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WO2024163870A1 - Développement de vecteurs de sérotype de vaa monocaténaires de génération z (genz) - Google Patents

Développement de vecteurs de sérotype de vaa monocaténaires de génération z (genz) Download PDF

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Publication number
WO2024163870A1
WO2024163870A1 PCT/US2024/014220 US2024014220W WO2024163870A1 WO 2024163870 A1 WO2024163870 A1 WO 2024163870A1 US 2024014220 W US2024014220 W US 2024014220W WO 2024163870 A1 WO2024163870 A1 WO 2024163870A1
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sequence
genome
aav
raav
raav genome
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Keyun Qing
Jakob SHOTI
Arun Srivastava
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University of Florida
University of Florida Research Foundation Inc
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University of Florida
University of Florida Research Foundation Inc
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2750/14121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • 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

Definitions

  • the present disclosure is based at least in part on the realization that inefficient second- strand synthesis of single-stranded AAV genomes impedes efficient expression of transgenes delivered by AAV vectors.
  • Modification of D-sequences of inverted terminal repeats (ITRs) within AAV genomes can increase second-strand synthesis, and thereby improve various aspects of AAV packaging, replication, transduction efficiency, and transgene expression.
  • ITRs inverted terminal repeats
  • rAAV recombinant AAV
  • rAAV recombinant adeno-associated virus genomes
  • an rAAV genome comprises a heterologous nucleotide T18934 Attorney Docket No.
  • the heterologous nucleotide sequence is (i) inserted within a first D- sequence of the rAAV genome, (ii) inserted within the rAAV genome at a position adjacent to a 3' or 5' end of the first D-sequence, or (iii) substituted in place of a portion of the first D- sequence in the rAAV genome, wherein the first D-sequence is proximal to the 3' terminus of the rAAV genome, and wherein the rAAV genome is single-stranded.
  • substitution of the heterologous nucleotide sequence in place of 5, 6, 7, 8, 9, 10, or more nucleotides at the 3' terminus of the first D-sequence does not substantially inhibit packaging of the rAAV genome.
  • the heterologous nucleotide sequence is substituted in place of a portion of the first D-sequence.
  • the heterologous nucleotide sequence is substituted in place of a portion of the first D-sequence, and wherein the portion of the first D-sequence: (i) is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length; and/or (ii) is equal in length to the heterologous nucleotide sequence.
  • the portion of the first D-sequence comprises consecutive nucleotides including the 3'-most nucleotide of the first D-sequence.
  • the heterologous nucleotide sequence comprises, consists of, or consists essentially of a sequence of 5'-ATGTGCTTGA-3' (SEQ ID NO: 26) or 5'-TCAAGCACAT-3' (SEQ ID NO: 27).
  • an rAAV genome further comprises a second heterologous nucleotide sequence, wherein the second heterologous nucleotide sequence is (i) inserted within a second D-sequence of the rAAV genome, (ii) inserted within the rAAV genome at a position adjacent to a 3' or 5' end of the second D-sequence, or (iii) substituted in place of a portion of the second D-sequence in the rAAV genome, wherein the second D-sequence is proximal to the 5' terminus of the rAAV genome.
  • the second heterologous nucleotide sequence is substituted in place of a portion of the second D-sequence.
  • the second heterologous nucleotide sequence is substituted in place of a portion of the second D-sequence, and wherein the portion of the second D- sequence: T18934 Attorney Docket No. U1202.70129WO00 (i) is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length; and/or (ii) is equal in length to the second heterologous nucleotide sequence.
  • the portion of the second D-sequence comprises consecutive nucleotides including the 5'-most nucleotide of the second D-sequence.
  • the second heterologous nucleotide sequence comprises, consists of, or consists essentially of a sequence of 5'-ATGTGCTTGA-3' (SEQ ID NO: 26) or 5'-TCAAGCACAT-3' (SEQ ID NO: 27).
  • the rAAV genome is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, AAVrh74, or a combination thereof.
  • the rAAV genome is of serotype AAV2, AAV3, or AAV6.
  • the rAAV genome comprises AAV2 inverted terminal repeats (ITRs).
  • the rAAV genome further comprises a nucleic acid sequence comprising a gene of interest.
  • the gene of interest encodes a therapeutic agent and/or a diagnostic agent.
  • the rAAV genome further comprises a regulatory element.
  • the regulatory element comprises a promoter, an enhancer, a silencer, an insulator, a response element, an initiation site, a termination signal, or a ribosome binding site.
  • the promoter is a constitutive promoter.
  • the promoter is an inducible promoter.
  • the promoter is a tissue-specific promoter, a cell type-specific promoter, or a synthetic promoter.
  • the rAAV genome is at least 4 kilobases (kb), 4.5 kb, 5 kb, 5.5 kb, 6 kb, 6.5 kb, 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, 10 kb, 10.5 kb, 11 kb, 11.5 kb, 12 kb, 12.5 kb, 13 kb, 13.5 kb, 14 kb or more in length.
  • rAAV particles are provided herein.
  • an rAAV particle comprises an rAAV genome disclosed herein and a capsid.
  • the capsid comprises a modified capsid protein, wherein the modified capsid protein comprises an amino acid substitution at a position corresponding to T491, Y444, Y500, and/or Y730 of SEQ ID NO: 2.
  • the modified capsid protein comprises amino acid substitutions at positions corresponding to each of T491, Y444, T18934 Attorney Docket No. U1202.70129WO00 Y500, and Y730 of SEQ ID NO: 2, optionally wherein the amino acid substitutions correspond to T491V, Y444F, Y500F, and Y730F substitutions in SEQ ID NO: 2.
  • the capsid is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, AAVrh74, or a combination thereof.
  • the capsid is of serotype AAVrh74.
  • plasmids comprising a nucleic acid sequence corresponding to an rAAV genome disclosed herein are provided.
  • compositions are provided herein, wherein the composition comprises an rAAV genome disclosed herein or a plasmid disclosed herein.
  • compositions are provided herein, wherein the composition comprises an rAAV particle disclosed herein.
  • a composition disclosed herein further comprises a pharmaceutically acceptable carrier.
  • methods comprising contacting a cell with a composition disclosed herein are provided.
  • the cell is a mammalian cell.
  • the contacting is in vivo.
  • the method further comprises administering the composition comprising the rAAV particle to a subject.
  • the cell is in the subject.
  • the subject is human.
  • the subject is at risk of or has been diagnosed with a disease, disorder, or condition.
  • the composition is administered to the subject by intravenous injection, by subcutaneous injection, by intramuscular injection, by intraperitoneal injection, or orally.
  • the contacting is in vitro or ex vivo.
  • FIG.1 shows a schematic of an inverted terminal repeat (ITR) at the 3' end of an AAV genome.
  • ITR inverted terminal repeat
  • FIG.2 shows a schematic for the preparation and assembly of an N10 sequence library, in which the proximal 10 nucleotides of the D-sequence (relative to the terminus of the genome) are replaced with random nucleotides (shown in bold in the nucleotide sequences). Sequences with N10 substitutions are prepared and processed using restriction enzymes, then assembled into pSub201 plasmids. After processing by EcoRV restriction enzyme, the N10 library is processed and rescue, replication, and packaging are evaluated. [0043] FIG.3 shows a schematic for a screen of the N10 sequence library, with different types of modifications in the left and right ITRs. Only the N10-L1 version of the library demonstrated AAV packaging.
  • FIG.4 shows a schematic of the plasmid used to generate GenZ vectors (top), with N10 sequences replacing the D10 sequences in both ITRs (cross-hatched boxes).
  • the plasmid and corresponding AAV genome comprise a chicken ⁇ -actin promoter (“CBAp”) operably linked to nucleic acid sequences encoding firefly luciferase (“FLuc”) and a yellow fluorescent T18934 Attorney Docket No. U1202.70129WO00 protein (“EYFP”) with a poly A tail (“pA”), situated between the N10-modified ITRs.
  • CBAp chicken ⁇ -actin promoter
  • EYFP firefly luciferase
  • pA poly A tail
  • FIG.5 shows a schematic of a GenZ ssAAV-CBAp-FLuc-EYFP vector. In the schematic, both ITRs are modified with the N10 substitution.
  • the schematic on the left side of the figure shows an overview of the vector for illustrative purposes, to show the overall structure rather than a specific sequence of the vector.
  • Each segment (indicated by a shaded block) in the schematic on the left side represents a different portion of an AAV genome, including the ITR, TRS-N10, and D10 segments, an enhancer segment, a promoter segment, an intron segment, a coding sequence segment (encoding firefly luciferase), a second coding sequence segment (encoding EYFP), a polyA tail segment, and second D10, N10-TRS, and ITR segments.
  • FIGs.6A and 6B show results of transduction of primary human skeletal muscle cells in vitro with GenZ ssAAV-CBAp-FLuc-EYFP vectors.
  • FIG.6A shows fluorescence micrographs of EYFP in mock-treated cells (left panel, “Mock”), cells treated with 3,000 viral genomes (vgs) per cell (middle panel, “3,000 vgs/cell”), and cells treated with 10,000 vgs per cell (right panel, “10,000 vgs/cell”).
  • FIG. 6B shows quantification of the transgene expression in the skeletal muscle cells, measured as EYFP-positive pixels 2 per visual field.
  • FIGs.7A and 7B show transduction efficiency of wild-type control (ssAAV-FLuc- EYFP) and GenZ (GenZ ssAAV-Fluc-EYFP) vectors in HeLa cells in vitro.
  • FIG.7A shows a fluorescence micrograph of EYFP in cells treated with the control vector (not comprising the GenZ genome modifications).
  • FIG.7B shows a fluorescence micrograph of EYFP in cells treated with the GenZ vector. The micrographs show about 20-fold higher transgene expression in the GenZ- treated cells relative to the control-treated cells.
  • FIGs.8A and 8B show transduction efficiency of wild-type control (ssAAV-FLuc- EYFP) and GenZ (GenZ ssAAV-FLuc-EYFP) vectors in C57BL6/J mice in vivo. Approximately 1x10 8 viral genomes (vgs) of each vector were administered intravenously via the tail vein and whole-body bioluminescence images were obtained 8 days after administration.
  • FIG.8A shows bioluminescence imaging of mice administered the control T18934 Attorney Docket No. U1202.70129WO00 vector (not comprising the GenZ genome modifications).
  • FIG.8B shows bioluminescence imaging of mice administered the GenZ vector.
  • FIGs.9A-9D show transduction efficiency of wild-type (WT, left panels) and GenZ (right panels) ssAAVrh74 (FIG.9A), AAV3 (FIG.9B), AAV2 (FIG.9C), and AAV6 (FIG. 9D) vectors in human HeLa cells in vitro.
  • Cells were transduced with each vector at 1,000 viral genomes (vgs)/cell and transgene expression was visualized via fluorescence microscopy 72 hours post-transduction.
  • FIG.10 shows transgene expression in HeLa cells transduced with wild-type (WT) or GenZ ssAAVrh74 vectors (3,000 vgs/cell), with pre-incubation (“Before”) or co-incubation (“During”) with AAVrh74 empty capsids (“+AAVrh74 empty capsids”), AAV2 empty capsids (“+AAV2 empty capsids”), AAV3 empty capsids (“+AAV3 empty capsids”), or AAV6 empty capsids (“+AAV6 empty capsids”).
  • WT wild-type
  • GenZ ssAAVrh74 vectors 3,000 vgs/cell
  • the present disclosure is based at least in part on the development of adeno-associated virus (AAV) genomes and particles useful in the delivery of various cargoes to cells, facilitating efficient transgene expression therein.
  • AAV adeno-associated virus
  • the disclosure relates, at least in part, to the finding that incorporation of sequence modifications into AAV genomes results in improvements in various characteristics of AAVs, such as packaging, transduction efficiency, transgene expression, etc. These improvements may result from increased second-strand synthesis of the AAV genome, resulting from modifications therein.
  • the AAV genomes, particles, etc., disclosed herein may be used in a variety of applications including but not limited to compositions and methods (e.g., therapeutic and diagnostic methods).
  • compositions comprising AAV particles (e.g., infectious AAV particles), AAV genomes (e.g., genomes comprising sequence modifications), and methods of using the compositions for transducing cells of interest (e.g., for treating or diagnosing a disease or condition in a subject.
  • AAV particles e.g., infectious AAV particles
  • AAV genomes e.g., genomes comprising sequence modifications
  • methods of using the compositions for transducing cells of interest e.g., for treating or diagnosing a disease or condition in a subject.
  • nucleic acid vectors e.g., AAV genomes, e.g., recombinant AAV (rAAV) genomes
  • rAAV recombinant AAV
  • a nucleic acid vector (e.g., an AAV genome) provided herein may comprise AAV inverted terminal repeat(s) modified to improve (e.g., increase the rate, efficiency, etc.) second- strand synthesis.
  • an ITR as provided herein is a 5' ITR, i.e. an ITR that is 5' from a transgene in a nucleic acid vector (e.g., an AAV genome).
  • An ITR serves as an origin of replication and is comprised of two arm palindromes (B-B' and C-C') embedded in a larger stem palindrome (A-A').
  • An AAV ITR can be in flip or flop configurations.
  • an ITR has the B-B' and the C-C' palindrome closest to the 3' end.
  • the D- sequence is present only once at each end of the genome thus remaining single-stranded.
  • the D-sequence is also referred to as the “D-element” in the art.
  • the D-sequence consists of 20 or approximately 20 (e.g., 19, 20, 21, 22, 23, 24, etc.) nucleotides adjacent to the terminal resolution site (trs), and generally consists or consists essentially of the medial 20 (or approximately 20) nucleotides of the ITR (where “medial” indicates the segment of the ITR that is adjacent to the center of the AAV genome).
  • the first nucleotide of the D-sequence is generally at position 126 or approximately position 126 from the terminus of the AAV genome.
  • a nucleic acid vector e.g., an AAV genome
  • a nucleic acid vector as provided herein comprises a first inverted terminal repeat (ITR) and a second ITR.
  • ITR inverted terminal repeat
  • the first ITR is modified.
  • the second ITR is modified.
  • a modification of an ITR comprises substitution of the entire D-sequence or substitution of part of a D-sequence.
  • a modification of an ITR comprises deletion of an entire D-sequence (e.g., the D-sequence of the left ITR or the right ITR) or deletion of part of a D-sequence (e.g., the proximal 10 nucleotides of the ITR, relative to the terminus of the nucleic acid vector).
  • a modification of an ITR may in some embodiments comprise deletion or substitution of 1-20 nucleotides of the D-sequence.
  • the proximal 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides of the D-sequence, relative to the terminus of the nucleic acid vector are deleted or substituted.
  • the proximal 10 nucleotides of the D-sequence, relative to the terminus of the nucleic acid vector, are deleted or substituted.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in the middle of the D-sequence are deleted or substituted (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous nucleotides beginning 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the 3' or 5' end of the D-sequence).
  • the distal 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides of the D- sequence, relative to the terminus of the nucleic acid vector are deleted or substituted.
  • the distal 10 nucleotides of the D-sequence, relative to the terminus of the nucleic acid vector are deleted or substituted.
  • a D-sequence comprises the sequence CTCCATCACTAGGGGTTCCT (SEQ ID NO: 16) of the wild-type AAV2 ITR, or a corresponding sequence of a different serotype ITR.
  • a D-sequence is defined by the sequence CTCCATCACTAGGGGTTCCT (SEQ ID NO: 16).
  • the substituted sequence may be any alternative sequence described herein, such as a sequence described as a “heterologous nucleotide sequence.”
  • a “heterologous” nucleotide sequence or nucleic acid sequence refers to a sequence that is not native to an AAV or naturally-occurring in an AAV.
  • a heterologous sequence can be a short sequence (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides in length, or of a similar length), or can be a longer sequence (e.g., about 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 nucleotides in length, or longer).
  • U1202.70129WO00 heterologous sequence can comprise a coding sequence (e.g., encoding a gene of interest) or can be a non-coding sequence (e.g., inserted in or replacing a regulatory or structural portion of an AAV nucleic acid, such as an AAV genome).
  • a nucleic acid vector e.g., an AAV genome
  • ITR inverted terminal repeat
  • a nucleic acid vector (e.g., an AAV genome) is encapsidated within an AAV capsid forming an AAV particle.
  • a nucleic acid vector disclosed herein is encapsidated by a wild-type AAV capsid disclosed herein or another AAV capsid disclosed herein, such as an AAV capsid comprising one or more amino acid substitutions.
  • a nucleic acid vector (e.g., an AAV genome) comprises native AAV genes or native AAV nucleotide sequences.
  • one or more native AAV genes or native AAV nucleotide sequences may be removed from a nucleic acid vector (e.g., an AAV genome). In some embodiments, one or more native AAV genes or native AAV nucleotide sequences may be removed from a nucleic acid vector (e.g., an AAV genome) and replaced with a gene or interest.
  • a nucleic acid vector (e.g., an AAV genome) can be of any AAV serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, or AAVrh74, or a combination of serotypes.
  • a nucleic acid vector (e.g., an AAV genome) encapsidated within an AAV capsid forms a pseudotyped AAV particle, such that the genome is of a serotype distinct from the capsid in which it is encapsidated.
  • a nucleic acid vector (e.g., an AAV genome) of serotype AAV2 may be encapsidated within a capsid of serotype AAVrh74.
  • a nucleic acid vector e.g., an AAV genome
  • the first ITR refers to the ITR at the 5' terminus of the nucleic acid vector (e.g., AAV genome)
  • the second ITR refers to the ITR at the 3' terminus of the nucleic acid vector (e.g., AAV genome).
  • Each ITR in its native or wild-type form is or is about 145 nucleotides in length (e.g., about 140 nucleotides, about 145 nucleotides, about 150 nucleotides, about 155 nucleotides, about 160 nucleotides, or about 165 nucleotides) and comprises a D-sequence.
  • Each ITR can independently be of any AAV serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, T18934 Attorney Docket No.
  • U1202.70129WO00 AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, or AAVrh74), or both ITRs may be of the same serotype.
  • ITRs are described, for example, in Grimm et al. J. Virol. 80(1):426-439 (2006).
  • Exemplary left ITR sequences are provided below. In each ITR sequence, the D-sequence is underlined.
  • a right ITR has a nucleotide sequence which is the reverse complement of the corresponding left ITR (e.g., the AAV2 right ITR has a nucleotide sequence which is the reverse complement of the AAV2 left ITR).
  • Example of wild-type AAV1 left ITR TTGCCCACTCCCTCTCTGCGCTCGCTCGGTGGGGCCTGCGGACCAAAGGTCCGCAGACGGCAG AGGTCTCCTCTGCCGGCCCCACCGAGCGAGCGAGCGCGCAGAGAGGGAGTGGGCAACTCCATCACTAGG GGTAA (SEQ ID NO: 28)
  • Example of wild-type AAV2 left ITR TTGGCCACTCCCTCTCTGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCG GGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGG GGTTCCT (SEQ ID NO: 29)
  • Example of wild-type AAV3 left ITR TTGGCCACTCCCTCTATGCGCACTCGCTCGCTCGGTGGGGCCTGGCGACCAAAGGTCGCCAGACGGACG TGCTTTGCACGTCCGGCC
  • a nucleic acid vector (e.g., an AAV genome) comprises a modification (e.g., a deletion, a substitution, or an insertion) of a D-sequence of an ITR.
  • a nucleic acid vector (e.g., an AAV genome) comprises a modification (e.g., a deletion, a substitution, or an insertion) of a D-sequence of a left ITR.
  • a nucleic acid vector (e.g., an AAV genome) comprises a modification (e.g., a deletion, a substitution, or an insertion) of a D-sequence of a right ITR.
  • a nucleic acid vector (e.g., an AAV genome) comprises a modification (e.g., a deletion, a substitution, or an insertion) of a D-sequence of both a left ITR and a right ITR.
  • a nucleic acid vector (e.g., an AAV genome) comprises a modification (e.g., a deletion, a substitution, or an insertion) of either a left ITR or a right ITR, but not both (i.e., the nucleic acid vector comprises a modification of only one ITR).
  • the ITR sequence comprises a terminal sequence at the 5' or 3' end of the nucleic acid vector (e.g., AAV genome) which forms a palindromic double-stranded T-shaped hairpin structure, and an additional sequence which remains single-stranded (i.e., is not part of the T- shaped hairpin structure), termed the D-sequence.
  • the D-sequence of an ITR is typically approximately 20 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides located at the distal (relative to the terminus of the nucleic acid vector) end of the ITR (e.g., the 3' end of the ITR at the 5' end of the genome, or the 5' end of the ITR at the 3' end of the genome), and corresponds to the sequence of CTCCATCACTAGGGGTTCCT (SEQ ID NO: 16) of the wild-type AAV2 left ITR or a corresponding sequence in an ITR of another serotype.
  • the D- sequence of an ITR in some embodiments comprises, consists essentially of, or consists of the nucleic acid sequence CTCCATCACTAGGGGTTCCT (SEQ ID NO: 16) of the wild-type AAV2 left ITR or a corresponding sequence in an ITR of another serotype.
  • the D-sequence of an ITR e.g., the first ITR or the second ITR
  • a nucleic acid vector e.g., an AAV genome
  • the D-sequence of both ITRs of a nucleic acid vector (e.g., an AAV genome) disclosed herein is entirely or partially removed.
  • the D- sequence of an ITR (e.g., the first ITR or the second ITR) is entirely or partially replaced with a non-AAV sequence (i.e., a nucleotide sequence that is not from an AAV nucleic acid).
  • the D-sequence of an ITR (e.g., the first ITR or the second ITR) is entirely or partially replaced with a heterologous nucleotide sequence.
  • the heterologous nucleotide sequence comprises, consists essentially of, or consists of the nucleic acid sequence 5'-ATGTGCTTGA-3' (SEQ ID NO: 26), 5'-TCAAGCACAT-3' (SEQ ID NO: T18934 Attorney Docket No. U1202.70129WO00 27), or a reverse or reverse complement of either 5'-ATGTGCTTGA-3' (SEQ ID NO: 26) or 5'-TCAAGCACAT-3' (SEQ ID NO: 27).
  • the heterologous nucleotide sequence has at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity) with the sequence 5'-ATGTGCTTGA-3' (SEQ ID NO: 26), 5'-TCAAGCACAT-3' (SEQ ID NO: 27), or a reverse or reverse complement of either 5'-ATGTGCTTGA-3' (SEQ ID NO: 26) or 5'-TCAAGCACAT-3' (SEQ ID NO: 27).
  • identity e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least
  • the heterologous nucleotide sequence has less than 95% identity (e.g., less than 90% identity, less than 85% identity, less than 80% identity, less than 75% identity, or less than 70% identity) with the sequence 5'-ATGTGCTTGA-3' (SEQ ID NO: 26), 5'-TCAAGCACAT-3' (SEQ ID NO: 27), or a reverse or reverse complement of either 5'-ATGTGCTTGA-3' (SEQ ID NO: 26) or 5'-TCAAGCACAT-3' (SEQ ID NO: 27).
  • the heterologous nucleotide sequence has about 70% to about 95% identity (e.g., about 95% identity, about 90% identity, about 85% identity, about 80% identity, about 75% identity, or about 70% identity) with the sequence 5'-ATGTGCTTGA-3' (SEQ ID NO: 26), 5'-TCAAGCACAT-3' (SEQ ID NO: 27), or a reverse or reverse complement of either 5'-ATGTGCTTGA-3' (SEQ ID NO: 26) or 5'-TCAAGCACAT-3' (SEQ ID NO: 27).
  • the heterologous nucleotide sequence has fewer than 6 mismatches (e.g., fewer than 5, fewer than 4, fewer than 3, fewer than 2, 1, or no mismatches) relative to the sequence 5'-ATGTGCTTGA-3' (SEQ ID NO: 26), 5'-TCAAGCACAT-3' (SEQ ID NO: 27), or a reverse or reverse complement of either 5'-ATGTGCTTGA-3' (SEQ ID NO: 26) or 5'-TCAAGCACAT-3' (SEQ ID NO: 27).
  • mismatches e.g., fewer than 5, fewer than 4, fewer than 3, fewer than 2, 1, or no mismatches
  • the heterologous nucleotide sequence has 1, 2, 3, 4, 5, or 6 mismatches relative to the sequence 5'-ATGTGCTTGA-3' (SEQ ID NO: 26), 5'-TCAAGCACAT-3' (SEQ ID NO: 27), or a reverse or reverse complement of either 5'-ATGTGCTTGA-3' (SEQ ID NO: 26) or 5'-TCAAGCACAT-3' (SEQ ID NO: 27).
  • the heterologous nucleotide sequence has a length of or about 10 nucleotides (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides).
  • a heterologous nucleotide sequence is inserted into a nucleic acid vector (e.g., an AAV genome) (i.e., instead of substituting a portion of an ITR).
  • a heterologous nucleotide sequence may be inserted inside the D-sequence of an ITR, upstream of the D-sequence of an ITR, or downstream of the D-sequence of an ITR.
  • substitution of a D-sequence comprises substitution of at least 5 nucleotides (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides) of the D- sequence with a different nucleotide sequence (e.g., a heterologous nucleotide sequence or portion thereof). In some embodiments, substitution of a D-sequence comprises substitution of 10 nucleotides of the D-sequence.
  • substitution of a D-sequence comprises substitution of the 3'-most 10 nucleotides of the D-sequence (e.g., of the D-sequence of the ITR at the 3' end of the nucleic acid vector (e.g., AAV genome)). In some embodiments, substitution of a D-sequence comprises substitution of the 5'-most 10 nucleotides of the D- sequence (e.g., of the D-sequence of the ITR at the 5' end of the nucleic acid vector (e.g., AAV genome)).
  • substitution of a D-sequence comprises substitution of an internal portion (i.e., not comprising a terminal nucleotide) of the D-sequence, such as 10 nucleotides of the internal portion of the D-sequence.
  • an ITR comprising a substitution, insertion, or deletion as disclosed herein comprises one or more additional modifications, such as an additional substitution, modification, or deletion in another portion of the ITR.
  • modification of a nucleic acid vector e.g., an AAV genome
  • wild-type AAV genomes are approximately 4.7 kilobases (kb) in length; recombinant AAV genomes have typically been limited to approximately this same length. See, e.g., Wu, et al., Mol Ther. (2010) 18(1): 80-86. Self-complementary AAV genomes are often even more limited, with maximum packaging capacities of about 2.3 kb.
  • the modifications provided herein can, in some embodiments, enable substantially larger AAV genomes (comprising the modification(s)) to be useful in generating AAV particles for delivery of genes of interest.
  • an AAV genome disclosed herein comprising a modification can be generated having a length greater than a corresponding AAV genome not comprising the modification, without substantial negative impacts on AAV genome rescue, replication, and/or packaging.
  • an AAV genome disclosed herein is about 4 kilobases (kb), 4.5 kb, 5 kb, 5.5 kb, 6 kb, 6.5 kb, 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, 10 kb, 10.5 kb, 11 kb, 11.5 kb, 12 kb, 12.5 kb, 13 kb, 13.5 kb, 14 kb, 14.5 kb, 15 kb, 15.5 kb, 16 kb, 16.5 kb, 17 kb, 17.5 kb, or more in length.
  • an AAV genome disclosed herein is at least 4 kilobases (kb), 4.5 kb, 5 kb, 5.5 kb, 6 kb, 6.5 kb, 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, 10 kb, 10.5 kb, 11 kb, 11.5 kb, 12 kb, 12.5 kb, 13 kb, 13.5 kb, 14 kb, 14.5 kb, 15 kb, 15.5 kb, 16 T18934 Attorney Docket No.
  • an AAV genome disclosed herein is less than 4 kilobases (kb), 4.5 kb, 5 kb, 5.5 kb, 6 kb, 6.5 kb, 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, 10 kb, 10.5 kb, 11 kb, 11.5 kb, 12 kb, 12.5 kb, 13 kb, 13.5 kb, 14 kb, 14.5 kb, 15 kb, 15.5 kb, 16 kb, 16.5 kb, 17 kb, or 17.5 kb in length.
  • a nucleic acid vector e.g., an AAV genome as disclosed herein in some embodiments comprises one or more regulatory elements, such as regulatory elements operably linked to a transgene.
  • a regulatory element is located between two ITRs, a 5' ITR and a 3' ITR.
  • a regulatory element is located upstream of or 5' relative to a transgene.
  • a regulatory element is located downstream of or 3' relative to the 5' ITRs as described herein. In some embodiments, a regulatory element is located upstream of or 5' relative to a transgene and downstream of or 3' relative to a 5' ITR.
  • a regulatory element refers to a nucleotide sequence or structural component of a nucleic acid vector which is involved in the regulation of expression of components of the nucleic acid vector (e.g., a gene of interest comprised therein). Regulatory elements include, but are not limited to, promoters, enhancers, silencers, insulators, response elements, initiation sites, termination signals, and ribosome binding sites.
  • Promoters include constitutive promoters, inducible promoters, tissue-specific promoters, cell type-specific promoters, and synthetic promoters.
  • a nucleic acid vector disclosed herein may include viral promoters or promoters from mammalian genes that are generally active in promoting transcription.
  • constitutive viral promoters include the Herpes Simplex virus (HSV), thymidine kinase (TK), Rous Sarcoma Virus (RSV), Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV), Ad E1A and cytomegalovirus (CMV) promoters.
  • Non-limiting examples of constitutive mammalian promoters include various housekeeping gene promoters, as exemplified by the ⁇ -actin promoter.
  • Inducible promoters or other inducible regulatory elements may also be used to achieve desired expression levels of a gene of interest (e.g., a protein or polypeptide of interest).
  • suitable inducible promoters include those from genes such as cytochrome P450 genes, heat shock protein genes, metallothionein genes, and hormone- T18934 Attorney Docket No. U1202.70129WO00 inducible genes, such as the estrogen gene promoter.
  • Another example of an inducible promoter is the tetVP16 promoter that is responsive to tetracycline.
  • a nucleic acid vector e.g., an AAV genome
  • a nucleic acid vector comprises a nucleotide sequence encoding a product (e.g., a protein or polypeptide product).
  • a nucleotide sequence comprises a nucleotide sequence of a gene of interest.
  • a gene of interest encodes a therapeutic and/or diagnostic agent (e.g., protein or polypeptide).
  • a therapeutic or diagnostic agent is an antibody, a peptibody, a growth factor, a clotting factor, a hormone, a membrane protein, a cytokine, a chemokine, an activating or inhibitory peptide acting on cell surface receptors or ion channels, a cell-permeant peptide targeting intracellular processes, a thrombolytic agent, an enzyme, a bone morphogenetic protein, a nuclease, a protein used for gene editing, an Fc- fusion protein, an anticoagulant, or a protein or polypeptide that can be detected using a laboratory test.
  • a nucleic acid vector (e.g., an AAV genome) provided herein comprises a nucleotide sequence encoding a guide RNA or other nucleic acid used for gene editing, optionally in addition to a protein used for gene editing.
  • a product encoded by a nucleic acid vector (e.g., an AAV genome) disclosed herein is a detectable molecule.
  • a detectable molecule is a molecule that can be visualized (e.g., using a naked eye, under a microscope, or using a light detection device such as a camera).
  • the detectable molecule is a fluorescent molecule, a bioluminescent molecule, or a molecule that provides color (e.g., ⁇ -galactosidase, ⁇ -lactamase, ⁇ -glucuronidase, or spheroidenone).
  • the detectable molecule is a fluorescent, bioluminescent or enzymatic protein or functional peptide or polypeptide thereof.
  • fluorescent protein is a blue fluorescent protein, a cyan fluorescent protein, a green fluorescent protein, a yellow fluorescent protein, an orange fluorescent protein, a red fluorescent protein, or a functional peptide or polypeptide thereof.
  • a blue fluorescent protein may be azurite, EBFP, EBFP2, mTagBFP, or Y66H.
  • a cyan fluorescent protein may be ECFP, AmCyan1, Cerulean, CyPet, mECFP, Midori-ishi Cyan, mTFP1, or TagCFP.
  • a Green fluorescent protein may be AcGFP, Azami Green, EGFP, T18934 Attorney Docket No. U1202.70129WO00 Emarald, GFP or a mutated form of GFP (e.g., GFP-S65T, mWasabi, Stemmer, Superfolder GFP, TagGFP, TurboGFP, or ZsGreen).
  • a yellow fluorescent protein may be EYFP, mBanana, mCitrine, PhiYFp, TagYFP, Topaz, Venus, YPet, or ZsYellow1.
  • An orange fluorescent protein may be DsRed, RFP, DsRed2, DsRed-Express, Ds-Red-monomer, Tomato, tdTomato, Kusabira Orange, mKO2, mOrange, mOrange2, mTangerine, TagRFP, or TagRFP- T.
  • a red fluorescent protein may be AQ142, AsRed2, dKeima-Tandem, HcRed1, tHcRed, Jred, mApple, mCherry, mPlum, mRasberry, mRFP1, mRuby or mStrawberry.
  • a detectable molecule is a bioluminescent protein or a functional peptide or polypeptide thereof.
  • bioluminescent proteins are firefly luciferase, click-beetle luciferase, Renilla luciferase, and luciferase from Oplophorus gracilirostris.
  • a detectable molecule may be any polypeptide or protein that can be detected using methods known in the art. Non-limiting methods of detection are fluorescence imaging, luminescent imaging, bright filed imaging, and include imaging facilitated by immunofluorescence or immunohistochemical staining. [0087] Additional features of AAV particles, nucleic acid vectors, and capsid proteins are described in Patent Application Publication No. US2017/0356009, the contents of which are incorporated herein by reference in their entirety. [0088] In some embodiments, a nucleic acid vector (e.g., an AAV genome) is comprised within or encoded by a plasmid.
  • Nucleic acid vectors as disclosed herein, e.g., comprising modified ITRs, can be prepared by one of ordinary skill in the art by known methods.
  • AAV Particles [0090] According to some aspects, provided herein are AAV particles that comprise any of the nucleic acid vectors (e.g., AAV genomes) disclosed herein.
  • An AAV particle is a supramolecular assembly of 60 individual capsid protein subunits forming a non-enveloped T- 1 icosahedral lattice capable of protecting a single-stranded DNA genome.
  • a mature AAV particle is approximately 20 nm in diameter, and its capsid is formed from three structural capsid proteins VP1, VP2, and VP3, with molecular masses of 87, 73, and 62 kDa, respectively, in a ratio of approximately 1:1:18.
  • the 60 capsid proteins are arranged in an anti- Attorney Docket No. U1202.70129WO00 parallel ⁇ -strand barreloid arrangement, resulting in a defined tropism and a high resistance to degradation.
  • an AAV particle comprises an empty capsid (e.g., a capsid without a cargo).
  • an AAV particle comprises a capsid encapsidating a nucleic acid (e.g., a nucleic acid vector that comprises a gene of interest, such as a nucleic acid vector disclosed herein).
  • a nucleic acid encapsidated within an AAV capsid to generate an AAV particle comprises a nucleic acid vector disclosed herein.
  • an AAV particle disclosed herein comprises a capsid protein comprising one or more mutations, e.g., one or more amino acid substitutions.
  • an AAV particle described herein may have an AAV capsid protein (e.g., a wild-type AAV capsid protein or one comprising one or more amino acid substitutions) and an AAV nucleic acid vector comprising a modification (e.g., a deletion or substitution of a D-sequence, and/or an insertion of a non-AAV sequence).
  • AAV capsid protein e.g., a wild-type AAV capsid protein or one comprising one or more amino acid substitutions
  • an AAV nucleic acid vector comprising a modification (e.g., a deletion or substitution of a D-sequence, and/or an insertion of a non-AAV sequence).
  • an AAV particle disclosed herein comprises a capsid protein comprising amino acid substitutions at one or more positions corresponding to T491, Y444, Y500, and/or Y730 of SEQ ID NO: 2.
  • an AAV particle disclosed herein comprises a capsid protein comprising one or more amino acid substitutions corresponding to T491V, Y444F, Y500F, and/or Y730F substitutions in SEQ ID NO: 2.
  • an AAV particle disclosed herein comprises a capsid protein comprising amino acid substitutions at one or more positions corresponding to T491, Y444, Y500, and/or Y730 of SEQ ID NO: 2 and further comprises a nucleic acid vector comprising modification (e.g., a deletion, a substitution, or an insertion) of a D-sequence of an ITR (e.g., a modification of a D-sequence of a right ITR, a left ITR, or both a right ITR and a left ITR).
  • modification e.g., a deletion, a substitution, or an insertion
  • the AAV particle comprises a capsid protein comprising amino acid substitutions at one or more positions corresponding to T491, Y444, Y500, and/or Y730 of SEQ ID NO: 2 and a nucleic acid vector comprising a substitution of a portion of a D- sequence of an ITR with a heterologous nucleotide sequence.
  • the amino acid substitutions correspond to T491V, Y444F, Y500F, and/or Y730F substitutions in SEQ ID NO: 2.
  • the heterologous nucleotide sequence comprises, consists essentially of, or consists of the nucleotide sequence 5'-ATGTGCTTGA-3' (SEQ ID NO: 26), Attorney Docket No. U1202.70129WO00 5'-TCAAGCACAT-3' (SEQ ID NO: 27), or a reverse or reverse complement of either 5'-ATGTGCTTGA-3' (SEQ ID NO: 26) or 5'-TCAAGCACAT-3' (SEQ ID NO: 27).
  • a portion or the entirety of a D-sequence of an ITR is substituted with the heterologous nucleotide sequence.
  • an AAV particle disclosed herein is replicative.
  • a replicative AAV particle is capable of replicating within a host cell (e.g., a host cell within a subject or a host cell in culture).
  • an AAV particle disclosed herein is non- replicating.
  • a non-replicating AAV particle is not capable of replicating within a host cell (e.g., a host cell within a subject or a host cell in culture), but can infect the host and incorporate a genetic components into the host’s genome for expression.
  • an AAV particle disclosed herein is capable of infecting a host cell.
  • an AAV particle disclosed herein is capable of facilitating stable integration of genetic components into the genome of a host cell. In some embodiments, an AAV particle disclosed herein is not capable of facilitating integration of genetic components into the genome of a host cell.
  • an AAV particle disclosed herein comprises a nucleic acid vector (e.g., an AAV genome) provided herein.
  • a nucleic acid vector e.g., AAV genome
  • the nucleic acid vector e.g., AAV genome
  • the nucleic acid vector is a single-stranded DNA vector.
  • an AAV particle disclosed herein comprises one single-stranded DNA. In some embodiments, an AAV particle disclosed herein comprises two complementary DNA strands, forming a self-complementary AAV (scAAV).
  • a nucleic acid vector that may be comprised in an AAV particle comprises an ITR comprising a modification (e.g., a deletion, substitution, or insertion) of part or all of the ITR’s D-sequence.
  • part or all of the ITR’s D-sequence is substituted with a heterologous nucleotide sequence. In some embodiments, part or all of the ITR’s D-sequence is deleted. Further description of such modifications (e.g., deletions, substitutions, and insertions) is provided elsewhere herein.
  • an ITR comprising a substitution, insertion, or deletion of a nucleic acid vector as disclosed herein comprises one or more additional modifications, such as an additional substitution, modification, or deletion in another portion of the ITR. T18934 Attorney Docket No.
  • An AAV particle disclosed herein may be of any AAV serotype (e.g., AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13), including any derivative (including non-naturally occurring variants of a serotype) or pseudotype.
  • Non-limiting examples of derivatives and pseudotypes include AAV2-AAV3 hybrid, AAVrh.10, AAVhu.14, AAV3a/3b, AAVrh32.33, AAV-HSC15, AAV-HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15/17, AAVM41, AAV9.45, AAV2.5T, AAV-HAE1/2, AAV clone 32/83, AAVShH10, AAV2.15, AAV2.4, AAVM41, and AAVr3.45.
  • the AAV particle is a pseudotyped AAV particle, which comprises a nucleic acid vector comprising ITRs from one serotype (e.g., AAV2 or AAV3) and a capsid comprised of capsid proteins derived from another serotype (i.e., a serotype other than AAV2 or AAV3, respectively).
  • SEQ ID NOs: 1-14 provide examples of amino acid sequences of AAV capsid proteins of different serotypes.
  • Methods for producing and using pseudotyped rAAV vectors are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J.
  • an AAV particle disclosed herein is a recombinant AAV (rAAV) particle, e.g., comprising a recombinant nucleic acid or transgene.
  • rAAV recombinant AAV
  • Any combination of modifications described herein may result in an additive or synergistic effect, in which the beneficial properties of the resulting combination are equal to or greater than, respectively, the sum of the effects of the individual modifications.
  • an AAV particle comprising a modified capsid protein and a modified genome may have improvements in transduction efficiency, transgene expression, and/or packaging efficiency relative to a corresponding wild-type AAV particle that are equal to the sum of the improvements conferred by the individual capsid protein modification and the genome modification, or that are greater than the sum of the improvements conferred by the individual modifications.
  • AAV particles as disclosed herein, e.g., comprising modified ITRs can be prepared by one of ordinary skill in the art by known methods. T18934 Attorney Docket No.
  • AAV particles e.g., comprising modified genome and/or capsid protein
  • a plasmid such as a pSub201 plasmid, which includes sequences encoding AAV Rep and capsid proteins, and in which a gene of interest can be inserted.
  • the pSub201 sequence is provided below, in which the open reading frame encoding ampicillin-resistance marker is bolded and the D-sequences are underlined. The portions of the underlined D-sequences that are also bolded are the preferred portion that can be replaced by a heterologous nucleotide sequence.
  • a corresponding position for replacement by a heterologous nucleotide sequence in a different sequence can be identified by methods known in the art.
  • an AAV capsid protein disclosed herein comprises amino acid substitutions at one or more positions corresponding to T491, Y444, Y500, and/or Y730 of SEQ ID NO: 2.
  • the amino acid substitutions correspond to T491V, Y444F, Y500F, and/or Y730F substitutions in SEQ ID NO: 2. It should be understood that an amino acid substitution at a position corresponding to a position of SEQ ID NO: 2 can be an amino acid substitution in a capsid protein of any serotype.
  • the corresponding position in a capsid protein having a different baseline amino acid sequence can be determined by methods known in the art, such as by constructing structural alignments of the amino acid sequences and identifying corresponding amino acids.
  • a “corresponding” amino acid to be substituted is one which is at the corresponding position, and may have the same amino acid identity (i.e., the amino acid at the corresponding position in the second capsid protein sequence is the same as the amino acid in the reference capsid protein sequence), or may be an amino acid with similar properties T18934 Attorney Docket No. U1202.70129WO00 (e.g., similar hydrophobicity, size, charge, etc.) as the amino acid in the reference capsid protein.
  • an amino acid substitution at a position corresponding to T491 of SEQ ID NO: 2, or corresponding to a T491V substitution in SEQ ID NO: 2 may be a substitution at a position corresponding to position 491 of SEQ ID NO: 2 in a second capsid protein, which may also be a threonine, or which may be a similar amino acid (e.g., another amino acid with a polar uncharged side chain, such as serine, asparagine, or glutamine).
  • an AAV capsid protein disclosed herein comprises amino acid substitutions as described in Patent Application Publication Nos.
  • an AAV capsid protein as disclosed herein is a VP1 protein, a VP2 protein, or a VP3 protein.
  • the VP1, VP2, and VP3 capsid proteins are each encoded from the same segment of the AAV genome, and differ in their N termini based on alternative mRNA splicing.
  • the different capsid proteins VP1, VP2, and VP3 are defined according to numbering of the full-length VP1 protein.
  • a VP1 capsid protein is defined by amino acids 1-735 of SEQ ID NO: 2; a VP2 capsid protein is defined by amino acids 138-735 of SEQ ID NO: 2; and a VP3 capsid protein is defined by amino acids 203-735 of SEQ ID NO: 2.
  • Numbering of AAV capsid proteins is provided according to the VP1 sequence.
  • T491 refers to the threonine at position 491 of SEQ ID NO: 2 in a VP1 protein or the corresponding threonine in a VP2 or VP3 protein.
  • Y444, Y500, and Y730 refer to the tyrosines at positions 444, 500, and 730 of SEQ ID NO: 2, respectively, in a VP1 protein, or the corresponding tyrosines in a VP2 or VP3 protein.
  • An AAV capsid protein disclosed herein can be of any serotype, or can be a chimeric capsid protein (i.e., comprising segments from capsid proteins of two or more serotypes).
  • a capsid protein disclosed herein is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, or AAVrh74 capsid protein.
  • an AAV capsid protein as provided herein is of serotype 2, serotype 3, serotype 6, or serotype rh74.
  • Amino acid sequences of capsid proteins of other AAV serotypes are known and can be aligned with SEQ ID NO: 2 (AAV2 capsid protein) using techniques known in the art. Examples of amino acid sequences of AAV capsid proteins of various serotypes are provided below.
  • a nucleic acid may comprise a sequence that encodes a capsid protein disclosed here (e.g., a capsid protein comprising one or more amino acid substitutions).
  • a sequence encoding a capsid protein disclosed herein can be determined by one of ordinary skill in the art by known methods.
  • a nucleic acid encoding a capsid protein may comprise a promoter or other regulatory sequence operably linked to the coding sequence.
  • a nucleic acid encoding a capsid protein may be in the form of a plasmid, an mRNA, or another nucleic acid capable of being used by enzymes or machinery of a host cell to produce a capsid protein.
  • Nucleic acids encoding capsid proteins as provided herein can be used to make AAV particles that can be used for delivering a gene to a cell. Methods of making AAV particles are known in the art. For example, see Scientific Reports volume 9, Article number: 13601 (2019); Methods Mol Biol.2012; 798: 267–284; and thermofisher.com/us/en/home/clinical/cell-gene-therapy/gene-therapy/aav-production- workflow.html. Example sequences of nucleic acids encoding capsid proteins are provided below.
  • Second-strand synthesis can be measured by one of ordinary skill in the art by known methods.
  • second-strand synthesis of an AAV genome disclosed herein is increased relative to a corresponding T18934 Attorney Docket No. U1202.70129WO00 wild-type AAV genome.
  • second-strand synthesis of an AAV genome disclosed herein is increased as a result of a decrease in binding of a host-cell protein (e.g., a phosphorylated host-cell protein, such as FKBP52) to the AAV genome (e.g., to a D-sequence of the AAV genome).
  • a host-cell protein e.g., a phosphorylated host-cell protein, such as FKBP52
  • the second-strand synthesis of an AAV genome as disclosed herein is at least 5% higher (e.g., at least 10% higher, at least 15% higher, at least 20% higher, at least 25% higher, at least 30% higher, at least 35% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 100% higher, at least 150% higher, at least 200% higher, at least 250% higher, or more) than the second-strand synthesis of a corresponding wild-type AAV genome.
  • 5% higher e.g., at least 10% higher, at least 15% higher, at least 20% higher, at least 25% higher, at least 30% higher, at least 35% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 100% higher, at least 150% higher, at least 200% higher, at least 250% higher, or more
  • the second-strand synthesis of an AAV genome as disclosed herein is at least 1.5-fold higher (e.g., at least 2-fold higher, at least 2.5-fold higher, at least 3-fold higher, at least 3.5-fold higher, at least 4-fold higher, at least 4.5-fold higher, at least 5-fold higher, at least 5.5-fold higher, at least 6-fold higher, at least 6.5-fold higher, at least 7-fold higher, at least 7.5-fold higher, at least 8-fold higher, at least 8.5-fold higher, at least 9-fold higher, at least 9.5-fold higher, at least 10-fold higher, at least 10.5-fold higher, at least 11-fold higher, at least 11.5-fold higher, at least 12-fold higher, at least 12.5-fold higher, at least 13-fold higher, at least 13.5-fold higher, at least 14-fold higher, at least 14.5-fold higher, at least 15-fold higher, at least 15.5-fold higher, at least 16-fold higher, at least 16.5-fold higher, at least 17- fold
  • second- strand synthesis of an AAV particle as disclosed herein is not modified relative to a corresponding wild-type AAV particle.
  • Transduction Efficiency e.g., transduction efficiency of an AAV particle disclosed herein (e.g., comprising a modification in a nucleic acid vector and/or in a capsid protein) is modified relative to a corresponding wild-type AAV particle (e.g., not comprising the modification in the nucleic acid vector and/or in the capsid protein).
  • Transduction efficiency of an AAV particle can be determined, for example, by comparing expression of a gene of interest in a cell following contacting the cell with the AAV particle, or by measuring the number of viral genome copies per cell following contacting a population of cells with the AAV particle.
  • transduction efficiency of an AAV particle as disclosed herein is higher than the transduction efficiency of a corresponding wild-type AAV particle (e.g., not comprising the modified capsid protein or nucleic acid modification).
  • the transduction efficiency of an AAV particle as disclosed herein is at least 5% higher (e.g., at least 10% higher, at least 15% higher, at least 20% higher, at least 25% higher, at least 30% higher, at least 35% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 100% higher, at least 150% higher, at least 200% higher, at least 250% higher, or more) than the transduction efficiency of a corresponding wild-type AAV particle.
  • the transduction efficiency of an AAV particle as disclosed herein is at least 1.5-fold higher (e.g., at least 2-fold higher, at least 2.5-fold higher, at least 3-fold higher, at least 3.5-fold higher, at least 4-fold higher, at least 4.5-fold higher, at least 5-fold higher, at least 5.5-fold higher, at least 6-fold higher, at least 6.5-fold higher, at least 7-fold higher, at least 7.5-fold higher, at least 8-fold higher, at least 8.5-fold higher, at least 9-fold higher, at least 9.5-fold higher, at least 10-fold higher, at least 10.5-fold higher, at least 11-fold higher, at least 11.5-fold higher, at least 12-fold higher, at least 12.5-fold higher, at least 13-fold higher, at least 13.5-fold higher, at least 14-fold higher, at least 14.5-fold higher, at least 15-fold higher, at least 15.5- fold higher, at least 16-fold higher, at least 16.5-fold higher, at least 17-fold higher,
  • transduction efficiency of an AAV particle as disclosed herein is not modified relative to a corresponding wild-type AAV particle.
  • Transgene expression [0138] According to some aspects, expression of a transgene encoded by a nucleic acid vector comprising a modification (e.g., a deletion or substitution of a sequence, such as a D-sequence, or insertion of a sequence) disclosed herein is altered relative to expression of the transgene T18934 Attorney Docket No. U1202.70129WO00 encoded by a nucleic acid vector that does not comprise the modification.
  • transgene expression is, in some embodiments, on a per nucleic acid vector copy number basis (e.g., transgene expression in a cell, when normalized to the total amount of nucleic acid vector in the cell, is altered).
  • a modified AAV particle as disclosed herein results in greater transgene expression relative to a corresponding AAV particle not comprising the same modification but that delivers a comparable number of viral genomes to a cell.
  • Relative transgene expression levels can be determined, for example, by measuring expression of the transgene in a cell by methods known in the art following contacting the cell with an AAV particle comprising the modified nucleic acid vector encoding the transgene and comparing an equivalent measurement in another cell contacted with an AAV particle comprising a nucleic acid vector that does not comprise the modification.
  • transgene expression from a modified nucleic acid vector as disclosed herein is higher than the transgene expression from a corresponding nucleic acid vector that does not comprise the modification.
  • the transgene expression from a modified nucleic acid vector as disclosed herein is at least 5% higher (e.g., at least 10% higher, at least 15% higher, at least 20% higher, at least 25% higher, at least 30% higher, at least 35% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 100% higher, at least 150% higher, at least 200% higher, at least 250% higher, or more) than the transgene expression from a corresponding nucleic acid vector that does not comprise the modification.
  • the transgene expression from a modified nucleic acid vector as disclosed herein is at least 1.5-fold higher (e.g., at least 2-fold higher, at least 2.5-fold higher, at least 3-fold higher, at least 3.5-fold higher, at least 4-fold higher, at least 4.5-fold higher, at least 5-fold higher, at least 5.5-fold higher, at least 6-fold higher, at least 6.5-fold higher, at least 7-fold higher, at least 7.5-fold higher, at least 8-fold higher, at least 8.5-fold higher, at least 9-fold higher, at least 9.5-fold higher, at least 10-fold higher, at least 10.5-fold higher, at least 11-fold higher, at least 11.5-fold higher, at least 12-fold higher, at least 12.5-fold higher, at least 13-fold higher, at least 13.5-fold higher, at least 14-fold higher, at least 14.5-fold higher, at least 15-fold higher, at least 15.5-fold higher, at least 16-fold higher, at least 16.5- fold
  • transgene expression from a modified nucleic acid vector as disclosed herein is not changed relative to transgene expression from a corresponding nucleic acid vector that does not comprise the modification.
  • Packaging efficiency [0142] According to some aspects, packaging efficiency of an AAV particle disclosed herein is modified relative to a corresponding wild-type AAV particle. Packaging efficiency of an AAV particle refers to the capability of a particular AAV capsid to encapsidate a particular viral genome. Packaging efficiency can be measured by one of ordinary skill in the art, such as by quantifying the ratio of capsids to viral genomes (see, e.g., Grimm, et al.
  • the packaging efficiency of an AAV particle as disclosed herein is higher than the packaging efficiency of a corresponding wild-type AAV particle.
  • the packaging efficiency of an AAV particle as disclosed herein is at least 5% higher (e.g., at least 10% higher, at least 15% higher, at least 20% higher, at least 25% higher, at least 30% higher, at least 35% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 100% higher, at least 150% higher, at least 200% higher, at least 250% higher, or more) than the packaging efficiency of a corresponding wild-type AAV particle.
  • the packaging efficiency of an AAV particle as disclosed herein is at least 1.5-fold higher (e.g., at least 2-fold higher, at least 2.5-fold higher, at least 3-fold higher, at least 3.5-fold higher, at least 4-fold higher, at least 4.5-fold higher, at least 5-fold higher, at least 5.5-fold higher, at least 6-fold higher, at least 6.5-fold higher, at least 7-fold higher, at least 7.5-fold higher, at least 8-fold higher, at least 8.5-fold higher, at least 9-fold higher, at least 9.5-fold higher, at least 10-fold higher, at least 10.5-fold higher, at least 11-fold higher, at least 11.5-fold higher, at least 12-fold higher, at least 12.5-fold higher, at least 13-fold higher, at least 13.5-fold higher, at least 14-fold higher, at least 14.5-fold higher, at least 15-fold higher, at least 15.5-fold higher, at least 16-fold higher, at least 16.5-fold higher, at least 17-fold higher, at least 1.5-fold
  • the packaging efficiency of an AAV particle as disclosed herein is lower than the packaging efficiency of a corresponding wild-type AAV particle.
  • the packaging efficiency of an AAV particle as disclosed herein is decreased by at least 5% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or more) relative to the packaging efficiency of a corresponding wild-type AAV particle.
  • packaging efficiency of an AAV particle disclosed herein is not modified relative to a corresponding wild-type AAV particle.
  • both the transduction efficiency and the packaging efficiency of an AAV particle as disclosed herein is modified (i.e., increased or decreased) relative to a corresponding unmodified or wild-type AAV particle (e.g., of the same serotype).
  • the immunogenicity of an AAV particle as disclosed herein is modified relative to a corresponding unmodified or wild-type AAV particle (e.g., of the same serotype).
  • Pharmaceutical compositions [0147] Any one of the AAV particles, capsid proteins, or nucleic acids disclosed herein may be comprised within a pharmaceutical composition comprising a pharmaceutically-acceptable carrier or may be comprised within a pharmaceutically-acceptable carrier.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the AAV particle, capsid protein, or nucleic acid is comprised or administered to a subject.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum oil such as mineral oil, vegetable oil such as peanut oil, soybean oil, and sesame oil, animal oil, or oil of synthetic origin. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers.
  • Non-limiting examples of pharmaceutically acceptable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, polyacrylic acids, lubricating agents (such as talc, magnesium stearate, and mineral oil), wetting agents, emulsifying agents, suspending agents, preserving agents (such as methyl-, ethyl-, and propyl-hydroxy-benzoates), and pH adjusting agents (such as inorganic and organic T18934 Attorney Docket No.
  • U1202.70129WO00 acids and bases and solutions or compositions thereof.
  • Other examples of carriers include phosphate buffered saline, HEPES-buffered saline, and water for injection, any of which may be optionally combined with one or more of calcium chloride dihydrate, disodium phosphate anhydrous, magnesium chloride hexahydrate, potassium chloride, potassium dihydrogen phosphate, sodium chloride, or sucrose.
  • carriers that might be used include saline (e.g., sterilized, pyrogen-free saline), saline buffers (e.g., citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (for example, serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. USP grade carriers and excipients are particularly useful for delivery of AAV particles to human subjects.
  • saline e.g., sterilized, pyrogen-free saline
  • saline buffers e.g., citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer
  • amino acids e.g., citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer
  • amino acids e.g., citrate buffer, phosphate buffer, acetate
  • compositions may contain at least about 0.1% of the therapeutic agent (e.g., AAV particle) or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 70% or 80% or more of the weight or volume of the total formulation.
  • the amount of therapeutic agent(s) (e.g., AAV particle) in each therapeutically-useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound.
  • compositions as disclosed herein comprising nucleic acid vectors comprising modified ITRs and/or AAV particles comprising nucleic acid vectors comprising modified ITRs, can be prepared by one of ordinary skill in the art by known methods.
  • Methods of contacting a cell According to some aspects, methods of contacting a cell with an AAV particle or nucleic acid vector are provided herein.
  • Methods of contacting a cell may comprise, for example, contacting a cell in a culture with a composition comprising an AAV particle or nucleic acid vector.
  • contacting a cell comprises adding a composition comprising an AAV particle or nucleic acid vector to the supernatant of a cell culture (e.g., a cell culture on a tissue culture plate or dish) or mixing a composition comprising an AAV particle or nucleic acid vector with a cell culture (e.g., a suspension cell culture).
  • contacting a cell comprises mixing a composition comprising an AAV particle T18934 Attorney Docket No.
  • contacting a cell with an AAV particle or nucleic acid vector comprises administering a composition comprising an AAV particle or nucleic acid vector to a subject or device in which the cell is located. In some embodiments, contacting a cell comprises injecting a composition comprising an AAV particle or nucleic acid vector into a subject in which the cell is located. In some embodiments, contacting a cell comprises administering a composition comprising an AAV particle or nucleic acid vector directly to a cell, or into or substantially adjacent to a tissue of a subject in which the cell is present.
  • administering means providing a material to a subject in a manner that is pharmacologically useful.
  • an rAAV particle is administered to a subject enterally.
  • an enteral administration of the essential metal element/s is oral.
  • a rAAV particle is administered to the subject parenterally.
  • a rAAV particle is administered to a subject subcutaneously, intraocularly, intravitreally, subretinally, intravenously (IV), intracerebro- ventricularly, intramuscularly, intrathecally (IT), intracisternally, intraperitoneally, via inhalation, topically, or by direct injection to one or more cells, tissues, or organs.
  • a rAAV particle is administered to the subject by injection into the hepatic artery or portal vein.
  • a compositions of AAV particles is administered to a subject to treat a disease or condition.
  • compositions described above or elsewhere herein are typically administered to a subject in an effective amount, that is, an amount capable of producing a desirable result.
  • the desirable result will depend upon the active agent being administered.
  • an effective amount of rAAV particles may be an amount of the particles that are capable of transferring an expression construct to a host organ, tissue, or cell.
  • a therapeutically acceptable amount may be an amount that is capable of treating a disease, e.g., a muscular dystrophy.
  • a cell disclosed herein is a cell isolated or derived from a subject.
  • a cell is a mammalian cell (e.g., a cell isolated or derived from a mammal).
  • a cell is a human cell.
  • a cell is isolated or derived from a particular tissue of a subject, such as muscle tissue.
  • a cell is a muscle cell. In some embodiments, a cell is a skeletal muscle cell or a smooth muscle cell. In some embodiments, a cell is in vitro. In some embodiments, a cell is ex vivo. In some embodiments, a cell in in vivo. In some embodiments, a cell is within a subject (e.g., within a tissue or organ of a subject). In some embodiments, a cell is a primary cell. In some embodiments, a cell is from a cell line (e.g., an immortalized cell line). In some embodiments a cell is a cancer cell or an immortalized cell.
  • administering means providing a material to a subject in a manner that is pharmacologically useful.
  • administering means providing a material to a subject in a manner that is pharmacologically useful.
  • administering means providing a material to a subject in a manner that is pharmacologically useful.
  • an AAV particle disclosed herein in a suitably formulated pharmaceutical composition disclosed herein either subcutaneously, intraocularly, intravitreally, subretinally, parenterally, intravenously (IV), intracerebro- ventricularly, intramuscularly, intrathecally (IT), intracisternally, orally, intraperitoneally, by oral or nasal inhalation, or by direct injection to one or more cells, tissues, or organs by direct injection.
  • the administration is a route suitable for systemic delivery, such as by intravenous injection.
  • the administration is a route suitable for local delivery, such as by intramuscular injection.
  • “administering” or “administration” means providing a material to a subject in a manner that is pharmacologically useful.
  • the concentration of AAV particles administered to a subject may be on the order ranging from 10 6 to 10 14 particles/ml or 10 3 to 10 15 particles/ml, or any values therebetween for either range, such as for example, about 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or 10 14 particles/ml.
  • AAV particles of a higher concentration than 10 13 particles/ml are administered.
  • the concentration of AAV particles administered to a subject may be on the order ranging from 10 6 to 10 14 vector genomes (vgs)/ml or 10 3 to 10 15 vgs/ml, or any values therebetween for either range (e.g., 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or 10 14 vgs/ml).
  • AAV particles of higher concentration than 10 13 vgs/ml are administered.
  • the AAV particles can be administered as a single dose, or divided into two or more administrations as may be required to achieve therapy of the particular disease or disorder being treated.
  • T18934 Attorney Docket No.
  • U1202.70129WO00 0.0001 ml to 10 ml are delivered to a subject.
  • the number of AAV particles administered to a subject may be on the order ranging from 10 6 -10 14 vgs/kg body mass of the subject, or any values therebetween (e.g., 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or 10 14 vgs/kg).
  • the dose of AAV particles administered to a subject may be on the order ranging from 10 12 -10 14 vgs/kg.
  • the volume of AAVrh74 composition delivered to a subject is 0.0001 ml to 10 ml.
  • a composition disclosed herein e.g., comprising an AAV particle
  • the composition is administered to a subject multiple times (e.g., twice, three times, four times, five times, six times, or more).
  • Repeated administration to a subject may be conducted at a regular interval (e.g., daily, every other day, twice per week, weekly, twice per month, monthly, every six months, once per year, or less or more frequently) as necessary to treat (e.g., improve or alleviate) one or more symptoms of a disease, disorder, or condition in the subject.
  • a regular interval e.g., daily, every other day, twice per week, weekly, twice per month, monthly, every six months, once per year, or less or more frequently
  • a regular interval e.g., daily, every other day, twice per week, weekly, twice per month, monthly, every six months, once per year, or less or more frequently
  • a host cell in situ in a subject e.g., ex vivo or in vitro.
  • Non-limiting examples of non-human primate subjects include macaques (e.g., cynomolgus or rhesus macaques), marmosets, tamarins, spider monkeys, owl monkeys, vervet monkeys, squirrel monkeys, baboons, gorillas, chimpanzees, and orangutans.
  • the subject is a human subject.
  • Other exemplary subjects include domesticated animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters.
  • the subject has or is suspected of having a disease or disorder that may be treated with gene therapy.
  • a disease or disorder that may be treated with gene therapy may be characterized by one or more mutation(s) in the genome that results in abnormal structure or function of one or more proteins associated with development, health, maintenance and/or function of a cell and/or organ. Diseases and disorders can be characterized and identified, e.g., through laboratory tests and/or evaluation by a clinician.
  • the subject has or is suspected of having a disease (e.g., a disease caused by a defect, such as a genetic mutation, in one or more cells or genes).
  • U1202.70129WO00 nucleic acid isolated or derived from the subject is identified via sequencing (e.g., Sanger or next-generation sequencing) to comprise a mutation (e.g., in a gene associated with development, health, maintenance, or function of a cell and/or organ).
  • sequencing e.g., Sanger or next-generation sequencing
  • a subject comprises a mutant form of one or more genes associated with development, health, maintenance and/or function of a cell and/or organ.
  • methods disclosed herein provide a cell of a subject with a functional form of a gene.
  • Naturally-occurring adeno-associated viruses do not express their own genes efficiently, and viral second-strand DNA synthesis is required before gene expression can occur in a host cell.
  • Mammalian host cells do not have an RNA polymerase that is capable of transcribing the single-stranded DNA genome of an AAV.
  • RNA polymerase capable of transcribing the single-stranded DNA genome of an AAV.
  • proximal 10 nucleotides the 10 nucleotides closest to the terminus of the ITR, i.e., adjacent the hairpin structure of the ITR
  • deletion of the full 3' D-sequence substantially impedes rescue of the proviral genome, AAV replication, and AAV packaging (see, e.g., Wang, et al., J Virol. (1997) 71(4):3077-82).
  • ssAAV genomes comprising a sequence replacing the proximal 10 nucleotides of the 3' D-sequence allows successful packaging, while avoiding the negative impacts of the natural D-sequence (e.g., inhibited second-strand DNA synthesis).
  • a sequence library was generated with random 10-nucleotide sequences in place of the proximal 10 nucleotides (“D10-sequence random library”; FIG.1).
  • the library of sequences was cloned into plasmids to generate plasmid libraries comprising the random 10- nucleotide sequences in place of either the left ITR or the right ITR (FIG.2 and FIG.3).
  • the plasmids were transfected into HEK293 cells and rescue, replication, and packaging were observed.
  • FIG.3 only construct “N10-L1” resulted in successful generation of AAV particles, indicating that features of the other three constructs were incompatible with AAV rescue and/or packaging.
  • the failure of the other three constructs to successfully undergo rescue, replication, and packaging underscores the criticality of the D-sequence in the 3'-ITR to AAV packaging.
  • GenZ ssAAV particles were generated comprising this sequence substituted in place of the proximal 10 nucleotides (closest to the 5' and 3' ends of the AAV genome) of each D-sequence (FIG.4 and FIG.5).
  • GenZ ssAAV particles comprising a nucleic acid encoding firefly luciferase fused to EYFP with a chicken ⁇ -actin (CBA) promoter.
  • GenZ ssAAV particles achieve T18934 Attorney Docket No. U1202.70129WO00 strong transgene expression in host cells in a dose-dependent manner ( ⁇ 7,000 pixels 2 /visual field for cells treated with 3,000 vgs/cell and ⁇ 12,500 pixels 2 /visual field for cells treated with 10,000 vgs/cell; relative to no measurable EYFP expression in mock-treated cells; FIG.6A and 6B).
  • transgene expression from recombinant ssAAV vectors is also largely sub-optimal since the viral second-strand DNA synthesis is strongly inhibited by binding of phosphorylated forms of host cell chaperone protein, FKBP52, to the D-sequence at the 3'-end of the ssAAV genome (Proc Natl Acad Sci USA, 94(20): 10879-10884, 1997). It has not been possible to delete the D-sequence at the 3'-ITR as it serves as the “packaging signal” for the AAV genome (J. Virol., 70: 1668-1677, 1996).
  • 5'-ATGTGCTTGA-3' SEQ ID NO: 26
  • This sequence was inserted in a recombinant AAV2 genome replacing the proximal 10- nts in D-sequence at both ITRs flanking an expression cassette containing a firefly luciferase- enhanced yellow fluorescent protein (FLuc-EYFP) under the control of the chicken ⁇ -actin (CBA) promoter, designated as generation Z (“GenZ”) ssAAV vector.
  • FLuc-EYFP firefly luciferase- enhanced yellow fluorescent protein
  • CBA chicken ⁇ -actin
  • Transduction efficiencies of wild-type (WT) and GenZ ssAAVrh74-CBAp-FLuc-EYFP vectors were evaluated in human HeLa cells in vitro, the results of which are shown in FIGs.7A and 7B.
  • T18934 Attorney Docket No. U1202.70129WO00
  • the extent of transgene expression from the GenZ AAVrh74 vector was ⁇ 20-fold higher than that from the WT AAVrh74 vectors.
  • Transduction efficiencies of WT and GenZ ssAAVrh74-CBAp-FLuc-EYFP vectors were also evaluated in vivo in C57BL6/J mice following intravenous administration.
  • GenZ ssAAVrh74 vectors averaged ⁇ 5-fold increase in transgene expression in the liver, compared with that from the WT ssAAVrh74 vectors (FIGs.8A and 8B).
  • the observed increase in transgene expression was not as pronounced in vivo since AAVrh74 vectors are closely related to AAV8, and ssAAV8 vectors transduce mouse liver very efficiently.
  • the GenZ ssAAV DNA genome can be packaged into any AAV serotype capsid vector; and packaging of the GenZ ssAAV DNA genomes in capsid-modified NextGen AAV serotype vectors (comprising amino acid substitutions in their capsid proteins that correspond to substitutions Y444F, Y500F, Y730F, and/or T491V in an AAV2 capsid protein) should further enhance the performance of Opt Z vectors (which comprise both the amino acid substitutions corresponding to Y444F, Y500F, Y730F, and/or T491V and the GenZ genome modification), the availability of which has significant implications for their potential use in achieving high- efficiency transgene expression of larger genes.
  • Example 3 [0174] The GenZ ssAAV genome improves viral second-strand DNA synthesis, and mediates significantly higher levels of transgene expression. Thus, it behaves more like a scAAV genome, but without scAAV’s inherent size limitation. However, the extent of transgene expression from different GenZ AAV serotype vectors varies.
  • GenZ ssAAVrh74 and GenZ ssAAV3 vectors were ⁇ 20-fold and ⁇ 22-fold, respectively, compared with that of their wild-type (WT) counterparts (FIGs.9A and 9B, respectively)
  • the increase in transduction efficiency of GenZ ssAAV2 and GenZ ssAAV6 vectors was only ⁇ 3-fold and ⁇ 4-fold, respectively, compared with that of their WT counterparts (FIG.9C and 9D, respectively).
  • AAV2 J Virol., 88:10711079, 2014; J Virol 2016;90:7196-7204
  • AAV9 Mol. Ther., 28:1373-1380, 2020; T18934 Attorney Docket No. U1202.70129WO00 Hum. Gene Ther., 31:1155-1168, 2020
  • capsids play a role in transcription and second-strand DNA synthesis.
  • Experiments were conducted as explained below to test whether AAV2 and AAV6, but not AAVrh74 and AAV3 capsids, negatively impact transgene expression from GenZ ssAAV vectors.
  • GenZ ssAAVrh74 vectors were used to transduce HeLa cells in triplicates at 3x10 3 viral genomes (vgs)/cell, incubated with and without highly purified empty capsids of AAVrh74, AAV2, AAV3, and AAV6, either before or during transduction. Transgene expression was visualized via fluorescence microscopy 72 hours post-transduction. The results demonstrate that, while AAVrh74 and AAV3 empty capsids had no effect on transduction efficiency, both AAV2 and AAV6 empty capsids led to a significant decrease in transgene expression from GenZ ssAAVrh74 vectors (FIG.10).
  • any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.
  • All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
  • All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.
  • a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • “or” should be understood to have the same meaning as “and/or” as defined above.
  • At least one of A and B can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
  • transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of” and “consisting essentially of” the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B,” the disclosure T18934 Attorney Docket No. U1202.70129WO00 also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B.”

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Abstract

L'invention concerne des génomes de VAA modifiés, des particules de VAA comprenant les génomes modifiés, des compositions de ceux-ci, ainsi que des méthodes d'utilisation de ceux-ci.
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