WO2025006829A9 - Vecteurs viraux adéno-associés améliorés - Google Patents

Vecteurs viraux adéno-associés améliorés

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Publication number
WO2025006829A9
WO2025006829A9 PCT/US2024/035946 US2024035946W WO2025006829A9 WO 2025006829 A9 WO2025006829 A9 WO 2025006829A9 US 2024035946 W US2024035946 W US 2024035946W WO 2025006829 A9 WO2025006829 A9 WO 2025006829A9
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Prior art keywords
amino acid
substitution
vector
serotype
aav
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WO2025006829A1 (fr
Inventor
Bhargavi KONDRAGUNTA
Ruigong WANG
Siddhartha PAUL
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RP Scherer Technologies LLC
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RP Scherer Technologies LLC
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Priority to EP24832975.7A priority Critical patent/EP4735468A1/fr
Priority to CN202480043770.6A priority patent/CN121532412A/zh
Publication of WO2025006829A1 publication Critical patent/WO2025006829A1/fr
Publication of WO2025006829A9 publication Critical patent/WO2025006829A9/fr
Anticipated expiration legal-status Critical
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    • 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
    • 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
    • C12N2750/14151—Methods of production or purification of viral material
    • 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
    • C12N2750/14151—Methods of production or purification of viral material
    • C12N2750/14152—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles

Definitions

  • the present invention relates to AAV genomes, vectors and nucleic acids containing variant capsid protein sequences that allow for increased titer during recombinant AAV vector particle production.
  • Adeno-associated virus is one of the most actively investigated gene therapy vehicles. It was initially discovered as a contaminant of adenovirus preparations. AAV is a protein shell surrounding and protecting a small, single-stranded DNA genome of approximately 4.8 kilobases (kb). AAV belongs to the parvovirus family and is dependent on co-infection with other viruses, mainly adenoviruses, in order to replicate. Initially distinguished serologically, molecular cloning of AAV genes has identified hundreds of unique AAV strains in numerous species. Its single-stranded genome contains three genes, Rep (Replication), Cap (Capsid), and aap (Assembly).
  • ITRs inverted terminal repeats
  • Rep gene encoded proteins Rep78, Rep68, Rep52, and Rep40
  • Cap expression genes gives rise to the viral capsid proteins (VP; VP1/VP2/VP3), which form the outer capsid shell that protects the viral genome, as well as being actively involved in cell binding and internalization.
  • the viral coat is comprised of 60 proteins arranged into an icosahedral structure with the capsid proteins in a molar ratio of 1 : 1 : 10 (VP1 : VP2: VP3)
  • the aap gene encodes the assembly-activating protein (AAP) in an alternate reading frame overlapping the cap gene.
  • AAP assembly-activating protein
  • This nuclear protein is thought to provide a scaffolding function for capsid assembly.
  • AAP is essential for nucleolar localization of VP proteins and capsid assembly in AAV2, the subnuclear localization of AAP varies among 11 other serotypes recently examined, and is nonessential in AAV4, AAV5, and AAV11.
  • MAAP membrane- associated accessory protein
  • a variety of AAV genomes and production systems for producing recombinant AAV vector particles for use in gene therapies are known in the art. However, there is a need for systems which provide for higher titer and thus more efficient production of recombinant AAV vector particles.
  • the present invention relates to AAV genomes, vectors and nucleic acids containing variant capsid protein sequences that allow for increased titer, without an increase in encapsidated residual host cell DNA during recombinant AAV vector particle production.
  • the present invention provides an adeno-associated virus (AAV) genome, comprising a variant membrane associated accessory protein (MAAP) nucleic acid sequence having a mutation selected from the group consisting of a stop codon substitution at a codon position corresponding to positions W19, C20, S27, S57 and S88, an amino acid substitution at position L1, an amino acid substitution at position C20, an amino acid substitution at position C21, and combinations thereof, wherein the positions correspond to consensus sequence SEQ ID NO:1.
  • AAV adeno-associated virus
  • MAAP membrane associated accessory protein
  • the genome comprises a substitution mutation in at least one of amino acid positions L1, C20 and C21 and/or a stop codon substitution at an amino acid position selected from the group consisting of W19, C20, S27, S57 and S88.
  • the genome comprises a substitution mutation at amino acid position C20 and a stop codon substitution at an amino acid position selected from the group consisting of W19, C20, S27, S57 and S88.
  • the genome comprises a substitution mutation at amino acid position C21 and a stop codon substitution at an amino acid position selected from the group consisting of W19, C20, S27, S57 and S88.
  • the genome comprises a substitution mutation at amino acid position L1 and a stop codon substitution at a position selected from the group consisting of amino acid positions W19, C20, S27, S57 and S88. In some preferred embodiments, the genome comprises a substitution mutation at one of amino acid positions C20 and C21 and a stop codon substitution at an amino acid position selected from the group consisting of S27, S57 and S88. In some preferred embodiments, the genome comprises a substitution mutation at both of amino acid positions C20 and C21 and a stop codon substitution at an amino acid position selected from the group consisting of S27, S57 and S88. In some preferred embodiments, the substitution mutation at position L1 is an L1P substitution mutation.
  • the substitution mutation at position C20 is selected from the group consisting of C20S, C20Y and C20F substitution mutations.
  • the substitution mutation at amino acid position C21 is a C21S substitution mutation.
  • the stop codon substitution is at position W19.
  • the stop codon substitution is at position C20.
  • the stop codon substitution is at position S27.
  • the stop codon substitution is at position S57.
  • the stop codon substitution is at position S88.
  • the AAV genome is selected from the group consisting of a naturally-occurring serotype and a non-naturally-occurring serotype. In some preferred embodiments, the AAV genome is a non-naturally-occurring serotype. In some preferred embodiments, is a naturally-occurring serotype. In some preferred embodiments, the AAV genome comprises a serotype 6 genome. In some preferred embodiments, the AAV genome comprises a serotype 2 genome.
  • the AAV genome is selected from a serotype 1, serotype 3, serotype 4, serotype 5, serotype 7, serotype 8, serotype 9, serotype 10, serotype 11, serotype 12, serotype 13, serotype rh8 and serotype rh10 genome. In some preferred embodiments, the AAV genome is not an AAV serotype 2 genome.
  • the AAV genome comprises a VP1 nucleic sequence
  • the MAAP and VP1 nucleotide sequences encode MAAP and VP1 peptide sequences that each have at least 80% homology to wild type, with the proviso that the MAAP nucleotide sequence consists of at least one mutation as defined above.
  • the MAAP and VP1 peptide sequences each have at least 90% homology to wild type.
  • the MAAP and VP1 peptide sequences each have at least 95% homology to wild type.
  • the present invention provides a producer cell that produces adeno-associated virus, the producer cell comprising the adeno-associated virus genome as described above.
  • the producer cell is eukaryotic.
  • the producer cell is mammalian.
  • the producer cell is a human cell.
  • the producer cell is selected from yeast cells and insect cells.
  • the present invention provides methods for producing adeno-associated virus, the method comprising: introducing the AAV genome as described above a cell to create a producer cell and then culturing the producer cell whereby said producer cell produces adeno-associated virus.
  • the methods further comprise harvesting said adeno-associated virus, where said harvested adeno-associated virus comprises a transgene.
  • the titer of the produced adeno- associated virus is greater than the titer achieved with an adeno-associated virus genome comprising a corresponding wild-type MAAP sequence.
  • the present invention provides adeno-associated virus produced by the process described above.
  • the present invention provides a vector, comprising a variant AAV capsid gene sequence, the variant AAV capsid gene sequence comprising a mutated membrane associated accessory protein (MAAP) nucleic acid sequence having a mutation selected from the group consisting of a stop codon substitution at a codon position corresponding to positions W19, C20, S27, S57 and S88, an amino acid substitution at position L1, an amino acid substitution at position C20, an amino acid substitution at position C21, and combinations thereof, wherein the positions align with consensus sequence SEQ ID NO:1.
  • MAAP mutated membrane associated accessory protein
  • the variant AAV capsid gene sequence comprises a substitution mutation in at least one of amino acid positions L1, C20 and C21 and a stop codon substitution at an amino acid position selected from the group consisting of W19, C20, S27, S57 and S88. In some preferred embodiments, the variant AAV capsid gene sequence comprises a substitution mutation at amino acid position C20 and a stop codon substitution at an amino acid position selected from the group consisting of W19, C20, S27, S57 and S88.
  • the variant AAV capsid gene sequence comprises a substitution mutation at amino acid position C21 and a stop codon substitution at an amino acid position selected from the group consisting of W19, C20, S27, S57 and S88. In some preferred embodiments, the variant AAV capsid gene sequence comprises a substitution mutation at amino acid position L1 and a stop codon substitution at a position selected from the group consisting of amino acid positions W19, C20, S27, S57 and S88. In some preferred embodiments, the variant AAV capsid gene sequence comprises a substitution mutation at one of amino acid positions C20 and C21 and a stop codon substitution at an amino acid position selected from the group consisting of S27, S57 and S88.
  • the variant AAV capsid gene sequence comprises a substitution mutation at both of amino acid positions C20 and C21 and a stop codon substitution at an amino acid position selected from the group consisting of S27, S57 and S88.
  • the substitution mutation at position L1 is an L1P substitution mutation.
  • the substitution mutation at position C20 is selected from the group consisting of C20S, C20Y and C20F substitution mutations.
  • the substitution mutation at amino acid position C21 is a C21S substitution mutation.
  • the stop codon substitution is at position W19.
  • the stop codon substitution is at position C20.
  • the stop codon substitution is at position S27.
  • the stop codon substitution is at position S57.
  • the stop codon substitution is at position S88.
  • the variant capsid gene sequence is selected from the group consisting of a naturally-occurring serotype and a non-naturally-occurring serotype. In some preferred embodiments, the variant capsid gene sequence is a non-naturally-occurring serotype. In some preferred embodiments, the variant capsid gene sequence is a naturally- occurring serotype. In some preferred embodiments, the variant capsid gene sequence is a serotype 6 capsid gene sequence. In some preferred embodiments, the variant capsid gene sequence is a serotype 2 capsid gene sequence.
  • the variant capsid gene sequence is selected from a serotype 1, serotype 3, serotype 4, serotype 5, serotype 7, serotype 8, serotype 9, serotype 10, serotype 11, serotype 12, serotype 13, serotype rh8 and serotype rh10 and AAVRH.8 capsid gene sequences.
  • the vector comprises a VP1 nucleic sequence
  • the MAAP and VP1 nucleotide sequences encode MAAP and VP1 peptide sequences that each have at least 80% homology to wild type, with the proviso that the modified capsid nucleotide sequence consists of at least one mutation as defined above.
  • the MAAP and VP1 peptide sequences each have at least 90% homology to wild type.
  • the MAAP and VP1 peptide sequences each have at least 95% homology to wild type.
  • the vector further comprises a nucleic acid sequence encoding a rep gene.
  • the vector is a plasmid vector.
  • the present invention provides a vector system comprising the vector as described above and a second vector comprising a transgene flanked by ITR sequences.
  • the system further comprises a third vector comprising adenovirus helper sequences.
  • the second and third vectors are plasmid vectors.
  • the present invention provides a producer cell line comprising the vector or vector system as described above.
  • the producer cell is eukaryotic.
  • the producer cell is mammalian.
  • the producer cell is a human cell.
  • the producer cell is selected from of yeast cells and insect cells.
  • the present invention provides methods of for producing adeno-associated virus, the method comprising: introducing the vector or vector system of any of as described above into a cell to create a producer cell and then culturing the producer cell whereby the producer cell produces adeno-associated virus.
  • the titer of the produced adeno-associated virus is greater than the titer achieved with an adeno-associated virus genome comprising a corresponding wild-type MAAP sequence.
  • Fig. 1 provides a process flow diagram for viral particle production.
  • the present invention relates to AAV genomes, vectors and nucleic acids containing variant capsid protein sequences that allow for several fold increased titer during recombinant AAV vector particle production, without an increase in encapsidated residual host cell DNA.
  • the packaging efficiency measured by AUC for full capsid maintained at a similar or higher level as the standard capsid
  • a number of vector backbones for the production of recombinant AAV vector particles are known.
  • production of recombinant AAV vector particles can be inefficient.
  • the present invention addresses this problem by providing AAV capsid protein sequences containing variant MAAP sequences.
  • the variant capsid polypeptides described herein allow for higher-titer production of recombinant AAV particles.
  • each intervening number there between with the same degree of precision is explicitly contemplated.
  • the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
  • the term “genome” refers to the genetic material (e.g., chromosomes) of an organism.
  • vector refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells.
  • vector includes cloning and expression vehicles, as well as viral vectors.
  • viral vector refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral particle.
  • the viral vector can contain a nucleic acid (e.g., a payload) encoding a polypeptide as described herein in place of non-essential viral genes.
  • the vector and/or particle may be utilized for the purpose of transferring nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.
  • an "AAV virus” or "AAV viral particle” refers to a viral particle composed of at least one AAV capsid protein such as VP1 (typically by all of the capsid proteins of a wild- type AAV) and an encapsidated polynucleotide rAAV vector. If the particle comprises a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as a "recombinant AAV vector particle" or simply a "rAAV vector". Thus, production of a rAAV particle necessarily includes production of a rAAV vector, as such a vector is contained within a rAAV particle.
  • AAV capsid protein such as VP1 (typically by all of the capsid proteins of a wild- type AAV) and an encapsidated polynucleotide
  • membrane-associated accessory protein refers to an AAV MAAP protein of any AAV serotype.
  • wild- type MAAP refers to naturally occurring, AAV MAAP.
  • a specific amino acid residue or residues preferably refers to the residue with the corresponding number in the sequence of the MAAP sequences of SEQ ID NOs 1 to 16 and may be referred to a “position” within the designated sequence.
  • residue or residues or a position or positions in consensus SEQ ID NO: 1 or to a residue or residues or a position or positions aligning with MAAP polypeptide consensus sequence SEQ ID NO: 1 residue number the residue or residues in one of the MAAP amino acids sequences of AAV serotypes (as depicted in SEQ ID NOs 2 to 16) that correspond to the indicated residue or residues in the consensus sequence of SEQ ID NO: 1 are also encompassed.
  • Residue (or position) number X is defined as a mutation in a MAAP polypeptide at a residues or position corresponding to amino acid residue or position number X of the MAAP polypeptide consensus sequence SEQ ID NO. 1. This can be either the indicated residue number in the sequence of SEQ ID NO: 1 or a corresponding residue number in any AAV MAAP, in particular a corresponding residue number in a MAAP sequence having a sequence of any of SEQ ID NOs: 2-16.
  • a skilled person is well capable of determining the residue in any MAAP or a MAAP having a sequence of any of SEQ ID NOs: 2 to 16 corresponding to a particular residue in SEQ ID NO: 1, e.g., by performing an alignment of the MAAP sequence and the sequence of SEQ ID NO: 1.
  • a C to S mutation of the MAAP polypeptide is introduced at a polypeptide residue aligning with MAAP polypeptide consensus sequence SEQ ID NO. 1 residue number 20.
  • this refers to a mutation in a MAAP polypeptide at a position corresponding to amino acid residue number 20 of the MAAP polypeptide consensus sequence SEQ ID NO. 1, as well as sequence aligned with the consensus sequence, for example SEQ ID NO:7 where the C to S mutation would also be at residue number 20.
  • the term “adeno-associated virus genome” refers to a polynucleotide molecule comprising at least one polynucleotide sequence encoding AAV MAAP.
  • the AAV genome may be present in a vector.
  • the AAV genome or AAV vector comprises at least a gene encoding a variant mutant) MAAP.
  • the AAV genome or AAV vector preferably further comprises one or more polynucleotides sequences encoding one or more further AAV genes.
  • the genes other than the gene encoding MAAP may be wild-type or contain one or more mutations.
  • the AAV genome comprises a polynucleotide molecule comprising an AAV polynucleotide sequence flanked at both ends by AAV Inverted Terminal Repeats (ITRs).
  • ITRs AAV Inverted Terminal Repeats
  • the AAV genome is encompassed by an AAV expression vector or is an AAV expression vector.
  • the AAV genome or AAV expression vector thus preferably comprises at least one AAV polynucleotide, either wild-type or containing one or more mutations, flanked by AAV ITRs, as long as it contains a variant MAAP.
  • the AAV genome of the invention comprises a polynucleotide molecule comprising an AAV polynucleotide sequence that allows the production of AAV when introduced into a suitable host cell.
  • the AAV genome of the invention is combined with one or more further polynucleotide molecules comprising AAV polynucleotide sequences, such as an AAV helper construct comprising a polynucleotide sequence encoding AAV capsid proteins and other AAV helper functions and/or an AAV gene of interest (GOI, also referred to as a transgene) construct comprising a gene of interest flanked by AAV ITRs, so that the combined AAV genome and one or more further polynucleotide molecules allows the production of AAV when introduced into a suitable host cell.
  • a transgene is a therapeutic gene.
  • the AAV genome or AAV vector may be of any AAV serotype, either a naturally occurring serotype or a non-naturally-occurring serotype.
  • the cap genes encoding the MAAP are well known for each AAV serotype and a skilled person is therefore well capable of preparing a mutant AAV genome as described herein of any AAV serotype MAAP.
  • Mutations in MAAP can for instance be introduced by site-directed mutagenesis.
  • Site- directed mutagenesis is well known in the art and can be used to introduce one or more point mutations, including a mutation according to the invention (including substitution, insertion or deletion) into a viral polynucleotide or genome.
  • a skilled person is well capable of introducing a mutation according to the invention. Suitable techniques for site-directed mutagenesis are described in Sambrook's et al. Molecular Cloning A Laboratory Manual, second edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), Ausubel et al. Current Protocols in Molecular Biology(Greene Publishing Associates (1992)) and Bachman et al. (J. Methods Enzymol. 2013; 529:241-248), the content of which references is incorporated herein by reference.
  • viral capsid polypeptide refers to the proteinaceous shell or coat of a viral particle.
  • a viral capsid polypeptide as described herein permits packaging or assembly of the capsid polypeptide into a viral particle that is competent for delivery of nucleic acid to the host cell.
  • Capsids function to encapsidate, protect, transport, and release into a host cell a viral genome.
  • Capsids are generally comprised of oligomeric structural subunits of a polypeptide of the viral capsid polypeptides.
  • encapsidated means enclosed within a viral capsid.
  • the AAV genome comprises three overlapping sequences which encode capsid proteins, VP1, VP2 and VP3, which start from one promoter, p40.
  • the AAV capsid is composed of a mixture of VP1, VP2, and VP3 totaling 60 monomers arranged in icosahedral symmetry in a ratio of 1 : 1 : 10.
  • packetaging refers to a series of intracellular events that result in the assembly and encapsidation of an AAV particle.
  • payload refers to a nucleic acid which is encapsidated within a viral vector, e.g., an AAV vector.
  • a payload nucleic acid can encode, e.g., a polypeptide, an inhibitory RNA, an antibody or antibody reagent, an oligonucleotide, or a miRNA.
  • producer cell refers to any cell line suitable for the production of AAV vectors.
  • polypeptide refers to a polymer of amino acids.
  • protein and “polypeptide” are used interchangeably herein.
  • a peptide is a relatively short polypeptide, typically between about 2 and 60 amino acids in length.
  • Polypeptides used herein typically contain amino acids such as the 20 L-amino acids that are most commonly found in proteins. However, other amino acids and/or amino acid analogs known in the art can be used.
  • One or more of the amino acids in a polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a fatty acid group, a linker for conjugation, functionalization, etc.
  • polypeptide that has a nonpolypeptide moiety covalently or noncovalently associated therewith is still considered a "polypeptide.”
  • exemplary modifications include glycosylation and palmitoylation.
  • Polypeptides can be purified from natural sources, produced using recombinant DNA technology or synthesized through chemical means such as conventional solid phase peptide synthesis, etc.
  • the term "polypeptide sequence” or "amino acid sequence” as used herein can refer to the polypeptide material itself and/or to the sequence information (i.e., the succession of letters or three letter codes used as abbreviations for amino acid names) that biochemically characterizes a polypeptide.
  • a polypeptide sequence presented herein is presented in an N-terminal to C- terminal direction unless otherwise indicated.
  • a given amino acid can be replaced by a residue having similar physicochemical characteristics, e.g., substituting one aliphatic residue for another (such as lie, Val, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn).
  • Other such conservative substitutions e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known.
  • Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity, e.g. ligand-mediated receptor activity and specificity of a native or reference polypeptide is retained.
  • Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Val (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H).
  • Naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Val; Leu into He or into Val; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Val, into He or into Leu.
  • a polypeptide described herein can be a functional fragment of one of the amino acid sequences described herein.
  • a “functional fragment” is a fragment or segment of a peptide which retains at least 50% of the wild-type reference polypeptide’s activity according to an assay known in the art or described below herein.
  • a functional fragment described herein would retain at least 50% of the VP1 capsid function.
  • a functional fragment can comprise conservative substitutions of the sequences disclosed herein.
  • a polypeptide described herein can be a variant of a polypeptide or molecule as described herein.
  • the variant is a conservatively modified variant.
  • Conservative substitution variants can be obtained by mutations of native nucleotide sequences, for example.
  • a “variant,” as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, e.g., SEQ ID NO: 1, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions.
  • a variant capsid polypeptide as the term is used herein retains the capacity for assembly into a viral capsid.
  • Variant polypeptide- encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, e.g., SEQ ID NO: 1, but that encode a variant protein or fragment thereof that retains activity of the non- variant polypeptide.
  • a native or reference DNA sequence e.g., SEQ ID NO: 1
  • a wide variety of PCR-based site- specific mutagenesis approaches are known in the art and can be applied by the ordinarily skilled artisan.
  • a variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence.
  • the degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using computer programs commonly employed for this purpose, e.g., that are freely available on the world wide web (e.g. BLASTp or BLASTn with default settings). Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art.
  • Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites permitting ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.
  • oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations are well established and include, for example, those disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Pat. Nos.
  • Any cysteine residue not involved in maintaining the proper conformation of a polypeptide also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to a polypeptide to improve its stability or facilitate oligomerization.
  • DNA is defined as deoxyribonucleic acid.
  • polynucleotide is used herein interchangeably with “nucleic acid” to indicate a polymer of nucleosides.
  • a polynucleotide is composed of nucleosides that are naturally found in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxy cytidine) joined by phosphodiester bonds.
  • nucleosides or nucleoside analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not found in naturally occurring nucleic acids, and such molecules may be preferred for certain applications.
  • this specification refers to a polynucleotide it is understood that both DNA, RNA, and in each case both single- and double-stranded forms (and complements of each single- stranded molecule) are provided.
  • Polynucleotide sequence as used herein can refer to the polynucleotide material itself and/or to the sequence information (i.e. the succession of letters used as abbreviations for bases) that biochemically characterizes a specific nucleic acid. A polynucleotide sequence presented herein is presented in a 5' to 3' direction unless otherwise indicated.
  • the term “corresponding to,” when used in reference to an amino acid or polynucleotide sequence means that a given amino acid or polynucleotide sequence in one polypeptide or polynucleotide molecule has structural properties, functional properties, or both that are similar relative to an amino acid or polynucleotide sequence in a similar location in another polypeptide or polynucleotide molecule.
  • Homologues of a given polypeptide in different species “correspond to” each other, as do regions or domains of homologous polypeptides from different species.
  • capsid polypeptides of different serotypes of viral vectors including but not limited to adeno-associated virus (AAV) vectors, “correspond to” each other, as do regions of such polypeptides, defined, for example by alignment of their amino acid sequences. While other alignment parameters can be used to define such regions, for the avoidance of doubt, alignment can be performed using BLAST® (Basic Local Alignment Search Tool) using default parameters of version BLAST+ 2.8.0 released March 28, 2018.
  • BLAST® Basic Local Alignment Search Tool
  • a “transgene” is a polynucleotide that is delivered to a cell by a vector.
  • a “gene” refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular gene product after being transcribed, and sometimes also translated.
  • the term “gene” or “coding sequence” refers to a nucleotide sequence in vitro or in vivo that encodes a gene product. In some instances, the gene consists or consists essentially of coding sequence, that is, sequence that encodes the gene product. In other instances, the gene comprises additional, non-coding, sequence.
  • the gene may or may not include regions preceding and following the coding region, e.g. 5' untranslated (5' UTR) or “leader” sequences and 3' UTR or “trailer” sequences, as well as intervening sequences (introns) between individual coding segments (exons).
  • 5' UTR 5' untranslated
  • leader leader
  • 3' UTR or “trailer” sequences as well as intervening sequences (introns) between individual coding segments (exons).
  • a “gene product” is a molecule resulting from expression of a particular gene.
  • Gene products include, e.g., a polypeptide, an aptamer, an interfering RNA, an mRNA, and the like.
  • a “gene product” is a polypeptide, peptide, protein or interfering RNA including short interfering RNA (siRNA), miRNA or small hairpin RNA (shRNA).
  • a gene product is a therapeutic gene product, e.g., a therapeutic protein.
  • a “therapeutic gene” refers to a gene that, when expressed, produces a therapeutic gene product that confers a beneficial effect on the cell or tissue in which it is present, or on a mammal in which the gene is expressed. Examples of beneficial effects include amelioration of a sign or symptom of a condition or disease, prevention or inhibition of a condition or disease, or conferral of a desired characteristic.
  • Therapeutic genes include, but are not limited to, genes that correct a genetic deficiency in a cell or mammal.
  • cell culture refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro, including oocytes and embryos.
  • the term "gene of interest” refers to any nucleotide sequence (e.g., RNA or DNA), the manipulation of which may be deemed desirable for any reason (e.g., treat disease, confer improved qualities, expression of a protein of interest in a host cell, expression of a ribozyme, etc.), by one of ordinary skill in the art.
  • nucleotide sequences include, but are not limited to, coding sequences of structural genes (e.g., reporter genes, selection marker genes, oncogenes, drug resistance genes, growth factors, etc.), and non-coding regulatory sequences which do not encode an mRNA or protein product (e.g., promoter sequence, polyadenylation sequence, termination sequence, enhancer sequence, etc.).
  • the term “protein of interest” refers to a protein encoded by a gene of interest.
  • the terms “nucleic acid molecule encoding,” “DNA sequence encoding,” “DNA encoding,” “RNA sequence encoding,” and “RNA encoding” refer to the order or sequence of deoxyribonucleotides or ribonucleotides along a strand of deoxyribonucleic acid or ribonucleic acid. The order of these deoxyribonucleotides or ribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA or RNA sequence thus codes for the amino acid sequence.
  • promoter refers to a DNA sequence which when ligated to a nucleotide sequence of interest is capable of controlling the transcription of the nucleotide sequence of interest into mRNA.
  • a promoter is typically, though not necessarily, located 5' (i.e., upstream) of a nucleotide sequence of interest whose transcription into mRNA it controls, and provides a site for specific binding by RNA polymerase and other transcription factors for initiation of transcription.
  • Transcriptional control signals in eukaryotes comprise "promoter” and “enhancer” elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription (Maniatis et al., Science 236:1237 [1987]). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect and mammalian cells, and viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest.
  • eukaryotic promoters and enhancers have a broad host range while others are functional in a limited subset of cell types (for review see, Voss et al., Trends Biochem. Sci., 11:287 [1986]; and Maniatis et al., supra).
  • the SV40 early gene enhancer is very active in a wide variety of cell types from many mammalian species and has been widely used for the expression of proteins in mammalian cells (Dijkema et al., EMBO J. 4:761 [1985]).
  • promoter/enhancer denotes a segment of DNA which contains sequences capable of providing both promoter and enhancer functions (i.e., the functions provided by a promoter element and an enhancer element, see above for a discussion of these functions).
  • promoter/promoter may be "endogenous” or “exogenous” or “heterologous.”
  • An “endogenous” enhancer/promoter is one that is naturally linked with a given gene in the genome.
  • an “exogenous” or “heterologous” enhancer/promoter is one that is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques such as cloning and recombination) such that transcription of that gene is directed by the linked enhancer/promoter.
  • the terms “complementary” or “complementarity” are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules.
  • sequence “5'-A-G-T-3'” is complementary to the sequence “3'-T-C-A-5'.”
  • Complementarity may be “partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids.
  • the percentage of identity of an amino acid sequence or nucleic acid sequence is defined herein as the percentage of residues of the full length of an amino acid sequence or nucleic acid sequence that is identical with the residues in a reference amino acid sequence or nucleic acid sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity.
  • the percentage homology of an amino acid sequence or the term "% homology to” is defined herein as the percentage of amino acid residues in a particular sequence that are homologous with the amino acid residues in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence homology. This method takes into account conservative amino acid substitutions.
  • amino acids can be similar in several characteristics, for example, size, shape, hydrophobicity, hydrophilicity, charge, isoelectric point, polarity, aromaticity, etc.
  • operable combination refers to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced.
  • the term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
  • the present invention provides AAV sequences, genomes and vectors comprising variant MAAP sequences.
  • the variants comprise, for example, a stop codon substitution at a codon position corresponding to positions W19, C20, S27, S57 and S88, an amino acid substitution at position L1, an amino acid substitution at position C20, an amino acid substitution at position C21, and combinations thereof, wherein the positions correspond or are aligned to consensus sequence SEQ ID NO:1.
  • the MAAP sequence comprises a substitution mutation at position L1 and a stop codon substitution at a position selected from the group consisting of W19 (or L19 of AAV2 MAAP), C20, S27, S57 and S88.
  • the MAAP sequence comprises a substitution mutation at position C20 and/or C21 and a stop codon substitution at a position selected from the group consisting of W19, S27, S57 and S88. In some preferred embodiments, the MAAP sequence comprises a substitution mutation at both of positions C20 and C21 and a stop codon substitution at a position selected from the group consisting of S27, S57 and S88.
  • the substitution mutation at position L1 is an L1P substitution mutation.
  • the substitution mutation at position C20 is selected from the group consisting of C20S, C20Y and C20F substitution mutations. In some preferred embodiments, the substitution mutation at position C20 is a C20S substitution mutation.
  • the stop codon substitution is at position W19 (or L19 of AAV2 MAAP). In some preferred embodiments, the variant with a stop codon substitution at position W19 (or L19 of AAV2 MAAP) further comprises a substitution mutation at position L1. In some preferred embodiments, the stop codon substitution is at position C20. In some preferred embodiments, the variant with a stop codon substitution at position C20 further comprises a substitution mutation as described at position L1. In some preferred embodiments, the stop codon substitution is at position S27. In some preferred embodiments, the variant with a stop codon substitution at position S27 further comprises a substitution mutation as described at position L1 and/or C20 and/or C21.
  • the stop codon substitution is at position S57. In some preferred embodiments, the variant with a stop codon substitution at position S57 further comprises a substitution mutation as described at position L1 and/or C20 and/or C21. In some preferred embodiments, the stop codon substitution is at position S88. In some preferred embodiments, the variant with a stop codon substitution at position S88 further comprises a substitution mutation as described at position L1 and/or C20 and/or C21.
  • SEQ ID NO:1 is a MAAP consensus sequence.
  • SEQ ID NOs: 2-16 are wild-type MAAP sequences for AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh8 and AAVrh10.
  • SEQ ID NOs: 17-30 are exemplary variant MAAP polypeptide sequences for AAV6 MAAP.
  • SEQ ID NOs:31-44 are exemplary variant MAAP polypeptide sequences for AAV2 MAAP.
  • SEQ ID NOs:73-87 are exemplary variant MAAP polypeptide sequences for AAV5 MAAP.
  • SEQ ID NOs: 103-114 are exemplary variant MAAP polypeptide sequences for AAV8 MAAP.
  • SEQ ID NOs:127-140 are exemplary variant MAAP polypeptide sequences for AAVrh10 MAAP. It will be understood that the same mutations may be introduced at the corresponding sites in other MAAP polypeptide sequences, e.g., in MAAPs from AAV1, AAV3, AAV4, AAV7, AAV9, AAV10, AAV11, AAV12, AAV13, or AAVrh8.
  • the AAV genome is not an AAV serotype 2 genome.
  • SEQ ID NOs: 45-50 are nucleic acid sequences for the variant AAV6 MAAP polypeptides (i.e., SEQ ID NOs: 17-30).
  • SEQ ID NOs: 59-72 are nucleic acid sequences for the variant AAV2 MAAP polypeptides (i.e., SEQ ID NOs: 31-44).
  • SEQ ID NOs: 88-102 are nucleic acid sequences for the variant AAV5 MAAP polypeptides (i.e., SEQ ID NOs: 73-87).
  • SEQ ID NOs: 141-154 are nucleic acid sequences for the variant AAVrh10 MAAP polypeptides (i.e., SEQ ID NOs: 127-140).
  • the in the sequence represents the amino acid position at which a stop codon has been substituted in the corresponding nucleic acid coding sequence.
  • the amino acid position corresponding to a substituted stop codon in the coding nucleic acid sequence, if present, is indicated by an X. It will be understood by those of ordinary skill in the art that since the MAAP polypeptide is expressed in an alternative reading frame of the wild-type AAV capsid protein sequence, a stop codon that is substituted into a MAAP sequence (e.g., SEQ ID NO:1) will not introduce a stop codon in the reading frame for the capsid protein.
  • any MAAP polypeptide that is translated where a stop codon is inserted into the reading frame of the MAAP polypeptide coding sequence will be truncated (i.e., translation will stop) at the amino acid position immediately upstream (or 5’) to the position at which the stop codon is introduced (e.g., at the amino acid just before position W19 (L19), C20, S27, S57 or S88).
  • the variant amino acids sequences in Table 1 are all presented in relation to the full length MAAP polypeptide coding sequence even though in some instances a stop codon has been introduced at a position within the MAAP polypeptide coding sequence.
  • Mutations at corresponding locations in, for example homologous MAAP polypeptides would be expected to have similar effects on viral function.
  • Corresponding locations can include, for example, a location relative to the consensus MAAP polypeptide sequence (e.g., SEQ ID NO: 1) or indeed any of SEQ ID NOs:2-16).
  • a variant MAAP polypeptide or nucleic acid sequence encoding the variant MAAP polypeptide is provided herein that bears a mutation that corresponds to a mutation of the polypeptide of any of SEQ ID NOs: 17-44, 73-87, 103-1 14 or 127-140, as described herein.
  • a homologous MAAP polypeptide or nucleic acid sequence encoding a MA AP polypeptide that is encoded in or included in an AAV genome or vector of the present invention has at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98%, sequence identity to a consensus MAAP sequence (i.e., SEQ ID NO:1) or AAV MAAP wild-type sequence (i.e, SEQ ID NOs:2-16), with the proviso that the MAAP polypeptide sequence or nucleic acid sequence encoding the variant MAAP polypeptide comprises or consists of a one or more substitutions or mutations as described above.
  • a consensus MAAP sequence i.e., SEQ ID NO:1
  • AAV MAAP wild-type sequence i
  • the MAAP polypeptides of AAV serotypes 1, 3-5 and 7-13 are homologous to an AAV6 or AAV2 capsid polypeptide.
  • the present invention provides viral genomes and vectors comprising a variant MAAP nucleic acid sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% 99% or 100% sequence identity to the variant nucleic acid sequences listed in Table 1, with the proviso that the variant sequences encode, comprise or consist of one or more of the mutations described above (i.e., a stop codon substitution at a codon position corresponding to amino positions W19, C20, S27, S57 and S88, an amino acid substitution at position L1, an amino acid substitution at position C20, an amino acid substitution at position C21, and combinations thereof).
  • viral capsid polypeptide comprising a region corresponding to the amino acid sequence of SEQ ID NO: 1 , 3, 6, 7, 9 or 16, wherein the region corresponding to the amino acid sequence of SEQ ID NO: 1, 3, 6, 7, 9 or 16 comprises a mutation relative to SEQ ID NO: 1, 3, 6, 7, 9 or 16 as described above.
  • a homologous viral capsid polypeptide has a region with at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 1 , 3, 6, 7, 9 or 16, with the proviso that region comprises one or more substitutions as described above.
  • the identification of amino acid sites or regions of amino add sequence in the AAV2, AAV5, AAV6, AAV8 and AAVrh10 capsid polypeptides that tolerate change in general, i.e., that permit functions of the viral vector can be used to guide changes in the capsid polypeptides of other AAV serotypes to similarly influence function or provide for modified properties.
  • a mutation or set of mutations that provides a desired change in function for AAV2, AAV5, AAV6, AAV8 or AAVrh10 can be introduced to the corresponding location of the capsid polypeptide of another AAV serotype to similarly influence those properties in that serotype.
  • mutation refers to any change in the amino acid sequence, e.g., a substitution, insertion, or deletion of at least one amino acid.
  • Site-directed mutations in the nucleic acid sequence can be generated by one skilled in the art using techniques known in the art or described herein.
  • Mutations that are at equivalent positions in other homologous viruses can be made.
  • examples of other homologous viruses include any of AAV serotypes 1 to 13, as well as other natural isolates or synthetic sequences.
  • Corresponding positions in homologous viruses can be inferred from sequence homology to AAV2, AAV5, AAV6, AAV8 or AAVrh10.
  • the mutations described herein are introduced into the corresponding amino acid sequence of an AAV1 , AAV3, AAV3B, AAV4, AAV7, AAV9, AAV10, AAV11, AAV12, AAV13, or AAVrh8 capsid polypeptide.
  • nucleic acid e.g., a polynucleotide encoding any of the variant viral capsid polypeptides described herein.
  • One skilled in the art can alter the wild-type MAAP sequence (e.g., SEQ ID NOs. 2-16), e.g,, by introducing at least one base pair substitution, such that the altered sequence encodes a variant amino acid sequence.
  • the nucleic acid sequence for each codon is known in the art.
  • nucleic acid sequence may be altered to introduce a stop codon by substitution of nucleotides in a codon to produce the stop codon.
  • Site-directed mutagenesis is known in the art and can be used to introduce a point mutation(s) (e.g., amino acid substitutions, insertions, or deletions) or other mutations or combinations thereof to a viral capsid polypeptide.
  • a polynucleotide encoding a variant viral capsid polypeptide described herein is operably linked to a promoter sequence.
  • a promoter sequence that drives expression of the polynucleotide in a cell further comprises a sequence that encodes a REP protein, e.g., an AAV rep protein.
  • the polynucleotide further comprises a sequence that encodes a CAP protein, e.g., an AAV CAP protein.
  • a viral particle comprising any of the viral capsid polypeptides described herein. It is noted that a viral particle comprising a variant capsid polypeptides described herein can be replication-competent or replication- incompetent. Thus, in some embodiments, the viral particle comprising a variant capsid polypeptides described herein is replication competent.
  • replication competent refers to a virus or viral particle that is infectious, and is also capable of being replicated in an infected cell (i.e., in the presence of a helper virus or helper virus functions). In the case of AAV, replication competence generally requires the presence of functional AAV packaging genes.
  • the viral particle comprising a variant capsid polypeptide described herein is replication incompetent or replication defective.
  • replication incompetent and “replication defective” refer to a virus or viral particle that cannot independently replicate and package its genome. For example, when a cell is infected with the virus or viral particle, the heterologous gene is expressed in the infected cells, however, the virus or the viral particle is not able to replicate further.
  • the viral particle further comprises a polynucleotide encoding a transgene, e.g., a polynucleotide comprising a sequence that encodes a gene product such as a therapeutic gene product.
  • a polynucleotide comprising a sequence that encodes a gene product such as a therapeutic gene product.
  • the polynucleotide is flanked on the 5' and 3' ends by functional AAV inverted terminal repeat (ITR) sequences.
  • ITR AAV inverted terminal repeat
  • AAV ITR sequences By “functional AAV ITR sequences” is meant that the ITR sequences function as intended for the rescue, replication, and packaging of the AAV virion, Hence, AAV ITRs for use in the gene delivery vectors need not have a wild-type nucleotide sequence, and can be altered by the insertion, deletion, or substitution of nucleotides or the AAV ITRs can be derived from any of several AAV serotypes, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, etc.
  • the viral particles have the wild type Rep and Cap genes deleted in whole or part, but retain functional flanking ITR sequences.
  • the transgene may be any suitable gene or GOI. If the vector plasmid is for use in gene therapy, the transgene may be any gene that comprises or encodes a protein or nucleotide sequence that can be used to treat a disease.
  • the transgene may encode an enzyme, a metabolic protein, a signaling protein, an antibody, an antibody fragment, an antibody like protein, an antigen, or a non-translated RNA such as an miRNA, siRNA, snRNA, or antisense RNA.
  • the GOI may encode all or a portion of a gene editing system, such as Cas9, an sgRNA and donor template DNA.
  • a dual AAV system is utilized for CRISPR gene editing where CAS9 is included as the GOI in one GOI plasmid and sgRNA and donor template DNA as the GOI in a second GOI plasmid.
  • viral particles comprising a variant capsid polypeptide described herein using standard methods.
  • an AAV expression vector comprising a polynucleotide cassette can be introduced into a producer cell, followed by introduction of an AAV helper construct comprising a polynucleotide sequence encoding a variant capsid polypeptide described herein, and where the helper construct includes AAV coding regions capable of being expressed in the producer cell and which complement AAV helper functions absent in the AAV vector.
  • helper virus and/or additional vectors into the producer cell, wherein the helper virus and/or additional vectors provide accessory functions capable of supporting efficient rAAV virus production.
  • the producer cells are then cultured to produce rAAV.
  • viral particles comprising a variant polypeptide described herein can be produced by first generating a DNA library of AAV capsid variants.
  • libraries of AAV6 capsid gene sequence variants are cloned into a plasmid containing the AAV Inverted Terminal Repeat regions (ITRs).
  • the final ITR plasmids contain, e.g., a cytomegalovirus (CMV) promoter upstream of the Cap gene.
  • CMV cytomegalovirus
  • the capsid library' plasmids, AAV pHelper plasmids, and plasmids containing, for example, the AAV2 Rep gene are, for example, co-transfected into HEK-293T cells using PEI.
  • Capsids are purified using standard techniques for cell lysis (freeze-thaw or addition of 5 M NaCI), treatment with benzonase to remove unpackaged genomes, and purification and concentration by iodixanol ultra centrifugation.
  • any host cells for producing recombinant viral particles can be employed, including, for example, mammalian cells, insect cells, plant cells. microorganisms and yeast.
  • Host cells can also be packaging cells in which the AAV rep and cap genes are stably maintained in the host cell or producer cells in which the AAV vector genome is stably maintained and packaged.
  • Exemplary packaging and producer cells include but are not limited to HeLa cell, COS cell, COS- 1 cell, COS-7 cell, HEK293 cell, A549 cell, BHK cell, BSC-1 cell, BSC-40 cell, Vero cell, Sfc9 cell, Sf -21 cell, Tn-368 cell, BTI ⁇ Tn ⁇ 5B1- 4 (High- Five) cell, Saos cell, C2C12 cell, L cell, HT1080 cell, HepG2 cell, WEHI cell, 3T3 cell, 10T1/2 cell, MDCK cell, BMT-10 cell, WI38 cell, or primary fibroblast, hepatocyte or myoblast cell derived from mammals,
  • a cell comprising a variant viral capsid polypeptide described herein or a polynucleotide encoding a variant viral capsid polypeptide described herein.
  • the cell can be prokaryotic cell or eukaryotic cell.
  • the cell can be a mammalian cell, insect cell, plant cell, bacterial cell, or yeast cell.
  • Some exemplary cells include, but are not limited to, alveolar cells, basophils, cardiac smooth muscle cells), cardiomyocyte, collecting duct intercalated cells, collecting duct principal cells, ectodermal cells, endocardial cells, endoderm cells, eosinophils, epithelial cells, hepatic stellate cells, interstitial kidney cells, mtrahepatic lymphocytes, kidney distal tubule cells, kidney glomerulus parietal cells, kidney glomerulus podocytes, kidney proximal tubule brush border cells, loop of Henle thin segment cells, lung epithelial cells, lung smooth muscle cells, lymphocytes, monocytes, muscle cells, neutrophils, non-parenchymal cells, parenchymal cells, phagocytic Kupffer cells, platelets, red blood cells, sinusoidal endothelial cells, splenic endothelial cells, splenic fibroblasts, splenocytes, and thick ascending limb cells
  • the cell cart be a cell used for producing a viral particle, e,g., a producer cell.
  • the cell can be a cell which has been transduced, infected, transfected or transformed with a viral vector described herein.
  • a cell is referred to as “transduced”, “infected”; “transfected” or “transformed” dependent on the means used for administration, introduction or insertion of heterologous DNA (he., the viral vector) into the cell,
  • compositions comprising a variant capsid polypeptide described herein or a polynucleotide encoding same and one or more pharmaceutically acceptable diluent, carrier, or excipient.
  • the composition can comprise a viral particle comprising a variant capsid polypeptide described herein.
  • the composition can comprise a cell, wherein the cell comprises a variant capsid polypeptide described herein or a polynucleotide encoding same.
  • the variant capsid polypeptide or a polynucleotide encoding same can be combined with pharmaceutically- acceptable carriers, diluents, and reagents useful in preparing a formulation that is generally safe, non-loxic, and desirable, and includes excipients that are acceptable for primate use.
  • excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.
  • carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Supplementary' active compounds can also be incorporated into the formulations.
  • Solutions or suspensions used for the formulations can include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid DTA); buffers such as acetates, citrates or phosphates; detergents such as Tween 20 to prevent aggregation; and compounds for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the pharmaceutical compositions are sterile.
  • the subject polynucleotide cassettes or gene delivery vectors comprising the subject polynucleotide cassette can be treated as appropriate for delivery to the eye.
  • compositions can further include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the composition is sterile and should be fluid to the extent that easy syringability exists. In certain embodiments, it is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be, e.g., a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can he achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the internal compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • a method of delivering a payload to a cell comprising contacting a cell with an AAV virus or viral particle comprising any of the viral capsid polypeptides described herein.
  • the contacting occurs in vitro. In one embodiment, the contacting occurs ex vivo.
  • contacting or “contact” as used herein in connection with contacting a cell with an AAV virus or viral particle includes subjecting the cell to an appropriate culture medium which comprises the AAV virus or viral particle. Where the cell is in vivo, “contacting” or “contact” includes administering the AAV virus or viral particle in a pharmaceutical composition to a subject via an appropriate administration route such that the virus or viral particle contacts the cell in vivo.
  • a method of delivering a payload in vivo to a target cell comprising administering an AAV virus or viral particle comprising any of the viral capsid polypeptides described herein to a subject.
  • in vivo is systemic delivery.
  • Exemplary target or host cells include blood cells (e.g., red blood cells, platelets, neutrophils, eosinophils, basophils, lymphocytes, or monocytes); heart cells (e.g., cardiomyocyte, endocardial cells, or cardiac smooth muscle cells); muscle cells; epithelial cells; endoderm cells; ectodermal cells; kidney cells (e.g., kidney glomerulus parietal cells, kidney glomerulus podocytes, kidney proximal tubule brush border cells, loop of Henle thin segment cells, thick ascending limb cells, kidney distal tubule cells, collecting duct principal cells, collecting duct intercalated cells, and interstitial kidney cells); liver cells (e.g., parenchymal cells, nun-parenchymal cells, sinusoidal endothelial cells, phagocytic Kupffer cells, hepatic stellate cells, and intrahepatic lymphocytes); lung cells (e.g., lung epithelial cells,
  • fa vivo delivery of the AAV virus or viral particle can be, for example, by injection, infusion, instillation, inhalation, or ingestion.
  • injection includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, intrastemal injection and infusion.
  • the AAV virus or viral can be administered as a single bolus or multiple boluses, as a continuous infusion, or a combination thereof.
  • the AAV virus or viral particle can be administration into the blood stream of the subject.
  • the dose of AAV virions or viral particles required to achieve a particular "therapeutic effect,” e.g., the units of dose in vector genomes/per kilogram of body weight (vg/kg), will vary based on several factors including, but not limited to: the route of AAV virus or viral particle administration, the level of gene expression required to achieve a therapeutic effect, the specific disease or disorder being treated, a host immune response to the AAV virus or viral particle, a host immune response to the gene expression product, and the stability of the gene product.
  • One of skill in the art can readily determine a AAV virus or viral particle dose range to treat a patient having a particular disease or disorder based on the aforementioned factors, as well as other factors that are well known in the art.
  • therapeutic effect is meant a level of expression of one or more transgenes in the AAV virus or viral particle sufficient to alter a component of a disease (or disorder) toward a desired outcome or clinical endpoint, such that a patient’s disease or disorder shows clinical improvement, often reflected by the amelioration of a clinical sign or symptom relating to the disease or disorder.
  • exemplary doses for achieving therapeutic effects are AAV virus or viral particle titers of at least about 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 transducing units or more. For example, from about 10 8 to about 10 13 transducing units. It is noted that more than one administration (e.g., two, three, four, or more administrations) can be employed to achieve desired (e.g. therapeutic) levels of gene expression.
  • Example 1 provides a comparison of viral particle titer and cell growth for different mutations in the AAV6 MAAP polypeptide.
  • AAV plasmids (CAT pRepCap, CAT pHelper,CAT pGOI (gene of interest), CAPEXCEL MAAP CAPSID variants)
  • Upstream AAV viral particles production Production cell lines were thawed and expanded at least 5 passages in their respective culture medium. One day prior transfection, cells were passaged to half the target cell densities required for the transfection. On the day of transfection, cells reached their target cell densities for transfection as per the manufacturer’s protocol depending on the transfection reagent used for the experiment. Transfections were setup in Erlenmeyer shake flasks. For transfection, a molar ratio of 1 : 1 : 1 of pRepCap : pHelper : pGOI plasmid DNAs were used and transfection was performed as per the manufacturer’s protocol.
  • Viral titer following production in two different production cell lines was compared with production of vectors with Control AAV6 MAAP (CAPSID-STD-AAV6 (STD)). Analysis was performed for viral genome by ddPCR, and viral particles by ELISA. See Tables 2, 3 and 4. As can be seen in Tables 2 and 3, the variant capsids provided for an approximately 4 to 5-fold increase in titer as compared to control. Furthermore, the % of full viral particles was similar by Analytical Ultracentrifugation (AUC) between the mutant and wild-type particles demonstrating that a similar particle quality was maintained.
  • AUC Analytical Ultracentrifugation
  • This example provides data on production of AAV particles of additional serotypes with mutations in the MAAP sequence.
  • the production of viral particles with the mutant MAAP sequences was compared to the production of viral particles with wild-type MAAP sequences.
  • a process flow diagram for viral particle production is provided as FIG. 1.
  • AAV plasmids (CAT pRepCap, CAT pHelper,CAT pGOI (gene of interest), CAPEXCEL MAAP CAPSID mutants)
  • POROSTM CaptureSelectTM AAVX Affinity Resin (Cat# A36740, Thermofisher) • 10 % Lysis Buffer (10% Tween 20, 500 mM Tris Base, 20 mM MgC12, pH 8.0 ⁇ 0.1)
  • AUC analytical ultracentrifuge
  • hcDNA host cell DNA
  • TCID50 infectivity assays TCID50 infectivity assays.
  • Table 5 provides a summary of the mutations that were evaluated in this example.
  • Table 5 List of CAPEXCEL Mutations for various AAV serotypes
  • Table 7b AAV6 variant downstream summary data of capsid purity.
  • Table 10a AAV5 downstream summary data.
  • Table 14a AAV8 variant downstream summary data.

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Abstract

La présente invention concerne des génomes de VAA, des vecteurs et des acides nucléiques contenant des séquences de protéines capsidiques de variants qui permettent un titre accru, ainsi qu'une encapsidation améliorée pendant la production de particules de vecteur de VAA recombinant. Les titres ont été amélioré plusieurs fois, sans augmentation similaire de l'ADNhcA encapsidé résiduel, ainsi que pour maintenir un pourcentage de capside complet. De plus, l'expression et les rapports de protéines virales restent inchangés avec les variants.
PCT/US2024/035946 2023-06-30 2024-06-28 Vecteurs viraux adéno-associés améliorés Ceased WO2025006829A1 (fr)

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