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WO2008124015A1 - Procédés de purification de virions de virus adéno-associés - Google Patents

Procédés de purification de virions de virus adéno-associés Download PDF

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Publication number
WO2008124015A1
WO2008124015A1 PCT/US2008/004312 US2008004312W WO2008124015A1 WO 2008124015 A1 WO2008124015 A1 WO 2008124015A1 US 2008004312 W US2008004312 W US 2008004312W WO 2008124015 A1 WO2008124015 A1 WO 2008124015A1
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Prior art keywords
aav
virion
affinity peptide
substitution
fold
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David V. Schaffer
James T. Koerber
Andrew G. Stapleton
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Bio Rad Laboratories Inc
University of California Berkeley
University of California San Diego UCSD
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Bio Rad Laboratories Inc
University of California Berkeley
University of California San Diego UCSD
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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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • 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
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • 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

Definitions

  • Adeno-associated virus is nonpathogenic human parvovirus that requires a helper virus to replicate.
  • the single-stranded 4.6-kb DNA genome of AAV contains two open reading frames (ORFs): rep, which encodes a set of four proteins (Rep78, Rep68, Rep52, and Rep40) essential for replication of the viral genome, and cap, which encodes the structural proteins (VP 1-3).
  • rep which encodes a set of four proteins (Rep78, Rep68, Rep52, and Rep40) essential for replication of the viral genome
  • cap which encodes the structural proteins (VP 1-3).
  • VP 1-3 self-assemble into the virus' icosahedral capsid, 25 nm in diameter, into which the genome is inserted. The capsid then escorts the viral genome from the point of entry into tissue to the final viral destination in a target cell nucleus.
  • Methods for purifying AAV include precipitation followed by 2-3 rounds of ultracentrifugation through an isopycnic cesium chloride (CsCl) gradient; chromatographic methods, such as affinity chromatography and ion exchange chromatography.
  • CsCl cesium chloride
  • chromatographic methods such as affinity chromatography and ion exchange chromatography.
  • heparin and sialic acid affinity resins have been used.
  • not all AAV serotypes can bind to existing heparin or sialic acid affinity resins.
  • the present invention provides methods for purifying adeno-associated virus (AAV) virions, including recombinant AAV virions, generally involving binding of an AAV virion comprising a variant capsid protein comprising a purification peptide to a binding moiety that binds the purification peptide.
  • AAV adeno-associated virus
  • the present invention further provides nucleic acids comprising nucleotide sequences encoding the variant capsid proteins; as well as recombinant AAV virions comprising the variant capsid proteins.
  • Figure IA depicts a map of an AAV VP3 monomer; and Figure IB depicts amino acid sequences of wild-type (WT) AA V2, His6 AA V2 clones, and a His6 AAV8 clone.
  • Figures 2 A and 2B depict Ni-NTA purification of His6 AAV mutants.
  • Figures 3A-C depict in vitro characterization of Hi6 AAV gene delivery properties.
  • Figures 4A and 4B depict in vivo comparison of GFP-expressing vectors with either wild-type (WT) AA V2 or His6 AA V2 capsid.
  • Figures 5A-F depict amino acid sequences of a subtilisin prodomain and variants of same.
  • Figure 6 depicts a subtilisin (mature protein) amino acid sequence.
  • Figure 7 depicts amino acid sequences of variants of a subtilisin polypeptide.
  • Figure 8 depicts the amino acid sequence of an AAV VP2 capsid-subtilisin fusion protein, and the amino acid sequence of an AAV VP2 capsid protein.
  • Figure 9 depicts a nucleotide sequence encoding a subtilisin prodomain.
  • Figure 10 depicts an amino acid sequence of an AA V2 VPl capsid protein.
  • Figure 11 depicts viral production titer from parent AA V2 and an AA V2 variant
  • FIG. 12 depicts infectious elution profile of parent AA V2 and PaIAA V2 from a subtilisin affinity column.
  • affinity peptide high affinity peptide
  • metal ion affinity peptide are used interchangeably herein to refer to a histidine-rich peptide that binds to a metal ion.
  • metal ion refers to any metal ion for which the affinity peptide has affinity and that can be used for purification or immobilization of a fusion protein.
  • metal ions include, but are not limited to, Ni +2 , Co +2 , Fe +3 , Al +3 , Zn +2 and Cu +2 .
  • hard metal ion refers to a metal ion that shows a binding preference for oxygen. Hard metal ions include Fe 3+ , Ca 2+ , and Al 3+ .
  • soft metal ion refers to a metal ion that shows a binding preference of sulfur.
  • Soft metal ions include Cu + , Hg 2+ , and Ag + .
  • intermediate metal ion refers to a metal ion that coordinates nitrogen, oxygen, and sulfur.
  • Intermediate metal ions include Cu 2+ , Ni 2+ , Zn 2+ , and Co 2+ .
  • a "vector” as used herein refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide and which can be used to mediate delivery of the polynucleotide to a cell.
  • Illustrative vectors include, for example, plasmids, viral vectors, liposomes, and other gene delivery vehicles.
  • AAV is an abbreviation for adeno-associated virus, and may be used to refer to the virus itself or derivatives thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise.
  • the abbreviation “rAAV” refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or "rAAV vector”).
  • AAV includes any of a variety of AAV serotypes, including, but not limited to, AAV type 1 (AAV-I), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AA V-4), AAV type 5 (AAV-5), AAV type 6 (AA V-6), AAV type 7 (AA V-7), AAV type 8 (AA V-8), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV.
  • Primary AAV refers to AAV that infect primates
  • non-primate AAV refers to AAV that infect non-primate mammals
  • biovine AAV refers to AAV that infect bovine mammals, etc.
  • rAAV vector refers to an AAV vector comprising a polynucleotide sequence not of AAV origin (i.e., a polynucleotide heterologous to AAV), typically a sequence of interest for the genetic transformation of a cell.
  • the heterologous polynucleotide is flanked by at least one, and generally by two AAV inverted terminal repeat sequences (ITRs).
  • ITRs AAV inverted terminal repeat sequences
  • An "AAV virus” or “AAV viral particle” or “rAAV vector particle” refers to a viral particle composed of at least one AAV capsid protein (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 an "rAAV vector particle” or simply an "rAAV vector". Thus, production of rAAV particle necessarily includes production of rAAV vector, as such a vector is contained within an rAAV particle.
  • Packaging refers to a series of intracellular events that result in the assembly and encapsidation of an AAV particle.
  • AAV "rep” and “cap” genes refer to polynucleotide sequences encoding replication and encapsidation proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes.”
  • a "helper virus” for AAV refers to a virus that allows AAV (e.g. wild-type AAV) to be replicated and packaged by a mammalian cell.
  • a variety of such helper viruses for AAV are known in the art, including adenoviruses, herpesviruses and poxviruses such as vaccinia.
  • the adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C is most commonly used.
  • Numerous adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the ATCC.
  • Viruses of the herpes family include, for example, herpes simplex viruses (HSV) and Epstein- Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV); which are also available from depositories such as ATCC.
  • HSV herpes simplex viruses
  • EBV Epstein- Barr viruses
  • CMV cytomegaloviruses
  • PRV pseudorabies viruses
  • Helper virus function(s) refers to function(s) encoded in a helper virus genome which allow AAV replication and packaging (in conjunction with other requirements for replication and packaging described herein). As described herein, "helper virus function” may be provided in a number of ways, including by providing helper virus or providing, for example, polynucleotide sequences encoding the requisite function(s) to a producer cell in trans.
  • infectious virus or viral particle is one that comprises a polynucleotide component which it is capable of delivering into a cell for which the viral species is trophic. The term does not necessarily imply any replication capacity of the virus. Assays for counting infectious viral particles are described elsewhere in this disclosure and in the art. Viral infectivity can be expressed as the P:I ratio, or the ratio of total viral particles to infective viral particles.
  • a "replication-competent" virus e.g. a replication-competent AAV refers to a phenotypically wild-type virus 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).
  • replication competence generally requires the presence of functional AAV packaging genes.
  • rAAV vectors as described herein are replication-incompetent in mammalian cells (especially in human cells) by virtue of the lack of one or more AAV packaging genes.
  • rAAV vector preparations as described herein are those which contain few if any replication competent AAV (rcAAV, also referred to as RCA) (e.g., less than about 1 rcAAV per 10 2 rAAV particles, less than about 1 rcAAV per 10 4 rAAV particles, less than about 1 rcAAV per 10 8 rAAV particles, less than about 1 rcAAV per 10 12 rAAV particles, or no rcAAV).
  • rcAAV also referred to as RCA
  • polynucleotide refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • polynucleotide refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
  • a "small interfering” or “short interfering RNA” or siRNA is a RNA duplex of nucleotides that is targeted to a gene interest (a "target gene”).
  • An “RNA duplex” refers to the structure formed by the complementary pairing between two regions of a RNA molecule.
  • siRNA is "targeted” to a gene in that the nucleotide sequence of the duplex portion of the siRNA is complementary to a nucleotide sequence of the targeted gene.
  • the length of the duplex of siRNAs is less than 30 nucleotides.
  • the duplex can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 nucleotides in length.
  • the length of the duplex is 19-25 nucleotides in length.
  • the RNA duplex portion of the siRNA can be part of a hairpin structure.
  • the hairpin structure may contain a loop portion positioned between the two sequences that form the duplex.
  • the loop can vary in length. In some embodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides in length.
  • the hairpin structure can also contain 3' or 5' overhang portions. In some embodiments, the overhang is a 3' or a 5' overhang 0, 1, 2, 3, 4 or 5 nucleotides in length.
  • Recombinant as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature.
  • a recombinant virus is a viral particle comprising a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct.
  • control element or "control sequence” is a nucleotide sequence involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature.
  • Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers.
  • a promoter is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region usually located downstream (in the 3' direction) from the promoter.
  • “Operatively linked” or “operably linked” refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a promoter is operatively linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained.
  • An "expression vector” is a vector comprising a region which encodes a polypeptide of interest, and is used for effecting the expression of the protein in an intended target cell.
  • An expression vector also comprises control elements operatively linked to the encoding region to facilitate expression of the protein in the target.
  • the combination of control elements and a gene or genes to which they are operably linked for expression is sometimes referred to as an "expression cassette,” a large number of which are known and available in the art or can be readily constructed from components that are available in the art.
  • Heterologous means derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared.
  • a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous polynucleotide.
  • a promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linked is a heterologous promoter.
  • an rAAV that includes a heterologous nucleic acid encoding a heterologous gene product is an rAAV that includes a nucleic acid not normally included in a naturally-occurring, wild-type AAV, and the encoded heterologous gene product is a gene product not normally encoded by a naturally-occurring, wild-type AAV.
  • genetic alteration and “genetic modification” (and grammatical variants thereof), are used interchangeably herein to refer to a process wherein a genetic element (e.g., a polynucleotide) is introduced into a cell other than by mitosis or meiosis.
  • a genetic element e.g., a polynucleotide
  • the element may be heterologous to the cell, or it may be an additional copy or improved version of an element already present in the cell.
  • Genetic alteration may be effected, for example, by transfecting a cell with a recombinant plasmid or other polynucleotide through any process known in the art, such as electroporation, calcium phosphate precipitation, or contacting with a polynucleotide- liposome complex. Genetic alteration may also be effected, for example, by transduction or infection with a DNA or RNA virus or viral vector. Generally, the genetic element is introduced into a chromosome or mini-chromosome in the cell; but any alteration that changes the phenotype and/or genotype of the cell and its progeny is included in this term.
  • a cell is said to be “stably” altered, transduced, genetically modified, or transformed with a genetic sequence if the sequence is available to perform its function during extended culture of the cell in vitro.
  • a cell is "heritably” altered (genetically modified) in that a genetic alteration is introduced which is also inheritable by progeny of the altered cell.
  • polypeptide refers to polymers of amino acids of any length.
  • the terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component.
  • Polypeptides such as “CFTR,” “p53,” “EPO” and the like, when discussed in the context of delivering a gene product to a mammalian subject, and compositions therefor, refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, that retains the desired biochemical function of the intact protein.
  • references to CFTR, p53, EPO genes, and other such genes for use in delivery of a gene product to a mammalian subject include polynucleotides encoding the intact polypeptide or any fragment or genetically engineered derivative possessing the desired biochemical function.
  • An "isolated" plasmid, nucleic acid, vector, virus, host cell, or other substance refers to a preparation of the substance devoid of at least some of the other components that may also be present where the substance or a similar substance naturally occurs or is initially prepared from.
  • an isolated substance may be prepared by using a purification technique to enrich it from a source mixture. Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured in relation to a second, potentially interfering substance present in the source mixture. Increasing enrichments of the embodiments of this invention are increasingly more isolated.
  • An isolated plasmid, nucleic acid, vector, virus, host cell, or other substance is in some embodiments purified, e.g., from about 80% to about 90% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99%, or more, pure.
  • the terms "individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, human and non-human primates, including simians and humans; mammalian sport animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).
  • mammalian sport animals e.g., horses
  • mammalian farm animals e.g., sheep, goats, etc.
  • mammalian pets dogs, cats, etc.
  • rodents e.g., mice, rats, etc.
  • the present invention provides methods for purifying AAV virions, including recombinant AAV virions, from a composition comprising an AAV virion.
  • the methods generally involve binding of an AAV virion comprising a variant capsid protein to a moiety that binds a purification peptide, where the variant capsid protein comprises the purification peptide. Binding of the AAV virion to the binding moiety allows separation of the AAV virion from undesired molecules that may be present in a preparation comprising the AAV virion.
  • a subject purification method provides for highly-purified, infectious AAV virion particles.
  • the present invention further provides nucleic acids comprising nucleotide sequences encoding a variant AAV capsid protein, e.g., a variant AAV capsid protein comprising a purification peptide.
  • the present invention further provides a recombinant AAV virion comprising a variant capsid protein.
  • a recombinant AAV virion can comprise a recombinant AAV vector comprising a heterologous nucleic acid, e.g., a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product of interest, such as a protein that provides a detectable signal, a therapeutic protein, or an interfering nucleic acid.
  • the present invention provides methods for purifying AAV virions, including recombinant AAV virions, from a composition comprising an AAV virion.
  • the methods generally involve contacting a composition comprising an AAV virion with a binding moiety, where the AAV virion comprises a variant ("recombinant") capsid protein comprising a heterologous purification peptide (e.g., an affinity peptide), and where the purification peptide (e.g., the affinity peptide) binds to the binding moiety, forming a bound AAV virion; and collecting the AAV virion.
  • a variant recombinant
  • a subject purification method provides for infectious, highly purified AAV virions, e.g., the collected virions are at least 90% pure, at least 95% pure, at least 98% pure, at least 99% pure, or greater than 99% pure. "Infectious" AAV virions are capable of infecting at least one cell type.
  • a subject purification method provides for high yield of purified, infectious AAV virions.
  • the recovery of the AAV virions is at least about 10%, at least about 15%, at least about 20%, at least about 25%, 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or more.
  • the binding moiety is immobilized on an insoluble support.
  • the nature of the insoluble support can depend, in part, on the nature of the binding moiety.
  • Suitable insoluble supports include, but are not limited to, cross-linked dextrans, polystyrenes, nylon, agarose, cellulose, silica, ceramic, poly(sytrenedivinyl)benzene, and polyacrylamides.
  • the insoluble support can be in any of a variety of forms, including, e.g., a bead, a membrane, a filter, and the like.
  • the insoluble support is in the form of a bead or other particle retained in a column.
  • the affinity peptide is a heterologous peptide, e.g., a peptide not normally found in an
  • affinity peptides include, but are not limited to, metal ion affinity peptides, and protease substrate peptides.
  • Affinity peptides can be synthetic or recombinant, e.g., the affinity peptides have amino acid sequences not normally found in nature.
  • Affinity peptides can have a length of from about 5 amino acids to about 150 amino acids, e.g. from about 5 amino acids to about 10 amino acids, from about 10 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to about 25 amino acids, from about 25 amino acids to about 30 amino acids, from about 30 amino acids to about 40 amino acids, from about 40 amino acids to about 50 amino acids, from about 50 amino acids to about 60 amino acids, from about 60 amino acids to about 70 amino acids, from about 70 amino acids to about 80 amino acids, from about 80 amino acids to about 90 amino acids, from about 90 amino acids to about 100 amino acids, from about 100 amino acids to about 125 amino acids, or from about 125 amino acids to about 150 amino acids.
  • the affinity peptide is present in a fusion protein with an AAV capsid protein.
  • the affinity peptide will in some embodiments be fused at the amino terminus of an AAV capsid protein. In other embodiments, the affinity peptide will be fused at the carboxyl terminus of an AAV capsid protein. In still other embodiments, the affinity peptide will be present at an internal site within the AAV capsid protein.
  • An AAV capsid protein comprising one or more heterologous affinity peptides can be referred to herein as a "variant AAV capsid protein" a "recombinant AAV capsid protein” or "an affinity peptide-containing AAV capsid protein.”
  • a variant AAV capsid protein will in some embodiments comprise a single heterologous affinity peptide.
  • an affinity peptide-containing AAV virion comprising the variant capsid protein will comprise a single type of variant capsid protein, e.g., all of the variant capsid proteins in the affinity peptide-containing AAV virion will comprise an affinity peptide of the same amino acid sequence.
  • an affinity peptide-containing AAV virion comprising a variant capsid protein having a single heterologous affinity peptide will comprise two or more different types of variant capsid proteins.
  • an affinity peptide-containing AAV virion comprising a variant capsid protein having a single heterologous affinity peptide will comprise a first variant AAV capsid protein having a first single affinity peptide; and at least a second variant AAV capsid protein having a second affinity peptide.
  • an affinity peptide-containing AAV virion comprising a variant capsid protein having a single heterologous affinity peptide will comprise a first variant AAV capsid protein having a first single affinity peptide that is a metal ion affinity peptide; and at least a second variant AAV capsid protein having a second affinity peptide that is a protease substrate.
  • a variant AAV capsid protein will comprise multiple affinity peptides, e.g., two, three, four, five, or more heterologous affinity peptides, which can be in tandem or dispersed within the AAV capsid protein.
  • affinity peptide-containing AAV capsid protein comprises two or more affinity peptides
  • each of the two or more affinity peptides has the same amino acid sequence.
  • affinity peptide-containing AAV capsid protein comprises two or more affinity peptides
  • the two or more affinity peptides can include affinity peptides differing in amino acid sequence from one another.
  • an AAV capsid protein can include a first affinity peptide that is a metal ion affinity peptide; and a second affinity peptide that is a protease substrate.
  • An AAV virion that includes an affinity peptide in a capsid protein of the virion can be referred to herein as an "affinity peptide-containing AAV virion"; and the corresponding AAV virion without an affinity peptide in a capsid protein, e.g., an AAV virion that is identical to the affinity peptide-containing AAV virion but for the affinity peptide, can be referred to herein as the "parent AAV virion.”
  • addition of the affinity peptide to the amino terminus, the carboxyl terminus, or at an internal site of an AAV capsid protein in a virion does not significantly reduce infectivity of the affinity peptide-containing AAV virion for a cell, compared to the infectivity of a parent AAV virion.
  • the presence of the affinity peptide in the affinity peptide-containing AAV virion reduces the infectivity less than about 10-fold, less than about 5-fold, less than 2-fold, less than about 90%, less than 75%, less than about 50%, or less than about 25%, compared to the infectivity of the parent AAV virion for the same cell.
  • an affinity peptide can be present at an internal site of an AAV capsid protein.
  • an affinity peptide can be present at a site that is one, two, three, four, five, six, seven, eight, nine, 10, or more, amino acids carboxyl-terminal to the amino terminus of an AAV capsid protein; or an affinity peptide can be present at a site that is one, two, three, four, five, six, seven, eight, nine, 10, or more, amino acids amino-terminal to the carboxyl terminus of an AAV capsid protein.
  • each of the capsid proteins present in the affinity peptide- containing AAV virion comprises an affinity peptide.
  • the proportion of affinity peptide-containing capsid proteins present in an affinity peptide-containing AAV virion ranges from 10% to about 99%, e.g., from about 10% to about 20%, from about 20% to about 30%, from about 30% to about 40%, from about 40% to about 50%, from about 50% to about 60%, from about 60% to about 70%, from about 70% to about 80%, from about 80% to about 90%, or from about 90% to about 99%, where the remainder of the capsid proteins present in the virion do not include an affinity peptide.
  • An affinity peptide-containing AAV capsid protein can be an AAV VP 1 , VP2, or VP3 polypeptide comprising a heterologous affinity peptide.
  • an affinity peptide can be present at any of a number of locations.
  • an affinity peptide can be inserted in loop III of an AAV capsid protein.
  • an affinity peptide can be inserted in loop IV of an AAV capsid protein (e.g, at a site from amino acid 583-590).
  • the affinity peptide can be inserted between amino acids 585 and 586, between amino acids 586 and 587, or between amino acids 587 and 588 of an AAV VP2 capsid protein.
  • the purified AAV virion comprises an affinity peptide- containing capsid protein.
  • the affinity peptide is a metal ion affinity peptide
  • the metal ion affinity peptide is present in the purified AAV virion.
  • the affinity peptide is cleaved during the purification process, such that the purified AAV virion does not include the affinity peptide, e.g., the affinity peptide, if present in the purified AAV virion, is present in fewer than about 50%, fewer than about 40%, fewer than about 30%, fewer than about 25%, fewer than about 20%, fewer than about 10%, or fewer than about 5% of the capsid polypeptides present in the AAV virion.
  • a composition (also referred to herein as a "sample") comprising an AAV virion comprising an affinity peptide-AAV capsid fusion protein (an “affinity peptide-AAV virion”) can be any of a variety of samples comprising an affinity peptide-AAV virion in an unpurified state.
  • the sample is a lysate of cells that produce an affinity peptide- AAV virion.
  • a cell lysate is processed, to generate a processed cell lysate, where "processed” can include one or more of centrifugation to remove cellular debris; and the like.
  • the affinity peptide is a metal ion affinity peptide
  • the binding moiety comprises a metal ion.
  • Metal ion affinity peptides, metal ions, insoluble supports comprising a metal ion, and conditions for use of same are described below.
  • Metal ion affinity peptides are known in the art, and any metal ion binding peptide, including any known metal ion binding peptide, can be used in a subject recombinant envelope protein. See, e.g., Itakura, et al., Science 198:1056-63 (1977); Germino, et al., Proc. Natl. Acad. ScL USA 80:6848-52 (1983); Nilsson et al., Nucleic Acids Res. 13:1151-62 (1985); Smith et al., Gene 32:321-27 (1984); Dobeli, et al., U.S. Pat. No.
  • Metal ion affinity peptides include peptides that bind to a metal ion with an affinity of from about 10 3 M '1 to about 10 9 M “1 , e.g., from about 10 3 M “1 to about 10 4 M '1 , from about 10 4 M “1 to about 10 5 M “1 , from about 10 5 M “1 to about 10 6 M “1 , from about 10 6 M “1 to about 10 7 M ' ', from about 10 7 M '1 to about 10 8 M “1 , or from about 10 8 M “1 to about 10 9 M '1 , or greater than 10 9 M '1 .
  • Metal ion affinity peptides can contain from about 30% to about 50%, from about 33% to about 45%, from about 35% to about 43%, or from about 37% to about 40%, histidine residues.
  • a metal ion affinity peptide 18 amino acids in length can contain 6, 7, or 8 histidine residues.
  • Metal ion affinity peptides can be from about 6 to about 30, from about 7 to about 25, from about 8 to about 20, from about 9 to about 18, from about 10 to about 16, or from about 12 to about 14 amino acids in length.
  • a metal ion affinity peptide comprises the amino acid sequence
  • the metal ion affinity peptide comprises an amino acid sequence as set forth in U.S. Patent Publication No. 2002/0164718.
  • a metal ion affinity peptide comprises a peptide of the formula:
  • each of Xi and X 2 is independently selected from Leu, He, VaI,
  • each of Xi and X 2 is independently selected from Leu, VaI, Asn, and He.
  • each of X 3 , X 4 , X 5 is independently selected from Lys, Arg, Asp, and GIu.
  • each of X 3 , X 4 , X 5 is independently selected from Lys and GIu.
  • each X 6 is independently selected from Leu, He, VaI, Ala, GIy, Asn, and GIn. In other embodiments, each X 6 is independently selected from Ala and Asn.
  • the affinity peptide has the amino acid sequence NH 2 -HiS-LeU-IIe-HiS-ASn-VaI-HiS-LyS-GIu-GIu-HiS-AIa-HiS-AIa-HiS-ASn-COOH (SEQ ID NO: 17).
  • the affinity peptide comprises the sequence (His-Asp-Asp) 6 (SEQ ID NO: 18).
  • the affinity peptide comprises the sequence (HiS-GIu-GIu) 6 (SEQ ID NO: 19).
  • the affinity peptide comprises the sequence (His-Asp-Glu) 6 (SEQ ID NO:20).
  • the affinity peptide comprises the sequence (His- Glu-Asp) 6 (SEQ ID NO:21).
  • the binding moiety comprises a metal ion.
  • the metal ion is immobilized on an insoluble support.
  • Suitable insoluble supports include metal ion chelating resins. Any of a variety of metal ion chelating resins can be used, including commercially available metal ion chelating resins.
  • a metal ion chelating resin can include a carrier matrix, optionally a spacer, and a moiety that comprises a metal ion, e.g., an organic ligand that immobilizes a metal ion.
  • Suitable carrier matrices include, but are not limited to, cross-linked dextrans, polystyrenes, nylon, agarose, and polyacrylamides.
  • Metal chelating ligands include, but are not limited to, carboxymethyl aspartate (CM-Asp); iminodiacetic acid (IDA); tris(carboxymethyl)ethylene diamine (TED); nitrilo triacetic acid (NTA).
  • CM-Asp carboxymethyl aspartate
  • IDA iminodiacetic acid
  • TED tris(carboxymethyl)ethylene diamine
  • NTA nitrilo triacetic acid
  • a nickel-NTA resin is described in, e.g., U.S. Patent Nos. 4,877,830 and 5,047,513.
  • the metal ion chelating resin can be provided in the form of a chromatography column, e.g., wherein the resin is packed in a column.
  • the resin can also comprise a matrix that is a solid support of any shape or configuration.
  • the term "resin,” as used herein, refers to a resin comprising a matrix in any form, e.g., a bead, a sheet, a well, and the like.
  • the affinity peptide is a metal ion affinity peptide
  • the binding moiety comprises a metal ion, where the metal ion is immobilized on an insoluble support, e.g., a metal ion chelating resin.
  • an AAV virion comprising a capsid protein comprising a metal ion affinity peptide also referred to herein as a "metal ion affinity AAV virion"
  • metal ion affinity resin also referred to herein as a "metal ion affinity AAV virion
  • the sample comprising a metal ion affinity AAV virion is applied to the metal ion affinity resin, and the resin is equilibrated with a solution.
  • “Conditions for binding” include a condition of the sample being applied, as well as any equilibration conditions. Those skilled in the art can readily determine appropriate conditions for binding of a metal ion affinity AAV virion in a sample to a metal ion affinity resin.
  • the pH conditions suitable for applying a sample comprising a metal ion affinity AAV virion (an AAV virion comprising a capsid protein comprising a metal ion affinity peptide) to a metal ion affinity resin range from about 3.5 to about 11, from about 4.0 to about 10.0, from about 4.5 to about 9.5, from about 5.0 to about 9.0, from about 5.5 to about 8.5, from about 6.0 to about 8.0, or from about 6.5 to about 7.5.
  • Temperature conditions suitable for applying a sample comprising a metal ion affinity peptide- AAV virion to a metal ion affinity resin range from about 15°C to about 40 0 C, from about 2O 0 C to about 37 0 C, or from about 22°C to about 25°C.
  • Salt conditions generally range from about 0.01 M to about 1 M, e.g., from about 0.01 M to about 0.05 M, from about 0.05 M to about 0.1 M, from about 0.1 M to about 0.25 M. from about 0.25 M to about 0.5 M, or from about 0.5 M to about 1 M.
  • non-ionic detergents e.g., Tween 20TM, and the like
  • chaotropic agents and denaturants e.g., urea, and guanidinium HCl
  • buffers e.g., Tris-based buffers, borate -based buffers, phosphate-based buffers, imidazole, HEPES, PIPES, MOPS, PIPES, TES, and the like.
  • washing steps may be included, to remove undesired components.
  • a washing step may be performed after a metal ion affinity AAV virion is immobilized on a resin.
  • the composition and temperature of a washing solution may vary according to the desired result. The optimal composition and temperature of a washing solution can readily be determined by those skilled in the art, based on known properties of the immobilized metal ion affinity AAV virion. Wash solutions may comprise a buffer, and may further comprise additional components, as necessary, including, but not limited to, a detergent. Eluting
  • the immobilized metal ion affinity AAV virion can be eluted using a pH gradient; addition of a competitor, e.g., an organic acid, phosphates; addition of a displacer such as imidazole; and the like.
  • a competitor e.g., an organic acid, phosphates
  • a displacer such as imidazole
  • a heterologous affinity peptide is a substrate for a protease.
  • the binding moiety is a protease that specifically binds to an affinity peptide present in an affinity peptide-containing AAV virion, and that, when activated, cleaves the affinity peptide from the bound AAV virion.
  • the affinity peptide is a protease substrate, where the protease substrate is a substrate for subtilisin.
  • suitable subtilisin substrates include a subtilisin prodomain, or a variant or a fragment of a subtilisin prodomain that is specifically bound by and, under certain conditions, is cleaved by subtilisin from a fusion protein comprising the prodomain.
  • a subtilisin prodomain has a binding affinity for a subtilisin of from about 10 3 M “1 to about 10 9 M “1 , e.g., from about 10 3 M '1 to about 10 4 M '1 , from about 10 4 M '1 to about 10 5 M “1 , from about 10 5 M “1 to about 10 6 M “1 , from about 10 6 M '1 to about 10 7 M “ ', from about 10 7 M “1 to about 10 8 M '1 , or from about 10 8 M “1 to about 10 9 M “1 , or greater than 10 9 M "1 .
  • the affinity of the subtilisin prodomain for subtilisin is from about 10 8 M “1 to about 10 9 M “1 , from about 10 9 M “1 to about 10 10 M '1 , from about 10 10 M “1 to about 10 11 M “1 , or from about 10 12 M “1 , or greater than 10 12 M “1 .
  • a subtilisin prodomain can comprise the amino acid sequence depicted in Figure 5A and designated SEQ ID NO:1), or a variant or fragment thereof, where the variant or fragment is specifically bound by and, under certain conditions, is cleaved by subtilisin from a fusion protein comprising the prodomain.
  • a suitable subtilisin prodomain comprises an amino acid sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% amino acid identity to a contiguous stretch of from about 10 amino acids (aa) to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 40 aa, from about 40 aa to about 50 aa, from about 50 aa to about 60 aa, from about 60 aa to about 70 aa, or from about 70 aa to about 77 aa, of the amino acid sequence depicted in Figure 5 A (SEQ ID NO:1).
  • Suitable subtilisin prodomains are described in, e.g., U.S. Patent Publication No. 2006/0134740; and Ruan et al. (2004) Biochemistry 43:145
  • a suitable subtilisin prodomain has a length of from about 9 amino acids to about 77 amino acids, e.g., from about 9 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 40 aa, from about 40 aa to about 50 aa, from about 50 aa to about 60 aa, from about 60 aa to about 70 aa, or from about 70 aa to about 80 aa.
  • a suitable subtilisin prodomain comprises the amino acid sequence EEDKL(F/Y)(Q/K)(S/A)(M/L/Y) (SEQ ID NO:22). In some embodiments, a suitable subtilisin prodomain comprises the amino acid sequence
  • a suitable subtilisin prodomain comprises, compared to the amino acid sequence set forth in SEQ ID NO:1, from one to about 20, or from one to ten, amino acid substitutions and/or deletions and/or insertions.
  • a suitable subtilisin prodomain comprises, compared to the amino acid sequence set forth in SEQ ID NO:1, one or more of the following amino acid sequence changes: 1) a substitution of QTMSTM (SEQ ID NO:23; amino acids 16-21) with SGIK (SEQ ID NO:24); 2) an A23C substitution; 3) a K27Q substitution; 4) a V37L substitution; 5) a Q40C substitution; 6) an H72K substitution; 7) an H75K substitution; 8) an A74F substitution; 9) an H75K substitution; and 10) a Y77M substitution.
  • a suitable subtilisin prodomain comprises, compared to the amino acid sequence set forth in SEQ ID NO:1, the following changes: 1) a substitution of QTMSTM (SEQ ID NO:23; amino acids 16-21) with SGIK (SEQ ID NO:24); 2) an A23C substitution; 3) a K27Q substitution; 4) a V37L substitution; and 5) a Q40C substitution.
  • a suitable subtilisin prodomain comprises, compared to the amino acid sequence set forth in SEQ ID NO:1, the following changes: 1) a substitution of QTMSTM (SEQ ID NO:23; amino acids 16-21) with SGIK (SEQ ID NO:24); 2) an A23C substitution; 3) a K27Q substitution; 4) a V37L substitution; 5) a Q40C substitution; and 6) substitutions of one or more of amino acids 74-77 (e.g., substitution of A74-H75-A76-Y77 with one of: i) FKAM (SEQ ID NO:25); ii) FKAF SEQ ID NO:26); iii) FKAY (SEQ ID NO:27); or iv) FKAL (SEQ ID NO:28).
  • a suitable subtilisin prodomain comprises, compared to the amino acid sequence set forth in SEQ ID NO:1, the following changes: 1) a substitution of QTMSTM (SEQ ID NO:23; amino acids 16- 21) with SGIK (SEQ ID NO:24); 2) an A23C substitution; 3) a K27Q substitution; 4) a V37L substitution; 5) a Q40C substitution; and 6) substitution of A74-H75-A76-Y77 with FKAL (SEQ ID NO:28).
  • a suitable subtilisin prodomain comprises, compared to the amino acid sequence set forth in SEQ ID NO:1, one or more of the amino acid substitutions depicted in bold in Figures 5B-5F.
  • a suitable subtilisin prodomain comprises an amino acid sequence as set forth in one of Figures 5B-5F.
  • subtilisin prodomain is fused to the amino terminus of an
  • an affinity peptide-containing AAV virion comprises a VP2 capsid fusion protein with a subtilisin prodomain at the amino terminus of the VP2 polypeptide.
  • the AAV is AA V2.
  • the AAV is AAV8.
  • the AAV is a serotype other than AA V2 or AAV8.
  • the binding moiety is a protease that specifically binds to the affinity peptide and, under certain conditions, cleaves the affinity peptide from an affinity peptide-containing AAV virion or the affinity peptide-containing AAV capsid protein.
  • a suitable protease catalyzes cleavage of the affinity peptide from an affinity peptide-containing AAV capsid protein comprising the affinity peptide; and does not cleave internally within the capsid protein itself.
  • a suitable protease specifically cleaves only at the junction between the affinity peptide and the capsid protein.
  • a suitable protease binds specifically to an affinity peptide present in an affinity peptide-AAV capsid fusion polypeptide, but does not substantially cleave the affinity peptide from the affinity peptide-AAV capsid fusion polypeptide except under certain conditions, e.g., "activation" conditions.
  • activation e.g., addition of an activator can trigger the cleavage of the affinity peptide from the affinity peptide-AAV capsid fusion polypeptide.
  • the binding moiety is a subtilisin polypeptide.
  • suitable subtilisin polypeptides include polypeptides having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98, or at least about 99% amino acid identity to a contiguous stretch of from about 50 amino acids to about 100 amino acids, from about 100 amino acids to about 150 amino acids, from about 150 amino acids to about 200 amino acids, or from about 200 amino acids to about 275 amino acids, of the amino acid sequence depicted in Figure 6 (SEQ ID NO:7).
  • Suitable subtilisin polypeptides are described in, e.g., U.S. Patent Publication No. 2006/0134740; and Ruan et al. (2004) Biochemistry 43: 14539.
  • a suitable subtilisin polypeptide is a variant subtilisin and comprises, compared to the amino acid sequence set forth in Figure 6 (SEQ ID NO:7), one or more of the following changes: 1) a Q2K substitution; 2) an S3C substitution; 3) a P5S substitution; 4) an S9A substitution; 5) an I31L substitution; 6) a K43N substitution; 7) an M50F substitution; 8) an A73L substitution; 9) deletion of amino acids 75-83; 10) an E156S substitution; 11) a G166S substitution; 12) a G169A substitution; 13) an S188P substitution; 14) a Q206C substitution; 15) an N212G substitution; 16) a Y217L substitution; 17) an N218S substitution; 18) a T254A substitution; 19) a S271E substitution; 20) a Y104A substitution; 21) a G128S substitution; 22) a D32A substitution; 23) a
  • a suitable subtilisin polypeptide is a variant subtilisin and comprises, compared to the amino acid sequence set forth in Figure 6 (SEQ ID NO:7), all of the following changes: 1) a Q2K substitution; 2) an S3C substitution; 3) a P5S substitution; 4) an S9A substitution; 5) an I31L substitution; 6) a K43N substitution; 7) an M50F substitution; 8) an A73L substitution; 9) deletion of amino acids 75-83; 10) an E156S substitution; 1 1) a G166S substitution; 12) a G169A substitution; 13) an S188P substitution; 14) a Q206C substitution; 15) an N212G substitution; 16) a Y217L substitution; 17) an N218S substitution; 18) a T254A substitution; and 19) a S271E substitution.
  • the amino acid sequence such a variant is depicted in Figure 7 A (SEQ ID NO: 8), where the underlined residues 75-83
  • a suitable subtilisin polypeptide is a variant subtilisin and comprises, compared to the amino acid sequence set forth in Figure 6 (SEQ ID NO:7), all of the following changes: 1) a Q2K substitution; 2) an S3C substitution; 3) a P5S substitution; 4) an S9A substitution; 5) an 13 IL substitution; 6) a K43N substitution; 7) an M50F substitution; 8) an A73L substitution; 9) deletion of amino acids 75-83; 10) an E156S substitution; 11) a G166S substitution; 12) a G169A substitution; 13) an S188P substitution; 14) a Q206C substitution; 15) an N212G substitution; 16) a Y217L substitution; 17) an N218S substitution; 18) a T254A substitution; 19) a S271E substitution; 20) a Y104A substitution; and 21) a G128S substitution.
  • the amino acid sequence such a variant is depicted in Figure 6 (SEQ ID NO
  • a suitable subtilisin polypeptide is a variant subtilisin and comprises, compared to the amino acid sequence set forth in Figure 6 (SEQ ID NO:7), all of the following changes: 1) a Q2K substitution; 2) an S3C substitution; 3) a P5S substitution; 4) an S9A substitution; 5) an 13 IL substitution; 6) a K43N substitution; 7) an M50F substitution; 8) an A73L substitution; 9) deletion of amino acids 75-83; 10) an E156S substitution; 1 1) a G166S substitution; 12) a G169A substitution; 13) an S188P substitution; 14) a Q206C substitution; 15) an N212G substitution; 16) a Y217L substitution; 17) an N218S substitution; 18) a T254A substitution; 19) a S271E substitution; 20) a Y104A substitution; 21) a G128S substitution; and 22) a D32A substitution.
  • the amino acid sequence such
  • a suitable subtilisin polypeptide is a variant subtilisin and comprises, compared to the amino acid sequence set forth in Figure 6 (SEQ ID NO:7), all of the following changes: 1) a Q2K substitution; 2) an S3C substitution; 3) a P5S substitution; 4) an S9A substitution; 5) an 131 L substitution; 6) a K43N substitution; 7) an M50F substitution; 8) an A73L substitution; 9) deletion of amino acids 75-83; 10) an E156S substitution; 11) a G166S substitution; 12) a G169A substitution; 13) an S188P substitution; 14) a Q206C substitution; 15) an N212G substitution; 16) a Y217L substitution; 17) an N218S substitution; 18) a T254A substitution; 19) a S271E substitution; 20) a Y104A substitution; 21) a G128S substitution; and 22) a D32S substitution.
  • the amino acid sequence such
  • a suitable subtilisin polypeptide is a variant subtilisin and comprises, compared to the amino acid sequence set forth in Figure 6 (SEQ ID NO:7), all of the following changes: 1) a Q2K substitution; 2) an S3C substitution; 3) a P5S substitution; 4) an S9A substitution; 5) an 131 L substitution; 6) a K43N substitution; 7) an M50F substitution; 8) an A73L substitution; 9) deletion of amino acids 75-83; 10) an E156S substitution; 1 1) a G166S substitution; 12) a G169A substitution; 13) an S188P substitution; 14) a Q206C substitution; 15) an N212G substitution; 16) a Y217L substitution; 17) an N218S substitution; 18) a T254A substitution; 19) a S271E substitution; 20) a Y104A substitution; 21) a G128S substitution; and 22) a D32V substitution.
  • the amino acid sequence such
  • the subtilisin polypeptide is immobilized on an insoluble support. Any of a variety of standard chemical reactions can be carried out to immobilize a subtilisin polypeptide onto an insoluble support.
  • the insoluble support can include a moiety suitable for attachment of a protein, e.g., a moiety that allows for formation of an amide bond between the moiety and the subtilisin polypeptide. Immobilization of a subtilisin polypeptide onto an insoluble support can be carried out using a carbodiimide amidation reagent.
  • a suitable amidation reagent is N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride (EDC), which can be supplemented by N- hydroxysuccinimide (NHS) to facilitate the reaction.
  • EDC N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride
  • NHS N- hydroxysuccinimide
  • conjugation can be effected by reductive animation with a suitable reducing reagent, such as sodium cyanoborohydride.
  • cross-linking agents that are suitable for use include, e.g., l,l-bis(diazoacetyl)- 2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4- azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'- dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8- octane.
  • Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light, and can also be used.
  • reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and reactive substrates are employed for protein immobilization.
  • a subtilisin polypeptide can be immobilized directly, or via a linker, where suitable linkers can be of a flexible nature, although other chemical linkages are not excluded.
  • suitable linkers include peptides of between about 6 and about 40 amino acids in length, or between about 6 and about 25 amino acids in length. These linkers are generally produced by using synthetic, linker-encoding oligonucleotides to couple the proteins. Binding
  • a composition (also referred to as a "sample") comprising an affinity peptide- containing AAV virion (e.g., an AAV virion comprising a subtilisin prodomain-AAV capsid fusion protein; or a subtilisin prodomain-AAV virion) is contacted with an immobilized subtilisin polypeptide under conditions that promote binding of the subtilisin prodomain-AAV virion to the immobilized substilisin, but do not promote cleavage of the subtilisin prodomain from a subtilisin prodomain-AAV capsid protein present in the subtilisin prodomain-AAV virion.
  • AAV virion e.g., an AAV virion comprising a subtilisin prodomain-AAV capsid fusion protein; or a subtilisin prodomain-AAV virion
  • a sample comprising a subtilisin prodomain-AAV virion is applied to the immobilized subtilisin, and the resin is equilibrated with a solution.
  • “Conditions for binding” include a condition of the sample being applied, as well as any equilibration conditions. Those skilled in the art can readily determine appropriate conditions for binding of a subtilisin prodomain-AAV virion in a sample to an immobilized subtilisin.
  • AAV virion to an immobilized subtilisin range from about 5.5 to about 8.5, from about 6.0 to about 8.0, from about 7.0 to about 8.0, from about 7.5 to about 8.0, or from about 7.0 to about 7.5.
  • Temperature conditions suitable for applying a sample comprising a subtilisin prodomain- AAV virion to an immobilized subtilisin range from about 15°C to about 37°C, from about 2O 0 C to about 35 0 C, or from about 22°C to about 25 0 C.
  • Salt conditions generally range from about 0.01 M to about 1 M, e.g., from about 0.01 M to about 0.05 M, from about 0.05 M to about 0.1 M, from about 0.1 M to about 0.25 M. from about 0.25 M to about 0.5 M, or from about 0.5 M to about 1 M.
  • non-ionic detergents e.g., Tween 20TM, and the like
  • chaotropic agents and denaturants e.g., urea, and guanidinium HCl
  • buffers e.g., Tris-based buffers, borate -based buffers, phosphate-based buffers, imidazole, HEPES, PIPES, MOPS, PIPES, TES, and the like.
  • equilibration conditions and binding conditions include 0.1 M potassium phosphate buffer, pH 7.0-8.0 (e.g., pH 7.2), and a temperature of about 20°C to about 25°C.
  • equilibration conditions and binding conditions include 100 mM sodium phosphate buffer, 20 mM Tris-HCl, pH 7.0-8.0 (e.g., pH 7.2), and a temperature of about 20°C to about 25°C. Washinfi conditions
  • washing steps may be included, to remove undesired (e.g., unbound) components.
  • a washing step may be performed after a subtilisin prodomain-AAV virion is bound to an immobilized subtilisin polypeptide.
  • the composition and temperature of a washing solution may vary according to the desired result. The optimal composition and temperature of a washing solution can readily be determined by those skilled in the art, based on known properties of the bound subtilisin prodomain-AAV virion.
  • Wash solutions may comprise a buffer, and may further comprise additional components, as necessary, including, but not limited to, a detergent. In some embodiments, the wash conditions will be the same as the binding conditions. Elution
  • subtilisin prodomain-AAV virion is released upon application of conditions that activate catalytic cleavage of a subtilisin prodomain from a subtilisin prodomain-AAV capsid fusion protein present in the subtilisin prodomain-AAV virion.
  • Conditions that activate catalytic cleavage of a subtilisin prodomain from a subtilisin prodomain-AAV capsid fusion protein present in the subtilisin prodomain-AAV virion include, but are not limited to, fluoride ions, chloride ions, and high pH (e.g., pH higher than about 8.0, higher than about 8.5, or higher than about 9.0).
  • Exemplary suitable activation conditions include, e.g., potassium fluoride in a concentration range of from about 10 mM to about 200 mM, e.g., from about 10 mM to about 50 mM, from about 50 mM to about 100 mM, from about 100 mM to about 150 mM, or from about 150 mM to about 200 mM; potassium fluoride in a concentration range of from about 50 mM to about 1 M, e.g., from about 50 mM to about 100 mM, from about 100 mM to about 500 mM, or from about 500 mM to about 1 M.
  • activation conditions can include, e.g., 0.1 M KF, 0.1 M potassium phosphate, pH 7.0-8.0 (e.g., pH 7.5), and a temperature of from about 20°C to about 25°C.
  • AAV capsid proteins can include, e.g., 0.1 M KF, 0.1 M potassium phosphate, pH 7.0-8.0 (e.g., pH 7.5), and a temperature of from about 20°C to about 25°C.
  • an affinity peptide-containing AAV virion comprises a fusion protein comprising an AAV capsid protein and a heterologous affinity peptide.
  • Amino acid sequences of AAV capsid proteins are known in the art. See, e.g., GenBank Accession No. YP_680427 (AA V-2 VP2); GenBank Accession No. YP_680426 (AA V-2 VPl); GenBank Accession No. YP_068409 (AAV-5 VPl); GenBank Accession No. YP_077178 (AAV-7 VPl); and GenBank Accession No. YP_077180 (AAV-8 VPl).
  • the amino acid sequence of the AAV capsid protein present in the affinity peptide- AAV capsid fusion protein is a wild-type amino acid sequence, e.g., an amino acid sequence naturally found in an AAV capsid protein.
  • the amino acid sequence of the AAV capsid protein present in the affinity peptide- AAV capsid fusion protein has at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% amino acid sequence identity to the amino acid sequence depicted in Figure 10.
  • the amino acid sequence of the AAV capsid protein present in the affinity peptide-AAV capsid fusion protein has at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% amino acid sequence identity to the AA V2 VP2 amino acid sequence depicted in Figure 8 ("AAV2 VP2"; SEQ ID NO: 14).
  • the amino acid sequence of the AAV capsid protein comprises from about 1 to about 20 amino acid substitutions compared to the amino acid sequence depicted in Figure 8 ("AAV2 VP2"; SEQ ID NO: 14) or Figure 10 (SEQ ID NO: 16), e.g., the amino acid sequence of the AAV capsid protein comprises from about 1 to about 5, from about 5 to about 10, from about 10 to about 15, or from about 15 to about 20, amino acid substitutions compared to the amino acid sequence depicted in Figure 8 or Figure 10.
  • the amino acid substitution(s) provide for one or more of: 1 ) increased capacity of an AAV virion comprising the capsid protein to cross an endothelial cell layer, compared to an AAV virion comprising a capsid protein without the substitution(s); 2) increased infectivity of a non-permissive cell type (e.g., a hepatocyte, a stem cell, a lung epithelial cell) compared to an AAV virion comprising a capsid protein without the substitution(s); and 3) decreased binding to neutralizing antibody, e.g., neutralizing antibody present in the serum of a mammal, where decreased binding to neutralizing antibody results in increased infectivity due to avoidance of neutralizing antibody-mediated reduction of infectivity.
  • a non-permissive cell type e.g., a hepatocyte, a stem cell, a lung epithelial cell
  • neutralizing antibody e.g., neutralizing antibody present in the serum of a mammal, where decreased binding to
  • the present invention provides nucleic acids (e.g., recombinant nucleic acids) comprising a nucleotide sequence encoding a variant AAV capsid protein, where the variant AAV capsid protein comprises a heterologous affinity peptide. Also provided are expression vectors comprising the nucleic acids; isolated host cells comprising the nucleic acids; and isolated host cells comprising the expression vectors.
  • nucleic acids e.g., recombinant nucleic acids
  • expression vectors comprising the nucleic acids; isolated host cells comprising the nucleic acids; and isolated host cells comprising the expression vectors.
  • variant AAV-2 capsid proteins or variant AAV-8 capsid proteins are exemplified.
  • AAV-2 or AAV-8 herein is in no way meant to be limiting.
  • Those skilled in the art can readily adapt the methods as discussed herein to generate capsid mutants of other AAV, including, e.g., AAV-3, AAV-4, AAV-5, etc.
  • a subject nucleic acid comprises a nucleotide sequence encoding at least one of VPl,
  • a subject nucleic acid comprises a nucleotide sequence encoding a fusion protein comprising an AAV capsid protein and an affinity peptide, where such fusion proteins are described above.
  • Nucleotide sequences encoding AAV capsid proteins are known in the art. See, e.g.,
  • GenBank Accession No. NC_001401 (e.g., nucleotides 2203-4410 of the sequence provided in NC_001401, which is an AAV-2 complete genome sequence); GenBank Accession No. NC_006152 (e.g., nucleotides 2207-4381 of the sequence provided in NC_006152, which is an AAV-5 complete genome sequence); GenBank Accession No. NC_006260 (e.g., nucleotides 2222-4435 of the sequence provided in NC_006260, which is an AAV-7 complete genome sequence); and GenBank Accession No. NC_006261 (e.g., nucleotides 2121-4337 of the sequence provided in NC_006261, which is an AAV-8 complete genome sequence).
  • Nucleotide sequences encoding a subtilisin prodomain as described above include nucleotide sequences that have at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% identity to the nucleotide sequence depicted in Figure 9 (e.g., nucleotides 183-413 of the sequence set forth in GenBank Accession No. XOOl 65; SEQ ID NO: 15).
  • Nucleotide sequences encoding a metal ion affinity peptide are readily derived from the amino acid sequences of such peptides.
  • the nucleic acid is present in an expression vector that provides for expression of the encoded affinity peptide- AA V capsid fusion protein, and production of the fusion protein in an appropriate host cell, e.g., a mammalian cell.
  • the nucleic acid further comprises nucleotide sequences encoding an AAV rep protein.
  • the present invention further provides host cells, e.g., isolated host cells, comprising a subject nucleic acid.
  • a subject host cell can be an isolated cell, e.g., a cell in in vitro culture.
  • a subject host cell is useful for producing an affinity peptide- AAV capsid fusion protein; and can also be used for producing an affinity peptide- AAV virion, as described below.
  • a subject host cell is used to produce an affinity peptide- AA V virion, it is referred to as a "packaging cell.”
  • a subject host cell is stably genetically modified with a subject nucleic acid.
  • a subject host cell is transiently genetically modified with a subject nucleic acid.
  • a subject nucleic acid is introduced stably or transiently into a host cell, using established techniques, including, but not limited to, electroporation, calcium phosphate precipitation, liposome-mediated transfection, and the like.
  • a subject nucleic acid will generally further include a selectable marker, e.g., any of several well- known selectable markers such as neomycin resistance, and the like.
  • a subject host cell is generated by introducing a subject nucleic acid into any of a variety of cells, e.g., mammalian cells, including, e.g., murine cells, and primate cells (e.g., human cells).
  • mammalian cells include, but are not limited to, primary cells and cell lines, where suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No.
  • Vero cells NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCLlO), PC12 cells (ATCC No. CRLl 721), COS cells, COS-7 cells (ATCC No. CRLl 651), RATl cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRLl 573), HLHepG2 cells, and the like.
  • a subject host cell includes, in addition to a nucleic acid comprising a nucleotide sequence encoding a mutant capsid protein, a nucleic acid that comprises a nucleotide sequence encoding one or more AAV rep proteins.
  • a subject host cell further comprises an rAAV vector, as described below.
  • an rAAV virion is generated using a subject host cell.
  • a subject variant capsid protein can be incorporated into an AAV virion that comprises a heterologous nucleic acid that provides for production of a heterologous gene product (e.g., a heterologous nucleic acid or a heterologous protein).
  • a subject recombinant AAV virion (“rAAV virion”) comprises a subject variant capsid protein, and includes a heterologous nucleic acid that comprises a nucleotide sequence encoding a heterologous gene product.
  • the present invention provides rAAV virions that comprise a subject variant capsid protein, as described above; and a heterologous nucleic acid.
  • a subject rAAV virion is infectious (e.g., can infect one or more cells or cell types in an individual), and is thus useful for introducing a gene product into an individual.
  • a subject rAAV virion comprises an affinity peptide-AAV capsid fusion protein that comprises a metal ion affinity peptide.
  • the metal ion affinity peptide is present in the rAAV virion in both unpurii ⁇ ed (e.g., cell lysate) and purified states.
  • a subject rAAV virion comprises an affinity peptide-AAV capsid fusion protein that comprises a protease substrate, e.g., a subtilisin prodomain.
  • the protease substrate is present in the rAAV virion in the unpurified state, but is cleaved from the rAAV virion during the purification process, and is not present in the purified rAAV virion. Additional properties
  • an affinity peptide-AAV capsid fusion protein present in an AAV virion can comprises one or more amino acid substitution(s) that provide for one or more of: 1 ) increased capacity of an AAV virion comprising the capsid protein to cross an endothelial cell layer, compared to an AAV virion comprising a capsid protein without the substitution(s); 2) increased infectivity of a non-permissive cell type (e.g., a hepatocyte, a stem cell, a lung epithelial cell) compared to an AAV virion comprising a capsid protein without the substitution(s); and 3) decreased binding to neutralizing antibody, e.g., neutralizing antibody present in the serum of a mammal, where decreased binding to neutralizing antibody results in increased infectivity due to avoidance of neutralizing antibody- mediated reduction of infectivity.
  • a non-permissive cell type e.g., a hepatocyte, a stem cell, a lung epithelial cell
  • a subject rAAV virion exhibits increased resistance to neutralizing antibodies compared to wild-type AAV or AAV comprising a wild-typo capsid protein.
  • a subject rAAV virion has from about 10-fold to about 10,000- fold greater resistance to neutralizing antibodies than wt AAV, e.g., a subject rAAV virion has from about 10-fold to about 25-fold, from about 25-fold to about 50-fold, from about 50-fold to about 75-fold, from about 75-fold to about 100-fold, from about 100-fold to about 150-fold, from about 150-fold to about 200-fold, from about 200-fold to about 250-fold, from about 250- fold to about 300-fold, at least about 350-fold, at least about 400-fold, from about 400-fold to about 450-fold, from about 450-fold to about 500-fold, from about 500-fold to about 550-fold, from about 550-fold to about 600-fold, from about 600
  • a subject rAAV virion exhibits decreased binding to a neutralizing antibody that binds a wild-type AAV capsid protein.
  • a subject mutant rAAV virion exhibits from about 10-fold to about 10,000-fold reduced binding to a neutralizing antibody that binds a wild-type AAV capsid protein.
  • a subject mutant rAAV virion exhibits from about 10-fold to about 25-fold, from about 25-fold to about 50-fold, from about 50-fold to about 75-fold, from about 75-fold to about 100-fold, from about 100-fold to about 150-fold, from about 150-fold to about 200-fold, from about 200-fold to about 250-fold, from about 250-fold to about 300-fold, at least about 350-fold, at least about 400-fold, from about 400-fold to about 450-fold, from about 450-fold to about 500-fold, from about 500-fold to about 550-fold, from about 550-fold to about 600-fold, from about 600-fold to about 700-fold, from about 700-fold to about 800-fold, from about 800-fold to about 900- fold, from about 900-fold to about 1000-fold, from about 1, 000-fold to about 2,000-fold, from about 2,000-fold to about 3,000-fold, from about 3,000-fold to about 4,000-fold, from about 4,000-fold to about
  • an anti-AAV neutralizing antibody binds to a subject rAAV virion with an affinity of less than about 10 "7 M, less than about 5 x 10 "6 M, less than about 10 ⁇ 6 M, less than about 5 x 10 ⁇ 5 M, less than about 10 "5 M, less than about 10 "4 M, or lower.
  • a subject rAAV virion that exhibits reduced binding to neutralizing antibodies has increased residence time in the body, compared to the residence time of an AAV virion comprising wild-type capsid proteins.
  • a subject rAAV virion has at least about 25%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 50-fold, or more, increased residence time in vivo compared to the residence time of an AAV virion comprising wild-type capsid proteins.
  • mutant rAAV virion exhibits reduced binding to a neutralizing antibody and/or increased resistance to neutralizing antibody can be determined using any known assay, including the assay described in the Example.
  • mutant rAAV virion is contacted with a permissive cell type, e.g., 293 cells, in the presence of neutralizing antibody.
  • a control sample contains the cells, mutant rAAV virion, and no neutralizing antibody.
  • the cells are contacted with adenovirus, and rAAV particles are detected. The level of rAAV particles is compared to the amount of rAAV particles that are generated in the absence of neutralizing antibody.
  • a subject rAAV virion exhibits increased ability to infect a cell that is relatively refractory to AAV infection (e.g., a non-permissive cell).
  • a subject mutant AAV exhibits at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 4-fold, at least about 10-fold, at least about 20-fold, or at least about 50- fold, or more, greater infectivity of a non- permissive cell than a wild-type AAV or an rAAV virion comprising wild-type capsid protein.
  • Examples of cells that are relatively refractory to AAV infection include, but are not limited to stem cells, hepatocytes, and lung epithelial cells.
  • stem cell is used herein to refer to a mammalian cell that has the ability both to self-renew, and to generate differentiated progeny (see, e.g., Morrison et al. (1997) Cell 88:287-298).
  • stem cells also have one or more of the following properties: an ability to undergo asynchronous, or symmetric replication, that is where the two daughter cells after division can have different phenotypes; extensive self-renewal capacity; capacity for existence in a mitotically quiescent form; and clonal regeneration of all the tissue in which they exist, for example the ability of hematopoietic stem cells to reconstitute all hematopoietic lineages.
  • Progenitor cells differ from stem cells in that they typically do not have the extensive self- renewal capacity, and often can only regenerate a subset of the lineages in the tissue from which they derive, for example only lymphoid, or erythroid lineages in a hematopoietic setting.
  • Stem cells may be characterized by both the presence of markers associated with specific epitopes identified by antibodies and the absence of certain markers as identified by the lack of binding of specific antibodies. Stem cells may also be identified by functional assays both in vitro and in vivo, particularly assays relating to the ability of stem cells to give rise to multiple differentiated progeny.
  • Stem cells of interest include hematopoietic stem cells and progenitor cells derived therefrom (U.S. Pat. No. 5,061,620); neural crest stem cells (see Morrison et al. (1999) Cell 96:737-749); adult neural stem cells and neural progenitor cells; embryonic stem cells; mesenchymal stem cells; mesodermal stem cells; induced pluripotent stem (iPS) cells; etc.
  • Other hematopoietic "progenitor" cells of interest include cells dedicated to lymphoid lineages, e.g. immature T cell and B cell populations.
  • a subject rAAV virion exhibits increased ability to cross an endothelial cell layer.
  • a subject rAAV virion exhibits at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2- fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, or at least about 50-fold increase in ability to cross an endothelial cell layer.
  • Whether a given rAAV virion exhibits an increased ability to cross an endothelial cell layer can be determined experimentally using well-known systems. Generation of subject rAAV virions
  • a host or "producer” cell for rAAV vector replication and packaging.
  • a producer cell usually a mammalian host cell
  • the first component is a recombinant adeno-associated viral (rAAV) vector genome (or "rAAV pro-vector”) that can be replicated and packaged into vector particles by the host packaging cell.
  • rAAV adeno-associated viral
  • the rAAV pro-vector will normally comprise a heterologous polynucleotide (or "transgene"), with which it is desired to genetically alter another cell in the context of gene therapy (since the packaging of such a transgene into rAAV vector particles can be effectively used to deliver the transgene to a variety of mammalian cells).
  • the transgene is generally flanked by two AAV inverted terminal repeats (ITRs) which comprise sequences that are recognized during excision, replication and packaging of the AAV vector, as well as during integration of the vector into a host cell genome.
  • ITRs AAV inverted terminal repeats
  • a second component is a helper virus that can provide helper functions for AAV replication.
  • helper virus can provide helper functions for AAV replication.
  • helper viruses can also be used as is known in the art.
  • the requisite helper virus functions can be isolated genetically from a helper virus and the encoding genes can be used to provide helper virus functions in trans.
  • the AAV vector elements and the helper virus (or helper virus functions) can be introduced into the host cell either simultaneously or sequentially in any order.
  • the final components for AAV production to be provided in the producer cell are:
  • AAV packaging genes such as AAV rep and cap genes that provide replication and encapsidation proteins, respectively.
  • AAV packaging genes such as AAV rep and cap genes that provide replication and encapsidation proteins, respectively.
  • AAV packaging genes can be provided (including rep-cap cassettes and separate rep and/or cap cassettes in which the rep and/or cap genes can be left under the control of the native promoters or operably linked to heterologous promoters.
  • AAV packaging genes can be introduced either transiently or stably into the host packaging cell, as is known in the art and described in more detail below.
  • Variant AAV capsid proteins e.g., affinity peptide- AAV capsid fusion proteins
  • a subject rAAV virion including the heterologous DNA of interest (where
  • heterologous DNA of interest is also referred to herein as “heterologous nucleic acid”
  • the methods generally involve the steps of (1) introducing a subject rAAV vector into a host cell; (2) introducing an AAV helper construct into the host cell, where the helper construct includes AAV coding regions capable of being expressed in the host cell to complement AAV helper functions missing from the AAV vector; (3) introducing one or more helper viruses and/or accessory function vectors into the host cell, wherein the helper virus and/or accessory function vectors provide accessory functions capable of supporting efficient recombinant AAV (“rAAV”) virion production in the host cell; and (4) culturing the host cell to produce rAAV virions.
  • AAV efficient recombinant AAV
  • AAV expression vector, AAV helper construct and the helper virus or accessory function vector(s) can be introduced into the host cell, either simultaneously or serially, using standard transfection techniques.
  • AAV expression vectors are constructed using known techniques to at least provide as operatively linked components in the direction of transcription, control elements including a transcriptional initiation region, the DNA of interest and a transcriptional termination region.
  • the control elements are selected to be functional in a mammalian muscle cell.
  • the resulting construct which contains the operatively linked components is bounded (5 1 and 3') with functional AAV ITR sequences.
  • AAV ITR regions are known. See, e.g., Kotin, R. M.
  • AAV ITRs used in the vectors of the invention need not have a wild-type nucleotide sequence, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides. Additionally, AAV ITRs may be derived from any of several AAV serotypes, including without limitation, AAV-I, AAV-2, AAV-3, AAV-4, AAV-5, AAV-7, etc.
  • 5' and 3' ITRs which flank a selected nucleotide sequence in an AAV expression vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i.e., to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the DNA molecule into the recipient cell genome when AAV Rep gene products are present in the cell.
  • ITRs allow replication of the vector sequence in the presence of an appropriate mixture of Rep proteins. ITRs also allow for the incorporation of the vector sequence into the capsid to generate an AAV particle.
  • a suitable heterologous DNA molecule for use in a subject rAAV vector will generally be less than about 5 kilobases (kb) in size and will include, for example, a gene (a nucleotide sequence) that encodes a protein that is defective or missing from a recipient subject; a gene that encodes a protein having a desired biological or therapeutic effect (e.g., an antibacterial, antiviral or antitumor function); a nucleotide sequence that encodes an RNA that inhibits or reduces production of a deleterious or otherwise undesired protein; a nucleotide sequence that encodes an antigenic protein; or a nucleotide sequence that encodes an RNA that inhibits or reduces production of a protein.
  • a gene a nucleotide sequence that encodes a protein that is defective or missing from a recipient subject
  • a gene that encodes a protein having a desired biological or therapeutic effect e.g., an antibacterial, antiviral or antitumor
  • Suitable heterologous nucleic acids include, but are not limited to, those encoding proteins used for the treatment of endocrine, metabolic, hematologic, cardiovascular, neurologic, musculoskeletal, urologic, pulmonary and immune disorders, including such disorders as inflammatory diseases, autoimmune, chronic and infectious diseases, such as acquired immunodeficiency syndrome (AIDS), cancer, hypercholestemia, insulin disorders such as diabetes, growth disorders, various blood disorders including various anemias, thalassemias and hemophilia; genetic defects such as cystic fibrosis, Gaucher's Disease, Hurler's Disease, adenosine deaminase (ADA) deficiency, emphysema, or the like.
  • AIDS acquired immunodeficiency syndrome
  • ADA adenosine deaminase
  • Suitable heterologous nucleic acids include, but are not limited to, those encoding any of a variety of proteins, including, but not limited to: a cytokine, a chemokine, an angiogenesis-inducing polypeptide, an apoptosis-inducing polypeptide, an angiogenesis- inhibiting polypeptide, an antibody, a growth factor, a polypeptide that induced cell differentiation, a polypeptide that increases cell proliferation, a colony stimulating factor, a blood clotting factor, and the like.
  • proteins including, but not limited to: a cytokine, a chemokine, an angiogenesis-inducing polypeptide, an apoptosis-inducing polypeptide, an angiogenesis- inhibiting polypeptide, an antibody, a growth factor, a polypeptide that induced cell differentiation, a polypeptide that increases cell proliferation, a colony stimulating factor, a blood clotting factor, and the like.
  • Suitable heterologous nucleic acids include, but are not limited to, those encoding any of a variety of proteins, including, but not limited to: an interferon (e.g., IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ ; IFN- ⁇ ); an insulin (e.g., Novolin, Humulin, Humalog, Lantus, Ultralente, etc.); an erythropoietin ("EPO"; e.g., Procrit®, Eprex®, or Epogen® (epoetin- ⁇ ); Aranesp® (darbepoietin- ⁇ ); NeoRecormon®, Epogin® (epoetin- ⁇ ); and the like); an antibody (e.g., a monoclonal antibody) (e.g., Rituxan® (rituximab); Remicade® (infliximab); Herceptin® (trastuzumab); HumiraTM (a) (e
  • a subject rAAV that comprises a heterologous nucleic acid that encodes an antigenic protein is suitable for stimulating an immune response to the antigenic protein in a mammalian host.
  • the antigenic protein is derived from an autoantigen, an allergen, a tumor-associated antigen, a pathogenic virus, a pathogenic bacterium, a pathogenic protozoan, a pathogenic helminth, or any other pathogenic organism that infects a mammalian host.
  • nucleic acid encoding an antigenic protein derived from includes nucleic acids encoding wild- type antigenic proteins, e.g., a nucleic acid isolated from a pathogenic virus that encodes a viral protein; synthetic nucleic acids generated in the laboratory that encode antigenic proteins that are identical in amino acid sequence to a naturally-occurring antigenic protein; synthetic nucleic acids generated in the laboratory that encode antigenic proteins that differ in amino acid sequence (e.g., by from one amino acid to about 15 amino acids) from a naturally- occurring antigenic protein, but that nonetheless induce an immune response to the corresponding naturally-occurring antigenic protein; synthetic nucleic acids generated in the laboratory that encode fragments of antigenic proteins (e.g., fragments of from about 5 amino acids to about 50 amino acids, which fragments comprises one or more antigenic epitopes), which fragments induce an immune response to the corresponding naturally-occurring antigenic protein; etc.
  • wild- type antigenic proteins e.g., a nucleic acid isolated from a path
  • an antigenic protein "derived from" an autoantigen, an allergen, a tumor- associated antigen, a pathogenic virus, a pathogenic bacterium, a pathogenic protozoan, a pathogenic helminth, or any other pathogenic organism that infects a mammalian host includes proteins that are identical in amino acid sequence to a naturally-occurring antigenic protein, and proteins that differ in amino acid sequence (e.g., by from one amino acid to about 15 amino acids) from a naturally-occurring antigenic protein, but that nonetheless induce an immune response to the corresponding naturally-occurring antigenic protein; and fragments of antigenic proteins (e.g., fragments of from about 5 amino acids to about 50 amino acids, which fragments comprises one or more antigenic epitopes), which fragments induce an immune response to the corresponding naturally-occurring antigenic protein.
  • proteins that are identical in amino acid sequence to a naturally-occurring antigenic protein and proteins that differ in amino acid sequence (e.g., by from one amino acid to
  • an immune response to an antigenic protein encoded by a subject rAAV will stimulate a protective immune response to a pathogenic organism that displays the antigenic protein or antigenic epitope (or a protein or an epitope that is cross- reactive with the rAAV-encoded antigenic protein or antigenic epitopes) in the mammalian host.
  • a cytotoxic T lymphocyte (CTL) response to the rAAV-encoded antigenic protein will be induced in the mammalian host.
  • CTL cytotoxic T lymphocyte
  • a humoral response to the rAAV-encoded antigenic protein will be induced in the mammalian host, such that antibodies specific to the antigenic protein are generated.
  • a THl immune response to the rAAV-encoded antigenic protein will be induced in the mammalian host.
  • Suitable antigenic proteins include tumor-associated antigens, viral antigens, bacterial antigens, and protozoal antigens; and antigenic fragments thereof.
  • the antigenic protein is derived from an intracellular pathogen.
  • the antigenic protein is a self-antigen.
  • the antigenic protein is an allergen.
  • Tumor-specific antigens include, but are not limited to, any of the various MAGEs
  • MAGE 1 e.g., GenBank Accession No. M77481
  • MAGE 2 e.g., GenBank Accession No. U03735
  • MAGE 3 MAGE 4, etc.
  • any of the various tyrosinases mutant ras
  • mutant p53 e.g., GenBank Accession No. X54156 and AA494311
  • p97 melanoma antigen e.g., GenBank Accession No. M12154.
  • tumor-specific antigens include the Ras peptide and p53 peptide associated with advanced cancers, the HPV 16/18 and E6/E7 antigens associated with cervical cancers, MUCIl-KLH antigen associated with breast carcinoma (e.g., GenBank Accession No. J03651), CEA (carcinoembryonic antigen) associated with colorectal cancer (e.g., GenBank Accession No. X98311), gplOO (e.g., GenBank Accession No. S73003) or MARTl antigens associated with melanoma, and the PSA antigen associated with prostate cancer (e.g., GenBank Accession No. X14810).
  • GenBank Accession No. J03651 GenBank Accession No. J03651
  • CEA carcinoembryonic antigen
  • gplOO e.g., GenBank Accession No. S73003
  • the p53 gene sequence is known (See e.g., Harris et al. (1986) MoI. Cell. Biol., 6:4650-4656) and is deposited with GenBank under Accession No. M 14694.
  • the present invention can be used as immunotherapeutics for cancers including, but not limited to, cervical, breast, colorectal, prostate, lung cancers, and for melanomas.
  • Viral antigens are derived from known causative agents responsible for diseases including, but not limited to, measles, mumps, rubella, poliomyelitis, hepatitis A, B (e.g., GenBank Accession No. E02707), and C (e.g., GenBank Accession No. E06890), as well as other hepatitis viruses, influenza, adenovirus (e.g., types 4 and 7), rabies (e.g., GenBank Accession No. M34678), yellow fever, Japanese encephalitis (e.g., GenBank Accession No. E07883), dengue (e.g., GenBank Accession No. M24444), hantavirus, and human immunodeficiency virus (e.g., GenBank Accession No. Ul 8552).
  • diseases including, but not limited to, measles, mumps, rubella, poliomyelitis, hepatitis A, B (e.g., Gen
  • Suitable bacterial and parasitic antigens include those derived from known causative agents responsible for diseases including, but not limited to, diphtheria, pertussis (e.g., GenBank Accession No. M35274), tetanus (e.g., GenBank Accession No. M64353), tuberculosis, bacterial and fungal pneumonias (e.g., Haemophilus influenzae, Pneumocystis carinii, etc.), cholera, typhoid, plague, shigellosis, salmonellosis (e.g., GenBank Accession No. L03833), Legionnaire's Disease, Lyme disease (e.g., GenBank Accession No.
  • malaria e.g., GenBank Accession No. X53832
  • hookworm e.g., GenBank Accession No. M27807
  • schistosomiasis e.g., GenBank Accession No. L08198
  • trypanosomiasis e.g., GenBank Accession No. L08198
  • trypanosomiasis e.g., GenBank Accession No. L08198
  • trypanosomiasis e.g., GenBank Accession No. L08198
  • trypanosomiasis e.g., GenBank Accession No. M33641
  • amoebiasis e.g., GenBank Accession No. J03266
  • borreliosis e.g., GenBank Accession No. J03266
  • Suitable heterologous nucleic acids that encode heterologous gene products include non-translated RNAs, such as an antisense RNA, a ribozyme, an RNAi and an siRNA.
  • Interfering RNA (RNAi) fragments, particularly double-stranded (ds) RNAi, can be used to inhibit gene expression.
  • siRNA short interfering RNA
  • siRNA short interfering RNA
  • the level of expression product of a target gene is reduced by specific double stranded siRNA nucleotide sequences that are complementary to at least a 19-25 nucleotide long segment (e.g., a 20-21 nucleotide sequence) of the target gene transcript, including the 5' untranslated (UT) region, the ORF, or the 3' UT region.
  • short interfering RNAs are about 19-25 nt in length.
  • the siRNA can be encoded by a nucleic acid sequence, and the nucleic acid sequence can also include a promoter.
  • the nucleic acid sequence can also include a polyadenylation signal.
  • the polyadenylation signal is a synthetic minimal polyadelylation signal.
  • Target genes include any gene encoding a target gene product (RNA or protein) that is deleterious (e.g., pathological); e.g., a target gene product that is malfunctioning, a target gene product that, when produced in a cell, has a pathological effect, etc.
  • Target gene products include, but are not limited to, huntingtin; hepatitis C virus; human immunodeficiency virus; amyloid precursor protein; tau; a protein that includes a polyglutamine repeat; a herpes virus (e.g., varicella zoster); any pathological virus; and the like.
  • rAAV that includes a heterologous nucleic acid encoding an siRNA is useful for treating a variety of disorders and conditions, including, but not limited to, neurodegenerative diseases, e.g., a trinucleotide-repeat disease, such as a disease associated with polyglutamine repeats, e.g., Huntington's disease , spinocerebellar ataxia, spinal and bulbar muscular atrophy (SBMA), dentatorubropallidoluysian atrophy (DRPLA), etc.; an acquired pathology (e.g., a disease or syndrome manifested by an abnormal physiological, biochemical, cellular, structural, or molecular biological state) such as a viral infection, e.g., hepatitis that occurs or may occur as a result of an HCV infection, acquired immunodeficiency syndrome, which occurs as a result of an HIV infection; and the like.
  • neurodegenerative diseases e.g., a trinucleotide-re
  • a heterologous nucleic acid encoding an siRNA is operably linked to a promoter.
  • Suitable promoters are known those skilled in the art and include the promoter of any protein-encoding gene, e.g., an endogenously regulated gene or a constitutively expressed gene.
  • the promoters of genes regulated by cellular physiological events e.g., heat shock, oxygen levels and/or carbon monoxide levels, e.g., in hypoxia, may be operably linked to an siRNA-encoding nucleic acid.
  • the selected heterologous nucleotide sequence such as EPO-encoding or nucleic acid of interest, is operably linked to control elements that direct the transcription or expression thereof in the nucleotide sequence in vivo.
  • control elements can comprise control sequences normally associated with the selected gene (e.g., endogenous cellular control elements).
  • heterologous control sequences can be employed.
  • Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes.
  • Examples include, but are not limited to, the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, an endogenous cellular promoter that is heterologous to the gene of interest, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like.
  • CMV cytomegalovirus
  • CMVIE CMV immediate early promoter region
  • RSV rous sarcoma virus
  • sequences derived from nonviral genes such as the murine metallothionein gene, will also find use herein.
  • Such promoter sequences are commercially available from, e.g., Stratagene (San Diego, Calif.).
  • cell type-specific or tissue-specific promoter will be operably linked to the heterologous nucleic acid encoding the heterologous gene product, such that the gene product is produced selectively or preferentially in a particular cell type(s) or tissue(s).
  • an inducible promoter will be operably linked to the heterologous nucleic acid.
  • muscle-specific and inducible promoters, enhancers and the like are useful for delivery of a gene product to a muscle cell.
  • control elements include, but are not limited to, those derived from the actin and myosin gene families, such as from the myoD gene family; the myocyte-specific enhancer binding factor MEF-2; control elements derived from the human skeletal actin gene and the cardiac actin gene; muscle creatine kinase sequence elements and the murine creatine kinase enhancer (mCK) element; control elements derived from the skeletal fast-twitch troponin C gene, the slow-twitch cardiac troponin C gene and the slow-twitch troponin I gene; hypoxia-inducible nuclear factors; steroid-inducible elements and promoters, such as the glucocorticoid response element (GRE); the fusion consensus element for RU486 induction; and elements that provide for tetracycline regulated gene expression.
  • GRE glucocorticoid
  • the AAV expression vector which harbors the DNA molecule of interest (the heterologous DNA) bounded by AAV ITRs can be constructed by directly inserting the selected sequence(s) into an AAV genome which has had the major AAV open reading frames ("ORFs") excised therefrom. Other portions of the AAV genome can also be deleted, so long as a sufficient portion of the ITRs remain to allow for replication and packaging functions.
  • ORFs major AAV open reading frames
  • Such constructs can be designed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941 ; International Publication Nos. WO 92/01070 (published Jan.
  • AAV ITRs can be excised from the viral genome or from an AAV vector containing the same and fused 5' and 3' of a selected nucleic acid construct that is present in another vector using standard ligation techniques, such as those described in Sambrook et al., supra.
  • ligations can be accomplished in 20 mM Tris-Cl pH 7.5, 10 mM MgCl 2 , 10 mM DTT, 33 ⁇ g/ml BSA, 10 mM-50 mM NaCl, and either 40 ⁇ M ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0 0 C to 16° C (for "sticky end” ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14° C (for "blunt end” ligation). Intermolecular "sticky end” ligations are usually performed at 30-100 ⁇ g/ml total DNA concentrations (5-100 nM total end concentration).
  • AAV vectors which contain ITRs have been described in, e.g., U.S. Pat. No. 5,139,941. In particular, several AAV vectors are described therein which are available from the American Type Culture Collection (“ATCC”) under Accession Numbers 53222, 53223, 53224, 53225 and 53226.
  • ATCC American Type Culture Collection
  • chimeric genes can be produced synthetically to include AAV ITR sequences arranged 5' and 3' of one or more selected nucleic acid sequences. Preferred codons for expression of the chimeric gene sequence in mammalian muscle cells can be used. The complete chimeric sequence is assembled from overlapping oligonucleotides prepared by standard methods. See, e.g., Edge, Nature (1981) 292:756; Nambair et al. Science (1984) 223:1299; Jay et al. J. Biol. Chem. (1984) 259:6311.
  • an AAV expression vector is introduced into a suitable host cell using known techniques, such as by transfection.
  • transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197.
  • Particularly suitable transfection methods include calcium phosphate co-precipitation (Graham et al. (1973) Virol.
  • suitable host cells for producing rAAV virions include microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of a heterologous DNA molecule.
  • the term includes the progeny of the original cell which has been transfected.
  • a "host cell” as used herein generally refers to a cell which has been transfected with an exogenous DNA sequence. Cells from the stable human cell line, 293 (readily available through, e.g., the American Type Culture Collection under Accession Number ATCC CRLl 573) are used in many embodiments.
  • the human cell line 293 is a human embryonic kidney cell line that has been transformed with adenovirus type-5 DNA fragments (Graham et al. (1977) J. Gen. Virol. 36:59), and expresses the adenoviral EIa and EIb genes (Aiello et al. (1979) Virology 94:460).
  • the 293 cell line is readily transfected, and provides a particularly convenient platform in which to produce rAAV virions. 2.
  • AAV helper functions are generally AAV-derived coding sequences which can be expressed to provide AAV gene products that, in turn, function in trans for productive AAV replication.
  • AAV helper functions are used herein to complement necessary AAV functions that are missing from the AAV expression vectors.
  • AAV helper functions include one, or both of the major AAV ORFs, namely the rep and cap coding regions, or functional homologues thereof.
  • the cap functions include one or more mutant capsid proteins, wherein at least one capsid protein comprises at least one mutation, as described above.
  • AAV rep coding region is meant the art-recognized region of the AAV genome which encodes the replication proteins Rep 78, Rep 68, Rep 52 and Rep 40. These Rep expression products have been shown to possess many functions, including recognition, binding and nicking of the AAV origin of DNA replication, DNA helicase activity and modulation of transcription from AAV (or other heterologous) promoters. The Rep expression products are collectively required for replicating the AAV genome.
  • HHV-6 human herpesvirus 6
  • AAV cap proteins include VPl , VP2, and VP3, where AAV capsid proteins are as described above.
  • an AAV capsid protein can be a fusion protein comprising an AAV capsid protein and an affinity peptide, as described above; and can further include one or more amino acid substitutions that provide for one or more of increased capacity to cross an endothelial cell layer, increased infectivity of a non-permissive cell, and decreased binding to a neutralizing antibody.
  • AAV helper functions are introduced into the host cell by transfecting the host cell with an AAV helper construct either prior to, or concurrently with, the transfection of the AAV expression vector.
  • AAV helper constructs are thus used to provide at least transient expression of AAV rep and/or cap genes to complement missing AAV functions that are necessary for productive AAV infection.
  • AAV helper constructs lack AAV ITRs and can neither replicate nor package themselves. These constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion.
  • a number of AAV helper constructs have been described, such as the commonly used plasmids pAAV/Ad and pIM29+45 which encode both Rep and Cap expression products.
  • Both AAV expression vectors and AAV helper constructs can be constructed to contain one or more optional selectable markers. Suitable markers include genes which confer antibiotic resistance or sensitivity to, impart color to, or change the antigenic characteristics of those cells which have been transfected with a nucleic acid construct containing the selectable marker when the cells are grown in an appropriate selective medium.
  • selectable marker genes that are useful in the practice of the invention include the hygromycin B resistance gene (encoding Aminoglycoside phosphotranferase (APH)) that allows selection in mammalian cells by conferring resistance to hygromycin; the neomycin phosphotranferase gene (encoding neomycin phosphotransferase) that allows selection in mammalian cells by conferring resistance to G418; and the like. Other suitable markers are known to those of skill in the art. 3. AAV Accessory Functions
  • the host cell (or packaging cell) must also be rendered capable of providing non AAV derived functions, or "accessory functions," in order to produce rAAV virions.
  • Accessory functions are non AAV derived viral and/or cellular functions upon which AAV is dependent for its replication.
  • accessory functions include at least those non AAV proteins and RNAs that are required in AAV replication, including those involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of Cap expression products and AAV capsid assembly.
  • Viral-based accessory functions can be derived from any of the known helper viruses.
  • accessory functions can be introduced into and then expressed in host cells using methods known to those of skill in the art.
  • accessory functions are provided by infection of the host cells with an unrelated helper virus.
  • helper viruses include adenoviruses; herpesviruses such as herpes simplex virus types 1 and 2; and vaccinia viruses.
  • Nonviral accessory functions will also find use herein, such as those provided by cell synchronization using any of various known agents. See, e.g., Buller et al. (1981) J. Virol. 40:241-247; McPherson et al. (1985) Virology 147:217-222; Schlehofer et al. (1986) Virology 152: 1 10-117.
  • accessory functions can be provided using an accessory function vector.
  • Accessory function vectors include nucleotide sequences that provide one or more accessory functions.
  • An accessory function vector is capable of being introduced into a suitable host cell in order to support efficient AAV virion production in the host cell.
  • Accessory function vectors can be in the form of a plasmid, phage, transposon, cosmid, or another virus.
  • Accessory vectors can also be in the form of one or more linearized DNA or RNA fragments which, when associated with the appropriate control elements and enzymes, can be transcribed or expressed in a host cell to provide accessory functions.
  • Nucleic acid sequences providing the accessory functions can be obtained from natural sources, such as from the genome of an adenovirus particle, or constructed using recombinant or synthetic methods known in the art.
  • adenovirus-derived accessory functions have been widely studied, and a number of adenovirus genes involved in accessory functions have been identified and partially characterized. See, e.g., Carter, B. J. (1990) "Adeno- Associated Virus Helper Functions," in CRC Handbook of Parvoviruses, vol. I (P. Tijssen, ed.), and Muzyczka, N. (1992) Curr. Topics. Microbiol, and Immun. 158:97-129.
  • EIa early adenoviral gene regions EIa, E2a, E4, VAI RNA and, possibly, EIb are thought to participate in the accessory process.
  • Herpesvirus-derived accessory functions have been described. See, e.g., Young et al. (1979) Prog. Med. Virol. 25:113.
  • Vaccinia virus-derived accessory functions have also been described. See, e.g., Carter, B. J. (1990), supra., Schlehofer et al. (1986) Virology 152:110- 117.
  • AAV helper construct to produce AAV Rep and/or Cap proteins.
  • the Rep expression products excise the recombinant DNA (including the DNA of interest, e.g., the heterologous nucleic acid) from the AAV expression vector.
  • the Rep proteins also serve to duplicate the AAV genome.
  • the expressed Cap proteins assemble into capsids, and the recombinant AAV genome is packaged into the capsids. Thus, productive AAV replication ensues, and the DNA is packaged into rAAV virions.
  • rAAV virions can be purified as described above. [00183] The resulting rAAV virions are then ready for use for DNA delivery, such as in gene therapy applications, or for the delivery of a gene product to a mammalian host.
  • Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pi, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c, subcutaneous(ly); and the like.
  • Example 1 Production of AAV virions including a histidine-tagged capsid protein MATERIALS AND METHODS Construction of AAV plasmids:
  • pXX2 a 1.8-kilobase linear fragment containing a portion backbone sequence of pXX2 (Xiao et al, (1998) J Virol 72, 2224-2232) was generated by PCR using the primers 5 '-GCGAAGCTTACGCGGCCGCTTGTTAATCAATAAACCGTTTAATTCG-S ' (SEQ ID NO:29) and 5'-CGGAATGGACGATATCCCGC-S ' (SEQ ID NO:30) with pXX2 (Xiao et al, (1998) supra) as template.
  • Both this product and pXX2 were digested with Hind III and CIa I, and the products were ligated to create the rAAV packaging plasmid, pXX2Not.
  • the cap sequence from AAV8 was cloned into this vector as well.
  • spliced overlap extension PCR was used to insert the His 6 peptide epitopes into the correct location, sometimes with additional flanking sequences. Primer sequences and cloning details are available upon request.
  • an AA V2 library containing a randomly inserted His 6 sequence within the cap gene was packaged (Maheshri et al (2006) Nat Biotechnol 24, 198-204) and selected as previously described (Yu and Schaffer (2006) J Virol 80, 3285-3292).
  • the chloroamphenical resistance (Cam R ) gene was randomly inserted into a plasmid containing the AA V2 cap gene by using a transposon kit (Finnyzmes, Espoo, Finland).
  • the resulting plasmid library was digested to excise the cap-CamR genes, which were subsequently cloned into pSub2 (Maheshri et ⁇ l., (2006) supra).
  • the pSub2 plasmid library was digested with Not /before ligation to His 6 fragments phosphorylated with T4 polynucleotide kinase.
  • the His 6 insert was constructed using the following oligonucleotides, with the histidine codons shown in bold: 5'- GGCCGGTC ACC ACCACC ACCACC ACTC-3 ' (SEQ ID NO:31) and 5'- GGCCGAGTGGTGGTGGTGGTGGTGACC-3' (SEQ ID NO:32).
  • HEK293T, HeLa, CHO Kl, CHO pgsA, and CHO pgsD were cultured in Iscove's modified Dulbecco's medium (IMDM) (Mediatech, Herndon, VA) with 10% fetal bovine serum (In vitro gen, Carlsbad, CA) and 1% penicillin and streptomycin (Invitrogen) at 37 0 C and 5% CO 2 . All cell lines were obtained from the ATCC.
  • AAV293 cells (Stratagene) were cultured in Dulbecco's modified medium (DMEM) with 10% fetal bovine serum and 1% penicillin and streptomycin at 37 0 C and 5% CO 2 .
  • DMEM Dulbecco's modified medium
  • rAAV was produced as previously described (Kaspar et al, (2003) Science 301, 839-
  • AA V2 and His 6 capsids were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose blot.
  • the blot was blocked in Tris-buffered saline with 3% bovine serum albumin and incubated with penta-his antibody (Qiagen) according to the manufacturer's instructions.
  • the blots were then developed by the ECL detection assay (Amersham Biosciences, Piscataway, NJ).
  • Recombinant AAV vectors were stereotaxically injected into the striatum region of the brain (AP, +0.2; ML, ⁇ 3.5; DV, -4.5 from skull) of adult female Fischer 344 rats (150 g, 6 weeks).
  • the animals were deeply anesthetized with a mixture of ketamine (90 mg/kg animal) and xylazine (10 mg/kg animal) prior to injection, and 3 ⁇ L of high-titer AAV vectors (1 x 10 9 vg/ ⁇ L) were injected using Hamilton syringe.
  • the retrieved brains were post-fixed by immersing in 4% PFA overnight at 4 0 C and subsequently stored in 30% sucrose for cryoprotection prior to sectioning.
  • Coronal sections were cut with 40 ⁇ m thickness using a freezing, sliding microtome.
  • Primary mouse-anti EDl (1 :100, Chemicon) and mouse-anti OX8 (1 :100, Chemicon) were utilized to detect antigens expressed by macrophages and T-cells, respectively. Additionally, primary mouse-anti NeuN (1 :200, Chemicon) and guinea pig-anti GFAP (1 : 1000, Advanced Immunochemical) were utilized to identify cell types (i.e., neurons and glial cells), and GFP expression was amplified using primary rabbit anti-GFP (1 :2000, Invitrogen). Corresponding secondary antibodies (labeled with AlexaFluor 488, 546, and 633) were used for detection.
  • a library of cap-Cam R insertion mutants was constructed using a MuA Transposase and a modified transposon insert containing a chloroamphenicol resistance gene (Cam ).
  • the resulting cap-CAM library was cloned into the AAV vector pSub2, and the Cam gene was subsequently replaced with a His 6 -encoding insert to create the final library.
  • Five nucleotides from cap become duplicated at the insertion site during the transposition reaction, and these together with the His 6 (SEQ ID NO:38) sequence yield a 13 amino acid (aa) insertion.
  • Restriction digest analysis of individual clones from the library confirmed that the insertions occurred throughout the entire cap gene and that each of these clones contained only one insert.
  • the estimated diversity of the library was ⁇ 5x 10 4 independent insertions into the 2.6- kb cap PCR product, which thus likely provides full coverage of all possible insertion sites.
  • the library was significantly diluted and used to package replication competent AAV (rcAAV), as previously described for AAV library packaging (Maheshri et al, (2006) supra).
  • flanking residues for the His 6 tag which consist of four alanine residues along with three random amino acids, may decrease infectivity due to the large insertion size or may not be optimal for display of the His 6 tag.
  • flanking residues for the His 6 tag which consist of four alanine residues along with three random amino acids, may decrease infectivity due to the large insertion size or may not be optimal for display of the His 6 tag.
  • a series of defined c ⁇ p-His 6 insertion mutants was constructed using spliced overlap extension PCR. These AA V2 insertion sites, previously shown to be permissive to the insertion of peptide sequences, were located in three regions: immediately after aa position 584 (Shi et al, 2001), after aa position 587 (Girod et al, 1999), and after aa position 588 (Grifman et al, 2001) (Fig.
  • FIGURE 1 Design of His 6 AAV Mutants.
  • A Map of VP3 monomer, constructed with Rasmol, indicating where insertions of His 6 tag occurred.
  • B Sequences of the insertion site of WT AA V2 (LQRGNRQA; SEQ ID NO:33) compared to sequences at the insertion sites of each of the His 6 AAV2 clones along with the His6 AA V8 clone. His 6 tags are shown in bold and flanking residues are shown in italics.
  • His 6 clones at the insertion sites are: His584: LQLGHHHHHHSRGNRQA (SEQ ID NO:34); His587: LQRGNLGHHHHHHSRQA (SEQ ID NO:35); His588: LQRGNRLGHHHHHHSQA (SEQ ID NO:36); HisAAV ⁇ : QQQNLGHHHHHHSTAP (SEQ ID NO:37). Analysis of Packaging and Ni-NTA Binding Ability of AAV Clones
  • the viral library was passed through a Ni-NTA column.
  • the virus present in the elution fractions was then amplified by a low multiplicity of infection (MOI) of 293T cells, followed by the addition of adenovirus serotype 5 (Ad5) to induce replication of the infectious AAV variants, which were again applied to the Ni-NTA column.
  • MOI multiplicity of infection
  • Ad5 adenovirus serotype 5
  • sequence analysis of the library indicated that the pool contained one dominant clone with an insertion after aa position 454, which bound to the Ni-NTA column at levels greatly exceeding that of WT AA V2.
  • FIGURE 2 Ni-NTA Purification of His 6 AAV mutant.
  • A Representative genomic titers of recombinant GFP vector packaged inside WT AA V2 capsid, His 6 AA V2, and His 6 AA V8 present within each column fraction. Titers were determined using quantitative PCR, and error bars represent the standard deviation of three independent trials.
  • B Representative transduction titers of recombinant GFP vector packaged with WT AA V2 capsid, His 6 AA V2, and His 6 AAV8 present within each column fraction. Titers were determined using flow cytometry, and error bars represent the standard deviation of three independent trials. In Vitro Characterization of HiS ⁇ -tagged AAV
  • FIGURE 3 In vitro characterization of His 6 AAV gene delivery properties.
  • Vector purification methods should yield viral vectors with high in vivo biological activity, while minimizing any immunogenic response arising from contaminants.
  • FIGURE 4 In vivo comparisons between injections of GFP-expressing vectors with either WT AA V2 or His 6 AA V2 capsid: GFP expression after 3 weeks.
  • A The anterior- posterier (AP) spread of GFP expression indicates no significant difference between the two vectors (P>0.05). The AP spread was the distance between the first and last tissue sections exhibiting GFP expression.
  • B The total volume of GFP expression (P>0.05). The error bars for (A), (B) indicate the standard deviations.
  • Example 2 Production of rAAV virions comprising an altered capsid protein with a protease substrate
  • a fusion protein comprising a subtilisin prodomain and an AA V2 VP2 capsid protein was generated.
  • the amino acid sequence of the subtilisin prodomain- AA V2 VP2 capsid protein is shown in Figure 8 (SEQ ID NO: 13).
  • An AAV virion comprising this fusion protein bound to immobilized subtilisin, and cleavage of the subtilisin prodomain from the AAV virion was activated by potassium fluoride. No substantial reduction in infectivity of the subtilisin prodomain-AAV virion, compared to an AAV virion with wild-type AA V2 capsid protein, was observed.
  • Figure 12 provides viral production titer from the parent AA V2 and the AA V2 variant ("PaIAA V2") with the subtilisin tag attached to the end of the viral protein VP2.
  • Figure 13 provides an infectious elution profile from the subtilisin affinity column. The parent AA V2 does not adhere to the column effectively; PaIAA V2 adheres to the subtilisin column in a manner such that it can be effectively recovered.

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Abstract

La présente invention concerne des procédés de purification de virions de virus adéno-associés (VAA), comportant des virions AAV recombinants, comprenant généralement la liaison d'un virion AAV comportant une protéine capsidique variante comportant un peptide de purification à un groupe fonctionnel de liaison qui se lie au peptide de purification. La présente invention concerne également des acides nucléiques comportant des séquences nucléotidiques codant pour des protéines capsidiques variantes ; ainsi que des virions AAV recombinants comportant les protéines capsidiques variantes.
PCT/US2008/004312 2007-04-09 2008-04-02 Procédés de purification de virions de virus adéno-associés Ceased WO2008124015A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017197355A2 (fr) 2016-05-13 2017-11-16 4D Molecular Therapeutics Inc. Variantes de capsides de virus adéno-associé et leurs procédés d'utilisation
WO2019060454A2 (fr) 2017-09-20 2019-03-28 4D Molecular Therapeutics Inc. Virus adéno-associé à variant de capsides et leurs procédés d'utilisation
WO2019104279A1 (fr) 2017-11-27 2019-05-31 4D Molecular Therapeutics Inc. Variants de capsides de virus adéno-associé et leurs procédés d'utilisation
WO2020174368A1 (fr) 2019-02-25 2020-09-03 Novartis Ag Compositions et procédés pour traiter une dystrophie cristalline de bietti
WO2020174369A2 (fr) 2019-02-25 2020-09-03 Novartis Ag Compositions et procédés pour traiter une dystrophie cristalline de bietti
CN114207438A (zh) * 2019-05-24 2022-03-18 阿维泰德有限责任公司 亲和剂
CN115135339A (zh) * 2020-02-21 2022-09-30 比亚分离公司 一种从完整的aav衣壳中分离或去除空的aav衣壳的方法
WO2023191827A1 (fr) * 2022-03-28 2023-10-05 President And Fellows Of Harvard College Purification à haut rendement de sérotypes d'aav divergents à l'aide d'une chromatographie d'affinité aavx

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060134740A1 (en) * 2003-08-06 2006-06-22 Bryan Philip N Engineered proteases for affinity purification and processing of fusion proteins

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060134740A1 (en) * 2003-08-06 2006-06-22 Bryan Philip N Engineered proteases for affinity purification and processing of fusion proteins

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KOERBER ET AL.: "Engineering Adeno-associated virus for one-step purification via immobilized metal affinity chromatography", HUMAN GENE THERAPY, vol. 18, 1 April 2007 (2007-04-01), pages 367 - 378 *

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WO2017197355A2 (fr) 2016-05-13 2017-11-16 4D Molecular Therapeutics Inc. Variantes de capsides de virus adéno-associé et leurs procédés d'utilisation
EP4206216A1 (fr) 2016-05-13 2023-07-05 4D Molecular Therapeutics Inc. Capsides de variants de virus adéno-associés et leurs procédés d'utilisation
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WO2020174368A1 (fr) 2019-02-25 2020-09-03 Novartis Ag Compositions et procédés pour traiter une dystrophie cristalline de bietti
WO2020174369A2 (fr) 2019-02-25 2020-09-03 Novartis Ag Compositions et procédés pour traiter une dystrophie cristalline de bietti
CN114207438A (zh) * 2019-05-24 2022-03-18 阿维泰德有限责任公司 亲和剂
CN115135339A (zh) * 2020-02-21 2022-09-30 比亚分离公司 一种从完整的aav衣壳中分离或去除空的aav衣壳的方法
WO2023191827A1 (fr) * 2022-03-28 2023-10-05 President And Fellows Of Harvard College Purification à haut rendement de sérotypes d'aav divergents à l'aide d'une chromatographie d'affinité aavx

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