 290), YTPS (SEQ ID NO: 291), YPAA (SEQ ID NO: 292), NPAE (SEQ ID NO: 293), CPAE (SEQ ID NO: 294), THAS(SEQ ID NO: 295) , PAEV (SEQ ID NO: 296), PPSL (SEQ ID NO: 297), KAEV (SEQ ID NO: 298), TAEV (SEQ ID NO: 299), PAEE (SEQ ID NO: 300), QAEV (SEQ ID NO: 301), TPSL (SEQ ID NO: 302), PAAV (SEQ ID NO: 303), or QAEE (SEQ ID NO: 304).
7. The AAV particle of any one of embodiments 1-6, wherein [N2] is or comprises YPAEV (SEQ ID NO: 39), YPPSL (SEQ ID NO: 40), NKAEV (SEQ ID NO: 41), YTAEV (SEQ ID NO: 42), YPAEE (SEQ ID NO: 43), YQAEV (SEQ ID NO: 44), YTPSL (SEQ ID NO: 45), YPAAV (SEQ ID NO: 46), NPAEV (SEQ ID NO: 47), CPAEV (SEQ ID NO: 48), or YQAEE(SEQ ID NO: 49). 8. The AAV particle of any one of embodiments 1-7, wherein [N3] comprises the amino acid sequence of VQK, EQK, or VKK.
9. The AAV particle of any one of embodiments 1-8, wherein [N3] comprises VQK.
10. The AAV particle of any one of embodiments 1-8, wherein [N3] comprises EQK.
11. The AAV particle of any one of embodiments 1-8, wherein [N3] comprises VKK
12. The AAV particle of any one of embodiments 1-11, wherein [N2] is or comprises the amino acid sequence of YPAEV (SEQ ID NO: 39) and [N3] is or comprises the amino acid sequence of VQK.
13. The AAV particle of any one of embodiments 1-, wherein:
(i) [N2] is or comprises the amino acid sequence of YTPSL (SEQ ID NO: 45) and [N3] is or comprises the amino acid sequence of VQK;
(ii) [N2] is or comprises the amino acid sequence of YPPSL (SEQ ID NO: 40) and [N3] is or comprises the amino acid sequence of VQK;
(iii) [N2] is or comprises the amino acid sequence of YPPSL (SEQ ID NO: 40) and [N3] is or comprises the amino acid sequence of EQK; or
(iv) [N2] is or comprises the amino acid sequence of YPPSL (SEQ ID NO: 40) and [N3] is or comprises the amino acid sequence of VKK.
14. The AAV particle of any one of embodiments lor 3-13, wherein [N2]-[N3] comprises:
(i) AEVVQK (SEQ ID NO: 50), PSLVQK (SEQ ID NO: 51), AEVEQK (SEQ ID NO: 52), AEEVQK (SEQ ID NO: 53), PSLEQK (SEQ ID NO: 54), PSLVKK (SEQ ID NO: 55), AEVVKK (SEQ ID NO: 56), AEVVHK (SEQ ID NO: 57), AAVVQK (SEQ ID NO: 58), AEVVQQ (SEQ ID NO: 59), or AEVLQK (SEQ ID NO: 60);
(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, or 5 amino acids, e.g., consecutive amino acids, thereof;
(iii) an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or
(iv) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
15. The AAV particle of any one of embodiments 1 or 3-14, wherein [N2]-[N3] comprises: (i) PAEVVQK (SEQ ID NO: 61) , PPSLVQK (SEQ ID NO: 62), KAEVVQK (SEQ ID NO: 63), TAEVVQK (SEQ ID NO: 64), PAEVEQK (SEQ ID NO: 65), PAEEVQK (SEQ ID NO: 66), QAEVVQK (SEQ ID NO: 67), TPSLVQK (SEQ ID NO: 68), PPSLEQK (SEQ ID NO: 69), PPSLVKK (SEQ ID NO: 70), PAEVVKK (SEQ ID NO: 71), PAEVVHK (SEQ ID NO: 72), PAAVVQK (SEQ ID NO; 73), PAEVVQQ (SEQ ID NO: 74), TAEVVKK (SEQ ID NO: 75), PAEVLQK (SEQ ID NO: 76), or QAEEVQK (SEQ ID NO: 77);
(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, or 6 amino acids, e.g., consecutive amino acids, thereof;
(iii) an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or
(iv) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
16. The AAV particle of any one of embodiments 1-15, wherein [N2]-[N3] is or comprises:
(i) YPAEVVQK (SEQ ID NO: 943), YPPSLVQK (SEQ ID NO: 946), NKAEVVQK, YTAEVVQK (SEQ ID NO: 948), YPAEVEQK (SEQ ID NO: 949), YPAEEVQK(SEQ ID NO: 950), YQAEVVQK (SEQ ID NO: 951), YTPSLVQK (SEQ ID NO: 952), YPPSLEQK (SEQ ID NO: 953), YPPSLVKK (SEQ ID NO: 954), YPAEVVKK (SEQ ID NO: 955), YPAEVVHK (SEQ ID NO: 956), YPAAVVQK (SEQ ID NO: 957), NPAEVVQK (SEQ ID NO: 958), YPAEVVQQ (SEQ ID NO: 959), CPAEVVQK (SEQ ID NO: 960), YTAEVVKK (SEQ ID NO: 961), YPAEVLQK (SEQ ID NO: 962), or YQAEEVQK (SEQ ID NO: 963);
(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, 6, or 7 amino acids, e.g., consecutive amino acids, thereof;
(iii) an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or
(iv) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
17. The AAV particle of any one of embodiments 1-16, wherein the AAV capsid variant further comprises one, two, three or all of an amino acid other than Q at position 574 (e.g., T, S, A, I, L, or H), an amino acid other than S at position 575 (e.g., G, A, or R), and/or an amino acid other than S at position 576 (e.g., K, L, R, A, or T), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. 18. The AAV particle of any one of embodiments 1-16, wherein the AAV capsid variant further comprises:
(i) a Q at position 574, an S at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 982;
(ii) a T at position 574, an S at position 575, and/or a L al position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 982;
(iii) an S at position 574, an S at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 982;
(iv) a Q at position 574, an S at position 575, and/or an R at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 982;
(v) a Q at position 574, an S at position 575, and/or a K at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 982;
(vi) an A at position 574, a G at position 575, and/or an A at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 982;
(vii) an I at position 574, a G at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 982;
(viii) a Q at position 574, an A at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 982;
(ix) an A at position 574, an S at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 982;
(x) an L at position 574, a G at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 982;
(xi) a Q at position 574, an S at position 575, and/or a T at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 982;
(xii) an H at position 574, an S at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 982;
(xiii) an L at position 574, an S at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 982; or
(xiv) a Q at position 574, an R at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 982.
19. The AAV particle of any one of embodiments 1 or 3-18, wherein the AAV capsid variant further comprises [Nl], wherein [Nl] comprises positions XD, XE, and XF, wherein:
(a) position XD is Q, T, S, A, I, L, or H;
(b) position XE is S, G, A, or R; and (c) position XF is S, K, L, R, A, or T; and optionally wherein the AAV capsid variant comprises an amino acid modification, e.g., a conservative substitution, of any of the aforesaid amino acids in (a)-(c).
20. The AAV particle of embodiment 19, wherein [Nl] comprises SK, SL, SS, SR, GA, GS, AS, ST, RS, QS, TS, AG, IG, QA, LG, HS, LS, or QR.
21. The AAV particle of any one of embodiments 2, 19, or 20, wherein [Nl] is or comprises QSS, QSK, TSL, SSS, QSR, AGA, IGS, QAS, ASS, LGS, QST, HSS, LSS, or QRS.
22. The AAV particle of any one of embodiments 19-21, wherein [N1]-[N2] comprises:
(i) SSYPA (SEQ ID NO: 78), SKYPA (SEQ ID NO: 79), SLYPA (SEQ ID NO: 80), SRYPA (SEQ ID NO: 81), SSYPP (SEQ ID NO: 82), GAYPA (SEQ ID NO: 83), GSYPA(SEQ ID NO: 84), ASYPA (SEQ ID NO: 85), STNKA (SEQ ID NO: 86), SSYTA (SEQ ID NO: 87), SSYQA (SEQ ID NO: 88), SSYTP (SEQ ID NO: 89), SSNPA (SEQ ID NO: 90), SLCPA (SEQ ID NO: 91), RSYTA (SEQ ID NO: 92), or SSTHA (SEQ ID NO: 93);
(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, or 4 amino acids, e.g., consecutive amino acids, thereof;
(iii) an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or
(iv) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
23. The AAV particle of any one of embodiments 19-22, wherein [N1]-[N2] comprises:
(i) SSYPAE (SEQ ID NO: 94), SKYPAE (SEQ ID NO: 95), SLYPAE (SEQ ID NO: 96), SRYPAE (SEQ ID NO: 97), SSYPPS (SEQ ID NO: 98), GAYPAE (SEQ ID NO: 99), GSYPAE (SEQ ID NO: 102), ASYPAE (SEQ ID NO: 103), STNKAE (SEQ ID NO: 104), SSYTAE (SEQ ID NO: 105), SSYQAE (SEQ ID NO: 106), SSYTPS (SEQ ID NO: 107), SSYPAA (SEQ ID NO: 108, SSNPAE (SEQ ID NO: 109), SLCPAE (SEQ ID NO: 110), RSYTAE (SEQ ID NO: 111), SSTHAS (SEQ ID NO: 112);
(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, or 5 amino acids, e.g., consecutive amino acids, thereof;
(iii) an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or
(iv) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
24. The AAV particle of any one of embodiments 2 or 19-23, wherein [N1]-[N2] is or comprises:
(i) QSSYPAEV (SEQ ID NO: 113), QSKYPAEV (SEQ ID NO: 114), TSLYPAEV (SEQ ID NO: 115), SSSYPAEV (SEQ ID NO: 116), QSRYPAEV (SEQ ID NO: 117), QSSYPPSL (SEQ ID NO: 118), AGAYPAEV (SEQ ID NO: 119), IGSYPAEV (SEQ ID NO: 120), QASYPAEV (SEQ ID NO: 121), ASSYPAEV (SEQ ID NO: 122), LGSYPAEV (SEQ ID NO: 123), QSTNKAEV (SEQ ID NO: 124), HSSYPAEV (SEQ ID NO: 125), SSSYTAEV (SEQ ID NO: 126), TSLYPAEE (SEQ ID NO: 127), ASSYQAEV (SEQ ID NO: 129), QSSYTPSL (SEQ ID NO: 129), QSRYPAEE (SEQ ID NO: 130), LSSYQAEV (SEQ ID NO: 131), HSSYPAAV (SEQ ID NO: 132), QSSNPAEV (SEQ ID NO: 100), QSSYTAEV (SEQ ID NO: 133), TSLCPAEV (SEQ ID NO: 134), QRSYTAEV (SEQ ID NO: 135), or QSSYQAEE (SEQ ID NO: 136);
(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, 6, or 7 amino acids, e.g., consecutive amino acids, thereof;
(iii) an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or
(iv) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
25. The AAV particle of any one of embodiments 19-24, wherein [N1]-[N2]-[N3] comprises:
(i) SSYPAEVVQ (SEQ ID NO: 142), SKYPAEVVQ (SEQ ID NO: 143), SLYPAEVVQ (SEQ ID NO: 101), SRYPAEVVQ (SEQ ID NO: 144), SSYPPSLVQ (SEQ ID NO: 145), GAYPAEVVQ (SEQ ID NO: 146), GSYPAEVVQ (SEQ ID NO: 147), ASYPAEVVQ (SEQ ID NO: 148), STNKAEVVQ (SEQ ID NO: 149), SSYTAEVVQ (SEQ ID NO: 150), SKYPAEVEQ (SEQ ID NO: 160), SLYPAEEVQ (SEQ ID NO: 161), SSYQAEVVQ (SEQ ID NO: 162), SSYTPSLVQ (SEQ ID NO: 163), SRYPAEEVQ (SEQ ID NO: 164), SSYPPSLEQ (SEQ ID NO: 165), SSYPPSLVK (SEQ ID NO: 166), SSYPAEVVK (SEQ ID NO: 167), SKYPAEVVH (SEQ ID NO: 168), SSYPAAVVQ (SEQ ID NO: 169), SSNPAEVVQ (SEQ ID NO: 170), SLCPAEVVQ (SEQ ID NO: 171), RSYTAEVVQ (SEQ ID NO: 172), SSYTAEVVK (SEQ ID NO: 173), SSYPAEVLQ (SEQ ID NO: 174), or SSYQAEEVQ (SEQ ID NO: 175);
(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, 6, 7, or 8 amino acids, e.g., consecutive amino acids, thereof;
(iii) an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or (iv) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
26. The AAV particle of any one of embodiments 2 or 19-25, wherein [N1]-[N2]-[N3] is or comprises:
(i) QSSYPAEVVQK (SEQ ID NO: 176), QSKYPAEVVQK (SEQ ID NO: 177), TSLYPAEVVQK (SEQ ID NO: 178), SSSYPAEVVQK (SEQ ID NO: 179), QSRYPAEVVQK (SEQ ID NO: 180), QSSYPPSLVQK (SEQ ID NO: 181), AGAYPAEVVQK (SEQ ID NO: 182), IGSYPAEVVQK (SEQ ID NO: 183), QASYPAEVVQK (SEQ ID NO: 184), ASSYPAEVVQK (SEQ ID NO: 186), LGSYPAEVVQK (SEQ ID NO: 187), QSTNKAEVVQK (SEQ ID NO: 188), HSSYPAEVVQK (SEQ ID NO: 189), SSSYTAEVVQK (SEQ ID NO: 190), QSKYPAEVEQK (SEQ ID NO: 191), TSLYPAEEVQK (SEQ ID NO: 192), ASSYQAEVVQK (SEQ ID NO: 193), QSSYTPSLVQK (SEQ ID NO: 194), QSRYPAEEVQK (SEQ ID NO: 195), QSSYPPSLEQ) (SEQ ID NO: 196), QSSYPPSLVKK (SEQ ID NO: 197), LSSYQAEVVQK (SEQ ID NO: 198), SSSYPAEVVKK (SEQ ID NO: 199), QSKYPAEVVHK (SEQ ID NO: 200), HSSYPAAVVQK (SEQ ID NO: 201), QSSNPAEVVQK (SEQ ID NO: 202), SSSYPAEVVQQ (SEQ ID NO: 203), QSSYTAEVVQK (SEQ ID NO: 204), TSLCPAEVVQK (SEQ ID NO: 205), QRSYTAEVVQK (SEQ ID NO: 206), QSSYTAEVVKK (SEQ ID NO: 207), HSSYPAEVLQK (SEQ ID NO: 208), or QSSYQAEEVQK (SEQ ID NO: 209);
(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, e.g., consecutive amino acids, thereof;
(iii) an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or
(iv) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
27. The AAV particle of any one of embodiments 1 or 3-26, wherein the AAV capsid variant further comprises [NO], wherein [NO] comprises positions XA, XB, and XC, wherein:
(a) position XA is T, I, or N;
(b) position XB is N;
(c) position XC is N, T, S, or K; and optionally, wherein the AAV capsid variant comprises an amino acid modification, e.g., a conservative substitution, of any of the aforesaid amino acids in (a)-(c).
28. The AAV particle of embodiment 27, wherein [NO] comprises TN, IN, NN, NT, NS, or NK. 29. The AAV particle of any one of embodiments 2, 27, or 28, wherein [NO] is or comprises TNN, TNT, INN, TNS, NNN, or TNK.
30. The AAV particle of any one of embodiments 2 or 27-29, wherein [NO]-[N1] is or comprises:
(i) TNNQSS (SEQ ID NO; 210), TNNQSK (SEQ ID NO: 211), TNNTSL (SEQ ID NO: 212), TNNSSS (SEQ ID NO: 213), TNNQSR (SEQ ID NO: 214), TNNAGA (SEQ ID NO: 215), TNNIGS (SEQ ID NO: 216), TNNQAS (SEQ ID NO: 217), TNTASS (SEQ ID NO: 218), TNNLGS (SEQ ID NO: 219), TNNQST (SEQ ID NO: 220), TNNHSS (SEQ ID NO: 221), TNNLSS (SEQ ID NO: 223), INNQSS (SEQ ID NO: 224), TNSQSS (SEQ ID NO: 225), NNNQSR (SEQ ID NO: 226), TNSTSL (SEQ ID NO: 227), TNNQRS (SEQ ID NO: 228), or TNKQAS (SEQ ID NO: 229);
(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, or 5 amino acids, e.g., consecutive amino acids, thereof;
(iii) an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or
(iv) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
31. The AAV particle of any one of embodiments 2 or 27-30, wherein [NO]-[N1]-[N2]-[N3] is or comprises:
(i) TNNQSSYPAEVVQK (SEQ ID NO: 230), TNNQSKYPAEVVQK (SEQ ID NO: 231), TNNTSL YPAEVVQK (SEQ ID NO: 232), TNNSSSYPAEVVQK (SEQ ID NO: 233), TNNQSRYPAEVVQK (SEQ ID NO: 234), TNNQSSYPPSLVQK (SEQ ID NO: 235), TNNAGA YPAEVVQK (SEQ ID NO: 236), TNNIGSYPAEVVQK (SEQ ID NO: 237), TNNQAS YPAEVVQK (SEQ ID NO: 238), TNTASS YPAEVVQK (SEQ ID NO: 239), TNNLGSYPAEVVQK (SEQ ID NO: 240), TNNQSTNKAEVVQK (SEQ ID NO: 241), TNNHSSYPAEVVQK (SEQ ID NO: 242), TNNSSSYTAEVVQK (SEQ ID NO: 243), TNNQSKYPAEVEQK (SEQ ID NO: 244), TNNTSLYPAEEVQK (SEQ ID NO: 245), TNTASSYQAEVVQK (SEQ ID NO: 246), TNNQSSYTPSLVQK (SEQ ID NO: 247), TNNQSRYPAEEVQK (SEQ ID NO: 248), TNNQSSYPPSLEQK (SEQ ID NO: 249), TNNQSS YPPSLVKK (SEQ ID NO: 250), TNNLSS YQAEVVQK (SEQ ID NO: 251), TNNSSSYPAEVVKK (SEQ ID NO: 252), TNNQSKYPAEVVHK (SEQ ID NO: 253), INNQSSYPAEVVQK (SEQ ID NO: 254), TNNHSSYPAAVVQK (SEQ ID NO: 255), TNSQSSNPAEVVQK (SEQ ID NO: 256), TNNSSSYPAEVVQQ (SEQ ID NO: 257), NNNQSRYPAEVVQK (SEQ ID NO: 258), TNNQSSYTAEVVQK (SEQ ID NO: 259), TNNTSLCPAEVVQK (SEQ ID NO: 260), TNSTSL YPAEVVQK (SEQ ID NO: 261), TNNQRSYTAEVVQK (SEQ ID NO: 262), TNNQSSYTAEVVKK (SEQ ID NO: 263), TNNHSSYPAEVLQK (SEQ ID NO: 264), TNNQSSYQAEEVQK (SEQ ID NO: 266), or TNKQASYPAEVVQK (SEQ ID NO: 267);
(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acids, e.g., consecutive amino acids, thereof;
(iii) an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or
(iv) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
32. The AAV particle of embodiment 2 or 27-31, wherein [NOJ-[N1J-[N2J-[N3J is or comprises TNNQSSYPAEVVQK (SEQ ID NO: 230).
33. The AAV particle of embodiment 2 or 27-31, wherein [NO]-[N1]-[N2]-[N3] is or comprises TNNAGAYPAEVVQK (SEQ ID NO: 236), TNNTSLYPAEVVQK (SEQ ID NO: 232), TNNQSKYPAEVVQK (SEQ ID NO: 2 1), TNNQSSYTPSLVQK (SEQ ID NO: 247), TNNQSSYPPSLVQK (SEQ ID NO: 235), TNNQSRYPAEVVQK (SEQ ID NO: 234), TNNQSSYPPSLEQK (SEQ ID NO: 249), TNNQSSYPPSLVKK (SEQ ID NO: 250), or INNQSSYPAEVVQK (SEQ ID NO: 254).
34. The AAV particle of any one of embodiments 1 or 3-33, wherein the AAV capsid variant further comprises [N4], wherein [N4] comprises positions XG and XH, wherein:
(a) position XG is T, P, or N; and
(b) position XH is A; and optionally wherein the AAV capsid variant comprises an amino acid modification, e.g., a conservative substitution, of any of the aforesaid amino acids in (a) or (b).
35. The AAV particle of embodiment 34, wherein [N4] is or comprises TA, PA, or NA.
36. The AAV particle of any one of embodiments 2, 34, or 35, wherein [N3]-[N4] is or comprises:
(i) VQKTA (SEQ ID NO: 268), EQKTA (SEQ ID NO: 269), VKKTA (SEQ ID NO: 270), VQKPA (SEQ ID NO: 271), VHKTA (SEQ ID NO: 272), VQQTA (SEQ ID NO: 273), VQKNA (SEQ ID NO: 274), or LQKTA (SEQ ID NO: 275);
(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, or 4 amino acids, e.g., consecutive amino acids, thereof; (iii) an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or
(iv) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
37. The AAV particle of any one of embodiments 2 or 34-36, wherein [NO]-[N1]-[N2]-[N3]-[N4] is or comprises:
(i) TNNQSSYPAEVVQKTA (SEQ ID NO: 1533), TNNQSKYPAEVVQKTA (SEQ ID NO: 1538), TNNTSLYPAEVVQKTA (SEQ ID NO: 1232), TNNSSSYPAEVVQKTA (SEQ ID NO: 1539), TNNQSRYPAEVVQKTA(SEQ ID NO: 1327), TNNQSSYPPSLVQKTA (SEQ ID NO: 1300), TNNAGAYPAEVVQKTA (SEQ ID NO: 1021), TNNIGSYPAEVVQKTA (SEQ ID NO: 1112), TNNQASYPAEVVQKTA (SEQ ID NO: 1586), TNTASSYPAEVVQKTA (SEQ ID NO: 1575), TNNLGSYPAEVVQKTA (SEQ ID NO: 1027), TNNQSTNKAEVVQKTA (SEQ ID NO: 1578), TNNHSSYPAEVVQKTA (SEQ ID NO: 1310), TNNQSKYPAEVVQKTA(SEQ ID NO: 1538, TNNSSSYTAEVVQKTA (SEQ ID NO: 1214), TNNQSKYPAEVEQKTA (SEQ ID NO: 1254), TNNTSLYPAEEVQKTA(SEQ ID NO: 1583), TNTASSYQAEVVQKTA (SEQ ID NO: 1584), TNNQSSYTPSLVQKTA (SEQ ID NO: 1585), TNNQSRYPAEEVQKTA (SEQ ID NO: 1342), TNNQSSYPPSLEQKTA (SEQ ID NO: 1590), TNNQSSYPPSLVKKTA (SEQ ID NO: 1591), TNNLSSYQAEVVQKTA (SEQ ID NO: 1592), TNNQSSYPPSLVQKPA (SEQ ID NO: 1593), TNNSSSYPAEVVKKTA (SEQ ID NO: 1331), TNNQSKYPAEVVHKTA (SEQ ID NO: 1453), TNNSSSYPAEVVQKPA (SEQ ID NO: 1142), INNQSSYPAEVVQKTA(SEQ ID NO: 1024), TNNHSSYPAAVVQKTA (SEQ ID NO: 1598), TNSQSSNPAEVVQKTA (SEQ ID NO: 1599), TNNSSSYPAEVVQQTA (SEQ ID NO: 1419), NNNQSRYPAEVVQKTA (SEQ ID NO: 1601), TNNQSSYTAEVVQKNA (SEQ ID NO: 1602), TNNTSLCPAEVVQKTA (SEQ ID NO: 1603), TNSTSLYPAEVVQKTA (SEQ ID NO: 1605), TNNQRSYTAEVVQKTA (SEQ ID NO: 1604), TNNQSSYTAEVVKKTA (SEQ ID NO: 1606), TNNHSSYPAEVLQKTA (SEQ ID NO: 1607), TNNQSSYQAEEVQKTA (SEQ ID NO: 1608), or TNKQASYPAEVVQKTA (SEQ ID NO: 1587);
(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids, e.g., consecutive amino acids, thereof;
(iii) an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or
(iv) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i). 38. The AAV particle of any one of embodiments 2 or 34-37, wherein [NO]-[N1]-[N2]-[N3]-[N4] is or comprises TNNQSSYPAEVVQKTA (SEQ ID NO: 1533).
39. The AAV particle of any one of embodiments 2 or 34-37, wherein [NO]-[N1]-[N2]-[N3]-[N4] is or comprises TNNAGAYPAEVVQKTA (SEQ ID NO: 1021), TNNTSLYPAEVVQKTA (SEQ ID NO: 1232), TNNQSKYPAEVVQKTA (SEQ ID NO: 1538), TNNQSSYTPSLVQKTA (SEQ ID NO: 1585), TNNQSSYPPSLVQKTA (SEQ ID NO: 1300), TNNQSRYPAEVVQKTA (SEQ ID NO: 1327), TNNQSSYPPSLEQKTA (SEQ ID NO: 1590), TNNQSSYPPSLVKKTA (SEQ ID NO: 1591), or INNQSSYPAEVVQKTA(SEQ ID NO: 1024) .
40. An AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant comprises an amino sequence comprising the following formula: [B]-[C], wherein
(i) [B] comprises positions XI, X2, and X3, wherein:
(a) position XI is Q, T, S, A, I, L, or H;
(b) position X2 is S, G, or A; and
(c) position X3 is S, K, L, R, or A; and
(ii) [C] comprises the amino acid sequence of YPAEVVQK (SEQ ID NO: 943); and optionally wherein the AAV capsid variant comprises an amino acid modification, e.g., a conservative substitution, of any of the aforesaid amino acids in (i) and/or (ii).
41. An AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant comprises one, two, three, four, or all of:
(i) an [A], wherein [A] comprises the amino acid sequence of TNN, TNT, INN, NNN, TNS, or TNK;
(ii) a [B], wherein [B] comprises the amino acid sequence of QSS, TSL, SSS, QSR, QSK, AGA, IGS, QAS, ASS, LGS, or HSS;
(iii) a [C], wherein [C] comprises the amino acid sequence of YPAEVVQK (SEQ ID NO: 943); and
(iv) a [D], wherein [D] comprises the amino acid sequence of TA or PA; and optionally wherein the AAV capsid variant comprises an amino acid modification, e.g., a conservative substitution, of any of the aforesaid amino acids in (i)-(v); and further optionally wherein [C] replaces position 577 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. 42. The AAV particle of embodiment 40, wherein:
(a) position XI is Q, T, S, A, or H;
(b) position X2 is S or G; and
(c) position X3 is S, K, L, or R.
43. The AAV particle of embodiment 40 or 42, wherein [B] comprises QS, TS, SS, AG, IG, QA, AS, LG, HS, SK, SL, SR, GA, or GS.
44. The AAV particle of any one of embodiments 40-43, wherein [B] is or comprises QSS, TSL, SSS, QSR, QSK, AGA, IGS, QAS, ASS, LGS, or HSS.
45. The AAV particle of any one of embodiments 40 or 42-44, wherein [B]-[C] comprises:
(i) SSYPAEVVQK (SEQ ID NO: 276), SKYPAEVVQK (SEQ ID NO: 277), SLYPAEVVQK (SEQ ID NO: 278), SRYPAEVVQK (SEQ ID NO: 279), GAYPAEVVQK (SEQ ID NO: 280), GSYPAEVVQK (SEQ ID NO: 281), or ASYPAEVVQK (SEQ ID NO: 282);
(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, 6, 7, 8, or 9 amino acids, e.g., consecutive amino acids, thereof;
(iii) an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or
(iv) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
46. The AAV particle of any one of embodiments 40-45, wherein [B]-[C] is or comprises:
(i) QSSYPAEVVQK (SEQ ID NO: 176), QSKYPAEVVQK (SEQ ID NO: 177), TSLYPAEVVQK (SEQ ID NO: 178), SSSYPAEVVQK (SEQ ID NO: 179), QSRYPAEVVQK (SEQ ID NO: 180), AGAYPAEVVQK (SEQ ID NO: 182), IGSYPAEVVQK (SEQ ID NO: 183), QASYPAEVVQK (SEQ ID NO: 184), ASSYPAEVVQK (SEQ ID NO: 186), LGSYPAEVVQK (SEQ ID NO: 187), or HSSYPAEVVQK (SEQ ID NO: 189);
(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, e.g., consecutive amino acids, thereof;
(iii) an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or (iv) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
47. The AAV particle of any one of embodiments 40-46, wherein [B]-[C] is or comprises QSSYPAEVVQK (SEQ ID NO: 176).
48. The AAV particle of any one of embodiments embodiment 40-46, wherein [B]-[C] is or comprises AGAYPAEVVQK (SEQ ID NO: 182), TSLYPAEVVQK (SEQ ID NO: 178), QSKYPAEVVQK (SEQ ID NO: 177), or QSRYPAEVVQK (SEQ ID NO: 180).
49. The AAV particle of any one of embodiments 1-48, wherein the AAV capsid variant further comprises one or both of an amino acid other than T at position 571 (e.g., I or N), and/or an amino acid other than N at position 573 (e.g., T, S, or K), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
50. The AAV particle of any one of embodiments 1-50, wherein the AAV capsid variant further comprises:
(i) a T at position 571, an N at position 572, and/or an N at position 573, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 982;
(ii) a T at position 571, an N at position 572, and/or a T at position 573, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 982;
(iii) an I at position 571, an N at position 572, and/or an N at position 573, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 982;
(iv) a T at position 571, an N at position 572, and/or an S at position 573, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 982;
(v) an N at position 571, an N at position 572, and/or an N at position 573, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 982; or
(vi) a T at position 571, an N at position 572, and/or a K at position 573, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 982.
51. The AAV particle of any one of embodiments 40 or 42-50, wherein the AAV capsid variant further comprises [A], wherein [A] comprises positions XA, XB, and Xc, wherein:
(a) position XA is T, I, or N;
(b) position XB is N; and
(c) position Xc is N, T, S, or K; and 1 optionally wherein the AAV capsid variant comprises an amino acid modification, e.g., a conservative substitution, of any of the aforesaid amino acids in (a)-(c).
52. The AAV particle of embodiment 51, wherein [A] comprises TN, IN, NN, NT, NS, or NK.
53. The AAV particle of any one of embodiments 41, 51, or 52, wherein [A] is or comprises TNN, TNT, INN, NNN, TNS, or TNK.
54. The AAV particle of any one of embodiments 41 or 51-53, wherein [A]-[B] is or comprises:
(i) TNNQSS (SEQ ID NO: 210), TNNQSK (SEQ ID NO: 211), TNNTSL (SEQ ID NO: 212), TNNSSS (SEQ ID NO: 213), TNNQSR (SEQ ID NO: 214), TNNAGA (SEQ ID NO: 215), TNNIGS (SEQ ID NO: 216), TNNQAS (SEQ ID NO: 217), TNTASS (SEQ ID NO: 218), TNNLGS (SEQ ID NO: 219), TNNHSS (SEQ ID NO: 220), INNQSS (SEQ ID NO: 224), NNNQSR (SEQ ID NO: 226), TNSTSL (SEQ ID NO: 227), or TNKQAS (SEQ ID NO: 229);
(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, or 5 amino acids, e.g., consecutive amino acids, thereof;
(iii) an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or
(iv) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
55. The AAV particle of any one of embodiments 41 or 51-54, wherein [A]-[B]-[C] is or comprises:
(i) TNNQSSYPAEVVQK (SEQ ID NO: 230), TNNQSKYPAEVVQK (SEQ ID NO: 231), TNNTSL YPAEVVQK (SEQ ID NO: 232), TNNSSSYPAEVVQK (SEQ ID NO: 233), TNNQSRYPAEVVQK (SEQ ID NO: 234), TNNAGAYPAEVVQK (SEQ ID NO: 236), TNNIGSYPAEVVQK (SEQ ID NO: 237), TNNQASYPAEVVQK (SEQ ID NO: 238), TNTASSYPAEVVQK (SEQ ID NO: 239), TNNLGSYPAEVVQK (SEQ ID NO: 240), TNNHSSYPAEVVQK (SEQ ID NO: 242), INNQSSYPAEVVQK (SEQ ID NO: 254), NNNQSRYPAEVVQK (SEQ ID NO: 258), TNSTSLYPAEVVQK (SEQ ID NO: 261), or TNKQAS YPAEVVQK (SEQ ID NO: 267);
(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acids, e.g., consecutive amino acids, thereof;
(iii) an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or (iv) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
56. The AAV particle of any one of embodiments 41 or 51-55, wherein [A]-[B]-[C] is or comprises TNNQSSYPAEVVQK (SEQ ID NO: 230).
57. The AAV particle of any one of embodiments 41 or 41-55, wherein [A]-[B]-[C] is or comprises TNNAGAYPAEVVQK, TNNTSLYPAEVVQK (SEQ ID NO: 232), TNNQSKYPAEVVQK (SEQ ID NO: 231), TNNQSRYPAEVVQK (SEQ ID NO: 234), or INNQSSYPAEVVQK (SEQ ID NO: 254).
58. The AAV particle of any one of embodiments 1-57, wherein the AAV capsid variant further comprises:
(i) an amino acid other than T at position 578 (e.g., P or N), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138; or
(ii) an amino acid other than T at position 585 (e.g., P or N), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 982.
59. The AAV particle of any one of embodiments 1-57, wherein the AAV capsid variant further comprises:
(i) a T at position 578 and/or an A at position 579, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138; ; or a T at position 585 and/or an A at position 586 relative to a reference sequence numbered according to SEQ ID NO: 982;
(ii) a P at position 578 and/or an A at position 579, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138; or a P at position 585 and/or an A at position 586 relative to a reference sequence numbered according to SEQ ID NO: 982; or
(iii) an N at position 578 and/or an A at position 579, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138; .or an N at position 585 and/or an A at position 586 relative to a reference sequence numbered according to SEQ ID NO: 982
60. The AAV particle of any one of embodiments 40 or 42-59, wherein the AAV capsid variant further comprises [D], wherein [D] comprises positions X4 and X5, wherein:
(a) position X4 is T or N; and
(b) position X5 is A; and optionally wherein the AAV capsid variant comprises an amino acid modification, e.g., a conservative substitution, of any of the aforesaid amino acids in (a) or (b).
61. The AAV particle of embodiment 41 or 60, wherein [D] is or comprises TA or PA. 62. The AAV particle of any one of embodiments 41, 60, or 61, wherein [C]-[D] is or comprises:
(i) YPAEVVQKTA (SEQ ID NO: 283) or YPAEVVQKPA (SEQ ID NO: 284);
(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, 6, 7, 8, or 9 amino acids, e.g., consecutive amino acids, thereof;
(iii) an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or
(iv) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
63. The AAV particle of any one of embodiments 41 or 60-62, wherein [AJ-|BJ-[CJ-[DJ is or comprises:
(i) TNNQSSYPAEVVQKTA (SEQ ID NO: 1533), TNNQSKYPAEVVQKTA (SEQ ID NO: 1538), TNNTSLYPAEVVQKTA (SEQ ID NO: 1232), TNNSSS YPAEVVQKTA (SEQ ID NO: 1539), TNNQSRYPAEVVQKTA (SEQ ID NO: 1327), TNNAGAYPAEVVQKTA (SEQ ID NO: 1021), TNNIGSYPAEVVQKTA (SEQ ID NO: 1112), TNNQ AS YPAEVVQKTA (SEQ ID NO: 1586), TNTASSYPAEVVQKTA (SEQ ID NO: 1575), TNNLGSYPAEVVQKTA (SEQ ID NO: 1027), TNNHSS YPAEVVQKTA (SEQ ID NO: 1310), TNNSSS YPAEVVQKPA (SEQ ID NO: 1142), INNQSSYPAEVVQKTA (SEQ ID NO: 1024) , NNNQSRYPAEVVQKTA (SEQ ID NO: 1601), TNSTSLYPAEVVQKTA (SEQ ID NO: 1605), or TNKQASYPAEVVQKTA (SEQ ID NO: 1587);
(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids, e.g., consecutive amino acids, thereof;
(iii) an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or
(iv) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the amino acid sequences in (i).
64. The AAV particle of any one of embodiments 41 or 60-63, wherein [A]-[B]-[C]-[D] is or comprises TNNQSSYPAEVVQKTA (SEQ ID NO: 1533).
65. The AAV particle of any one of embodiments 41 or 60-63, wherein [A]-[B]-[C]-[D] is or comprises TNNAGAYPAEVVQKTA (SEQ ID NO: 1021), TNNTSLYPAEVVQKTA (SEQ ID NO: 1232), TNNQSKYPAEVVQKTA (SEQ ID NO: 1538), TNNQSRYPAEVVQKTA (SEQ ID NO: 1327), or INNQSSYPAEVVQKTA (SEQ ID NO: 1024) . 66. The AAV particle of any one of the embodiments 1-39, 58, or 59, wherein [N2]-[N3] is present in loop VIII, optionally wherein loop VIII comprises positions 571-592 (e.g., amino acids TNNQSSTTAPATGTYNLQEIVP (SEQ ID NO: 285) numbered according to SEQ ID NO: 138, or positions 571-599, numbered according to SEQ ID NO: 982.
67. The AAV particle of any one of embodiments 2, 14-39, 58, 59, or 66, wherein [NO], [Nl], and/or [N4] is present in loop VIII, optionally wherein loop VIH comprises positions 571-592 (e.g., amino acids TNNQSSTTAPATGTYNLQEIVP (SEQ ID NO: 285) numbered according to SEQ ID NO: 138, or positions 571-599, numbered according to SEQ ID NO: 982.
68. The AAV particle of any one of embodiments 2, 14-39, 58, 59, 66, or 67, wherein [NO]-[N1]-[N2]- [N3J-[N4J is present in loop VIII, optionally wherein loop VIII comprises positions 571-592 (e.g., amino acids TNNQSSTTAPATGTYNLQEIVP (SEQ ID NO: 285) numbered according to SEQ ID NO: 138, or positions 571-599, numbered according to SEQ ID NO: 982.
69. The AAV particle of any one of embodiments 1-39, 58, 59, or 66-68, wherein [N2] is present immediately subsequent to position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or 982.
70. The AAV particle of any one of embodiments 1-39, 58, 59, or 66-69, wherein [N2] replaces position 577 (e.g., T577), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
71. The AAV particle of any one of embodiments 1-39, 58, 59, or 66-70, wherein [N2] is present immediately subsequent to position 576, and wherein [N2] replaces position 577 (e.g., T577), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
72. The AAV particle of any one of 1-39, 58, 59, or 66-71, wherein [N2] corresponds to positions 577- 581 (e.g., Y577, P578, A579, E580, V581) of SEQ ID NO: 982.
73. The AAV particle of any one of embodiments 1-72, which comprises an amino acid other than T at position 577, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
74. The AAV particle of any one of embodiments 1-73, which comprises a Y at position 577, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or 982. 75. The AAV particle of any one of embodiments 1-39, 58, 59, or 66-74, wherein XI of [N2] is present at position 577 (e.g., T577), and positions X2-X5 of [N2] are present immediately subsequent to position 577, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
76. The AAV particle of any one of embodiments 1-39, 58, 59, or 66-75, wherein XI of [N2] corresponds to position 577 (e.g., Y577), positions X2 corresponds to position 578 (e.g., P588), X3 of [N2] corresponds to position 579 (e.g., A579), X4 of [N2] corresponds to position 580 (e.g., E580), and X5 of [N2] corresponds to position 581 (e.g., V581) of SEQ ID NO: 982.
77. The AAV particle of any one of embodiments 1-39, 58, 59, or 66-76, wherein [N2J- [N3J is present immediately subsequent to position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or 982.
78. The AAV particle of any one of embodiments 1-39, 58, 59, or 66-77, wherein [N2]-[N3] replaces position 577 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
79. The AAV particle of any one of embodiments 1-39, 58, 59, or 66-78, wherein [N2]-[N3] is present immediately subsequent to position 576, and wherein [N2]-[N3] replaces position 577 (e.g., T577), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
80. The AAV particle of any one of embodiments 1-39, 58, 59, or 66-79, wherein [N2]-[N3] corresponds to positions 577-584 (e.g., Y577, P578, A579, E580, V581, V582, Q583, K584) of SEQ ID NO: 982.
81. The AAV particle of any one of embodiments 1-39, 58, 59, or 66-80, wherein [N2]-[N3]-[N4] is present immediately subsequent to position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or 982.
82. The AAV particle of any one of embodiments 1-39, 58, 59, or 66-81, wherein [N2]-[N3]-[N4] replaces positions 577-579 (e.g., T577, T578, and A579), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
83. The AAV particle of any one of embodiments 1-39, 58, 59, or 66-82, wherein [N2]-[N3]-[N4] is present immediately subsequent to position 576, and wherein [N2]-[N3]-[N4] replaces positions 577-579 (e.g., T577, T578, and A579), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
84. The AAV particle of any one of embodiments 1-39, 58, 59, or 66-83, wherein [N2]-[N3]-[N4] corresponds to positions 577-586 (e.g., Y577, P578, A579, E580, V581, V582, Q583, K584, T585, A586) of SEQ ID NO: 982.
85. The AAV particle of any one of embodiments 2, 19-39, 58, 59, or 66-84, wherein [Nl] is present immediately subsequent to position 573, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or 982.
86. The AAV particle of any one of embodiments 2, 19-39, 58, 59, or 66-85, wherein [N1J replaces positions 574-576 (e.g., Q574, S575, and S576), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
87. The AAV particle of any one of embodiments 2, 19-39, 58, 59, or 66-86, wherein [Nl] is present immediately subsequent to position 573, and wherein [Nl ] replaces positions 574-576 (e.g., Q574, S575, and S576), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
88. The AAV particle of any one of embodiments 2, 19-39, 58, 59, or 66-87, wherein [Nl] replaces positions 574-576 (e.g., Q574, S575, and S576), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 982.
89. The AAV particle of any one of embodiments 2, 19-39, 58, 59, or 66-88, wherein [Nl] is present immediately subsequent to position 573, and wherein [Nl] replaces positions 574-576 (e.g., Q574, S575, and S576), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 982.
90. The AAV particle of any one of embodiments 2, 19-39, 58, 59, or 66-89, wherein [Nl] corresponds to positions 574-576 (e.g., Q574, S575, and S576) of SEQ ID NO: 982.
91. The AAV particle of any one of embodiments 2, 34-39, 58, 59, or 66-90, wherein [N1]-[N2]-[N3]- [N4] is present immediately subsequent to position 573, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or 982. 92. The AAV particle of any one of embodiments 2, 34-39, 58, 59, or 66-91, wherein [N1]-[N2]-[N3]- [N4] replaces positions 574-579 (e.g., Q574, S575, S576, T577, T578, and A579), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
93. The AAV particle of any one of embodiments 2, 34-39, 58, 59, or 66-92, wherein [N1]-[N2]-[N3]- [N4] is present immediately subsequent to position 573, and wherein [N1]-[N2]-[N3]-[N4] replaces positions 574-579 (e.g., Q574, S575, S576, T577, T578, and A579), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
94. The AAV particle of any one of embodiments 2, 34-39, 58, 59, or 66-93, wherein [N1]-[N2]-[N3]- [N4] corresponds to positions 574-586 (e.g., Q574, S575, S576, Y577, P578, A579, E58O, V581, V582, Q583, K584, T585, A586) of SEQ ID NO: 982.
95. The AAV particle of any one of embodiments 2, 27-39, 59, 60, 68, 79-98, 139, 140, or 146-174 wherein [NO] is present immediately subsequent to position 570, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138 or 982.
96. The AAV particle of any one of embodiments 2, 27-39, 58, 59, or 66-95, wherein [NO] replaces positions 571-573 (e.g., T571, N572, and N573), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
97. The AAV particle of any one of embodiments 2, 27-39, 58, 59, or 66-96, wherein [NO] is present immediately subsequent to position 570, and wherein [NO] replaces positions 571-573 (e.g., T571, N572, and N573), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
98. The AAV particle of any one of embodiments 2, 27-39, 58, 59, or 66-96, wherein [NO] is present immediately subsequent to position 570, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 982.
99. The AAV particle of any one of embodiments 2, 27-39, 58, 59, or 66-98, wherein [NO] replaces positions 571-573 (e.g., T571, N572, and N573), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 982.
100. The AAV particle of any one of embodiments 2, 27-39, 58, 59, or 66-99, wherein [NO] is present immediately subsequent to position 570, and wherein [NO] replaces positions 571-573 (e.g., T571, N572, and N573), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 982.
101. The AAV particle of any one of embodiments 2, 27-39, 58, 59, or 66-100, wherein [NO] corresponds to positions 571-573 (e.g., T571, N572, and N573) of SEQ ID NO: 982.
102. The AAV particle of any one of embodiments 2, 34-39, 58, 59, or 66-101, wherein [NO]-[N1]-[N2]- [N3]-[N4] is present immediately subsequent to position 570, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
103. The AAV particle of any one of embodiments 2, 34-39, 58, 59, or 66-102, wherein [NO]-[N1]-[N2]- LN3J-LN4J replaces positions 571-579 (e.g., T571, N572, N573, Q574, S575, S576, T577, T578, and A579), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
104. The AAV particle of any one of embodiments 2, 34-39, 58, 59, or 66-103, wherein [N0]-[Nl]-[N2]- [N3]-[N4] is present immediately subsequent to position 570, and wherein [N0]-[N1]-[N2]-[N3]-[N4] replaces positions 571-579 (e.g., T571, N572, N573, Q574, S575, S576, T577, T578, and A579), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
105. The AAV particle of any one of embodiments 2, 34-39, 58, 59, or 66-104, wherein [NO]-[N1]-[N2]- [N3]-[N4] corresponds to positions 571-586 (e.g., T571, N572, N573, Q574, S575, S576, Y577, P578, A579, E580, V581, V582, Q583, K584, T585, A586), of SEQ ID NO: 982.
106. The AAV particle of any one of embodiments 2, 34-39, 58, 59, or 66-105, wherein [N4] is present immediately subsequent to position 584, numbered according to SEQ ID NO: 982.
107. The AAV particle of any one of embodiments 2, 34-39, 58, 59, or 66-106, wherein [N4] replaces position 578 and 579, numbered according to SEQ ID NO: 138; or positions 585 and 586 numbered according to SEQ ID NO: 982.
108. The AAV particle of any one of embodiments 2, 34-39, 58, 59, or 66-107, wherein [N4] is present immediately subsequent to position 584 and replaces positions 585 and 586 numbered according to SEQ ID NO: 982.
109. The AAV particle of any one of embodiments 2, 34-39, 58, 59, or 66-108, wherein: (i) XA of [NO] is present at position 571, XB of [NO] is present at position 572, and Xc of [NO] is present at position 573, numbered according to SEQ ID NO: 982;
(ii) XD of [Nl] is present at position 574, XE of [Nl] is present at position 575, and XF of [Nl] is present at position 576, numbered according to SEQ ID NO: 982;
(iii) XI of [N2] is present at position 577, X2 of [N2] is present at position 578, X3 of [N2] is present at position 579, X4 of [N2] is present at position 580, and X5 of [N2] is present at position 581, numbered according to SEQ ID NO: 982;
(iv) [N3] is present at positions 582-584, numbered according to SEQ ID NO: 982; and/or
(v) XQ of [N4] is present at position 585 and XH of [N4] is present at position 586, numbered according to SEQ ID NO: 982.
110. The AAV particle of any one of embodiments 2, 34-39, 58, 59, or 66-109, wherein:
(i) [NO] is present at positions 571-573, numbered according to SEQ ID NO: 982;
(ii) [Nl] is present at positions 574-576, numbered according to SEQ ID NO: 982;
(iii) [N2] is present at positions 577-581, numbered according to SEQ ID NO: 982;
(iv) [N3] is present at positions 582-584, numbered according to SEQ ID NO: 982;
(v) [N4] is present at positions 585-586, numbered according to SEQ ID NO: 982;
(vi) [N2]-[N3] is present at positions 577-584, numbered according to SEQ ID NO: 982; and/or
(vii) [NO]-[N1]-[N2]-[N3]-[N4] is present at positions 571-586, numbered according to SEQ ID NO: 982.
111. The AAV particle of any one of embodiments 1-39, 58, 59, or 66-110, wherein [N3] is present immediately subsequent to [N2].
112. The AAV particle of any one of embodiments 2, 34-39, 58, 59, or 66-111, wherein [N4] is present immediately subsequent to [N3].
113. The AAV particle of any one of embodiments 1-39, 58, 59, or 66-112, which comprises from N- terminus to C-terminus [N2]-[N3].
114. The AAV particle of any one of embodiments 1-39, 58, 59, or 66-113, which comprises from N- terminus to C-terminus [N1]-[N2]-[N3].
115. The AAV particle of any one of embodiments 2, 27-39, 58, 59, or 66-114, which comprises from N- terminus to C-terminus [NO]-[N1]-[N2]-[N3]. 116. The AAV particle of any one of embodiments 2, 34-39, 58, 59, or 66-115, which comprises from N- terminus to C-terminus [N1]-[N2]-[N3]-[N4].
117. The AAV particle of any one of embodiments 2, 34-39, 58, 59, or 66-116, which comprises from N- terminus to C-terminus [NO]-[N1]-[N2]-[N3]-[N4],
118. The AAV particle of any one of embodiments 40-65, wherein [B]-[C] is present in loop VIII, optionally wherein loop VUI comprises positions 571-592 (e.g., amino acids TNNQSSTTAPATGTYNLQEIVP (SEQ ID NO: 285) numbered according to SEQ ID NO: 138, or positions 571-599, numbered according to SEQ ID NO: 982.
119. The AAV particle of any one of embodiments 41, 51-65, or 118, wherein [AJ and/or [DJ is present in loop VIII, optionally wherein loop VIII comprises positions 571-592 (e.g., amino acids TNNQSSTTAPATGTYNLQEIVP (SEQ ID NO: 285)) numbered according to SEQ ID NO: 138, or positions 571-599, numbered according to SEQ ID NO: 982.
120. The AAV particle of any one of embodiments 41 , 51 -65, 1 18 or 1 19, wherein [A]-[B]-[C]-[D] is present in loop VIII, optionally wherein loop VUI comprises positions 571-592 (e.g., amino acids TNNQSSTTAPATGTYNLQEIVP (SEQ ID NO: 285) numbered according to SEQ ID NO: 138, or positions 571-599, numbered according to SEQ ID NO: 982.
121. The AAV particle of any one of embodiments 40-65 or 118-120, wherein [B] is present immediately subsequent to position 573, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
122. The AAV particle of any one of embodiments 40-65 or 118-121, wherein [B] replaces positions 574-576 (e.g., Q574, S575, and S576), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
123. The AAV particle of any one of embodiments 40-65 or 118-122, wherein [B] is present immediately subsequent to position 573, and wherein [B] replaces positions 574-576 (e.g., Q574, S575, and S576), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
124. The AAV particle of any one of embodiments 40-65 or 118-123, wherein [B] is present immediately subsequent to position 573, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 982. 125. The AAV particle of any one of embodiments 40-65 or 118-124, wherein [B] replaces positions 574-576 (e.g., Q574, S575, and S576), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 982.
126. The AAV particle of any one of embodiments 40-65 or 118-125, wherein [B] is present immediately subsequent to position 573, and wherein [B] replaces positions 574-576 (e.g., Q574, S575, and S576), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 982.
127. The AAV particle of any one of embodiments 40-65 or 118-126, wherein [B] corresponds to positions 574-576 (e.g., Q574, S575, and S576) of SEQ ID NO: 982.
128. The AAV particle of any one of embodiments 40-65 or 118-127, wherein [B]-(C] is present immediately subsequent to position 573, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
129. The AAV particle of any one of embodiments 40-65 or 1 18-128, wherein [B]-[C] replaces positions 574-577 (e.g., Q574, S575, S576, and T577), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
130. The AAV particle of any one of embodiments 40-65 or 118-129, wherein [B]-[C] is present immediately subsequent to position 573, and wherein [B]-[C] replaces positions 574-577 (e.g., Q574, S575, S576, and T577), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
131. The AAV particle of any one of embodiments 40-65 or 118-130, wherein [B]-[C] corresponds to positions 574-584 (e.g., Q574, S575, S576, Y577, P578, A579, E580, V581, V582, Q583, K584) of SEQ ID NO: 982.
132. The AAV particle of any one of embodiments 40-65 or 118-131, wherein [B]-[C]-[D] is present immediately subsequent to position 573, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
133. The AAV particle of any one of embodiments 40-65 or 118-132, wherein [B]-[C]-[D] replaces positions 574-579 (e.g., Q574, S575, S576, T577, T578, and A579), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. 134. The AAV particle of any one of embodiments 40-65 or 118-133, wherein [B]-[C]-[D] is present immediately subsequent to position 573, and wherein [B]-[C]-[D] replaces positions 574-579 (e.g., Q574, S575, S576, T577, T578, and A579), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
135. The AAV particle of any one of embodiments 40-65 or 118-134, wherein [B]-[C]-[D] corresponds to positions 574-586 (e.g., Q574, S575, S576, Y577, P578, A579, E580, V581, V582, Q583, K584, T585, A586) of SEQ ID NO: 982.
136. The AAV particle of any one of embodiments 40-65 or 118-135, wherein [C] is present immediately subsequent to position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
137. The AAV particle of any one of embodiments 40-65 or 118-136, wherein [C] replaces position 577 (e.g., T577), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
138. The AAV particle of any one of embodiments 40-65 or 118-137, wherein [C] is present immediately subsequent to position 576, and wherein [C] replaces 577 (e.g., T577), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
139. The AAV particle of any one of embodiments 40-65 or 118-137, wherein [C] corresponds to positions 577-584 (e.g., Y577, P578, A579, E580, V581, V582, Q583, K584) of SEQ ID NO: 982.
140. The AAV particle of any one of embodiments 41, 51-65 or 118-139, wherein [A] is present immediately subsequent to position 570, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
14E The AAV particle of any one of embodiments 41, 51-65 or 118-140, wherein [A] replaces positions 571-573 (e.g., T571, N572, and N573), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
142. The AAV particle of any one of embodiments 41, 51-65 or 118-141, wherein [A] is present immediately subsequent to position 570, and wherein [A] replaces positions 571-573 (e.g., T571, N572, and N573), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
143. The AAV particle of any one of embodiments 51, 51-65 or 118-142, wherein [A] is present immediately subsequent to position 570, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 982.
144. The AAV particle of any one of embodiments 41, 51-65 or 118-143, wherein [A] replaces positions 571-573 (e.g., T571, N572, and N573), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 982.
145. The AAV particle of any one of embodiments 41, 51-65 or 118-144, wherein [AJ is present immediately subsequent to position 570, and wherein [A] replaces positions 571-573 (e.g., T571, N572, and N573), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 982.
146. The AAV particle of any one of embodiments 41 , 51 -65 or 1 18-145, wherein [A] corresponds to positions 571-573 (e.g., T571, N572, and N573) of SEQ ID NO: 982.
147. The AAV particle of any one of embodiments 41, 51-65 or 118-146, wherein [D] is present immediately subsequent to position 584, numbered according to SEQ ID NO: 982.
148. The AAV particle of any one of embodiments 41, 51-65 or 118-147, wherein [D] replaces positions 578 and 579 numbered according to SEQ ID NO: 138; or positions 585 and 586, numbered according to SEQ ID NO: 982.
149. The AAV particle of any one of embodiments 41, 51-65 or 118-147, wherein [D] is present immediately subsequent to position 584 and replaces positions 585 and 586, numbered according to SEQ ID NO: 982.
150. The AAV particle of any one of embodiments 41, 51-65 or 118-149, wherein [A]-[B]-[C]-[D] is present immediately subsequent to position 570, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. 151. The AAV particle of any one of embodiments 41 or 118-150, wherein [A]-[B]-[C]-[D] replaces positions 571-579 (e.g., T571, N572, N573, Q574, S575, S576, T577, T578, and A579), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
152. The AAV particle of any one of embodiments 41 or 118-151, wherein [A]-[B]-[C]-[D] is present immediately subsequent to position 570, and wherein [A]-[B]-[C]-[D] replaces positions 571-579 (e.g., T571, N572, N573, Q574, S575, S576, T577, T578, and A579), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
153. The AAV particle of any one of embodiments 41, 51-65, or 118-152, wherein [A]-[B]-[C]-[D] corresponds to positions 571-586 (e.g., T571, N572, N573, Q574, S575, S576, Y577, P578, A579, E58O, V581, V582, Q583, K584, T585, A586) of SEQ ID NO: 982.
154. The AAV particle of any one of embodiments 41, 51-65, or 118-153, wherein:
(i) XA of [A] is present at position 571, XB of [A] is present at position 572, and Xc of [A] is present at position 573, numbered according to SEQ ID NO: 982;
(ii) X1 of [B] is present at position 574, X2 of [B] is present at position 575, and X3 of [B] is present at position 576, numbered according to SEQ ID NO: 982;
(iii) [C] is present at positions 577-584, numbered according to SEQ ID NO: 982; and/or
(iv) X4 of [D] is present at position 585 and position X5 of [D] is present at position 586, numbered according to SEQ ID NO: 982.
155. The AAV particle of any one of embodiments 41, 51-65, or 118-154, wherein:
(i) [A] is present at positions 571-573, numbered according to SEQ ID NO: 982;
(ii) [B] is present at positions 574-576, numbered according to SEQ ID NO: 982;
(iii) [C] is present at positions 577-584, numbered according to SEQ ID NO: 982;
(iv) [D] is present at positions 585-586, numbered according to SEQ ID NO: 982; and/or
(v) [A]-[B]-[C]-[D] is present at positions 571-586, numbered according to SEQ ID NO: 982.
156. The AAV particle of any one of embodiments 40-65 or 118-155, wherein [C] is present immediately subsequent to [B].
157. The AAV particle of any one of embodiments 41, 60-65, or 118-156, wherein [D] is present immediately subsequent to [C]. 158. The AAV particle of any one of embodiments 41-65 or 118-157, which comprises from N-terminus to C-terminus [B]-[C].
159. The AAV particle of any one of embodiments 41, 51-65, or 118-158, which comprises from N- terminus to C-terminus [A]-[B]-[C].
160. The AAV particle of any one of embodiments 41, 51-65, or 118-159, which comprises from N- terminus to C-terminus [B]-[C]-[D].
161. The AAV particle of any one of embodiments 41, 60-65, or 118-160, which comprises from N- terminus to C-terminus [A]-[B]-[C]-[D].
162. An AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant comprises:
(a) the amino acid sequence of any of the sequences provided in Tables 2A, 2B, 2C, 15 or 21;
(b) an amino acid sequence comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15 consecutive amino acids from any one of the sequences provided in Tables 2A, 2B, 2C, 15 or 21;
(c) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to the amino acid sequence of any one of the amino acid sequences provided in Tables 2A, 2B, 2C, 15 or 21; or
(d) an amino sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of any one of the amino acid sequences provided in Tables 2A, 2B, 2C, 15 or 21.
163. An AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant comprises:
(a) tire amino acid sequence of any of SEQ ID NOs: 943, 1021, 1024, 1027, 1112, 1142, 1214, 1232, 1254, 1300, 1310, 1327, 1331, 1342, 1419, 1453, 1533, 1538, 1539, 1575, 1578, 1583-1587, 1590, 1591-1593, 1598-1608, or 1610-1624;
(b) an amino acid sequence comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive amino acids from any one of SEQ ID NOs: 943, 1021, 1024, 1027, 1112, 1142, 1214, 1232, 1254, 1300, 1310, 1327, 1331, 1342, 1419, 1453, 1533, 1538, 1539, 1575, 1578, 1583-1587, 1590, 1591- 1593, 1598-1608, or 1610-1624; (c) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to the amino acid sequence of any one of SEQ ID NOs: 943, 1021, 1024, 1027, 1112, 1142, 1214, 1232, 1254, 1300, 1310, 1327, 1331, 1342, 1419, 1453, 1533, 1538, 1539, 1575, 1578, 1583- 1587, 1590, 1591-1593, 1598-1608, or 1610-1624; or
(d) an amino sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of any one of SEQ ID NOs: 943, 1021, 1024, 1027, 1112, 1142, 1214, 1232, 1254, 1300, 1310, 1327, 1331, 1342, 1419, 1453, 1533, 1538, 1539, 1575, 1578, 1583-1587, 1590, 1591-1593, 1598-1608, or 1610-1624.
164. An AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant comprises:
(a) the amino acid sequence of any of SEQ ID NOs: 1021, 1024, 1232, 1300, 1327, 1533, 1538, 1585, 1590, or 1591;
(b) an amino acid sequence comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive amino acids from any one of SEQ ID NOs: 1021 , 1024, 1232, 1300, 1327, 1533, 1538, 1585, 1590, or 1591;
(c) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to the amino acid sequence of any one of SEQ ID NOs: 1021, 1024, 1232, 1300, 1327, 1533, 1538, 1585, 1590, or 1591; or
(d) an amino sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of any one of SEQ ID NOs: 1021, 1024, 1232, 1300, 1327, 1533, 1538, 1585, 1590, or 1591.
165. The AAV particle of any one of embodiments 162-164, which comprises at least 3, 4, 5, 6, or 7 consecutive amino acids from of any one of SEQ ID NOs: 943 or 946-966.
166. The AAV particle of embodiment 162-165, wherein the 3 consecutive amino acids comprise YPA.
167. The AAV particle of embodiment 162-166, wherein the 4 consecutive amino acids comprise YPAE (SEQ ID NO: 286).
168. The AAV particle of embodiment 162-167, wherein the 5 consecutive amino acids comprise YPAEV (SEQ ID NO: 39). 169. The AAV particle of embodiment 162-168, wherein the 6 consecutive amino acids comprise YPAEVV (SEQ ID NO: 151).
170. The AAV particle of embodiment 162-169, wherein the 7 consecutive amino acids comprise YPAEVVQ (SEQ ID NO: 152).
171. The AAV particle of embodiment 162-170, wherein the amino acid sequence comprises YPAEVVQK (SEQ ID NO: 943).
172. The AAV particle of any one of embodiments 162-164, wherein:
(i) the 3 consecutive amino acids comprise YTP;
(ii) the 4 consecutive amino acids comprise YTPS (SEQ ID NO: 306);
(iii) the 5 consecutive amino acids comprise YTPSL (SEQ ID NO: 145) ;
(iv) the 6 consecutive amino acids comprise YTPSLV (SEQ ID NO: 153);
(v) the 7 consecutive amino acids comprise YTPSLVQ (SEQ ID NO: 154); and/or
(vi) the amino acid sequence comprises YTPSLVQK (SEQ ID NO: 952).
173. The AAV particle of any one of embodiments 162-164, wherein:
(i) the 3 consecutive amino acids comprise YPP;
(ii) the 4 consecutive amino acids comprise YPPS (SEQ ID NO: 307);
(iii) the 5 consecutive amino acids comprise YPPSL (SEQ ID NO: 140);
(iv) the 6 consecutive amino acids comprise YPPSLV (SEQ ID NO: 155);
(v) the 7 consecutive amino acids comprise YPPSLVQ (SEQ ID NO: 156); and/or
(vi) the amino acid sequence comprises YPPSLVQK (SEQ ID NO: 946).
174. The AAV particle of any one of embodiments 162-164, wherein:
(i) the 3 consecutive amino acids comprise YPP;
(ii) the 4 consecutive amino acids comprise YPPS (SEQ ID NO: 307);
(iii) the 5 consecutive amino acids comprise YPPSL (SEQ ID NO: 40);
(iv) the 6 consecutive amino acids comprise YPPSLE (SEQ ID NO: 157);
(v) the 7 consecutive amino acids comprise YPPSLEQ (SEQ ID NO: 158); and/or
(vi) the amino acid sequence comprises YPPSLEQK (SEQ ID NO: 953).
175. The AAV particle of any one of embodiments 162-164, wherein:
(i) the 3 consecutive amino acids comprise YPP;
(ii) the 4 consecutive amino acids comprise YPPS (SEQ ID NO: 307); (iii) the 5 consecutive amino acids comprise YPPSL (SEQ ID NO: 40);
(iv) the 6 consecutive amino acids comprise YPPSLV (SEQ ID NO: 155);
(v) the 7 consecutive amino acids comprise YPPSLVK (SEQ ID NO: 159); and/or
(vi) the amino acid sequence comprises YPPSLVKK (SEQ ID NO: 954).
176. The AAV particle of any one of embodiments 162-175, wherein the AAV capsid variant comprises an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to the amino acid sequence of YPAEVVQK (SEQ ID NO: 943).
177. The AAV particle of any one of embodiments 162-164 or 172-175, wherein the AAV capsid variant comprises an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to the amino acid sequence of any of SEQ ID NOs: 1021, 1024, 1232, 1300, 1327, 1533, 1538, 1585, 1590, or 1591.
178. The AAV particle of any one of embodiments 162-164 or 172-175, wherein the AAV capsid variant comprises an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to the amino acid sequence of any of SEQ ID NOs: 946, 952, 953, or 954.
179. The AAV particle of any one of embodiments 162-171 or 176, wherein the AAV capsid variant comprises an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of YPAEVVQK (SEQ ID NO: 943).
180. The AAV particle of any one of embodiments 162-164, 172-175, or 177, wherein the AAV capsid variant comprises an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of any one of SEQ ID NOs: 1021, 1024, 1232, 1300, 1327, 1533, 1538, 1585, 1590, or 1591.
181. The AAV particle of any one of embodiments 162-164, 172-175, or 178, wherein the AAV capsid variant comprises an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of any one of SEQ ID NOs: 946, 952, 953, or 954.
182. The AAV particle of any one of embodiments 162-171, 178 or 179, wherein the AAV capsid variant comprises an amino acid sequence encoded by: (i) a nucleotide sequence comprising at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to the nucleotide sequence of SEQ ID NO: 944; or
(ii) a nucleotide sequence comprising at least one, two, three, four, five, six, or seven, but no more than ten different nucleotides relative to the nucleotide sequence of SEQ ID NO: 944.
183. The AAV particle of any one of embodiments 162-171, 176, 179 or 182, wherein the nucleotide sequence encoding the amino acid sequence comprises:
(i) a nucleotide sequence comprising at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to the nucleotide sequence of SEQ ID NO: 944; or
(ii) a nucleotide sequence comprising at least one, two, three, four, five, six, or seven, but no more than ten different nucleotides relative to the nucleotide sequence of SEQ ID NO: 944.
184. The AAV particle of any one of embodiments 162-183, wherein the amino acid sequence is present in loop VIII.
185. The AAV particle of any one of embodiments 162-184, wherein the amino acid sequence is present immediately subsequent to position 570, 571, 572, 573, 574, 575, or 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
186. The AAV particle of any one of embodiments 162-185, wherein the amino acid sequence replaces one, two, three, four, five or all of positions 571, 572, 573, 574, 575, and/or 576 (e.g., positions T571, N572, N573, Q574, S575, S576, T577, T578, and/or A579), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
187. The AAV particle of any one of embodiments 162-186, wherein the amino acid sequence is present immediately subsequent to position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
188. The AAV particle of any one of embodiments 162-187, which comprises an amino acid residue other than T at position 577, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. 189. The AAV particle of any one of embodiments 162-188, wherein the AAV capsid variant comprises the amino acid Y at position 577, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
190. The AAV particle of any one of embodiments 162-189, wherein the AAV capsid variant comprises the substitution T577Y, numbered according to SEQ ID NO: 138.
191. The AAV particle of any one of embodiments 188-190, wherein the amino acid sequence is or comprises YPAEVVQK (SEQ ID NO: 943), and starts at position 577, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
192. The AAV particle of any one of embodiments 162-171, 176, 179, 182, 183, or 184-191, wherein the AAV capsid variant comprises an amino acid other than T at position 577, and further comprises the amino acid sequence of PAEVVQK (SEQ ID NO: 61), which is present immediately subsequent to position 577, numbered relative to SEQ ID NO: 138.
193. The AAV particle of any one of embodiments 162-171 , 176, 179, 182, 183, or 184-192, wherein the AAV capsid variant comprises the amino acid Y at position 577, and further comprises the amino acid sequence of PAEVVQK (SEQ ID NO: 61), which is present immediately subsequent to position 577, numbered relative to SEQ ID NO: 138.
194. The AAV particle of any one of embodiments 162-171, 176, 179, 182, 183, or 184-193, wherein the AAV capsid variant comprises the amino acid Y at position 577, and further comprises the amino acid sequence of PAEVVQK (SEQ ID NO: 61), which is present immediately subsequent to position 577, numbered relative to SEQ ID NO: 982.
195. The AAV particle of any one of embodiments 162-171, 176, 179, 182, 183, 184-193, wherein the AAV capsid variant comprises the amino acid sequence of YPAEVVQK (SEQ ID NO: 943), wherein the amino acid sequence replaces position 577 (e.g., T577), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
196. The AAV particle of any one of embodiments 162-171, 176, 179, 182, 183, or 184-195, wherein the AAV capsid variant comprises the amino acid sequence of YPAEVVQK (SEQ ID NO: 943), wherein the amino acid sequence:
(i) is present immediately subsequent to position 576; and
(ii) replaces position 577 (e.g., T577), wherein in (i) and (ii) are numbered relative to SEQ ID NO: 138.
197. The AAV particle of any one of embodiments 162-171, 176, 179, 182, 183, or 184-196, wherein the AAV capsid variant further comprises an amino acid other than T at position 571 (e.g., I), numbered relative to SEQ ID NO: 138 or SEQ ID NO: 982.
198. The AAV particle of any one of embodiments 162-171, 176, 179, 182, 183, or 184-197, wherein the AAV capsid variant further comprises I at position 571, numbered relative to SEQ ID NO: 138 or SEQ ID NO: 982.
199. The AAV particle of any one of embodiments 162-171, 176, 182, 183, or 184-198, wherein the AAV capsid variant further comprises one, two, or all of an amino acid other than Q at position 574 (e.g., A or T), S at position 575 (e.g., G), and/or S (e.g., A, L, K, or R) at position 576, numbered relative to SEQ ID NO: 138 or SEQ ID NO: 982.
200. The AAV particle of any one of embodiments 162-171, 176, 179, 182, 183, or 184-199, wherein the AAV capsid variant further comprises:
(i) A at position 574, G at position 575, and A at position 576, numbered relative to SEQ ID NO: 138 or 982; or
(ii) T at position 574 and L at position 576, numbered relative to SEQ ID NO: 138 or 982.
201. The AAV particle of any one of embodiments 162-171, 176, 179, 182, 183, or 184-199, wherein the AAV capsid variant further comprises:
(i) K at position 576, numbered relative to SEQ ID NO: 138 or 982; or
(ii) R at position 576, numbered relative to SEQ ID NO: 138 or 982.
202. The AAV particle of any one of embodiments 162-171, 176, 179, 182, 183, or 184-200, wherein the AAV capsid variant comprises:
(i) A at position 574, G at position 575, A at position 576, and Y at position 577, numbered relative to SEQ ID NO: 138 or 982; and
(ii) the amino acid sequence of PAEVVQK (SEQ ID NO: 61), which is present immediately subsequent to position 577, numbered according to SEQ ID NO: 138 or 982.
203. The AAV particle of any one of embodiments 162-171, 176, 179, 182, 183, or 184-200, wherein the AAV capsid variant comprises: (i) T at position 574, L at position 576, and Y at position 577, numbered relative to SEQ ID NO: 138 or 982; and
(ii) the amino acid sequence of PAEVVQK (SEQ ID NO: 61), which is present immediately subsequent to position 577, numbered according to SEQ ID NO: 138 or 982.
204. The AAV particle of any one of embodiments 162-171, 176, 179, 182, 183, 184-199 or 201, wherein the AAV capsid variant comprises:
(i) K at position 576 and Y at position 577, numbered relative to SEQ ID NO: 138 or 982; and
(ii) the amino acid sequence of PAEVVQK (SEQ ID NO: 61), which is present immediately subsequent to position 577, numbered according to SEQ ID NO: 138 or 982.
205. The AAV particle of any one of embodiments 162-171, 176, 179, 182, 183, 184-199 or 201, wherein the AAV capsid variant comprises:
(i) R at position 576 and Y at position 577, numbered relative to SEQ ID NO: 138 or 982; and
(ii) the amino acid sequence of PAEVVQK (SEQ ID NO: 61), which is present immediately subsequent to position 577, numbered according to SEQ ID NO: 138 or 982.
206. The AAV particle of any one of embodiments 162-171, 176, 179, 182, 183, 184-195, wherein the AAV capsid variant comprises:
(i) I at position 571 and Y at position 577, numbered relative to SEQ ID NO: 138 or 982; and
(ii) the amino acid sequence of PAEVVQK (SEQ ID NO: 61), which is present immediately subsequent to position 577, numbered according to SEQ ID NO: 138 or 982.
207. The AAV particle of any one of embodiments 162-164, 172, 177, 178, 180, or 181, wherein the AAV capsid variant comprises Y at position 577 and the amino acid sequence TPSLVQK (SEQ ID NO: 68), which is present immediately subsequent to position 577, all numbered according to SEQ ID NO: 138 or 982.
208. The AAV particle of any one of embodiments 162-164, 172, 177, 178, 180, or 181, wherein the AAV capsid variant comprises Y at position 577 and the amino acid sequence PPSLVQK (SEQ ID NO: 62), which is present immediately subsequent to position 577, all numbered according to SEQ ID NO: 138 or 982.
209. The AAV particle of any one of embodiments 162-164, 172, 177, 178, 180, or 181, wherein the
AAV capsid variant comprises Y at position 577 and the amino acid sequence PPSLEQK (SEQ ID NO: 69), which is present immediately subsequent to position 577, all numbered according to SEQ ID NO: 138 or 982.
210. The AAV particle of any one of embodiments 162-164, 172, 177, 178, 180, or 181, wherein the AAV capsid variant comprises Y at position 577 and the amino acid sequence PPSLVKK (SEQ ID NO:
70), which is present immediately subsequent to position 577, all numbered according to SEQ ID NO: 138 or 982.
211. The AAV particle of any one of the preceding embodiments, wherein the AAV capsid variant further comprises a modification, e.g., an insertion, substitution (e.g., conservative substitution), and/or deletion, in loop I, II, IV, and/or VI.
212. The AAV particle of any one of the preceding embodiments, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one, two or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but not more than 30, 20 or 10 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SEQ ID NO: 1 8.
213. The AAV particle of any one of the preceding embodiments, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one, two or three, but no more than 30, 20 or 10 different amino acids relative to the amino acid sequence of SEQ ID NO: 138.
214. The AAV particle of any one of the preceding embodiments, wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 138, or an amino acid sequence with at least 80% (e.g., at least about 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.
215. The AAV particle of any one of the preceding embodiments, wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 138.
216. The AAV particle of any one of the preceding embodiments, wherein the AAV capsid variant comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 137, or a sequence with at least 80% (e.g., at least about 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.
217. The AAV particle of any one of the preceding embodiments, wherein the nucleotide sequence encoding the AAV capsid variant comprises the nucleotide sequence of SEQ ID NO: 137, or a sequence with at least 80% (e.g., at least about 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto. 218. The AAV particle of any one of the preceding embodiments, wherein the AAV capsid variant comprises a VP1 protein, a VP2 protein, a VP3 protein, or a combination thereof.
219. The AAV particle of any one of embodiments 1-171, 176, 179, 182, 183, 184-196 or 211-218, wherein the AAV capsid variant comprises the amino acid sequence corresponding to positions 137-731, e.g., a VP2, of SEQ ID NO: 982, or a sequence with at least 80% (e.g., at least about 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.
220. The AAV particle of any one of embodiments 1-171, 176, 179, 182, 183, 184-196 or 211-219, wherein the AAV capsid variant comprises the amino acid sequence corresponding to positions 193-731, e.g., a VP3, of SEQ ID NO: 982, or a sequence with at least 80% (e.g., at least about 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.
221. The AAV particle of any one of embodiments 1-220, wherein the AAV capsid variant comprises the amino acid sequence corresponding to positions 137-724, e.g., a VP2, of SEQ ID NO: 138, or a sequence with at least 80% (e.g., at least about 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.
222. The AAV particle of any one of embodiments 1-220, wherein the AAV capsid variant comprises the amino acid sequence corresponding to positions 193-724, e.g., a VP3, of SEQ ID NO: 138, or a sequence with at least 80% (e.g., at least about 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.
223. The AAV capsid variant, of any one of embodiments 162-171, 176, 179, 182, 183, 184-196 or 211- 218, comprising an amino acid sequence comprising at least 3, 4, 5, or 6 consecutive amino acids from the amino acid sequence of YPAEVVQK (SEQ ID NO: 943), wherein:
(i) the 3 consecutive amino acids comprise YPA;
(ii) the 4 consecutive amino acids comprise YPAE (SEQ ID NO: 286);
(iii) the 5 consecutive amino acids comprise YPAEV (SEQ ID NO: 39);
(iv) the 6 consecutive amino acids comprise YPAEVV (SEQ ID NO: 151);
(v) the 7 consecutive amino acids comprise YPAEVVQ (SEQ ID NO: 152); wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 739, or an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 739.
224. The AAV particle of any one of embodiments 162-171, 176, 179, 182, 183, 184-196, 211-218 or 223, wherein the AAV capsid variant comprises an amino acid sequence comprising at least 3, 4, 5, or 6 consecutive amino acids from the amino acid sequence of YPAEVVQK (SEQ ID NO: 943), wherein: (i) the 3 consecutive amino acids comprise YPA;
(ii) the 4 consecutive amino acids comprise YPAE (SEQ ID NO: 286);
(iii) the 5 consecutive amino acids comprise YPAEV (SEQ ID NO: 39);
(iv) the 6 consecutive amino acids comprise YPAEVV (SEQ ID NO: 151);
(v) the 7 consecutive amino acids comprise YPAEVVQ (SEQ ID NO: 152); wherein the AAV capsid variant comprises: (a) a VP1 protein comprising the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 982; (b) a VP2 protein comprising the amino acid sequence of positions 137-724 of SEQ ID NO: 138 or positions 137-731 of SEQ ID NO: 982; (c) a VP3 protein comprising the amino acid sequence of positions 193-724 of SEQ ID NO: 138 or positions 193- 731 of SEQ ID NO: 982; or (d) an amino acid sequence with at least 90% (e.g., at least about 95, 96, 97, 98, or 99%) sequence identity to any of the amino acid sequences in (a)-(c).
225. The AAV particle of any one of embodiments 162-171, 176, 179, 182, 183, 184-196, 211-218, or 224, wherein the AAV capsid variant comprises one, two, or three but no more than four different amino acids, relative to the amino acid sequence of YPAEVVQK (SEQ ID NO: 943), wherein the AAV capsid variant comprises:
(a) a VP1 protein comprising the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 982;
(b) a VP2 protein comprising the amino acid sequence of positions 137-724 of SEQ ID NO: 138 or positions 137-731 of SEQ ID NO: 982;
(c) a VP3 protein comprising the amino acid sequence of positions 193-724 of SEQ ID NO: 138 or positions 193-731 of SEQ ID NO: 982; or
(d) an amino acid sequence with at least 90% (e.g., at least about 95, 96, 97, 98, or 99%) sequence identity to any of the amino acid sequences in (a)-(c).
226. The AAV particle of any one of embodiments 162-171, 176, 179, 182, 183, 184-196, 211-218, 224, or 225, wherein the AAV capsid variant comprises one or two, but no more than three substitutions relative to the amino acid sequence of YPAEVVQK (SEQ ID NO: 943), wherein the AAV capsid variant comprises an amino acid sequence at least 90% (e.g., at least about 95, 96, 97, 98, or 99%) identical to the amino acid sequence of SEQ ID NO: 739.
227. The AAV particle of any one of embodiments 162-171, 176, 179, 182, 183, 184-196, 211-218, or 224-226, wherein the AAV capsid variant comprises one or two, but no more than three substitutions relative to the amino acid sequence of YPAEVVQK (SEQ ID NO: 943), wherein the AAV capsid variant comprises an amino acid sequence at least 90% (e.g., at least about 95, 96, 97, 98, or 99%) identical to the amino acid sequence of SEQ ID NO: 982. 228. The AAV particle of any one of embodiments 162-171, 176, 179, 182, 183, 184-196, 211-218, or 224-227, wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 982, or an amino acid sequence with at least 80% (e.g., at least about 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.
229. The AAV particle of any one of embodiments 1-171, 176, 179, 182, 183, 184-196, 211-219, or 224-
228, wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 982 or an amino acid sequence with at least 95% identity thereto.
230. The AAV particle, of any one of embodiments 1-171, 176, 179, 182, 183, 184-196, 211-219, or 224-
229, wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 982 or an amino acid sequence with at least 98% identity thereto.
231. The AAV particle of any one of embodiments 1-171, 176, 179, 182, 183, 184-196, 211-219, or 224-
230, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one, two or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but not more than 30, 20 or 10 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SEQ ID NO: 982.
232. The AAV particle of any one of embodiments 1-171, 176, 179, 182, 183, 184-196, 211-219, or 224-
231, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one, two or three, but not more than 30, 20 or 10 different amino acids, relative to the amino acid sequence of SEQ ID NO: 982.
233. The AAV particle of any one of the preceding embodiments 1-171, 176, 179, 182, 183, 184-196, 211-219, or 224-232, wherein the nucleotide sequence encoding the AAV capsid variant comprises the nucleotide sequence of SEQ ID NO: 984, or a nucleotide sequence with at least 80% (e.g., at least about 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.
234 An AAV particle comprising an AAV capsid variant and a nucleic acid comprising a nucleotide sequence encoding a survival motor neuron (SMN) protein, (e.g., human SMN), fragment or variant thereof, wherein the AAV capsid variant comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 739, and wherein the AAV capsid variant comprises the amino acid sequence of YPAEVVQK (SEQ ID NO: 943). 235. The AAV particle of embodiment 234, wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 739.
236. The AAV particle of any one of the preceding embodiments, wherein the nucleotide sequence encoding the AAV capsid variant is codon optimized.
237. The AAV particle of any one embodiments 1-236, wherein the AAV capsid variant has an increased tropism for a CNS cell or tissue, e.g., a brain cell, brain tissue, spinal cord cell, or spinal cord tissue, relative to the tropism of a reference sequence comprising the amino acid sequence of SEQ ID NO: 138.
238. The AAV particle of any one embodiments 1-237, wherein the AAV capsid variant has an increased tropism for a CNS cell or tissue, e.g., a brain cell, brain tissue, spinal cord cell, or spinal cord tissue, relative to the tropism of a reference sequence comprising the amino acid sequence of SEQ ID NO: 982.
239. The AAV particle of any one embodiments 1-238, wherein the AAV capsid variant has an increased tropism for a CNS cell or tissue, e.g., a brain cell, brain tissue, spinal cord cell, or spinal cord tissue, relative to the tropism of a reference sequence comprising the amino acid sequence of SEQ ID NO: 1 9.
240. The AAV particle of any one of embodiments 1-239, wherein the AAV capsid variant transduces a brain region, e.g., a temporal cortex, perirhinal cortex, globus pallidus, putamen, caudate, thalamus, hippocampus, geniculate nucleus, Purkinje Layer, deep cerebellar nuclei, and/or cerebellum, optionally wherein the level of transduction is at least 1.5, 2.2, 2.4, 2.5, 2.6, 2.7, 3.0, 3.2, 3.5, 3.7, 4.0, 4.2, 4.5, 4.7, 4.9, 5, 10, 15, 20, 25, 30, 35-fold greater as compared to a reference sequence of SEQ ID NO: 139, e.g., when measured by an assay, e.g., an immunohistochemistry assay or a qPCR assay, e.g., as described in Example 5.
241. The AAV particle of any one of embodiments 1-240, wherein the AAV capsid variant is enriched at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 65-fold, in the brain compared to a reference sequence of SEQ ID NO: 138, e.g., when measured by an assay as described in Example 4.
242. The AAV particle of any one of embodiments 1-241, wherein the AAV capsid variant is enriched at least about 10, 12, 15, 17, 20, 25, 30, 35, 40, 45, 50, 55, 60, 61, 62, 63, 64, or 65-fold, in the brain compared to a reference sequence of SEQ ID NO: 138, e.g., when measured by an assay as described in Example 4. 243. The AAV particle of any one of embodiments 1-242, wherein the AAV capsid variant is enriched in the brain of at least two to three species, e.g., a non-human primate and rodent (e.g., rat and/or mouse), e.g., as compared to a reference sequence of SEQ ID NO: 138.
244. The AAV particle, of any one of embodiments 1-243, wherein the AAV capsid variant is enriched at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, 30, 35, 40, 45-fold, in the brain of at least two to three species, e.g., a non-human primate and rodent (e.g., rat and/or mouse), compared to a reference sequence of SEQ ID NO: 138, e.g., when measured by an assay as described in Examples 4-7.
245. The AAV particle of embodiment 243 or 244, wherein the at least two to three species are Macaca fascicularis , Chlorocebus sabaeus, Callithrixjacchus, rat, and/or mouse (e.g., BALB/c mice).
246. The AAV particle, of any one of embodiments 1-245, wherein the AAV capsid variant is enriched at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, or 225-fold, in the brain compared to a reference sequence of SEQ ID NO: 982, e.g., when measured by an assay as described in Example 6.
247. The AAV particle of any one of embodiments 1-246, wherein the AAV capsid variant delivers an increased level of a payload to a brain region, optionally wherein the level of the payload is increased by at least 20, 25, 30, 35-fold, as compared to a reference sequence of SEQ ID NO: 139, e.g., when measured by an assay, e.g., a qRT-PCR or a qPCR assay (e.g., as described in Example 5).
248. The AAV particle of any one of embodiments 1-247, wherein the AAV capsid variant delivers an increased level of viral genomes to a brain region, optionally wherein the level of viral genomes is increased by at least 1.5, 2.2, 2.4, 2.5, 2.6, 2.7, 3.0, 3.2, 3.5, 3.7, 4.0, 4.2, 4.5, 4.7, 4.9, or 5-fold, as compared to a reference sequence of SEQ ID NO: 139, e.g., when measured by an assay, e.g., a qRT- PCR or a qPCR assay (e.g., as described in Example 5).
249. The AAV particle of any one of embodiments 237-248, wherein the brain region is a temporal cortex, perirhinal cortex, globus pallidus, putamen, caudate, thalamus, hippocampus, geniculate nucleus, Purkinje Layer, deep cerebellar nuclei, cerebellum, or a combination thereof.
250. The AAV particle of any one of embodiments 1-249, wherein the AAV capsid variant is enriched at least about 3, 3.5, 4.0, 4.5, 5, 5.0, 6.0, or 6.5-fold, in a spinal cord region compared to a reference sequence of SEQ ID NO: 139, e.g., when measured by an assay as described in Example 5. 251. The AAV particle of any one of embodiments 250, wherein the spinal cord region is a cervical region, a lumbar region, a thoracic region, or a combination thereof.
252. The AAV particle of any one of the preceding embodiments, wherein the AAV capsid variant shows preferential transduction in a brain region relative to the transduction in the dorsal root ganglia (DRG).
253. The AAV particle of any one of embodiments 1-252, wherein the AAV capsid variant is capable of transducing neuronal cells.
254. The AAV particle of any one of embodiments 1-253, wherein the AAV capsid variant is capable of transducing non-neuronal cells, e.g., glial cells (e.g., oligodendrocytes).
255. The AAV particle of any one of embodiments 1-254, wherein the AAV capsid variant shows preferential transduction in a brain region relative to the transduction in the liver.
256. The AAV particle of any one of embodiments 1-255, wherein the encoded SMN protein is a human SMN protein.
257. The AAV particle of any one of embodiments 1-256, wherein the encoded SMN protein is an SMN1 protein, an SMN2 protein, or both.
258. The AAV particle of any one of embodiments 1-257, wherein the encoded SMN protein is an SMN1 protein.
259. The AAV particle of any one of embodiments 1-258, wherein the encoded SMN protein comprises an amino acid sequence having at least one, two, three or four, but no more than 30, 20 or 10 different amino acids relative to SEQ ID NO: 2000.
260. The AAV particle of any one of embodiments 1-259, wherein the encoded SMN protein comprises an amino acid sequence having at least one, two, three or four modifications, e.g., substitutions (e.g., conservative substitutions), but no more than 30, 20 or 10 modifications, e.g., substitutions (e.g., conservative substitutions), relative to SEQ ID NO: 2000.
261. The AAV particle of any one of embodiments 1-260, wherein the encoded SMN protein comprises the amino acid sequence of SEQ ID NO: 2000, or an amino acid sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto. 262. The AAV particle of any one of embodiments 1-261, wherein the encoded SMN protein comprises the amino acid sequence of SEQ ID NO: 2000.
263. The AAV particle of any one of embodiments 1-261, wherein the nucleotide sequence encoding the SMN protein comprises:
(i) the nucleotide sequence of any one of SEQ ID NOs: 6-8;
(ii) a nucleotide sequence comprising at least one, two, three, or four but no more than 30, 20, or 10 different nucleotides relative to any one of SEQ ID NOs: 6-8;
(iii) a nucleotide sequence comprising at least one, two, three, or four modifications, e.g., substitutions, but no more than 30, 20, or 10 different modifications, e.g., substitutions, relative to any one of SEQ ID NOs: 6-8; or
(iv) a nucleotide sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%) identical to any one of SEQ ID NOs: 6-8.
264. The AAV particle of any one of embodiments 1-263, wherein the nucleotide sequence encoding the SMN protein comprises SEQ ID NO: 8, or a nucleotide sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto.
265. The AAV particle of any one of embodiments 1-264, wherein the nucleotide sequence encoding the SMN protein comprises SEQ ID NO: 8, or a nucleotide sequence comprising at least one, two, three, or four but no more than 30, 20, or 10 different nucleotides relative to SEQ ID NO: 8.
266. The AAV particle of any one of embodiments 1-265, wherein the nucleotide sequence encoding the SMN protein comprises SEQ ID NO: 8, or a nucleotide sequence comprising at least one, two, three, or four modifications, e.g., substitutions, but no more than 30, 20, or 10 different modifications, e.g., substitutions, relative to SEQ ID NO: 8.
267. The AAV particle of any one of embodiments 1-265, wherein the nucleotide sequence encoding the SMN protein comprises SEQ ID NO: 8.
268. The AAV particle of any one of embodiments 1-262, wherein the nucleotide sequence encoding the SMN protein comprises the nucleotide sequence of any one of SEQ ID NOs: 2001-2004, or a nucleotide sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto. 269. The AAV particle of any one of embodiments 1-262, wherein the nucleotide sequence encoding the SMN protein comprises the nucleotide sequence of SEQ ID NO: 9, or a nucleotide sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto.
270. The AAV particle of any one of the preceding embodiments, wherein the nucleotide sequence encoding the SMN protein is codon optimized.
271. The AAV particle of any one of embodiments 1-265, 267, 268 or 270, wherein the encoded SMN protein is a murine SMN protein, optionally wherein:
(i) the encoded SMN protein comprises the amino acid sequence of SEQ ID NO: 2005 or an amino acid sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto; and/or
(ii) the nucleotide sequence encoding the SMN protein comprises SEQ ID NO: 2006, or a nucleotide sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto.
272. The AAV particle of any one of embodiments 1 -265, 267, 268 or 270, wherein the encoded SMN protein is a SMN protein from a non-human primate, e.g., Macacafascicularis, optionally wherein the encoded SMN protein comprises:
(i) the amino acid sequence of SEQ ID NO: 2008 or an amino acid sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto; or
(ii) the amino acid sequence of SEQ ID NO: 2008 or an amino acid sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto.
273. The AAV particle of any one of embodiments 1-272, wherein the nucleic acid encoding the SMN protein further comprises a nucleotide sequence encoding a splicing modulator element.
274. The AAV particle of embodiment 273, wherein the encoded splicing modulator element increases the level of exon 7-containing SMN2 mRNA.
275. The AAV particle of embodiment 273 or 274, wherein the encoded splicing modulator element is an antisense oligonucleotide.
276. The isolated particle of embodiment 275, wherein the antisense oligonucleotide targets the intronic silencer element ISSN-N1 located in exon 7 of the SMN2 gene. 277. The AAV particle of embodiment 275 or 276, wherein the antisense oligonucleotide comprises any one of SEQ ID NOs: 2010-2013.
278. The AAV particle of embodiment 273 or 274, wherein the encoded splicing modulator element is an UlsnRNA, e.g., an UlsnRNA capable of correcting the skipping of exon 7 of the SMN2 pre-mRNA.
279. The AAV particle of embodiment 278, wherein the UlsnRNA is exon specific (e.g., ExSpeUl).
280. The AAV particle of embodiment 278 or 279, wherein the UlsnRNA comprises one or more of SEQ ID NOs: 2014-2019.
281. The AAV particle of any one of embodiments 273-280, wherein:
(i) the nucleotide sequence encoding the splicing modulator is located 5’ relative to the nucleotide sequence encoding the SMN protein; and/or
(ii) the nucleotide sequence encoding the splicing modulator is located 3’ relative to the nucleotide sequence encoding the SMN protein.
282. The AAV particle of any one of the preceding embodiments, which comprises a viral genome comprising a promoter operably linked to the nucleic acid sequence encoding the SMN protein.
283. The AAV particle of embodiment 282, wherein the promoter is selected from human elongation factor la-subunit (EF-la), SMN1, chicken P-actin (CBA) and its derivative, a phosphoglycerate kinase 1 (PGK), methyl-CpG binding protein 2 (MeCP2), insulin, Hb9, synapsin (Syn), cytomegalovirus (CMV) immediate-early enhancer and/or promoter, CAG, glucuronidase (GUSB), or ubiquitin C (UBC), neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B- chain (PDGF-P), intercellular adhesion molecule 2 (ICAM-2), Ca2+/calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), neurofilament light (NFL) or heavy (NFH), P-globin minigene np2, preproenkephalin (PPE), enkephalin (Enk) and excitatory amino acid transporter 2 (EAAT2), glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), a cardiovascular promoter (e.g., aMHC, cTnT, and CMV-MLC2k), a liver promoter (e.g., hAAT, TBG), a skeletal muscle promoter (e.g., desmin, MCK, C512) or a fragment (e.g., a truncation) or a functional variant thereof.
284. The AAV particle of embodiment 282 or 283, wherein the promoter is an EF-la promoter or a fragment (e.g., a truncation) or a functional variant thereof. 285. The AAV particle of embodiment 282 or 283, wherein the promoter is a CBA promoter or a fragment (e.g., a truncation) or a functional variant thereof.
286. The AAV particle of embodiment 282 or 283, wherein the promoter is a PGK promoter or a fragment (e.g., a truncation) or a functional variant thereof.
287. The AAV particle of any one of embodiments 282-286, wherein the promoter comprises the nucleotide sequence of any one of SEQ ID NOs: 26-35 or 987, 988, 990, 991, 995, 996 998-1007, or any one of the nucleotide sequences in Table 7, a nucleotide sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions, relative to the nucleotide sequence of any one of SEQ ID NOs: 26-35 or 987, 988, 990, 991, 995-1007, or any one of the nucleotide sequences in Table 7, or a nucleotide sequence with at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 26-35 or 987, 988, 990, 991, 995-1007, or any one of the nucleotide sequences in Table 7.
288. The AAV particle of any one of embodiments 282-287, wherein the promoter comprises the nucleotide sequence of any one of SEQ ID NOs: 26-35, a nucleotide sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions, relative to the nucleotide sequence of any one of SEQ ID NOs: 26-35, or a nucleotide sequence with at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 26-35.
289. The AAV particle of any one of embodiments 282-284, 287, or 288, wherein the promoter comprises the nucleotide sequence of SEQ ID NO: 26, a nucleotide sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions, relative to the nucleotide sequence of SEQ ID NO: 26, or a nucleotide sequence with at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 26.
290. The AAV particle of any one of embodiments 282, 283, 285, 287, or288, wherein the promoter comprises the nucleotide sequence of SEQ ID NO: 27, a nucleotide sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions, relative to the nucleotide sequence of SEQ ID NO: 27, or a nucleotide sequence with at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 27.
291. The AAV particle of any one of embodiments 282, 283, 287, 288 or 288, wherein the promoter comprises the nucleotide sequence of SEQ ID NO: 29, a nucleotide sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions, relative to the nucleotide sequence of SEQ ID NO: 29, or a nucleotide sequence with at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 29.
292. The AAV particle of any one of embodiments 282, 283 or 284-288, wherein the promoter comprises the nucleotide sequence of SEQ ID NO: 31, a nucleotide sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions, relative to the nucleotide sequence of SEQ ID NO: 31, or a nucleotide sequence with at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 31.
293. The AAV particle of any one of embodiments 282-292, wherein the viral genome further comprises an enhancer.
294. The AAV particle of embodiment 293, wherein the enhancer comprises a CMVie enhancer.
295. The AAV particle of any one of embodiments 282-294, wherein the viral genome further comprises an intron.
296. The AAV particle of embodiment 295, wherein the intron is a chimeric intron or a variant thereof.
297. The AAV particle of embodiment 295 or 296, wherein the intron comprises the nucleotide sequence of SEQ ID NO: 3, a nucleotide sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions, relative to the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence with at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NOs: 3.
298. The AAV particle of embodiment 295, wherein the intron is an SV40 intron or a variant thereof.
299. The AAV particle of any one of embodiments 282-298, wherein the viral genome which further comprises an inverted terminal repeat (ITR) sequence.
300. The AAV particle of embodiment 299, wherein the ITR sequence is positioned 5’ relative to the nucleic acid comprising the transgene encoding the SMN protein.
301. The AAV particle of embodiment 299 or 300, wherein the ITR sequence is positioned 3’ relative to the nucleic acid comprising the transgene encoding the SMN protein. 302. The AAV particle of any one of embodiments 282-301, wherein the viral genome comprises an ITR positioned 5’ relative to the nucleic acid comprising the transgene encoding the SMN protein and an ITR positioned 3’ relative to the nucleic acid comprising the transgene encoding the SMN protein.
303. The AAV particle of any one of embodiments 299-302, wherein the ITR comprises a nucleic acid sequence of SEQ ID NO: 1 or 2, or a nucleotide sequence at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical thereto.
304. The AAV particle of any one of embodiments 299-303, wherein the ITR comprises the nucleotide sequence of SEQ ID NO: 1 or 2, or a nucleotide sequence having one, two, or three modifications (e.g., substitutions), but no more than four modifications (e.g., substitutions) relative to SEQ ID NO: 1 or 2.
305. The AAV particle of any one of embodiments 299-304, wherein the ITR is positioned 5’ relative to the nucleic acid comprising the transgene encoding the SMN protein and comprises the nucleotide sequence of SEQ ID NO: 1, a nucleotide sequence having at least one, two, or three modifications, but no more than four modifications of SEQ ID NO: 1, or a nucleotide sequence at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 1 .
306. The AAV particle of any one of embodiments 299-305, wherein the ITR is positioned 3’ relative to the nucleic acid comprising the transgene encoding the SMN protein and comprises the nucleotide sequence of SEQ ID NO: 2, a nucleotide sequence having at least one, two, or three modifications, but no more than four modifications of SEQ ID NO: 2, or a nucleotide sequence at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 2.
307. The AAV particle of any one of embodiments 299-306, wherein:
(i) the ITR positioned 5’ relative to the nucleic acid comprising the transgene encoding the SMN protein comprises the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence at least 80% (e.g., 85%, 90%', 95%, 96%, 97%, 98%', or 99%) identical thereto; and/or
(ii) the ITR positioned 3’ relative to the nucleic acid comprising the transgene encoding the SMN protein comprises the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical thereto.
308. The AAV particle of any one of embodiments 282-307, wherein the viral genome further comprises a polyA signal sequence. 309. The AAV particle of embodiment 308, wherein the polyA signal region comprises the nucleotide sequence of SEQ ID NO: 4, a nucleotide sequence having at least one, two, or three modifications, but no more than four modifications of SEQ ID NO: 4, or a nucleotide sequence at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 4.
310. The AAV particle of any one of embodiments 282-309, wherein the viral genome further comprises a Kozak sequence, and/or an exon region.
311. The AAV particle of any one of embodiments 282-310, wherein the viral genome further comprises an untranslated region.
312. The AAV particle of any one of embodiments 282-311, wherein the viral genome further comprises a first untranslated region and a second untranslated region, optionally wherein the first untranslated region is present 5’ relative to the nucleotide sequence encoding the SMN protein and the second untranslated region is located 3’ relative to the nucleotide sequence encoding the SMN protein.
313. The AAV particle of any one of embodiments 282-312, wherein the viral genome further comprises a nucleotide sequence encoding a miR binding site, e.g., a miR binding site that modulates, e.g., reduces, expression of the antibody molecule encoded by the viral genome in a cell or tissue where the corresponding miRNA is expressed.
314. The AAV particle of embodiment 313, wherein the encoded miRNA binding site is complementary, e.g., fully complementary or partially complementary, to a miRNA expressed in a cell or tissue of the DRG, liver, heart, hematopoietic, or a combination thereof.
315.The AAV particle of embodiment 313 or 314, wherein the encoded miR binding site modulates, e.g., reduces, expression of the encoded antibody molecule in a cell or tissue of the DRG, liver, heart, hematopoietic lineage, or a combination thereof.
316. The AAV particle of any one of embodiments 282-315, wherein the viral genome comprises at least 1-5 copies of the encoded miR binding site, e.g., at least 1, 2, 3, 4, or 5 copies.
317. The AAV particle of any one of embodiments 282-316, wherein the viral genome comprises at least 3 copies of an encoded miR binding sites, optionally wherein all three copies comprise the same miR binding site, or at least one, two, three, or all of the copies comprise a different miR binding site. 318. The AAV particle of embodiment 317, wherein the at least 3 copies of the encoded miR binding sites are continuous (e.g., not separated by a spacer), or are separated by a spacer, optionally wherein the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions, but no more than four modifications, e.g., substitutions, relative to GATAGTTA.
319. The AAV particle of any one of embodiments 282-318, wherein the viral genome comprises at least 4 copies of an encoded miR binding site, optionally wherein all four copies comprise the same miR binding site, or at least one, two, three, or all of the copies comprise a different miR binding site.
320. The AAV particle of embodiment 319, wherein the at least 4 copies of the encoded miR binding sites are continuous (e.g., not separated by a spacer), or are separated by a spacer, optionally wherein the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions, but no more than four modifications, e.g., substitutions, relative to GATAGTTA.
321 . The AAV particle of any one of embodiments 313-320, wherein the encoded miR binding site comprises a miR122 binding site, a miR183 binding site, a miR-1 binding site, a miR-142-3p, or a combination thereof, optionally wherein:
(i) the encoded miR122 binding site comprises the nucleotide sequence of SEQ ID NO: 1865, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions relative to SEQ ID NO: 1865;
(ii) the encoded miR183 binding site comprises the nucleotide sequence of SEQ ID NO: 1847, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 1847;
(iii) the encoded miR-1 binding site comprises the nucleotide sequence of SEQ ID NO: 4679, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 4679; and/or
(iv) the encoded miR-142-3p binding site comprises the nucleotide sequence of SEQ ID NO: 1869, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 1869.
322. The AAV particle of any one of embodiments 282-321, wherein the viral genome comprises an encoded miR122 binding site.
323. The AAV particle of any one of embodiments 282-322, wherein the viral genome comprises at least 1-5 copies, e.g., 1, 2, or 3 copies of a miR122 binding site, optionally wherein each copy is continuous (e.g., not separated by a spacer), or each copy is separated by a spacer, optionally wherein the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions, but no more than four modifications, e.g., substitutions, relative to GATAGTTA.
324. The AAV particle of any one of embodiments 321-323, wherein the encoded miR122 binding site comprises the nucleotide sequence of SEQ ID NO: 1865, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 1865.
325. The AAV particle of any one of embodiments 282-324, wherein the viral genome comprises:
(A) (i) a first encoded miR122 binding site comprising the nucleotide sequence of SEQ ID NO: 1865, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 1865;
(ii) a first spacer comprising the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions, but no more than four modifications, e.g., substitutions, relative to GATAGTTA; and
(iii) a second encoded miR122 binding site comprising the nucleotide sequence of SEQ ID NO: 1865, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 1865; or
(B) (i) a first encoded miR122 binding site comprising the nucleotide sequence of SEQ ID NO: 1865, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 1865;
(ii) a first spacer comprising the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions, but no more than four modifications, e.g., substitutions, relative to GATAGTTA;
(iii) a second encoded miR122 binding site comprising the nucleotide sequence of SEQ ID NO: 1865, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 1865;
(iv) a second spacer comprising the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions, but no more than four modifications, e.g., substitutions, relative to GATAGTTA; and
(v) a third encoded miR122 binding site comprising the nucleotide sequence of SEQ ID NO: 1865, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 1865.
326. The AAV particle of any one of embodiments 282-311, wherein the viral genome comprises an encoded miR183 binding site.
327. The AAV particle of any one of embodiments 282-326, wherein the viral genome comprises at least 1-5 copies, e.g., 1, 2, or 3 copies of a miR183 binding site, optionally wherein each copy is continuous (e.g., not separated by a spacer), or each copy is separated by a spacer, optionally wherein the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions, but no more than four modifications, e.g., substitutions, relative to GATAGTTA.
328. The AAV particle of embodiment 327, wherein the encoded miR183 binding site comprises the nucleotide sequence of SEQ ID NO: 1847, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 1847. 329. The AAV particle of any one of embodiments 282-328, wherein the viral genome comprises:
(A) (i) a first encoded miR183 binding site comprising the nucleotide sequence of SEQ ID NO: 1847, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, live, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 1847;
(ii) a first spacer comprising the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions, but no more than four modifications, e.g., substitutions, relative to GATAGTTA; and
(iii) a second encoded miR183 binding site comprising the nucleotide sequence of SEQ ID NO: 1847, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 1847; or
(B) (i) a first encoded miR183 binding site comprising the nucleotide sequence of SEQ ID NO: 1847, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 1847;
(ii) a first spacer comprising the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions, but no more than four modifications, e.g., substitutions, relative to GATAGTTA;
(iii) a second encoded miR183 binding site comprising the nucleotide sequence of SEQ ID NO: 1847, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 1847;
(iv) a second spacer comprising the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions, but no more than four modifications, e.g., substitutions, relative to GATAGTTA; and
(v) a third encoded miR183 binding site comprising the nucleotide sequence of SEQ ID NO: 1847, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 1847. 330. The AAV particle of any one of embodiments 282-329, wherein the viral genome comprises an encoded miR122 binding site and a miR-1 binding site.
331. The AAV particle of any one of embodiments 282-330, wherein the viral genome is single stranded or self-complementary.
332. The AAV particle of any one of embodiments 282-331, wherein the viral genome is self- complementary.
333. The AAV particle of any one of embodiments 282-332, wherein the viral genome further comprises a nucleotide sequence encoding a Rep protein, e.g., a non-structural protein, wherein the Rep protein comprises a Rep78 protein, a Rep68, Rep52 protein, and/or a Rep40 protein (e.g., a Rep78 and a Rep52 protein).
334. The AAV particle of any one of embodiments 282-332, wherein the AAV particle further comprises a nucleotide sequence encoding a Rep protein, e.g., a non-structural protein, wherein the Rep protein comprises a Rep78 protein, a Rep68, Rep52 protein, and/or a Rep40 protein (e.g., a Rep78 and a Rep52 protein).
335. The AAV particle of embodiment 333 or 334, wherein the Rep78 protein, the Rep68 protein, the Rep52 protein, and/or the Rep40 protein are encoded by at least one Rep gene.
336. The AAV particle of any one of embodiments 282-335, wherein the viral genome comprises in 5’ to 3’ order:
(i) a 5’ ITR, optionally wherein the 5’ ITR comprises the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical thereto;
(ii) a promoter, optionally wherein the promoter comprises the nucleotide sequence of any one of SEQ ID NOs: 26-35, or a nucleotide sequence at least 80%' (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical to any one of SEQ ID NOs: 26-35;
(iii) an intron, optionally wherein the intron comprises the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical thereto;
(iv) a nucleotide sequence encoding an SMN protein, optionally comprising the nucleotide sequence of any one of SEQ ID NOs: 6-9, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical to any one of SEQ ID NOs: 6-8; (v) a polyA sequence, optionally wherein the polyA sequence comprises the nucleotide sequence of SEQ ID NO: 4, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical thereto; and
(vi) a 3’ ITR, optionally wherein the 3’ ITR comprises the nucleotide sequence of SEQ ID NO:
2, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical thereto.
337. The AAV particle of any one of embodiments 282-336, wherein the viral genome comprises in 5’ to 3’ order:
(i) a 5’ ITR, optionally wherein the 5’ ITR comprises the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical thereto;
(ii) a promoter, optionally wherein the promoter comprises the nucleotide sequence of any one of SEQ ID NOs: 26, 27, 29, or 31, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical to any one of SEQ ID NOs: 26, 27, 29, or 31;
(iii) an intron, optionally wherein the intron comprises the nucleotide sequence of SEQ ID NO:
3, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical thereto;
(iv) a nucleotide sequence encoding an SMN protein, optionally comprising the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical thereto;
(v) a polyA sequence, optionally wherein the polyA sequence comprises the nucleotide sequence of SEQ ID NO: 4, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical thereto; and
(vi) a 3’ ITR, optionally wherein the 3’ ITR comprises the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical thereto.
338. The AAV particle of any one of embodiments 282-337, which comprises the nucleotide sequence of any one of SEQ ID NOs: 10-25 or 36-38, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical to any one of SEQ ID NOs: 10-25 or 36-38.
339. The AAV particle of any one of embodiments 282-338, which comprises the nucleotide sequence of any one of SEQ ID NOs: 20-22 or 24, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical to any one of SEQ ID NOs: 20-22 or 24.
340. A cell, e.g., a host cell, comprising the AAV particle of any one of the preceding embodiments.
341. The cell of embodiment 340, wherein the cell is a mammalian cell (e.g., an HEK293 cell) or an insect cell (e.g., an Sf9 cell). 342. The cell of embodiment 340 or 341, wherein the cell is a cell of the temporal cortex, perirhinal cortex, globus pallidus, putamen, caudate, thalamus, hippocampus, geniculate nucleus, Purkinje Layer, deep cerebellar nuclei, cerebellum, cervical spinal cord, thoracic spinal cord, lumbar spinal cord, or a combination thereof.
343. The cell of any one of embodiments 340-342, wherein the cell is a neuron, a motor neuron, a sensory neuron, an astrocyte, or a glial cell.
344. A method of making the AAV particle of any one of embodiments 1-339 comprising:
(i) providing a host cell comprising a viral genome; and
(ii) incubating the host cell under conditions suitable to enclose the viral genome in the AAV capsid variant; thereby making the AAV particle.
345. The method of embodiment 344, further comprising, prior to step (i), introducing a first nucleic acid molecule comprising the viral genome into the host cell.
346. The method of embodiment 344 or 345 , wherein the host cell comprises a second nucleic acid encoding the capsid variant.
347. The method of embodiment 346, wherein the second nucleic acid molecule is introduced into the host cell prior to, concurrently with, or after the first nucleic acid molecule.
348. A pharmaceutical composition comprising the AAV particle of any one of embodiments 1-339, and a pharmaceutically acceptable excipient.
349. A method of delivering a payload to a cell or tissue (e.g., a CNS cell or a CNS tissue), comprising administering an effective amount of the pharmaceutical composition of embodiment 334, or the AAV particle of any one of embodiments 1-339.
350. The method of embodiment 349, wherein the cell is a cell a cell of a brain region or a spinal cord region, optionally a cell of the temporal cortex, perirhinal cortex, globus pallidus, putamen, caudate, thalamus, hippocampus, geniculate nucleus, Purkinje Layer, deep cerebellar nuclei, cerebellum, cervical spinal cord, thoracic spinal cord, lumbar spinal cord, or a combination thereof. 351. The method of embodiment 349 or 350, wherein the cell is a neuron, a motor neuron, a sensory neuron, or an astrocyte.
352. The method of any one of embodiments 349-351, wherein the cell or tissue is within a subject.
353. The method of embodiment 352 wherein the subject has, has been diagnosed with having, or is at risk of having a neurological, e.g., a neurodegenerative, disorder.
354. The method of embodiment 352 or 353, wherein the subject has, has been diagnosed with having, or is at risk of having a disease associated with a reduction in the quantity or function of SMN protein, e.g., decreased SMN protein expression.
355. The method of any one of embodiments 352-354, wherein the subject has, has been diagnosed with having, or is at risk of having a Spinal Muscular Atrophy (SMA) or an SMA-related disorder.
356. The method of any one of embodiments 352-355, wherein the subject has, has been diagnosed with having, or is at risk of having Werdnig-Hoffman disease, Dubowitz disease, or Kugelberg-Welander disease.
357. A method of treating a subject having or diagnosed with having a neurological disorder, e.g., a neurodegenerative disorder, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 348, or the AAV particle of any one of embodiments 1-339.
358. A method of treating a subject having or diagnosed with having a disease related to decreased SMN protein expression, e.g., a mutation in an SMN1 gene, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 348, or the AAV particle of any one of embodiments 1-339.
359. A method of treating a subject having or diagnosed with Spinal Muscular Atrophy (SMA), comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 348, or the AAV particle of any one of embodiments 1-339.
360. The method of embodiment 353, 354, 357, or 358, wherein the neurological disorder, e.g., a neurodegenerative disorder or disease related to decreased SMN protein expression is SMA. 361. The method of any one of embodiments 353-360, wherein the neurological disorder, e.g., a neurodegenerative disorder, disease related to decreased SMN protein expression, or SMA is Werdnig- Hoffman disease, Dubowitz disease, or Kugelberg-Welander disease.
362. The method of any one of embodiments 353-360, wherein the neurological disorder, e.g., a neurodegenerative disorder, disease related to decreased SMN protein expression, or SMA is infantile onset SMA, type II SMA, or type III SMA.
363. The method of any one of embodiments 354-362, wherein the subject comprises at least 2 to 4 copies, e.g., at least 2 copies, at least 3 copies or at least 4 copies, of an SMN2 gene (e.g., a type II or a type III SMA).
364. The method of any one of embodiments 352-363, wherein the subject comprises 2-4 copies, e.g., 2 copies, 3 copies or at least 4 copies, of an SMN2 gene (e.g., a type II or a type III SMA).
365. The method of any one of embodiments 352-362, wherein the subject comprises 1 to 2 copies, e.g., 1 copy or 2 copies, of an SMN2 gene (e.g., a type I SMA).
366. The method of any one of embodiments 352-362, wherein the subject comprises no more than 2 copies, of an SMN2 gene (e.g., a type I SMA).
367. The method of any one of embodiments 357-366, where treating comprises prevention of progression of the disease or disorder in the subject.
368. The method of any one of embodiments 352-367, wherein the subject is a human.
369. The method of any one of embodiments 352-368, wherein the AAV particle is administered to the subject intravenously, via intra-cisterna magna injection (ICM), intracerebrally, intrathecally, intracerebroventricularly, via intraparenchymal administration, or intramuscularly.
370. The method of any one of embodiments 352-368, wherein the AAV particle is administered to the subject via focused ultrasound (FUS), e.g., coupled with the intravenous administration of microbubbles (FUS-MB), or MRI-guided FUS coupled with intravenous administration.
371. The method of any one of embodiments 352-368, wherein the AAV particle is administered to the subject intravenously. 372. The method of any one of embodiments 352-371, wherein administration of the AAV particle results in an increased presence, level, and/or activity of SMN protein.
373. The method of any one of embodiments 353-372, further comprising administration of an additional therapeutic agent and/or therapy suitable for treatment or prevention of the disease associated with a reduction in the quantity or function of an SMN protein, e.g., decreased SMN protein expression.
374. The method of embodiment 373, wherein the additional therapeutic agent comprises a splicing modifier, a small molecule inhibitor (e.g., RG7916 (e.g., EVRYSDI™ or risdiplam)), an antisense oligonucleotide (e.g., Nusinersen (e.g., SPINRAZA™)), a myostatin inhibitor (e.g., an anti-myostatin antibody, e.g., SRK-015), a fast skeletal muscle troponin activator (FSTA) (e.g., CK-2127107), a neuroprotective agent (e.g., olesoxime or TRO19622), a chloride channel antagonist (e.g., a small molecule antagonist, e.g., NMD-670), or a combination thereof.
375. The method of any one of embodiments 352-374, wherein the subject is between 2 months to 2 years of age, e.g., an early infantile subject.
376.The method of any one of embodiments 352-374, wherein the subject is between 1.5 years of age to 6 years of age, e.g., a late infantile subject.
377. The method of any one of embodiments 352-374, wherein the subject is between 6 years of age to 18 years of age, e.g., ajuvenile.
378. The method of any one of embodiments 352-374, wherein the subject is above 18 years of age, e.g., an adult.
379. The method of any one of embodiments 352-374, wherein the subject is 2 months of age or older.
380. The method of any one of embodiments 352-374, wherein the subject is 18 years of age or older.
381. The method of any one of embodiments 352-374, wherein the subject is 2 years of age or younger.
382. The AAV particle of any one of embodiments 1-339, or the pharmaceutical composition of embodiment 348, for use in a method of delivering a payload to a cell or tissue. 383. The AAV particle of any one of embodiments 51-339, or the pharmaceutical composition of embodiment 348, for use in a method of treating a neurological disorder, a neurodegenerative disorder, a disease associated with a mutation in the SMN1 gene and/or reduced SMN protein expression, or SMA.
384. The AAV particle of any one of embodiments 1-339, or the pharmaceutical composition of embodiment 348, for use in the manufacture of a medicament.
385. Use of the AAV particle of any one of embodiments 1-339, or the pharmaceutical composition of embodiment 348, in the manufacture of a medicament.
386. Use of the AAV particle of any one of embodiments 1-339, or the pharmaceutical composition of embodiment 348, in the manufacture of a medicament for treating a neurological disorder, a neurodegenerative disorder, a disease associated with a mutation in the SMN1 gene and/or a decrease in SMN protein expression, or SMA.
[0025] The details of various aspects or embodiments of the present disclosure are set forth below. Other features, objects, and advantages of the disclosure will be apparent from the description and the claims. In the description, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art in the field of this disclosure. In the case of conflict, the present description will control.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIGs. 1A-1D show immunohistochemistry images from various CNS and peripheral tissues isolated from NHPs (cynomolgus macaques) at 28 days post intravenous administration of AAV particles comprising the TTN-002 capsid variant (top panels) or AAV9 control capsid (bottom panels) and a self- complementary genome encoding a payload fused to an HA tag driven by a heterologous constitutive promoter. FIG 1A shows, from left to right, the cerebellum (Purkinje layer), spinal cord (cervical), cortex (temporal), and the brainstem. FIG. IB shows, from left to right, the globus pallidus, the hippocampus, the thalamus, the putamen, and the dentate. FIG. 1C shows, from left to right, the whole brain (level H), the whole brain (level K), and the cerebellum. FIG. ID shows, from left to right, the spinal cord (thoracic), the DRG (thoracic), the liver, and the heart.
DETAILED DESCRIPTION
Overview
[0027] Described herein, inter alia, are compositions comprising isolated, e.g., recombinant, viral particles, e.g., AAV particles, for delivery, e.g., vectorized delivery, of a protein, e.g., an SMN protein, and methods of making and using the same
[0028] Gene therapy presents an alternative approach for SMA and related diseases sharing single-gene etiology and related disorders. AAVs are commonly used in gene therapy approaches as a result of a number of advantageous features. Without wishing to be bound by theory, it is believed in some embodiments, that expression vectors, e.g., an adeno-associated viral vector (AAVs) or AAV particle, e.g., an AAV particle described herein, can be used to administer and/or deliver an SMN protein (e.g., SMN and related proteins), in order to achieve sustained, high concentrations, allowing for longer lasting efficacy, fewer dose treatments, broad biodistribution, and/or more consistent levels of the SMN protein, relative to a non- AAV therapy.
[0029] The compositions and methods described herein provides improved features compared to prior SMN gene replacement approaches, including (i) increased SMN activity in a cell, tissue, (e.g., a cell or tissue of the CNS, e.g., the cortex, striatum, thalamus, cerebellum, and/or brainstem), and/or fluid (e.g., CSF and/or serum), of the subject; (ii) increased biodistribution throughout the CNS (e.g., the cortex, striatum, thalamus, cerebellum, brainstem, and/or spinal cord), and the periphery (e.g., the liver), and/or (iii) elevated payload expression, e.g., SMN mRNA expression, in multiple brain regions (e.g., cortex, thalamus, and brain stem) and the periphery (e.g., the liver). In some embodiments, an AAV viral genome encoding an SMN protein described herein which comprise an optimized nucleotide encoding the SMN protein (e.g., SEQ ID NO: 2000) result in high biodistribution in the CNS; increased SMN activity in the CNS, peripheral tissues, and/or fluid; and successful transgene transcription and expression. The compositions and methods described herein can be used in the treatment of disorders associated with a lack of an SMN protein and/or SMN activity (e.g., SMA, Werdnig-Hoffman disease, Dubowitz disease, Kugelberg-Welander disease), such as SMA-related disorders associated with a mutation in an SMN gene.
[0030] As demonstrated in the Examples herein below, certain AAV capsid variants described herein show multiple advantages over wild-type AAV5 and/or wild-type AAV9, including (i) increased penetrance through the blood brain barrier following intravenous administration, (ii) wider distribution throughout the multiple brain regions, e.g., frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate, and/or hippocampus, (iii) elevated payload expression in multiple brain regions, (iv) wider distribution in one or more peripheral tissues, e.g., the heart, muscle, and/or liver, and/or (v) elevated payload expression in one or more peripheral tissues. Without wishing to be being bound by theory, it is believed that these advantages may be due, in part, to the dissemination of the AAV capsid variants through the brain vasculature. In some embodiments, the AAV capsids described herein enhance the delivery of a payload to multiple regions of the brain including, for example, a temporal cortex, perirhinal cortex, globus pallidus, putamen, caudate, thalamus, hippocampus, geniculate nucleus, Purkinje Layer, deep cerebellar nuclei, cerebellum, or a combination thereof. In some embodiments, enhance the expression of a payload, e.g., an SMN protein described herein, to multiple cell types in the CNS, e.g., neurons, oligodendrocytes, and/or glial cells. Without wishing to be bound by theory, an AAV particle comprising an AAV capsid polypeptide, e.g., an AAV capsid variant described herein, for the vectorized delivery of an SMN protein described here will result in increased penetrance through the blood brain barrier, e.g., following intravenous administration, and/or increased biodistribution of the SMN protein in the central nervous system, e.g., the brain and the spinal cord.
[0031] In some embodiments, AAV capsid variants disclosed herein comprise a modification in loop VIII of AAV5, e.g., at positions between 571-579, e.g., at position 577, numbered relative to SEQ ID NO: 138. Without wishing to be bound by theory, it is believed in some embodiments that the aforesaid region (e.g., positions between 571-579, e.g., at position 577) of the AAV5 capsid protrudes above the 3- fold axis of symmetry, e.g., is a surface-exposed location in the AAV5 capsid , e.g., as described in Govindasamy et al. “Structural Insights into Adeno-Associated Virus Serotype 5,” Journal of Virology, 2013, 87(20): 11187-11199 (the contents of which are hereby incorporated by reference in their entirety). In some embodiments, loop (e.g., loop VIII) is used interchangeably herein with the term variable region (e.g., variable region VIII), or VR (e.g., VR-VIII). In some embodiments loop VUI (e.g., VR-VIII) comprises positions 571-592 (e.g., amino acids TNNQSSTTAPATGTYNLQEIVP; SEQ ID NO: 285), numbered according to SEQ ID NO: 138. In some embodiments, loop VIII (e.g., VR-VIII) comprises positions 571-599 (e.g., amino acids TNNQSSYPAEVVQKTAPATGTYNLQEIVP (SEQ ID NO: 756)), numbered according to SEQ ID NO: 982 . In some embodiments, loop VIII or variable region VIII (VR- VIII) is as described in Govindasamy et al. (supra) (the contents of which are hereby incorporated by reference in their entirety).
[0032] SMA is caused by a reduction in the expression of the survival motor neuron (SMN) protein caused by mutations in the survival motor neuron 1 (SMN1) gene and loss of encoded SMN protein (Lefebvre et al., Cell (1995) 80:155-165). SMN is a ubiquitously expressed protein that functions in the assembly of the spliceosome and may also mediate mRNA trafficking in the axon and nerve terminus of neurons. The lack of SMN results in motor neuron degeneration in the ventral (anterior) horn of the spinal cord, which leads to weakness of the proximal muscles responsible for crawling, walking, neck control and swallowing, and the involuntary muscles that control breathing and coughing (Sumner C. J., NeuroRx (2006) 3:235-245). Consequently, SMA patients present with increased tendencies for pneumonia and other pulmonary problems such as restrictive lung disease.
[0033] In humans there are two very similar copies of the SMN gene termed SMN1 and SMN2. The amino acid sequence encoded by the two genes is identical. The SMN1 and SMN2 genes lie within the telomeric and centromeric halves, respectively, of a large, inverted duplication on chromosome 5ql3. These genes share more than 99% nucleotide identity, and both are capable of encoding SMN (a 294- amino acid RNA-binding protein). However, there is a single, silent nucleotide change in SMN 2 in exon 7 that results in exon 7 being excluded in 80-90% of transcripts from SMN2. The resulting truncated protein, called SMNA7, is less stable and rapidly degraded. The remaining 10-20% of transcript from SMN2 encodes the full length SMN protein. Disease results when all copies of SMN1 are lost, leaving only SMN2 to generate full length SMN protein. Accordingly, SMN2 acts as a phenotypic modifier in SMA in that patients with a higher SMN2 copy number generally exhibit later onset and less severe disease. For example, patients with a high SMN2 copy number (3-4 copies) exhibit a less severe form of the disease (referred to as Types II or III), whereas 1-2 copies of SMN2 typically result in the more severe Type I disease (Campbell et al., Am. J. Hum. Genet. (1997) 61:40-50; Lefebvre et al., Nat. Genet. (1997) 16:265-269).
[0034] More particularly, the SMN1 and SMN2 genes differ by live nucleotides; one of these differences - a translationally silent C to T substitution in an exonic splicing region - results in frequent exon 7 skipping during transcription of SMN2. As a result, the majority of transcripts produced from SMN2 lack exon 7 (SMNAEx7), and encode a truncated protein which is rapidly degraded (about 10% of the SMN2 transcripts are full length and encode a functional SMN protein).
I. Compositions
Adeno-associated viral (AAV) particles
[0035] In some embodiments, AAV are used as a biological tool due to a relatively simple structure, their ability to infect a wide range of cells (including quiescent and dividing cells) without integration into the host genome and without replicating, and their relatively benign immunogenic profile. In some embodiments, the genome, e.g., viral genome, of the virus may be manipulated to contain a minimum of components for the assembly of a functional recombinant virus, or viral particle, which is loaded with or engineered to target a particular tissue and express or deliver a desired payload, e.g., a polypeptide encoding polynucleotide, e.g., an SMN protein described herein, e.g., an SMN1 and/or SMN2 protein described herein.
[0036] In some embodiments, the AAV particle is a naturally occurring (e.g., wild-type) AAV or a recombinant AAV. In some embodiments, the wild-type AAV viral genome is a linear, single- stranded DNA (ssDNA) molecule approximately 5,000 nucleotides (nt) in length. In some embodiments, inverted terminal repeats (ITRs) cap the viral genome at both the 5’ and the 3’ end, providing origins of replication for the viral genome. In some embodiments, an AAV viral genome typically comprises two ITR sequences. These ITRs have a characteristic T-shaped hairpin structure defined by a self- complementary region (145nt in wild-type AAV) at the 5’ and 3’ ends of the ssDNA which form an energetically stable double stranded region. The double stranded hairpin structures comprise multiple functions including, but not limited to, acting as an origin for DNA replication by functioning as primers for the endogenous DNA polymerase complex of the host viral replication cell.
[0037] In some embodiments, the wild-type AAV viral genome further comprises nucleotide sequences for two open reading frames, one for the four non-structural Rep proteins (Rep78, Rep68, Rep52, Rep40, encoded by Rep genes) and one for the three capsid, or structural, proteins (VP1, VP2, VP3, encoded by capsid genes or Cap genes). The Rep proteins are used for replication and packaging, while the capsid proteins are assembled to create the protein shell of the AAV, or AAV capsid polypeptide, e.g., an AAV capsid variant. Alternative splicing and alternate initiation codons and promoters result in the generation of four different Rep proteins from a single open reading frame and the generation of three capsid proteins from a single open reading frame. Though it varies by AAV serotype, as a non-limiting example, for AAV5 (SEQ ID NO: 138 and 137) VP1 refers to amino acids 1-724, VP2 refers to amino acids 137- 724, and VP3 refers to amino acids 193-724. In some embodiments, for the amino acid sequence of SEQ ID NO: 982, VP1 comprises amino acids 1-731, VP2 comprises amino acids 137-731, and VP3 comprises amino acids 193-731. In other words, VP1 is the full-length capsid sequence, while VP2 and VP3 are shorter components of the whole. As a result, changes in the sequence in the VP3 region, are also changes to VP1 and VP2, however, the percent difference as compared to the parent sequence will be greatest for VP3 since it is the shortest sequence of the three. Though described here in relation to the amino acid sequence, the nucleic acid sequence encoding these proteins can be similarly described. Together, the three capsid proteins assemble to create the AAV capsid protein. While not wishing to be bound by theory, the AAV capsid protein typically comprises a molar ratio of 1:1:10 of VP1:VP2:VP3. [0038] In some embodiments, a viral genome of a wild-type, e.g., naturally occurring, AAV can be modified to replace the rep/cap sequences with a nucleic acid comprising a transgene encoding a payload, e.g., an antibody molecule, wherein the viral genome comprises at least one ITR region. In some embodiments, the viral genome of a recombinant AAV comprises two ITR regions, e.g., a 5TTR or a 3TTR. In some embodiments, the rep/cap sequences can be provided in trans during production to generate AAV particles. In some embodiments, the viral genome of an AAV is comprised in an AAV vector, which further encodes a capsid protein e.g., a structural protein, wherein the capsid protein comprises a VP1 polypeptide, a VP2 polypeptide, and/or a VP3 polypeptide; and/or a Rep protein, e.g., a non-structural protein, wherein the Rep protein comprises a Rep78 protein, a Rep68, Rep52 protein, and/or a Rep40 protein (e.g., a Rep25 protein and/or a Rep78 protein).
[0039] In some embodiments, in addition to the viral genome comprising a nucleic acid encoding a transgene encoding a payload (e.g., a therapeutic protein, e.g., an SMN protein), an AAV particle, e.g., an AAV particle described herein, may comprise the viral genome, in whole or in part, of any naturally occurring and/or recombinant AAV serotype nucleotide sequence or variant. In some embodiments, AAV variants may have sequences of significant homology at the nucleic acid (viral genome or capsid) and amino acid levels (capsids), to produce constructs which are generally physical and functional equivalents, replicate by similar mechanisms, and assemble by similar mechanisms. Chiorini et al., J. Vir. 71: 6823-33(1997); Srivastava et al., J. Vir. 45:555-64 (1983); Chiorini et al., J. Vir. 73:1309-1319 (1999); Rutledge et al., J. Vir. 72:309-319 (1998); and Wu et al., J. Vir. 74: 8635-47 (2000), the contents of each of which are incorporated herein by reference in their entirety. [0040] In some embodiments, AAV particles of the present disclosure are recombinant AAV particles which are replication defective and lacking the nucleotide sequences encoding functional Rep and Cap proteins. In some embodiments, these defective AAV particles may lack most or all parental coding sequences and carry only one or two AAV ITR sequences and the nucleic acid of interest for delivery to a cell, a tissue, an organ, or an organism.
[0041] In some embodiments, the viral genome or the AAV vector of the AAV particles described herein comprise at least one control element which provides for the replication, transcription, and translation of a coding sequence encoded therein. In some embodiments, a sufficient number of control elements are present such that the coding sequence of the transgene encoded by the viral genome is capable of being replicated, transcribed, and/or translated in a host cell. Non-limiting examples of expression control elements include sequences for transcription initiation and/or termination, promoter and/or enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation signals, sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficacy (e.g., Kozak consensus sequence), sequences that enhance protein stability, and/or sequences that enhance protein processing and/or secretion.
[0042] In some embodiments, the recombinant AAV particles of the present disclosure are capable of providing, e.g., delivering, a transgene to a mammalian cell. In some embodiments, the recombinant AAV particles of the present disclosure arc capable of vectorized delivery of an SMN protein (e.g., an SM 1 and/or SMN2 protein) or fragment thereof.
[0043] In some embodiments, the AAV particles, vectors, viral genomes, and/or nucleic acids of the present disclosure may be produced recombinantly and may be based on adeno-associated virus (AAV) parent or reference sequences. Methods for producing and/or modifying AAV particles are disclosed in the art such as pseudotyped AAV vectors (PCT Patent Publication Nos. W0200028004; W0200123001; W02004112727; W02005005610; and W02005072364, the content of each of which is incorporated herein by reference in its entirety). In some embodiments, the AAV particles described herein may be modified to enhance the efficiency of delivery, e.g., delivery of a transgene encoding a payload, e.g., an antibody molecule. Without wishing to be bound by theory, it is believed in some embodiments, that a modified, e.g., recombinant, AAV particle can be packaged efficiently and successfully infect target cells at high frequency and with minimal toxicity. In some embodiments, the capsid protein of the AAV particles is engineered according to the methods described in US Publication Number US20130195801, the contents of which are incorporated herein by reference in their entirety.
AAV Capsids and Variants thereof
[0044] In some embodiments, an AAV particle, e.g., an AAV particle for the vectorized delivery of protein described herein (e.g., an SMN protein), may comprise an AAV capsid polypeptide, e.g., an AAV capsid variant. [0045] In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto. In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence having at least 90% identity thereto. In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence having at least 95% identity thereto. In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence having at least 96% identity thereto. In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence having at least 97% identity thereto. In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence having at least 98% identity thereto. In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence having at least 99% identity thereto. In some embodiments the AAV capsid polypeptide or the AAV capsid variant, comprises an amino acid sequence comprising at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), but no more than 30, 20, or 10 modifications, e.g., substitutions (e.g., conservative substitutions), relative to the amino acid sequence of SEQ ID NO: 138. In some embodiments the AAV capsid polypeptide or the AAV capsid variant, comprises an amino acid sequence comprising at least one, two, or three, but no more than 30, 20, or 10 different amino acids, relative to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid polypeptide or the AAV capsid variant, comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 137 or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto. In some embodiments, the nucleotide sequence encoding the AAV capsid polypeptide or the AAV capsid variant comprises the nucleotide sequence of SEQ ID NO: 137 or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto.
[0046] In some embodiments, the AAV capsid variant, comprises immediately subsequent to position 570, 571, 572, 573, 574, 575, or 576, numbered relative to SEQ ID NO: 138 or corresponding to equivalent positions in any other AAV serotype (e.g., AAV1, AAV2, AAV3, AAV3b, AAV4, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrhlO, AAVrh32.33, AAVrh74, SEQ ID NO: 139, PHP.N, PHP.B, or an AAV serotype as provided in Table 6 of WO 2021/230987 (the contents of which are hereby incorporated by reference in their entirety)), at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive amino acids of any of amino acid sequence provided in Tables 2A, 2B, 2C, 15 or 21-23. In some embodiments, the at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive amino acids of any of amino acid sequence provided in Tables 2A, 2B, 2C, 15 or 21-23 replaces at least one, two, three, four, five, six, seven, eight, or all of positions T571, N572, N573, Q574, S575, S576, T577, T578, and/or A579, numbered according to SEQ ID NO: 138 or corresponding to equivalent positions in any other AAV serotype (e.g., AAV1, AAV2, AAV3, AAV3b, AAV4, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrhlO, AAVrh32.33, AAVrh74, SEQ ID NO: 139, PHP.N, PHP.B, or an AAV serotype as provided in Table 6 of WO 2021/230987 (the contents of which are hereby incorporated by reference in their entirety)). In some embodiments, the at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive amino acids of any of amino acid sequence provided in Tables 2A, 2B, 2C, 15 or 21-23 replaces positions T577, numbered according to SEQ ID NO: 138 or corresponding to equivalent positions in any other AAV serotype (e.g., AAV1, AAV2, AAV3, AAV3b, AAV4, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrhlO, AAVrh32.33, AAVrh74, SEQ ID NO: 139, PHP.N, PHP.B, or an AAV serotype as provided in Table 6 of WO 2021/230987 (the contents of which are hereby incorporated by reference in their entirety)). In some embodiments, the AAV capsid variant comprises an amino acid other than the wild-type, e.g., native, amino acid, at one, two, three, four, five, six, seven, eight, or all of positions T571, N572, N573, Q574, S575, S576, T577, T578, and/or A579, numbered according to SEQ ID NO: 138 or corresponding to equivalent positions in any other AAV serotype (e.g., AAV1, AAV2, AAV3, AAV3b, AAV4, AAV6, AAV7, AAV8, AAV9, AAVrhS, AAVrhlO, AAVrh32.33, AAVrh74, SEQ ID NO: 139, PHP.N, PHP.B, or an AAV serotype as provided in Table 6 of WO 2021/230987 (the contents of which are hereby incorporated by reference in their entirety)). In some embodiments, the AAV capsid variant comprises an amino acid other than the wild-type, e.g., native, amino acid, at position T577, numbered according to SEQ ID NO: 138 or corresponding to equivalent positions in any other AAV serotype (e.g., AAV1, AAV2, AAV3, AAV3b, AAV4, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrhlO, AAVrh32.33, AAVrh74, SEQ ID NO: 139, PHP.N, PHP.B, or an AAV serotype as provided in Table 6 of WO 2021/230987 (the contents of which are hereby incorporated by reference in their entirety)). In some embodiments, the AAV capsid variant comprises a modification, e.g., substitution, at one, two, three, four, five, six, seven, eight, or all of positions T571, N572, N573, Q574, S575, S576, T577, T578, and/or A579, numbered according to SEQ ID NO: 138 or corresponding to equivalent positions in any other AAV serotype (e.g., AAV1, AAV2, AAV3, AAV3b, AAV4, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrhlO, AAVrh32.33, AAVrh74, SEQ ID NO: 139, PHP.N, PHP.B, or an AAV serotype as provided in Table 6 of WO 2021/230987 (the contents of which are hereby incorporated by reference in their entirety). In some embodiments, the AAV capsid variant comprises a modification, e.g., substitution, at position T577, numbered according to SEQ ID NO: 138 or corresponding to equivalent positions in any other AAV serotype (e.g., AAV1, AAV2, AAV3, AAV3b, AAV4, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrhlO, AAVrh32.33, AAVrh74, SEQ ID NO: 139, PHP.N, PHP.B, or an AAV serotype as provided in Table 6 of WO 2021/230987 (the contents of which are hereby incorporated by reference in their entirety). [0047] In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence or is encoded by the nucleotide sequence of any of SEQ ID NO: 1 or 2 of US7427396; SEQ ID NO: 114 of US20030138772; SEQ ID NO: 199 of US20150315612; or SEQ ID NOs: 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 37, 38, 40, 41, 43, or 44 of US20160289275A1 (the contents of each are incorporated herein by reference in their entirety), or a sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto. [0048] In some embodiments, the AAV capsid variant described herein comprises a modification, e.g., substitution, at position 569 (e.g., M569V), 652 (e.g., D652A), 362 (e.g., T362M), 359 (e.g., Q359D), 350 (e.g., E350Q), 533 (e.g., P533S), 585 (e.g., Y585V), 587 (e.g., L587T), 581 (e.g., A581T), 582 (e.g., T582A), 584 (e.g., T584A), or a combination thereof, all numbered relative to SEQ ID NO: 138.
[0049] In some embodiments, an AAV capsid variant described herein comprises an amino acid from a wild-type AAV5 sequence, e.g., the amino acid sequence of SEQ ID NO: 138, at one or more of positions 581 to 589, numbered relative to SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises 1, 2, 3, 4, 5, 6, 7, 8, or all of: the amino acid from a wild-type AAV5 sequence (e.g., SEQ ID NO: 138) at position 581 (e.g., comprises the amino acid A at position 581); the amino acid from a wild-type AAV5 sequence (e.g., SEQ ID NO: 138) at position 582 (e.g., comprises the amino acid T at position 582); the amino acid from a wild-type AAV5 sequence (e.g., SEQ ID NO: 138) at position 583 (e.g., comprises the amino acid G at position 583); the amino acid from a wild-type AAV5 sequence (e.g., SEQ ID NO: 138) at position 584 (e.g., comprises the amino acid T at position 584); the amino acid from a wild-type AAV5 sequence (e.g., SEQ ID NO: 138) at position 585 (e.g., comprises the amino acid Y at position 585); the amino acid from a wild-type AAV5 sequence (e.g., SEQ ID NO: 138) at position 586 (e.g., comprises the amino acid N at position 586); the amino acid from a wild-type AAV5 sequence (e.g., SEQ ID NO: 138) at position 587 (e.g., comprises the amino acid L at position 587); the amino acid from a wild-type AAV5 sequence (e.g., SEQ ID NO: 138) at position 588 (e.g., comprises the amino acid Q at position 588); and/or the amino acid from a wild-type AAV5 sequence (e.g., SEQ ID NO: 138) at position 589 (e.g., comprises the amino acid E at position 589).
[0050] In certain embodiments, an AAV capsid described herein does not comprise a T at position 581, an A at position 582, an A at position 584, a V at position 585, a T at position 585, a V at position 569, an A at position 652, an M at position 362, a Q at position 359, a Q at position 350, an S at position 533, or a combination thereof, all numbered relative to SEQ ID NO: 138.
[0051] In some embodiments, an AAV capsid described herein does not comprise a modification, e.g., substitution, at positions 581-589 (numbered according to SEQ ID NO: 138), wherein the modification has the amino acid sequence of any of the sequences provided in Tables 2, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, or 71-86 of WO 2021/242909. [0052] In any of the embodiments described herein, a position numbered relative to SEQ ID NO: 138 can be identified by providing an alignment of a reference sequence and a query sequence, wherein the reference sequence is SEQ ID NO: 138, and identifying the residues corresponding to the positions in the query sequence that correspond to positions in the reference sequence.
Table 1. Exemplary full length capsid sequences
Figure imgf000079_0001
Figure imgf000080_0001
[0053] In some embodiments, an AAV capsid variant described herein (e.g., an AAV5 capsid variant) comprises an amino acid other than T at position 577 (e.g., Y, N, or C), numbered relative to SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises Y at position 577, numbered relative to SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises N at position 577, numbered relative to SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises C at position 577, numbered relative to SEQ ID NO: 138.
[0054] In some embodiments, an AAV capsid variant described herein (e.g., an AAV5 capsid variant) comprises more than one amino acid that replaces the threonine (T) at position 577, numbered relative to SEQ ID NO: 138. In some embodiments, an insert of two, three, four, five, six, seven, eight, nine, or ten amino acids replaces the T at position 577, numbered relative to SEQ ID NO: 138. In some embodiments an insert of eight amino acids replaces the T at position 577, numbered relative to SEQ ID NO: 138.
[0055] In some embodiments, an AAV particle described herein comprises an AAV capsid variant, e.g., an AAV capsid variant described herein (e.g., an AAV capsid variant comprising a peptide or an amino acid sequence described herein). In some embodiments, an AAV capsid variant comprises a peptide as set forth in any of Tables 2A, 2B, 2C, 15 or 21.
[0056] In some embodiments, the AAV capsid variant, comprises immediately subsequent to position 570, 571, 572, 573, 574, 575, or 576, numbered relative to SEQ ID NO: 138, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive amino acids of any of amino acid sequence provided in Tables 2A, 2B, 2C, 15 or 21.
Table 2A. Exemplary Peptide Sequences
Figure imgf000081_0001
Table 2B. Exemplary Peptide Sequences
Figure imgf000081_0002
Table 2C. Exemplary Peptide Sequences
Figure imgf000081_0003
[0057] In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence having the formula [N2]-[N3], wherein [N2] comprises positions XI, X2, X3, X4, and X5 and [N3] comprises the amino acid sequence of VQK, EQK, VKK, VHK, VQQ, or LQK. In some embodiments [N3] comprises the amino acid sequence of VQK, EQK, or VKK. In some embodiments [N3] comprises the amino acid sequence VQK. In some embodiments, position XI of [N2] is Y, N, or C. In some embodiments position XI of [N2] is Y or N. In some embodiments, position X2 of [N2] is P, K, T, or Q. In some embodiments position X2 of [N2] is P, T, or Q. In some embodiments, position X3 of [N2] is A or P. In some embodiments, position X3 of [N2] is A. In some embodiments, position X4 of [N2J is E, S, or A. In some embodiments, position X5 of [N2J is V, L, or E. In some embodiments, position X5 of [N2] is V or L. In some embodiments, [N2] comprises Y at position XI. In some embodiments, [N2] comprises P at position X2. In some embodiments, [N2] comprises A at position X3. In some embodiments, [N2] comprises E at position X4. In some embodiments, [N2] comprises V at position X5. In some embodiments, [N2] comprises YPA, YPP, NKA, YTA, YQA, YTP, NPA, CPA, THA, PAE, PPS, KAE, TAE, QAE, TPS, PAA, HAS, AEV, PSL, AEE, or AAV.
[0058] In some embodiments, [N2] comprises YPAE (SEQ ID NO: 286), YPPS (SEQ ID NO: 287), NKAE (SEQ ID NO: 288), YTAE (SEQ ID NO: 289), YQAE (SEQ ID NO: 290), YTPS (SEQ ID NO: 291), YPAA (SEQ ID NO: 292), NPAE (SEQ ID NO: 293), CPAE (SEQ ID NO: 294), THAS(SEQ ID NO: 295) , PAEV (SEQ ID NO: 296), PPSL (SEQ ID NO: 297), KAEV (SEQ ID NO: 298), TAEV (SEQ ID NO: 299), PAEE (SEQ ID NO: 300), QAEV (SEQ ID NO: 301), TPSL (SEQ ID NO: 302), PAAV (SEQ ID NO: 303), or QAEE (SEQ ID NO: 304). In some embodiments [N2] is or comprises YPAEV (SEQ ID NO: 39), YPPSL (SEQ ID NO: 40), NKAEV (SEQ ID NO: 41), YTAEV (SEQ ID NO: 42), YPAEE (SEQ ID NO: 43), YQAEV (SEQ ID NO: 44), YTPSL (SEQ ID NO: 45) , YPAAV (SEQ ID NO: 46), NPAEV (SEQ ID NO: 47), CPAEV (SEQ ID NO: 48), or YQAEE (SEQ ID NO: 49). In some embodiments [N2] is YPAEV (SEQ ID NO: 39).
[0059] In some embodiments, [N2]-[N3] comprises the amino acid sequence of AEVVQK (SEQ ID NO: 50), PSLVQK (SEQ ID NO: 51), AEVEQK (SEQ ID NO: 52), AEEVQK (SEQ ID NO:53), PSLEQK (SEQ ID NO: 54), PSLVKK (SEQ ID NO: 55), AEVVKK (SEQ ID NO:56), AEVVHK (SEQ ID NO: 57), AAVVQK (SEQ ID NO: 58), AEVVQQ (SEQ ID NO: 59), or AEVLQK (SEQ ID NO: 60). In some embodiments [N2]-[N3] comprises the amino acid sequence PAEVVQK (SEQ ID NO: 61), PPSLVQK (SEQ ID NO: 62), KAEVVQK (SEQ ID NO: 63), TAEVVQK (SEQ ID NO: 64), PAEVEQK (SEQ ID NO: 65), PAEEVQK (SEQ ID NO: 66), QAEVVQK (SEQ ID NO: 67), TPSLVQK (SEQ ID NO: 68), PPSLEQK (SEQ ID NO: 69) , PPSLVKK (SEQ ID NO: 70), PAEVVKK (SEQ ID NO: 71), PAEVVHK (SEQ ID NO: 72), PAAVVQK (SEQ ID NO: 73), PAEVVQQ (SEQ ID NO: 74), TAEVVKK (SEQ ID NO: 75), PAEVLQK (SEQ ID NO: 76), or QAEEVQK (SEQ ID NO: 77).
[0060] In some embodiments [N2]-[N3] is or comprises YPAEV VQK (SEQ ID NO: 943), YPPSLVQK (SEQ ID NO: 946), NKAEVVQK (SEQ ID NO: 947), YTAEVVQK (SEQ ID NO: 948), YP AEVEQK (SEQ ID NO: 949), YPAEEVQK (SEQ ID NO: 950), YQAEVVQK (SEQ ID NO: 951), YTPSLVQK (SEQ ID NO: 952), YPPSLEQK (SEQ ID NO: 953), YPPSLVKK (SEQ ID NO: 954), YPAEVVKK (SEQ ID NO: 955), YPAEVVHK (SEQ ID NO: 956), YPAAVVQK (SEQ ID NO: 957), NPAEVVQK (SEQ ID NO: 958), YPAEVVQQ (SEQ ID NO: 959), CPAEVVQK(SEQ ID NO: 960) , YTAEVVKK (SEQ ID NO: 961), YPAEVLQK (SEQ ID NO: 962), or YQAEEVQK (SEQ ID NO: 963); an amino acid sequence comprising any portion of any of the aforesaid amino acid sequences (e.g., any 2, 3, 4, 5, 6, or 7 amino acids, e.g., consecutive amino acids) thereof; an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforesaid amino acid sequences; or an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the aforesaid amino acid sequences. In some embodiments [N2]-[N3] is YPAEVVQK (SEQ ID NO: 943). [0061] In some embodiments, the AAV capsid variant comprising the amino acid sequence comprising the formula of [N2]-[N3], further comprises [Nl], which comprises positions XD, XE, and XF. In some embodiments, position XD of [Nl] is Q, T, S, A, I, L, or H. In some embodiments, position XE of [Nl] is S, G, A, or R. In some embodiments, position XF of [N1] is S, K, L, R, A, or T. In some embodiments, [Nl] comprises SK, SL, SS, SR, GA, GS, AS, ST, RS, QS, TS, AG, IG, QA, LG, HS, LS, or QR. In some embodiments, [Nl] is or comprises QSS, QSK, TSL, SSS, QSR, AGA, IGS, QAS, ASS, LGS, QST, HSS, LSS, or QRS.
[0062] In some embodiments, the amino acid sequence of [Nl] is QSS. In some embodiments, [Nl]- [N2] comprises SSYPA (SEQ ID NO: 78), SKYPA (SEQ ID NO: 79), SLYPA (SEQ ID NO: 80), SRYPA (SEQ ID NO: 81), SSYPP (SEQ ID NO: 82), GAYPA (SEQ ID NO: 83), GSYPA(SEQ ID NO: 84), ASYPA (SEQ ID NO: 85), STNKA (SEQ ID NO: 86), SSYTA (SEQ ID NO: 87), SSYQA (SEQ ID NO: 88), SSYTP (SEQ ID NO: 89), SSNPA (SEQ ID NO: 90), SLCPA (SEQ ID NO: 91), RSYTA (SEQ ID NO: 92), or SSTHA (SEQ ID NO: 93). In some embodiments, [N1]-[N2] comprises SSYPAE (SEQ ID NO: 94), SKYPAE (SEQ ID NO: 95), SLYPAE (SEQ ID NO: 96), SRYPAE (SEQ ID NO: 97), SSYPPS (SEQ ID NO: 98), GAYPAE (SEQ ID NO: 99), GSYPAE (SEQ ID NO: 102), ASYPAE (SEQ ID NO: 103), STNKAE (SEQ ID NO: 104), SSYTAE (SEQ ID NO: 105), SSYQAE (SEQ ID NO: 106), SSYTPS (SEQ ID NO: 107), SSYPAA (SEQ ID NO: 108), SSNPAE (SEQ ID NO: 109), SLCPAE (SEQ ID NO: 110), RSYTAE (SEQ ID NO: 111), SSTHAS (SEQ ID NO: 112). In some embodiments, [Nl]- [N2] is or comprises QSSYPAEV (SEQ ID NO: 113), QSKYPAEV (SEQ ID NO: 114), TSLYPAEV (SEQ ID NO: 115), SSSYPAEV (SEQ ID NO: 116), QSRYPAEV (SEQ ID NO: 117), QSSYPPSL (SEQ ID NO: 118), AGAYPAEV (SEQ ID NO: 119), IGSYPAEV (SEQ ID NO: 120), QASYPAEV (SEQ ID NO: 121), ASSYPAEV (SEQ ID NO: 122), LGSYPAEV (SEQ ID NO: 123), QSTNKAEV (SEQ ID NO: 124), HSSYPAEV (SEQ ID NO: 125), SSSYTAEV (SEQ ID NO: 126), TSLYPAEE (SEQ ID NO: 127), ASSYQAEV (SEQ ID NO: 128), QSSYTPSL (SEQ ID NO: 129), QSRYPAEE (SEQ ID NO: 130), LSSYQAEV (SEQ ID NO: 131), HSSYPAAV (SEQ ID NO: 132), QSSNPAEV (SEQ ID NO: 100), QSSYTAEV (SEQ ID NO: 133), TSLCPAEV (SEQ ID NO: 134), QRSYTAEV (SEQ ID NO: 135), or QSSYQAEE (SEQ ID NO: 136); an amino acid sequence comprising any portion of any of the aforesaid amino acid sequences (e.g., any 2, 3, 4, 5, 6, or 7 amino acids, e.g., consecutive amino acids) thereof; an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforesaid amino acid sequences; or an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the aforesaid amino acid sequences. In some embodiments, [N1]-[N2] is QSSYPAEV (SEQ ID NO: 113). In some embodiments, [N1]-[N2]-[N3] comprises SSYPAEVVQ (SEQ ID NO: 142), SKYPAEVVQ (SEQ ID NO: 143), SLYPAEVVQ (SEQ ID NO: 101), SRYPAEVVQ (SEQ ID NO: 144), SSYPPSLVQ (SEQ ID NO: 145), GAYPAEVVQ (SEQ ID NO: 146), GSYPAEVVQ (SEQ ID NO: 147), ASYPAEVVQ (SEQ ID NO: 148), STNKAEVVQ (SEQ ID NO: 149), SSYTAEVVQ (SEQ ID NO: 150), SKYPAEVEQ (SEQ ID NO: 160), SLYPAEEVQ (SEQ ID NO: 161), SSYQAEVVQ (SEQ ID NO: 162), SSYTPSLVQ (SEQ ID NO: 163), SRYPAEEVQ (SEQ ID NO: 164), SSYPPSLEQ (SEQ ID NO: 165), SSYPPSLVK (SEQ ID NO: 166), SSYPAEVVK (SEQ ID NO: 167), SKYPAEVVH (SEQ ID NO: 168), SSYPAAVVQ (SEQ ID NO: 169), SSNPAEVVQ (SEQ ID NO: 170), SLCPAEVVQ (SEQ ID NO: 171), RSYTAEVVQ (SEQ ID NO: 172), SSYTAEVVK (SEQ ID NO: 173), SSYPAEVLQ (SEQ ID NO: 174), or SSYQAEEVQ (SEQ ID NO: 175).
[0063] In some embodiments, [N1]-[N2]-[N3] is or comprises QSSYPAEVVQK (SEQ ID NO: 176), QSKYPAEVVQK (SEQ ID NO: 177), TSLYPAEVVQK (SEQ ID NO: 178), SSSYPAEVVQK (SEQ ID NO: 179), QSRYPAEVVQK (SEQ ID NO: 180), QSSYPPSLVQK (SEQ ID NO: 181), AGAYPAEVVQK (SEQ ID NO: 182), IGSYPAEVVQK (SEQ ID NO: 183), QASYPAEVVQK (SEQ ID NO: 184), ASSYPAEVVQK (SEQ ID NO: 186), LGSYPAEVVQK (SEQ ID NO: 187), QSTNKAEVVQK (SEQ ID NO: 188), HSSYPAEVVQK (SEQ ID NO: 189), SSSYTAEVVQK (SEQ ID NO: 190), QSKYPAEVEQK (SEQ ID NO: 191), TSLYPAEEVQK (SEQ ID NO: 192), ASSYQAEVVQK (SEQ ID NO: 193), QSSYTPSLVQK (SEQ ID NO: 194), QSRYPAEEVQK (SEQ ID NO: 195), QSSYPPSLEQK (SEQ ID NO: 196), QSSYPPSLVKK (SEQ ID NO: 197), LSSYQAEVVQK (SEQ ID NO: 198), SSSYPAEVVKK (SEQ ID NO: 199), QSKYPAEVVHK (SEQ ID NO: 200), HSSYPAAVVQK (SEQ ID NO: 201), QSSNPAEVVQK (SEQ ID NO: 202), SSSYPAEVVQQ (SEQ ID NO: 203), QSSYTAEVVQK (SEQ ID NO: 204), TSLCPAEVVQK (SEQ ID NO: 205), QRSYTAEVVQK (SEQ ID NO: 206), QSSYTAEVVKK (SEQ ID NO: 207), HSSYPAEVLQK (SEQ ID NO: 208), or QSSYQAEEVQK (SEQ ID NO: 209); an amino acid sequence comprising any portion of any of the aforesaid amino acid sequences (e.g., any 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, e.g., consecutive amino acids) thereof; an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforesaid amino acid sequences; or an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the aforesaid amino acid sequences. In some embodiments, [N1]-[N2]-[N3] is QSSYPAEVVQK (SEQ ID NO: 176).
[0064] In some embodiments, the AAV capsid variant comprising the amino acid sequence comprising the formula [N2]-[N3], further comprises [NO], wherein [NO] comprises positions XA, XB, and Xc- In some embodiments, position XA of [NO] is T, I, or N. In some embodiments, positions XB of [NO] is N. In some embodiments, position Xc of [NO] is N, T, S, or K. In some embodiments, [NO] comprises TN, IN, NN, NT, NS, or NK. In some embodiments, [NO] is or comprises TNN, TNT, INN, TNS, NNN, or TNK. In some embodiments, [NO] is TNN. In some embodiments, [NO]-[N1] is or comprises TNNQSS (SEQ ID NO: 210), TNNQSK (SEQ ID NO: 211), TNNTSL (SEQ ID NO: 212), TNNSSS (SEQ ID NO: 213), TNNQSR (SEQ ID NO: 214), TNNAGA (SEQ ID NO: 215), TNNIGS (SEQ ID NO: 216), TNNQAS (SEQ ID NO: 217), TNTASS (SEQ ID NO: 218), TNNLGS (SEQ ID NO: 219), TNNQST (SEQ ID NO: 220), TNNHSS (SEQ ID NO: 221), TNNLSS (SEQ ID NO: 223), INNQSS (SEQ ID NO: 224), TNSQSS (SEQ ID NO: 225), NNNQSR (SEQ ID NO: 226), TNSTSL (SEQ ID NO: 227), TNNQRS (SEQ ID NO: 228), or TNKQAS (SEQ ID NO: 229); an amino acid sequence comprising any portion of any of the aforesaid amino acid sequences (e.g., any 2, 3, 4, or 5 amino acids, e.g., consecutive amino acids) thereof; an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforesaid amino acid sequences; or an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the aforesaid amino acid sequences. In some embodiments, [NO]-[N1] is TNNQSS(SEQ ID NO: 210).
[0065] In some embodiments, [N0]-[Nl]-[N2]-[N3] is or comprises TNNQSSYPAEVVQK (SEQ ID NO: 230), TNNQSKYPAEVVQK (SEQ ID NO: 231), TNNTSLYPAEVVQK (SEQ ID NO: 232), TNNSSSYPAEVVQK (SEQ ID NO: 233), TNNQSRYPAEVVQK (SEQ ID NO: 234), TNNQSSYPPSLVQK (SEQ ID NO: 235), TNNAGAYPAEVVQK (SEQ ID NO: 236), TNNIGSYPAEVVQK (SEQ ID NO: 237), TNNQASYPAEVVQK (SEQ ID NO: 238), TNTASSYPAEVVQK (SEQ ID NO: 239), TNNLGSYPAEVVQK (SEQ ID NO: 240), TNNQSTNKAEVVQK (SEQ ID NO: 241), TNNHSS YPAEVVQK (SEQ ID NO: 242), TNNSSSYTAEVVQK (SEQ ID NO: 243), TNNQSKYPAEVEQK (SEQ ID NO: 244), TNNTSL YPAEEVQK (SEQ ID NO: 245), TNTASSYQAEVVQK (SEQ ID NO: 246), TNNQSSYTPSLVQK (SEQ ID NO: 247), TNNQSRYPAEEVQK (SEQ ID NO: 248), TNNQSSYPPSLEQK (SEQ ID NO: 249), TNNQSSYPPSLVKK (SEQ ID NO: 250), TNNLSSYQAEVVQK (SEQ ID NO: 251), TNNSSSYPAEVVKK (SEQ ID NO: 252), TNNQSKYPAEVVHK (SEQ ID NO: 253), INNQSSYPAEVVQK (SEQ ID NO: 254), TNNHSSYPAAVVQK (SEQ ID NO: 255), TNSQSSNPAEVVQK (SEQ ID NO: 256), TNNSSSYPAEVVQQ (SEQ ID NO: 257), NNNQSRYPAEVVQK (SEQ ID NO: 258), TNNQSSYTAEVVQK (SEQ ID NO: 259), TNNTSLCPAEVVQK (SEQ ID NO: 260), TNSTSLYPAEVVQK (SEQ ID NO: 261), TNNQRSYTAEVVQK (SEQ ID NO: 262), TNNQSSYTAEVVKK (SEQ ID NO: 263), TNNHSSYPAEVLQK (SEQ ID NO: 264), TNNQSSYQAEEVQK (SEQ ID NO: 266) or TNKQASYPAEVVQK (SEQ ID NO: 267); an amino acid sequence comprising any portion of any of the aforesaid amino acid sequences (e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acids, e.g., consecutive amino acids) thereof; an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforesaid amino acid sequences; or an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the aforesaid amino acid sequences. In some embodiments, [N0]-[Nl]-[N2]-[N3] is TNNQSSYPAEVVQK (SEQ ID NO: 230),.
[0066] In some embodiments, the AAV capsid variant comprising the amino acid sequence comprising the formula [N2]-[N3], further comprises [N4], which comprises positions Xc, and Xu. In some embodiments, position XG of [N4] is T, P, or N. In some embodiments, position Xu of [N4] is A. In some embodiments, [N4] is or comprises TA, PA, or NA. In some embodiments, [N4] is TA. In some embodiments, [N3]-[N4] is or comprises VQKTA (SEQ ID NO: 268), EQKTA (SEQ ID NO: 269), VKKTA (SEQ ID NO: 270), VQKPA (SEQ ID NO: 271), VHKTA (SEQ ID NO: 272), VQQTA (SEQ ID NO: 273), VQKNA (SEQ ID NO: 274), or LQKTA (SEQ ID NO: 275); an amino acid sequence comprising any portion of any of the aforesaid amino acid sequences (e.g., any 2, 3, or 4 amino acids, e.g., consecutive amino acids) thereof; an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforesaid amino acid sequences; or an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the aforesaid amino acid sequences. In some embodiments, [N3]-[N4] is VQKTA (SEQ ID NO: 268).
[0067] In some embodiments, [N0]-[Nl]-[N2]-[N3]-[N4] is or comprises TNNQSSYPAEVVQKTA (SEQ ID NO: 1533), TNNQSKYPAEVVQKTA (SEQ ID NO: 1538), TNNTSLYPAEV VQKTA (SEQ ID NO: 1232), TNNSSSYPAEV VQKTA (SEQ ID NO: 1539), TNNQSRYPAEVVQKTA (SEQ ID NO: 1327), TNNQSSYPPSLVQKTA (SEQ ID NO: 1300), TNNAGAYPAEVVQKTA (SEQ ID NO: 1021), TNNIGSYPAEVVQKTA (SEQ ID NO: 1112) , TNNQ AS YPAEV VQKTA (SEQ ID NO: 1586), TNTASSYPAEVVQKTA (SEQ ID NO: 1575), TNNLGSYPAEVVQKTA (SEQ ID NO: 1027), TNNQSTNKAEVVQKTA (SEQ ID NO: 1578), TNNHSSYPAEVVQKTA (SEQ ID NO: 1310), TNNSSSYTAEVVQKTA (SEQ ID NO: 1214), TNNQSKYPAEVEQKTA (SEQ ID NO: 1254), TNNTSLYPAEEVQKTA (SEQ ID NO: 1583), TNTASSYQAEV VQKTA (SEQ ID NO: 1584), TNNQSSYTPSLVQKTA (SEQ ID NO: 1585), TNNQSRYPAEEVQKTA (SEQ ID NO: 1342), TNNQSSYPPSLEQKTA (SEQ ID NO: 1590), TNNQSSYPPSLVKKTA (SEQ ID NO: 1591), TNNLSSYQAEVVQKTA (SEQ ID NO: 1592), TNNQSSYPPSLVQKPA (SEQ ID NO: 1593), TNNSSSYPAEVVKKTA (SEQ ID NO: 1331), TNNQSKYPAEVVHKTA (SEQ ID NO: 1453), TNNSSSYPAEVVQKPA (SEQ ID NO: 1142), INNQSSYPAEVVQKTA (SEQ ID NO: 1024), TNNHSSYPAAVVQKTA (SEQ ID NO: 1598), TNSQSSNPAEVVQKTA (SEQ ID NO: 1599), TNNSSSYPAEVVQQTA (SEQ ID NO: 1419), NNNQSRYPAEVVQKTA (SEQ ID NO: 1601), TNNQSSYTAEVVQKNA (SEQ ID NO: 1602), TNNTSLCPAEVVQKTA (SEQ ID NO: 1603), TNSTSLYPAEVVQKTA (SEQ ID NO: 1605), TNNQRSYTAEVVQKTA (SEQ ID NO: 1604), TNNQSSYTAEVVKKTA (SEQ ID NO: 1606), TNNHSSYPAEVLQKTA (SEQ ID NO: 1607), TNNQSSYQAEEVQKTA (SEQ ID NO: 1608), or TNKQASYPAEVVQKTA (SEQ ID NO: 1587); an amino acid sequence comprising any portion of any of the aforesaid amino acid sequences (e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids, e.g., consecutive amino acids) thereof; an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforesaid amino acid sequences; or an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the aforesaid amino acid sequences. In some embodiments, [NO]-[N1]-[N2]-[N3]- [N4] is TNNQSSYPAEVVQKTA (SEQ ID NO: 1533).
[0068] In some embodiments, [N2]-[N3] is present in loop VIII of the AAV capsid variant. In some embodiments [NO], [Nl], and/or [N4] are present in loop VIII of the AAV capsid variant. In some embodiments, [N0]-[Nl]-[N2]-[N3]-[N4] is present in loop VIII of the AAV capsid variant. In some embodiments, loop VIII comprises positions 571-592 numbered according to SEQ ID NO: 138. In some embodiments, loop VIII comprises positions 571-599, numbered according to SEQ ID NO: 982.
[0069] In some embodiments, [NO] is present immediately subsequent to position 570, numbered relative to SEQ ID NO: 138. In some embodiments, [NO] replaces positions 571-573 (e.g., T571, N572, and N573), numbered relative to SEQ ID NO: 138. In some embodiments, [NO] is present immediately subsequent to position 570, and [NO] replaces positions 571-573 (e.g., amino acids T571, N572, and N573), numbered relative to SEQ ID NO: 138. In some embodiments, [Nl] is present immediately subsequent to position 573, numbered relative to SEQ ID NO: 138. In some embodiments, [Nl] replaces positions 574-576 (e.g., Q574, S575, and S576), numbered relative to SEQ ID NO: 138. In some embodiments, [Nl] is present immediately subsequent to position 573, and [Nl] replaces positions 574- 576 (e.g., Q574, S575, and S576), numbered relative to SEQ ID NO: 138. In some embodiments, [N2] is present immediately subsequent to position 576, numbered relative to SEQ ID NO: 138. In some embodiments, [N2] replaces position 577 (e.g., T577), numbered relative to SEQ ID NO: 138. In some embodiments, [N2] is present immediately subsequent to position 576, and [N2] replaces position 577 (e.g., T577), numbered relative to SEQ ID NO: 138. In some embodiments, [N2]-[N3] is present immediately subsequent to position 576, numbered relative to SEQ ID NO: 138. In some embodiments, [N2]-[N3] replaces position 577 (e.g., T577), numbered relative to SEQ ID NO: 138. In some embodiments, [N2]-[N3] is present immediately subsequent to position 576, and [N2]-[N3] replaces position 577 (e.g., T577), numbered relative to SEQ ID NO: 138. In some embodiments, [N2]-[N3]-[N4] is present immediately subsequent to position 576, numbered relative to SEQ ID NO: 138. In some embodiments, [N2]-[N3]-[N4] replaces positions 577-579 (e.g., T577, T578, and A579), numbered relative to SEQ ID NO: 138. In some embodiments, [N2]-[N3]-[N4] is present immediately subsequent to position 576, and [N2]-[N3]-[N4] replaces positions 577-579 (e.g., T577, T578, and A579), numbered relative to SEQ ID NO: 138. In some embodiments, [N1]-[N2]-[N3]-[N4] is present immediately subsequent to position 573, numbered relative to SEQ ID NO: 138. In some embodiments, [N1]-[N2]- [N3]-[N4] replaces positions 574-579 (e.g., Q574, S575, S576, T577, T578, and A579), numbered relative to SEQ ID NO: 138. In some embodiments, [N1]-[N2]-[N3]-[N4] is present immediately subsequent to position 573, and [N1]-[N2]-[N3]-[N4] replaces positions 574-579 (e.g., Q574, S575, S576, T577, T578, and A579), numbered relative to SEQ ID NO: 138. In some embodiments, [NO]- [N1]-[N2]-[N3]-[N4] is present immediately subsequent to position 570. In some embodiments, [N0]- [N 1 J-LN2J- [N3]-[N4] replaces positions 571-579 (e.g., T571, N572, N573, Q574, S575, S576, T577, T578, and A579), numbered relative to SEQ ID NO: 138. In some embodiments, |N0|-[N I |-[N2|-[N3 |- [N4] is present immediately subsequent to position 570, and [N0]-[Nl]-[N2]-[N3]-[N4] replaces positions 571-579 (e.g., T571, N572, N573, Q574, S575, S576, T577, T578, and A579), numbered relative to SEQ ID NO: 138. In some embodiments, [N0]-[Nl]-[N2]-[N3]-[N4] is present immediately subsequent to position 570, numbered relative to SEQ ID NO: 138, wherein [N2]-[N3] replaces position 577 (e.g., T577), numbered relative to SEQ ID NO: 138.
[0070] In some embodiments, the AAV capsid variant comprises an amino acid other than T at position 577, relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises Y at position 577, relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, XI of [N2] is present at position 577 (e.g., T577), and positions X2 and X3 of [N2] arc present immediately subsequent to position 577, relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, [N3] is present immediately subsequent to [N2].
[0071] In some embodiments, XA of [NO] is present at position 571, XB of [NO] is present at position 572, and Xc of [NO] is present at position 573, numbered according to SEQ ID NO: 982. In some embodiments, XD of [Nl] is present at position 574, XE of [Nl] is present at position 575, and XF of [Nl] is present at position 576, numbered according to SEQ ID NO: 982. In some embodiments, XI of [N2] is present at position 577, X2 of [N2] is present at position 578, X3 of [N2] is present at position 579, X4 of [N2] is present at position 580, and of X5 of [N2] is present at position 581, numbered according to SEQ ID NO: 982. In some embodiments, [N3] is present at positions 582-584, numbered according to SEQ ID NO: 982. In some embodiments, XQ of [N4] is present at position 585 and Xu of [N4] is present at position 586, numbered according to SEQ ID NO: 982.
[0072] In some embodiments, [NO] is present at positions 571 -573, numbered according to SEQ ID NO: 982. In some embodiments, [Nl] is present at positions 574-576, numbered according to SEQ ID NO: 982. In some embodiments, [N2] is present at positions 577-581, numbered according to SEQ ID NO:
982. In some embodiments, [N3] is present at positions 582-584, numbered according to SEQ ID NO:
982. In some embodiments, [N4] is present at positions 585-586, numbered according to SEQ ID NO:
982. In some embodiments, [N2]-[N3] is present at positions 577-584, numbered according to SEQ ID
NO: 982. In some embodiments, [NO]-[N1]-[N2]-[N3]-[N4] is present at positions 571-586, numbered according to SEQ ID NO: 982.
[0073] In some embodiments, [Nl] is present immediately subsequent to [NO]. In some embodiments, [N2] is present immediately subsequent to [Nl]. In some embodiments, [N3] is present immediately subsequent to [N2J. In some embodiments, [N4J is present immediately subsequent to [N3J.
[0074] In some embodiments, the AAV capsid variant comprises from N-terminus to C-terminus, [N2]- [N3]. In some embodiments, the AAV capsid variant comprises from N-terminus to C-terminus, [Nl]- [N2]-[N3]. In some embodiments, the AAV capsid variant comprises from N-terminus to C-terminus, [N1]-[N2]-[N3]-[N4], In some embodiments, the AAV capsid variant comprises from N-terminus to C- terminus, [NO]-[N1]-[N2]-[N3], In some embodiments, the AAV capsid variant comprises from N- terminus to C-terminus, [N0]-[Nl]-[N2]-[N3]-[N4],
[0075] In some embodiments, [N2]-[N3] is YPAEVVQK (SEQ ID NO: 943), wherein YPAEVVQK (SEQ ID NO: 943) replaces position 577, numbered relative to SEQ ID NO: 138. In some embodiments, [NO]-[N1]-[N2]-[N3]-[N4] is TNNQSSYPAEVVQKTA (SEQ ID NO: 1533) and is present immediately subsequent to position 570, numbered relative to SEQ ID NO: 138, wherein [N2]-[N3] (YPAEVVQK; SEQ ID NO: 943) replaces position 577 (e.g., replaces T577) numbered relative to SEQ ID NO: 138. In some embodiments, [N2]-[N3] is YPAEVVQK, wherein [N2]-[N3] is present at positions 577-584, numbered according to SEQ ID NO: 982. In some embodiments, [NO]-[N1]-[N2]-[N3]-[N4] is TNNQSSYPAEVVQKTA (SEQ ID NO: 1533) and is present at positions 571-586, numbered according to SEQ ID NO: 982.
[0076] In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence having the formula [B]-[C], wherein [B] comprises positions XI, X2, and X3, and [C] comprises the amino acid sequence YPAEVVQK (SEQ ID NO: 943). In some embodiments, position XI of [B] is Q, T, S, A, I, L, or H. In some embodiments, position XI of [B] is Q, T, S, A, or H. In some embodiments, position X2 of [B] is S, G, or A. In some embodiments, position X2 of [B] is S or G. In some embodiments, position X3 of [B] is S, K, L, R, or A. In some embodiments, position X3 of [B] is S, K, L, or R. In some embodiments, [B] comprises Q at position XI. In some embodiments, [B] comprises S at position X2. In some embodiments, [B] comprises S at position X3. In some embodiments, [B] comprises QS, TS, SS, AG, IG, QA, AS, LG, HS, SK, SL, SR, GA, or GS. In some embodiments, [B] is or comprises QSS, TSL, SSS, QSR, QSK, AGA, IGS, QAS, ASS, LGS, or HSS. In some embodiments, [B] is QSS. [0077] In some embodiments, [B]-[C] comprises SSYPAEVVQK (SEQ ID NO: 276), SKYPAEVVQK (SEQ ID NO: 277), SLYPAEVVQK (SEQ ID NO: 278), SRYPAEVVQK (SEQ ID NO: 279), GAYPAEVVQK (SEQ ID NO: 280), GSYPAEVVQK (SEQ ID NO: 281), or ASYPAEVVQK (SEQ ID NO: 282). In some embodiments, [B]-[C] is or comprises QSSYPAEVVQK (SEQ ID NO: 176), QSKYPAEVVQK (SEQ ID NO: 177), TSLYPAEVVQK (SEQ ID NO: 178), SSSYPAEVVQK (SEQ ID NO: 179), QSRYPAEVVQK (SEQ ID NO: 180), AGAYPAEVVQK (SEQ ID NO: 182), IGSYPAEVVQK (SEQ ID NO: 183), QASYPAEVVQK (SEQ ID NO: 184), ASSYPAEVVQK (SEQ ID NO: 186), LGSYPAEVVQK (SEQ ID NO: 187), or HSSYPAEVVQK (SEQ ID NO: 189); an amino acid sequence comprising any portion of any of the aforesaid amino acid sequences (e.g., any 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, e.g., consecutive amino acids) thereof; an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforesaid amino acid sequences; or an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the aforesaid amino acid sequences. In some embodiments, [B]-[C] is QSSYPAEVVQK (SEQ ID NO: 176).
[0078] In some embodiments, an AAV capsid variant comprising the formula [B]-[C], further comprises [A], which comprises positions XA, XB, and Xc- In some embodiments, position XA of [A] is T, I, or N. In some embodiments, position XB of [A] is N. In some embodiments, position Xc of [A] is N, T, S, or K. In some embodiments, [A] comprises TN, IN, NN, NT, NS, or NK. In some embodiments, [A] is or comprises TNN, TNT, INN, NNN, TNS, or TNK. In some embodiments, [A] is TNN. In some embodiments, [A]-[B] is or comprises TNNQSS (SEQ ID NO: 210), TNNQSK (SEQ ID NO: 211), TNNTSL (SEQ ID NO: 212), TNNSSS (SEQ ID NO: 213), TNNQSR (SEQ ID NO: 214), TNNAGA (SEQ ID NO: 215), TNNIGS (SEQ ID NO: 216), TNNQAS (SEQ ID NO: 217), TNTASS (SEQ ID NO: 218), TNNLGS (SEQ ID NO: 219), TNNHSS (SEQ ID NO: 221), INNQSS (SEQ ID NO: 224), NNNQSR (SEQ ID NO: 226), TNSTSL (SEQ ID NO: 227), or TNKQAS (SEQ ID NO: 229); an amino acid sequence comprising any portion of any of the aforesaid amino acid sequences (e.g., any 2, 3, 4, or 5 amino acids, e.g., consecutive amino acids) thereof; an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforesaid amino acid sequences; or an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the aforesaid amino acid sequences. In some embodiments, [A]-[B] is TNNQSS (SEQ ID NO: 210). In some embodiments, [A]-[B]-[C] is or comprises TNNQSSYPAEVVQK (SEQ ID NO: 230), TNNQSKYPAEVVQK (SEQ ID NO: 231), TNNTSLYPAEVVQK (SEQ ID NO: 232), TNNS SSYPAEVVQK (SEQ ID NO: 233), TNNQSRYPAEVVQK (SEQ ID NO: 234), TNNAGAYPAEVVQK (SEQ ID NO: 236), TNNIGSYPAEVVQK (SEQ ID NO: 237), TNNQASYPAEVVQK (SEQ ID NO: 238), TNTASSYPAEVVQK (SEQ ID NO: 239), TNNLGSYPAEVVQK (SEQ ID NO: 240), TNNHSSYPAEVVQK (SEQ ID NO: 242), INNQSSYPAEVVQK (SEQ ID NO: 254), NNNQSRYPAEVVQK (SEQ ID NO: 258), TNSTSLYPAEVVQK (SEQ ID NO: 261), or TNKQASYPAEVVQK (SEQ ID NO: 267); an amino acid sequence comprising any portion of any of the aforesaid amino acid sequences (e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acids, e.g., consecutive amino acids) thereof; an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforesaid amino acid sequences; or an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the aforesaid amino acid sequences. In some embodiments, [A]-[B]-[C] is TNNQSSYPAEVVQK (SEQ ID NO: 230).
[0079] In some embodiments, an AAV capsid variant comprising the formula [B]- [C] , further comprises [D], wherein [D] comprises position X4 and X5. In some embodiments, position X4 of [D] is T or N. In some embodiments, position X5 of [D] is A. In some embodiments [D] is or comprises TA or PA. In some embodiments, [D] is TA. In some embodiments, [C]-[D] is or comprises YPAEVVQKTA (SEQ ID NO: 283) or YPAEVVQKPA (SEQ ID NO: 284); an amino acid sequence comprising any portion of any of the aforesaid amino acid sequences (e.g., any 2, 3, 4, 5, 6, 7, 8, or 9 amino acids, e.g., consecutive amino acids) thereof; an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforesaid amino acid sequences; or an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the aforesaid amino acid sequences. In some embodiments, [C]-[D] is YPAEVVQKTA (SEQ ID NO: 283).
[0080] In some embodiments, [A]-[B]-[C]-[D] is or comprises TNNQSSYPAEVVQKTA (SEQ ID NO: 1533), TNNQSKYPAEVVQKTA (SEQ ID NO: 1538), TNNTSLYPAEVVQKTA (SEQ ID NO: 1232), TNNSSSYPAEVVQKTA (SEQ ID NO: 1539), TNNQSRYPAEVVQKTA (SEQ ID NO: 1327), TNNAGAYPAEVVQKTA(SEQ ID NO: 1021), TNNIGSYPAEVVQKTA(SEQ ID NO: 1112), TNNQ AS YPAEVVQKTA (SEQ ID NO: 1586), TNTASSYPAEVVQKTA (SEQ ID NO: 1575), TNNLGSYPAEVVQKTA (SEQ ID NO: 1027), TNNHSSYPAEVVQKTA (SEQ ID NO: 1310), TNNSSSYPAEVVQKPA (SEQ ID NO: 1142), INNQSSYPAEVVQKTA (SEQ ID NO: 1024), NNNQSRYPAEVVQKTA (SEQ ID NO: 1601), TNSTSLYPAEVVQKTA (SEQ ID NO: 1605), or TNKQASYPAEVVQKTA (SEQ ID NO: 1587); an amino acid sequence comprising any portion of any of the aforesaid amino acid sequences (e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, or 15 amino acids, e.g., consecutive amino acids) thereof; an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforesaid amino acid sequences; or an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to any one of the aforesaid amino acid sequences. In some embodiments, [A]-[B]-[C]-[D] is TNNQSSYPAEVVQKTA (SEQ ID NO: 1533). [0081] In some embodiments, [B]-[C] is present in loop VIII of the AAV capsid variant. In some embodiments, [A] and/or [D] is present in loop VUI of the AAV capsid variant. In some embodiments,
[A]-[B]-[C]-[D] is present in loop VIII of the AAV capsid variant. In some embodiments, loop VIH comprises positions 571-592 numbered according to SEQ ID NO: 138. In some embodiments, loop VIII comprises positions 571-599, numbered according to SEQ ID NO: 982.
[0082] In some embodiments, [A] is present immediately subsequent to position 570, numbered relative to SEQ ID NO: 138. In any of these embodiments, [A] replaces positions 571-573 (e.g., T571, N572, and N573) numbered relative to SEQ ID NO: 138. In some embodiments, [A] is present immediately subsequent to position 570, and [A] replaces positions 571-573 (e.g., T571, N572, and N573) numbered relative to SEQ ID NO: 138. In some embodiments, [B] is present immediately subsequent to position 573, relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments,
[B] replaces positions 574-576 (e.g., Q574, S575, and S576), relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, [B] is present immediately subsequent to position 573, and [B] replaces positions 574-576 (e.g., Q574, S575, and S576), relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, [C] is present immediately subsequent to position 576, relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, [C] replaces position 577 (e.g., T577), relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, [C] is present immediately subsequent to position 576, wherein [C] replaces position 577 (e.g., T577), relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, [B]-[C] is present immediately subsequent to position 573, relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, [B]-[C] replaces positions 574-577 (e.g., Q574, S575, S576, and T577), relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, [B]-[C] is present immediately subsequent to position 573, and [B]-[C] replaces positions 574-577 (e.g., Q574, S575, S576, and T577), relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, XI of
[B] is present at position 574, X2 of [B] is present at position 575, and X3 of [B] is present at position 576, numbered according to SEQ ID NO: 982. In some embodiments, [B] is present at positions 574-576, numbered according to SEQ ID NO: 982. In some embodiments, [C] is present at positions 577-584, numbered according to SEQ ID NO: 982. In some embodiments, [B]-[C] is present at positions 574-584, numbered according to SEQ ID NO: 982.
[0083] In some embodiments, [C]-[D] is present immediately subsequent to position 576, relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, [C]-[D] replaces positions 577-579 (e.g., T577, T578, and A579), relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, [C]-[D] is present immediately subsequent to position 576, and
[C]-[D] replaces positions 577-579 (e.g., T577, T578, and A579), relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, [A] is present at positions 571-573, numbered according to SEQ ID NO: 982. In some embodiments, [B] is present at positions 574-576, numbered according to SEQ ID NO: 982. In some embodiments, [C] is present at positions 577-584, numbered according to SEQ ID NO: 982. In some embodiments, [D] is present at positions 585-586, numbered according to SEQ ID NO: 982. In some embodiments, [A]-[B]-[C]-[D] is present at positions 571-586, numbered according to SEQ ID NO: 982.
[0084] In some embodiments, [B]-[C]-[D] is present immediately subsequent to position 573, numbered relative to SEQ ID NO: 138. In some embodiments, [B]-[C]-[D] replaces positions 574-579 (e.g., Q574, S575, S576, T577, T578, and A579), numbered relative to SEQ ID NO: 138. In some embodiments [B]- [CJ-[DJ is present immediately subsequent to position 573, and [BJ-[CJ-[DJ replaces positions 574-579 (e.g., Q574, S575, S576, T577, T578, and A579), numbered relative to SEQ ID NO: 138. In some embodiments [A]-[B]-[C]-[D] is present immediately subsequent to position 570, numbered relative to SEQ ID NO: 138. In some embodiments, [A]-[B]-[C]-[D] replaces positions 571-579 (e.g., T571, N572, N573, Q574, S575, S576, T577, T578, and A579), numbered relative to SEQ ID NO: 138. In some embodiments [A] -[B] - [C] - [D] is present immediately subsequent to position 570, and [A]-[B]-[C]-[D] replaces positions 571 -579 (e.g., T571 , N572, N573, Q574, S575, S576, T577, T578, and A579), numbered relative to SEQ ID NO: 138.
[0085] In some embodiments, XA of [A] is present at position 571, XB of [A] is present at position 572, and Xc of [A] is present at position 573, numbered according to SEQ ID NO: 982. In some embodiments, XI of [B] is present at position 574, X2 of [B] is present at position 575, and X3 of [B] is present at position 576, numbered according to SEQ ID NO: 982. In some embodiments, [C] is present at positions 577-584, numbered according to SEQ ID NO: 982. In some embodiments, X4 of [D] is present at position 585 and position X5 of [D] is present at position 586, numbered according to SEQ ID NO: 982.
[0086] In some embodiments, [A] is present at positions 571-573, numbered according to SEQ ID NO: 982. In some embodiments, [B] is present at positions 574-576, numbered according to SEQ ID NO: 982. In some embodiments, [C] is present at positions 577-584, numbered according to SEQ ID NO: 982. In some embodiments, [D] is present at positions 585-586, numbered according to SEQ ID NO: 982. In some embodiments, [A]-[B]-[C]-[D] is present at positions 571-586, numbered according to SEQ ID NO: 982.
[0087] In some embodiments, [B] is present immediately subsequent to [A]. In some embodiments, [C] is present immediately subsequent to [B]. In some embodiments, [D] is present immediately subsequent to [C]. In some embodiments, the AAV capsid variant comprises from N-terminus to C-terminus, [B]- [C]. In some embodiments, the AAV capsid variant comprises from N-terminus to C-terminus, [B]-[C], In some embodiments, the AAV capsid variant comprises from N-terminus to C-terminus, [A]-[B]-[C], In some embodiments, the AAV capsid variant comprises from N-terminus to C-terminus, [B]-[C]-[D]. In some embodiments, the AAV capsid variant comprises from N-terminus to C-terminus, [A]-[B]-[C]- [D],
[0088] In some embodiments, [C] is YPAEVVQK (SEQ ID NO: 943), wherein YPAEVVQK (SEQ ID NO: 943) replaces position 577 (e.g., T577), numbered relative to SEQ ID NO: 138. In some embodiments, [A]-[B]-[C]-[D] is TNNQSSYPAEVVQKTA (SEQ ID NO: 1533)and is present immediately subsequent to position 570, numbered relative to SEQ ID NO: 138, wherein [C] (YPAEVVQK) replaces position 577 (e.g., replaces T577) numbered relative to SEQ ID NO: 138. In some embodiments, [C] is YPAEVVQK (SEQ ID NO: 943), wherein [C] is present at positions 577-584, numbered according to SEQ ID NO: 982.
[0089] In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive amino acids from any one of the sequences provided in Tables 2A, 2B, 2C, 15 and 21 . In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive amino acids from any one of SEQ ID NOs: 943, 1021, 1024, 1027, 1112, 1142, 1214, 1232, 1254, 1300, 1310, 1327, 1331, 1342, 1419, 1453, 1533, 1538, 1539, 1575, 1578, 1583- 1587, 1590, 1591-1593, 1598-1608, or 1610-1624. In some embodiments, the AAV capsid variant comprises at least 3, 4, 5, 6, or 7 consecutive amino acids from any one of SEQ ID NOs: 943 or 946-966. In some embodiments, the amino acid sequence is present in loop VIII. In some embodiments, the amino acid sequence is present immediately subsequent to position 570, 571, 572, 573, 574, 575, or 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
[0090] In some embodiments, the 3 consecutive amino acids comprise YPA. In some embodiments, the 4 consecutive amino acids comprise YPAE. In some embodiments, the 5 consecutive amino acids comprise YPAEV (SEQ ID NO: 39). In some embodiments, the 6 consecutive amino acids comprise YPAEVV (SEQ ID NO: 151). In some embodiments, the 7 consecutive amino acids comprise YPAEVVQ (SEQ ID NO: 152). In some embodiments, the amino acid sequence comprises YPAEVVQK (SEQ ID NO: 943). In some embodiments, the amino acid sequence consists of YPAEVVQK (SEQ ID NO: 943).
[0091] In In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of any one of the sequences provided in Tables 2A, 2B, 2C, 15 and 21. In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to the amino acid sequence of any one of the sequences provided in Tables 2A, 2B, 2C, 15 and 21. In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of any one of SEQ ID NOs: 943, 1021, 1024, 1027, 1112, 1142, 1214, 1232, 1254, 1300, 1310, 1327, 1331, 1342, 1419, 1453, 1533, 1538, 1539, 1575, 1578, 1583- 1587, 1590, 1591-1593, 1598-1608, or 1610-1624. In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to the amino acid sequence of any one of SEQ ID NOs: 943, 1021, 1024, 1027, 1112, 1142, 1214, 1232, 1254, 1300, 1310, 1327, 1331, 1342, 1419, 1453, 1533, 1538, 1539, 1575, 1578, 1583- 1587, 1590, 1591-1593, 1598-1608, or 1610-1624. In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising one, two, or three but no more than four different amino acids relative to the amino acid sequence of any one of SEQ ID NOs: 943 or 2064-2080. In some embodiments, the amino acid sequence is present in loop VIII. In some embodiments, loop VIII comprises positions 571-592 numbered according to SEQ ID NO: 138. In some embodiments, loop VIII comprises positions 571-599, numbered according to SEQ ID NO: 982. In some embodiments, the amino acid sequence is present immediately subsequent to position 570, 571, 572, 573, 574, 575, or 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces position 577 (e.g., T577), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. [0092] In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of YPAEVVQK (SEQ ID NO: 943). In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising one, two, or three, but no more than four different amino acids that relative to the amino acid sequence of YPAEVVQK (SEQ ID NO: 943).
[0093] In some embodiments, the AAV capsid variant comprises the amino acid sequence of any of the sequences provided in Tables 2A, 2B, 2C, 15 and 21. In some embodiments, the AAV capsid variant comprises the amino acid sequence of any of SEQ ID NOs: 943, 1021, 1024, 1027, 1112, 1142, 1214, 1232, 1254, 1300, 1310, 1327, 1331, 1342, 1419, 1453, 1533, 1538, 1539, 1575, 1578, 1583- 1587, 1590, 1591-1593, 1598-1608, or 1610-1624. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 943. In some embodiments, the amino acid sequence is present in loop VIII. In some embodiments, the amino acid sequence is present immediately subsequent to position 570, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces positions 571-579 (e.g., T571, N572, N573, Q574, S575, S576, T577, T578, and T579), relative to a reference sequence numbered according to SEQ ID NO 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 570, and the amino acid sequence replaces positions 571-579 (e.g., T571, N572, N573, Q574, S575, S576, T577, T578, and T579), relative to a reference sequence numbered according to SEQ ID NO 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 571, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces positions 572-579 (e.g., N572, N573, Q574, S575, S576, T577, T578, and T579), relative to a reference sequence numbered according to SEQ ID NO 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 571, and the amino acid sequence replaces positions 572-579 (e.g., N572, N573, Q574, S575, S576, T577, T578, and T579), relative to a reference sequence numbered according to SEQ ID NO 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 572, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces positions 573-579 (e.g., N573, Q574, S575, S576, T577, T578, and T579), relative to a reference sequence numbered according to SEQ ID NO 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 572, and the amino acid sequence replaces positions 573-579 (e.g., N573, Q574, S575, S576, T577, T578, and T579), relative to a reference sequence numbered according to SEQ ID NO 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 573, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces positions 574-579 (e.g., Q574, S575, S576, T577, T578, and T579), relative to a reference sequence numbered according to SEQ ID NO 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 573, and the amino acid sequence replaces positions 574-579 (e.g., Q574, S575, S576, T577, T578, and T579), relative to a reference sequence numbered according to SEQ ID NO 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 574, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces positions 575-579 (e.g., S575, S576, T577, T578, and T579), relative to a reference sequence numbered according to SEQ ID NO 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 574, and the amino acid sequence replaces positions 575-579 (e.g., S575, S576, T577, T578, and T579), relative to a reference sequence numbered according to SEQ ID NO 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 575, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces positions 576-579 (e.g., S576, T577, T578, and T579), relative to a reference sequence numbered according to SEQ ID NO 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 575, and the amino acid sequence replaces positions 576-579 (e.g., S575, S576, T577, T578, and T579), relative to a reference sequence numbered according to SEQ ID NO 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces position 577 (e.g., T577), numbered relative to SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces positions 577-579 (e.g., T577, T578, and T579), relative to a reference sequence numbered according to SEQ ID NO 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 576, and the amino acid sequence replaces position 577 (e.g., T577), relative to a reference sequence numbered according to SEQ ID NO 138. In some embodiments, the amino acid sequence is present immediately subsequent to position 576, and the amino acid sequence replaces positions 577-579 (e.g., T577, T578, and T579), relative to a reference sequence numbered according to SEQ ID NO 138.
[0094] In some embodiments, the AAV capsid variant comprises the amino acid sequence of any of SEQ ID NOs: 943 or 946-966, wherein the amino acid sequence replaces position 577 (e.g., T577), numbered relative to SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises the amino acid sequence of any of SEQ ID NOs: 943 or 946-966, wherein the amino acid sequence is present immediately subsequent to position 576, numbered relative to SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises the amino acid sequence of any of SEQ ID NOs: 943 or 946-966, wherein the amino acid sequence is present immediately subsequent to position 576, and wherein the amino acid sequence replaces position 577 (e.g., T577), numbered relative to SEQ ID NO: 138.
[0095] In some embodiments, the AAV capsid variant (e.g., an AAV capsid variant described herein), comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 944, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto. In some embodiments, the AAV capsid variant described herein, comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 944, or a nucleotide sequence comprising at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, insertions, or deletions, but no more than ten modifications, e.g., substitutions, insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 944. In some embodiments, the AAV capsid variant comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one, two, three, four, five, six, or seven, but no more than ten different nucleotides relative to the nucleotide sequence of SEQ ID NO: 944.
[0096] In some embodiments, the nucleotide sequence encoding the AAV capsid variant (e.g., an AAV capsid variant described herein), comprises the nucleotide sequence of SEQ ID NO: 944, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto. In some embodiments, the nucleic acid sequence encoding the AAV capsid variant comprises a nucleotide sequence comprising at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, insertions, or deletions, but no more than ten modifications, e.g., substitutions, insertions, or deletions, relative to the nucleotide sequences of SEQ ID NO: 944. In some embodiments, the nucleotide sequence encoding an AAV capsid variant described herein comprises a nucleotide sequence comprising at least one, two, three, four, five, six, or seven, but no more than ten different nucleotides relative to the nucleotide sequence of SEQ ID NO: 944. [0097] In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of YPAEVVQK (SEQ ID NO: 943), wherein the amino acid sequence is present in loop VIII. In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of YPAEVVQK (SEQ ID NO: 943), wherein the amino acid sequence is present immediately subsequent to position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of YPAEVVQK (SEQ ID NO: 943), wherein the amino acid sequence of YPAEVVQK (SEQ ID NO: 943) replaces position 577 (e.g., T577), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of YPAEVVQK (SEQ ID NO: 943), wherein the amino acid sequence of YPAEVVQK (SEQ ID NO: 943) is present immediately subsequent to position 576, and wherein the amino acid sequence of YPAEVVQK (SEQ ID NO: 943) replaces position 577 (e.g., T577), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. [0098] In some embodiments, an AAV capsid variant described herein comprises the amino acid Y at position 577, and further comprises the amino acid sequence of PAEVVQK (SEQ ID NO: 61), which is present immediately subsequent to position 577, numbered relative to SEQ ID NO: 138.
[0099] In some embodiments, an AAV capsid variant described herein comprises the amino acid Y at position 577 and the amino acid sequence of PAEVVQK (SEQ ID NO: 20) at positions 578-584, numbered relative to SEQ ID NO: 982.
[00100] In some embodiments, an AAV capsid variant described herein comprises the amino acid Y al position 577, and comprises the amino acid sequence of PAEVVQK (SEQ ID NO: 20), which is present immediately subsequent to position 577, numbered relative to SEQ ID NO: 982. In some embodiments, the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 739, or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto. In some embodiments, the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 738, or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto. In some embodiments, the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 982, or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.
[00101] In some embodiments, an AAV capsid variant described herein comprises the amino acid Y at position 577 and the amino acid sequence of PAEVVQK (SEQ ID NO: 20) at positions 578-584, numbered relative to SEQ ID NO: 982. In some embodiments, the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 739, or an amino acid sequence at least 95% (e.g., at least 96,
97, 98, or 99%) identical thereto. In some embodiments, the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 738, or an amino acid sequence at least 95% (e.g., at least 96, 97,
98, or 99%) identical thereto. In some embodiments, the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 982, or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.
[00102] In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of TNNQSSYPAEVVQKTA (SEQ ID NO: 1533), wherein the amino acid sequence is present in loop VIII. In some embodiments, the AAV capsid variant comprises TNNQSSYPAEVVQKTA (SEQ ID NO: 1533) and is present immediately subsequent to position 570, numbered relative to SEQ ID NO: 138 or 982, wherein YPAEVVQK (SEQ ID NO: 943) replaces position 577 (e.g., replaces T577) numbered relative to SEQ ID NO: 138.
[00103] In some embodiments, an AAV capsid variant described herein comprises the amino acid Y at position 577 and the amino acid sequence of PAEVVQK (SEQ ID NO: 61) at positions 578-584, numbered relative to SEQ ID NO: 982.
[00104] In some embodiments, the AAV capsid variant further one, two, three or all of an amino acid other than Q at position 574 (e.g., T, S, A, I, L, or H), an amino acid other than S at position 575 (e.g., G, A, L, T, or R), and/or an amino acid other than S at position 576 (e.g., K, L, R, A, Y, or T), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a Q at position 574, an S at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a T at position 574, an S at position 575, and/or a L at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an S at position 574, an S at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a Q at position 574, an S at position 575, and/or an R at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises Q at position 574, an S at position 575, and/or a K at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an A at position 574, a G at position 575, and/or an A at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an I at position 574, a G at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a Q at position 574, an A at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an A at position 574, an S at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an L at position 574, a G at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a Q at position 574, an S at position 575, and/or a T at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an H at position 574, an S at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an L at position 574, an S at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a Q at position 574, an R at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an S at position 574, an L at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an S at position 574, an L at position 575, and/or a Y at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an S at position 574, an A at position 575, and/or a T at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a Q at position 574, a T at position 575, and/or an S at position 576, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
[00105] In some embodiments, the AAV capsid variant comprises amino acid other than Q at position 574 (c.g., S), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises S at position 574, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
[00106] In some embodiments, the AAV capsid variant further comprises one or both of an amino acid other than T at position 571 (e.g., I or N), and/or an amino acid other than N at position 573 (e.g., T, S, or K), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an R at position 456, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a T at position 571, an N at position 572, and/or an N at position 573, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a T at position 571, an N at position 572, and/or a T at position 573, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an I at position 571, an N at position 572, and/or an N at position 573, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a T at position 571, an N at position 572, and/or an S at position 573, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an N at position 571, an N at position 572, and/or an N at position 573, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a T at position 571, an N at position 572, and/or a K at position 573, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
[00107] In some embodiments, the AAV capsid variant further comprises an amino acid other than T at position 578 (e.g., P or N), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises one or both of an amino acid other than T at position 578 (e.g., P or N), and/or an amino acid other than A at position 589 (e.g., D), relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a T at position 578 and/or an A at position 588, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a P at position 578 and/or an A at position 588, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an N at position 578 and/or an A at position 588, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a T at position 578 and/or a D at position 579, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.
[00108] In some embodiments, the AAV capsid variant further comprises an amino acid other than T (e.g., Y) at position 577, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises Y at position 577, relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. [00109] In some embodiments, the AAV capsid variant further comprises a modification, e.g., an insertion, substitution, and/or deletion in loop I, H, IV, and/or VI. In some embodiments, loop I, II, IV, VI, and VIII can be identified as described in Govindasamy et al. Structurally Mapping the Diverse Phenotype of Adeno-Associated Virus Serotype 4. Journal of Virology. 2006 Dec. 80(23): 11556- 11570; and Govindasamy et al. Structural Insights into Adeno-Associated Virus Serotype 5. Journal of Virology. 2013 Oct. 87(20): 11187-11199; the contents of which are each hereby incorporated by reference in their entirety.
[00110] In some embodiments, additional modifications, e.g., substitutions (e.g., conservative substitutions), insertions, and/or deletions can be introduced into an AAV capsid variant described herein at positions determined using a structural map of wild-type AAV5, e.g., a structural map described and generated by Govindasamy et al. et al. Structural Insights into Adeno- Associated Virus Serotype 5. Journal of Virology. 2013 Oct. 87(20): 11187-11199 (the contents of which are hereby incorporated herein by reference in their entirety) or Walters et al. “Structure of Adeno- Associated Virus Serotype 5,” Journal of Virology, 2004, 78(7):3361-3371 (the contents of which are hereby incorporated by reference in their entirety).
[00111] In some embodiments, an AAV capsid variant described herein comprises a modification as described in Jose et al. “High-Resolution Structural Characterization of a New Adenoassociated Virus Serotype 5 Antibody Epitope toward Engineering Antibody-Resistant Recombinant Gene Delivery Vectors,” Journal of Virology, 2020, 93(1): e01394-18; Qian et al. “Directed Evolution of AAV Serotype 5 for Increased Hepatocyte Transduction and Retained Low Humoral Seroreactivity,” Molecular Therapy: Methods and Clinical Development, 2021, 20: 122-132; Afione et al. “Identification and Mutagenesis of the Adeno-Associated Virus 5 Sialic Acid Binding Region,” Journal of Virology, 2015, 89(3): 1660-1672; and/or Wang et al. “Directed evolution of adeno-associated virus 5 capsid enables specific liver tropism,” Mol Ther Nucleic Acids, 2022, 28:293-306; the contents of each of which are hereby incorporated by reference in their entirety.
[00112] In some embodiments, the AAV capsid variant, further comprises an amino acid sequence comprising at least one, two or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but not more than 30, 20 or 10 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant, further comprises an amino acid sequence comprising at least one, two or three, but not more than 30, 20 or 10 different amino acids relative to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 138, or an amino acid sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.
[00113] In some embodiments, the AAV capsid variant further comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 137, or a sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto. In some embodiments, the AAV capsid variant further comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one, two or three modifications, e.g., substitutions, insertions, or deletions, but not more than 30, 20 or 10 modifications, e.g., substitutions, insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 137. In some embodiments, the AAV capsid variant further comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one, two or three, but not more than 30, 20 or 10 different nucleotides, relative to the amino acid sequence of SEQ ID NO: 137.
[00114] In some embodiments, the nucleotide sequence encoding the AAV capsid variant further comprises the nucleotide sequence of SEQ ID NO: 137, or a sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto. In some embodiments, the nucleotide sequence encoding the AAV capsid variant further comprises a nucleotide sequence comprising at least one, two or three modifications, e.g., substitutions, insertions, or deletions, but not more than 30, 20 or 10 modifications, e.g., substitutions, insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 137. In some embodiments, the nucleotide sequence encoding the AAV capsid variant further comprises a nucleotide sequence comprising at least one, two or three, but not more than 30, 20 or 10 different nucleotides, relative to the amino acid sequence of SEQ ID NO: 137.
[00115] In some embodiments, an AAV capsid variant of the present disclosure comprises an amino acid sequence as described herein, e.g., an amino acid sequence of an AAV capsid variant of TTN-002, TTN-003, TTN-004, TTN-005, or TTN-006, e.g., as described in Tables 3 and 4. In some embodiments, an AAV capsid variant of the present disclosure comprises an amino acid sequence as described herein, e.g., an amino acid sequence of an AAV capsid variant of TTN-002, e.g., as described in Tables 3 and 4. [00116] In some embodiments, an AAV capsid variant described herein comprises a VP1, VP2, and/or VP3 protein comprising an amino acid sequence described herein, e.g., an amino acid sequence of an AAV capsid variant of TTN-002, TTN-003, TTN-004, TTN-005, or TTN-006, e.g., as described in Tables 3 and 4. In some embodiments, an AAV capsid variant described herein comprises a VP1, VP2, and/or VP3 protein comprising an amino acid sequence described herein, e.g., an amino acid sequence of an AAV capsid variant of TTN-002, e.g., as described in Tables 3 and 4.
[00117] In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence encoded by a nucleotide sequence as described herein, e.g., a nucleotide sequence of an AAV capsid variant of TTN-002, e.g., as described in Tables 3 and 5.
[00118] In some embodiments, a polynucleotide or nucleic acid encoding an AAV capsid variant, of the present disclosure comprises a nucleotide sequence described herein, e.g., a nucleotide sequence of an AAV capsid variant of TTN-002, e.g., as described in Tables 3 and 5.
Table 3. Exemplary full length capsid sequences
Figure imgf000103_0001
Table 4. Exemplary full length capsid amino acid sequences
Figure imgf000103_0002
Figure imgf000104_0001
Figure imgf000105_0001
Table 5. Exemplary full length capsid nucleic acid sequences
Figure imgf000105_0002
Figure imgf000106_0001
[00119] In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 982, or an amino acid sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto. In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 982 or an amino acid sequence with at least 90% sequence identity thereto. In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 982 or an amino acid sequence with at least 95% sequence identity thereto. In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 982 or an amino acid sequence with at least 96% sequence identity thereto. In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 982 or an amino acid sequence with at least 97% sequence identity thereto. In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 982 or an amino acid sequence with at least 98% sequence identity thereto. In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 982 or an amino acid sequence with at least 99% sequence identity thereto. In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence comprising at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but not more than 30, 20 or 10 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SEQ ID NO: 982. In some embodiments, the AAV capsid variant, comprises an amino acid sequence comprising at least one, two or three, but not more than 30, 20 or 10 different amino acids, relative to the amino acid sequence of SEQ ID NO: 982.
[00120] In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 984, or a nucleotide sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto. In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one, two or three, but not more than 30, 20 or 10 different nucleotides, relative to the amino acid sequence of SEQ ID NO: 984. In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one, two or three modifications, e.g., substitutions, insertions, or deletions, but not more than 30, 20 or 10 modifications, e.g., substitutions, insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 984. [00121] In some embodiments, the nucleotide sequence encoding an AAV capsid variant, described herein comprises the nucleotide sequence of SEQ ID NO: 984, or a nucleotide sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto. In some embodiments, the nucleotide sequence encoding an AAV capsid variant described herein comprises the nucleotide sequence of SEQ ID NO: 984, or a nucleotide sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto. In some embodiments, the nucleotide sequence encoding an AAV capsid variant described herein, comprises a nucleotide sequence comprising at least one, two or three modifications, e.g., substitutions, insertions, or deletions, but not more than 30, 20 or 10 modifications, e.g., substitutions, insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 984. In some embodiments, the nucleotide sequence encoding an AAV capsid variant described herein, comprises a nucleotide sequence comprising at least one, two or three, but not more than 30, 20 or 10 different nucleotides, relative to the amino acid sequence of SEQ ID NO: 984. In some embodiments, the nucleic acid sequence encoding an AAV capsid variant described herein is codon optimized.
[00122] In some embodiments, an AAV capsid variant described herein comprises a VP1, VP2, VP3 protein, or a combination thereof. In some embodiments, an AAV capsid variant comprises the amino acid sequence corresponding to positions 137-731, e.g., a VP2, of SEQ ID NO: 982, or a sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto. In some embodiments, the AAV capsid protein comprises the amino acid sequence corresponding to positions 193-731, e.g., a VP3, of SEQ ID NO: 982, or a sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto. In some embodiments, the AAV capsid variant comprises the amino acid sequence corresponding to positions 1-731, e.g., a VP1, of SEQ ID NO: 982, or an amino acid sequence with at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.
[00123] In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 739, or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 738, or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 982, or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto. [00124] In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 739 (e.g., VP3). In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 738 (e.g., VP2). In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 982 (e.g., VP1). [00125] In some embodiments, an AAV capsid variant, described herein has an increased tropism for a CNS cell or tissue, e.g., a brain cell, brain tissue, spinal cord cell, or spinal cord tissue, relative to the tropism of a reference sequence comprising the amino acid sequence of SEQ ID NO: 138.
[00126] In some embodiments, an AAV capsid variant described herein has an increased tropism for a CNS cell or tissue, e.g., a brain cell, brain tissue, spinal cord cell, or spinal cord tissue, relative to the tropism of a reference sequence comprising the amino acid sequence of SEQ ID NO: 982.
[00127] In some embodiments, an AAV capsid variant described herein has an increased tropism for a CNS cell or tissue, e.g., a brain cell, brain tissue, spinal cord cell, or spinal cord tissue, relative to the tropism of a reference sequence comprising the amino acid sequence of SEQ ID NO: 139.
[00128] In some embodiments, an AAV capsid variant described herein transduces a brain region, e.g., a temporal cortex, perirhinal cortex, globus pallidus, putamen, caudate, thalamus, hippocampus, geniculate nucleus, Purkinje Layer, deep cerebellar nuclei, and/or cerebellum. In some embodiments, the level of transduction is at least .5, 2.2, 2.4, 2.5, 2.6, 2.7, 3.0, 3.2, 3.5, 3.7, 4.0, 4.2, 4.5, 4.7, 4.9, 5, 10, 15, 20, 25, 30, or 35-fold greater as compared to a reference sequence of SEQ ID NO: 139.
[00129] In some embodiments, an AAV capsid variant described herein is enriched at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 65-fold in the brain compared to a reference sequence of SEQ ID NO: 138. In some embodiments, an AAV capsid variant described herein is enriched at least about 10, 12, 15, 17, 20, 25, 30, 35, 40, 45, 50, 55, 60, 61, 62, 63, 64, or 65-fold in the brain compared to a reference sequence of SEQ ID NO: 138.
[00130] In some embodiments, an AAV capsid variant described herein is enriched in the brain of at least two to three species, e.g., a non-human primate and rodent (e.g., mouse and/or rat) species, compared to a reference sequence of SEQ ID NO: 138. In some embodiments, an AAV capsid variant described herein is enriched at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100-fold in the brain of at least two to three species, e.g., a non-human primate and rodent (e.g., mouse and/or rat) species, compared to a reference sequence of SEQ ID NO: 138 or 982. In some embodiments, the at least two to three species are Macaca fascicularis, Chlorocebus sabaeus, Callithrix jacchus, rat and/or mouse (e.g., BALB/c mice).
[00131] In some embodiments, an AAV capsid variant described herein is enriched about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, or 225-fold in the brain compared to a reference sequence of SEQ ID NO: 982.
[00132] In some embodiments, an AAV capsid variant described herein delivers an increased level of viral genomes to a brain region. In some embodiments, the level of viral genomes is increased by at least 1.5, 2.2, 2.4, 2.5, 2.6, 2.7, 3.0, 3.2, 3.5, 3.7, 4.0, 4.2, 4.5, 4.7, 4.9, or 5-fold, as compared to a reference sequence of SEQ ID NO: 139. In some embodiments, the brain region comprises a midbrain region (e.g., the hippocampus or thalamus) and/or the brainstem. [00133] In some embodiments, an AAV capsid variant described herein delivers an increased level of a payload to a brain region. In some embodiments, the level of the payload is increased by at least 20, 25, 30, 35-fold, as compared to a reference sequence of SEQ ID NO: 139. In some embodiments, the brain region comprises a temporal cortex, perirhinal cortex, globus pallidus, putamen, caudate, thalamus, hippocampus, geniculate nucleus, Purkinje Layer, deep cerebellar nuclei, cerebellum, or a combination thereof.
[00134] In some embodiments an AAV capsid variant described herein is enriched at least about 3, 3.5, 4.0, 4.5, 5, 5.0, 6.0, or 6.5-fold, in a spinal cord region compared to a reference sequence of SEQ ID NO: 139. In some embodiments, the spinal cord region comprises a cervical spinal cord region, a lumbar spinal cord region, a thoracic spinal cord region, or a combination thereof
[00135] In some embodiments an AAV capsid variant described herein shows preferential transduction in a brain region relative to the transduction in the dorsal root ganglia (DRG). In some embodiments an AAV capsid variant described herein shows preferential transduction in a brain region relative to the transduction in the liver.
[00136] In some embodiments, an AAV capsid variant described herein is capable of transducing neuronal cells.
[00137] In some embodiments, an AAV capsid variant described herein is capable of transducing nonneuronal cells, e.g., glial cells (e.g., oligodendrocytes or astrocytes). In some embodiments, the AAV capsid variant is capable of transducing neuronal cells and non-neuronal cells, e.g., glial cells (e.g., oligodendrocytes or astrocytes). In some embodiments, the non-neuronal cells are glial cells (e.g., oligodendrocytes or astrocytes).
[00138] In some embodiments, an AAV capsid variant described herein has decreased tropism for the liver. In some embodiments, an AAV capsid variant comprises a modification, e.g., substitution, insertion, or deletion, that results in reduced tropism (e.g., de-targeting) and/or activity in the liver. In some embodiments, the reduced tropism in the liver is compared to an otherwise similar capsid that does not comprise the modification, e.g., a wild-type capsid polypeptide. In some embodiments, an AAV capsid variant described comprises a modification, e.g., substitution, insertion, or deletion, that results in one or more of the following properties: (1) reduced tropism in the liver; (2) de -targeted expression in the liver; (3) reduced activity in the liver; and/or (4) reduced binding to galactose. In some embodiments, the reduction in any one, or all of properties (1 )-(3) is compared to an otherwise similar AAV capsid variant that does not comprise the modification. In some embodiments, the AAV capsid variant, e.g., the AAV capsid variant having reduced tropism in the liver, comprises one or more of: an amino acid other than A, G, K, M, N, Q, R, S, and/or T at position 581; an amino acid other than A, C, H, I, K, S, T, and/or V at position 582; an amino acid other than A, G, H, K, M, N, Q, R, S, T, and/or V at position 583; an amino acid other than L, M, P, Q, R. T and/or W at position 584; an amino acid other than F, H, I, K, M, T and/or Y at position 585; an amino acid other than E, G, H, L, M, N, Q, T, and/or W at position 586; an amino acid other than A, C, G, H, L, M, R, and/or S at position 587; an amino acid other than A, C, D, F, G, H, M, Q, S, V, W, and/or Y at position 588; and/or an amino acid other than A, C, E, G, H, M, N, P, Q, S, V, and/or W at position 589, all numbered relative to SEQ ID NO: 138.
[00139] In some embodiments, an AAV capsid variant of the present disclosure is isolated, e.g., recombinant. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, of the present disclosure is isolated, e.g., recombinant.
[00140] Also provided herein are polynucleotide sequences encoding any of the AAV capsid variants described above and AAV particles, vectors, and cells comprising the same.
[00141] In some embodiments, an, AAV capsid variant of the present disclosure is isolated, e.g., recombinant. In some embodiments, a polynucleotide encoding an AAV capsid variant of the present disclosure is isolated, e.g., recombinant.
[00142] The present disclosure refers to structural capsid proteins (including VP1, VP2 and VP3) which are encoded by capsid (Cap) genes. These capsid proteins form an outer protein structural shell (i.e., capsid) of a viral vector such as AAV. VP capsid proteins synthesized from Cap polynucleotides generally include a methionine as the first amino acid in the peptide sequence (Metl), which is associated with the start codon (AUG or ATG) in the corresponding Cap nucleotide sequence. However, it is common for a first-methionine (Metl) residue or generally any first amino acid (AA1) to be cleaved off after or during polypeptide synthesis by protein processing enzymes such as Met-aminopeptidases. This “Met/AA-clipping” process often correlates with a corresponding acetylation of the second amino acid in the polypeptide sequence (e.g., alanine, valine, serine, threonine, etc.). Met-clipping commonly occurs with VP1 and VP3 capsid proteins but can also occur with VP2 capsid proteins.
[00143] Where the Met/AA-clipping is incomplete, a mixture of one or more (one, two or three) VP capsid proteins comprising the viral capsid may be produced, some of which may include a Metl/AAl amino acid (Met+/AA+) and some of which may lack a Metl/AAl amino acid as a result of Met/AA- clipping (Met-/AA-). For further discussion regarding Met/AA-clipping in capsid proteins, see Jin, et al. Direct Liquid Chromatography/Mass Spectrometry Analysis for Complete Characterization of Recombinant Adeno- Associated Virus Capsid Proteins. Hum Gene Ther Methods. 2017 Oct. 28(5):255- 267; Hwang, et al. N-Terminal Acetylation of Cellular Proteins Creates Specific Degradation Signals. Science. 2010 February 19. 327(5968): 973-977; the contents of which are each incorporated herein by reference in their entirety.
[00144] According to the present disclosure, references to capsid proteins is not limited to either clipped (Met-/AA-) or unclipped (Met+/AA+) and may, in context, refer to independent capsid proteins, viral capsids comprised of a mixture of capsid proteins, and/or polynucleotide sequences (or fragments thereof) which encode, describe, produce or result in capsid proteins of the present disclosure. A direct reference to a “capsid protein” or “capsid polypeptide” (such as VP1, VP2 or VP2) may also comprise VP capsid proteins which include a Metl/AAl amino acid (Met+/AA+) as well as corresponding VP capsid proteins which lack the Metl/AAl amino acid as a result of Met/AA-clipping (Met-/AA-). [00145] Further according to the present disclosure, a reference to a specific “SEQ ID NO:” (whether a protein or nucleic acid) which comprises or encodes, respectively, one or more capsid proteins which include a Metl/AAl amino acid (Met+/AA+) should be understood to teach the VP capsid proteins which lack the Metl/AAl amino acid as upon review of the sequence, it is readily apparent any sequence which merely lacks the first listed amino acid (whether or not Metl/AAl).
[00146] As a non-limiting example, reference to a VP1 polypeptide sequence which is 736 amino acids in length and which includes a “Metl” amino acid (Met+) encoded by the AUG/ATG start codon may also be understood to teach a VP1 polypeptide sequence which is 735 amino acids in length and which does not include the “Metl” amino acid (Met-) of the 736 amino acid Met+ sequence. As a second nonlimiting example, reference to a VP1 polypeptide sequence which is 736 amino acids in length and which includes an “AA1 ” amino acid (AA1 +) encoded by any NNN initiator codon may also be understood to teach a VP1 polypeptide sequence which is 735 amino acids in length and which does not include the “AA1” amino acid (AA1-) of the 736 amino acid AA1+ sequence.
[00147] References to viral capsids formed from VP capsid proteins (such as reference to specific AAV capsid serotypes), can incorporate VP capsid proteins which include a Metl/AAl amino acid (Met+/AA1+), corresponding VP capsid proteins which lack the Metl/AAl amino acid as a result of Mct/AAl -clipping (Mct-/AA1-), and combinations thereof (Mct+/AA1+ and Mct-/AA1-).
[00148] As a non-limiting example, an AAV capsid serotype can include VP1 (Met+/AA1+), VP1 (Met- /AA1-), or a combination of VP1 (Met+/AA1+) and VP1 (Met-/AA1-). An AAV capsid serotype can also include VP3 (Met+/AA1+), VP3 (Met-/AA1-), or a combination of VP3 (Met+/AA1+) and VP3 (Met-/AA1-); and can also include similar optional combinations of VP2 (Met+/AA1) and VP2 (Met- /AA1-).
[00149] Also provided herein are polynucleotide sequences encoding any of the AAV capsid variants described above and AAV particles, vectors, and cells comprising the same.
AAV Viral Genome
[00150] In some aspects, the AAV particle of the present disclosure serves as an expression vector comprising a viral genome which encodes an SMN protein. In some embodiments, expression vectors are not limited to AAV and may be adenovirus, retrovirus, lentivirus, plasmid, vector, or any variant thereof. [00151] In some embodiments, an AAV particle, e.g., an AAV particle for the vectorized delivery of an SMN protein described herein, comprises a viral genome, e.g., an AAV viral genome (e.g., a vector genome or AAV vector genome). In some embodiments, the viral genome, e.g., the AAV viral genome, further comprises an inverted terminal repeat (ITR) region, an enhancer, a promoter, an intron region, a Kozak sequence, an exon region, a nucleic acid encoding a transgene encoding a payload (e.g., an SMN protein described herein) with or without an enhancement element, a nucleotide sequence encoding a miR binding site (e.g., a miR183 binding site), a poly A signal region, or a combination thereof. Viral Genome Component: Inverted Terminal Repeats (ITRs)
[00152] In some embodiments, the viral genome may comprise at least one inverted terminal repeat (ITR) region. The AAV particles of the present disclosure comprise a viral genome with at least one ITR region and a payload region. In some embodiments, the viral genome has two ITRs. These two ITRs flank the payload region at the 5’ and 3’ ends. In some embodiments, the ITR functions as an origin of replication comprising a recognition site for replication. In some embodiments, the ITR comprises a sequence region which can be complementary and symmetrically arranged. In some embodiments, the ITR incorporated into a viral genome described herein may be comprised of a naturally occurring polynucleotide sequence or a recombinantly derived polynucleotide sequence.
[00153] The ITRs may be derived from the same serotype as the capsid, selected from any of the serotypes listed in Table 1, or a derivative thereof. The ITR may be of a different serotype than the capsid. In some embodiments, the AAV particle has more than one ITR. In a non-limiting example, the AAV particle has a viral genome comprising two ITRs. In some embodiments, the ITRs are of the same serotype as one another. In another embodiment, the ITRs are of different serotypes. Non-limiting examples include zero, one or both of the ITRs having the same serotype as the capsid. In some embodiments both ITRs of the viral genome of the AAV particle are AAV2 ITRs. In some embodiments, each ITR may be 141 nucleotides in length. In some embodiments, each ITR may be 130 nucleotides in length.
[00154] In some embodiments, the ITR comprises the nucleotide sequence of SEQ ID NO: 1 or 2, or a nucleotide sequence substantially identical (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical) to any of the aforesaid sequences. In some embodiments, the ITR comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a nucleotide sequence having one, two, or three but no more than four modifications, e.g., substitutions, relative to SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, a viral genome encoding an SMN protein described herein comprises an ITR comprising the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence substantially identical (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical), which is present 5’ relative to the nucleotide sequence encoding the SMN protein. In some embodiments, a viral genome encoding an SMN protein described herein comprises an ITR comprising the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence substantially identical (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical), thereto which is present 3’ relative to the nucleotide sequence encoding the SMN protein.
Table 6. Exemplary ITR sequences
Figure imgf000112_0001
Viral Genome Component: Promoters and Expression Enhancers
[00155] In some embodiments, the payload region of the viral genome comprises at least one element to enhance the transgene target specificity and expression. See, e.g., Powell et al. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, 2015; the contents of which are herein incorporated by reference in their entirety. Non-limiting examples of elements to enhance the transgene target specificity and expression include promoters, endogenous miRNAs, post- transcriptional regulatory elements (PREs), polyadenylation (Poly A) signal sequences, upstream enhancers (USEs), CMV enhancers, and introns.
[00156] In some embodiments, expression of the polypeptides in a target cell may be driven by a promoter, including but not limited to, a promoter that is species specific, inducible, tissue-specific, ubiquitous, or cell cycle-specific (e.g., as described in Parr et al., Nat. Med. '.1145-9 (1997); the contents of which are herein incorporated by reference in their entirety).
[00157] In some embodiments, the viral genome comprises a that is sufficient for expression, e.g., in a target cell, of a payload (e.g., an SMN protein) encoded by a transgene. In some embodiments, the promoter is deemed to be efficient when it drives expression of the polypeptide(s) encoded in the payload region of the viral genome of the AAV particle. In some embodiments, the promoter is a promoter deemed to be efficient when it drives expression in the cell or tissue being targeted.
[00158] Promoters may be naturally occurring or non-naturally occurring. Non-limiting examples of promoters include viral promoters, plant promoters and mammalian promoters. In some embodiments, the promoters may be human promoters. In some embodiments, the promoter may be truncated.
[00159] In some embodiments, the viral genome comprises a promoter that results in expression in one or more, e.g., multiple, cells and/or tissues, e.g., a ubiquitous promoter. In some embodiments, a promoter which drives or promotes expression in most mammalian tissues includes, but is not limited to, human elongation factor la-subunit (EFla), cytomegalovirus (CMV) immediate-early enhancer and/or promoter, chicken 0-actin (CBA) and its derivative CAG, glucuronidase (GUSB), and ubiquitin C (UBC). Tissue-specific expression elements can be used to restrict expression to certain cell types such as, but not limited to, CNS-specific promoters, B cell promoters, monocyte promoters, leukocyte promoters, macrophage promoters, pancreatic acinar cell promoters, endothelial cell promoters, lung tissue promoters, astrocyte promoters, or various specific nervous system cell- or tissue-type promoters which can be used to restrict expression to neurons, astrocytes, or oligodendrocytes, for example.
[00160] In some embodiments, the viral genome comprises a nervous system specific promoter, e.g., a promoter that results in expression of a payload in a neuron, an astrocyte, and/or an oligodendrocyte. Non-limiting examples of tissue-specific expression elements for neurons include neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B-chain (PDGF-0), synapsin (Syn), synapsin 1 (Synl), methyl-CpG binding protein 2 (MeCP2), Ca2+/calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), neurofilament light (NFL) or heavy (NFH), -globin minigene n [ 2, preproenkephalin (PPE), enkephalin (Enk) and excitatory amino acid transporter 2 (EAAT2) promoters. Non-limiting examples of tissue-specific expression elements for astrocytes include glial fibrillary acidic protein (GFAP) and EAAT2 promoters. A non-limiting example of a tissue-specific expression element for oligodendrocytes includes the myelin basic protein (MBP) promoter. Prion promoter represents an additional tissue specific promoter useful for driving protein expression in CNS tissue (see Loftus, Stacie K., et al. Human molecular genetics 11.24 (2002): 3107- 3114, the disclosure of which is incorporated by reference in its entirety).
[00161] In some embodiments, the viral genome comprises a ubiquitous promoter. Non-limiting examples of ubiquitous promoters include CMV, CBA (including derivatives CAG, CB6, CBh, etc.), EFla, PGK, UBC, GUSB (hGBp), and UCOE (promoter of HNRPA2B1-CBX3). In some embodiments, the viral genome comprises an EF-lapromoter or EF-la promoter variant, e.g., as provided in Table 7. In some embodiments, the EF-lapromoter comprises the nucleotide sequence of any one of SEQ ID NOs: 26-35 or 987, 988, 990, 991, 995, 996, 998-1007, or any one of the nucleotide sequences in Table 7, a nucleotide sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions, relative to the nucleotide sequence of SEQ ID NOs: 26-35 or 987-1007, or a nucleotide sequence with at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 26-35 or 995, 996, 998-1007, or any one of the nucleotide sequences in Table 7.
Table 7. Exemplary promoter sequences and variants thereof
Figure imgf000114_0001
Figure imgf000115_0001
Figure imgf000116_0001
Figure imgf000117_0001
[00162] In some embodiments, the promoter is a ubiquitous promoter. In some embodiments, the ubiquitous promoter is as described in Yu et al. (Molecular Pain 2011, 7:63), Soderblom et al. (E. Neuro 2015), Gill et al., (Gene Therapy 2001, Vol. 8, 1539-1546), and Husain et al. (Gene Therapy 2009), each of which are incorporated by reference in their entirety. In some embodiments, a viral genome encoding an SMN protein described herein comprises a ubiquitous promoter, e.g., an EF- la promoter, an insulin promoter, a PGK promoter, a CBA promoter, or a functional variant, e.g., truncation, thereof.
[00163] In some embodiments, the promoter is a tissue specific promoter. In some embodiments, the promoter is a cell specific promoter. In some embodiments, a viral genome encoding an SMN protein described herein comprises a neuron-specific promoter, e.g., a synapsin promoter, an Hb9 promoter, an MeCP2 promoter, or a functional variant thereof. In some embodiments the promoter is a motor neuron specific promoter, e.g., an Hb9 promoter or a functional variant thereof.
[00164] In some embodiments, the promoter is not cell specific.
[00165] In some embodiments, the promoter is a ubiquitin c (UBC) promoter. In some embodiments, the promoter is a 0-glucuronidase (GUSB) promoter. In some embodiments, the promoter is a neurofilament light (NFL) promoter. In some embodiments, the promoter is a neurofilament heavy (NFH) promoter. In some embodiments, the promoter is a scn8a promoter.
[00166] In some embodiments, the promoter is a phosphoglycerate kinase 1 (PGK) promoter, or a functional variant thereof. [00167] In some embodiments, the promoter is a synapsin promoter or a functional variant thereof. [00168] In some embodiments, the promoter is a MeCP2 promoter or a functional variant thereof. [00169] In some embodiments, the promoter is a chicken P-actin (CBA) promoter, or a functional variant thereof.
[00170] In some embodiments, the promoter is a CB6 promoter, or a functional variant thereof.
[00171] In some embodiments, the promoter is a CB promoter, or a functional variant thereof . In some embodiments, the promoter is a minimal CB promoter, or a functional variant thereof.
[00172] In some embodiments, the promoter is a CBA promoter, or functional variant thereof. In some embodiments, the promoter is a minimal CBA promoter, or functional variant thereof.
[00173] In some embodiments, the promoter is a cytomegalovirus (CMV) promoter, or a functional variant thereof.
[00174] In some embodiments, the promoter is a CAG promoter, or a functional variant thereof.
[00175] In some embodiments, the promoter is an EFla promoter or functional variant thereof, e.g., a truncated EFla promoter.
[00176] In some embodiments, the promoter is a GFAP promoter (e.g., as described, for example, in Zhang, Min, et al. Journal of neuroscience research 86.13 (2008): 2848-2856, the disclosure of which is incorporated by reference in its entirety) to drive expression of an SMN polypeptide, or an SMN polypeptide and a splicing modulator (e.g., SMN and a UlsnRNA, SMN and an antisense molecule)) in astrocytes.
[00177] In some embodiments, the promoter is an endogenous promoter, e.g., endogenous SMN1 (e.g., human SMN1) promoter. In some embodiments, the promoter is an SMN promoter, e.g., an SMN1 promoter. In some embodiments the promoter is an SMN1 promoter.
[00178] In some embodiments, the promoter is an RNA pol III promoter. As a non-limiting example, the RNA pol III promoter is U6. As a non-limiting example, the RNA pol HI promoter is Hl.
[00179] In some embodiments, the viral genome comprises two promoters. As a non-limiting example, the promoters are an EFl a promoter and a CMV promoter.
[00180] In some embodiments, the viral genome comprises an enhancer element, a promoter and/or a 5’UTR intron. The enhancer element may be, but is not limited to, a CMV enhancer, the promoter may be, but is not limited to, a CMV, CBA, UBC, GUSB, NSE, Synapsin, MeCP2, and GFAP promoter and the 5’UTR/intron may be, but is not limited to, SV40, and CBA-MVM. As a non-limiting example, the enhancer, promoter and/or intron used in combination may be: (1) CMV enhancer, CMV promoter, SV40 5’UTR intron; (2) CMV enhancer, CBA promoter, SV 40 5’UTR intron; (3) CMV enhancer, CBA promoter, CBA-MVM 5’UTR intron; (4) UBC promoter; (5) GUSB promoter; (6) NSE promoter; (7) Synapsin promoter; (8) MeCP2 promoter; and (9) GFAP promoter.
[00181] In some embodiments, the viral genome comprises an enhancer. In some embodiments, the enhancer comprises a CMVie enhancer.
[00182] In some embodiments the viral genome comprises a CMVie enhancer and a CB promoter. In some embodiments, the viral genome comprises a CMVie enhancer and a CMV promoter (e.g., a CMV promoter region). In some embodiments, the viral genome comprises a CMVie enhancer, a CBA promoter or functional variant thereof, and an intron (e.g., a CAG promoter).
[00183] In some embodiments, the viral genome comprises an engineered promoter. In another embodiments, the viral genome comprises a promoter from a naturally expressed protein.
Viral Genome Component: Introns
[00184] In some embodiments, the vector genome comprises at least one intron or a fragment or derivative thereof. In some embodiments, the at least one intron may enhance expression of an SMN protein and/or an splicing modulator or variant thereof Non-limiting examples of introns include, MVM (67-97 bps), F.IX truncated intron 1 (300 bps), P-globin SD/immunoglobulin heavy chain splice acceptor (250 bps), adenovirus splice donor/immunoglobin splice acceptor (500 bps), SV40 late splice donor/splice acceptor (19S/16S) (180 bps), and hybrid adenovirus splice donor/IgG splice acceptor (230 bps).
[00185] In some embodiments, the viral genome, e.g., a viral genome encoding an SMN protein described herein, may comprise an SV40 intron or fragment or variant thereof.
[00186] In some embodiments, the viral genome, e.g., a viral genome encoding an SMN protein described herein, may comprise a beta-globin intron or a fragment or variant thereof. In some embodiments, the intron comprises one or more human beta-globin sequences (e.g., including fragments/variants thereof).
[00187] In some embodiments, a viral genome, e.g., a viral genome encoding an SMN protein described herein, comprises a chimeric intron or functional variant thereof. In some embodiments, the viral genome, e.g., a viral genome encoding an SMN protein described herein comprises an intron comprising the nucleotide sequence of SEQ ID NO: 3, a nucleotide sequence substantially identical (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical) to SEQ ID NO: 3, a nucleotide sequence comprising one, two, or three but no more than four modifications, e.g., substitutions, relative to the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence comprising one, two, or three but no more than four different nucleotides relative to the nucleotide sequence of SEQ ID NO: 3.
Table 8. Exemplary intron sequences
Figure imgf000119_0001
Viral Genome Component: Untranslated Regions (UTRs)
[00188] In some embodiments, a wild type untranslated region (UTR) of a gene is transcribed but not translated. Generally, the 5’ UTR starts at the transcription start site and ends at the start codon and the 3’ UTR starts immediately following the stop codon and continues until the termination signal for transcription.
[00189] Features typically found in abundantly expressed genes of specific target organs may be engineered into UTRs to enhance the stability and protein production. As a non-limiting example, a 5’ UTR from mRNA normally expressed in the liver (e.g. , albumin, serum amyloid A, Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, or Factor VIII) may be used in the viral genomes of the AAV particles of the disclosure to enhance expression in hepatic cell lines or liver.
[00190] In some embodiments, the viral genome encoding a transgene described herein (e.g., a transgene encoding an SMN protein) comprises a Kozak sequence. While not wishing to be bound by theory, wild- type 5' untranslated regions (UTRs) include features that play roles in translation initiation. Kozak sequences, which are commonly known to be involved in the process by which the ribosome initiates translation of many genes, are usually included in 5’ UTRs. Kozak sequences have the consensus CCR(A/G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (ATG), which is followed by another ’G’.
[00191] In some embodiments, the 5’ UTR in the viral genome includes a Kozak sequence.
[00192] In some embodiments, the 5’ UTR in the viral genome does not include a Kozak sequence. [00193] While not wishing to be bound by theory, wild-type 3' UTRs are known to have stretches of adenosines and uridines embedded therein. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al, 1995, the contents of which are herein incorporated by reference in their entirety): Class I AREs, such as, but not limited to, c-Myc and MyoD, contain several dispersed copies of an AUUUA motif within U-rich regions. Class II AREs, such as, but not limited to, GM-CSF and TNF-a, possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Class 111 ARES, such as, but not limited to, c-Jun and Myogenin, are less well defined. These U rich regions do not contain an AUUUA motif. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo.
[00194] Introduction, removal or modification of 3' UTR AU rich elements (AREs) can be used to modulate the stability of polynucleotides. When engineering specific polynucleotides, e.g., payload regions of viral genomes, one or more copies of an ARE can be introduced to make polynucleotides less stable and thereby curtail translation and decrease production of the resultant protein. Likewise, AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein.
[00195] In some embodiments, the 3’ UTR of the viral genome may include an oligo(dT) sequence for templated addition of a poly-A tail.
[00196] Any UTR from any gene known in the art may be incorporated into the viral genome of the AAV particle. These UTRs, or portions thereof, may be placed in the same orientation as in the gene from which they were selected or they may be altered in orientation or location. In some embodiments, the UTR used in the viral genome of the AAV particle may be inverted, shortened, lengthened, or made with one or more other 5' UTRs or 3' UTRs known in the art. As used herein, the term “altered,” as it relates to a UTR, means that the UTR has been changed in some way in relation to a reference sequence. For example, a 3' or 5' UTR may be altered relative to a wild type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides.
[00197] In some embodiments, the viral genome of the AAV particle comprises at least one artificial UTR, which is not a variant of a wild type UTR.
[00198] In some embodiments, the viral genome of the AAV particle comprises UTRs which have been selected from a family of transcripts whose proteins share a common function, structure, feature, or property.
Viral Genome Component: miR Binding Site
[00199] Tissue- or cell-specific expression of the AAV viral particles of the invention can be enhanced by introducing tissue- or cell-specific regulatory sequences, e.g., promoters, enhancers, microRNA binding sites, e.g., a detargeting site. Without wishing to be bound by theory, it is believed that an encoded miR binding site can modulate, e.g., prevent, suppress, or otherwise inhibit, the expression of a gene of interest on the viral genome of the invention, based on the expression of the corresponding endogenous microRNA (miRNA) or a corresponding controlled exogenous miRNA in a tissue or cell, e.g., a non-targeting cell or tissue. In some embodiments, a miR binding site modulates, e.g., reduces, expression of the payload encoded by a viral genome of an AAV particle described herein in a cell or tissue where the corresponding mRNA is expressed. In some embodiments, the miR binding site modulates, e.g., reduces, expression of the encoded SMN protein in a cell or tissue of the DRG, liver, hematopoietic lineage, or a combination thereof.
[00200] In some embodiments, the viral genome of an AAV particle described herein comprises a nucleotide sequence encoding a microRNA binding site, e.g., a detargeting site. In some embodiments, the viral genome of an AAV particle described herein comprises a nucleotide sequence encoding a miR binding site, a microRNA binding site series (miR BSs), or a reverse complement thereof.
[00201] In some embodiments, the nucleotide sequence encoding the miR binding site series or the miR binding site is located in the 3’-UTR region of the viral genome (e.g., 3’ relative to the nucleic acid sequence encoding a payload), e.g., before the polyA sequence, 5’ -UTR region of the viral genome (e.g., 5’ relative to the nucleic acid sequence encoding a payload), or both. [00202] In some embodiments, the encoded miR binding site series comprise at least 1-5 copies, e.g., at least 1-3, 2-4, 3-5, 1, 2, 3, 4, 5 or more copies of a miR binding site (miR BS). In some embodiments, the encoded miR binding site series comprises 4 copies of a miR binding site. In some embodiments, all copies are identical, e.g., comprise the same miR binding site. In some embodiments, the miR binding sites within the encoded miR binding site series are continuous and not separated by a spacer. In some embodiments, the miR binding sites within an encoded miR binding site series are separated by a spacer, e.g. , a non-coding sequence. In some embodiments, the spacer is about 1 to 6 nucleotides or about 5 to 10 nucleotides, e.g., about 7-8 nucleotides, nucleotides in length. In some embodiments, the spacer is about 8 nucleotides in length. In some embodiments, the spacer sequence comprises one or more of (i) GGAT; (ii) CACGTG; (iii) GCATGC, or a repeat of one or more of (i)-(iii). In some embodiments, the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, but no more than four modifications of GATAGTTA.
[00203] In some embodiments, the encoded miR binding site series comprise at least 1 -5 copies, e.g., at least 1-3, 2-4, 3-5, 1, 2, 3, 4, 5 or more copies of a miR binding site (miR BS). In some embodiments, at least 1, 2, 3, 4, 5, or all of the copies are different, e.g., comprise a different miR binding site. In some embodiments, the miR binding sites within the encoded miR binding site series are continuous and not separated by a spacer. In some embodiments, the miR binding sites within an encoded miR binding site series are separated by a spacer, e.g., a non-coding sequence. In some embodiments, the spacer is about 1 to 6 nucleotides or about 5 to 10 nucleotides, e.g., about 7-8 nucleotides or about 8 nucleotides, in length. In some embodiments, the spacer sequence comprises one or more of (i) GGAT; (ii) CACGTG; (iii) GCATGC, or a repeat of one or more of (i)-(iii). In some embodiments, the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, but no more than four modifications of GATAGTTA.
[00204] In some embodiments, the encoded miR binding site is substantially identical (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical), to the miR in the host cell. In some embodiments, the encoded miR binding site comprises at least 1, 2, 3, 4, or 5 mismatches or no more than 6, 7, 8, 9, or 10 mismatches to a miR in the host cell. In some embodiments, the mismatched nucleotides are contiguous. In some embodiments, the mismatched nucleotides are non-contiguous. In some embodiments, the mismatched nucleotides occur outside the seed region- binding sequence of the miR binding site, such as at one or both ends of the miR binding site. In some embodiments, the encoded miR binding site is 100% identical to the miR in the host cell.
[00205] In some embodiments, the nucleotide sequence encoding the miR binding site is substantially complimentary (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% complementary), to the miR in the host cell. In some embodiments, the sequence complementary to the nucleotide sequence encoding the miR binding site comprises at least 1, 2, 3, 4, or 5 mismatches or no more than 6, 7, 8, 9, or 10 mismatches relative to the corresponding miR in the host cell. In some embodiments, the mismatched nucleotides are contiguous. In some embodiments, the mismatched nucleotides are non-contiguous. In some embodiments, the mismatched nucleotides occur outside the seed region-binding sequence of the miR binding site, such as at one or both ends of the miR binding site. In some embodiments, the encoded miR binding site is 100% complementary to the miR in the host cell.
[00206] In some embodiments, the encoded miR binding site or the encoded miR binding site series is about 10 to about 125 nucleotides in length, e.g., about 10 to 50 nucleotides, 10 to 100 nucleotides, 50 to 100 nucleotides, 50 to 125 nucleotides, or 100 to 125 nucleotides in length. In some embodiments, an encoded miR binding site or the encoded miR binding site series is about 7 to about 28 nucleotides in length, e.g., about 8-28 nucleotides, 7-28 nucleotides, 8-18 nucleotides, 12-28 nucleotides, 20-26 nucleotides, 22 nucleotides, 24 nucleotides, or 26 nucleotides in length, and optionally comprises at least one consecutive region (e.g., 7 or 8 nucleotides) complementary (e.g., full complementary or partially complementary) to the seed sequence of a miRNA (e.g., a miR122, a miR142, a miR-1, a miR183). [00207] In some embodiments, the encoded miR binding site is complementary (e.g., fully complementary or partially complementary) to a miR expressed in liver or hepatocytes, such as miR122. In some embodiments, the encoded miR binding site or encoded miR binding site series comprises a miR122 binding site sequence. In some embodiments, the encoded miR122 binding site comprises the nucleotide sequence of ACAAACACCATTGTCACACTCCA (SEQ ID NO: 1865), or a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95%, at least 99%, or 100% sequence identity, or having at least one, two, three, four, five, six, or seven modifications but no more than ten modifications to SEQ ID NO: 1865, e.g., wherein the modification can result in a mismatch between the encoded miR binding site and the corresponding miRNA. In some embodiments, the viral genome comprises at least 3, 4, or 5 copies of the encoded miR122 binding site, e.g., an encoded miR122 binding site series, optionally wherein the encoded miR122 binding site series comprises the nucleotide sequence of:
AC AAAC AC C AT T G T C AC AC T C C C AC AAAC AC C AT T G T C AC AC T C C C AC AAAC AC C AT T G T C AC AC T C C A (SEQ ID NO: 1866), or a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95%, at least 99%, or 100% sequence identity, or having at least one, two, three, four, five, six, or seven modifications but no more than ten modifications to SEQ ID NO: 1866, e.g., wherein the modification can result in a mismatch between the encoded miR binding site and the corresponding miRNA. In some embodiments, at least two of the encoded miR122 binding sites are connected directly, e.g., without a spacer. In other embodiments, at least two of the encoded miR122 binding sites are separated by a spacer, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length, which is located between two or more consecutive encoded miR122 binding site sequences. In embodiments, the spacer is about 1 to 6 nucleotides or about 5 to 10 nucleotides, e.g., about 7-8 nucleotides or about 8 nucleotides, in length. In some embodiments, the spacer sequence comprises one or more of (i) GGAT; (ii) CACGTG; (iii) GCATGC, or a repeat of one or more of (i)-(iii). In some embodiments, the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, but no more than four modifications of GATAGTTA.
[00208] In some embodiments, the encoded niiR binding site is complementary (e.g., fully or partially complementary) to a miR expressed in the heart. In embodiments, the encoded miR binding site or encoded miR binding site series comprises a miR-1 binding site. In some embodiments, the encoded miR- 1 binding site comprises the nucleotide sequence of ATACATACTTCTTTACATTCCA (SEQ ID NO: 4679), a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95%, at least 99%, or 100% sequence identity, or having at least one, two, three, four, five, six, or seven modifications but no more than ten modifications to SEQ ID NO: 4679, e.g., wherein the modification can result in a mismatch between the encoded miR binding site and the corresponding miRNA. In some embodiments, the viral genome comprises at least 2, 3, 4, or 5 copies of the encoded miR-1 binding site, e.g., an encoded miR-1 binding site series. In some embodiments, the at least 2, 3, 4, or 5 copies (e.g., 2 or 3 copies) of the encoded miR-1 binding site are continuous (e.g., not separated by a spacer) or separated by a spacer. In some embodiments, the spacer is about 1 to 6 nucleotides or about 5 to 10 nucleotides, e.g., about 7-8 nucleotides or about 8 nucleotides, in length. In some embodiments, the spacer sequence comprises one or more of (i) GGAT; (ii) CACGTG; (iii) GCATGC, or a repeat of one or more of (i)-(iii). In some embodiments, the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, but no more than four modifications of GATAGTTA.
[00209] In some embodiments, the encoded miR binding site is complementary (e.g., fully complementary or partially complementary) to a miR expressed in hematopoietic lineage, including immune cells (e.g., antigen presenting cells or APC, including dendritic cells (DCs), macrophages, and B-lymphocytes). In some embodiments, the encoded miR binding site is complementary (e.g., fully complementary or partially complementary) to a miR expressed in hematopoietic lineage comprises a nucleotide sequence disclosed, e.g., in US 2018/0066279, the contents of which are incorporated by reference herein in its entirety.
[00210] In some embodiments, the encoded miR binding site or encoded miR binding site series comprises a miR-142-3p binding site sequence. In some embodiments, the encoded miR-142-3p binding site comprises the nucleotide sequence of TCCATAAAGTAGGAAACACTACA (SEQ ID NO: 1869), a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95%, at least 99%, or 100% sequence identity, or having at least one, two, three, four, five, six, or seven modifications but no more than ten modifications to SEQ ID NO: 1842, e.g., wherein the modification can result in a mismatch between the encoded miR binding site and the corresponding miRNA. In some embodiments, the viral genome comprises at least 3, 4, or 5 copies of an encoded miR-142-3p binding site, e.g., an encoded miR-142-3p binding site series. In some embodiments, the at least 3, 4, or 5 copies (e.g., 4 copies) of the encoded miR-142-3p binding site are continuous (e.g., not separated by a spacer) or separated by a spacer. In some embodiments, the spacer is about 1 to 6 nucleotides or about 5 to 10 nucleotides, e.g., about 7-8 nucleotides or about 8 nucleotides, in length. In some embodiments, the spacer sequence comprises one or more of (i) GGAT; (ii) CACGTG; (iii) GCATGC, or a repeat of one or more of (i)-(iii). In some embodiments, the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, but no more than four modifications of GATAGTTA.
[00211] In some embodiments, the encoded miR binding site is complementary (e.g., fully complementary or partially complementary) to a miR expressed in a DRG (dorsal root ganglion) neuron, e.g., a miR183, a miR182, and/or miR96 binding site. In some embodiments, the encoded miR binding site is complementary (e.g., fully complementary or partially complementary) to a miR expressed in expressed in a DRG neuron. In some embodiments, the encoded miR binding site comprises a nucleotide sequence disclosed, e.g., in W02020/132455, the contents of which are incorporated by reference herein in its entirety.
[00212] In some embodiments, the encoded miR binding site or encoded miR binding site series comprises a miR183 binding site sequence. In some embodiments, the encoded miR183 binding site comprises the nucleotide sequence of AG T GAAT T C T AC C AG T G C CAT A (SEQ ID NO: 1847), or a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95%, at least 99%, or 100% sequence identity, or having at least one, two, three, four, five, six, or seven modifications but no more than ten modifications to SEQ ID NO: 1847, e.g., wherein the modification can result in a mismatch between the encoded miR binding site and the corresponding miRNA. In some embodiments, the sequence complementary (e.g., fully complementary or partially complementary) to the seed sequence corresponds to the double underlined of the encoded miR- 183 binding site sequence. In some embodiments, the viral genome comprises at least comprises at least 3, 4, or 5 copies (e.g., 4 copies) of the encoded miR183 binding site, e.g., an encoded miR183 binding site. In some embodiments, the viral genome comprises at least comprises 4 copies of the encoded miR183 binding site, e.g., an encoded miR183 binding site comprising 4 copies of a miR183 binding site. In some embodiments, the at least 3, 4, or 5 copies (e.g., 4 copies) of the encoded miR183 binding site are continuous (e.g., not separated by a spacer) or separated by a spacer. In some embodiments, the spacer is about 1 to 6 nucleotides or about 5 to 10 nucleotides, e.g., about 7-8 nucleotides or about 8 nucleotides, in length. In some embodiments, the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, but no more than four modifications of GATAGTTA. In some embodiments, the encoded miRl 83 binding site series comprises the nucleotide sequence of SEQ ID NO: 1847, or a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95%, at least 99%, or 100% sequence identity, or having at least one, two, three, four, five, six, or seven modifications but no more than ten modifications to SEQ ID NO: 1847. [00213] In some embodiments, the encoded miR binding site or encoded miR binding site series comprises a miR182 binding site sequence. In some embodiments, the encoded miR182 binding site comprises, the nucleotide sequence of AGTGTGAGTTCTACCATTGCCAAA (SEQ ID NO: 1867), a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95%, at least 99%, or 100% sequence identity, or having at least one, two, three, four, five, six, or seven modifications but no more than ten modifications to SEQ ID NO: 1867, e.g., wherein the modification can result in a mismatch between the encoded miR binding site and the corresponding miRNA. In some embodiments, the viral genome comprises at least 3, 4, or 5 copies of the encoded miR182 binding site, e.g., an encoded miR182 binding site series. In some embodiments, the at least 3, 4, or 5 copies (e.g., 4 copies) of the encoded miR182 binding site are continuous (e.g., not separated by a spacer) or separated by a spacer. In some embodiments, the spacer is about 1 to 6 nucleotides or about 5 to 10 nucleotides, e.g., about 7-8 nucleotides or about 8 nucleotides, in length. In some embodiments, the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, but no more than four modifications of GATAGTTA.
[00214] In some embodiments, the encoded miR binding site or encoded miR binding site series comprises a miR96 binding site sequence. In some embodiments, the encoded miR96 binding site comprises the nucleotide sequence of AGCAAAAATGTGCTAGTGCCAAA (SEQ ID NO: 1868), a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95%, at least 99%, or 100% sequence identity, or having at least one, two, three, four, five, six, or seven modifications but no more than ten modifications to SEQ ID NO: 1868, e.g., wherein the modification can result in a mismatch between the encoded miR binding site and the corresponding miRNA. In some embodiments, the viral genome comprises at least 3, 4, or 5 copies of the encoded miR96 binding site, e.g., an encoded miR96 binding site series. In some embodiments, the at least 3, 4, or 5 copies (e.g., 4 copies) of the encoded miR96 binding site are continuous (e.g., not separated by a spacer) or separated by a spacer. In some embodiments, the spacer is about 1 to 6 nucleotides or about 5 to 10 nucleotides, e.g., about 7-8 nucleotides or about 8 nucleotides, in length. In some embodiments, the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, but no more than four modifications of GATAGTTA.
[00215] In some embodiments, the encoded miR binding site series comprises a miR122 binding site, a miRl 42 binding site, a miRl 83 binding site, a miRl 82 binding site, a miR 96 binding site, or a combination thereof. In some embodiments, the encoded miR binding site series comprises at least 3, 4, or 5 copies of a miR122 binding site, a miR-1, a miR142 binding site, a miR183 binding site, a miR182 binding site, a miR 96 binding site, or a combination thereof. In some embodiments, at least two of the encoded miR binding sites are connected directly, e.g., without a spacer. In other embodiments, at least two of the encoded miR binding sites are separated by a spacer, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length, which is located between two or more consecutive encoded miR binding site sequences. In embodiments, the spacer is at least about 5 to 10 nucleotides, e.g., about 7-8 nucleotides or about 8 nucleotides, in length. In some embodiments, the spacer sequence comprises one or more of (i) GGAT; (ii) CACGTG; (iii) GCATGC, or a repeat of one or more of (i)-(iii). In some embodiments, the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, but no more than four modifications of GATAGTTA.
[00216] In some embodiments, an encoded miR binding site series comprises at least 3-5 copies (e.g., 4 copies) of a combination of at least two, three, four, five, or all of a miR122 binding site, a miR-1 a miR142 binding site, a miR183 binding site, a miR182 binding site, a miR96 binding site, wherein each of the miR binding sites within the series are continuous (e.g., not separated by a spacer) or separated by a spacer. In some embodiments, the spacer is about 1 to 6 nucleotides or about 5 to 10 nucleotides, e.g., about 7-8 nucleotides or about 8 nucleotides, in length. In some embodiments, the spacer sequence comprises one or more of (i) GGAT; (ii) CACGTG; (iii) GCATGC, or a repeat of one or more of (i)-(iii). In some embodiments, the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, but no more than four modifications of GATAGTTA.
Viral Genome Component: Poly adenylation Sequence
[00217] In some embodiments, the viral genome of the AAV particles of the present disclosure comprises at least one polyadenylation (poly A) sequence. The viral genome of the AAV particle may comprise a polyadenylation sequence between the 3’ end of the payload coding sequence and the 5’ end of the 3’UTR. In some embodiments, the poly A signal region is positioned 3’ relative to the nucleic acid comprising the transgcnc encoding the payload, e.g., an SMN protein described herein.
[00218] In some embodiments, the viral genome comprises a human growth hormone (hGH) polyA sequence. In some embodiments, the viral genome comprises a rabbit beta globin polyA (rBG) sequence. [00219] In some embodiments, the viral genome, e.g., a viral genome encoding an SMN protein described herein comprises a polyA sequence comprising the nucleotide sequence of SEQ ID NO: 4, a nucleotide sequence substantially identical (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical) to SEQ ID NO: 4, a nucleotide sequence comprising one, two, or three but no more than four modifications, e.g., substitutions, relative to the nucleotide sequence of SEQ ID NO: 4, or a nucleotide sequence comprising one, two, or three but no more than four different nucleotides relative to the nucleotide sequence of SEQ ID NO: 4.
Table 9. Exemplary polyA sequences
Figure imgf000127_0001
Viral Genome Component: Filler Sequence
[00220] In some embodiments, an AAV particle, e.g., an AAV particle for the vectorized delivery of an SMN protein described herein, comprises a filler sequence. In some embodiments, the viral genome, e.g., a viral genome encoding an SMN protein described herein comprises a filler comprising the nucleotide sequence of SEQ ID NO: 5; a nucleotide sequence substantially identical (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical) to SEQ ID NO: 5: a nucleotide sequence comprising one, two, or three but no more than four modifications, e.g., substitutions, relative to the nucleotide sequence of SEQ ID NO: 5; or a nucleotide sequence comprising one, two, or three but no more than four different nucleotides relative to the nucleotide sequence of SEQ ID NO: 5.
Table 10. Exemplary filler sequences
Figure imgf000128_0001
Viral Genome Component: Payloads
[00221] In some embodiments, an AAV particle, e.g., an AAV particle for the vectorized delivery of an
SMN protein described herein, comprises a payload. In some embodiments, an AAV particle, e.g., an AAV particle for the vectorized delivery of an SMN protein described herein, comprises a viral genome encoding a payload. In some embodiments, the viral genome comprises a promoter operably linked to a nucleic acid comprising a transgene encoding a payload. In some embodiments, the payload comprises an SMN protein.
[00222] In some embodiments, the disclosure herein provides constructs that allow for improved expression of an SMN protein delivered by gene therapy vectors.
[00223] In some embodiments, the disclosure provides constructs that allow for improved biodistribution of SMN protein delivered by gene therapy vectors.
[00224] In some embodiments, the disclosure provides constructs that allow for improved sub-cellular distribution or trafficking of an SMN protein delivered by gene therapy vectors.
[00225] In some embodiments, the disclosure provides constructs that allow for improved trafficking of an SMN protein to lysosomal membranes delivered by gene therapy vectors.
[00226] In some aspects, the present disclosure relates to a composition containing or comprising a nucleic acid sequence encoding an SMN protein or functional fragment or variants thereof and methods of administering the composition in vitro or in vivo in a subject, e.g., a humans and/or an animal model of disease, e.g., a disease related to expression of SMN.
[00227] AAV particles of the present disclosure may comprise a nucleic acid sequence encoding at least one “payload.” As used herein, “payload” or “payload region” refers to one or more polynucleotides or polynucleotide regions encoded by or within a viral genome or an expression product of such polynucleotide or polynucleotide region, e.g., a transgene, a polynucleotide encoding a polypeptide or multi-polypeptide, e.g., SMN protein or fragment or variant thereof. The pay load may comprise any nucleic acid known in the art that is useful for the expression (by supplementation of the protein product or gene replacement using a modulatory nucleic acid) of SMN protein in a target cell transduced or contacted with the AAV particle carrying the payload.
[00228] Specific features of a transgene encoding SMN for use in an AAV genome as described herein include the use of a wildtype SMN-encoding sequence and enhanced SMN-encoding constructs. In some instances, the SMN-encoding sequence is a recombinant and/or modified SMN sequence as described in , the contents of which are herein incorporated by reference in their entirety. In some embodiments, the SMN-encoding sequence is as provided by GenBank Accession Nos. NM_001297715.1; NM_000344.3; NM_022874.2., DQ894095, NM_ 000344, NM_ 022874, and BC062723 (for example, as described in W02010129021 Al, and W02009151546A2, the entire contents of which are incorporated herein by reference).
[00229] In some embodiments, the SMN-encoding sequence is codon optimized. In some aspects, the entire length of the open reading frame (ORF) is modified. However, in some aspects, only a fragment of the ORF is altered. By using one of these methods, one can apply the frequencies to any given polypeptide sequence, and produce a nucleic acid fragment of a codon-optimized coding region which encodes the polypeptide. Accordingly, in some aspects a codon optimized SMN1 coding sequence is used (e.g., a codon optimized hSMNl ORF). In some aspects, one or more portions of the SMN1 coding sequence (e.g., up to the entire ORF) are codon optimized for expression in humans.
[00230] The payload construct may comprise a combination of coding and non-coding nucleic acid sequences.
[00231] Any segment, fragment, or the entirety of the viral genome and therein, the payload region, may be codon optimized.
[00232] In some embodiments, the viral genome encodes more than one payload. As a non-limiting example, a viral genome encoding more than one payload may be replicated and packaged into a viral particle. A target cell transduced with a viral particle comprising more than one payload may express each of the payloads in a single cell.
[00233] In some embodiments, the viral genome may encode a coding or non-coding RNA. In certain embodiments, the adeno-associated viral vector particle further comprises at least one cis-element selected from the group consisting of a Kozak sequence, a backbone sequence, and an intron sequence. [00234] In some embodiments, the payload is a polypeptide which may be a peptide or protein. A protein encoded by the payload construct may comprise a secreted protein, an intracellular protein, an extracellular protein, and/or a membrane protein. The encoded proteins may be structural or functional. Proteins encoded by the viral genome include, but are not limited to, mammalian proteins. In certain embodiments, the AAV particle contains a viral genome that encodes SMN protein or a fragment or variant thereof. The AAV particles described herein may be useful in the fields of human disease, veterinary applications, and a variety of in vivo and in vitro settings.
[00235] In some embodiments, a payload may comprise polypeptides that serve as marker proteins to assess cell transformation and expression, fusion proteins, polypeptides having a desired biological activity, gene products that can complement a genetic defect, RNA molecules, transcription factors, and other gene products that are of interest in regulation and/or expression. In some embodiments, a payload may comprise nucleotide sequences that provide a desired effect or regulatory function (e.g., transposons, transcription factors).
[00236] The encoded payload may comprise a gene therapy product. A gene therapy product may include, but is not limited to, a polypeptide, RNA molecule, or other gene product that, when expressed in a target cell, provides a desired therapeutic effect. In some embodiments, a gene therapy product may comprise a substitute for a non-functional gene or a gene that is absent, expressed in insufficient amounts, or mutated. In some embodiments, a gene therapy product may comprise a substitute for a nonfunctional protein or polypeptide or a protein or polypeptide that is absent, expressed in insufficient amounts, misfolded, degraded too rapidly, or mutated. For example, a gene therapy product may comprise an SMN protein or a polynucleotide encoding an SMN protein to treat SMN deficiency or SMA-related disorders (e.g., SMA, Werdnig-Hoffman disease, Dubowitz disease, Kugelberg-Welander disease).
[00237] In some embodiments, the payload encodes a messenger RNA (mRNA). As used herein, the term “messenger RNA” (mRNA) refers to any polynucleotide that encodes a polypeptide of interest and that is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ, or ex vivo. Certain embodiments provide the mRNA as encoding SMN or a variant thereof.
[00238] The components of an mRNA include, but are not limited to, a coding region, a 5'-UTR (untranslated region), a 3'-UTR, a 5 '-cap and a poly -A tail. In some embodiments, the encoded mRNA or any portion of the AAV genome may be codon optimized.
[00239] In some embodiments, a nucleic acid for expression of a payload in a target cell will be incorporated into the viral genome and located between two TTR sequences.
[00240] In some embodiments, a payload construct further comprises a nucleic acid sequence encoding a peptide that binds to the cation-independent mannose 6- phosphate (M6P) receptor (CI-MPR) with high affinity, as described in Int’l Pat. App. Pub. No. W02019213180A1, the disclosure of which is incorporated herein by reference in its entirety. The peptide that binds CI-MPR can be, e.g., an IGF2 peptide or variant thereof. Binding of CI-MPR can facilitate cellular uptake or delivery and intracellular or sub-cellular targeting of therapeutic proteins provided by gene therapy vectors.
[00241] In some embodiments, the payload, e.g., of a viral genome described herein, is an SMN protein, e.g., a wild-type SMN protein, or a functional variant thereof. In some embodiments, a functional variant is a variant that retains some or all of the activity of its wild-type counterpart, so as to achieve a desired therapeutic effect. For example, in some embodiments, a functional variant is effective to be used in gene therapy to treat a disorder or condition, for example, an SMN gene product deficiency (e.g., SMA, e.g., Werdnig-Hoffman disease, Dubowitz disease, Kugelberg- Welander disease), or an SMA-related disorders, a neurodegenerative disorder, and/or a neuromuscular disorder. Unless indicated otherwise, a variant of an SMN protein as described herein (e.g., in the context of the constructs, vectors, genomes, methods, kits, compositions, etc. of the disclosure) is a functional variant.
[00242] As used herein, “associated with decreased SMN protein levels” or “associated with decreased expression” means that one or more symptoms of a disease are caused by lower-than-normal SMN protein levels in a target tissue or in a biofluid such as blood. A disease or condition associated with decreased SMN protein levels or expression may be a disorder of the central nervous system. Also specifically contemplated herein are Spinal Muscular Atrophy (SMA) and related disorders arising from expression of defective SMN gene product, e.g., an SMA associated with an SMN mutation. Such a disease or condition may be a neuromuscular or a neurological disorder or condition. For example, a disease associated with decreased SMN protein levels may be Spinal Muscular Atrophy (SMA) or related disorder.
[00243] The present disclosure addresses the need for new technologies by providing SMN protein related treatment deliverable by AAV-based compositions and complexes for the treatment of SMA and SMA-related disorders.
[00244] While delivery is exemplified in the AAV context, other viral vectors, non-viral vectors, nanoparticles, or liposomes may be similarly used to deliver the therapeutic SMN protein(s) and include, but are not limited to, vector genomes of any of the AAV serotypes or other viral delivery vehicles or lentivirus, etc. The observations and teachings extend to any macromolecular structure, including modified cells, introduced into the CNS in the manner as described herein.
[00245] Given in Table 11A and 11B are the sequence identifiers of exemplary polynucleotide and polypeptide sequences for SMN proteins that may be used in the viral genomes disclosed herein and which may constitute an SMN protein payload. Functional variants, e.g., those retaining at least about 90% or at least 95% sequence identity to a sequence shown in Table 11 A and 1 IB, may also be used. In some embodiments, a codon-optimized and other variants that encode the same or essentially the same SMN protein amino acid sequence (e.g., those having at least about 90% amino acid sequence identity) may also be used.
[00246] In some embodiments, the viral genome comprises a nucleic acid comprising a transgene encoding an SMN protein, or functional variant thereof. In some embodiments, the encoded SMN protein, or functional variant thereof comprises an amino acid sequence from an SMN protein described herein, e.g., as described in Table 11 A or Table 1 IB, or an amino acid sequence substantially identical (e.g., having at least about 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) to any of the aforesaid sequences. In some embodiments, the encoded SMN protein or functional variant thereof comprises an amino acid sequence from an SMN protein described herein, e.g., as described in Table 11A or 11B, or an amino acid sequence having at least one, two or three modifications, e.g., substitutions (e.g., conservative substitutions), but not more than 30, 20 or 10 modifications, e.g., substitutions (e.g., conservative substitutions), relative to any of the aforesaid amino acid sequences. In some embodiments, the encoded SMN protein or functional variant thereof, comprises an amino acid sequence encoded by a nucleotide sequence encoding an SMN protein described herein, e.g., as described in Table 11 A or 1 IB, or a nucleotide sequence substantially identical (e.g., having at least about 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) to any of the aforesaid sequences.
[00247] In some embodiments, the nucleotide sequence encoding the SMN protein or functional variant thereof comprises a nucleotide sequence encoding an SMN protein described herein, e.g., as described in Table 11A or 11B, or a nucleotide sequence substantially identical (e.g., having at least about 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) to any of the aforesaid sequences. In some embodiments, the nucleotide sequence encoding the SMN protein or functional variant thereof comprises a nucleotide sequence encoding an SMN protein described herein, e.g., as described in Table 11A or 11B, or a nucleotide sequence having at least one, two or three modifications, e.g., substitutions (e.g., conservative substitutions), but not more than 30, 20 or 10 modifications, e.g., substitutions (e.g., conservative substitutions), relative to any of the aforesaid nucleotide sequences. In some embodiments, the nucleotide sequence encoding an SMN protein or functional variant thereof is a codon optimized nucleotide sequence.
[00248] A non-limiting example of an amino acid sequence for wild-type human SMN protein is provided in UniProtKB/Swiss-Prot: Q16637.1. Conservative nucleotide substitutions of SMN DNA are also contemplated (e.g., a guanine to adenine change at position 625 of GenBank Accession Number NM.sub.— 000344.2).
[00249] In some embodiments, the nucleotide sequence encoding the SMN protein or functional variant thereof comprises the nucleotide sequence of SEQ ID NO: 8 or a nucleotide sequence substantially identical (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical) thereto. In some embodiments, the nucleotide sequence encoding the SMN protein or functional variant thereof comprises a nucleotide sequence having at least one, two or three modifications, e.g., substitutions (e.g., conservative substitutions), but not more than 30, 20 or 10 modifications, e.g., substitutions (e.g., conservative substitutions), relative to SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding the SMN protein or functional variant thereof comprises a nucleotide sequence having at least one, two or three, but no more than 30, 20, or 10 different nucleotides relative to the nucleotide sequence of SEQ ID NO: 8.
Table 11A. Exemplary SMN1 Sequences
Figure imgf000133_0001
Figure imgf000134_0001
Table 11B. Exemplary SMN1/SMN2 Sequences
Figure imgf000134_0002
Figure imgf000135_0001
Figure imgf000136_0001
Figure imgf000137_0001
[00250] In some embodiments, the encoded an SMN protein or functional variant thereof comprises the amino acid sequence of SEQ ID NO: 2000, or an amino acid sequence substantially identical (e.g., having at least about 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) to any of the aforesaid sequences. In some embodiments, the encoded SMN protein or functional variant thereof comprises the amino acid sequence of SEQ ID NO: 2000, or an amino acid having at least one, two or three modifications but not more than 30, 20 or 10 modifications relative to any of the aforesaid amino acid sequences. In some embodiments, the encoded an SMN protein or functional variant thereof comprises an amino acid sequence encoded by the nucleotide sequence of any of SEQ ID NOs: 6-9 or 2001-2004, or a nucleotide sequence substantially identical (e.g., having at least about 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) to any of the aforesaid sequences.
[00251] In some embodiments, the nucleotide sequence encoding an SMN protein or functional variant thereof comprises the nucleotide sequence of any one of SEQ ID NOs: SEQ ID NOs: 6-9 or 2001-2004, or a nucleotide sequence substantially identical (e.g., having at least about 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) to any of the aforesaid sequences. In some embodiments, the nucleic acid sequence encoding an SMN protein or functional variant thereof comprises the nucleotide sequence of any one of SEQ ID NOs: SEQ ID NOs: 6-9 or 2001-2004, or a nucleotide sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications relative to any of the aforesaid nucleotide sequences. In some embodiments, the nucleotide sequence encoding the an SMN protein or functional variant thereof comprises the nucleotide sequence of SEQ ID NO: 2001, a nucleotide sequence substantially identical (e.g., having at least about 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 2001, or a nucleotide sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications relative to SEQ ID NO: 2001. In some embodiments, the nucleotide sequence encoding the SMN protein or functional variant thereof does not comprise a stop codon. In some embodiments, the nucleotide sequence encoding an SMN protein or functional variant thereof is a codon optimized nucleotide sequence.
[00252] In some embodiments, a codon optimized nucleotide sequence encoding an SMN protein described herein (e.g., SEQ ID NO: 2000) contains more than 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140 or more unique modifications, e.g., mutations, compared to the nucleotide sequence of SEQ ID NO: 9 or 2001. In some embodiments, a codon optimized nucleotide sequence of an SMN protein described herein (e.g., SEQ ID NO: 2000) comprises a unique GC content profile. Without wishing to be bound by theory, it is believed in some embodiments, that altering the GC-content of a nucleotide sequence of an SMN protein described herein enhances the expression of the codon optimized nucleotide sequence in a cell (e.g., a human cell or a neuronal cell). [00253] In some embodiments, the viral genome comprises a payload region encoding an SMN protein. The encoded SMN protein may be derived from any species, such as, but not limited to human, nonhuman primate, or rodent.
[00254] In some embodiments, the viral genome comprises a payload region encoding a human (Homo sapiens) SMN protein, or a variant thereof.
[00255] Various embodiments of the disclosure herein provide an adeno-associated viral (AAV) particle comprising a viral genome, the viral genome comprising at least one inverted terminal repeat region and a nucleic acid sequence encoding a polypeptide having at least 90% sequence identity to a human SMN protein sequence, or a fragment thereof, as provided in Table 11 A or 1 IB. In some embodiments, the AAV viral genome comprises at least one inverted terminal repeat region and a nucleic acid sequence encoding a polypeptide having at least 95% sequence identity to an SMN protein sequence, or a fragment thereof, as provided in Table 11 A or 1 IB. In some embodiments, the AAV viral genome comprises at least one inverted terminal repeat region and a nucleic acid sequence encoding a polypeptide having at least 98% sequence identity to an SMN protein sequence, or a fragment thereof, as provided in Table 11 A or 1 IB. In some embodiments, the AAV viral genome comprises at least one inverted terminal repeat region and a nucleic acid sequence encoding a polypeptide having at least 99% sequence identity to an SMN protein sequence, or a fragment thereof, as provided in Table 11A or 11B. In some embodiments, the AAV viral genome comprises at least one inverted terminal repeat region and a nucleic acid sequence encoding an SMN protein sequence, or a fragment thereof, provided in Table 11A or 11B.
[00256] In some embodiments, the viral genome comprises a nucleic acid sequence encoding a recombinant, a recombinant SMN for use in treating Spinal Muscular Atrophy (SMA) (e.g., Werdnig- Hoffman disease, Dubowitz disease, Kugelberg-Welander disease), .
[00257] In some embodiments, the SMN protein is derived from an SMN protein encoding sequence of a non-human primate, such as the cynomolgus monkey, Macaca fascicularis (UniProtKB/Swiss-Prot: Q4R4F8.1; SEQ ID NO: 2008) Certain embodiments provide the SMN protein as a humanized version of a Macaca fascicularis sequence. In some embodiments, the viral genome comprises a payload region encoding a rhesus macaque (Macaca mulattcr, XP_015002432.2; SEQ ID NO: 2009) SMN protein, or a variant thereof.
[00258] In some embodiments, the SMN protein may comprise an amino acid sequence with 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,
70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of the those described above and provided in Table 11 A or 1 IB.
[00259] In some embodiments, the SMN protein may be encoded by a nucleic acid sequence with 50%,
51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,
69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of the those described above and provided in Table 11 A or 1 IB.
[00260] The SMN protein payloads as described herein can encode any SMN protein, or any portion or derivative of an SMN protein, and are not limited to the SMN proteins or protein-encoding sequences provided in Table 11A or 11B.
[00261] In some embodiments, the nucleotide sequence of an SMN protein described herein is modified to reduce the number of CpG di-nucleotides in order to produce a viral vector with a reduced toll-like receptor 9-mediated innate immune response in a subject when compared to an unmodified AAV vector. Modifications to reduce the number of CpG di-nucleotides can be in one or more of transgene, promoter, enhancer, intron, miRNA and polyA signal sequence. Methods for reducing CpG di-nucleotide numbers have been described, for example, by Faust et al. (J. Clin. Inv. 123(7):2994-3001, 2013).
Payload Component: Splicing Modulator
[00262] In some embodiments, the SMN payload includes a nucleotide sequence which encodes an element which modulates splicing of the SMN2 pre-mRNA sequence to enhance the level of exon 7- containing SMN2 mRNA relative to exon-deleted SMN2 mRNA in a cell or cell extract. The 54- nucleotide-long exon 7 of human SMN genes contains .about.65% of A+U residues. Hence, exon 7 fits into the typical definition of a cassette exon that generally contains a low percentage of G+C residues (Clark, F., and T. A. Thanaraj. 2002. Hum. Mol. Genet. 11:451-64). In addition to the exon 7 sequence, intronic sequences located immediately upstream of the 3' ss or downstream of the 5' splice site (5' ss) of SMN2 exon 7 have been demonstrated as functionally important in splicing (Miriami, E., et al. 2003. Nucleic Acids Res. 31:1974-1983; Zhang, X. H., and L. A. Chasin. 2004. Genes Dev. 18: 1241-1250). These sequences are highly diverse and can be broadly categorized into G+C -rich and A+U-rich regions that constitute distinct pentamer motifs (Zhang, X. H., et al. 2005. Genome Res. 15:768-779). Intron 7 sequence downstream of the 5' ss is rich in A and U residues, but lacks characteristic pentamer motifs. [00263] In some embodiments, the splicing modulator is an antisense oligonucleotide (ASO) designed to base pair with splicing regulator sequences. Suitable antisense oligonucleotides which reduce inclusion of exon 7 in SMN2 pre-mRNA are known in the art, for example, in WO/2020/037161).
[00264] Modulation of expression of SMN2 can be measured in a bodily fluid, which may or may not contain cells, tissue; or organ of the animal. In some aspects, by comparing the level of full-length SMN2 mRNA in a sample obtained from a subject receiving an SMN2 ASO treatment to a level of full-length SMN2 mRNA in a subject not treated with the SMN2 ASO, the extent to which the SMN2 ASO increased full-length SMN2 mRNA can be determined. In some aspects, the reference level of full-length SMN2 mRNA is obtained from the same subject prior to receiving SMN2 ASO. In some aspects, the reference level of full-length SMN2 mRNA is a range determined by a population of subjects not receiving SMN2 ASO.
[00265] In some aspects, an increased level of full-length SMN2 mRNA is, for example, greater than 1 fold, 1.5-5 fold, 5-10 fold, 10-50 fold, 50-100 fold, about 1.1-, 1.2-, 1.5-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 30-, 40-, 50-, 60-, 70-, 80-, 90-, 100-fold or more higher than a reference value.
[00266] In some embodiments, the target of the splicing modulator is the intronic splicing silencer element ISSN-N1 located in intron 7 of SMN2, which can be blocked to promote exon 7 inclusion and restore SMN expression in vitro and in vivo. As used herein, the term "intronic splicing silencer-Nl" or "ISS-N1" refers to the sequence 5'-CCAGCAUUAUGAAAG-3' (SEQ ID NO: 2010), or any sequence or variant thereof capable of inhibiting the inclusion of exon 7 during splicing of the SMN2 pre-mRNA. One such effective sequence thereof is 5'-CCAGCAUU-3’ (SEQ ID NO: 2011). Critical residues that mediate the splice site inhibitory activity of the ISS-N1 sequence can also be represented by the sequence 5'-CCAGCNNNNNGAAAG-3' (SEQ ID NO: 2012). Thus, any such sequence that acts to inhibit inclusion of exon 7 during splicing of the SMN2 pre-mRNA can be referred to simply as an "ISSN-N1 sequence."
[00267] In some embodiments the ASO is Nusinersen (Spinraza®). Nusinersen is an antisense oligonucleotide in which the 2’ -hydroxy groups of the ribofuranosyl rings are replaced with 2’ -O-2- methoxyethyl groups and the phosphate linkages are replaced with phosphorothioate linkages, as described for example in US Patent Nos. 7101993, 78110560, 7838657, 8361977, 8980853, 9929669, 9717750, 10266822, 10436802 (the contents of which are hereby incorporated herein in their entirety). [00268] Nusinersen was designed to pair with a specific target sequence on the SMN2 pre-mRNA to displace heterogeneous ribonucleoproteins (hnRNPs) at the intronic splice silencing site-1 (ISS-1) between exons 7 and 8 to allow for more complete translation of SMN protein from the paralogous gene SMN2. Intrathecal injection of nusinersen into the cerebrospinal fluid (CSF) allows it to be distributed from the CSF to the target central nervous system (CNS) tissues. The full sequence of nusinersen is [2’- O-(2-methoxyethyl)](3’-5')(P-thio) (T-5mC-A-5mC-T-T-T-5mC-A-T-A-A-T-G-5mC-T-G-G) (SEQ ID NO:2013). The antisense oligonucleotide contains 2‘-O-(2-methoxy ethyl) (2’-MOE)- oligoribonucleotides to reduce nuclease degradation and to enhance binding affinity towards the complementary RNA.
[00269] In other embodiments, the splicing modulator is an UlsnRNA. As described in U.S. Patent No. 9669109 (incorporated herein by reference), during the splicing process, the small nuclear RNAs (snRNAs) play a primary role as essential components of the spliceosome, the cell machinery appointed to mediate the entire mRNA maturation process. In particular, the small U1 RNA (UlsnRNA), 164 ribonucleotides in length, is encoded by genes that occur in several copies within the human genome and represents the ribonucleic component of the nuclear particle UlsnRNP. The UlsnRNA molecules have a stem and loop tridimensional structure and within the 5' region they include a single-stranded sequence, generally 9 nucleotides in length, capable of binding by complementary base pairing the splicing donor site on the pre-mRNA molecule The sequence in the 5' region capable of recognizing the splicing donor site is shown paired with the consensus sequence of the splicing donor site in the primary transcripts of eukaryotic genes. Such a sequence exhibits varying degrees of conservation and is located at the exon/intron junction. The recognition mediated by the UlsnRNA 5' region is critical for defining the exon/intron junctions on the primary transcript and for a correct assembly of the spliceosome complex. [00270] In some embodiments, the nucleic acid comprises a human UlsnRNA molecule capable of correcting the skipping of exon 7 of the SMN2 pre-mRNA. During the splicing process, the small nuclear RNAs (snRNAs) play a primary role as essential components of the spliceosome, the cell machinery appointed to mediate the entire mRNA maturation process. In particular, the small U1 RNA (UlsnRNA), 164 ribonucleotides in length, is encoded by genes that occur in several copies within the human genome and represents the ribonucleic component of the nuclear particle UlsnRNP. The UlsnRNA molecules have a stem and loop tridimensional structure and within the 5' region they include a single-stranded sequence, generally 9 nucleotides in length, capable of binding by complementary base pairing the splicing donor site on the pre-mRNA molecule. The sequence in the 5' region capable of recognizing the splicing donor site is shown paired with the consensus sequence of the splicing donor site in the primary transcripts of eukaryotic genes. Such a sequence exhibits varying degrees of conservation and is located at the exon/intron junction. The recognition mediated by the UlsnRNA 5' region is critical for defining the exon/intron junctions on the primary transcript and for a correct assembly of the spliceosome complex.
[00271] In some embodiments, the UlsnRNA is a modified UlsnRNA molecule that is complementary to intron sequences downstream of the 5’ splicing site of the pre-mRNA. Exon specific UlsnRNA (ExSpeUl), restore the splicing process of the SMN2 pre-mRNA and inclusion of exon 7 (Donadon et al., NAR 47:7618-7632, 2019). Suitable ExSpeUl molecules which restore splicing of the SMN2 pre- mRNA to include exon 7 are known in the art and are described, for example, in U.S. Patent No. 9074207 and US 2017/0143847. In some embodiments, the Ul snRNA is ExSpeUl sma (Donadon, supra). [00272] In some embodiments, the UlsnRNA is ExSpeUlsm25 Fw New_2 (5’-
TACAAAAGTAAGATTCAGCAG-3’; SEQ ID NO: 2014), which binds to SMN2 intron 7 at nucleotides 25-42, UlsnRNA comprises 5’ACUUAUCC3’ (SEQ ID NO: 2015), SMN_SH2 (5’-
GCAGACUUA-3’; SEQ ID NO: 2016), SMN_SH17 (5’-ACUUUCAUA-3’; SEQ ID NO: 2017);
SMN_SH 37 (AAACCAUAAAGUUUUACAA; SEQ ID NO: 2018), SMN_SH40 (5’CAAACCAUAAAGUUUUA-3’; SEQ ID NO: 2019) or a combination thereof.
PLS3 Modulation ofSMN
[00273] In some embodiments, a viral genome, e.g., an AAV viral genome or vector genome, described herein, comprises a nucleotide sequence encoding Plastin 3 (PLS3) or a variant thereof. Plastin 3 (PLS3) is a Ca2+-dependent F-actin-binding and -bundling protein that influences the G/F-actin ratio.
Overexpression of PLS3, either in primary MN culture rom SMA mice or in zebrafish smn morphants, significantly restored the impaired axonal growth and motor-axon truncation. (Oprea et al., Science
2008;320:524-527; Walsh et al., BMC Biol. 2020;18:127).
[00274] In some embodiments, the AAV particle further includes a nucleotide sequence encoding
Plastin-3 (PLS3). In some embodiments, the nucleotide sequence encodes PLS3 having the amino acid sequence:
MDEMATTQISKDELDELKEAFAKVDLNSNGFICDYELHELFKEANMPLPGYKVREIIQKLMLDG DRNKDGKISFDEFVYIFQEVKSSDIAKTFRKAINRKEGICALGGTSELSSEGTQHSYSEEEKYAFV NWINKALENDPDCRHVIPMNPNTDDLFKAVGDGIVLCKMINLSVPDTIDERAINKKKLTPFIIQEN LNLALNSASAIGCHVVNIGAEDLRAGKPHLVLGLLWQIIKIGLFADIELSRNEALAALLRDGETLE ELMKLSPEELLLRWANFHLENSGWQKINNFSADIKDSKAYFHLLNQIAPKGQKEGEPRIDINMSG FNETDDLKRAESMLQQADKLGCRQFVTPADVVSGNPKLNLAFVANLFNKYPALTKPENQDIDW TLLEGETREERTFRNWMNSLGVNPHVNHLYADLQDALVILQLYERIKVPVDWSKVNKPPYPKL GANMKKLENCNYAVELGKHPAKFSLVGIGGQDLNDGNQTLTLALVWQLMRRYTLNVLEDLG DGQKANDDIIVNWVNRTLSEAGKSTSIQSFKDKTISSSLAVVDLIDAIQPGCINYDLVKSGNLTED DKHNNAKYAVSMARRIGARVYALPEDLVEVKPKMVMTVFACLMGRGMKRV (NP_005023.2;
SEQ ID NO: 2020).
[00275] In some embodiments, the nucleotide sequence encoding PLS3 has NCBI Accession No.
NM_005032:
ACAGCTGCAAAGATTCCGAGGTGCAGAAGTTGTCTGAGTGGGTTGGTCGGCGGCAGTCGGGCCAGACCCA GGACTCTGC G AC T T T AC AT C T T T AAAT GGATGAGATGGCTACCACTCAGATTTC C AAAGAT GAG C T T GAT GAAC T C AAAG AG G C C T T T G C AAAAGT T GAT CTCAACAGCAACGGATTCATTTGT G AC TAT GAAC T T C AT G AGCTCTTCAAGGAAGCTAATATGCCATTACCAGGATATAAAGTGAGAGAAATTATTCAGAAACTCATGCT G GAT G G T G AC AG G AAT AAAG AT G G G AAAAT AAG T T T T G AC G AAT TTGTTTATATTTTT C AAGAG G T AAAA AGTAGTGATATTGCCAAGACCTTCCGCAAAGCAATCAACAGGAAAGAAGGTATTTGTGCTCTGGGTGGAA CTTCAGAGTTGTCCAGCGAAGGAACACAGCATTCTTACTCAGAGGAAGAAAAATATGCTTTTGTTAACTG GATAAACAAAGCTTTGGAAAATGATCCTGATTGTAGACATGTTATACCAATGAACCCTAACACCGATGAC CTGTTCAAAGCTGTTGGTGATGGAATTGTGCTTTGTAAAATGATTAACCTTTCAGTTCCTGATACCATTG ATGAAAGAGCAATCAACAAGAAGAAACTTACACCCTTCATCATTCAGGAAAACTTGAACTTGGCACTGAA CTCTGCTTCTGCCATTGGGTGTCATGTTGTGAACATTGGTGCAGAAGATTTGAGGGCTGGGAAACCTCAT CTGGTTTTGGGACTGCTTTGGCAGATCATTAAGATCGGTTTGTTCGCTGACATTGAATTAAGCAGGAATG AAGCCTTGGCTGCTTTACTCCGAGATGGTGAGACTTTGGAGGAACTTATGAAATTGTCTCCAGAAGAGCT TCTGCTTAGATGGGCAAACTTTCATTTGGAAAACTCGGGCTGGCAAAAAATTAACAACTTTAGTGCTGAC ATCAAGGATTCCAAAGCCTATTTCCATCTTCTCAATCAAATCGCACCAAAAGGACAAAAGGAAGGTGAAC C AC GG AT AGAT AT T AAC AT GTCAGGTTT C AAT G AAAC AG AT GAT T T G AAG AGAG C T G AGAG T AT G C T T C A ACAAGCAGATAAATTAGGTTGCAGACAGTTTGTTACCCCTGCTGATGTTGTCAGTGGAAACCCCAAACTC AACTTAGCTTTCGTGGCTAACCTGTTTAATAAATACCCAGCACTAACTAAGCCAGAGAACCAGGATATTG ACTGGACTCTATTAGAAGGAGAAACTCGTGAAGAAAGAACCTTCCGTAACTGGATGAACTCTCTTGGTGT CAATCCTCACGTAAACCATCTCTATGCTGACCTGCAAGATGCCCTGGTAATCTTACAGTTATATGAACGA ATTAAAGTTCCTGTTGACTGGAGTAAGGTTAATAAACCTCCATACCCGAAACTGGGAGCCAACATGAAAA AGCTAGAAAACTGCAACTATGCTGTTGAATTAGGGAAGCATCCTGCTAAATTCTCCCTGGTTGGCATTGG AGGGCAAGACCTGAATGATGGGAACCAAACCCTGACTTTAGCTTTAGTCTGGCAGCTGATGAGAAGATAT AC C C T C AAT G T C C T G GAAG AT C T T GG AG AT G G T C AGAAAG C C AAT G AC GAC AT CAT T G T G AAC T G GG T GA AC AGAAC G T T G AG T GAAGC T G GAAAAT C AAC T T C CAT T C AG AG T T T T AAG G AC AAGAC GAT C AG C T C C AG TTTGGCAGTTGTGGATTTAATTGATGCCATCCAGCCAGGCTGTATAAACTATGACCTTGTGAAGAGTGGC AATCTAACAGAAGATGACAAGCACAATAATGCCAAGTATGCAGTGTCAATGGCTAGAAGAATCGGAGCCA GAGTGTATGCTCTCCCTGAAGACCTTGTGGAAGTAAAGCCCAAGATGGTCATGACTGTGTTTGCATGTTT GATGGGCAGGGGAATGAAGAGAGTGTAAAATAACCAATCTGAATAAAACAGCCATGCTCCCAGGTGCATG ATTCGCAGGTCAGCTATTTCCAGGTGAAGTGCTTATGGCTTAAGGAACTCTTGGCCATTCAAAGGACTTT TCATTTTGATTAACAGGACTAGCTTATCATGAGAGCCCTCAGGGGAAAGGGTTTAAGAAAAACAACTCCT CTTTCCCATAGTCAGAGTTGAATTTGTCAGGCACGCCTGAAATGTGCTCATAGCCAAAACATTTTACTCT CTCCTCCTAGAATGCTGCCCTTGACATTTCCCATTGCTGTATGTTATTTCTTGCTCTGTTATCTTTTGCC CTCTTAGAATGTCCCTCTCTTGGGACTTGCTTAGATGATGGGATATGAATATTATTAGACAGTAATTTTG CTTTCCATCCAGTATGCTAGTTCTTATTCGAGAACTATGGTCAGAGCGTATTTGGATATGAGTATCCTTT GCTTATCTTTGTAGTACTGAAAATTTGCCGAAGTAACTGGCTGTGCAGAATGTAATAGAAGCTTTTCTTA TTCTTTTATTCTTAAGATCAGTATCTTTTTACAGTATTCTTTCTACATGATCCTTTTTTGTACATTTAAG AATATTTTGATTATATTAAACAAGACTGCTGATTTTGCTACTTTTTTTAAGGGGTCTTCAAGTAAGTAAA ACATACATCGTAGC T AG AAG AAAAAT G T AC C T T AAAT TTGCATCTTCCCTCTCATACC C AAGC T G T AAAC AATTGAAATATTTTGTCTTAAATCACTTGGTTCAATACATGCTTATTTGTTTTAAAACCTGTATCATCAA ACTCTCTCTCTAAATTTAAAATGCTGTTGAATATGATACTTTTGAGGAGAGAGTGTGCTCAGAACTTAGA CGGGATTTGGTAGGCCAAGTATGCTAAGTGTACAATATATTTTTTAATTTTACACCTGAAACAAAGAAAT GTGGTCACTAAAAATAAAAGTATATATGTAGGAATTAATGTACTCTTGCTTTGTCAAGCTGTTTGCTATA GTTTCCAAGGTATTATGTTACTCTAACTCTGAAAAGTGATGTAATCTGGTAGCAATGTAGTAGTTCAAAT AAAGGCATTTACATAATAATTAGTCTGTTCTTCATGCTTTTGTCTCTTAGGAAGTATGCCAATGTTTGTC AGGATTTTTTTCTTTTTGTTTTTCTGATGTATTCTGTAAAATGGTGTTTGTTAAATTTGAGTTTTGGGAG CTGAATTAGAGGTACTGAATTAAGGACAGTACAAATGAAGTAAAAAGGTTTTCTCCAATTTACCAAAA ( SEQ ID NO : 2021 ) .
[00276] In some embodiments, the nucleotide sequence encoding PLS3 is provided in the same viral genome, e.g., an AAV viral genome or vector genome, described herein comprising a transgene encoding SMN1. In some embodiments, the nucleotide sequence encoding PLS3 is provided in the second viral genome, e.g., an AAV viral genome or vector genome, which is separately encapsulated in a viral particle described herein and co-administered with an AAV particle comprising viral genome comprising a transgene encoding an SMN.
Exemplary SMN AAV Viral Genome Sequence Regions and ITR to ITR Sequences
[00277] In some embodiments, a viral genome, e.g., an AAV viral genome or vector genome, described herein, comprises a promoter operably linked to a transgene encoding an SMN protein. In some embodiments, the viral genome further comprises an inverted terminal repeat region, an enhancer, an intron, a miR binding site, a polyA region, or a combination thereof.
[00278] In some embodiments, the viral genome comprises an inverted terminal repeat sequence region (ITR) provided in Table 6, or a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to any of the ITR sequences in Table 6.
[00279] This disclosure also provides in some embodiments, an SMN protein encoded by any one of SEQ ID NOs: 6, 7, 8, 9, 2001, 2002, 2003, 2004, or a nucleotide sequence substantially identical (e.g., having at least about 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) to any of the aforesaid sequences.
[00280] In some embodiments, the viral genome of an AAV particle described herein comprises the nucleotide sequence, e g., the nucleotide sequence from the 5’ ITR to the 3’ ITR, of the nucleotide sequences of SMN, e.g., as described in Tables 12 and 13, or a nucleotide sequence substantially identical (e.g., having at least about 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) to any of the aforesaid sequences. In some embodiments, the viral genome of an AAV particle described herein comprises the nucleotide sequence, e.g., the nucleic acid sequence from the 5’ ITR to the 3’ ITR, of any of the nucleotide sequences in Tables 12 and 13, or a nucleotide sequence substantially identical (e.g., having at least about 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) to any of the aforesaid sequences.
[00281] In some embodiments, a viral genome encoding an SMN protein is a wtSMN viral genome, wherein the viral genome comprises a transgene encoding an SMN protein (optionally wherein the nucleotide sequence encoding the SMN protein is a codon optimized nucleotide sequence), but does not encode splicing modulator element, e.g., a splicing modulator element described herein. In some embodiments, a viral genome encoding an SMN protein is an enSMN viral genome, wherein the viral genome comprises a transgene encoding a an SMN protein (optionally wherein the nucleotide sequence encoding an SMN protein is a codon optimized nucleotide sequence), and further encodes a splicing modulator, e.g., a splicing modulator described herein.
Table 12. Exemplary ITR- ITR sequences
Figure imgf000144_0001
Figure imgf000145_0001
Figure imgf000146_0001
Figure imgf000147_0001
Figure imgf000148_0001
Figure imgf000149_0001
Figure imgf000150_0001
Figure imgf000151_0001
Figure imgf000152_0001
Figure imgf000153_0001
Table 13. Exemplary Viral Genome (ITR to ITR) sequences
Figure imgf000154_0001
[00282] In some embodiments, the viral genome of an AAV particle described herein comprises a nucleotide sequence comprising all of the components or a combination of the components as described, e.g., in Tables 12 and 13, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any of the aforesaid sequences.
Self- Complementary and Single Strand Vectors
[00283] In some embodiments, an AAV viral genome, e.g., an AAV viral genome encoding an SMN protein as described herein, is single stranded (ssAAV).
[00284] In some embodiments, the AAV viral genome may be self-complementary (scAAVs). See, e.g., US Patent No. 7,465,583. In some embodiments, scAAV vectors contain both DNA strands that anneal together to form double stranded DNA. By skipping second strand synthesis, scAAVs allow for rapid expression in the cell.
[00285] In some embodiments, an AAV viral genome, e.g., an AAV viral genome encoding an SMN protein as described herein is a scAAV.
[00286] Methods for producing and/or modifying AAV viral genomes and/or AAV vectors are disclosed in the art such as pseudotyped AAV vectors (International Patent Publication Nos. W0200028004;
W0200123001; W02004112727; WO 2005005610 and WO 2005072364, the content of each of which are incorporated herein by reference in their entirety).
Viral Genome Size
[00287] In some embodiments, the viral genome of the AAV particles of the present disclosure may be single or double stranded. The size of the vector genome may be small, medium, large or the maximum size.
[00288] In some embodiments, the vector genome, which comprises a nucleic acid sequence encoding SMN protein described herein, may be a small single stranded vector genome. A small single stranded vector genome may be about 2.7 kb to about 3.5 kb in size such as about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, or about 3.5 kb in size. In some embodiments, the small single stranded vector genome may be 3.2 kb in size.
[00289] In some embodiments, the vector genome, which comprises a nucleic acid sequence encoding SMN protein described herein, may be a small double stranded vector genome. A small double stranded vector genome may be about 1.3 to about 1.7 kb in size such as about 1.3, about 1.4, about 1.5, about 1.6, or about 1.7 kb in size. In some embodiments, the small double stranded vector genome may be 1.6 kb in size.
[00290] In some embodiments, the vector genome, which comprises a nucleic acid sequence encoding SMN protein described herein, may be a medium single stranded vector genome. A medium single stranded vector genome may be about 3.6 to about 4.3 kb in size such as about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, or about 4.3 kb in size. In some embodiments, the medium single stranded vector genome may be 4.0 kb in size.
[00291] In some embodiments, the vector genome, which comprises a nucleic acid sequence encoding SMN protein described herein, may be a medium double stranded vector genome. A medium double stranded vector genome may be about 1.8 to about 2.1 kb in size such as about 1.8, about 1.9, about 2.0, or about 2.1 kb in size. In some embodiments, the medium double stranded vector genome may be 2.0 kb in size. Additionally, the vector genome may comprise a promoter and a polyA tail.
[00292] In some embodiments, the vector genome which comprises a nucleic acid sequence encoding SMN protein described herein may be a large single stranded vector genome. A large single stranded vector genome may be 4.4 to 6.0 kb in size such as about 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 and 6.0 kb in size. As a non-limiting example, the large single stranded vector genome may be 4.7 kb in size. As another non-limiting example, the large single stranded vector genome may be 4.8 kb in size. As yet another non-limiting example, the large single stranded vector genome may be 6.0 kb in size.
[00293] In some embodiments, the vector genome which comprises a nucleic acid sequence encoding SMN protein described herein may be a large double stranded vector genome. A large double stranded vector genome may be 2.2 to 3.0 kb in size such as about 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 and 3.0 kb in size. As a non-limiting example, the large double stranded vector genome may be 2.4 kb in size.
II. Viral production
General Viral Production Process
[00294] Cells for the production of AAV, e.g., rAAV, particles may comprise, in some embodiments, mammalian cells (such as HEK293 cells) and/or insect cells (such as Sf9 cells).
[00295] In various embodiments, AAV production includes processes and methods for producing AAV particles and vectors which can contact a target cell to deliver a pay load, e.g., a recombinant viral construct, which includes a nucleotide encoding a payload molecule. In certain embodiments, the viral vectors are adeno-associated viral (AAV) vectors such as recombinant adeno-associated viral (rAAV) vectors. In certain embodiments, the AAV particles are adeno-associated viral (AAV) particles such as recombinant adeno-associated viral (rAAV) particles.
[00296] In some embodiments, disclosed herein is a vector comprising a viral genome of the present disclosure. In some embodiments, disclosed herein is a cell comprising a viral genome of the present disclosure. In some embodiments, the cell is a bacterial cell, a mammalian cell (e.g., a HEK293 cell), or an insect cell (e.g., an Sf9 cell).
[00297] In some embodiments, disclosed herein is a method of making a viral genome. The method comprising providing a nucleic acid encoding a viral genome described herein and a backbone region suitable for replication of the viral genome in a cell, e.g., a bacterial cell (e.g., wherein the backbone region comprises one or both of a bacterial origin of replication and a selectable marker), and excising the viral genome from the backbone region, e.g., by cleaving the nucleic acid molecule at upstream and downstream of the viral genome. In some embodiments, the viral genome comprising a promoter operably linked to nucleic acid comprising a transgene encoding an SMN protein (e.g., an SMN protein described herein), will be incorporated into an AAV particle produced in the cell. In some embodiments, the cell is a bacterial cell, a mammalian cell (e.g., a HEK293 cell), or an insect cell (e.g., an Sf9 cell). [00298] In some embodiments, disclosed herein is a method of making a recombinant AAV particle of the present disclosure, the method comprising (i) providing a host cell comprising a viral genome described herein and incubating the host cell under conditions suitable to enclose the viral genome in a capsid protein, e.g., a capsid protein described herein (e.g., an AAV capsid variant described herein (e.g.,, an AAV capsid comprising a peptide sequence set forth in any one of Tables 2A, 2B, 2C, 15 or 21), an AAV capsid variant as set forth in Tables 3-5, or an AAV capsid as listed in Table 1), thereby making the recombinant AAV particle. In some embodiments, the method comprises prior to step (i), introducing a first nucleic acid comprising the viral genome into a cell. In some embodiments, the host cell comprises a second nucleic acid encoding the capsid protein. In some embodiments, the second nucleic acid is introduced into the host cell prior to, concurrently with, or after the first nucleic acid molecule. In some embodiments, the host cell is a bacterial cell, a mammalian cell (e.g., a HEK293 cell), or an insect cell (e.g., an Sf9 cell).
[00299] In various embodiments, methods are provided herein of producing AAV particles or vectors by (a) contacting a viral production cell with one or more viral expression constructs encoding at least one AAV capsid protein, and one or more payload constructs encoding a payload molecule, which can be selected from: a transgene, a polynucleotide encoding protein, and a modulatory nucleic acid; (b) culturing the viral production cell under conditions such that at least one AAV particle or vector is produced, and (c) isolating the AAV particle or vector from the production stream.
[00300] In these methods, a viral expression construct may encode at least one structural protein and/or at least one non-structural protein. The structural protein may include any of the native or wild type capsid proteins VP1, VP2, and/or VP3, or a chimeric protein thereof. The non-structural protein may include any of the native or wild type Rep78, Rep68, Rep52, and/or Rep40 proteins or a chimeric protein thereof.
[00301] In certain embodiments, contacting occurs via transient transfection, viral transduction, and/or electroporation.
[00302] In certain embodiments, the viral production cell is selected from a mammalian cell and an insect cell. In certain embodiments, the insect cell includes a Spodoptera frugiperda insect cell. In certain embodiments, the insect cell includes a Sf9 insect cell. In certain embodiments, the insect cell includes a Sf21 insect cell.
[00303] The payload construct vector of the present disclosure may include, in various embodiments, at least one inverted terminal repeat (ITR) and may include mammalian DNA.
[00304] Also provided are AAV particles and viral vectors produced according to the methods described herein.
[00305] In various embodiments, the AAV particles of the present disclosure may be formulated as a pharmaceutical composition with one or more acceptable excipients.
[00306] In certain embodiments, an AAV particle or viral vector may be produced by a method described herein.
[00307] In certain embodiments, the AAV particles may be produced by contacting a viral production cell (e.g., an insect cell or a mammalian cell) with at least one viral expression construct encoding at least one capsid protein and at least one payload construct vector. The viral production cell may be contacted by transient transfection, viral transduction, and/or electroporation. The payload construct vector may include a payload construct encoding a payload molecule such as, but not limited to, a transgene, a polynucleotide encoding protein, and a modulatory nucleic acid. The viral production cell can be cultured under conditions such that at least one AAV particle or vector is produced, isolated (e.g., using temperature-induced lysis, mechanical lysis and/or chemical lysis) and/or purified (e.g., using filtration, chromatography, and/or immunoaffinity purification). As a non-limiting example, the payload construct vector may include mammalian DNA.
[00308] In certain embodiments, the AAV particles are produced in an insect cell (e.g., Spodoptera frugiperda (Sf9) cell) using a method described herein. As a non-limiting example, the insect cell is contacted using viral transduction which may include baculoviral transduction.
[00309] In certain embodiments, the AAV particles are produced in an mammalian cell (e.g., HEK293 cell) using a method described herein. As a non-limiting example, the mammalian cell is contacted using viral transduction which may include multiplasmid transient transfection (such as triple plasmid transient transfection).
[00310] In certain embodiments, the AAV particle production method described herein produces greater than 101, greater than 102, greater than 103, greater than 104, or greater than 10s AAV particles in a viral production cell. [00311] In certain embodiments, a process of the present disclosure includes production of viral particles in a viral production cell using a viral production system which includes at least one viral expression construct and at least one payload construct. The at least one viral expression construct and at least one payload construct can be co-transfected (e.g., dual transfection, triple transfection) into a viral production cell. The transfection is completed using standard molecular biology techniques known and routinely performed by a person skilled in the art. The viral production cell provides the cellular machinery necessary for expression of the proteins and other biomaterials necessary for producing the AAV particles, including Rep proteins which replicate the payload construct and Cap proteins which assemble to form a capsid that encloses the replicated payload constructs. The resulting AAV particle is extracted from the viral production cells and processed into a pharmaceutical preparation for administration.
[00312] In various embodiments, once administered, an AAV particle disclosed herein may, without being bound by theory, contact a target cell and enter the cell, e.g., in an endosome. The AAV particles, e.g. , those released from the endosome, may subsequently contact the nucleus of the target cell to deliver the payload construct. The payload construct, e.g., recombinant viral construct, may be delivered to the nucleus of the target cell wherein the payload molecule encoded by the payload construct may be expressed.
[00313] In certain embodiments, the process for production of viral particles utilizes seed cultures of viral production cells that include one or more baculoviruses (e.g., a Baculoviral Expression Vector (BEV) or a baculovirus infected insect cell (BIIC) that has been transfected with a viral expression construct and a payload construct vector). In certain embodiments, the seed cultures are harvested, divided into aliquots and frozen, and may be used at a later time point to initiate an infection of a naive population of production cells.
[00314] In some embodiments, large scale production of AAV particles utilizes a bioreactor. Without being bound by theory, the use of a bioreactor may allow for the precise measurement and/or control of variables that support the growth and activity of viral production cells such as mass, temperature, mixing conditions (impellor RPM or wave oscillation), CO2 concentration, O2 concentration, gas sparge rates and volumes, gas overlay rates and volumes, pH, Viable Cell Density (VCD), cell viability, cell diameter, and/or optical density (OD). In certain embodiments, the bioreactor is used for batch production in which the entire culture is harvested at an experimentally determined time point and AAV particles are purified. In some embodiments, the bioreactor is used for continuous production in which a portion of the culture is harvested at an experimentally determined time point for purification of AAV particles, and the remaining culture in the bioreactor is refreshed with additional growth media components.
[00315] In various embodiments, AAV viral particles can be extracted from viral production cells in a process which includes cell lysis, clarification, sterilization and purification. Cell lysis includes any process that disrupts the structure of the viral production cell, thereby releasing AAV particles. In certain embodiments, cell lysis may include thermal shock, chemical, or mechanical lysis methods. Clarification can include the gross purification of the mixture of lysed cells, media components, and AAV particles. In certain embodiments, clarification includes centrifugation and/or filtration, including but not limited to depth end, tangential flow, and/or hollow fiber filtration.
[00316] In various embodiments, the end result of viral production is a purified collection of AAV particles which include two components: (1) a payload construct (e.g., a recombinant AAV vector genome construct) and (2) a viral capsid.
[00317] In certain embodiments, a viral production system or process of the present disclosure includes steps for producing baculovirus infected insect cells (BIICs) using Viral Production Cells (VPC) and plasmid constructs. Viral Production Cells (VPCs) from a Cell Bank (CB) are thawed and expanded to provide a target working volume and VPC concentration. The resulting pool of VPCs is split into a Rep/Cap VPC pool and a Payload VPC pool. One or more Rep/Cap plasmid constructs (viral expression constructs) are processed into Rep/Cap Bacmid polynucleotides and transfected into the Rep/Cap VPC pool. One or more Payload plasmid constructs (payload constructs) are processed into Payload Bacmid polynucleotides and transfected into the Payload VPC pool. The two VPC pools are incubated to produce Pl Rep/Cap Baculoviral Expression Vectors (BEVs) and Pl Payload BEVs. The two BEV pools are expanded into a collection of Plaques, with a single Plaque being selected for Clonal Plaque (CP) Purification (also referred to as Single Plaque Expansion). The process can include a single CP Purification step or can include multiple CP Purification steps either in series or separated by other processing steps. The one-or-more CP Purification steps provide a CP Rep/Cap BEV pool and a CP Payload BEV pool. These two BEV pools can then be stored and used for future production steps, or they can be then transfected into VPCs to produce a Rep/Cap BIIC pool and a Payload BIIC pool.
[00318] In certain embodiments, a viral production system or process of the present disclosure includes steps for producing AAV particles using Viral Production Cells (VPC) and baculovirus infected insect cells (BIICs). Viral Production Cells (VPCs) from a Cell Bank (CB) are thawed and expanded to provide a target working volume and VPC concentration. The working volume of Viral Production Cells is seeded into a Production Bioreactor and can be further expanded to a working volume of 200-2000 L with a target VPC concentration for BIIC infection. The working volume of VPCs in the Production Bioreactor is then co-infected with Rep/Cap BIICs and Payload BIICs, with a target VPC:BHC ratio and a target BIIC:BIIC ratio. VCD infection can also utilize BEVs. The co-infected VPCs are incubated and expanded in the Production Bioreactor to produce a bulk harvest of AAV particles and VPCs. Viral Expression Constructs
[00319] In various embodiments, the viral production system of the present disclosure includes one or more viral expression constructs that can be transfected/transduced into a viral production cell. In certain embodiments, a viral expression construct or a payload construct of the present disclosure can be a bacmid, also known as a baculovirus plasmid or recombinant baculovirus genome. In certain embodiments, the viral expression includes a protein-coding nucleotide sequence and at least one expression control sequence for expression in a viral production cell. In certain embodiments, the viral expression includes a protein-coding nucleotide sequence operably linked to least one expression control sequence for expression in a viral production cell. In certain embodiments, the viral expression construct contains parvoviral genes under control of one or more promoters. Parvoviral genes can include nucleotide sequences encoding non-structural AAV replication proteins, such as Rep genes which encode Rep52, Rep40, Rep68, or Rep78 proteins. Parvoviral genes can include nucleotide sequences encoding structural AAV proteins, such as Cap genes which encode VP1, VP2, and VP3 proteins.
[00320] Viral expression constructs of the present disclosure may include any compound or formulation, biological or chemical, which facilitates transformation, transfection, or transduction of a cell with a nucleic acid. Exemplary biological viral expression constructs include plasmids, linear nucleic acid molecules, and recombinant viruses including baculovirus. Exemplary chemical vectors include lipid complexes. Viral expression constructs are used to incorporate nucleic acid sequences into virus replication cells in accordance with the present disclosure. (O'Reilly, David R., Lois K. Miller, and Verne A. Luckow. Baculovirus expression vectors: a laboratory manual. Oxford University Press, 1994.);
Maniatis et al., eds. Molecular Cloning. CSH Laboratory, NY, N.Y. (1982); and, Philiport and Scluber, eds. Liposomes as tools in Basic Research and Industry. CRC Press, Ann Arbor, Mich. (1995), the contents of each of which are herein incorporated by reference in their entirety as related to viral expression constructs and uses thereof.
[00321] In certain embodiments, the viral expression construct is an AAV expression construct which includes one or more nucleotide sequences encoding non-structural AAV replication proteins, structural AAV capsid proteins, or a combination thereof.
[00322] In certain embodiments, the viral expression construct of the present disclosure may be a plasmid vector. In certain embodiments, the viral expression construct of the present disclosure may be a baculoviral construct.
[00323] The present disclosure is not limited by the number of viral expression constructs employed to produce AAV particles or viral vectors. In certain embodiments, one, two, three, four, live, six, or more viral expression constructs can be employed to produce AAV particles in viral production cells in accordance with the present disclosure. In certain embodiments of the present disclosure, a viral expression construct may be used for the production of an AAV particles in insect cells. In certain embodiments, modifications may be made to the wild type AAV sequences of the capsid and/or rep genes, for example to improve attributes of the viral particle, such as increased infectivity or specificity, or to enhance production yields.
[00324] In certain embodiments, the viral expression construct may contain a nucleotide sequence which includes start codon region, such as a sequence encoding AAV capsid proteins which include one or more start codon regions. In certain embodiments, the start codon region can be within an expression control sequence. The start codon can be ATG or a non-ATG codon (i.e., a suboptimal start codon where the start codon of the AAV VP1 capsid protein is a non-ATG).
[00325] In certain embodiments, the viral expression construct used for AAV production may contain a nucleotide sequence encoding the AAV capsid proteins where the initiation codon of the AAV VP1 capsid protein is a non-ATG, i.e. , a suboptimal initiation codon, allowing the expression of a modified ratio of the viral capsid proteins in the production system, to provide improved infectivity of the host cell. In a non-limiting example, a viral construct vector may contain a nucleic acid construct comprising a nucleotide sequence encoding AAV VP1, VP2, and VP3 capsid proteins, wherein the initiation codon for translation of the AAV VP1 capsid protein is CTG, TTG, or GTG, as described in US Patent No. US 8,163,543, the contents of which are herein incorporated by reference in their entirety as related to AAV capsid proteins and the production thereof.
[00326] In certain embodiments, the viral expression construct of the present disclosure may be a plasmid vector or a baculoviral construct that encodes the parvoviral rep proteins for expression in insect cells. In certain embodiments, a single coding sequence is used for the Rep78 and Rep52 proteins, wherein start codon for translation of the Rep78 protein is a suboptimal start codon, selected from the group consisting of ACG, TTG, CTG, and GTG, that effects partial exon skipping upon expression in insect cells, as described in US Patent No. 8,512,981, the contents of which are herein incorporated by reference in their entirety, for example to promote less abundant expression of Rep78 as compared to Rep52, which may promote high vector yields.
[00327] In certain embodiments, a VP-coding region encodes one or more AAV capsid proteins of a specific AAV serotype. The AAV serotypes for VP-coding regions can be the same or different. In certain embodiments, a VP-coding region can be codon optimized. In certain embodiments, a VP-coding region or nucleotide sequence can be codon optimized for a mammal cell. In certain embodiments, a VP- coding region or nucleotide sequence can be codon optimized for an insect cell. In certain embodiments, a VP-coding region or nucleotide sequence can be codon optimized for a Spodoptera frugiperda cell. In certain embodiments, a VP-coding region or nucleotide sequence can be codon optimized for Sf9 or SI21 cell lines.
[00328] In certain embodiments, a nucleotide sequence encoding one or more VP capsid proteins can be codon optimized to have a nucleotide homology with the reference nucleotide sequence of less than 100%. In certain embodiments, the nucleotide homology between the codon-optimized VP nucleotide sequence and the reference VP nucleotide sequence is less than 100%, less than 99%, less than 98%, less than 97%, less than 96%, less than 95%, less than 94%, less than 93%, less than 92%, less than 91%, less than 90%, less than 89%, less than 88%, less than 87%, less than 86%, less than 85%, less than 84%, less than 83%, less than 82%, less than 81%, less than 80%, less than 78%, less than 76%, less than 74%, less than 72%, less than 70%, less than 68%, less than 66%, less than 64%, less than 62%, less than 60%, less than 55%, less than 50%, and less than 40%. [00329] In certain embodiments, a viral expression construct or a payload construct of the present disclosure can be a bacmid, also known as a baculovirus plasmid or recombinant baculovirus genome. In certain embodiments, a viral expression construct or a payload construct of the present disclosure (e.g., bacmid) can include a polynucleotide incorporated by homologous recombination (transposon do nor/ acceptor system) into the bacmid by standard molecular biology techniques known and performed by a person skilled in the art.
[00330] In certain embodiments, the polynucleotide incorporated into the bacmid (i.e., polynucleotide insert) can include an expression control sequence operably linked to a protein-coding nucleotide sequence. In certain embodiments, the polynucleotide incorporated into the bacmid can include an expression control sequence which includes a promoter, such as plO or polh, and which is operably linked to a nucleotide sequence which encodes a structural AAV capsid protein (e.g., VP1, VP2, VP3 or a combination thereof). In certain embodiments, the polynucleotide incorporated into the bacmid can include an expression control sequence which includes a promoter, such as plO or polh, and which is operably linked to a nucleotide sequence which encodes a non-structural AAV capsid protein (e.g., Rep78, Rep52, or a combination thereof).
[00331] The method of the present disclosure is not limited by the use of specific expression control sequences. However, when a certain stoichiometry of VP products are achieved (close to 1:1: 10 for VP1, VP2, and VP3, respectively) and also when the levels of Rcp52 or Rcp40 (also referred to as the pl9 Reps) are significantly higher than Rep78 or Rep68 (also referred to as the p5 Reps), improved yields of AAV in production cells (such as insect cells) may be obtained. In certain embodiments, the p5/pl9 ratio is below 0.6 more, below 0.4, or below 0.3, but always at least 0.03. These ratios can be measured at the level of the protein or can be implicated from the relative levels of specific mRNAs.
[00332] In certain embodiments, AAV particles are produced in viral production cells (such as mammalian or insect cells) wherein all three VP proteins are expressed at a stoichiometry approaching, about or which is: 1:1:10 (VP1:VP2:VP3); 2:2:10 (VP1:VP2:VP3); 2:0:10 (VP1:VP2:VP3); 1-2:0-2:10 (VP1:VP2:VP3); 1-2:1-2:10 (VP1:VP2:VP3); 2-3:0-3:10 (VP1:VP2:VP3); 2-3:2-3:10 (VP1:VP2:VP3); 3:3:10 (VP1:VP2:VP3); 3-5:0-5:10 (VP1:VP2:VP3); or 3-5:3-5:10 (VP1:VP2:VP3).
[00333] In certain embodiments, the expression control regions are engineered to produce a VP1:VP2:VP3 ratio selected from the group consisting of: about or exactly 1:0:10; about or exactly 1 :1 :10; about or exactly 2:1 :10; about or exactly 2:1 :10; about or exactly 2:2: 10; about or exactly 3:0: 10; about or exactly 3:1:10; about or exactly 3:2:10; about or exactly 3:3:10; about or exactly 4:0:10; about or exactly 4: 1 : 10; about or exactly 4:2: 10; about or exactly 4:3:10; about or exactly 4:4: 10; about or exactly 5:5:10; about or exactly 1-2:0-2:10; about or exactly 1-2:1-2:10; about or exactly 1-3:0-3:10; about or exactly 1-3:1-3:10; about or exactly 1-4:0-4:10; about or exactly 1-4:1-4:10; about or exactly 1- 5:1-5:10; about or exactly 2-3:0-3:10; about or exactly 2-3:2-3:10; about or exactly 2-4:2-4:10; about or exactly 2-5:2-5:10; about or exactly 3-4:3-4:10; about or exactly 3-5:3-5:10; and about or exactly 4-5:4- 5:10.
[00334] In certain embodiments of the present disclosure, Rep52 or Rep78 is transcribed from the baculoviral derived polyhedron promoter (polh). Rep52 or Rep78 can also be transcribed from a weaker promoter, for example a deletion mutant of the ie-1 promoter, the Aie-1 promoter, has about 20% of the transcriptional activity of that ie-1 promoter. A promoter substantially homologous to the Aie-1 promoter may be used. In respect to promoters, a homology of at least 50%, 60%, 70%, 80%, 90% or more, is considered to be a substantially homologous promoter.
Mammalian Cells
[00335] Viral production of the present disclosure disclosed herein describes processes and methods for producing AAV particles or viral vector that contacts a target cell to deliver a payload construct, e.g., a recombinant AAV particle or viral construct, which includes a nucleotide encoding a payload molecule. The viral production cell may he selected from any biological organism, including prokaryotic (e.g., bacterial) cells, and eukaryotic cells, including, insect cells, yeast cells and mammalian cells.
[00336] In certain embodiments, the AAV particles of the present disclosure may be produced in a viral production cell that includes a mammalian cell. Viral production cells may comprise mammalian cells such as A549, WEH1, 3T3, 10T1/2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, HEK293, HEK293T (293T), Saos, C2C12, L cells, HT1080, Huh7, HepG2, C127, 3T3, CHO, HeLa cells, KB cells, BHK and primary fibroblast, hepatocyte, and myoblast cells derived from mammals. Viral production cells can include cells derived from any mammalian species including, but not limited to, human, monkey, mouse, rat, rabbit, and hamster or cell type, including but not limited to fibroblast, hepatocyte, tumor cell, cell line transformed cell, etc.
[00337] AAV viral production cells commonly used for production of recombinant AAV particles include, but is not limited to other mammalian cell lines as described in U.S. Pat. Nos. 6,156,303, 5,387,484, 5,741,683, 5,691,176, 6,428,988 and 5,688,676; U.S. patent application 2002/0081721, and International Patent Publication Nos. WO 00/47757, WO 00/24916, and WO 96/17947, the contents of each of which are herein incorporated by reference in their entireties insofar as they do no conflict with the present disclosure. In certain embodiments, the AAV viral production cells are trans-complementing packaging cell lines that provide functions deleted from a replication-defective helper virus, e.g., HEK293 cells or other Ea trans-complementing cells.
[00338] In certain embodiments, the packaging cell line 293-10-3 (ATCC Accession No. PTA-2361) may be used to produce the AAV particles, as described in US Patent No. US 6,281,010, the contents of which are herein incorporated by reference in their entirety as related to the 293-10-3 packaging cell line and uses thereof.
[00339] In certain embodiments, of the present disclosure a cell line, such as a HeLA cell line, for trans- complementing El deleted adenoviral vectors, which encoding adenovirus Ela and adenovirus Elb under the control of a phosphoglycerate kinase (PGK) promoter can be used for AAV particle production as described in US Patent No. 6365394, the contents of which are incorporated herein by reference in their entirety as related to the HeLa cell line and uses thereof.
[00340] In certain embodiments, AAV particles are produced in mammalian cells using a multiplasmid transient transfection method (such as triple plasmid transient transfection). In certain embodiments, the multiplasmid transient transfection method includes transfection of the following three different constructs: (i) a payload construct, (ii) a Rep/Cap construct (parvoviral Rep and parvoviral Cap), and (iii) a helper construct. In certain embodiments, the triple transfection method of the three components of AAV particle production may be utilized to produce small lots of virus for assays including transduction efficiency, target tissue (tropism) evaluation, and stability. In certain embodiments, the triple transfection method of the three components of AAV particle production may be utilized to produce large lots of materials for clinical or commercial applications.
[00341] AAV particles to be formulated may be produced by triple transfection or baculovirus mediated virus production, or any other method known in the art. Any suitable permissive or packaging cell known in the art may be employed to produce the vectors. In certain embodiments, trans-complementing packaging cell lines are used that provide functions deleted from a replication-defective helper virus, e.g., 293 cells or other Ela trans-complementing cells.
[00342] The gene cassette may contain some or all of the parvovirus (e.g., AAV) cap and rep genes. In certain embodiments, some or all of the cap and rep functions arc provided in trans by introducing a packaging vector(s) encoding the capsid and/or Rep proteins into the cell. In certain embodiments, the gene cassette does not encode the capsid or Rep proteins. Alternatively, a packaging cell line is used that is stably transformed to express the cap and/or rep genes.
[00343] Recombinant AAV virus particles are, in certain embodiments, produced and purified from culture supernatants according to the procedure as described in US2016/0032254, the contents of which are incorporated by reference in their entirety as related to the production and processing of recombinant AAV virus particles. Production may also involve methods known in the art including those using 293T cells, triple transfection or any suitable production method.
[00344] In certain embodiments, mammalian viral production cells (e.g., 293T cells) can be in an adhesion/adherent state (e.g., with calcium phosphate) or a suspension state (e.g., with polyethyleneimine (PEI)). The mammalian viral production cell is transfected with plasmids required for production of AAV, (i.e., AAV rep/cap construct, an adenoviral helper construct, and/or ITR flanked payload construct). In certain embodiments, the transfection process can include optional medium changes (e.g., medium changes for cells in adhesion form, no medium changes for cells in suspension form, medium changes for cells in suspension form if desired). In certain embodiments, the transfection process can include transfection mediums such as DMEM or F17. In certain embodiments, the transfection medium can include serum or can be serum-free (e.g., cells in adhesion state with calcium phosphate and with serum, cells in suspension state with PEI and without serum). [00345] Cells can subsequently be collected by scraping (adherent form) and/or pelleting (suspension form and scraped adherent form) and transferred into a receptacle. Collection steps can be repeated as necessary for full collection of produced cells. Next, cell lysis can be achieved by consecutive freezethaw cycles (-80C to 37C), chemical lysis (such as adding detergent triton), mechanical lysis, or by allowing the cell culture to degrade after reaching ~0% viability. Cellular debris is removed by centrifugation and/or depth filtration. The samples are quantified for AAV particles by DNase resistant genome titration by DNA qPCR.
[00346] AAV particle titers are measured according to genome copy number (genome particles per milliliter). Genome particle concentrations are based on DNA qPCR of the vector DNA as previously reported (Clark et al. (1999) Hum. Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol. Ther., 6:272- 278, the contents of which are each incorporated by reference in their entireties as related to the measurement of particle concentrations).
Insect cells
[00347] Viral production of the present disclosure includes processes and methods for producing AAV particles or viral vectors that contact a target cell to deliver a payload construct, e.g.. a recombinant viral construct, which includes a nucleotide encoding a payload molecule. In certain embodiments, the AAV particles or viral vectors of the present disclosure may be produced in a viral production cell that includes an insect cell.
[00348] Growing conditions for insect cells in culture, and production of heterologous products in insect cells in culture are well-known in the art, see U.S. Pat. No. 6,204,059, the contents of which are herein incorporated by reference in their entirety as related to the growth and use of insect cells in viral production.
[00349] Any insect cell which allows for replication of parvovirus and which can be maintained in culture can be used in accordance with the present disclosure. AAV viral production cells commonly used for production of recombinant AAV particles include, but is not limited to, Spodoptera frugiperda, including, but not limited to the Sf9 or Sf21 cell lines, Drosophila cell lines, or mosquito cell lines, such as Aedes albopictus derived cell lines. Use of insect cells for expression of heterologous proteins is well documented, as are methods of introducing nucleic acids, such as vectors, e.g., insect-cell compatible vectors, into such cells and methods of maintaining such cells in culture. See, for example, Methods in Molecular Biology, ed. Richard, Humana Press, NJ (1995); O'Reilly et al., Baculovirus Expression Vectors, A Laboratory Manual, Oxford Univ. Press (1994); Samulski et al., J. Vir.63:3822-8 (1989); Kajigaya et al., Proc. Nat’l. Acad. Sci. USA 88: 4646-50 (1991); Ruffing et al., J. Vir. 66:6922-30 (1992); Kimbauer et a/.,Vir.219:37-44 (1996); Zhao et al., Vir.272:382-93 (2000); and Samulski et al., U.S. Pat. No. 6,204,059, the contents of each of which are herein incorporated by reference in their entirety as related to the use of insect cells in viral production.
[00350] In some embodiments, the AAV particles arc made using the methods described in W02015/191508, the contents of which are herein incorporated by reference in their entirety insofar as they do not conflict with the present disclosure.
[00351] In certain embodiments, insect host cell systems, in combination with baculoviral systems (e.g., as described by Luckow etal., Bio/Technology 6: 47 (1988)) may be used. In certain embodiments, an expression system for preparing chimeric peptide is Trichoplusia ni, Tn 5B1-4 insect cells/baculoviral system, which can be used for high levels of proteins, as described in US Patent No. 6660521, the contents of which are herein incorporated by reference in their entirety as related to the production of viral particles.
[00352] Expansion, culturing, transfection, infection and storage of insect cells can be carried out in any cell culture media, cell transfection media or storage media known in the art, including Hyclone™ SFX- Insect™ Cell Culture Media, Expression System ESF AF™ Insect Cell Culture Medium, ThermoFisher Sf-900IT™ media, ThermoFisher Sf-900III™ media, or ThermoFisher Grace’s Insect Media. Insect cell mixtures of the present disclosure can also include any of the formulation additives or elements described in the present disclosure, including (but not limited to) salts, acids, bases, buffers, surfactants (such as Poloxamer 188/Pluronic F-68), and other known culture media elements. Formulation additives can be incorporated gradually or as “spikes” (incorporation of large volumes in a short time). Baculovirus-production systems
[00353] In certain embodiments, processes of the present disclosure can include production of AAV particles or viral vectors in a baculoviral system using a viral expression construct and a payload construct vector. In certain embodiments, the baculoviral system includes Baculovirus expression vectors (BEVs) and/or baculovirus infected insect cells (BIICs). In certain embodiments, a viral expression construct or a payload construct of the present disclosure can be a bacmid, also known as a baculovirus plasmid or recombinant baculovirus genome. In certain embodiments, a viral expression construct or a payload construct of the present disclosure can be polynucleotide incorporated by homologous recombination (transposon donor/acceptor system) into a bacmid by standard molecular biology techniques known and performed by a person skilled in the art. Transfection of separate viral replication cell populations produces two or more groups (e.g. two, three) of baculo viruses (BEVs), one or more group which can include the viral expression construct (Expression BEV), and one or more group which can include the pay load construct (Payload BEV). The baculoviruses may be used to infect a viral production cell for production of AAV particles or viral vector.
[00354] In certain embodiments, the process includes transfection of a single viral replication cell population to produce a single baculovirus (BEV) group which includes both the viral expression construct and the payload construct. These baculoviruses may be used to infect a viral production cell for production of AAV particles or viral vector.
[00355] In certain embodiments, BEVs are produced using a Bacmid Transfection agent, such as Promega FuGENE® HD, WFI water, or ThermoFisher Cellfectin® II Reagent. In certain embodiments, BEVs are produced and expanded in viral production cells, such as an insect cell.
[00356] In certain embodiments, the method utilizes seed cultures of viral production cells that include one or more BEVs, including baculovirus infected insect cells (BIICs). The seed BIICs have been transfected/transduced/infected with an Expression BEV which includes a viral expression construct, and also a Payload BEV which includes a payload construct. In certain embodiments, the seed cultures are harvested, divided into aliquots and frozen, and may be used at a later time to initiate transfection/transduction/infection of a naive population of production cells. In certain embodiments, a bank of seed BIICs is stored at -80 °C or in LN2 vapor.
[00357] Baculoviruses are made of several essential proteins which are essential for the function and replication of the Baculovirus, such as replication proteins, envelope proteins and capsid proteins. The Baculovirus genome thus includes several essential-gene nucleotide sequences encoding the essential proteins. As a non-limiting example, the genome can include an essential-gene region which includes an essential-gene nucleotide sequence encoding an essential protein for the Baculovirus construct. The essential protein can include: GP64 baculovirus envelope protein, VP39 baculovirus capsid protein, or other similar essential proteins for the Baculovirus construct.
[00358] Baculovirus expression vectors (BEV) for producing AAV particles in insect cells, including but not limited to Spodoptera frugiperda (Sf9) cells, provide high titers of viral vector product. Recombinant baculovirus encoding the viral expression construct and payload construct initiates a productive infection of viral vector replicating cells. Infectious baculovirus particles released from the primary infection secondarily infect additional cells in the culture, exponentially infecting the entire cell culture population in a number of infection cycles that is a function of the initial multiplicity of infection, see Urabe, M. et al. J Virol. 2006 Feb;80(4): 1874-85, the contents of which are herein incorporated by reference in their entirety as related to the production and use of BEVs and viral particles.
[00359] Production of AAV particles with baculovirus in an insect cell system may address known baculovirus genetic and physical instability.
[00360] In certain embodiments, the production system of the present disclosure addresses baculovirus instability over multiple passages by utilizing a titerless infected-cells preservation and scale-up system. Small scale seed cultures of viral producing cells are transfected with viral expression constructs encoding the structural and/or non- structural components of the AAV particles. Baculovirus-infected viral producing cells are harvested into aliquots that may be cryopreserved in liquid nitrogen; the aliquots retain viability and infectivity for infection of large scale viral producing cell culture. Wasilko DI et al. Protein Expr Purif. 2009 Iun:65(2): 122-32, the contents of which are herein incorporated by reference in their entirety as related to the production and use of BEVs and viral particles.
[00361] A genetically stable baculovirus may be used to produce a source of the one or more of the components for producing AAV particles in invertebrate cells. In certain embodiments, defective baculovirus expression vectors may be maintained episomally in insect cells. In such embodiments, the corresponding bacmid vector is engineered with replication control elements, including but not limited to promoters, enhancers, and/or cell-cycle regulated replication elements.
[00362] In certain embodiments, stable viral producing cells permissive for baculovirus infection are engineered with at least one stable integrated copy of any of the elements necessary for AAV replication and vector production including, but not limited to, the entire AAV genome, Rep and Cap genes, Rep genes, Cap genes, each Rep protein as a separate transcription cassette, each VP protein as a separate transcription cassette, the AAP (assembly activation protein), or at least one of the baculovirus helper genes with native or non-native promoters.
[00363] In some embodiments, the AAV particle of the present disclosure may be produced in insect cells (e.g., Sf9 cells).
[00364] In some embodiments, the AAV particle of the present disclosure may be produced using triple transfection.
[00365] In some embodiments, the AAV particle of the present disclosure may be produced in mammalian cells.
[00366] In some embodiments, the AAV particle of the present disclosure may be produced by triple transfection in mammalian cells.
[00367] In some embodiments, the AAV particle of the present disclosure may be produced by triple transfection in HEK293 cells.
[00368] The AAV viral genomes encoding SMN protein described herein may be useful in the fields of human disease, veterinary applications and a variety of in vivo and in vitro settings. The AAV particles of the present disclosure may be useful in the field of medicine for the treatment, prophylaxis, palliation, or amelioration of neurological or neuromuscular diseases and/or disorders. In some embodiments, the AAV particles of the disclosure are used for the prevention and/or treatment of SMA-related disorders. [00369] Various embodiments of the disclosure herein provide a pharmaceutical composition comprising the AAV particle described herein and a pharmaceutically acceptable excipient.
[00370] Various embodiments of the disclosure herein provide a method of treating a subject in need thereof comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition described herein.
[00371] Certain embodiments of the method provide that the subject is treated by a route of administration of the pharmaceutical composition selected from the group consisting of: intravenous, intracerebroventricular, intraparenchymal, intrathecal, subpial, and intramuscular, or a combination thereof. Certain embodiments of the method provide that the subject is treated for SMA-related disorders and/or other neurological disorder arising from a deficiency in the quantity or function of SMN gene products. In one aspect of the method, a pathological feature of the SMA-related disorders or the other neurological disorder is alleviated and/or the progression of the SMA-related disorders or the other neurological disorder is halted, slowed, ameliorated, or reversed. [00372] Various embodiments of the disclosure herein describe a method of increasing the level of SMN protein in the central nervous system of a subject in need thereof comprising administering to said subject via infusion, an effective amount of the pharmaceutical composition described herein.
[00373] Also described herein are compositions, methods, processes, kits and devices for the design, preparation, manufacture and/or formulation of AAV particles. In some embodiments, payloads, such as but not limited to payloads comprising SMN protein, may be encoded by payload constructs or contained within plasmids or vectors or recombinant adeno-associated viruses (AAVs).
[00374] The present disclosure also provides administration and/or delivery methods for vectors and viral particles, e.g., AAV particles, for the treatment or amelioration of SMA-related disorders. Such methods may involve gene replacement or gene activation. Such outcomes are achieved by utilizing the methods and compositions taught herein.
III. Pharmaceutical Compositions
[00375] The present disclosure additionally provides a method for treating SMA-related disorders and disorders related to deficiencies in the function or expression of SMN protein(s) in a mammalian subject, including a human subject, comprising administering to the subject any of the AAV polynucleotides or AAV genomes described herein (i.e., “vector genomes,” “viral genomes,” or “VGs”) or administering to the subject a particle comprising said AAV polynucleotide or AAV genome, or administering to the subject any of the described compositions, including pharmaceutical compositions.
[00376] As used herein the term “composition” comprises an AAV polynucleotide or AAV genome or AAV particle and at least one excipient.
[00377] As used herein the term “pharmaceutical composition” comprises an AAV polynucleotide or AAV genome or AAV particle and one or more pharmaceutically acceptable excipients.
[00378] Although the descriptions of pharmaceutical compositions, e.g., AAV comprising a payload encoding an SMN protein to be delivered, provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g., non-human mammals. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.
[00379] In some embodiments, compositions are administered to humans, human patients, or subjects. [00380] In some embodiments, the AAV particle formulations described herein may contain a nucleic acid encoding at least one payload. In some embodiments, the formulations may contain a nucleic acid encoding 1, 2, 3, 4, or 5 payloads. In some embodiments, the formulation may contain a nucleic acid encoding a payload construct encoding proteins selected from categories such as, but not limited to, human proteins, veterinary proteins, bacterial proteins, biological proteins, antibodies, immunogenic proteins, therapeutic peptides and proteins, secreted proteins, plasma membrane proteins, cytoplasmic proteins, cytoskeletal proteins, intracellular membrane bound proteins, nuclear proteins, proteins associated with human disease, and/or proteins associated with non-human diseases. In some embodiments, the formulation contains at least three payload constructs encoding proteins. Certain embodiments provide that at least one of the payloads is SMN protein or a variant thereof.
[00381] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
IV. Formulations
[00382] Formulations of the AAV pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.
[00383] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
[00384] For example, the composition may comprise between 0.1% and 99% (w/w) of the active ingredient. By way of example, the composition may comprise between 0.1% and 100%, e.g., between .5% and 50%, between 1-30%, between 5-80%, or at least 80% (w/w) active ingredient.
[00385] The AAV particles of the disclosure can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection or transduction; (3) permit the sustained or delayed release; (4) alter the biodistribution (e.g., target the viral particle to specific tissues or cell types); (5) increase the translation of encoded protein in vivo; (6) alter the release profile of encoded protein in vivo and/or (7) allow for regulatable expression of the payload.
[00386] Formulations of the present disclosure can include, without limitation, saline, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with viral vectors (e.g., for transplantation into a subject), nanoparticle mimics and combinations thereof. Further, the viral vectors of the present disclosure may be formulated using selfassembled nucleic acid nanoparticles.
[00387] In some embodiments, the viral vectors encoding SMN protein may be formulated to optimize baricity and/or osmolality. In some embodiments, the baricity and/or osmolality of the formulation may be optimized to ensure optimal drug distribution in the central nervous system or a region or component of the central nervous system.
[00388] In some embodiments, the AAV particles of the disclosure may be formulated in PBS with 0.001% of pluronic acid (F-68) at a pH of about 7.0.
[00389] In some embodiments, the AAV particles of the disclosure may be formulated in PBS, in combination with an ethylene oxide/propylene oxide copolymer (also known as pluronic or poloxamer). [00390] In some embodiments, the AAV particles of the disclosure may be formulated in PBS with 0.001% pluronic acid (F-68) (poloxamer 188) at a pH of about 7.0.
[00391] In some embodiments, the AAV particles of the disclosure may be formulated in PBS with 0.001% pluronic acid (F-68) (poloxamer 188) at a pH of about 7.3.
[00392] In some embodiments, the AAV particles of the disclosure may be formulated in PBS with 0.001% pluronic acid (F-68) (poloxamer 188) at a pH of about 7.4.
[00393] In some embodiments, the AAV particles of the disclosure may be formulated in a solution comprising sodium chloride, sodium phosphate and an ethylene oxide/propylene oxide copolymer. [00394] In some embodiments, the AAV particles of the disclosure may be formulated in a solution comprising sodium chloride, sodium phosphate dibasic, potassium chloride, potassium phosphate monobasic, and poloxamer 188/pluronic acid (F-68).
[00395] In some embodiments, the AAV particles of the disclosure may be formulated in a solution comprising 192 mM sodium chloride, 10 mM sodium phosphate (dibasic), 2.7 mM potassium chloride, 2 mM potassium phosphate (monobasic) and 0.001% pluronic F-68 (v/v), at pH 7.4. This formulation is referred to as Formulation 1 in the present disclosure.
[00396] In some embodiments, the AAV particles of the disclosure may be formulated in a solution comprising about 192 mM sodium chloride, about lOmM sodium phosphate dibasic and about 0.001% poloxamer 188, at a pH of about 7.3. The concentration of sodium chloride in the final solution may be 150 mM-200 mM. As non-limiting examples, the concentration of sodium chloride in the final solution may be 150 mM, 160 mM, 170 mM, 180 mM, 190 mM or 200 mM. The concentration of sodium phosphate dibasic in the final solution may be 1 mM-50 mM. As non-limiting examples, the concentration of sodium phosphate dibasic in the final solution may be 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, or 50 mM. The concentration of poloxamer 188 (pluronic acid (F-68)) may be 0.0001%-l%. As non-limiting examples, the concentration of poloxamer 188 (pluronic acid (F-68)) may be 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, or 1%. The final solution may have a pH of 6.8-7.7. Non-limiting examples for the pH of the final solution include a pH of 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, or 7.7.
[00397] In some embodiments, the AAV particles of the disclosure may be formulated in a solution comprising about 1.05% sodium chloride, about 0.212% sodium phosphate dibasic, heptahydrate, about 0.025% sodium phosphate monobasic, monohydrate, and 0.001% poloxamer 188, at a pH of about 7.4. As a non-limiting example, the concentration of AAV particle in this formulated solution may be about 0.001%. The concentration of sodium chloride in the final solution may be 0.1-2.0%, with non-limiting examples of 0.1%, 0.25%, 0.5%, 0.75%, 0.95%, 0.96%, 0.97%, 0.98%, 0.99%, 1.00%, 1.01%, 1.02%, 1.03%, 1.04%, 1.05%, 1.06%, 1.07%, 1.08%, 1.09%, 1.10%, 1.25%, 1.5%, 1.75%, or 2%. The concentration of sodium phosphate dibasic in the final solution may be 0.100-0.300% with non-limiting examples including 0.100%, 0.125%, 0.150%, 0.175%, 0.200%, 0.210%, 0.211%, 0.212%, 0.213%, 0.214%, 0.215%, 0.225%, 0.250%, 0.275%, 0.300%. The concentration of sodium phosphate monobasic in the final solution may be 0.010-0.050%, with non-limiting examples of 0.010%, 0.015%, 0.020%, 0.021%, 0.022%, 0.023%, 0.024%, 0.025%, 0.026%, 0.027%, 0.028%, 0.029%, 0.030%, 0.035%, 0.040%, 0.045%, or 0.050%. The concentration of poloxamer 188 (pluronic acid (F-68)) may be 0.0001 %-1 %. As non-limiting examples, the concentration of poloxamer 188 (pluronic acid (F-68)) may be 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, or 1%. The final solution may have a pH of 6.8-7.7. Non-limiting examples for the pH of the final solution include a pH of 6.8, 6.9, 7.0, 7.1, 7.2, 73, 7.4, 7.5, 7.6, or 7.7.
Excipients
[00398] The formulations of the disclosure can include one or more excipients, each in an amount that together increases the stability of the AAV particle, increases cell transfection or transduction by the viral particle, increases the expression of viral particle encoded protein, and/or alters the release profile of AAV particle encoded proteins. In some embodiments, a pharmaceutically acceptable excipient may be at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use for humans and for veterinary use. In some embodiments, an excipient may be approved by United States Food and Drug Administration. In some embodiments, an excipient may be of pharmaceutical grade. In some embodiments, an excipient may meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
[00399] Excipients, which, as used herein, include, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21 st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006; the contents of which are herein incorporated by reference in their entirety). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.
Inactive Ingredients
[00400] In some embodiments, AAV formulations may comprise at least one excipient which is an inactive ingredient. As used herein, the term “inactive ingredient” refers to one or more agents that do not contribute to the activity of the pharmaceutical composition included in formulations. In some embodiments, all, none, or some of the inactive ingredients which may be used in the formulations of the present disclosure may be approved by the US Food and Drug Administration (FDA).
[00401] Formulations of AAV particles disclosed herein may include cations or anions. In some embodiments, the formulations include metal cations such as, but not limited to, Zn2+, Ca2+, Cu2+, Mg+, or combinations thereof. In some embodiments, formulations may include polymers or polynucleotides complexed with a metal cation (see, e.g., U.S. Pat. Nos. 6,265,389 and 6,555,525, the contents of each of which are herein incorporated by reference in their entirety).
V. Uses and Applications
[00402] The compositions of the disclosure may be administered to a subject or used in the manufacture of a medicament for administration to a subject having a deficiency in the quantity or function of SMN protein or having a disease or condition associated with decreased SMN protein expression. In some embodiments, the disease is SMA with a mutation in an SMN1 gene. In certain embodiments, the AAV particles including SMN protein may be administered to a subject to treat SMA e.g., as SMA associated with a mutation in an SMN gene. In some embodiments, administration of the AAV particles comprising viral genomes that encode SMN protein may protect central nervous system pathways from degeneration. The compositions and methods described herein are also useful for treating other SMA-related disorders. [00403] In some embodiments, the delivery of the AAV particles may halt or slow progression of SMA- related disorders. In certain embodiments, the delivery of the AAV particles improves symptoms of SMA-related disorders, including, for example, physical, and sensory symptoms of SMA-related disorders.
[00404] In some embodiments, the present disclosure encompasses the delivery of pharmaceutical, prophylactic, diagnostic, or imaging compositions in combination with agents that may improve their bioavailability, reduce and/or modify their metabolism, and/or modify their distribution within the body. [00405] In certain embodiments, the pharmaceutical compositions described herein are used as research tools, particularly in in vitro investigations using human cell lines such as HEK293T and in vivo testing in nonhuman primates which will occur prior to human clinical trials.
CNS diseases
[00406] The present disclosure provides a method for treating a disease, disorder and/or condition in a mammalian subject, including a human subject, comprising administering to the subject any of the viral particles e.g., AAV, AAV particle, or AAV genome that produces SMN protein described herein (z'.e., viral genomes or “VG”) or administering to the subject a particle comprising said AAV particle or AAV genome, or administering to the subject any of the described compositions, including pharmaceutical compositions.
[00407] In some embodiments, AAV particles of the present disclosure, through delivery of a functional payload that is a therapeutic product comprising an SMN protein or variant thereof that can modulate the level or function of a gene product in the CNS.
[00408] A functional payload may alleviate or reduce symptoms that result from abnormal level and/or function of a gene product (e.g., an absence or defect in a protein) in a subject in need thereof or that otherwise confers a benefit to a CNS disorder in a subject in need thereof.
[00409] As non-limiting examples, companion or combination therapeutic products delivered by AAV particles of the present disclosure may include, but are not limited to, growth and trophic factors, cytokines, hormones, neurotransmitters, enzymes, anti-apoptotic factors, angiogenic factors, SMN proteins, and any protein known to be mutated in pathological disorders such as SMA-related disorders. [00410] In some embodiments, AAV particles of the present disclosure may be used to treat diseases that are associated with impairments of the growth and development of the CNS, i.e., neurodevelopmental disorders. In some aspects, such neurodevelopmental disorders may be caused by genetic mutations.
[00411] In some embodiments, the neurological disorders may be functional neurological disorders with motor and/or sensory symptoms which have neurological origin in the CNS. As non-limiting examples, functional neurological disorders may be chronic pain, seizures, speech problems, involuntary movements, or sleep disturbances.
[00412] In some embodiments, the AAV particles herein are administered to subjects having SMA, treatment of spinal muscular atrophy (SMA), such as SMA is infantile SMA, intermediate SMA, juvenile SMA or adult-onset SMA.
[00413] Neonatal SMA (Type 0 SMA; before birth): Type 0, also known as very severe SMA, is the most severe form of SMA and begins before birth. Usually, the first symptom of type 0 is reduced movement of the fetus that is first seen between 30 and 36 weeks of the pregnancy. After birth, these newborns have little movement and have difficulties with swallowing and breathing.
[00414] Infantile SMA (Type 1 SMA or Werdnig-Hoffmann disease; generally 0-6 months): Type 1 SMA, also known as severe infantile SMA or Werdnig Hoffmann disease, is very severe, and manifests at birth or within 6 months of life. Patients have profound flaccid symmetrical weakness and hypotonia and are unable to sit without support. Bulbar denervation results in tongue weakness and fasciculation with poor suckling and swallowing. Death usually occurs within the first 2 years without ventilatory support. [00415] Intermediate SMA (Type 2 SMA or Dubowitz disease; generally 6-18 months): Patients with Type 2 SMA, or intermediate SMA, achieve the ability to sit unsupported, but are unable to stand or walk unaided. The onset of weakness is usually recognized sometime between 6 and 18 months. Prognosis in this group is largely dependent on the degree of respiratory involvement.
[00416] Juvenile SMA (Type 3 or Kugelberg- Welander disease; generally >18 months): Type 3 SMA describes those who are able to walk independently at some point during their disease course, but often become wheelchair bound during youth or adulthood.
[00417] Adult SMA (Type 4 SMA): Weakness usually begins in late adolescence in tongue, hands, or feet then progresses to other areas of the body. The course of adult disease is much slower and has little or no impact on life expectancy.
[00418] AAV Particles and methods of using the AAV particles described herein may be used to prevent, manage and/or treat SMA, e.g., an SMA associated with a mutation in an SMN gene.
[00419] In some aspects, a subject having SMA has one or more symptoms of SMA (e.g., atrophy of the limb muscles, difficulty or inability walking, difficulty breathing, or other symptom of SMA). In some aspects, a subject having SMA has two mutant alleles of the genomic SMN1 gene. In some aspects, the subject has a deletion or a loss of function point mutation in each SMN1 allele. In some aspects, the subject is homozygous for an SMN1 gene mutation. In some aspects, the subject is heterozygous for two different SMN1 gene mutations.
[00420] In some aspects, the subject is a human subject. In some aspects, the subject is selected from the pediatric and adult population. In some aspects, the subject is greater than or equal to 18 years of age (e.g., 18 years of age or older). In some aspects, the subject is younger than 18 years of age, younger than 10 years of age, or younger than 6 years of age.
[00421] In some aspects, the subject is around 2 weeks, 1 month, 3 months, 6 months, 1 year, 2 years, 3 years, 4 years, or 5 years of age.
[00422] Methods of determining if a subject has SMA or is pre-disposed to develop SMA are known in the art. For example, in order to identify subjects who would benefit from treatment with an SMA therapy (such as the one or more of the SMA therapies described above), there are several known biomarkers for SMA. These include prognostic biomarkers such as SMN2 copy number as an indicator of disease severity; disease progression biomarkers such as Compound Muscle Action Potential (CMAP) amplitude that serves as an indicator of motor neuron loss; predictive biomarkers such as reduced CMAP amplitude that is indicative of less response to SMN restoring therapies; pharmacodynamic biomarkers such as increased full-length SMN transcripts and/or increased SMN protein as indicators of effective induction of the SMN2 gene; and surrogate end point biomarkers such as increased Motor unit number estimation (MUNE) as an indicator of improved physical function. WO 2019/147960 discloses neurofilament levels, the predominant cytoskeletal element of nerve cells, as a novel biomarker for SMA. [00423] The compositions provided herein (e.g., AAV viral particles) can also be used to treat SMA- related disorders, e.g., in which increased SMN expression improves nerve regeneration. For example, increased SMN expression improved nerve regeneration in mice following sciatic nerve injury, and SMN overexpression can reduce aging-related motor unit losses and improve regeneration and maintenance at the neuromuscular junction (NMJ). For example, sarcopenia, the age-related wasting and loss of strength, is an important neuromuscular problem of aging. It affects up to 50% of individuals by the 8th decade, and can lead to impaired mobility, loss of independence, and increased risk of mortality (WO/2017/08748).
[00424] The individual having sarcopenia which can be treated with the composition provided herein can be of any age. Specifically, the individual can be 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 years old, or younger, older, or any amount in between. For example, the subject can be 35 years old or older. In one embodiment, the subject has not been diagnosed with spinal muscular atrophy (SMA). In another embodiment, the subject has been tested for SMA and it has been determined that the subject does not have SMA. SMA differs from sarcopenia and nerve injuries in multiple ways. Spinal muscular atrophy ( SMA) is a neurological disorder characterized by loss of function of the anterior horn cells in the spinal cord that results from reduced levels of SMN protein as a result of homozygous mutation of the SMN1 gene.
[00425] In some embodiments, the neurological or neuromuscular disease, disorder, and/or condition is SMA-related disorders. In some embodiments, the delivery of the AAV particles may halt or slow the disease progression of SMA-related disorders by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95% using a known analysis method and comparator group for SMA-related disorders. As a non-limiting example, the delivery of the AAV particles may halt or slow progression of SMA-related disorders as measured according to the assays described herein.
[00426] In some embodiments, the AAV particles described herein increase the amount of SMN protein in a tissue by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more than 100%. In some embodiments, the AAV particle encoding a payload may increase the amount of SMN protein in a tissue to be comparable to (e.g., approximately the same as) the amount of SMN protein in the corresponding tissue of a healthy subject. In some embodiments, the AAV particle encoding a payload may increase the amount of SMN protein in a tissue effective to reduce one or more symptoms of a disease associated with decreased SMN protein expression or a deficiency in the quantity and/or function of SMN protein.
[00427] In some embodiments, the AAV particles and AAV vector genomes described herein, upon administration to subject or introduction to a target cell, increase SMN activity 2-3 fold over baseline SMN activity. In the case of subjects or target cells with deficient SMN activity, as in the case of subjects having an SMA-related disorder or cells or tissues harboring a mutation in an SMN gene, the AAV particles and AAV vector genomes described herein restore SMN activity to normal levels, as defined by SMN activity levels in subjects, tissues, and cells not afflicted with an SMA-related disorder or not harboring an SMN gene mutation. In some embodiments, the AAV particles and AAV vector genomes described herein effectively reduce a-synuclein levels in subjects having an SMA-related disorder or cells or tissues harboring a mutation in an SMN gene. In some embodiments, the AAV particles and AAV vector genomes described herein effectively prevent a-synuclein mediated pathology.
Therapeutic applications
[00428] The present disclosure additionally provides methods for treating non-infectious diseases and/or disorders in a mammalian subject, including a human subject, comprising administering to the subject any of the AAV particles or pharmaceutical compositions described herein. In some embodiments, non- infectious diseases and/or disorders treated according to the methods described herein include, but are not limited to, Spinal muscular atrophy (SMA), (e.g., SMA associated with a mutation in an SMN gene). [00429] The present disclosure provides a method for administering to a subject in need thereof, including a human subject, a therapeutically effective amount of the AAV particles of the invention to slow, stop or reverse disease progression. As a non-limiting example, disease progression may be measured by tests or diagnostic tool(s) known to those skilled in the art. As another non-limiting example, disease progression may be measured by change in the pathological features of the brain, CSF, or other tissues of the subject.
[00430] In another aspect, a peptide described herein is fused or coupled, e.g., conjugated, to an active agent. In some embodiments, the active agent is a therapeutic agent. In some embodiments, the active agent comprises a therapeutic protein (e.g., an SMN protein), an antibody molecule, an enzyme, one or more components of a genome editing system, an Fc polypeptide fused or coupled (e.g., covalently or non-covalently) to a therapeutic agent, and/or an RNAi agent (e.g., a dsRNA, antisense oligonucleotide (ASO), siRNA, shRNA, pre-miRNA, pri-miRNA, miRNA, stRNA, IncRNA, piRNA, or snoRNA). In some embodiments, the therapeutic agent is an antibody. In some embodiments, a peptide described herein is fused or coupled, e.g., conjugated (e.g., directly or indirectly) to the Fc region of the antibody, e.g., at the C-terminus of the Fc region or the N-terminus of the Fc region. In some embodiments, the therapeutic agent is an RNAi agent. In some embodiments, the RNAi agent is a siRNA or an ASO. In some embodiments, the ASO or siRNA comprises at least one (e.g., one or more or all) modified nucleotides. In some embodiments, a peptide described herein is fused or coupled, e.g., conjugated (e.g., directly or indirectly via a linker), to at least one strand of the RNAi agent. In some embodiments, a peptide described herein is conjugated, e.g., directly or indirectly via a linker, to the C-terminus of at least one strand of the RNAi agent. In some embodiments, a peptide described herein is conjugated, e.g., directly or indirectly via a linker, to an internal nucleotide of at least one strand of the RNAi agent. In some embodiments, the at least one strand is the sense strand. In some embodiments, the therapeutic agent modulates, e.g., inhibits, decreases, or increases, expression of a CNS related gene, mRNA, and/or protein.
[00431] In some embodiments, the active agent is a diagnostic agent. In some embodiments, the diagnostic agent is or comprises an imaging agent (e.g., a protein or small molecule compound coupled to a detectable moiety). In some embodiments, the imaging agent comprises a PET or MRI ligand, or an antibody molecule coupled to a detectable moiety. In some embodiments, the detectable moiety is or comprises a radiolabel, a fluorophore, a chromophore, or an affinity tag. In some embodiments, the radiolabel is or comprises tc99m, iodine-123, a spin label, iodine-131, indium-111, fluorine-19, carbon- 13, nitrogen- 15, oxygen-17, gadolinium, manganese, or iron. In some embodiments, the active agent is a small molecule. In some embodiments, the active agent is a ribonucleic acid complex (e.g., a Cas9/gRNA complex), a plasmid, a closed-end DNA, a circ-RNA, or an mRNA.
[00432] In some embodiments, at least 1-5, e.g., at least 1, 2, 3, 4, or 5, peptides are fused or coupled, e.g., conjugated, to an active agent, e.g., a therapeutic agent or a diagnostic agent. In some embodiments, the at least 1-5, e.g., at least 1, 2, 3, 4, or 5, peptides comprise the same amino acid sequence. In some embodiments, the at least 1-5, e.g., at least 1, 2, 3, 4, or 5, peptides comprise different amino acid sequences. In some embodiments, the at least 1-5, e.g., at least 1, 2, 3, 4, or 5, peptides are present in tandem (e.g., connected directly or indirectly via a linker) or in a multimeric configuration. In some embodiments, the peptide comprises an amino acid sequence of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, or 35 amino acids in length.
[00433] In some embodiments, the peptide covalently linked, e.g., directly or indirectly via a linker, to the active agent. In some embodiments, the peptide is conjugated to the active agent via a linker. In some embodiments, the linker is a cleavable linker or a non-cleavable linker. In some embodiments, the cleavable linker is a pH sensitive linker or an enzyme sensitive linker. In some embodiments, the pH sensitive linker comprises a hydrazine/hydrazone linker or a disulfide linker. In some embodiments, the enzyme sensitive linker comprises a peptide based linker, e.g., a peptide linker sensitive to a protease (e.g., a lysosomal protease); or a beta-glucuronide linker. In some embodiments, the non-cleavable linker is a linker comprising a thioether group or a maleimidocaproyl group. In some embodiments, the peptide and the active agent are fused or coupled post-translationally, e.g., using click chemistry. In some embodiments, the peptide and the active agent are fused or couple via chemically induced dimerization. In some embodiments, the peptide is present N-terminal relative to the active agent. In some embodiments, the peptide is present C-terminal relative to the active agent.
[00434] In some embodiments, the peptide is present or coupled to a carrier. In some embodiments, the carrier comprises an exosome, a microvesicle, or a lipid nanoparticle (LNP). In some embodiments, the carrier comprises a therapeutic agent (e.g., an RNAi agent (e.g., an dsRNA, a siRNA, a shRNA, a pre- miRNA, a pri-miRNA, a miRNA, a stRNA, a IncRNA, a piRNA, an antisense oligonucleotide agent (ASO), or a snoRNA), an mRNA, a ribonucleoprotein complex (e.g., a Cas9/gRNA complex), or a
Yin circRNA). In some embodiments, the peptide is present on the surface of the carrier. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the surface of the carrier comprises at least 1-5, e.g., at least 1, 2, 3, 4, or 5, peptides described herein.
[00435] The present disclosure also provides a nucleic acid or polynucleotide encoding any of the peptides described herein and AAV capsid variants, AAV particles, vectors, and cells comprising the same.
VI. Dosing and Administration
Administration
[00436] In some aspects, the present disclosure provides administration and/or delivery methods for vectors and viral particles, e.g., AAV particles, encoding SMN protein or a variant thereof, for the prevention, treatment, or amelioration of diseases or disorders of the CNS. For example, administration of the AAV particles prevents, treats, or ameliorates SMA-related disorders. Thus, robust widespread SMN protein distribution throughout the CNS and periphery is desired for maximal efficacy. Particular target tissues for administration or delivery include CNS tissues, brain tissue, and, more specifically, caudate-putamen, thalamus, superior colliculus, cortex, and corpus collosum. Particular embodiments provide administration and/or delivery of the AAV particles and AAV vector genomes described herein to caudate-putamen and/or substantia nigra. Other particular embodiments provide administration and/or delivery of the AAV particles and AAV vector genomes described herein to thalamus.
[00437] The AAV particles of the present disclosure may be administered by any route which results in a therapeutically effective outcome. These include, but are not limited to, enteral (into the intestine), gastroenteral, epidural (into the dura matter), oral (by way of the mouth), transdermal, peridural, intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), intracranial (into the skull), picutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intraparenchymal (into the substance of), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intravesicular infusion, intravitreal, (through the eye), intracavernous injection (into a pathologic cavity) intracavitary (into the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through the intact skin for systemic distribution), transmucosal (diffusion through a mucous membrane), transvaginal, insufflation (snorting), sublingual, sublabial, enema, eye drops (onto the conjunctiva), in ear drops, auricular (in or by way of the ear), buccal (directed toward the cheek), conjunctival, cutaneous, dental (to a tooth or teeth), electro-osmosis, endocervical, endosinusial, endotracheal, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, intra-amniotic, intraarticular, intrabiliary, intrabronchial, intrabursal, intracartilaginous (within a cartilage), intracaudal (within the cauda equine), intracisternal (within the cisterna magna cerebellomedularis), intracorneal (within the cornea), dental intracoronal, intracoronary (within the coronary arteries), intracorporus cavernosum (within the dilatable spaces of the corporus cavernosa of the penis), intradiscal (within a disc), intraductal (within a duct of a gland), intraduodenal (within the duodenum), intradural (within or beneath the dura), intraepidermal (to the epidermis), intraesophageal (to the esophagus), intragastric (within the stomach), intragingival (within the gingivae), intraileal (within the distal portion of the small intestine), intralesional (within or introduced directly to a localized lesion), intraluminal (within a lumen of a tube), intralymphatic (within the lymph), intramedullary (within the marrow cavity of a bone), intrameningeal (within the meninges), intraocular (within the eye), intraovarian (within the ovary), intrapericardial (within the pericardium), intrapleural (within the pleura), intraprostatic (within the prostate gland), intrapulmonary (within the lungs or its bronchi), intrasinal (within the nasal or periorbital sinuses), intraspinal (within the vertebral column), intrasynovial (within the synovial cavity of a joint), intratendinous (within a tendon), intratesticular (within the testicle), intrathecal (within the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (within the thorax), intratubular (within the tubules of an organ), intratumor (within a tumor), intratympanic (within the aurus media), intravascular (within a vessel or vessels), intraventricular (within a ventricle), iontophoresis (by means of electric current where ions of soluble salts migrate into the tissues of the body), irrigation (to bathe or flush open wounds or body cavities), laryngeal (directly upon the larynx), nasogastric (through the nose and into the stomach), occlusive dressing technique (topical route administration which is then covered by a dressing which occludes the area), ophthalmic (to the external eye), oropharyngeal (directly to the mouth and pharynx), parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (within the respiratory tract by inhaling orally or nasally for local or systemic effect), retrobulbar (behind the pons or behind the eyeball), soft tissue, subarachnoid, subconjunctival, submucosal, subpial, topical, transplacental (through or across the placenta), transtracheal (through the wall of the trachea), transtympanic (across or through the tympanic cavity), ureteral (to the ureter), urethral (to the urethra), vaginal, caudal block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, photopheresis or spinal.
[00438] In some embodiments, AAV particles of the present disclosure are administered so as to be delivered to a target cell or tissue. Delivery to a target cell results in SMN protein expression. A target cell may be any cell in which it is considered desirable to increase SMN protein expression levels. A target cell may be a CNS cell. Non-limiting examples of such cells and/or tissues include, dorsal root ganglia and dorsal columns, proprioceptive sensory neurons, Clark’s column, gracile and cuneate nuclei, cerebellar dentate nucleus, corticospinal tracts and the cells comprising the same, Betz cells, and cells of the heart.
[00439] In some embodiments, compositions may be administered in a way that allows them to cross the blood-brain barrier, vascular barrier, or other epithelial barrier.
[00440] In some embodiments, delivery of SMN protein by adeno-associated virus (AAV) particles to cells of the central nervous system (e.g., parenchyma) comprises infusion into cerebrospinal fluid (CSF). CSF is produced by specialized ependymal cells that comprise the choroid plexus located in the ventricles of the brain. CSF produced within the brain then circulates and surrounds the central nervous system including the brain and spinal cord. CSF continually circulates around the central nervous system, including the ventricles of the brain and subarachnoid space that surrounds both the brain and spinal cord, while maintaining a homeostatic balance of production and reabsorption into the vascular system. The entire volume of CSF is replaced approximately four to six times per day or approximately once every four hours, though values for individuals may vary.
[00441] In some embodiments, the AAV particles may be delivered by systemic delivery. In some embodiments, the systemic delivery may be by intravascular administration. In some embodiments, the systemic delivery may be by intravenous (IV) administration.
[00442] In some embodiments, the AAV particles may be delivered by intravenous delivery.
[00443] In some embodiments, the AAV particle is administered to the subject via focused ultrasound (FUS), e.g., coupled with the intravenous administration of microbubbles (FUS-MB), or MRI-guided FUS coupled with intravenous administration, e.g., as described in Terstappen et al. (Nat Rev Drug Discovery, https://doi.org/10.1038/s41573-021-00139-y (2021)), Burgess et al. (Expert Rev Neurother. 15(5): 477^191 (2015)), and/or Hsu et al. (PLOS One 8(2): 1-8), the contents of which are incorporated herein by reference in its entirety. In some embodiments, an AAV capsid variant described herein allows for blood brain barrier penetration following intravenous administration. In some embodiments, the AAV capsid, e.g., AAV capsid variant, allows for blood brain barrier penetration following focused ultrasound (FUS), e.g., coupled with the intravenous administration of microbubbles (FUS-MB), or MRI- guided FUS coupled with intravenous administration. In some embodiments the AAV capsid, e.g., AAV capsid variant allows for increased distribution to a brain region. In some embodiments, the brain region comprises a temporal cortex, perirhinal cortex, globus pallidus, putamen, caudate, thalamus, hippocampus, geniculate nucleus, Purkinje Layer, deep cerebellar nuclei, dentate, brainstem, and cerebellum, or a combination thereof. In some embodiments, the AAV capsid, e.g., AAV capsid variant allows for preferential transduction in a brain region relative to the transduction in the dorsal root ganglia (DRG). In some embodiments, the AAV capsid variant allows for preferential transduction in a brain region relative to the transduction in the liver. In some embodiments, the AAV capsid variant allows for transduction in neuronal cells. In some embodiments, the AAV capsid variant allows for transduction in a non-neuronal cell, e.g., a glial cell (e.g., an astrocyte, an oligodendrocyte, or a combination thereof). In some embodiments, the AAV capsid variant allows for transduction in both neuronal cells and non- neuronal cell, e.g., a glial cell (e.g., an astrocyte, an oligodendrocyte, or a combination thereof).
[00444] In some embodiments, the AAV particles may be delivered by injection into the CSF pathway. Non-limiting examples of delivery to the CSF pathway include intrathecal and intracerebroventricular administration. [00445] In some embodiments an AAV capsid variant described herein allows for increased distribution to a spinal cord region. In some embodiments, the spinal region comprises a cervical spinal cord region, thoracic spinal cord region, and/or lumbar spinal cord region.
[00446] In some embodiments, the AAV particles may be delivered by thalamic delivery.
[00447] In some embodiments, the AAV particles may be delivered by intracerebral delivery.
[00448] In some embodiments, the AAV particles may be delivered by intracardiac delivery.
[00449] In some embodiments, the AAV particles may be delivered by intracranial delivery.
[00450] In some embodiments, the AAV particles may be delivered by intra cisterna magna (ICM) delivery.
[00451] In some embodiments, the AAV particles may be delivered by direct (intraparenchymal) injection into an organ (e.g., CNS (brain or spinal cord)). In some embodiments, the intraparenchymal delivery may be to any region of the brain or CNS.
[00452] In some embodiments, the AAV particles may be delivered by intrastriatal injection.
[00453] In some embodiments, the AAV particles may be delivered into the putamen.
[00454] In some embodiments, the AAV particles may be delivered into the spinal cord.
[00455] In some embodiments, the AAV particles of the present disclosure may be administered to the ventricles of the brain.
[00456] In some embodiments, the AAV particles of the present disclosure may be administered to the ventricles of the brain by intracerebroventricular delivery.
[00457] In some embodiments, the AAV particles of the present disclosure may be administered by intramuscular delivery.
[00458] In some embodiments, the AAV particles of the present disclosure are administered by more than one route described above. As a non-limiting example, the AAV particles may be administered by intravenous delivery and thalamic delivery.
[00459] In some embodiments, the AAV particles of the present disclosure are administered by more than one route described above. As a non-limiting example, the AAV particles may be administered by intravenous delivery and intracerebral delivery.
[00460] In some embodiments, the AAV particles of the present disclosure are administered by more than one route described above. As a non-limiting example, the AAV particles may be administered by intravenous delivery and intracranial delivery.
[00461] In some embodiments, the AAV particles of the present disclosure are administered by more than one route described above. In some embodiments, the AAV particles of the present disclosure may be delivered by intrathecal and intracerebroventricular administration.
[00462] In some embodiments, the AAV particles may be delivered to a subject to improve and/or correct mitochondrial dysfunction. [00463] In some embodiments, the AAV particles may be delivered to a subject to preserve neurons. The neurons may be primary and/or secondary sensory neurons. In some embodiments, AAV particles are delivered to dorsal root ganglia and/or neurons thereof.
[00464] In some embodiments, administration of the AAV particles may preserve and/or correct function in the sensory pathways.
[00465] In some embodiments, the AAV particles may be delivered via intravenous (IV), intracerebroventricular (ICV), intraparenchymal, and/or intrathecal (IT) infusion and the therapeutic agent may also be delivered to a subject via intramuscular (IM) limb infusion in order to deliver the therapeutic agent to the skeletal muscle. Delivery of AAVs by intravascular limb infusion is described by Gruntman and Flotte, Human Gene Therapy Clinical Development, 2015, 26(3), 159-164, the contents of which are herein incorporated by reference in their entirety.
[00466] In some embodiments, delivery of viral vector pharmaceutical compositions in accordance with the present disclosure to cells of the central nervous system (e.g., parenchyma) comprises a rate of delivery defined by VG/hour = mL/hour * VG/mL, wherein VG is viral genomes, VG/mL is composition concentration, and mL/hour is rate of infusion.
[00467] In some embodiments, delivery of AAV particle pharmaceutical compositions in accordance with the present disclosure to cells of the central nervous system (e.g., parenchyma) comprises infusion of up to 1 rnL. In some embodiments, delivery of viral vector pharmaceutical compositions in accordance with the present disclosure to cells of the central nervous system (e.g., parenchyma) may comprise infusion of 0.0001, 0.0002, 0.001, 0.002, 0.003, 0.004, 0.005, 0.008, 0.010, 0.015, 0.020, 0.025, 0.030, 0.040, 0.050, 0.060, 0.070, 0.080, 0.090, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 mL.
[00468] In some embodiments, delivery of AAV particle pharmaceutical compositions in accordance with the present disclosure to cells of the central nervous system (e.g., parenchyma) comprises infusion of between about 1 mL to about 120 mL. In some embodiments, delivery of viral vector pharmaceutical compositions in accordance with the present disclosure to cells of the central nervous system (e.g., parenchyma) may comprise an infusion of 0.1, 1, 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, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,
65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,
116, 117, 118, 119, or 120 mL. In some embodiments delivery of AAV particles to cells of the central nervous system (e.g., parenchyma) comprises infusion of at least 3 mL. In some embodiments, delivery of AAV particles to cells of the central nervous system (e.g., parenchyma) consists of infusion of 3 mL. In some embodiments, delivery of AAV particles to cells of the central nervous system (e.g., parenchyma) comprises infusion of at least 10 mL. In some embodiments, delivery of AAV particles to cells of the central nervous system (e.g., parenchyma) consists of infusion of 10 rnL. [00469] In some embodiments, the volume of the AAV particle pharmaceutical composition delivered to the cells of the central nervous system (e.g., parenchyma) of a subject is 2 pl, 20 pl, 50 pl, 80 pl, 100 pl, 200 pl, 300 pl, 400 pl, 500 pl, 600 pl, 700 pl, 800 pl, 900 pl, 1000 pl, 1100 pl, 1200 pl, 1300 pl, 1400 pl, 1500 pl, 1600 pl, 1700 pl, 1800 pl, 1900 pl, 2000 pl, or more than 2000 pl.
[00470] In some embodiments, the volume of the AAV particle pharmaceutical composition delivered to a region in both hemispheres of a subject brain is 2 pl, 20 pl, 50 pl, 80 pl, 100 pl, 200 pl, 300 pl, 400 pl, 500 pl, 600 pl, 700 pl, 800 pl, 900 pl, 1000 pl, 1100 pl, 1200 pl, 1300 pl, 1400 pl, 1500 pl, 1600 pl, 1700 pl, 1800 pl, 1900 pl, 2000 pl, or more than 2000 pl. In some embodiments, the volume delivered to a region in both hemispheres is 200 pl. As another non-limiting example, the volume delivered to a region in both hemispheres is 900 pl. As yet another non-limiting example, the volume delivered to a region in both hemispheres is 1800 pl.
[00471] In certain embodiments, AAV particle or viral vector pharmaceutical compositions in accordance with the present disclosure may be administered at about 10 to about 600 pl/site, about 50 to about 500 pl/site, about 100 to about 400 pl/site, about 120 to about 300 pl/site, about 140 to about 200 pl/site, or about 160 pl/site.
[00472] In some embodiments, the total volume delivered to a subject may be split between one or more administration sites e.g., 1, 2, 3, 4, 5, or more than 5 sites. In some embodiments, the total volume is split between administration to the left and right hemisphere.
Delivery of AAV Particles
[00473] In some embodiments, the AAV particles or pharmaceutical compositions of the present disclosure may be administered or delivered using the methods for treatment of disease described in US Patent No. 8,999,948, or International Publication No. WO2014178863, the contents of which are herein incorporated by reference in their entirety.
[00474] In some embodiments, the AAV particles or pharmaceutical compositions of the present disclosure may be administered or delivered using the methods for delivering gene therapy in Alzheimer’s Disease or other neurodegenerative conditions as described in US Application No. 20150126590, the contents of which are herein incorporated by reference in their entirety.
[00475] In some embodiments, the AAV particles or pharmaceutical compositions of the present disclosure may be administered or delivered using the methods for delivery of a CNS gene therapy as described in US Patent Nos. 6436708, and 8946152, and International Publication No. WO2015168666, the contents of which are herein incorporated by reference in their entirety.
[00476] In some embodiments, the AAV particles of the present disclosure may be administered or delivered using the methods for the delivery of AAV virions described in European Patent Application No. EP1857552, the contents of which are herein incorporated by reference in their entirety.
[00477] In some embodiments, the AAV particle or pharmaceutical compositions of the present disclosure may be administered or delivered using the methods for delivering proteins using AAV vectors described in European Patent Application No. EP2678433, the contents of which are herein incorporated by reference in their entirety.
[00478] In some embodiments, the viral vector encoding SMN protein may be administered or delivered using the methods for delivering DNA molecules using AAV vectors described in US Patent No. US 5858351, the contents of which are herein incorporated by reference in their entirety.
[00479] In some embodiments, the AAV particle or pharmaceutical compositions of the present disclosure may be administered or delivered using the methods for delivering DNA to the bloodstream described in US Patent No. US 6211163, the contents of which are herein incorporated by reference in their entirety.
[00480] In some embodiments, the viral vector encoding SMN protein may be administered or delivered using the methods for delivering AAV virions described in US Patent No. US 6325998, the contents of which are herein incorporated by reference in their entirety.
[00481] In some embodiments, the viral vector encoding SMN protein may be administered or delivered using the methods for delivering DNA to muscle cells described in US Patent No. US 6335011, the contents of which are herein incorporated by reference in their entirety.
[00482] In some embodiments, the viral vector encoding SMN protein may be administered or delivered using the methods for delivering DNA to muscle cells and tissues described in US Patent No. US 6610290, the contents of which are herein incorporated by reference in their entirety.
[00483] In some embodiments, the viral vector encoding SMN protein may be administered or delivered using the methods for delivering DNA to muscle cells described in US Patent No. US 7704492, the contents of which are herein incorporated by reference in their entirety.
[00484] In some embodiments, the viral vector encoding SMN protein may be administered or delivered using the methods for delivering a payload to skeletal muscles described in US Patent No. US 7112321, the contents of which are herein incorporated by reference in their entirety.
[00485] In some embodiments, the AAV particle or pharmaceutical compositions of the present disclosure may be administered or delivered using the methods for delivering a payload to the central nervous system described in US Patent No. US 7588757, the contents of which are herein incorporated by reference in their entirety.
[00486] In some embodiments, the AAV particle or pharmaceutical compositions of the present disclosure may be administered or delivered using the methods for delivering a payload described in US Patent No. US 8,283,151, the contents of which are herein incorporated by reference in their entirety.
[00487] In some embodiments, the AAV particle or pharmaceutical compositions of the present disclosure may be administered or delivered using the methods for delivering a payload described in International Patent Publication No. WO2012144446, the contents of which are herein incorporated by reference in their entirety. [00488] In some embodiments, the AAV particle or pharmaceutical compositions of the present disclosure may be administered or delivered using the methods for delivering a payload using a glutamic acid decarboxylase (GAD) delivery vector described in International Patent Publication No. W02001089583, the contents of which are herein incorporated by reference in their entirety.
[00489] In some embodiments, the AAV particle or pharmaceutical compositions of the present disclosure may be administered or delivered using the methods for delivering a payload to neural cells described in International Patent Publication No. WO2012057363, the contents of which are herein incorporated by reference in their entirety.
[00490] In some embodiments, the AAV particle or pharmaceutical compositions of the present disclosure may be administered or delivered using the methods for delivering a payload described in International Patent Publication No. W02001096587, the contents of which are herein incorporated by reference in their entirety.
[00491] In some embodiments, the AAV particle or pharmaceutical compositions of the present disclosure may be administered or delivered using the methods for delivering a payload to muscle tissue described in International Patent Publication No. W02002014487, the contents of which are herein incorporated by reference in their entirety.
[00492] In some embodiments, a catheter may be used to administer the AAV particles. In certain embodiments, the catheter or cannula may be located at more than one site in the spine for multi-site delivery. The viral particles encoding may be delivered in a continuous and/or bolus infusion. Each site of delivery may be a different dosing regimen or the same dosing regimen may be used for each site of delivery. In some embodiments, the sites of delivery may be in the cervical and the lumbar region. In some embodiments, the sites of delivery may be in the cervical region. In some embodiments, the sites of delivery may be in the lumbar region.
[00493] In some embodiments, a subject may be analyzed for spinal anatomy and pathology prior to delivery of the AAV particles described herein. As a non-limiting example, a subject with scoliosis may have a different dosing regimen and/or catheter location compared to a subject without scoliosis.
[00494] In some embodiments, the delivery method and duration is chosen to provide broad transduction in the spinal cord. In some embodiments, intrathecal delivery is used to provide broad transduction along the rostral-caudal length of the spinal cord. In some embodiments, multi-site infusions provide a more uniform transduction along the rostral-caudal length of the spinal cord.
Delivery to Cells
[00495] In some aspects, the present disclosure provides a method of delivering to a cell or tissue any of the above-described AAV particles, comprising contacting the cell or tissue with said AAV particle or contacting the cell or tissue with a formulation comprising said AAV particle, or contacting the cell or tissue with any of the described compositions, including pharmaceutical compositions. The method of delivering the AAV particle to a cell or tissue can be accomplished in vitro, ex vivo, or in vivo. Delivery to Subjects
[00496] In some aspects, the present disclosure additionally provides a method of delivering to a subject, including a mammalian subject, any of the above-described AAV particles comprising administering to the subject said AAV particle, or administering to the subject a formulation comprising said AAV particle, or administering to the subject any of the described compositions, including pharmaceutical compositions.
[00497] In some embodiments, the AAV particles may be delivered to bypass anatomical blockages such as, but not limited to the blood brain barrier.
[00498] In some embodiments, the AAV particles may be formulated and delivered to a subject by a route which increases the speed of drug effect as compared to oral delivery.
[00499] In some embodiments, the AAV particles may be delivered by a method to provide uniform transduction of the spinal cord and dorsal root ganglion (DRG). In some embodiments, the AAV particles may be delivered using intrathecal infusion.
[00500] In some embodiments, a subject may be administered the AAV particles described herein using a bolus infusion. As used herein, a “bolus infusion” means a single and rapid infusion of a substance or composition.
[00501] In some embodiments, the AAV particles encoding SMN protein may be delivered in a continuous and/or bolus infusion. Each site of delivery may be a different dosing regimen or the same dosing regimen may be used for each site of delivery. As a non-limiting example, the sites of delivery may be in the cervical and the lumbar region. As another non-limiting example, the sites of delivery may be in the cervical region. As another non-limiting example, the sites of delivery may be in the lumbar region.
[00502] In some embodiments, the AAV particles may be delivered to a subject via a single route administration.
[00503] In some embodiments, the AAV particles may be delivered to a subject via a multi-site route of administration. For example, a subject may be administered the AAV particles at 2, 3, 4, 5, or more than 5 sites.
[00504] In some embodiments, a subject may be administered the AAV particles described herein using sustained delivery over a period of minutes, hours or days. The infusion rate may be changed depending on the subject, distribution, formulation or another delivery parameter known to those in the art.
[00505] In some embodiments, if continuous delivery (continuous infusion) of the AAV particles is used, the continuous infusion may be for 1 hour, 2, hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or more than 24 hours.
[00506] In some embodiments, the intracranial pressure may be evaluated prior to administration. The route, volume, AAV particle concentration, infusion duration and/or vector titer may be optimized based on the intracranial pressure of a subject.
[00507] In some embodiments, the AAV particles may be delivered by systemic delivery. In some embodiments, the systemic delivery may be by intravascular administration.
[00508] In some embodiments, the AAV particles may be delivered by injection into the CSF pathway. Non-limiting examples of delivery to the CSF pathway include intrathecal and intracerebroventricular administration.
[00509] In some embodiments, the AAV particles may be delivered by direct (intraparenchymal) injection into the substance of an organ, e.g., one or more regions of the brain.
[00510] In some embodiments, the AAV particles may be delivered by subpial injection into the spinal cord. For example, subjects may be placed into a spinal immobilization apparatus. A dorsal laminectomy may be performed to expose the spinal cord. Guiding tubes and XYZ manipulators may be used to assist catheter placement. Subpial catheters may be placed into the subpial space by advancing the catheter from the guiding tube and AAV particles may be injected through the catheter (Miyanohara et al., Mol Ther Methods Clin Dev. 2016; 3: 16046). In some cases, the AAV particles may be injected into the cervical subpial space. In some cases, the AAV particles may be injected into the thoracic subpial space. [00511] In some embodiments, the AAV particles may be delivered by direct injection to the CNS of a subject. In some embodiments, direct injection is intracerebral injection, intraparenchymal injection, intrathecal injection, intra-cisterna magna injection, or any combination thereof. In some embodiments, direct injection to the CNS of a subject comprises convection enhanced delivery (CED). In some embodiments, administration comprises peripheral injection. In some embodiments, peripheral injection is intravenous injection.
[00512] In some embodiments, the AAV particles may be delivered to a subject in order to increase the SMN protein levels in the caudate -putamen, thalamus, superior colliculus, cortex, and/or corpus callosum as compared to endogenous levels. The increase may be O.lx to 5x, 0.5x to 5x, lx to 5x, 2x to 5x, 3x to 5x, 4x to 5x, O.lx to 4x, 0.5x to 4x, lx to 4x, 2x to 4x, 3x to 4x, O.lx to 3x, 0.5x to 3x, lx to 3x, 2x to 3x, 0.1 x to 2x, 0.5x to 2x, 0.1 x to l x, 0.5x to 1 x, 0.1 x to 0.5x, 1 x to 2x, 0.1 x, 0.2x, 0.3x, 0.4x, 0.5x, 0.6x, 0.7x, 0.8x, 0.9x, l.Ox, l.lx, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2.0x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3.0x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4.0x, 4.1x, 4.2x, 4.3x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, 4.9x or more than 5x as compared to endogenous levels.
[00513] In some embodiments, the AAV particles may be delivered to a subject in order to increase the SMN protein levels in the caudate, putamen, thalamus, superior colliculus, cortex, and/or corpus callosum by transducing cells in these CNS regions. Transduction may also be referred to as the amount of cells that are positive for SMN protein. The transduction may be greater than or equal to 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of cells in these CNS regions. [00514] In some embodiments, delivery of AAV particles comprising a viral genome encoding SMN protein described herein to neurons in the caudate-putamen, thalamus, superior colliculus, cortex, and/or corpus callosum will lead to an increased expression of SMN protein. The increased expression may lead to improved survival and function of various cell types in these CNS regions and subsequent improvement of SMA-related disorder symptoms.
[00515] In particular embodiments, the AAV particles may be delivered to a subject in order to establish widespread distribution of the SMN throughout the nervous system by administering the AAV particles to the thalamus of the subject.
[00516] Specifically, in some embodiments, the increased expression of SMN protein may lead to improved gait, sensory capability, coordination of movement and strength, functional capacity, cognition, and/or quality of life.
Dosing
[00517] In some aspects, the present disclosure provides methods comprising administering viral vectors and their payloads in accordance with the disclosure to a subject in need thereof. Viral vector pharmaceutical, imaging, diagnostic, or prophylactic compositions thereof, may be administered to a subject using any amount and any route of administration effective for preventing, treating, diagnosing, or imaging a disease, disorder, and/or condition (e.g., a disease, disorder, and/or condition associated with decreased SMN protein expression or a deficiency in the quantity and/or function of SMN protein). In some embodiments, the disease, disorder, and/or condition is SMA-related disorders. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. Compositions in accordance with the disclosure are typically formulated in unit dosage form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present disclosure may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex, and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific peptide(s) employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
[00518] In certain embodiments, AAV particle pharmaceutical compositions in accordance with the present disclosure may be administered at dosage levels sufficient to deliver SMN protein from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic, diagnostic, prophylactic, or imaging effect. It will be understood that the above dosing concentrations may be converted to VG or viral genomes per kg or into total viral genomes administered by one of skill in the art.
[00519] In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When multiple administrations are employed, split dosing regimens such as those described herein may be used. As used herein, a “split dose” is the division of single unit dose or total daily dose into two or more doses, e.g., two or more administrations of the single unit dose. As used herein, a “single unit dose” is a dose of any therapeutic composition administered in one dose/at one time/single route/single point of contact, i.e., single administration event. In some embodiments, a single unit dose is provided as a discrete dosage form (e.g., a tablet, capsule, patch, loaded syringe, vial, etc.). As used herein, a “total daily dose” is an amount given or prescribed in 24-hour period. It may be administered as a single unit dose. The viral particles may be formulated in buffer only or in a formulation described herein.
[00520] A pharmaceutical composition described herein can be formulated into a dosage form described herein, such as a topical, intranasal, pulmonary, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, and/or subcutaneous).
[00521] In some embodiments, delivery of the AAV particles described herein results in minimal serious adverse events (SAEs) as a result of the delivery of the AAV particles.
[00522] In some embodiments, delivery of AAV particle pharmaceutical compositions in accordance with the present disclosure to cells of the central nervous system (e.g., parenchyma) may comprise a total concentration between about 1x10s VG/mL and about IxlO16 VG/mL. In some embodiments, delivery may comprise a composition concentration of about IxlO6, 2x10s, 3xl06, 4x10s, 5x10s, 6x10s, 7x10s, 8x10s, 9x10s, IxlO7, 2xl07, 3xlO7, 4xl07, 5xl07, 6xl07, 7xl07, 8xl07, 9xl07, IxlO8, 2xl08, 3xlO8, 4xl08, 5xl08, 6xlO8, 7xl08, 8xlO8, 9xlO8, IxlO9, 2xl09, 3xlO9, 4xl09, 5xl09, 6xl09, 7xl09, 8xl09, 9xl09, IxlO10, 2xlO10, 3xl010, 4xlO10, 5xlO10, 6xlO10, 7xlO10, 8xl010, 9xlO10, IxlO11, 1.6x10", 1.8x10", 2x10", 3x10", 4x10", 5x10", 5.5x10", 6x10", 7x10", 8x10", 9x10", 0.8xl012, 0.83xl012, IxlO12, l.lxlO12, 1.2xl012, 1.3xl012, 1.4xl012, 1.5xl012, 1.6xl012, 1.7xl012, 1.8xl012, 1.9xl012, 2xl012, 2.1xl012, 2.2xl012, 2.3xl012, 2.4xl012, 2.5xl012, 2.6xl012, 2.7xl012, 2.8xl012, 2.9xl012, 3xl012, 3. IxlO12, 3.2xl012, 3.3xl012, 3.4xl012, 3.5xl012, 3.6xl012, 3.7xl012, 3.8xl012, 3.9xl012, 4xl012, 4.1xl012, 4.2xl012, 4.3xl012, 4.4xl012, 4.5xl012, 4.6xl012, 4.7xl012, 4.8xl012, 4.9xl012, 5xl012, 6xl012, 7xl012, 8xl012, 9xl012, IxlO13, 2xl013, 2.3xl013, 3xl013, 4xl013, 5xl013, 6xl013, 7xl013, 8xl013, 9xl013, IxlO14, 1.9xl014, 2xl014, 3xl014, 4xl014, 5xl014, 6xl014, 7xl014, 8xl014, 9xl014, IxlO15, 2xl015, 3xl015, 4xl015, 5xlO15, 6xl015, 7xl015, 8xl015, 9xl015, or IxlO16 VG/mL. In some embodiments, the concentration of the viral vector in the composition is IxlO13 VG/mL. In some embodiments, the concentration of the viral vector in the composition is l.lxlO12 VG/mL. In some embodiments, the concentration of the viral vector in the composition is 3.7xl012 VG/mL. In some embodiments, the concentration of the viral vector in the composition is 8x10" VG/mL. In some embodiments, the concentration of the viral vector in the composition is 2.6xl012 VG/mL. In some embodiments, the concentration of the viral vector in the composition is 4.9xl012 VG/mL. In some embodiments, the concentration of the viral vector in the composition is 0.8xl012 VG/mL. In some embodiments, the concentration of the viral vector in the composition is 0.83xl012 VG/mL. In some embodiments, the concentration of the viral vector in the composition is the maximum final dose which can be contained in a vial. In some embodiments, the concentration of the viral vector in the composition is 1.6x10" VG/mL. In some embodiments, the concentration of the viral vector in the composition is 5x10" VG/mL. In some embodiments, the concentration of the viral vector in the composition is 2.3xl013 VG/mL. In some embodiments, the concentration of the viral vector in the composition is 1.9xl014 VG/mL.
[00523] In some embodiments, delivery of AAV particle pharmaceutical compositions in accordance with the present disclosure to cells of the central nervous system (e.g., parenchyma) may comprise a total concentration per subject between about IxlO6 VG and about IxlO16 VG. In some embodiments, delivery may comprise a composition concentration of about IxlO6, 2xl06, 3xl06, 4xl06, 5xl06, 6xl06, 7xl06, 8xl06, 9xl06, IxlO7, 2xl07, 3xlO7, 4xl07, 5xl07, 6xl07, 7xl07, 8xl07, 9xl07, IxlO8, 2xl08, 3xlO8, 4xl08, 5xlO8, 6xlO8, 7xl08, 8xlO8, 9xlO8, IxlO9, 2xl09, 3xlO9, 4xl09, 5xl09, 6xl09, 7xl09, 8xl09, 9xl09, IxlO10, 2xlO10, 3xl010, 4xlO10, 5xlO10, 6xlO10, 7xlO10, 8xl010, 9xlO10, IxlO11, 1.6x10", 2x10", 2.1x10", 2.2xlOn, 2.3x10", 2.4x10", 2.5x10", 2.6x10", 2.7x10", 2.8x10", 2.9x10", 3x10", 4x10", 4.6x10", 5x10", 6x10", 7x10", 7.1x10", 7.2x10", 7.3x10", 7.4x10", 7.5x10", 7.6x10", 7.7x10", 7.8x10", 7.9x10", 8x10", 9x10", IxlO12, 1.1 xlO12, 1.2xl012, 1.3xl012, 1.4xl012, 1.5xl012, 1.6xl012, 1.7xl012, 1.8xl012, 1.9xl012, 2xl012, 2.3xl012, 3xl012, 4xl012, 4.1xl012, 4.2xl012, 4.3xl012, 4.4xl012, 4.5X1012,4.6X1012, 4.7X1012, 4.8X1012, 4.9X1012, 5X1012, 6X1012, 7X1012, 8X1012, 8. IxlO12, 8.2xl012, 8.3xl012, 8.4xl012, 8.5xl012, 8.6xl012, 8.7xl012, 8.8 xlO12, 8.9xl012, 9xl012, IxlO13, 2xl013, 3xl013, 4xl013, 5xl013, 6xl013, 7xl013, 8xl013, 9xl013, IxlO14, 2xl014, 3xl014, 4xl014, 5xl014, 6xl014, 7xl014, 8x10", 9xl014, IxlO15, 2xl015, 3xl015, 4xl015, 5xl015, 6xl015, 7xl015, 8xl015, 9xl015, or IxlO16 VG/subject. In some embodiments, the concentration of the viral vector in the composition is 2.3x10" VG/ subject. In some embodiments, the concentration of the viral vector in the composition is 7.2x10" VG/ subject. In some embodiments, the concentration of the viral vector in the composition is 7.5x10" VG/ subject. In some embodiments, the concentration of the viral vector in the composition is 1.4xl012 VG/ subject. In some embodiments, the concentration of the viral vector in the composition is 4.8xl012 VG/ subject. In some embodiments, the concentration of the viral vector in the composition is 8.8xl012 VG/ subject. In some embodiments, the concentration of the viral vector in the composition is 2.3xl012 VG/ subject. In some embodiments, the concentration of the viral vector in the composition is 2xlO10 VG/ subject. In some embodiments, the concentration of the viral vector in the composition is 1.6x10" VG/ subject. In some embodiments, the concentration of the viral vector in the composition is 4.6x10' 1 VG/ subject.
[00524] In some embodiments, delivery of AAV particles to cells of the central nervous system (e.g., parenchyma) may comprise a total dose between about 1 x 106 VG and about 1 x 1016 VG. In some embodiments, delivery may comprise a total dose of about 1 x 106, 2 x 106, 3 x 106, 4 x 106, 5 x 106, 6 x 106, 7 x 106, 8 x 106, 9 x 106, 1 x 107, 2 x 107, 3 x 107, 4 x 107, 5 x 107, 6 x 107, 7 x 107, 8 x 107, 9 x 107, 1 x 108, 2 x 108, 3 x 108, 4 x 108, 5 x 108, 6 x 108, 7 x 108, 8 x 108, 9 x 108, 1 x 109, 2 x 109, 3 x 109, 4 x 109, 5 x 109, 6 x 109, 7 x 109, 8 x 109, 9 x 109, 1 x 10'°, 1.9 x 10'°, 2 x 10'°, 3 x 10'°, 3.73 x 10'°, 4 x 10'°, 5 x 10'°, 6 x 10'°, 7 x 10'°, 8 x 10'°, 9 x 10'°, 1 x 10", 2 x 10", 2.5 x 10", 3 x 10", 4 x 10", 5 x
10", 6 x 10", 7 x 10", 8 x 10", 9 x 10", 1 x 1012, 2 x 1012, 3 x 1012, 4 x 1012, 5 x 1012, 6 x 1012, 7 x
1012, 8 x 1012, 9 x 1012, 1 x 1013, 2 x 1013, 3 x 1013, 4 x 1013, 5 x 1013, 6 x 1013, 7 x 1013, 8 x 1013, 9 x
1013, 1 x 1014, 2 x 1014, 3 x 1014, 4 x 1014, 5 x 1014, 6 x 1014, 7 x 1014, 8 x 1014, 9 x 1014, 1 x 1015, 2 x
1015, 3 x 1015, 4 x 1015, 5 x 1015, 6 x 1015, 7 x 1015, 8 x 1015, 9 x 1015, or 1 x 1016 VG. In some embodiments, the total dose is 1 x 1013 VG. In some embodiments, the total dose is 3 x 1013 VG. In some embodiments, the total dose is 3.73 x 10'° VG. In some embodiments, the total dose is 1.9 x 10'° VG. In some embodiments, the total dose is 2.5 x 10" VG. In some embodiments, the total dose is 5 x 10" VG. In some embodiments, the total dose is 1 x 1012 VG. In some embodiments, the total dose is 5 x 1012 VG. [00525] Efficacy
[00526] Response to administration of the AAV particles provided herein can be determined using a number of criteria including, but not limited to, the Hammersmith Functional Motor Scale (HFMSE) (O’Hagen et al., Neuro. Dis. 17:693-697, 2007), CHOP INTEND (Glanzman et al., Neuromuscul. Disord. 2010; 20: 155-161) and Revised Upper Limb Module (Pera et aL, Muscle Nerve. 2019;59:426- 430), and other criteria such as survival (including need for permanent ventilation).
Combinations
[00527] The AAV particles may be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. The phrase “in combination with,” is not intended to require that the agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope of the present disclosure. Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent. In some embodiments, the present disclosure encompasses the delivery of pharmaceutical, prophylactic, diagnostic, or imaging compositions in combination with agents that may improve their bioavailability, reduce and/or modify their metabolism, and/or modify their distribution within the body.
[00528] The therapeutic agents may be approved by the US Food and Drug Administration or may be in clinical trial or at the preclinical research stage. The therapeutic agents may utilize any therapeutic modality known in the art, with non-limiting examples including gene silencing or interference (i.e., miRNA, siRNA, RNAi, shRNA), gene editing (i.e., TALEN, CRISPR/Cas9 systems, zinc finger nucleases), and gene, protein or enzyme replacement.
[00529] In some embodiments, the AAV particles provided herein are administered in combination with compounds that increase SMN levels such as histone deacetylase inhibitors, aminoglycosides, and quinazoline derivatives. Histone deacetylase inhibitors such as valproic acid, sodium butyrate, phenylbutyrate, and trichotomic A activate the SMN2 promoter, resulting in increased full-length SMN protein. Other therapies include CK-2127107 (a fast skeletal muscle troponin activator), LM1070 (branaplan, formerly known as NVS-SM1), olesoxime (a cholesterol oximes family member), and Risdiplam (RG7916).
[00530] In some embodiments, the AAV particles provided herein are administered in combination with a small molecule splicing modulators (SMSMs). An SMSMs is a small molecule compound that binds to a cell component (e.g., DNA, RNA, pre-mRNA, protein, RNP, snRNA, carbohydrates, lipids, co-factors, nutrients and/or metabolites) and modulates splicing of a target polynucleotide, e.g., a pre-mRNA. For example, an SMSM can bind directly or indirectly to a target polynucleotide, e.g., RNA (e.g., a pre- mRNA) with a mutated, non-mutated, bulged and/or aberrant splice site, resulting in modulation of splicing of the target polynucleotide. Suitable SMSMs include but are not limited those described in US 2021/0171519 (incorporated herein), and RG7916 (disclosed in US Patent Nos. 9586955 and 9969754), which is a pyridazine derivative that modifies the splicing of SMN2 messenger RNA to include exon 7, resulting in an increase in the concentration of the functional SMN protein in vivo.
Measurement of Expression
[00531] Expression of SMN protein from viral genomes may be determined using various methods known in the art such as, but not limited to immunochemistry (e.g., IHC), enzyme-linked immunosorbent assay (ELISA), affinity ELISA, ELISPOT, flow cytometry, immunocytology, surface plasmon resonance analysis, kinetic exclusion assay, liquid chromatography-mass spectrometry (LCMS), high-performance liquid chromatography (HPLC), BCA assay, immunoelectrophoresis, Western blot, SDS-PAGE, protein immunoprecipitation, PCR, and/or in situ hybridization (ISH). In some embodiments, transgenes encoding SMN protein delivered in different AAV capsids may have different expression levels in different CNS tissues.
[00532] In certain embodiments, the SMN protein is detectable by Western blot.
[00533] Alternatively, methods of detecting SMN expression are known, including, for example, use of the methods and compounds as described in Int’l Pub. No. WO2019136484, incorporated herein by reference in its entirety. VII. Kits and Devices
Kits
[00534] In some aspects, the present disclosure provides a variety of kits for conveniently and/or effectively carrying out methods of the present disclosure. Typically, kits will comprise sufficient amounts and/or numbers of components to allow a user to perform multiple treatments of a subject(s) and/or to perform multiple experiments.
[00535] Any of the vectors, constructs, or SMN proteins of the present disclosure may be comprised in a kit. In some embodiments, kits may further include reagents and/or instructions for creating and/or synthesizing compounds and/or compositions of the present disclosure. In some embodiments, kits may also include one or more buffers. In some embodiments, kits of the disclosure may include components for making protein or nucleic acid arrays or libraries and thus, may include, for example, solid supports. [00536] In some embodiments, kit components may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and suitably aliquoted. Where there is more than one kit component, (labeling reagent and label may be packaged together), kits may also generally contain second, third or other additional containers into which additional components may be separately placed. In some embodiments, kits may also comprise second container means for containing sterile, pharmaceutically acceptable buffers and/or other diluents. In some embodiments, various combinations of components may be comprised in one or more vial. Kits of the present disclosure may also typically include means for containing compounds and/or compositions of the present disclosure, e.g., proteins, nucleic acids, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which desired vials are retained.
[00537] In some embodiments, kit components are provided in one and/or more liquid solutions. In some embodiments, liquid solutions are aqueous solutions, with sterile aqueous solutions being particularly used. In some embodiments, kit components may be provided as dried powder(s). When reagents and/or components are provided as dry powders, such powders may be reconstituted by the addition of suitable volumes of solvent. In some embodiments, it is envisioned that solvents may also be provided in another container means. In some embodiments, labeling dyes are provided as dried powders. In some embodiments, it is contemplated that 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000 micrograms or at least or at most those amounts of dried dye are provided in kits of the disclosure. In such embodiments, dye may then be resuspended in any suitable solvent, such as DMSO.
[00538] In some embodiments, kits may include instructions for employing kit components as well the use of any other reagent not included in the kit. Instructions may include variations that may be implemented. Devices
[00539] In some embodiments, compounds and/or compositions of the present disclosure may be combined with, coated onto or embedded in a device. Devices may include, but are not limited to, dental implants, stents, bone replacements, artificial joints, valves, pacemakers and/or other implantable therapeutic device.
[00540] The present disclosure provides for devices which may incorporate viral vectors that encode one or more SMN protein molecules. These devices contain in a stable formulation the viral vectors which may be immediately delivered to a subject in need thereof, such as a human patient.
[00541] Devices for administration may be employed to deliver the viral vectors encoding SMN protein of the present disclosure according to single, multi- or split-dosing regimens taught herein.
[00542] Method and devices known in the art for multi-administration to cells, organs and tissues are contemplated for use in conjunction with the methods and compositions disclosed herein as embodiments of the present disclosure.
VIII. Definitions
[00543] At various places in the present specification, substituents of compounds of the present disclosure are disclosed in groups or in ranges. It is specifically intended that the present disclosure include each and every individual sub-combination of the members of such groups and ranges. The following is a non-limiting list of term definitions.
[00544] Adeno-associated virus: As used herein, the term “adeno-associated virus” or “AAV” refers to members of the dependovirus genus or a variant, e.g., a functional variant, thereof. In some embodiments, the AAV is wildtype, or naturally occurring. In some embodiments, the AAV is recombinant.
[00545] AA V Particle: As used herein, an “AAV particle” refers to an AAV capsid, e.g., an AAV capsid variant, and a polynucleotide, e.g., a viral genome. In some embodiments, the viral genome of the AAV particle comprises at least one payload region and at least one TTR. In some embodiments, an AAV particle of the disclosure is an AAV particle comprising an AAV variant. In some embodiments, the AAV particle is capable of delivering a nucleic acid, e.g., a payload region, encoding a payload to cells, typically, mammalian, e.g., human, cells. In some embodiments, an AAV particle of the present disclosure may be produced recombinantly. In some embodiments, an AAV particle may be derived from any serotype, described herein or known in the art, including combinations of serotypes (e.g., “pseudotyped” AAV) or from various genomes (e.g., single stranded or self-complementary). In some embodiments, the AAV particle may be replication defective and/or targeted. It is to be understood that reference to the AAV particle of the disclosure also includes pharmaceutical compositions thereof, even if not explicitly recited.
[00546] Administered in combination: As used herein, the term “administered in combination” or “delivered in combination” or “combined administration” refers to exposure of two or more agents (e.g., AAV) administered at the same time or within an interval such that the subject is at some point in time exposed to both agents and/or such that there is an overlap in the effect of each agent on the patient. In some embodiments, at least one dose of one or more agents is administered within about 24 hours, 12 hours, 6 hours, 3 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or 1 minute of at least one dose of one or more other agents. In some embodiments, administration occurs in overlapping dosage regimens. As used herein, the term “dosage regimen” refers to a plurality of doses spaced apart in time. Such doses may occur at regular intervals or may include one or more hiatuses in administration. In some embodiments, the administration of individual doses of one or more compounds and/or compositions of the present disclosure, as described herein, are spaced sufficiently closely together such that a combinatorial (e.g., a synergistic) effect is achieved.
[00547] Amelioration: As used herein, the term “amelioration” or “ameliorating” refers to a lessening of severity of at least one indicator of a condition or disease. For example, in the context of a neurodegenerative disorder, amelioration includes the reduction or stabilization of neuron loss.
[00548] Approximately: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. When referring to a measurable value such as an amount, a temporal duration, and the like, the term is meant to encompass is meant to encompass variations of ±20% or in some instances ±10%, or in some instances ±5%, or in some instances ±1%, or in some instances ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
[00549] Capsid: As used herein, the term “capsid” refers to the exterior, e.g., a protein shell, of a virus particle, e.g., an AAV particle, that is substantially (e.g., >50%, >90%, or 100%) protein. In some embodiments, the capsid is an AAV capsid comprising an AAV capsid protein described herein, e.g., a VP1, VP2, and/or VP3 polypeptide. The AAV capsid protein can be a wild-type AAV capsid protein or a variant, e.g., a structural and/or functional variant from a wild-type or a reference capsid protein, referred to herein as an “AAV capsid variant.” In some embodiments, the AAV capsid variant described herein has the ability to enclose, e.g., encapsulate, a viral genome and/or is capable of entry into a cell, e.g., a mammalian cell. In some embodiments, the AAV capsid variant described herein may have modified tropism compared to that of a wild-type AAV capsid, e.g., the corresponding wild-type capsid. [00550] Encapsulate: As used herein, the term “encapsulate” means to enclose, surround or encase. As an example, a capsid protein, e.g., an AAV capsid variant, often encapsulates a viral genome. In some embodiments, encapsulate within a capsid, e.g., an AAV capsid variant, encompasses 100% coverage by a capsid, as well as less than 100% coverage, e.g., 95%, 90%, 85%, 80%, 70%, 60% or less. For example, gaps or discontinuities may be present in the capsid so long as the viral genome is retained in the capsid, e.g., prior to entry into a cell.
[00551] Codon optimization: As used herein, the term “codon optimization” refers to a process of changing codons of a given gene in such a manner that the polypeptide sequence encoded by the gene remains the same while the changed codons improve the process of expression of the polypeptide sequence. For example, if the polypeptide is of a human protein sequence and expressed in E. coli, expression will often be improved if codon optimization is performed on the DNA sequence to change the human codons to codons that are more effective for expression in E. coli.
[00552] Codon-optimized coding regions can be designed by various different methods. This optimization may be performed using methods which are available online (e.g., GeneArt), published methods, or a company which provides codon optimizing services, e.g., DNA2.0 (Menlo Park, CA). One codon optimizing method is described, e.g., in US International Patent Publication No. WO 2015/012924, which is incorporated by reference herein in its entirety. See also, e.g., US Patent Publication No. 2014/0032186 and US Patent Publication No. 2006/0136184.
[00553] Suitably, the entire length of the open reading frame (ORF) for the product is modified. However, in some embodiments, only a fragment of the ORF may be altered. By using one of these methods, one can apply the frequencies to any given polypeptide sequence, and produce a nucleic acid fragment of a codon-optimized coding region which encodes the polypeptide.
[00554] Conservative amino acid substitution'. As used herein, a "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
[00555] Conserved'. As used herein, the term “conserved” refers to nucleotides or amino acid residues of polynucleotide or polypeptide sequences, respectively, that are those that occur unaltered in the same position of two or more sequences being compared. Nucleotides or amino acids that are relatively conserved are those that are conserved among more related sequences than nucleotides or amino acids appearing elsewhere in the sequences.
[00556] In some embodiments, two or more sequences are said to be “completely conserved” if they are 100% identical to one another. In some embodiments, two or more sequences are said to be “highly conserved” if they are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to one another. In some embodiments, two or more sequences are said to be “highly conserved” if they are about 70% identical, about 80% identical, about 90% identical, about 95%, about 98%, or about 99% identical to one another. In some embodiments, two or more sequences are said to be “conserved” if they are at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to one another. In some embodiments, two or more sequences are said to be “conserved” if they are about 30% identical, about 40% identical, about 50% identical, about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical to one another. Conservation of sequence may apply to the entire length of an oligonucleotide or polypeptide or may apply to a portion, region or feature thereof.
[00557] In some embodiments, conserved sequences are not contiguous. Those skilled in the art are able to appreciate how to achieve alignment when gaps in contiguous alignment are present between sequences, and to align corresponding residues not withstanding insertions or deletions present.
[00558] Delivery: As used herein, “delivery” refers to the act or manner of delivering a parvovirus e.g., AAV compound, substance, entity, moiety, cargo or payload to a target. Such target may be a cell, tissue, organ, organism, or system (whether biological or production).
[00559] Delivery Agent: As used herein, “delivery agent” refers to any agent which facilitates, at least in part, the delivery of one or more substances (including, but not limited to a compounds and/or compositions of the present disclosure, e.g., viral particles or AAV vectors) to targeted cells.
Delivery route As used herein, the term “delivery route” and the synonymous term “administration route” refers to any of the different methods for providing a therapeutic agent to a subject. Routes of administration are generally classified by the location at which the substance is applied and may also be classified based on where the target of action is. Examples include, but are not limited to: intravenous administration, subcutaneous administration, oral administration, parenteral administration, enteral administration, topical administration, sublingual administration, inhalation administration, and injection administration, or other routes of administration described herein.
[00560] Effective amount: As used herein, the term “effective amount” of an agent is that amount sufficient to effect beneficial or desired results, for example, upon single or multiple dose administration to a subject or a cell, in curing, alleviating, relieving or improving one or more symptoms of a disorder and, as such, an “effective amount” depends upon the context in which it is being applied. For example, in the context of administering an agent that treats (collectively, “SMA-related disorders”), an effective amount of an agent is, for example, an amount sufficient to achieve treatment, as defined herein, of an SMA-related disorder as compared to the response obtained without administration of the agent.
[00561] Expression: As used herein, “expression” of a nucleic acid sequence refers to production of an RNA template from a DNA sequence (e.g., by transcription). In some embodiments, expression further comprises one or more of: (1 ) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end processing); (2) translation of an RNA into a polypeptide or protein; and (3) post-translational modification of a polypeptide or protein.
[00562] Fragment: A “fragment,” as used herein, refers to a portion. For example, an antibody fragment may comprise a CDR, or a heavy chain variable region, or a scFv, etc. In some embodiments, a fragment is a nucleic acid fragment.
[00563] SMA-related disorder: The terms “SMA-related disorder,” “SMA-related disease,” “an SMN patient,” and the like refer to diseases or disorders having a deficiency in the SMN gene, such as a heritable, e.g., autosomal recessive, mutation in SMN resulting in deficient or defective SMN protein expression in patient cells. SMA-related disorders expressly include, but are not limited to SMA, and related disorders. SMN patients are individuals harboring one or more mutation in the SMN gene, including, e.g., biallelic mutations, making them more susceptible to SMA-related disorders.
[00564] SMN protein'. As used herein, the terms “SMN”, “SMN protein,” “SMN proteins,” and the like refer to protein products or portions of protein products including peptides of the SMN gene, homologs or variants thereof, and orthologs thereof, including non-human proteins and homologs thereof. SMN proteins include fragments, derivatives, and modifications of SMN gene products.
[00565] Homology. As used herein, the term “homology” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules, in some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar. The term “homologous” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). In accordance with the disclosure, two polynucleotide sequences are considered to be homologous if the polypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%, 95%, or even 99% identical for at least one stretch of at least about 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is typically determined by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. In accordance with the disclosure, two protein sequences are considered to be homologous if the proteins are at least about 50%, 60%, 70%, 80%, or 90% identical for at least one stretch of at least about 20 amino acids. In many embodiments, homologous protein may show a large overall degree of homology and a high degree of homology over at least one short stretch of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more amino acids. In many embodiments, homologous proteins share one or more characteristic sequence elements. As used herein, the term “characteristic sequence element” refers to a motif present in related proteins. In some embodiments, the presence of such motifs correlates with a particular activity (such as biological activity).
[00566] Humanized As used herein, the term “humanized” refers to a non-human sequence of a polynucleotide or a polypeptide which has been altered to increase its similarity to a corresponding human sequence.
[00567] Identity. As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between oligonucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of the percent identity of two polynucleotide sequences, for example, may be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 ; each of which is incorporated herein by reference in its entirety. For example, the percent identity between two nucleotide sequences can be determined, for example using the algorithm of Meyers and Miller (CAB IOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by reference in its entirety. Techniques for determining identity are codified in publicly available computer programs. Computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molecular Biol., 215, 403 (1990)).
[00568] Isolated'. As used herein, the term “isolated” refers to a substance or entity that is altered or removed from the natural state, e.g., altered or removed from at least some of the components with which it is associated in the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of the environment in which it is found in nature. In some embodiments, an isolated nucleic acid is recombinant or may be incorporated into a vector.
[00569] miR binding site series: As used herein, the “miR binding site series” or the “miR binding site” includes an RNA sequence on the RNA transcript produced by transcribing the AAV vector genome. The “miR binding site series” or the “miR binding site” also includes the DNA sequence corresponding to the RNA sequence, in that they differ only by the T in DNA and the U in RNA. The reverse complement of such DNA is the coding sequence for the RNA sequence. That is, in some embodiments, in an expression cassette containing a DNA positive strand, the miR binding site sequence is the reverse complement of the miRNA to which it binds.
[00570] Variant: The term “variant” refers to a polypeptide or polynucleotide that has an amino acid or a nucleotide sequence that is substantially identical, e.g., having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to a reference sequence. In some embodiments, the variant is a functional variant.
[00571] Functional Variant: The term “functional variant” refers to a polypeptide variant or a polynucleotide variant that has at least one activity of the reference sequence.
[00572] Insertional Variant: "Insertional variants" when referring to polypeptides are those with one or more amino acids inserted, e.g., immediately adjacent or subsequent, to a position in an amino acid sequence. "Immediately adjacent" or “immediately subsequent” to an amino acid means connected to either the alpha-carboxy or alpha-amino functional group of the amino acid.
[00573] Nucleic acid: As used herein, the terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” refer to any nucleic acid polymers composed of either polydeoxyribonucleotides (containing 2-deoxy-D- ribose), or polyribonucleotides (containing D-ribose), or any other type of polynucleotide that is an N glycoside of a purine or pyrimidine base, or modified purine or pyrimidine bases. There is no intended distinction in length between the term “nucleic acid,” “polynucleotide,” and “oligonucleotide,” and these terms will be used interchangeably. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA.
[00574] Operably linked: As used herein, the phrase “operably linked” refers to a functional connection between two or more molecules, constructs, transcripts, entities, moieties or the like.
[00575] Payload As used herein, “payload” or “payload region” refers to one or more polynucleotides or polynucleotide regions encoded by or within a viral genome or an expression product of such polynucleotide or polynucleotide region, e.g., a transgcnc, a polynucleotide encoding a polypeptide. [00576] Payload construct: As used herein, “payload construct” is one or more polynucleotide regions encoding or comprising a payload that is flanked on one or both sides by an inverted terminal repeat (ITR) sequence. The payload construct is a template that is replicated in a viral production cell to produce a viral genome.
[00577] Payload construct vector. As used herein, “payload construct vector” is a vector encoding or comprising a payload construct, and regulatory regions for replication and expression in bacterial cells. The payload construct vector may also comprise a component for viral expression in a viral replication cell.
[00578] Pharmaceutically acceptable: The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are suitable for use in contact with the tissues of human beings and animals.
[00579] Polypeptide: As used herein, “polypeptide” means a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. If the polypeptide is a peptide, it will be at least about 2, 3, 4, or at least 5 amino acid residues long. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer. They may also comprise single chain or multichain polypeptides and may be associated or linked. The term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.
[00580] Polypeptide variant: The term “polypeptide variant” refers to molecules which differ in their amino acid sequence from a native or reference sequence. The amino acid sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence. In some embodiments, a variant comprises a sequence having at least about 50%, at least about 80%, or at least about 90%, identical (homologous) to a native or a reference sequence.
[00581] Peptide: As used herein, “peptide” is less than or equal to 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
[00582] Preventing: As used herein, the term “preventing” refers to partially or completely delaying onset of an infection, disease, disorder and/or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying progression from an infection, a particular disease, disorder and/or condition; and/or decreasing the risk of developing pathology associated with the infection, the disease, disorder, and/or condition.
[00583] Region: As used herein, the term “region” refers to a zone or general area. In some embodiments, when referring to a protein or protein module, a region may comprise a linear sequence of amino acids along the protein or protein module or may comprise a three-dimensional area, an epitope and/or a cluster of epitopes. In some embodiments, regions comprise terminal regions. As used herein, the term “terminal region” refers to regions located at the ends or termini of a given agent. When referring to proteins, terminal regions may comprise N- and/or C-termini.
[00584] In some embodiments, when referring to a polynucleotide, a region may comprise a linear sequence of nucleic acids along the polynucleotide or may comprise a three-dimensional area, secondary structure, or tertiary structure. In some embodiments, regions comprise terminal regions. As used herein, the term “terminal region” refers to regions located at the ends or termini of a given agent. When referring to polynucleotides, terminal regions may comprise 5’ and/or 3’ termini.
[00585] RNA or RNA molecule: As used herein, the term “RNA” or “RNA molecule” or “ribonucleic acid molecule” refers to a polymer of ribonucleotides; the term “DNA” or “DNA molecule” or “deoxyribonucleic acid molecule” refers to a polymer of deoxyribonucleotides. DNA and RNA can be synthesized naturally, e.g., by DNA replication and transcription of DNA, respectively; or be chemically synthesized. DNA and RNA can be single-stranded (i.e., ssRNA or ssDNA, respectively) or multistranded (e.g., double stranded, i.e., dsRNA and dsDNA, respectively). The term “mRNA” or “messenger RNA”, as used herein, refers to a single stranded RNA that encodes the amino acid sequence of one or more polypeptide chains.
[00586] Sample : As used herein, the term “sample” or “biological sample” refers to a subset of its tissues, cells, nucleic acids, or component parts (e.g., body fluids, including but not limited to blood, serum, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen).
[00587] Similarity: As used herein, the term “similarity” refers to the overall relatedness between polymeric molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of percent similarity of polymeric molecules to one another can be performed in the same manner as a calculation of percent identity, except that calculation of percent similarity takes into account conservative substitutions as is understood in the art.
[00588] Spacer: As used herein, a “spacer” is generally any selected nucleic acid sequence of, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length, which is located between two or more consecutive miR binding site sequences.
[00589] Subject: As used herein, the term “subject” or “patient” refers to any organism to which a composition in accordance with the disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants.
[00590] Suffering from: An individual who is “suffering from” a disease, disorder, and/or condition has been diagnosed with or displays one or more symptoms of a disease, disorder, and/or condition.
[00591] Susceptible to: An individual who is “susceptible to” a disease, disorder, and/or condition has not been diagnosed with and/or may not exhibit symptoms of the disease, disorder, and/or condition but harbors a propensity to develop a disease or its symptoms. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition (for example, cancer) may be characterized by one or more of the following: (1) a genetic mutation associated with development of the disease, disorder, and/or condition; (2) a genetic polymorphism associated with development of the disease, disorder, and/or condition; (3) increased and/or decreased expression and/or activity of a protein and/or nucleic acid associated with the disease, disorder, and/or condition; (4) habits and/or lifestyles associated with development of the disease, disorder, and/or condition; (5) a family history of the disease, disorder, and/or condition; and (6) exposure to and/or infection with a microbe associated with development of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.
[00592] Targeted Cells: As used herein, “target cells” or “targeted cells” refers to any one or more cells of interest. The cells may be found in vitro, in vivo, in situ or in the tissue or organ of an organism. The organism may be an animal, a mammal, a human and/or a patient. The target cells may be CNS cells or cells in CNS tissue.
[00593] Therapeutic Agent: The term “therapeutic agent” refers to any agent that, when administered to a subject has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect.
[00594] Therapeutically effective amount: As used herein, the term “therapeutically effective amount” means an amount of an agent to be delivered (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc. ) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is provided in a single dose. In some embodiments, a therapeutically effective amount is administered in a dosage regimen comprising a plurality of doses. Those skilled in the art will appreciate that in some embodiments, a unit dosage form may be considered to comprise a therapeutically effective amount of a particular agent or entity if it comprises an amount that is effective when administered as part of such a dosage regimen.
[00595] Treating: As used herein, the term “treating” refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a particular infection, disease, disorder, and/or condition. For example, “treating” cancer may refer to inhibiting survival, growth, and/or spread of a tumor. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
[00596] Vector. As used herein, the term “vector” refers to any molecule or moiety which transports, transduces, or otherwise acts as a carrier of a heterologous molecule. In some embodiments, vectors may be plasmids. Vectors of the present disclosure may be produced recombinantly. The heterologous molecule may be a polynucleotide and/or a polypeptide.
[00597] Viral genome: As used herein, the term “viral genome” or “vector genome” refers to the nucleic acid sequence(s) encapsulated in an AAV particle. A viral genome comprises a nucleic acid sequence with at least one payload region encoding a payload and at least one ITR.
EXAMPLES
[00598] The present disclosure is further illustrated by the following non-limiting examples. Experiments described in the Examples establish that enhanced AAV-based SMN gene therapy treatments are superior to and/or additive with wild-type SMN-based treatment in ameliorating SMA- related disorders.
Cell Lines, Tissues, and Animal Models
[00599] In vitro experiments: Human fibroblasts from SMA patients (all 3 types) are obtained. SMN primary neurons can be obtained from SMA animal models, for example, as provided herein.
[00600] Animal models: Therapeutic effectiveness and safety using the AAV virions including transgenes as described above can be tested in an appropriate animal model. For example, animal models which appear most similar to human disease include animal species which either spontaneously develop a high incidence of the particular disease or those that have been induced to do so. Tn particular, several animal models for SMA are known and have been generated. See, e.g., Sumner C. J., NeuroRx (2006) 3:235-245; Schmid et al., J. Child Neurol. (2007) 22: 1004-1012. As explained above, the molecular basis of SMA, an autosomal recessive neuromuscular disorder, is the homozygous loss of the survival motor neuron gene 1 (SMN1).
[00601] Caenorhabditis elegans models containing a null mutation of smn-1, smn-l( k355'), that deletes most of the smn-1 coding region, causes developmental arrest, reduced lifespan and progressive loss of motor function (Briese, et al., Hum. Mol. Genet. 18 (2009) 97-104). In addition, a C. elegans model containing a point mutation in smn-1 that mimics a human SMA disease mutation has been reported providing an efficient tool, for example, for large-scale screening for modifiers of SMN function (Sleigh et al., Hum. Mol. Genet. 20 (2011) 245-260.
[00602] Drosophila models carrying different Smn mutations have been developed and extensively studied. Mutant animals carrying an Smn point mutation similar to that in human SMA patients exhibit reduced excitatory post-synaptic currents, disorganized motor neuron boutons, loss of glutamate receptors at the neuromuscular junctions and compromised motor abilities (Chan et al., Hum. Mol. Genet. 12 (2003) 1367-1376).
[00603] The zebrafish (Danio rerio spinal cord primary motor neurons are commonly used to study changes in axon morphology, protein aggregation, and neuromuscular junction formation, which are highly relevant to SMA pathogenesis (McWhorter et al., J. Cell Biol. 162 (2003) 919-931. Zebrafish models with two stop mutations (smnY267stop and smnL265stop), and one exon 7 missense mutation (smnG264D) which was found to correspond to a human mutation (SMNG279V) known to cause SMA have also been reported (Boon et al., Hum. Mol. Genet. 18 (2009) 3615-3625). Additional zebrafish models of SMA carrying the human SMN2 gene also have been reported (Hao et al., Mol.
Neurodegener. 6 (2011) 24).
[00604] SMA mouse models have been generated by expressing different copies of human SMN2 through gene targeting or transgenesis on the background of homozygous disruption of Smnl gene. Phenotypic manifestations of severe (type I), intermediate (type II), and mild (type HI) forms of SMA in different mouse models correlate directly with SMN expression levels (Park et al., Curr. Neurol.
Neurosci. Rep. 10 (2010) 108-117; Schrank et al., PNAS (1997) 9920-9925), which is consistent with clinical observations in human patients. Among the most widely used SMA mouse models are the Smnl-/- ;SMN2tg/tg; SMNA7tg/tg (the A7 SMA mice or Jackson Laboratory stock #005025) (Le et al., Hum. Mol. Genet. 14 (2005) 845-857), and the Smnlhung-/-; ;SMN2Hungtg/tg (the Hung-Li SMA mice or Jackson laboratory stock #005058) (Hsieh-Li et al., Nat. Genet. 24 (2006) 66-70). Both models have an average lifespan of approximately 13 days and exhibit symptoms and neuropathology similar to patients afflicted with intermediate type II SMA.
[00605] Another mouse model, Smnl-/-;SMN2tg/tg (Jackson Laboratory stock #005024) mice that express a transgene encoding the full-length SMN2, are born with normal numbers of spinal motor neuron, but die around postnatal day 5 with a 40% loss of motor neurons and represent severe type I SMA (Monani et al., Hum. Mol. Genet. 9 (2000) 333-339). Smnl-/-;SMN2tg/tg;SMNA7tg/tg (the A7 SMA mice or Jackson Laboratory stock #005025) mice, serving as a model for type II intermediate SMA. The A7 SMA mice die around 13.3 days after birth, with the loss of approximately 50% of spinal cord a-motor neurons.
[00606] Another mouse model in which the SMNA7 transgene was replaced with an SMN1 A2G missense mutation, a mouse model (Smnl-/-;SMN2tg/tg;SMNlA2G, stock #005026) was generated (Monani et al., J. Cell. Biol. 160 (2003) 41-52). These mice exhibit mild type III SMA phenotypes characterized by motor axon sprouting and loss, muscle atrophy and abnormal EMG patterns.
[00607] In another mouse model, a high copy number SMN2 transgene SMN2(566) was incorporated into the Smnl knockout mice, the resulting mouse strain (Smnl-/-;SMN2(566) or Jackson laboratory stock #008206) expresses 16 copies of SMN2 transgene when made homozygous [Monani et al., Hum. Mol. Genet. 9 (2000) 333-339]. The increased copies of SMN2 rescue these mice from overt SMA phenotypes except that mice homozygous for both Smnl targeted mutation and SMN2(566) transgene show a shorter and thicker tail.
[00608] Large animal models of SMA also have been reported include pigs (Duque et al. (2015) Ann Neurol. 77(3): 399-414, 2015).
Example 1. Vector Design and Synthesis
[00609] Viral genomes were designed for AAV delivery of a SMN protein, e.g., a SMN1 protein. The nucleotide sequences from 5’ ITR to 3’ ITR are provided herein in Tables 12 and 13, as SEQ ID NOs: 10-24.
[00610] Each of these viral genome constructs comprise nucleic acid comprising a nucleotide sequence encoding an SMN protein. The nucleotide sequence was designed to comprise one of three codon- optimized sequences for expressing the SMN protein, which are provided in Table 11 A as SEQ ID NOs: 6-8. In designing these SMN constructs, several promoters were selected (e.g., the promoters described in Table 7), including an EF-1 a promoter variant (e.g., a truncated EF- la promoter) (SEQ ID NO: 26), a CBA promoter (SEQ ID NO: 29), a CBA promoter variant (e.g., a truncated CBA promoter) (SEQ ID NO: 27), a PGK promoter (SEQ ID NO: 31), an insulin promoter variant (SEQ ID NO: 30), a synapsin promoter (SEQ ID NO: 32), an Hb9 promoter (SEQ ID NO: 33), an MeCP2 promoter (SEQ ID NO: 34), and an MeCP2 promoter variant (SEQ ID NO: 35).
[00611] The viral genome constructs also all comprised a chimeric intron comprising the nucleotide sequence of SEQ ID NO: 3 and a polyA sequence comprising the nucleotide sequence of SEQ ID NO: 4. The 5’ ITR for all constructs comprised the nucleotide sequence of SEQ ID NO: 1 and the 3' ITR for all constructs comprised the nucleotide sequence of SEQ ID NO: 2.
Example 2. In vitro screen of SMN1 expressing viral genome constructs
[00612] Viral genome constructs expressing the SMN1 protein under different promoters and codon- optimized nucleotide sequences (SEQ ID NOs: 10-14 and 20-24) were evaluated in vitro for their relative expression of SMN1 in HEK293 cells compared to a control viral genome construct expressing SMN1 of SEQ NO: 25.
[00613] HEK293 cells were transfected with the SMN expressing viral genomes and the SMN viral genome control of SEQ ID NO: 25. After 48 hours, cell lysates were harvested for Western blot analysis of SMN1 protein expression as well as qPCR for SMN1 mRNA expression. The levels of SMN1 protein and mRNA were quantified as percent overexpression (% O.E.) relative to the control construct of SEQ ID NO: 25 (Table 14).
[00614] As provided in Table 14, viral genome constructs comprising the codon-optimized nucleotide sequence of SEQ ID NO: 8 expressing SMN (ITR to ITR constructs of SEQ ID NOs: 20-24) all led to increased expression of SMN relative to control construct of SEQ ID NO: 25, particular under the control of the CBA promoter, the CBA promoter variant, EF-1 a promoter variant, PGK promoter, and insulin promoter variant. [00615] Without wishing to be bound by theory, it is believed that viral genome constructs comprising the codon-optimized nucleotide sequence of SEQ ID NO: 8 expressing the SMN1 protein under the control of the CBA promoter, the CBA promoter variant, EF-la promoter variant, PGK promoter, and insulin promoter variant will result in differential, ubiquitous expression in disease-relevant cells populations.
Table 14. Percent overexpression (% O.E.) of SMN protein and mRNA by various viral genome constructs encoding an SMN protein relative to a viral genome control construct of SEQ ID NO: 25
Figure imgf000208_0001
Example 3. In vivo screen of AAV particles comprising viral genomes expressing an SMN protein [00616] In vivo target engagement in SMN disease model: After In vitro screening, target engagement in an SMN mouse model as described herein is demonstrated. In vivo evaluations determine whether AAV constructs expressing SMN proteins (e.g., a construct from Tablesl2 or 13) selected during in vitro evaluations result in comparable/significantly higher SMN expression and activity (e.g., increased motor neuron survival, decreased SMA-associated physical symptoms), as compared to AAV control (e.g., without an SMN payload) construct in an SMN mouse model.
[00617] SMN expressing constructs with favorable attributes as compared to control construct using the SMN mouse model are tested. AAV-untreated non transgenic (NT) mice are used as controls for biochemical analyses. SMN expressing candidates lead to at least a modest increase or greater (-30% over baseline) of SMN activity in SMN animal models. Untreated strain- and age-matched WT mice are included to compare physiological levels of SMN in healthy animals.
Example 4, High-throughput screen of TRACER AAV library in NHP and Rats
[00618] A TRACER based method as described in WO 2020/072683, WO 2021/202651, and WO 2021/230987, the contents of which are herein incorporated by reference in their entirety, was used to generate the AAV capsid variants described herein. An orthogonal evolution approach was combined with high throughput screening by NGS. Briefly, the library of AAV capsid variants was generated using a mutagenesis approach, where sequences of 7 to 8 amino acids in length were inserted into different positions across loop VIII of AAV5, including between residues 570-584, relative to a reference sequence numbered according to SEQ ID NO: 138. The initial library was passed three times through non-human primates (NHP), specifically cynomolgus macaques (Macaca fascicularis), rats, or human brain microvascular endothelial cells (hBMVECs). Following the third passage in each system, 572 variants from the NHPs, 80 variants from the rats, and 99 variants from the hBMVECs were pooled into a passage 3 synthetic library of 747 total variants. This library was then passaged in NHPs and rats. After this passage (e.g., one-month post injection into two NHPs and the rats), RNA was extracted from three brain regions. Following RNA recovery and RT-PCR amplification, a systematic NGS enrichment analysis was performed to calculate fold enrichment relative to an AAV5 wild-type control in NHPs (Table 15, left column) and rats (Table 15, right column) and the peptides comprised within the variants were identified in both animals. Fold enrichment values above 1 indicate an increase in expression relative to AAV5. All libraries of AAV5 variants generated and passaged in the different hosts were under the control of the synapsin promoter.
[00619] Approximately 288 variants were identified with an average fold change greater than wild-type AAV5 in the brain of NHPs. Of the 288 NHP variants, 27 demonstrated a fold-change of greater than 5 compared to wild-type, with 1 variant demonstrating a fold change of greater than 60. As shown in Table 15, the variant comprising YPAEVVQK (SEQ ID NO: 943) demonstrated a 64.9-fold enrichment in the brain of NHPs. [00620] Approximately 98 variants were identified with an average fold change greater than wild-type AAV5 in the brain of rats. Of the 98 variants, 33 demonstrated a fold-change of greater than 2 compared to wild-type, with one variant demonstrating a fold change of greater than 40. For instance, as shown in Table 15, the variant comprising YPAEVVQK (SEQ ID NO: 943) demonstrated a 41.1-fold enrichment in the brain of rats.
[00621] The variant comprising YPAEVVQK (SEQ ID NO: 943) which demonstrated a high fold enrichment in the brains of NHPs relative to wild-type AAV5 (64.9-fold enrichment), also demonstrated a high fold-change in the brains of rats (41.1 -fold enrichment). This indicates that this AAV capsid variant comprising SEQ ID NO: 943 is able to cross species, as evidenced by increased expression and tropism in both the NHP and rat brain.
Tabic 15. NGS fold-cnrichmcnt of AAV capsid variants in NHPs and rats
Figure imgf000210_0001
[00622] Taken together, these results demonstrate that after 3 rounds of screening of this AAV5 variant library with loop VIII modifications in NHP and rats, many AAV capsid variants outperformed the wildtype AAV5, for example, in penetration of the blood brain barrier (BBB) and expression in the brain. Also, capsid variants were identified that could infect both rats and NHPs, indicating cross-species tropism and compatibility.
Example 5. Individual capsid characterization in NHPs, rats, and mice
[00623] This Example describes the transduction level, tropism, ability to cross the blood brain barrier, and overall spatial distribution in the central nervous system (CNS) of an AAV capsid variant selected from the study described in Example 1, relative to wild-type AAV5, following intravenous injection in cynomolgus macaques (Macacafascicularis , Norway rats, and BALB/c mice. The capsid variant was TTN-002 (SEQ ID NO: 982 (amino acid) and 984 (DNA), comprising SEQ ID NO: 943 (encoded by SEQ ID NO: 944)), as outlined in Table 3 above. The amino acid and DNA sequence of TTN-002 is provided, e.g., in Tables 4 and 5, respectively.
[00624] AAV particles were generated with this capsid variant encapsulating a luciferase-EGFP transgene or a payload-HA tag driven by a heterologous constitutive promoter. Each capsid variant and control AAV5 and/or AAV9 capsids were tested by intravenously administering the AAV particle formulation at 5el3 VG/kg to NHPs (n=2), lel3 VG per rat (n=3; 3el3 VG/kg), and/or 5el l VG per mouse (n=3; 2el3 VG/kg). The in-life period was 28 days for NHPs and mice, and 25 days for rats. Various CNS and peripheral tissues were then collected for measuring transgene mRNA, transgene protein, and/or viral DNA (biodistribution). The AAV particles administered to the NHPs and rats comprised self-complementary viral genomes and the AAV particles administered to mice comprised a single-stranded viral genome.
A. Individual capsid characterization in NHPs
[00625] The brains, spinal cord, and peripheral tissues including the heart, liver, and quadriceps, were isolated from NHPs (cynomolgus macaques (Macaca fascicularis)) at 28 days post intravenous administration of the AAV particles comprising the TTN-002 capsid variant and were assayed by qPCR for the presence of transgene RNA as a measure of transgene expression and compared to the AAV9 control. Data were provided as average mRNA fold change for the transgene relative to a housekeeping gene as well as the fold change relative to the AAV9 control (Table 16). As shown in Table 16, mRNA transgene expression from the TTN-002 capsid variant, which is an AAV5 capsid variant, was significantly higher in the brain of NHPs relative to the wild-type AAV9 control. More specifically, mRNA expression was approximately 20-25-fold higher from the TTN-002 capsid variant compared to wild-type AAV9 in the brain of NHPs. Additionally, mRNA expression was approximately 4-5-fold higher from the TTN-002 capsid variants compared to wild-type AAV9 in the spinal cord of the NHPs. TTN-002 also demonstrated lower mRNA expression in the liver and DRG relative the AAV9 control (Table 16).
[00626] The brains, spinal cord, and peripheral tissues isolated from the NHPs were also assayed for the presence of viral DNA as a measure of viral genome levels. Data are provided in Table 17 as average DNA (viral genome (VG)) copies per diploid genome as well as fold change relative to the AAV9 control. As shown in Table 17, biodistribution of the AAV5 capsid variant, TTN-002, was significantly higher in the NHP brain relative to the wild-type AAV9 control. Biodistribution of TTN-002 was lower in the NHP liver relative to the wild-type AAV9 control.
[00627] The brain tissues and spinal cords of the NHPs were also subjected to immunohistochemistry staining to evaluate overall CNS tropism and biodistribution in various regions (FIGs. 1A-1D). Immunohistochemical staining correlated with the qPCR analysis, as TTN-002 showed significantly stronger staining and payload expression in the brain (e.g., across the entire cerebrum and cerebellum, FIG. 1A-1C) and spinal cord (FIG. 1A and ID), as compared to the AAV9 control. More specifically, TTN-002 demonstrated localization, strong payload expression, and transduction in both neurons and glial cells in the temporal cortex, perirhinal cortex, globus pallidus, putamen, caudate, thalamus, hippocampus, geniculate nucleus, Purkinje Layer, deep cerebellar nuclei, dentate, nucleus, brainstem, and cerebellum (FIGs. 1A-1C). Payload expression was also observed in astrocytes in the dentate nucleus. Additionally, quantification of co-expression of the payload and the pan-neuronal marker SMI-311 showed payload expression in approximately 73.4% of the large neurons in the dentate nucleus of the deep cerebral nucleus in the cerebellum at 28-days post intravenous administration the AAV particles comprising the TTN-002 capsid variant. In the spinal cord, TTN-002 demonstrated localization, strong payload expression, and transduction in the cervical region (e.g., C2), thoracic region (e.g., T10), and lumbar region (e.g., L2) (FIG. 1A and ID). Additionally, TTN-002 showed less staining in the DRG relative to the wild-type AAV9 control (approximately 2-fold less) (FIG. ID). Both TTN-002 and AAV9 appeared to transduce the liver and heart with similarly high efficiency, by IHC analysis (FIG. ID). Additionally, the histopathology of these samples isolated from the NHPs showed no signs of toxicity in the NHPs, following intravenous administration of AAV particles comprising the TTN-002 capsid variant at a dose of 5el3 VG/kg with a self-complementary viral genome.
Table 16. Transgene mRNA expression with the TTN-002 capsid variant in NHPs
Figure imgf000212_0001
Table 17. Viral DNA biodistribution with the TTN-002 capsid variant in NHPs
Figure imgf000212_0002
Figure imgf000213_0001
B. Individual capsid characterization in rats
[00628] The brains and spinal cords were isolated from rats at 25 days post intravenous administration of AAV particles comprising the TTN-002 capsid variant (AAV5 variant) and assayed for the presence of transgene RNA as a measure of transgene expression, relative to a wild-type AAV5 control capsid or a wild-type AAV9 control capsid (Table 18). Data were provided as average mRNA fold change for the transgene relative to a housekeeping gene as well as the fold change relative to the AAV5 and AAV9 controls (Table 18). As shown in Table 18, mRNA transgene expression from the TTN-002 capsid variant was higher in both the brains and spinal cords relative to the AAV5 and AAV9 controls. More specifically, transgene mRNA expression was approximately 40-67-fold higher from the TTN-002 variant in the rat brain and spinal cord regions (cervical, thoracic and lumbar) compared to wild-type AAV5 and transgene mRNA expression was approximately 5-7-fold higher in the rat brain and spinal cord regions (cervical, thoracic and lumbar) compared to wild-type AAV9.
[00629] The brain and spinal cord tissues, as well as the heart peripheral tissue were subject to immunohistochemistry staining to evaluate overall tropism and biodistribution. Immunohistochemical staining correlated with the qPCR analysis, as TTN-002 showed increased staining relative to both AAV9 and AAV5 in the cortex, hippocampus, cerebellum, and spinal cord of the rat. TTN-002 showed increased staining in the heart of the rat relative to AAV5 but decreased staining relative to AAV9.
Table 18. Transgene mRNA expression with TTN-002 capsid variant in rats
Figure imgf000213_0002
C. Individual capsid characterization in mice
[00630] The brains and livers were isolated from BALB/c mice 28 days post intravenous injection of following intravenous administration of AAV particles comprising the TTN-002 capsid variant and were assayed by qPCR for the presence of transgene RNA as a measure of transgene expression and compared to an AAV9 and AAV5 control. Data were provided as average mRNA fold change for the transgene relative to a housekeeping gene (Table 19) and as fold change in transgene mRNA expression relative AAV9 and AAV5 controls (Table 20). As shown in Table 19 and Table 20, the AAV5 capsid variant TTN-002 demonstrated similar levels of transgene expression relative to AAV9 in the brain and higher expression than wild-type AAV5. Transgene mRNA expression in the mouse brain was 265.9-fold higher with the TTN-002 capsid variant as compared to wild-type AAV5 (Table 20). Additionally, wildtype AAV5 and the AAV5 capsid variant, TTN-002 both resulted in lower transgene expression in the liver, as compared to wild-type AAV9.
[00631] The brains and livers isolated from the mice were also assayed for the presence of viral DNA as a measure of viral genome levels. Data are provided in Table 19 as average DNA (viral genome (VG)) copies per diploid genome and in Table 20 as fold change in DNA copies per diploid genome relative AAV9 and AAV5 controls. The AAV5 capsid variant TTN-002 demonstrated comparable biodistribution relative to AAV9 in the mouse brain and increased biodistribution and viral genome levels than wild-type AAV5. More specifically, in the brain, the TTN-002 capsid variant led to 9-fold higher DNA (viral genome (VG)) copies per diploid genome relative to the AAV5 control (Table 20). Furthermore, wild-type AAV5 and the AAV5 capsid variant, TTN-002 resulted in decreased biodistribution and DNA (viral genome (VG)) copies per diploid genome in the liver relative to AAV9 (Table 20).
Table 19. Transgene mRNA expression with the TTN-002 capsid variant in mice
Figure imgf000214_0001
Figure imgf000215_0001
Table 20. Fold-change in transgene mRNA expression and DNA copies per diploid genome relative to AAV9 and AAV5 in the brain and liver of mice
Figure imgf000215_0002
Conclusions
[00632] Taken together, these data demonstrate that TTN-002, which is an AAV5 capsid variant, is an enhanced CNS tropic capsid, in both NHPs and rodents (c.g., rats and mice), and was able to successfully penetrate the blood brain barrier. More specifically, TTN-002 demonstrated the ability to cross species, demonstrating enhanced CNS tropism and biodistribution in both NHPs and rats, and improvement relative to AAV5 in mice. Additionally, the TTN-002 capsid variant was able to successfully penetrate the blood brain barrier.
Example 6. Maturation of the TTN-002 capsid in NHPs
[00633] This Example describes maturation of TTN-002 (SEQ ID NO: 982 (amino acid) and 984 (DNA), comprising SEQ ID NO: 943 (encoded by SEQ ID NO: 944)) capsid variant in NHPs, specifically cynomolgus macaques (Macacafascicularis), to further enhance its transduction and biodistribution in the central nervous system and peripheral tissues and to evolve the AAV capsid variant further. Two approaches were used to mature the TTN-002 capsid sequences in order to randomize and mutate within and around the peptide insert comprised within loop VIII of the capsid variant. In the first maturation approach, mutagenic primers were used to introduce point mutations at a low frequency, scattered across the mutagenesis region in the TTN-002 sequence ranging from approximately position 571 to position 586, numbered according to SEQ ID NO: 982. In the second maturation approach, sets of three contiguous amino acids were randomized across the mutagenesis region in the TTN-002 sequence, which spanned from approximately position 571 to position 586, numbered according to SEQ ID NO: 982. AAV capsid variants arising from each maturation approach for TTN-002 were pooled together, for subsequent testing and characterization in NHPs.
[00634] The library of pooled matured AAV capsid variants generated from TTN-002 using the first maturation approach and the library of pooled matured AAV capsid variants generated from TTN-002 using the second maturation approach were each injected into two NHPs. After a period in life, the brains, heart, liver, muscle, and DRG of the NHPs were isolated and RNA was extracted. Following RNA recovery and RT-PCR amplification, a systematic NGS enrichment analysis was performed to calculate the fold enrichment ratio relative to the TTN-002 control, and the peptides comprised within the variants were identified.
[00635] Following the RNA recovery and NGS analysis from the second maturation approach, the matured capsid variants were filtered based on their coefficient of variance (CV), which was calculated for each peptide across the brain, heart, liver, muscle and DRG samples taken from the two NHPs. Those that had a CV value <2 were identified, as these were the peptides that were reliably detected in the majority of samples isolated from the brains of the two NHPs.
[00636] Table 21 provides the peptide sequences of these matured capsid variants and the fold enrichment of the matured capsid variant relative to the non-matured TTN-002 control that demonstrated the greatest fold-change in expression relative to the non-matured TTN-002 capsid variant in the brain of NHPs, following the first maturation approach and the second maturation approach. As shown in Table 21, following the first maturation approach, approximately 5 TTN-002 matured capsid variants demonstrated an increase in expression relative to the non-matured TTN-002 control, which demonstrated at least a 5-fold to 53-fold increase in expression in the NHP brain relative to the nonmatured TTN-002 control. Following the second maturation approach, approximately 38 TTN-002 matured capsid variants demonstrated an increase in expression in the NHP brain relative to the nonmatured TTN-002 control, with at least 27 demonstrating at least a 2-fold increase in expression (Table 21). Several variants demonstrated at least a 12-fold to 222-fold increase in expression in the NHP brain relative to the non-matured TTN-002 control (Table 21).
[00637] Fold-change in expression for the TTN-002 matured variants in Table 21 were also calculated for the DRG, muscle, liver (RNA and DNA), and heart of the NHPs following each maturation approach. Several variants that led to increased expression relative to the non-matured TTN-002 variant in the brain of the NHP also led to increased expression in other tissues. For instance, the matured TTN-002 capsid variant comprising the amino acid sequence TNNQSSYTPSLVQKTA (SEQ ID NO: 1585) demonstrated increased expression in the brain, heart, and liver relative to the non-matured TTN-002 control. Similarly, the matured TTN-002 capsid variants comprising the amino acid sequence TNNQSSYPPSLVKKTA (SEQ ID NO: 1591) and TNNQSSYPPSLVQKPA (SEQ ID NO: 1593), demonstrated increased expression in the brain and heart relative to the non-matured TTN-002 control. Additionally, the matured TTN-002 capsid variant comprising the amino acid sequence INNQSSYPAEVVQKTA (SEQ ID NO: 1024) demonstrated increased expression in the brain and the muscle relative to the non-matured TTN-002 control. Also, as shown in Table 21, many of the TTN-002 capsid variants that had increased expression in the brain, were de-targeted in the DRG. Therefore, several matured variants demonstrated increased tropism in more than one tissue type in the NHPs, with many showing reduced expression in the DRG.
Table 21. NGS fold-enrichment of the TTN-002 matured AAV capsid variants in the brain of NHPs following first and second mutagenesis approaches as compared to other NHP and mouse tissues
Figure imgf000217_0001
Figure imgf000218_0001
[00638] Furthermore, several of the TTN-002 matured capsid variants demonstrating an increase in expression relative to the non-matured TTN-002 control following the first and second maturation approaches in the brain of NHPs as shown in Table 21, also demonstrated an increase in expression in the brain of mice following the first and second maturation approach in mice. For instance, the matured TTN-002 capsid variant comprising the amino acid sequence TNNQSKYPAEVVQKTA (SEQ ID NO: 1538) demonstrated a 53.7-fold increase in expression relative to the non-matured TTN-002 following the first maturation approach in brain of NHPs, demonstrated a 20.86-fold increase in expression relative to the non-matured TTN-002 following the second maturation approach in brain of NHPs, a 10.26-fold increase in expression relative to the non-matured TTN-002 following the first maturation approach in brain of mice, and a 5.47-fold increase in increase in expression relative to the non-matured TTN-002 following the second maturation approach in brain of mice. Similarly, the matured TTN-002 capsid variant comprising the amino acid sequence TNNSSSYPAEVVQKTA (SEQ ID NO: 1539) demonstrated an 18.997-fold increase in expression relative to the non-matured TTN-002 following the first maturation approach in brain of NHPs, an 8.093-fold increase in expression relative to the nonmatured TTN-002 following the second maturation approach in brain of NHPs, an 8.539-fold increase in expression relative to the non-matured TTN-002 following the first maturation approach in brain of mice, and a 5.903-fold increase in increase in expression relative to the non-matured TTN-002 following the second maturation approach in brain of mice. Matured TTN-002 capsid variants comprising the amino acid sequences of SEQ ID NOs: 1021, 1024, 1027, 1112, 1142, 1214, 1232, 1254, 1300, 1310, 1327, 1331, 1342, and 1593 also demonstrated an increase in expression in the brain of both NHPs and mice, relative to the non-matured TTN-002 control. Therefore, several matured variants demonstrated increased expression relative to the non-matured controls in at least two different species, indicating cross-species tropism. [00639] These data demonstrate that following two maturation approaches, matured TTN-002 capsid variants (AAV5 capsid variants) with loop VIII modifications were generated with significantly enhanced CNS tropism in NHPs compared to the corresponding non-matured capsid variants, which already exhibited a significant fold enrichment over AAV5 and/or AAV9 in the brain of mice, rats, and/or NHPs. Also, several of the resulting matured variants demonstrated cross-species CNS tropism in both NHPs and mice.
Example 7. Evaluation of TTN-002 AAV capsid variant in diverse primate species
[00640] This Example evaluates the tropism and cross-species compatibility of the TTN-002 (SEQ ID NO: 982 (amino acid) and 984 (DNA), comprising SEQ ID NO: 943) capsid variant in two diverse primate species, marmosets (CcilUthrix jacchii. ) and African green monkeys (Chlorocebus sabaeus), as compared to their tropism in cynomolgus macaques (Macaca fascicularis) provided in Example 1 and 2. The amino acid and DNA sequences of the TTN-002 capsid variant are provided, e.g., in Tables 4 and 5, respectively.
[00641] To investigate tropism in African green monkeys, AAV particles comprising the TTN-002 capsid variant or an AAV5 control under the control of a synapsin promoter, were intravenously injected into the African green monkeys (n=2, 3-12 years of age) at a dose of 2E13 vg/kg. After 14-days in life, the brains and tissues (liver, DRG, quadriceps, and heart) of the NHPs were collected and RNA was extracted. Following RNA recovery and RT-PCR amplification, a systematic NGS enrichment analysis was performed to calculate the fold enrichment ratio relative to the AAV5 wild-type control.
[00642] To investigate tropism in marmoset monkeys, AAV particles comprising the TTN-002 capsid variant, or an AAV5 control, were intravenously injected into marmosets (n=2, >10 months of age) at a dose of 2E13 vg/kg. After 28-days in life, the brains and tissues (liver quadriceps, and heart) of the NHPs were collected and RNA was extracted. Following RNA recovery and RT-PCR amplification, a systematic NGS enrichment analysis was performed to calculate the fold enrichment ratio relative to the AAV5 wild- type control.
[00643] As provided in Table 22 (African green monkeys) and Table 23 (marmosets), the TTN-002 capsid variant demonstrated increased CNS tropism in diverse primate species. The TTN-002 capsid variant demonstrated a 64.9-fold increase in expression relative to AAV5 in the brain of cynomolgus macaques (Table 15, Example 4), a 7.5-fold increase in expression relative to AAV5 in the brain of African green monkeys, and a 40.4-fold increase in expression relative to AAV5 in the brain of marmosets. Furthermore, TTN-002 also resulted in increased expression in the brain of rats (Table 15, Example 4), demonstrating an average fold change in expression relative to AAV5 of 41.1.
Table 22. NGS-Fold Enrichment of TTN-002 in African green monkeys
Figure imgf000219_0001
Table 23. NGS-Fold Enrichment of TTN-002 in Marmosets
Figure imgf000220_0001
[00644] Taken together, these data demonstrate that the AAV5 capsid variant TTN-002 demonstrated increased CNS tropism relative to the AAV5 control in the CNS across three diverse primate species and rats, providing evidence of strong cross-species capacity.
IX. Equivalents and Scope
[00645] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to certain embodiments, it is apparent that further embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

We claim:
1. An AAV particle comprising an AAV capsid variant and a nucleic acid encoding a human survival motor neuron protein 1 (SMN1), fragment or variant thereof and/or a human SMN2 protein, fragment or variant thereof, wherein the AAV capsid variant comprises an amino acid sequence having the following formula: [N2]-[N3], wherein:
(i) [N2] comprises positions XI, X2, X3, X4, and X5, wherein:
(a) position XI is Y, N, or C;
(b) position X2 is P, K, T, or Q;
(c) position X3 is A or P;
(d) position X4 is E, S, or A; and
(e) position X5 is V, L, or E; and
(ii) [N3J comprises the amino acid sequence of VQK, EQK, VKK, VHK, VQQ, or LQK; wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 739, or an amino acid sequence at least 95% identical thereto.
2. The AAV particle of claim 1, wherein:
(a) [N2] comprises YP, NK, YT, YQ, NP, CP, TH, AE, PS, AA, AS, PA, PP, KA, TA, QA, TP, HA, EV, SL, EE, AV, or SH:
(b) [N2] comprises YPA, YPP, NKA, YTA, YQA, YTP, NPA, CPA, THA, PAE, PPS, KAE, TAE, QAE, TPS, PAA, HAS, AEV, PSL, AEE, or AAV;
(c) [N2] comprises YPAE (SEQ ID NO: 286), YPPS (SEQ ID NO: 287), NKAE (SEQ ID NO: 288), YTAE (SEQ ID NO: 289), YQAE (SEQ ID NO: 290), YTPS (SEQ ID NO: 291), YPAA (SEQ ID NO: 292), NPAE (SEQ ID NO: 293), CPAE (SEQ ID NO: 294), THAS(SEQ ID NO: 295) , PAEV (SEQ ID NO: 296), PPSL (SEQ ID NO: 297), KAEV (SEQ ID NO: 298), TAEV (SEQ ID NO: 299), PAEE (SEQ ID NO: 300), QAEV (SEQ ID NO: 301), TPSL (SEQ ID NO: 302), PAAV (SEQ ID NO: 303), or QAEE (SEQ ID NO: 304); and/or
(d) [N] is or comprises YPAEV (SEQ ID NO: 39), YPPSL (SEQ ID NO: 40), NKAEV (SEQ ID NO: 41), YTAEV (SEQ ID NO: 42), YPAEE (SEQ ID NO: 43), YQAEV (SEQ ID NO: 44), YTPSL (SEQ ID NO: 45), YPAAV (SEQ ID NO: 46), NPAEV (SEQ ID NO: 47), CP AEV (SEQ ID NO: 48), or YQAEE(SEQ ID NO: 49).
3. The AAV particle of claim 1 or 2, wherein [N2]-[N3] comprises:
(i) AEVVQK (SEQ ID NO: 50), PSLVQK (SEQ ID NO: 51), AEVEQK (SEQ ID NO: 52), AEEVQK (SEQ ID NO: 53), PSLEQK (SEQ ID NO: 54), PSLVKK (SEQ ID NO: 55), AEVVKK (SEQ ID NO: 56), AEVVHK (SEQ ID NO: 57), AAVVQK (SEQ ID NO: 58), AEVVQQ (SEQ ID NO: 59), or AEVLQK (SEQ ID NO: 60); (ii) PAEVVQK (SEQ ID NO: 61) , PPSLVQK (SEQ ID NO: 62), KAEVVQK (SEQ ID NO: 63), TAEVVQK (SEQ ID NO: 64), PAEVEQK (SEQ ID NO: 65), PAEEVQK (SEQ ID NO: 66), QAEVVQK (SEQ ID NO: 67), TPSLVQK (SEQ ID NO: 68), PPSLEQK (SEQ ID NO: 69), PPSLVKK (SEQ ID NO: 70), PAEVVKK (SEQ ID NO: 71), PAEVVHK (SEQ ID NO: 72), PAAVVQK (SEQ ID NO; 73), PAEVVQQ (SEQ ID NO: 74), TAEVVKK (SEQ ID NO: 75), PAEVLQK (SEQ ID NO: 76), or QAEEVQK (SEQ ID NO: 77); or
(iii) YPAEVVQK (SEQ ID NO: 943), YPPSLVQK (SEQ ID NO: 946), NKAEVVQK, YTAEVVQK (SEQ ID NO: 948), YPAEVEQK (SEQ ID NO: 949), YPAEEVQK(SEQ ID NO: 950), YQAEVVQK (SEQ ID NO: 951), YTPSLVQK (SEQ ID NO: 952), YPPSLEQK (SEQ ID NO: 953), YPPSLVKK (SEQ ID NO: 954), YPAEVVKK (SEQ ID NO: 955), YPAEVVHK (SEQ ID NO: 956), YPAAVVQK (SEQ ID NO: 957), NPAEVVQK (SEQ ID NO: 958), YPAEVVQQ (SEQ ID NO: 959), CPAEVVQK (SEQ ID NO: 960), YTAEVVKK (SEQ ID NO: 961), YPAEVLQK (SEQ ID NO: 962), or YQAEEVQK (SEQ ID NO: 963).
4. The AAV particle of any one of claims 1-3, wherein the AAV capsid variant further comprises [Nl], wherein [Nl] comprises positions XD, XE, and XF, wherein:
(a) position XD is Q, T, S, A, I, L, or H;
(b) position XE is S, G, A, or R; and
(c) position XF is S, K, L, R, A, or T.
5. The AAV particle of claim 4, wherein:
(i) [Nl] comprises QSS, QSK, TSL, SSS, QSR, AGA, IGS, QAS, ASS, LGS, QST, HSS, LSS, or QRS;
(ii) [N1]-[N2] comprises QSSYPAEV (SEQ ID NO: 113), QSKYPAEV (SEQ ID NO: 114), TSLYPAEV (SEQ ID NO: 115), SSSYPAEV (SEQ ID NO: 116), QSRYPAEV (SEQ ID NO: 117), QSSYPPSL (SEQ ID NO: 118), AGAYPAEV (SEQ ID NO: 119), IGSYPAEV (SEQ ID NO: 120), QASYPAEV (SEQ ID NO: 121), ASSYPAEV (SEQ ID NO: 122), LGSYPAEV (SEQ ID NO: 123), QSTNKAEV (SEQ ID NO: 124), HSSYPAEV (SEQ ID NO: 125), SSSYTAEV (SEQ ID NO: 126), TSLYPAEE (SEQ ID NO: 127), ASSYQAEV (SEQ ID NO: 129), QSSYTPSL (SEQ ID NO: 129), QSRYPAEE (SEQ ID NO: 130), LSSYQAEV (SEQ ID NO: 131), HSSYPAAV (SEQ ID NO: 132), QSSNPAEV (SEQ ID NO: 100), QSSYTAEV (SEQ ID NO: 133), TSLCPAEV (SEQ ID NO: 134), QRSYTAEV (SEQ ID NO: 135), or QSSYQAEE (SEQ ID NO: 136); and/or
(iii) [N1]-[N2]-[N3] comprises QSSYPAEVVQK (SEQ ID NO: 176), QSKYPAEVVQK (SEQ ID NO: 177), TSLYPAEVVQK (SEQ ID NO: 178), SSSYPAEVVQK (SEQ ID NO: 179), QSRYPAEVVQK (SEQ ID NO: 180), QSSYPPSLVQK (SEQ ID NO: 181), AGAYPAEVVQK (SEQ ID NO: 182), IGSYPAEVVQK (SEQ ID NO: 183), QASYPAEVVQK (SEQ ID NO: 184), ASSYPAEVVQK (SEQ ID NO: 186), LGSYPAEVVQK (SEQ ID NO: 187), QSTNKAEVVQK (SEQ ID NO: 188), HSSYPAEVVQK (SEQ ID NO: 189), SSSYTAEVVQK (SEQ ID NO: 190), QSKYPAEVEQK (SEQ ID NO: 191), TSLYPAEEVQK (SEQ ID NO: 192), ASSYQAEVVQK (SEQ ID NO: 193), QSSYTPSLVQK (SEQ ID NO: 194), QSRYPAEEVQK (SEQ ID NO: 195), QSSYPPSLEQ) (SEQ ID NO: 196), QSSYPPSLVKK (SEQ ID NO: 197), LSSYQAEVVQK (SEQ ID NO: 198), SSSYPAEVVKK (SEQ ID NO: 199), QSKYPAEVVHK (SEQ ID NO: 200), HSSYPAAVVQK (SEQ ID NO: 201), QSSNPAEVVQK (SEQ ID NO: 202), SSSYPAEVVQQ (SEQ ID NO: 203), QSSYTAEVVQK (SEQ ID NO: 204), TSLCPAEVVQK (SEQ ID NO: 205), QRSYTAEVVQK (SEQ ID NO: 206), QSSYTAEVVKK (SEQ ID NO: 207), HSSYPAEVLQK (SEQ ID NO: 208), or QSSYQAEEVQK (SEQ ID NO: 209).
6. The AAV particle of any one of claims 1-5, which further comprises:
(i) [NO], wherein [NO] comprises TNN, TNT, INN, TNS, NNN, or TNK; and/or
(ii) [N4], wherein [N4] comprises TA, PA, or NA.
7. The AAV particle of claim 6, wherein:
(i) [N0]-[N1 ] comprises TNNQSS (SEQ ID NO: 210), TNNQSK (SEQ ID NO: 21 1 ), TNNTSL (SEQ ID NO: 212), TNNSSS (SEQ ID NO: 213), TNNQSR (SEQ ID NO: 214), TNNAGA (SEQ ID NO: 215), TNNIGS (SEQ ID NO: 216), TNNQAS (SEQ ID NO: 217), TNTASS (SEQ ID NO: 218), TNNLGS (SEQ ID NO: 219), TNNQST (SEQ ID NO: 220), TNNHSS (SEQ ID NO: 221), TNNLSS (SEQ ID NO: 223), INNQSS (SEQ ID NO: 224), TNSQSS (SEQ ID NO: 225), NNNQSR (SEQ ID NO: 226), TNSTSL (SEQ ID NO: 227), TNNQRS (SEQ ID NO: 228), or TNKQAS (SEQ ID NO: 229);
(ii) [N0]-[Nl]-[N2]-[N3] comprises TNNQSSYPAEVVQK (SEQ ID NO: 230), TNNQSKYPAEVVQK (SEQ ID NO: 231), TNNTSL YPAEVVQK (SEQ ID NO: 232), TNNSSSYPAEVVQK (SEQ ID NO: 233), TNNQSRYPAEVVQK (SEQ ID NO: 234), TNNQSSYPPSLVQK (SEQ ID NO: 235), TNNAGAYPAEVVQK (SEQ ID NO: 236), TNNIGSYPAEVVQK (SEQ ID NO: 237), TNNQASYPAEVVQK (SEQ ID NO: 238), TNTASSYPAEVVQK (SEQ ID NO: 239), TNNLGSYPAEVVQK (SEQ ID NO: 240), TNNQSTNKAEVVQK (SEQ ID NO: 241), TNNHSS YPAEVVQK (SEQ ID NO: 242), TNNSSSYTAEVVQK (SEQ ID NO: 243), TNNQSKYPAEVEQK (SEQ ID NO: 244), TNNTSL YPAEEVQK (SEQ ID NO: 245), TNTASSYQAEVVQK (SEQ ID NO: 246), TNNQSSYTPSLVQK (SEQ ID NO: 247), TNNQSRYPAEEVQK (SEQ ID NO: 248), TNNQSSYPPSLEQK (SEQ ID NO: 249), TNNQSSYPPSLVKK (SEQ ID NO: 250), TNNLSSYQAEVVQK (SEQ ID NO: 251), TNNSSSYPAEVVKK (SEQ ID NO: 252), TNNQSKYPAEVVHK (SEQ ID NO: 253), INNQSSYPAEVVQK (SEQ ID NO: 254), TNNHSSYPAAVVQK (SEQ ID NO: 255), TNSQSSNPAEVVQK (SEQ ID NO: 256), TNNSSSYPAEVVQQ (SEQ ID NO: 257), NNNQSRYPAEVVQK (SEQ ID NO: 258), TNNQSSYTAEVVQK (SEQ ID NO: 259), TNNTSLCPAEVVQK (SEQ ID NO: 260), TNSTSLYPAEVVQK (SEQ ID NO: 261), TNNQRSYTAEVVQK (SEQ ID NO: 262), TNNQSSYTAEVVKK (SEQ ID NO: 263), TNNHSSYPAEVLQK (SEQ ID NO: 264), TNNQSSYQAEEVQK (SEQ ID NO: 266), or TNKQASYPAEVVQK (SEQ ID NO: 267); and/or
(iii) [NO]-[N1]-[N2]-[N3]-[N4] comprises TNNQSSYPAEVVQKTA (SEQ ID NO: 1533), TNNQSKYPAEVVQKTA (SEQ ID NO: 1538), TNNTSLYPAEVVQKTA (SEQ ID NO: 1232), TNNSSSYPAEVVQKTA (SEQ ID NO: 1539), TNNQSRYPAEVVQKTA(SEQ ID NO: 1327), TNNQSSYPPSLVQKTA (SEQ ID NO: 1300), TNNAGAYPAEVVQKTA (SEQ ID NO: 1021), TNNIGSYPAEVVQKTA (SEQ ID NO: 1112), TNNQASYPAEVVQKTA (SEQ ID NO: 1586), TNTASSYPAEVVQKTA (SEQ ID NO: 1575), TNNLGSYPAEVVQKTA (SEQ ID NO: 1027), TNNQSTNKAEVVQKTA (SEQ ID NO: 1578), TNNHSSYPAEVVQKTA (SEQ ID NO: 1310), TNNQSKYPAEVVQKTA(SEQ ID NO: 1538, TNNSSSYTAEVVQKTA (SEQ ID NO: 1214), TNNQSKYPAEVEQKTA (SEQ ID NO: 1254), TNNTSLYPAEEVQKTA(SEQ ID NO: 1583), TNTASSYQAEVVQKTA (SEQ ID NO: 1584), TNNQSSYTPSLVQKTA (SEQ ID NO: 1585), TNNQSRYPAEEVQKTA (SEQ ID NO: 1342), TNNQSSYPPSLEQKTA (SEQ ID NO: 1590), TNNQSSYPPSLVKKTA (SEQ ID NO: 1591), TNNLSSYQAEVVQKTA (SEQ ID NO: 1592), TNNQSSYPPSLVQKPA (SEQ ID NO: 1593), TNNSSSYPAEVVKKTA (SEQ ID NO: 1331), TNNQSKYPAEVVHKTA (SEQ ID NO: 1453), TNNSSSYPAEVVQKPA (SEQ ID NO: 1142), INNQSSYPAEVVQKTA(SEQ ID NO: 1024), TNNHSSYPAAVVQKTA (SEQ ID NO: 1598), TNSQSSNPAEVVQKTA (SEQ ID NO: 1599), TNNSSSYPAEVVQQTA (SEQ ID NO: 1419), NNNQSRYPAEVVQKTA (SEQ ID NO: 1601), TNNQSSYTAEVVQKNA (SEQ ID NO: 1602), TNNTSLCPAEVVQKTA (SEQ ID NO: 1603), TNSTSLYPAEVVQKTA (SEQ ID NO: 1605), TNNQRSYTAEVVQKTA (SEQ ID NO: 1604), TNNQSSYTAEVVKKTA (SEQ ID NO: 1606), TNNHSSYPAEVLQKTA (SEQ ID NO: 1607), TNNQSSYQAEEVQKTA (SEQ ID NO: 1608), or TNKQASYPAEVVQKTA (SEQ ID NO: 1587).
8. The AAV particle of claim 6 or 7, wherein:
(i) [N2]-[N3] comprises YPAEVVQK (SEQ ID NO: 943);
(ii) [N1]-[N2] comprises QSSYPAEV (SEQ ID NO: 113);
(iii) [N1]-[N2]-[N3] comprises QSSYPAEVVQK (SEQ ID NO: 176);
(iv) [N0]-[Nl] comprises TNNQSS (SEQ ID NO; 210);
(v) [N0]-[Nl]-[N2]-[N3] comprises TNNQSSYPAEVVQK (SEQ ID NO: 230); and/or
(vi) [NO]-[N1]-[N2]-[N3]-[N4] comprises TNNQSSYPAEVVQKTA (SEQ ID NO: 1533).
9. The AAV particle of any one of claims 6-8, wherein: (A) (i) [N2]-[N3] is present in loop VIII; and/or
(ii) [NO], [Nl], and [N4] are present in loop VIII; wherein loop VIII comprises positions 571-599 of SEQ ID NO: 982; and/or
(B) (i) XA of [NO] is present at position 571, XB of [NO] is present at position 572, and Xc of [NO] is present at position 573, numbered according to SEQ ID NO: 982;
(ii) XD of [Nl] is present at position 574, XE of [Nl] is present at position 575, and XF of [Nl] is present at position 576, numbered according to SEQ ID NO: 982;
(iii) XI of [N2] is present at position 577, X2 of [N2] is present at position 578, X3 of [N2] is present at position 579, X4 of [N2] is present at position 580, and X5 of [N2] is present at position 581, numbered according to SEQ ID NO: 982;
(iv) [N3] is present at positions 582-584, numbered according to SEQ ID NO: 982; and/or
(v) XQ of [N4J is present at position 585 and XH of [N4J is present at position 586, numbered according to SEQ ID NO: 982.
10. An AAV particle comprising an AAV capsid variant and a nucleic acid encoding a human survival motor neuron protein 1 (SMN1), fragment or variant thereof or a human SMN2 protein, fragment or variant thereof, wherein the AAV capsid variant comprises:
(a) the amino acid sequence of any of the sequences provided in Tables 2A, 2B, 2C, 15 or 21;
(b) an amino acid sequence comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive amino acids from any one of the sequences provided in Tables 2A, 2B, 2C, 15 or 21;
(c) an amino acid sequence comprising one, two, or three but no more than four different amino acids, relative to the amino acid sequence of any one of the amino acid sequences provided in Tables 2A, 2B, 2C, 15 or 21; or
(d) an amino sequence comprising one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of any one of the amino acid sequences provided in Tables 2A, 2B, 2C, 15 or 21.
11. The AAV particle of claim 10, wherein:
(i) the 3 consecutive amino acids comprise YPA;
(ii) the 4 consecutive amino acids comprise YPAE (SEQ ID NO: 286);
(iii) the 5 consecutive amino acids comprise YPAEV (SEQ ID NO: 39);
(iv) the 6 consecutive amino acids comprise YPAEVV (SEQ ID NO: 151);
(v) the 7 consecutive amino acids comprise YPAEVVQ (SEQ ID NO: 152); and/or
(vi) the amino acid sequence comprises YPAEVVQK (SEQ ID NO: 943).
12. The AAV particle of claim 10 or 11, wherein the AAV capsid variant comprises the amino acid sequence of YPAEVVQK (SEQ ID NO: 943), or an amino acid sequence comprising one, two, or three but no more than four different amino acids relative to the amino acid sequence of YPAEVVQK (SEQ ID NO: 943).
13. The AAV particle of any one of claims 1-11, wherein:
(i) the AAV capsid variant comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 944; or a nucleotide sequence comprising at least one, two, three, four, five, six, or seven, but no more than ten different nucleotides relative to the nucleotide sequence of SEQ ID NO: 944; and/or
(ii) the nucleotide sequence encoding the AAV capsid variant comprises the nucleotide sequence of SEQ ID NO: 944; or a nucleotide sequence comprising at least one, two, three, four, five, six, or seven, but no more than ten different nucleotides relative to the nucleotide sequence of SEQ ID NO: 944.
14. The AAV particle of any one of claims 1-13, wherein the AAV capsid variant comprises the amino acid Y at position 577, and the amino acid sequence of PAEVVQK (SEQ ID NO: 61) at positions 578- 584, numbered relative to SEQ ID NO: 982.
15. An AAV particle comprising an AAV capsid variant and a nucleic acid encoding a human survival motor neuron protein 1 (SMN1), fragment or variant thereof or a human SMN2 protein, fragment or variant thereof, wherein the AAV capsid variant comprises an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical to SEQ ID NO: 739, and wherein the AAV capsid variant comprises the amino acid Y at position 577, and comprises the amino acid sequence of PAEVVQK (SEQ ID NO: 61), at positions 578-584, numbered relative to SEQ ID NO: 982, numbered relative to SEQ ID NO: 982.
17. The AAV particle of any one of claims 1-15, wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 739.
18. The AAV particle of any one of claims 1-17, wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 738, or a sequence with at least 95% sequence identity thereto, optionally wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 738.
19. The AAV particle of any one of claims 1-18, wherein:
(i) the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 982, or an amino acid sequence with at least 95% sequence identity thereto, optionally wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 982; and/or
(ii) the nucleotide sequence encoding the AAV capsid variant comprises the nucleotide sequence of SEQ ID NO: 984, or a nucleotide sequence with at least 90% sequence identity thereto, optionally wherein the nucleotide sequence is codon optimized.
20. An AAV particle comprising an AAV capsid variant and a nucleic acid encoding a human survival motor neuron protein 1 (SMN1), fragment or variant thereof or a human SMN2 protein, fragment or variant thereof, wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 982.
21. The AAV particle of any one of the preceding claims, wherein the AAV capsid variant:
(i) has an increased tropism for a CNS cell or tissue, e.g., a brain cell, brain tissue, spinal cord cell, or spinal cord tissue, relative to the tropism of a reference sequence comprising the amino acid sequence of SEQ ID NO: 138, 139, or 982;
(ii) transduces a brain region, e.g., a temporal cortex, perirhinal cortex, globus pallidus, putamen, caudate, thalamus, hippocampus, geniculate nucleus, Purkinje Layer, deep cerebellar nuclei, and/or cerebellum, optionally wherein the level of transduction is at least 1.5, 2.2, 2.4, 2.5, 2.6, 2.7, 3.0, 3.2, 3.5, 3.7, 4.0, 4.2, 4.5, 4.7, 4.9, 5, 10, 15, 20, 25, 30, 35-fold greater as compared to a reference sequence of SEQ ID NO: 139 , e.g., when measured by an assay, e.g., an immunohistochemistry assay or a qPCR assay, e.g., as described in Example 2;
(iii) is enriched at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 65-fold, in the brain compared to a reference sequence of SEQ ID N O: 138, e.g., when measured by an assay as described in Example 1 ;
(iv) is enriched at least about 10, 12, 15, 17, 20, 25, 30, 35, 40, 45, 50, 55, 60, 61, 62, 63, 64, or 65-fold, in the brain compared to a reference sequence of SEQ ID NO: 138, e.g., when measured by an assay as described in Example 1;
(v) is enriched at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, 30, 35, 40, 45-fold, in the brain of at least two to three species, e.g., a non-human primate and rodent (e.g., rat or mouse), compared to a reference sequence of SEQ ID NO: 138, e.g., when measured by an assay as described in Examples 1, 2, and 4;
(vi) is enriched at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, or 225-fold, in the brain compared to a reference sequence of SEQ ID NO: 982, e.g., when measured by an assay as described in Example 4;
(vii) delivers an increased level of a payload to a brain region, optionally wherein the level of the payload is increased by at least 20, 25, 30, 35-fold, as compared to a reference sequence of SEQ ID NO: 139, e.g., when measured by an assay, e.g., a qRT-PCR or a qPCR assay (e.g., as described in Example 2), optionally wherein the brain region is a temporal cortex, perirhinal cortex, globus pallidus, putamen, caudate, thalamus, hippocampus, geniculate nucleus, Purkinje Layer, deep cerebellar nuclei, and/or cerebellum; (viii) delivers an increased level of viral genomes to a brain region, optionally wherein the level of viral genomes is increased by at least 1.5, 2.2, 2.4, 2.5, 2.6, 2.7, 3.0, 3.2, 3.5, 3.7, 4.0, 4.2, 4.5, 4.7, 4.9, or 5-fold, as compared to a reference sequence of SEQ ID NO: 139, e.g., when measured by an assay, e.g., a qRT-PCR or a qPCR assay (e.g., as described in Example 2), optionally wherein the brain region is a temporal cortex, perirhinal cortex, globus pallidus, putamen, caudate, thalamus, hippocampus, geniculate nucleus, Purkinje Layer, deep cerebellar nuclei, and/or cerebellum;
(ix) enriched at least about 3, 3.5, 4.0, 4.5, 5, 5.0, 6.0, or 6.5-fold, in a spinal cord region compared to a reference sequence of SEQ ID NO: 139, e.g., when measured by an assay as described in Example 2, optionally wherein the spinal cord region is a cervical region, a lumbar region, a thoracic region, or a combination thereof;
(x) shows preferential transduction in a brain region relative to the transduction in the dorsal root ganglia (DRG);
(xi) shows preferential transduction in a brain region relative to the transduction in the liver; and/or
(xii) is capable of transducing neuronal cell and non-neuronal cells, e.g., glial cells (e.g., oligodendrocytes or astrocytes).
22. The AAV particle of any one of claims 1-21, wherein the encoded human SMN1 protein comprises:
(i) the amino acid sequence of SEQ ID NO: 2000, or an amino acid sequence at least 70% (e g., at least 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto;
(ii) an amino acid sequence having at least one, two, three or four, but no more than 30, 20 or 10 different amino acids relative to SEQ ID NO: 2000; and/or
(iii) an amino acid sequence having at least one, two, three or four modifications, e.g., substitutions (e.g., conservative substitutions), but no more than 30, 20 or 10 modifications, e.g., substitutions (e.g., conservative substitutions), relative to SEQ ID NO: 2000.
23. The AAV particle of any one of claims 1-22, wherein the nucleotide sequence encoding the human SMN1 protein comprises:
(i) the nucleotide sequence of any one of SEQ ID NOs: 6-8; a nucleotide sequence comprising at least one, two, three, or four but no more than 30, 20, or 10 different nucleotides relative to any one of SEQ ID NOs: 6-8; or a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 6-8;
(ii) the nucleotide sequence of SEQ ID NO: 9; a nucleotide sequence comprising at least one, two, three, or four but no more than 30, 20, or 10 different nucleotides relative to SEQ ID NO: 9; or a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 9; (iii) the nucleotide sequence of any one of SEQ ID NOs: 2001-2004; a nucleotide sequence comprising at least one, two, three, or four but no more than 30, 20, or 10 different nucleotides relative to any one of SEQ ID NOs: 2001-2004; or a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 2001-2004; optionally wherein the nucleotide sequence encoding the human SMN1 protein is codon optimized.
24. The AAV particle of any one claims 1-23, wherein the nucleotide sequence encoding the SMN1 protein comprises the nucleotide sequence of SEQ ID NO: 8; a nucleotide sequence comprising at least one, two, three, or four but no more than 30, 20, or 10 different nucleotides relative to SEQ ID NO: 8; or a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 8.
25. The AAV particle of any one of claims 1-24, wherein the nucleic acid encoding the human SMN1 protein or the human SMN2 protein further comprises a nucleotide sequence encoding a splicing modulator element, optionally wherein:
(i) the encoded splicing modulator element increases the level of exon 7-containing SMN2 mRNA; and/or
(ii) the encoded splicing modulator element is an antisense oligonucleotide.
26. The AAV particle of claim 25, wherein:
(i) the antisense oligonucleotide targets the intronic silencer element ISSN-N1 located in exon 7 of the SMN2 gene;
(ii) the antisense oligonucleotide comprises any one of SEQ ID NOs: 2010-2013;
(iii) the encoded splicing modulator element is an UlsnRNA (e.g., an UlsnRNA capable of correcting the skipping of exon 7 of the SMN2 pre-mRNA), optionally wherein the UlsnRNA is exon specific (e.g., ExSpeUl) or comprises one or more of SEQ ID NOs: 2014-2019.
27. The AAV particle of any one of claims 1-26, which comprises a viral genome comprising a promoter operably linked to the nucleic acid sequence encoding the SMN protein.
28. The AAV particle of claim 27, wherein:
(i) the promoter is selected from human elongation factor la-subunit (EF-la), chicken P-actin (CBA) and its derivative, a phosphoglycerate kinase 1 (PGK), methyl-CpG binding protein 2 (MeCP2), insulin, Hb9, synapsin (Syn), cytomegalovirus (CMV) immediate-early enhancer and/or promoter, CAG, glucuronidase (GUSB), or ubiquitin C (UBC), neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B-chain (PDGF-P), intercellular adhesion molecule 2 (ICAM-2), Ca2+/calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), neurofilament light (NFL) or heavy (NFH), P-globin minigene n 2, preproenkephalin (PPE), enkephalin (Enk) and excitatory amino acid transporter 2 (EAAT2), glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), a cardiovascular promoter (e.g., aMHC, cTnT, and CMV- MLC2k), a liver promoter (e.g., hAAT, TBG), a skeletal muscle promoter (e.g., desmin, MCK, C512) or a fragment (e.g., a truncation) or a functional variant thereof;
(ii) the promoter is an EF-la promoter or a fragment (e.g., a truncation) or a functional variant thereof, optionally wherein the promoter comprises the nucleotide sequence of SEQ ID NO: 26, a nucleotide sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions, relative to the nucleotide sequence of SEQ ID NO: 26, or a nucleotide sequence with at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NOs: 26;
(ii) the promoter is a CBA promoter or a fragment (e.g., a truncation) or a functional variant thereof, optionally wherein the promoter comprises the nucleotide sequence of SEQ ID NO: 27 or 29, a nucleotide sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions, relative to the nucleotide sequence of SEQ ID NO: 27 or 29, or a nucleotide sequence with at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 27 or 29;
(iii) the promoter is a PGK promoter or a fragment (e.g., a truncation) or a functional variant thereof, optionally wherein the promoter comprises the nucleotide sequence of SEQ ID NO: 31, a nucleotide sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions, relative to the nucleotide sequence of SEQ ID NO: 31, or a nucleotide sequence with at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NOs: 31; or
(iv) the promoter comprises the nucleotide sequence of any one of SEQ ID NOs: 26-34 or 26-35 or 987, 988, 990, 991, 995, 996 998-1007, or any one of the nucleotide sequences in Table 7, a nucleotide sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions, relative to the nucleotide sequence of any one of SEQ ID NOs: 26-35 or 987, 988, 990, 991, 995, 996 998-1007, or any one of the nucleotide sequences in Table 7, or a nucleotide sequence with at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 26-35 or 987, 988, 990, 991, 995, 996998-1007, or any one of the nucleotide sequences in Table 7.
29. The AAV particle of claim 27 or 28, wherein the viral genome further comprises:
(i) a polyA signal sequence, optionally wherein the polyA signal region comprises the nucleotide sequence of SEQ ID NO: 4, a nucleotide sequence having at least one, two, or three modifications, but no more than four modifications of SEQ ID NO: 4, or a nucleotide sequence at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 4; (ii) an inverted terminal repeat (ITR) sequence, optionally wherein the ITR sequence is positioned 5’ relative to the encoded payload and/or the ITR sequence is positioned 3’ relative to the encoded payload;
(iii) an enhancer, optionally wherein the enhancer is a CMVie enhancer;
(iv) a Kozak sequence;
(v) an intron region, optionally wherein the intron is an SV40 intron or variant thereof; the intron is a chimeric intron or a variant thereof; or the intron comprises the nucleotide sequence of SEQ ID NO: 3, a nucleotide sequence comprising at least one, two, or three but no more than four modifications, e.g., substitutions, relative to the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence with at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NOs: 3;
(vi) an exon region; and/or
(vii) a nucleotide sequence encoding a miR binding site, e.g., a miR binding site that modulates, e.g., reduces, expression of the antibody molecule encoded by the viral genome in a cell or tissue where the corresponding miRNA is expressed, optionally wherein the encoded miR binding site modulates, e.g., reduces, expression of the encoded antibody molecule in a cell or tissue of the DRG, liver, heart, hematopoietic lineage, or a combination thereof.
30. The AAV particle of any one of claims 27-29, wherein the viral genome comprises:
(i) at least 1-5 copies of the encoded miR binding site, e.g., at least 1, 2, 3, 4, or 5 copies;
(ii) at least 3 copies of an encoded miR binding sites, optionally wherein:
(a) all three copies comprise the same miR binding site, or at least one, two, three, or all of the copies comprise a different miR binding site; and/or
(b) the 3 copies of the encoded miR binding sites are continuous (e.g., not separated by a spacer), or are separated by a spacer, optionally wherein the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), relative to GATAGTTA; or
(iii) at least 4 copies of an encoded miR binding site, optionally wherein
(a) all four copies comprise the same miR binding site, or at least one, two, three, or all of the copies comprise a different miR binding site; and/or
(b) the 4 copies of the encoded miR binding sites are continuous (e.g., not separated by a spacer), or are separated by a spacer, optionally wherein the spacer comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), relative to GATAGTTA.
31. The AAV particle of claim 29 or 30, wherein the encoded miR binding site comprises a miR122 binding site, a miR183 binding site, a miR-1 binding site, a miR-142-3p, or a combination thereof, optionally wherein:
(i) the encoded miR122 binding site comprises the nucleotide sequence of SEQ ID NO: 1865, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions relative to SEQ ID NO: 1865;
(ii) the encoded miR183 binding site comprises the nucleotide sequence of SEQ ID NO: 1847, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, live, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 1847;
(iii) the encoded miR-1 binding site comprises the nucleotide sequence of SEQ ID NO: 4679, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 4679; and/or
(iv) the encoded miR-142-3p binding site comprises the nucleotide sequence of SEQ ID NO: 1869, or a nucleotide sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) thereto; or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, e.g., substitutions, but no more than ten modifications, e.g., substitutions, relative to SEQ ID NO: 1869.
32. The AAV particle of any one of claims 27-31, wherein the viral genome comprises in 5’ to 3’ order:
(A) (i) a 5’ ITR, optionally wherein the 5’ ITR comprises the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical thereto;
(ii) a promoter, optionally wherein the promoter comprises the nucleotide sequence of any one of SEQ ID NOs: 26-34, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical to any one of SEQ ID NOs: 26-34;
(iii) an intron, optionally wherein the intron comprises the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical thereto;
(iv) a nucleotide sequence encoding an SMN protein, optionally comprising the nucleotide sequence of any one of SEQ ID NOs: 6-9, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical to any one of SEQ ID NOs: 6-8; (v) a polyA sequence, optionally wherein the polyA sequence comprises the nucleotide sequence of SEQ ID NO: 4, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical thereto; and
(vi) a 3’ ITR, optionally wherein the 3’ ITR comprises the nucleotide sequence of SEQ ID NO:
2, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical thereto; or
(B) (i) a 5’ ITR, optionally wherein the 5’ ITR comprises the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical thereto;
(ii) a promoter, optionally wherein the promoter comprises the nucleotide sequence of any one of SEQ ID NOs: 26, 27, 29, or 31, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical to any one of SEQ ID NOs: 26, 27, 29, or 31;
(iii) an intron, optionally wherein the intron comprises the nucleotide sequence of SEQ ID NO:
3, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical thereto;
(iv) a nucleotide sequence encoding an SMN protein, optionally comprising the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical thereto;
(v) a polyA sequence, optionally wherein the polyA sequence comprises the nucleotide sequence of SEQ ID NO: 4, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical thereto; and
(vi) a 3’ ITR, optionally wherein the 3’ ITR comprises the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical thereto.
33. The AAV particle of any one of claims 27-32, wherein the viral genome comprises:
(i) the nucleotide sequence of any one of SEQ ID NOs: 10-25, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical to any one of SEQ ID NOs: 10-25; or
(ii) the nucleotide sequence of any one of SEQ ID NOs: 20-22 or 24, or a nucleotide sequence at least 80% (e.g., 85, 90, 95, 96, 97, 98, or 99%) identical to any one of SEQ ID NOs: 20-22 or 24.
34. The AAV particle of any one of claims 27-33, wherein the viral genome:
(i) is single stranded or self-complementary;
(ii) comprises a nucleic acid encoding the AAV capsid variant and/or a Rep protein, e.g., a non- structural protein, wherein the Rep protein comprises a Rep78 protein, a Rep68, Rep52 protein, and/or a Rep40 protein (e.g., a Rep52 protein and/or Rep78 protein), optionally wherein the Rep78 protein, the Rep68 protein, the Rep52 protein, and/or the Rep40 protein are encoded by at least one Rep gene.
35. A cell comprising the AAV particle of any one of claims 1-34.
36. A method of making the AAV particle of any one of claims 1-34, the method comprising
(i) providing a host cell comprising a viral genome; and
(ii) incubating the host cell under conditions suitable to enclose the viral genome in the AAV capsid variant; thereby making the isolated AAV particle.
37. A pharmaceutical composition comprising an AAV particle of any one of claims 1-34, and a pharmaceutically acceptable excipient.
38. A method of delivering an exogenous human SMN1 protein and/or human SMN2 protein to a subject comprising administering to the subject an effective amount of the AAV particle of any one of claims 1- 34 or the pharmaceutical composition of claim 37.
39. The method of claim 38, wherein:
(i) the subject has, has been diagnosed with having, or is at risk of having a disease associated with a reduction in the quantity or function of an SMN1 and/or SMN2 protein, e.g., decreased SMN1 and/or SMN2 protein expression; and/or
(ii) the subject has, has been diagnosed with having, or is at risk of having a Spinal Muscular Atrophy (SMA) or an SMA-related disorder; Werdnig-Hoffman disease, Dubowitz disease, or Kugelberg- Welander disease.
40. A method of treating a subject having or diagnosed with having a disease related to decreased SMN1 and/or SMN2 protein expression, e.g., a mutation in an SMN1 and/or SMN2 gene, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 37, or the AAV particle of any one of claims 1-34.
41. A method of treating a subject having or diagnosed with Spinal Muscular Atrophy (SMA), comprising administering to the subject an effective amount of the pharmaceutical composition of claim 37, or the AAV particle of any one of claims 1-40.
42. The method of any one of claims 39-41, wherein the neurological disorder, e.g., a neurodegenerative disorder or disease related to decreased SMN1 and/or SMN2 protein expression is SMA, infantile onset SMA, type II SMA, type III SMA, Werdnig-Hoffman disease, Dubowitz disease, or Kugelberg-Welander disease.
43. The method of any one of claims 39-42, wherein the subject comprises: (i) at least 2 to 4 copies, e.g., at least 2 copies, at least 3 copies or at least 4 copies, of an SMN2 gene (e.g., a type II or a type III SMA);
(ii) 2-4 copies, e.g., 2 copies, 3 copies or at least 4 copies, of an SMN2 gene (e.g., a type II or a type III SMA);
(iii) 1 to 2 copies, e.g., 1 copy or 2 copies, of an SMN2 gene (e.g., a type I SMA);
(iv) no more than 2 copies, of an SMN2 gene (e.g., a type I SMA).
44. The method of any one of claims 38-43, wherein the AAV particle or the pharmaceutical composition is administered to the subject intravenously, via intra-cisterna magna injection (ICM), intracerebrally, intrathecally, intracerebroventricularly, via intraparenchymal administration, or intramuscularly.
45. The method of any one of claims 38-44, wherein administration of the AAV particle or the pharmaceutical composition results in an increased presence, level, and/or activity of SMN protein.
46. The method of any one of claims 38-45, further comprising administration of an additional therapeutic agent and/or therapy suitable for treatment or prevention of the disease associated with a reduction in the quantity or function of an SMN1 and/or SMN2 protein, e.g., decreased SMN1 and/or SMN2 protein expression, optionally wherein the additional therapeutic agent comprises a splicing modifier, a small molecule inhibitor (e.g., RG7916 (e.g., EVRYSDI™ or risdiplam)), an antisense oligonucleotide (e.g., Nusinersen (e.g., SPINRAZA™)), a myostatin inhibitor (e.g., an anti-myostatin antibody, e.g., SRK-015), a fast skeletal muscle troponin activator (FSTA) (e.g., CK-2127107), a neuroprotective agent (e.g., olesoxime or TRO19622), a chloride channel antagonist (e.g., a small molecule antagonist, e.g., NMD-670), or a combination thereof.
47. The method of any one of claims 38-46, wherein the subject is:
(i) between 2 months to 2 years of age, e.g., an early infantile subject;
(ii) between 1.5 years of age to 6 years of age, e.g., a late infantile subject;
(iii) between 6 years of age to 18 years of age, e.g., a juvenile;
(iv) 18 years of age or older, e.g., an adult;
(v) 2 months of age or older;
(vi) 2 years of age or younger; optionally wherein the subject is a human.
48. The pharmaceutical composition of claim 37 or the AAV particle of any one of claims 1-34, for use in a method of delivering an SMN1 protein and/or an SMN2 protein to a subject.
49. The pharmaceutical composition of claim 37 or the AAV particle of any one of claims 1-34, for use in a method of treating a neurological disorder, a neurodegenerative disorder, a disease associated with a mutation in the SMN1 gene and/or reduced SMN1 and/or SMN2 protein expression, or SMA.
50. The pharmaceutical composition of claim 37 or the AAV particle of any one of claims 1-34, for use in the manufacture of a medicament.
51. Use of the pharmaceutical composition of claim 37 or the AAV particle of any one of claims 1-34, in the manufacture of a medicament.
52. Use of the pharmaceutical composition of claim 37 or the AAV particle of any one of claims 1-34, in the manufacture of a medicament for treating a neurological disorder, a neurodegenerative disorder, a disease associated with a mutation in the SMN1 gene and/or reduced SMN1 and/or SMN2 protein expression, or SMA.
PCT/US2023/068202 2022-06-10 2023-06-09 Compositions and methods for the treatment of spinal muscular atrophy related disorders WO2023240236A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202263351058P 2022-06-10 2022-06-10
US63/351,058 2022-06-10

Publications (1)

Publication Number Publication Date
WO2023240236A1 true WO2023240236A1 (en) 2023-12-14

Family

ID=87136827

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2023/068202 WO2023240236A1 (en) 2022-06-10 2023-06-09 Compositions and methods for the treatment of spinal muscular atrophy related disorders

Country Status (1)

Country Link
WO (1) WO2023240236A1 (en)

Citations (78)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5387484A (en) 1992-07-07 1995-02-07 International Business Machines Corporation Two-sided mask for patterning of materials with electromagnetic radiation
WO1996017947A1 (en) 1994-12-06 1996-06-13 Targeted Genetics Corporation Packaging cell lines for generation of high titers of recombinant aav vectors
US5688676A (en) 1995-06-07 1997-11-18 Research Foundation Of State University Of New York In vitro packaging of adeno-associated virus DNA
US5691176A (en) 1990-10-30 1997-11-25 Applied Immune Sciences, Inc. Recombinant adeno-associated virus vector packaging cells and methods for use
US5741683A (en) 1995-06-07 1998-04-21 The Research Foundation Of State University Of New York In vitro packaging of adeno-associated virus DNA
US5858351A (en) 1996-01-18 1999-01-12 Avigen, Inc. Methods for delivering DNA to muscle cells using recombinant adeno-associated virus vectors
WO2000024916A1 (en) 1998-10-27 2000-05-04 Crucell Holland B.V. Improved aav vector production
WO2000028004A1 (en) 1998-11-10 2000-05-18 The University Of North Carolina At Chapel Hill Virus vectors and methods of making and administering the same
WO2000047757A1 (en) 1999-02-10 2000-08-17 Medigene Ag Method of producing a recombinant adeno-associated virus, suitable means for producing the same and use thereof for producing a medicament
US6156303A (en) 1997-06-11 2000-12-05 University Of Washington Adeno-associated virus (AAV) isolates and AAV vectors derived therefrom
US6204059B1 (en) 1994-06-30 2001-03-20 University Of Pittsburgh AAV capsid vehicles for molecular transfer
US6265389B1 (en) 1995-08-31 2001-07-24 Alkermes Controlled Therapeutics, Inc. Microencapsulation and sustained release of oligonucleotides
US6281010B1 (en) 1995-06-05 2001-08-28 The Trustees Of The University Of Pennsylvania Adenovirus gene therapy vehicle and cell line
WO2001089583A2 (en) 2000-05-23 2001-11-29 Neurologix, Inc. Glutamic acid decarboxylase (gad)_delivery system for treating neurodegenerative diseases
US6325998B1 (en) 1996-01-18 2001-12-04 Avigen, Inc. Methods of treating disease using recombinant adeno-associated virus virions administered to muscle
WO2001096587A2 (en) 2000-06-13 2001-12-20 The Children's Hospital Of Philadelphia Methods for administering recombinant adeno-associated virus virions to humans previously exposed to adeno-associated virus
US6335011B1 (en) 1996-01-18 2002-01-01 Avigen, Inc. Methods for delivering DNA to muscle cells using recombinant adeno-associated virus virions to treat lysosomal storage disease
WO2002014487A2 (en) 2000-08-17 2002-02-21 Keiya Ozawa Adeno-associated virus-mediated delivery of angiogenic factors
WO2001023001A3 (en) 1999-09-29 2002-03-14 Univ Pennsylvania Rapid peg-modification
US6365394B1 (en) 1999-09-29 2002-04-02 The Trustees Of The University Of Pennsylvania Cell lines and constructs useful in production of E1-deleted adenoviruses in absence of replication competent adenovirus
US20020081721A1 (en) 1996-12-18 2002-06-27 James M. Allen Aav split-packaging genes and cell lines comprising such genes for use in the production of recombinant aav vectors
US6428988B1 (en) 1996-09-06 2002-08-06 The Trustees Of The University Of Pennsylvania Methods using cre-lox for production of recombinant adeno-associated viruses
US6436708B1 (en) 1997-04-17 2002-08-20 Paola Leone Delivery system for gene therapy to the brain
US20030138772A1 (en) 2001-11-13 2003-07-24 Guangping Gao Method of detecting and/or identifying adeno-associated virus (AAV) sequences and isolating novel sequences identified thereby
US6660521B2 (en) 1998-12-04 2003-12-09 Genvec, Inc. Use of trans-activation and CIS-activation to increase the persistence of a transgene in an at least E4-deficient adenovirus
WO2004112727A2 (en) 2003-06-19 2004-12-29 Avigen, Inc. Aav virions with decreased immunoreactivity and uses therefor
WO2005005610A2 (en) 2003-06-30 2005-01-20 The Regents Of The University Of California Mutant adeno-associated virus virions and methods of use thereof
WO2005072364A2 (en) 2004-01-27 2005-08-11 University Of Florida A modified baculovirus expression system for production of pseudotyped raav vector
US20060136184A1 (en) 2003-08-01 2006-06-22 Dna Twopointo Inc. Systems and methods for antibody engineering
US7101993B1 (en) 1990-01-11 2006-09-05 Isis Pharmaceuticals, Inc. Oligonucleotides containing 2′-O-modified purines
US7112321B2 (en) 2001-12-19 2006-09-26 Genzyme Corporation Adeno-associated virus-mediated delivery of GDNF to skeletal muscles
EP1857552A1 (en) 2006-05-20 2007-11-21 Cargill Incorporated Thermostable xylose isomerase enzyme
US7427396B2 (en) 2004-06-03 2008-09-23 Genzyme Corporation AAV vectors for gene delivery to the lung
US7465583B2 (en) 2000-06-01 2008-12-16 The University Of North Carolina At Chapel Hill Duplexed parvovirus vectors
US7588757B2 (en) 2001-03-14 2009-09-15 Genzyme Corporation Methods of treating Parkinson's disease using recombinant adeno-associated virus virions
WO2009151546A2 (en) 2008-05-27 2009-12-17 Ptc Therapeutics, Inc. Methods for treating spinal muscular atrophy
WO2010129021A1 (en) 2009-05-02 2010-11-11 Genzyme Corporation Gene therapy for neurodegenerative disorders
US7838657B2 (en) 2004-12-03 2010-11-23 University Of Massachusetts Spinal muscular atrophy (SMA) treatment via targeting of SMN2 splice site inhibitory sequences
US8163543B2 (en) 2005-10-20 2012-04-24 Amsterdam Molecular Therapeutics B.V. AAV vectors produced in insect cells
WO2012057363A1 (en) 2010-10-27 2012-05-03 学校法人自治医科大学 Adeno-associated virus virions for transferring genes into neural cells
US8283151B2 (en) 2005-04-29 2012-10-09 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Isolation, cloning and characterization of new adeno-associated virus (AAV) serotypes
WO2012144446A1 (en) 2011-04-18 2012-10-26 独立行政法人国立精神・神経医療研究センター Drug delivery particles and method for producing same
US8361977B2 (en) 2005-06-23 2013-01-29 Isis Pharmaceuticals, Inc. Compositions and methods for modulation of SMN2 splicing
US20130195801A1 (en) 2010-04-23 2013-08-01 University Of Massachusetts Cns targeting aav vectors and methods of use thereof
US8512981B2 (en) 2006-06-21 2013-08-20 Amsterdam Molecular Therapeutics B.V. Vectors with modified initiation codon for the translation of AAV-Rep78 useful for production of AAV
EP2678433A1 (en) 2011-02-22 2014-01-01 California Institute of Technology Delivery of proteins using adeno-associated virus (aav) vectors
US20140032186A1 (en) 2003-08-01 2014-01-30 Dna Twopointo, Inc. Systems and methods for antibody engineering
WO2014178863A1 (en) 2013-05-01 2014-11-06 Genzyme Corporation Compositions and methods for treating spinal muscular atrophy
WO2015012924A2 (en) 2013-04-29 2015-01-29 The Trustees Of The University Of Pennsylvania Tissue preferential codon modified expression cassettes, vectors containing same, and use thereof
US8946152B2 (en) 2003-02-24 2015-02-03 Amgen Inc. Method of treating parkinson's disease in humans by convection-enhanced infusion of glial cell-line derived neurotrophic factor to the putamen
US8980853B2 (en) 2009-06-17 2015-03-17 Isis Pharmaceuticals, Inc. Compositions and methods for modulation of SMN2 splicing in a subject
US8999948B2 (en) 2010-06-10 2015-04-07 Laboratorios Del Dr. Esteve, S.A. Vectors and sequences for the treatment of diseases
US20150126590A1 (en) 2013-11-01 2015-05-07 Cornell University Gene therapy for alzheimer's and other neurodegenerative diseases and conditions
US9074207B2 (en) 2010-10-15 2015-07-07 Universita′ Degli Studi Di Ferrara Modified human U1snRNA molecule, a gene encoding for the modified human U1snRNA molecule, an expression vector including the gene, and the use thereof in gene therapy
US20150315612A1 (en) 2003-09-30 2015-11-05 The Trustees Of The University Of Pennsylvania Adeno-associated virus (aav) clades, sequences, vectors containing same, and uses therefor
WO2015168666A2 (en) 2014-05-02 2015-11-05 Genzyme Corporation Aav vectors for retinal and cns gene therapy
WO2015191508A1 (en) 2014-06-09 2015-12-17 Voyager Therapeutics, Inc. Chimeric capsids
US20160032254A1 (en) 2007-07-26 2016-02-04 Uniqure Ip B.V. Aav vectors produced by insect cells comprising rep52 and rep78 coding sequences with differential codon biases
US20160289275A1 (en) 2015-04-06 2016-10-06 The Usa, As Represented By The Secretary, Dept. Of Health And Human Services Adeno-Associated Vectors for Enhanced Transduction and Reduced Immunogenicity
WO2017008748A1 (en) 2015-07-14 2017-01-19 The Hong Kong University Of Science And Technology Passive thermal diode
US9586955B2 (en) 2012-02-10 2017-03-07 Ptc Therapeutics, Inc. Compounds for treating spinal muscular atrophy
US20170143847A1 (en) 2015-11-24 2017-05-25 Universita Di Ferrara Modified human u1snrna molecule, a gene encoding for the modified human u1snrna molecule, an expression vector including the gene, and the use thereof in gene therapy of familial dysautonomia and spinal muscular atrophy
WO2017100671A1 (en) * 2015-12-11 2017-06-15 California Institute Of Technology TARGETING PEPTIDES FOR DIRECTING ADENO-ASSOCIATED VIRUSES (AAVs)
US20180066279A9 (en) 2014-04-25 2018-03-08 University Of Massachusetts Recombinant aav vectors useful for reducing immunity against transgene products
US9929669B2 (en) 2016-02-24 2018-03-27 Lsis Co., Ltd. Inverter
US9969754B2 (en) 2014-05-15 2018-05-15 Hoffmann-La Roche Inc. Compounds for treating spinal muscular atrophy
WO2019136484A1 (en) 2018-01-08 2019-07-11 Prevail Therapeutics, Inc. Pet imaging ligands for in vivo detection of gba1
WO2019147960A1 (en) 2018-01-25 2019-08-01 Biogen Ma Inc. Methods of treating spinal muscular atrophy
US10436802B2 (en) 2014-09-12 2019-10-08 Biogen Ma Inc. Methods for treating spinal muscular atrophy
WO2019213180A1 (en) 2018-04-30 2019-11-07 Amicus Therapeutics, Inc. Gene therapy constructs and methods of use
WO2020014471A1 (en) * 2018-07-11 2020-01-16 The Brigham And Women's Hospital, Inc. Methods and compositions for delivery of agents across the blood-brain barrier
WO2020037161A1 (en) 2018-08-15 2020-02-20 Biogen Ma Inc. Combination therapy for spinal muscular atrophy
WO2020072683A1 (en) 2018-10-02 2020-04-09 Voyager Therapeutics, Inc. Redirection of tropism of aav capsids
WO2020117898A1 (en) * 2018-12-05 2020-06-11 Abeona Therapeutics Inc. Recombinant adeno-associated viral vector for gene delivery
WO2020132455A1 (en) 2018-12-21 2020-06-25 The Trustees Of The University Of Pennsylvania Compositions for drg-specific reduction of transgene expression
US20210171519A1 (en) 2017-08-04 2021-06-10 Skyhawk Therapeutics, Inc. Methods and compositions for modulating splicing
WO2021202651A1 (en) 2020-04-01 2021-10-07 Voyager Therapeutics, Inc. Redirection of tropism of aav capsids
WO2021230987A1 (en) 2020-05-13 2021-11-18 Voyager Therapeutics, Inc. Redirection of tropism of aav capsids

Patent Citations (85)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7101993B1 (en) 1990-01-11 2006-09-05 Isis Pharmaceuticals, Inc. Oligonucleotides containing 2′-O-modified purines
US5691176A (en) 1990-10-30 1997-11-25 Applied Immune Sciences, Inc. Recombinant adeno-associated virus vector packaging cells and methods for use
US5387484A (en) 1992-07-07 1995-02-07 International Business Machines Corporation Two-sided mask for patterning of materials with electromagnetic radiation
US6204059B1 (en) 1994-06-30 2001-03-20 University Of Pittsburgh AAV capsid vehicles for molecular transfer
WO1996017947A1 (en) 1994-12-06 1996-06-13 Targeted Genetics Corporation Packaging cell lines for generation of high titers of recombinant aav vectors
US6281010B1 (en) 1995-06-05 2001-08-28 The Trustees Of The University Of Pennsylvania Adenovirus gene therapy vehicle and cell line
US5741683A (en) 1995-06-07 1998-04-21 The Research Foundation Of State University Of New York In vitro packaging of adeno-associated virus DNA
US5688676A (en) 1995-06-07 1997-11-18 Research Foundation Of State University Of New York In vitro packaging of adeno-associated virus DNA
US6265389B1 (en) 1995-08-31 2001-07-24 Alkermes Controlled Therapeutics, Inc. Microencapsulation and sustained release of oligonucleotides
US6555525B2 (en) 1995-08-31 2003-04-29 Alkermes Controlled Therapeutics, Inc. Microencapsulation and sustained release of oligonucleotides
US7704492B2 (en) 1996-01-18 2010-04-27 Genzyme Corporation Methods for delivering DNA to muscle cells using recombinant adeno-associated virus virions
US5858351A (en) 1996-01-18 1999-01-12 Avigen, Inc. Methods for delivering DNA to muscle cells using recombinant adeno-associated virus vectors
US6610290B2 (en) 1996-01-18 2003-08-26 Avigen, Inc. Adeno associated virus vectors for the treatment of a cardiomyopathy
US6335011B1 (en) 1996-01-18 2002-01-01 Avigen, Inc. Methods for delivering DNA to muscle cells using recombinant adeno-associated virus virions to treat lysosomal storage disease
US6211163B1 (en) 1996-01-18 2001-04-03 Avigen, Inc. Methods for delivering DNA to the bloodstream using recombinant adeno-associated virus vectors
US6325998B1 (en) 1996-01-18 2001-12-04 Avigen, Inc. Methods of treating disease using recombinant adeno-associated virus virions administered to muscle
US6428988B1 (en) 1996-09-06 2002-08-06 The Trustees Of The University Of Pennsylvania Methods using cre-lox for production of recombinant adeno-associated viruses
US20020081721A1 (en) 1996-12-18 2002-06-27 James M. Allen Aav split-packaging genes and cell lines comprising such genes for use in the production of recombinant aav vectors
US6436708B1 (en) 1997-04-17 2002-08-20 Paola Leone Delivery system for gene therapy to the brain
US6156303A (en) 1997-06-11 2000-12-05 University Of Washington Adeno-associated virus (AAV) isolates and AAV vectors derived therefrom
WO2000024916A1 (en) 1998-10-27 2000-05-04 Crucell Holland B.V. Improved aav vector production
WO2000028004A1 (en) 1998-11-10 2000-05-18 The University Of North Carolina At Chapel Hill Virus vectors and methods of making and administering the same
US6660521B2 (en) 1998-12-04 2003-12-09 Genvec, Inc. Use of trans-activation and CIS-activation to increase the persistence of a transgene in an at least E4-deficient adenovirus
WO2000047757A1 (en) 1999-02-10 2000-08-17 Medigene Ag Method of producing a recombinant adeno-associated virus, suitable means for producing the same and use thereof for producing a medicament
US6365394B1 (en) 1999-09-29 2002-04-02 The Trustees Of The University Of Pennsylvania Cell lines and constructs useful in production of E1-deleted adenoviruses in absence of replication competent adenovirus
WO2001023001A3 (en) 1999-09-29 2002-03-14 Univ Pennsylvania Rapid peg-modification
WO2001089583A2 (en) 2000-05-23 2001-11-29 Neurologix, Inc. Glutamic acid decarboxylase (gad)_delivery system for treating neurodegenerative diseases
US7465583B2 (en) 2000-06-01 2008-12-16 The University Of North Carolina At Chapel Hill Duplexed parvovirus vectors
WO2001096587A2 (en) 2000-06-13 2001-12-20 The Children's Hospital Of Philadelphia Methods for administering recombinant adeno-associated virus virions to humans previously exposed to adeno-associated virus
WO2002014487A2 (en) 2000-08-17 2002-02-21 Keiya Ozawa Adeno-associated virus-mediated delivery of angiogenic factors
US7588757B2 (en) 2001-03-14 2009-09-15 Genzyme Corporation Methods of treating Parkinson's disease using recombinant adeno-associated virus virions
US20030138772A1 (en) 2001-11-13 2003-07-24 Guangping Gao Method of detecting and/or identifying adeno-associated virus (AAV) sequences and isolating novel sequences identified thereby
US7112321B2 (en) 2001-12-19 2006-09-26 Genzyme Corporation Adeno-associated virus-mediated delivery of GDNF to skeletal muscles
US8946152B2 (en) 2003-02-24 2015-02-03 Amgen Inc. Method of treating parkinson's disease in humans by convection-enhanced infusion of glial cell-line derived neurotrophic factor to the putamen
WO2004112727A2 (en) 2003-06-19 2004-12-29 Avigen, Inc. Aav virions with decreased immunoreactivity and uses therefor
WO2005005610A2 (en) 2003-06-30 2005-01-20 The Regents Of The University Of California Mutant adeno-associated virus virions and methods of use thereof
US20060136184A1 (en) 2003-08-01 2006-06-22 Dna Twopointo Inc. Systems and methods for antibody engineering
US20140032186A1 (en) 2003-08-01 2014-01-30 Dna Twopointo, Inc. Systems and methods for antibody engineering
US20150315612A1 (en) 2003-09-30 2015-11-05 The Trustees Of The University Of Pennsylvania Adeno-associated virus (aav) clades, sequences, vectors containing same, and uses therefor
WO2005072364A2 (en) 2004-01-27 2005-08-11 University Of Florida A modified baculovirus expression system for production of pseudotyped raav vector
US7427396B2 (en) 2004-06-03 2008-09-23 Genzyme Corporation AAV vectors for gene delivery to the lung
US10266822B2 (en) 2004-12-03 2019-04-23 University Of Massachusetts Spinal muscular atrophy (SMA) treatment via targeting of SMN2 splice site inhibitory sequences
US7838657B2 (en) 2004-12-03 2010-11-23 University Of Massachusetts Spinal muscular atrophy (SMA) treatment via targeting of SMN2 splice site inhibitory sequences
US8283151B2 (en) 2005-04-29 2012-10-09 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Isolation, cloning and characterization of new adeno-associated virus (AAV) serotypes
US8361977B2 (en) 2005-06-23 2013-01-29 Isis Pharmaceuticals, Inc. Compositions and methods for modulation of SMN2 splicing
US8163543B2 (en) 2005-10-20 2012-04-24 Amsterdam Molecular Therapeutics B.V. AAV vectors produced in insect cells
EP1857552A1 (en) 2006-05-20 2007-11-21 Cargill Incorporated Thermostable xylose isomerase enzyme
US8512981B2 (en) 2006-06-21 2013-08-20 Amsterdam Molecular Therapeutics B.V. Vectors with modified initiation codon for the translation of AAV-Rep78 useful for production of AAV
US20160032254A1 (en) 2007-07-26 2016-02-04 Uniqure Ip B.V. Aav vectors produced by insect cells comprising rep52 and rep78 coding sequences with differential codon biases
WO2009151546A2 (en) 2008-05-27 2009-12-17 Ptc Therapeutics, Inc. Methods for treating spinal muscular atrophy
WO2010129021A1 (en) 2009-05-02 2010-11-11 Genzyme Corporation Gene therapy for neurodegenerative disorders
US8980853B2 (en) 2009-06-17 2015-03-17 Isis Pharmaceuticals, Inc. Compositions and methods for modulation of SMN2 splicing in a subject
US9717750B2 (en) 2009-06-17 2017-08-01 Biogen Ma Inc. Compositions and methods for modulation of SMN2 splicing in a subject
US20130195801A1 (en) 2010-04-23 2013-08-01 University Of Massachusetts Cns targeting aav vectors and methods of use thereof
US8999948B2 (en) 2010-06-10 2015-04-07 Laboratorios Del Dr. Esteve, S.A. Vectors and sequences for the treatment of diseases
US9074207B2 (en) 2010-10-15 2015-07-07 Universita′ Degli Studi Di Ferrara Modified human U1snRNA molecule, a gene encoding for the modified human U1snRNA molecule, an expression vector including the gene, and the use thereof in gene therapy
WO2012057363A1 (en) 2010-10-27 2012-05-03 学校法人自治医科大学 Adeno-associated virus virions for transferring genes into neural cells
EP2678433A1 (en) 2011-02-22 2014-01-01 California Institute of Technology Delivery of proteins using adeno-associated virus (aav) vectors
WO2012144446A1 (en) 2011-04-18 2012-10-26 独立行政法人国立精神・神経医療研究センター Drug delivery particles and method for producing same
US9586955B2 (en) 2012-02-10 2017-03-07 Ptc Therapeutics, Inc. Compounds for treating spinal muscular atrophy
WO2015012924A2 (en) 2013-04-29 2015-01-29 The Trustees Of The University Of Pennsylvania Tissue preferential codon modified expression cassettes, vectors containing same, and use thereof
WO2014178863A1 (en) 2013-05-01 2014-11-06 Genzyme Corporation Compositions and methods for treating spinal muscular atrophy
US20150126590A1 (en) 2013-11-01 2015-05-07 Cornell University Gene therapy for alzheimer's and other neurodegenerative diseases and conditions
US20180066279A9 (en) 2014-04-25 2018-03-08 University Of Massachusetts Recombinant aav vectors useful for reducing immunity against transgene products
WO2015168666A2 (en) 2014-05-02 2015-11-05 Genzyme Corporation Aav vectors for retinal and cns gene therapy
US9969754B2 (en) 2014-05-15 2018-05-15 Hoffmann-La Roche Inc. Compounds for treating spinal muscular atrophy
WO2015191508A1 (en) 2014-06-09 2015-12-17 Voyager Therapeutics, Inc. Chimeric capsids
US10436802B2 (en) 2014-09-12 2019-10-08 Biogen Ma Inc. Methods for treating spinal muscular atrophy
US20160289275A1 (en) 2015-04-06 2016-10-06 The Usa, As Represented By The Secretary, Dept. Of Health And Human Services Adeno-Associated Vectors for Enhanced Transduction and Reduced Immunogenicity
WO2017008748A1 (en) 2015-07-14 2017-01-19 The Hong Kong University Of Science And Technology Passive thermal diode
US9669109B1 (en) 2015-11-24 2017-06-06 Universita Di Ferrara Modified human U1snRNA molecule, a gene encoding for the modified human U1snRNA molecule, an expression vector including the gene, and the use thereof in gene therapy of familial dysautonomia and spinal muscular atrophy
US20170143847A1 (en) 2015-11-24 2017-05-25 Universita Di Ferrara Modified human u1snrna molecule, a gene encoding for the modified human u1snrna molecule, an expression vector including the gene, and the use thereof in gene therapy of familial dysautonomia and spinal muscular atrophy
WO2017100671A1 (en) * 2015-12-11 2017-06-15 California Institute Of Technology TARGETING PEPTIDES FOR DIRECTING ADENO-ASSOCIATED VIRUSES (AAVs)
US9929669B2 (en) 2016-02-24 2018-03-27 Lsis Co., Ltd. Inverter
US20210171519A1 (en) 2017-08-04 2021-06-10 Skyhawk Therapeutics, Inc. Methods and compositions for modulating splicing
WO2019136484A1 (en) 2018-01-08 2019-07-11 Prevail Therapeutics, Inc. Pet imaging ligands for in vivo detection of gba1
WO2019147960A1 (en) 2018-01-25 2019-08-01 Biogen Ma Inc. Methods of treating spinal muscular atrophy
WO2019213180A1 (en) 2018-04-30 2019-11-07 Amicus Therapeutics, Inc. Gene therapy constructs and methods of use
WO2020014471A1 (en) * 2018-07-11 2020-01-16 The Brigham And Women's Hospital, Inc. Methods and compositions for delivery of agents across the blood-brain barrier
WO2020037161A1 (en) 2018-08-15 2020-02-20 Biogen Ma Inc. Combination therapy for spinal muscular atrophy
WO2020072683A1 (en) 2018-10-02 2020-04-09 Voyager Therapeutics, Inc. Redirection of tropism of aav capsids
WO2020117898A1 (en) * 2018-12-05 2020-06-11 Abeona Therapeutics Inc. Recombinant adeno-associated viral vector for gene delivery
WO2020132455A1 (en) 2018-12-21 2020-06-25 The Trustees Of The University Of Pennsylvania Compositions for drg-specific reduction of transgene expression
WO2021202651A1 (en) 2020-04-01 2021-10-07 Voyager Therapeutics, Inc. Redirection of tropism of aav capsids
WO2021230987A1 (en) 2020-05-13 2021-11-18 Voyager Therapeutics, Inc. Redirection of tropism of aav capsids

Non-Patent Citations (89)

* Cited by examiner, † Cited by third party
Title
"Biocomputing: Informatics and Genome Projects", 1993, ACADEMIC PRESS
"GenBank", Database accession no. NM.sub.--000344.2
"Liposomes as tools in Basic Research and Industry", 1995, HUMANA PRESS
"Molecular Cloning", 1982, CSH LABORATORY
"Sequence Analysis Primer", 1991, M STOCKTON PRESS
"UniProtKB/Swiss-Prot", Database accession no. Q4R4F8.1
AFIONE ET AL.: "Identification and Mutagenesis of the Adeno-Associated Virus 5 Sialic Acid Binding Region", JOURNAL OF VIROLOGY, vol. 89, no. 3, 2015, pages 1660 - 1672
ALTSCHUL, S. F. ET AL., J. MOLECULAR BIOL., vol. 215, 1990, pages 403
BRIESE ET AL., HUM. MOL. GENET., vol. 18, 2009, pages 3615 - 3625
BURGESS ET AL., EXPERT REV NEUROTHER., vol. 15, no. 5, 2015, pages 477 - 491
CAMPBELL ET AL., AM. J. HUM. GENET., vol. 61, 1997, pages 40 - 50
CARILLO, H.LIPMAN, D., SIAM J APPLIED MATH., vol. 48, 1988, pages 1073
CHAN ET AL., HUM. MOL. GENET., vol. 12, 2003, pages 1367 - 1376
CHAYTOW ET AL., CELL MOL LIFE SCI., vol. 75, no. 21, 2018, pages 3877 - 3894
CHIORINI ET AL., J. VIR., vol. 71, 1997, pages 6823 - 33
CHIORINI ET AL., J. VIR., vol. 73, 1999, pages 1309 - 1319
CHOUDHURY SOURAV R ET AL: "Viral vectors for therapy of neurologic diseases", NEUROPHARMACOLOGY, ELSEVIER, AMSTERDAM, NL, vol. 120, 21 February 2016 (2016-02-21), pages 63 - 80, XP085066795, ISSN: 0028-3908, DOI: 10.1016/J.NEUROPHARM.2016.02.013 *
CLARK ET AL., HUM. GENE THER., vol. 10, 1999, pages 1031 - 1039
CLARK, F.T. A. THANARAJ, HUM. MOL. GENET., vol. 11, 2002, pages 451 - 64
DEVEREUX, J. ET AL., NUCLEIC ACIDS RESEARCH, vol. 12, no. 1, 1984, pages 387
DONADON ET AL., NAR, vol. 47, 2019, pages 7618 - 7632
DUQUE ET AL., ANN NEUROL., vol. 77, no. 3, 2015, pages 399 - 414
FAUST ET AL., J. CLIN. INV., vol. 123, no. 7, 2013, pages 2994 - 3001
GILL ET AL., GENE THERAPY, vol. 8, 2001, pages 1539 - 1546
GLANZMAN ET AL., NEUROMUSCUL. DISORD., vol. 20, 2010, pages 155 - 161
GOVINDASAMY ET AL.: "Structural Insights into Adeno-Associated Virus Serotype 5", JOURNAL OF VIROLOGY, vol. 7, no. 20, 8 October 2013 (2013-10-08), pages 11187 - 11199, XP055865805, DOI: 10.1128/JVI.00867-13
GOVINDASAMY ET AL.: "Structural Insights into Adeno-Associated Virus Serotype 5", JOURNAL OF VIROLOGY, vol. 87, no. 20, 2013, pages 11187 - 11199, XP055865805, DOI: 10.1128/JVI.00867-13
GOVINDASAMY ET AL.: "Structural Insights into Adeno-Associated Virus Serotype 5", JOURNAL OF VIROLOGY, vol. 87, no. 20, October 2013 (2013-10-01), pages 11187 - 11199, XP055865805, DOI: 10.1128/JVI.00867-13
GOVINDASAMY ET AL.: "Structurally Mapping the Diverse Phenotype of Adeno-Associated Virus Serotype 4", JOURNAL OF VIROLOGY, vol. 80, no. 23, December 2006 (2006-12-01), pages 11556 - 11570
GRUNTMANFLOTTE, HUMAN GENE THERAPY CLINICAL DEVELOPMENT, vol. 26, no. 3, 2015, pages 159 - 164
HAO ET AL., MOL. NEURODEGENER., vol. 6, 2011, pages 24
HSIEH-LI ET AL., NAT. GENET., vol. 24, 2006, pages 66 - 70
HSU ET AL., PLOS ONE, vol. 8, no. 2, pages 1 - 8
HUSAIN ET AL., GENE THERAPY, 2009
HWANG ET AL.: "N-Terminal Acetylation of Cellular Proteins Creates Specific Degradation Signals", SCIENCE, vol. 327, no. 5968, 19 February 2010 (2010-02-19), pages 973 - 977, XP055369420, DOI: 10.1126/science.1183147
JIN ET AL.: "Direct Liquid Chromatography/Mass Spectrometry Analysis for Complete Characterization of Recombinant Adeno-Associated Virus Capsid Proteins", HUM GENE THER METHODS., vol. 28, no. 5, October 2017 (2017-10-01), pages 255 - 267
JOSE ET AL.: "High-Resolution Structural Characterization of a New Adenoassociated Virus Serotype 5 Antibody Epitope toward Engineering Antibody-Resistant Recombinant Gene Delivery Vectors", JOURNAL OF VIROLOGY, vol. 93, no. 1, 2020, pages e01394 - 18
KAJIGAYA ET AL., PROC. NAT'1. ACAD. SCI. USA, vol. 88, 1991, pages 4646 - 50
KIMBAUER ET AL., VIR, vol. 219, 1996, pages 37 - 44
LE ET AL., HUM. MOL. GENET., vol. 14, 2005, pages 845 - 857
LEFEBVRE ET AL., CELL, vol. 80, 1995, pages 155 - 165
LEFEBVRE ET AL., NAT. GENET., vol. 16, 1997, pages 265 - 269
LOFTUS, STACIE K. ET AL., HUMAN MOLECULAR GENETICS, vol. 11, no. 24, 2002, pages 3107 - 3114
LUCKOW ET AL., BIO/TECHNOLOGY, vol. 6, 1988, pages 47
MCWHORTER ET AL., J. CELL BIOL., vol. 162, 2003, pages 919 - 931
MEYERSMILLER, CABIOS, vol. 4, 1989, pages 11 - 17
MIRIAMI, E. ET AL., NUCLEIC ACIDS RES., vol. 31, 2003, pages 1974 - 1983
MIYANOHARA ET AL., MOL THER METHODS CLIN DEV., vol. 3, 2016, pages 16046
MONANI ET AL., HUM. MOL. GENET., vol. 9, 2000, pages 333 - 339
MONANI ET AL., J. CELL. BIOL., vol. 160, 2003, pages 41 - 52
NONNENMACHER MATHIEU ET AL: "Rapid evolution of blood-brain-barrier-penetrating AAV capsids by RNA-driven biopanning", MOLECULAR THERAPY- METHODS & CLINICAL DEVELOPMENT, vol. 20, 1 March 2021 (2021-03-01), GB, pages 366 - 378, XP055822711, ISSN: 2329-0501, Retrieved from the Internet <URL:https://www.cell.com/action/showPdf?pii=S2329-0501(20)30254-0> DOI: 10.1016/j.omtm.2020.12.006 *
O'HAGEN ET AL., NEURO. DIS., vol. 17, 2007, pages 693 - 697
OPREA ET AL., SCIENCE, vol. 320, 2008, pages 524 - 527
O'REILLYDAVID R.LOIS K. MILLERVERNE A. LUCKOW: "Baculovirus Expression Vectors, A Laboratory Manual", 1994, OXFORD UNIVERSITY PRESS
PARK ET AL., CURR. NEUROL. NEUROSCI. REP., vol. 10, 2010, pages 108 - 117
PARR ET AL., NAT. MED., vol. 3, 1997, pages 1145 - 9
PASSINI ET AL., J CLIN INVEST, vol. 120, 2010, pages 1253 - 1264
PERA ET AL., MUSCLE NERVE., vol. 59, 2019, pages 426 - 430
POWELL ET AL., VIRAL EXPRESSION CASSETTE ELEMENTS TO ENHANCE TRANSGENE TARGET SPECIFICITY AND EXPRESSION IN GENE THERAPY, 2015
QIAN ET AL.: "Directed Evolution of AAV Serotype 5 for Increased Hepatocyte Transduction and Retained Low Humoral Seroreactivity", MOLECULAR THERAPY: METHODS AND CLINICAL DEVELOPMENT, vol. 20, 2021, pages 122 - 132, XP055864144, DOI: 10.1016/j.omtm.2020.10.010
RAVINDRA KUMAR SRIPRIYA ET AL: "Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types", NATURE METHODS, NATURE PUBLISHING GROUP US, NEW YORK, vol. 17, no. 5, 20 April 2020 (2020-04-20), pages 541 - 550, XP037112784, ISSN: 1548-7091, [retrieved on 20200420], DOI: 10.1038/S41592-020-0799-7 *
RUFFING ET AL., J. VIR., vol. 66, 1992, pages 6922 - 30
RUTLEDGE ET AL., J. VIR., vol. 72, 1998, pages 309 - 319
SAMULSKI ET AL., J. VIR., vol. 63, 1989, pages 3822 - 8
SCHMID ET AL., J. CHILD NEUROL., vol. 22, 2007, pages 1004 - 1012
SCHRANK ET AL., PNAS, 1997, pages 9920 - 9925
SLEIGH ET AL., HUM. MOL. GENET., vol. 20, 2011, pages 245 - 260
SODERBLOM ET AL., E. NEURO, 2015
SRIVASTAVA ET AL., J. VIR., vol. 45, 1983, pages 555 - 64
STEVEN JAMES GRAY: "Gene therapy and neurodevelopmental disorders", NEUROPHARMACOLOGY, vol. 68, 1 May 2013 (2013-05-01), pages 136 - 142, XP055099736, ISSN: 0028-3908, DOI: 10.1016/j.neuropharm.2012.06.024 *
SUMNER C. J., NEURORX, vol. 3, 2006, pages 235 - 245
TERSTAPPEN ET AL., NAT REV DRUG DISCOVERY, 2021, Retrieved from the Internet <URL:https://doi.org/10.1038/s41573-021-00139-y>
THE BRITISH PHARMACOPOEIA
THE EUROPEAN PHARMACOPOEIA (EP
THE INTERNATIONAL PHARMACOPOEIA
UNITED STATES PHARMACOPOEIA (USP
URABE, M. ET AL., J VIROL., vol. 80, no. 4, 2006, pages 1874 - 85
VELDWIJK ET AL., MOL. THER., vol. 6, 2002, pages 272 - 278
VON HEINJE, G.: "Sequence Analysis in Molecular Biology", 1987, ACADEMIC PRESS
WALSH ET AL., BMC BIOL., vol. 18, 2020, pages 127
WALTERS ET AL.: "Structure of Adeno-Associated Virus Serotype 5", JOURNAL OF VIROLOGY, vol. 78, no. 7, 2004, pages 3361 - 3371, XP002297595
WANG ET AL.: "Directed evolution of adeno-associated virus 5 capsid enables specific liver tropism", MOL THER NUCLEIC ACIDS, vol. 28, 2022, pages 293 - 306, XP055980992, DOI: 10.1016/j.omtn.2022.03.017
WASILKO DJ ET AL., PROTEIN EXPR PURIF., vol. 65, no. 2, June 2009 (2009-06-01), pages 122 - 32
WU ET AL., J. VIR., vol. 74, 2000, pages 8635 - 47
YU ET AL., MOLECULAR PAIN, vol. 7, 2011, pages 63
ZHANG, MIN ET AL., JOURNAL OF NEUROSCIENCE RESEARCH, vol. 86, no. 13, 2008, pages 2848 - 2856
ZHANG, X. H. ET AL., GENOME RES., vol. 15, 2005, pages 768 - 779
ZHANG, X. H.L. A. CHASIN, GENES DEV., vol. 18, 2004, pages 1241 - 1250
ZHAO ET AL., VIR, vol. 272, 2000, pages 382 - 93

Similar Documents

Publication Publication Date Title
US11752181B2 (en) Compositions and methods of treating Huntington&#39;s disease
US20230295663A1 (en) Compositions and methods of treating amyotrophic lateral sclerosis (als)
US20220333131A1 (en) Modulatory polynucleotides
US11951121B2 (en) Compositions and methods for treating Huntington&#39;s disease
ES2768763T3 (en) Enhanced rAAV vectors and methods for transduction of photoreceptors and EPR cells
US20230323388A1 (en) Treatment of amyotrophic lateral sclerosis (als)
US11931375B2 (en) Treatment of amyotrophic lateral sclerosis (ALS)
US20210395776A1 (en) Frataxin expression constructs having engineered promoters and methods of use thereof
US20230399642A1 (en) Compositions and methods of treating huntington&#39;s disease
US20210254103A1 (en) Treatment of amyotrophic lateral sclerosis and disorders associated with the spinal cord
CA3190309A1 (en) Compositions and methods for the treatment of neurological disorders related to glucosylceramidase beta deficiency
US20230285596A1 (en) Compositions and methods for the treatment of niemann-pick type c1 disease
WO2023240236A1 (en) Compositions and methods for the treatment of spinal muscular atrophy related disorders
TWI813851B (en) Frataxin expression constructs having engineered promoters and methods of use thereof
WO2021247995A2 (en) Compositions and methods of treating neuropathic pain

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23738400

Country of ref document: EP

Kind code of ref document: A1