US20070015238A1 - Production of pseudotyped recombinant AAV virions - Google Patents

Production of pseudotyped recombinant AAV virions Download PDF

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US20070015238A1
US20070015238A1 US10/456,423 US45642303A US2007015238A1 US 20070015238 A1 US20070015238 A1 US 20070015238A1 US 45642303 A US45642303 A US 45642303A US 2007015238 A1 US2007015238 A1 US 2007015238A1
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nucleic acid
aav
cell
protein
acid molecule
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Richard Snyder
Sergie Zolotukhin
Yoshihisa Sakai
Barry Byrne
Mark Potter
Irine Zolotukhin
Scott Loiler
Vince Chiodo
Nicholas Muzyczka
William Hauswirth
Terence Flotte
Corinna Burger
Edgardo Rodriguez
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Priority to US10/798,192 priority patent/US7094604B2/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10322—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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron

Definitions

  • the invention relates to the fields of molecular biology, gene therapy, microbiology and virology. More particularly, the invention relates to compositions and methods for producing and purifying recombinant Adeno-Associated Virus (rAAV) virions.
  • rAAV Adeno-Associated Virus
  • AAV a non-pathogenic, helper-dependent virus
  • Adenovirus (Ad) or Herpesvirus a suitable helper virus
  • the host and tissue tropism of AAV is determined by the ability of its capsid to bind to specific cellular receptors and/or co-receptors. Due to the broad host and tissue range, however, delivery of conventional AAV preferentially to a particular tissue of interest has been problematic.
  • serotype 2 AAV has been the most extensively studied and characterized. Accordingly, serotype 2 rAAV vectors (i.e., nucleic acid constructs) and virions (i.e., encapsidated vectors) have been proposed as the vector of choice for gene transfer protocols. Animal experiments, however, have shown that dramatic differences exist in the transduction efficiency and cell specificity of rAAV virions of different serotypes (Chao et al., Mol. Ther. 2:619-623, 2000; Davidson et al., PNAS 97:3428-3432, 2000; and Rabinowitz et al., J. Virol.
  • non-serotype 2 AAV virions were able to transduce certain tissues more efficiently and specifically than serotype 2 virions. Accordingly, an AAV virion including a well-characterized serotype 2 genome and a non-serotype 2 capsid would be useful for certain tissue-specific gene transfer applications. Methods that facilitate preparing such pseudotyped AAV virions would also be useful. Current methods involve the use of multiple vectors to provide the replication, packaging, and helper functions that are required for the formation of recombinant virions. These methods are inefficient and inadequate for large-scale production of pseudotyped recombinant virions.
  • the invention relates to the development of reagents and methods for producing purified AAV2 vectors pseudotyped with a non-serotype 2 AAV capsid.
  • AAV helper vectors were constructed for pseudotyping AAV serotype 2 DNA with capsids from AAV serotypes 1 and 5. These helper vectors encode AAV gene products necessary for AAV virion production (i.e., Rep and Cap proteins), as well as transcription products having Ad helper function.
  • a helper vector encoding a serotype 2 Rep protein and a serotype 1 Cap protein was used.
  • helper vector useful for pseudotyping AAV serotype 2 DNA with an AAV5 capsid encodes a serotype 2 Rep protein and a serotype 5 Cap protein.
  • methods were developed that result in highly purified and concentrated virion stocks. These methods involve applying a virus-containing sample to an iodixanol gradient centrifugation step followed by a chromatography step.
  • the helper vectors and purification methods described herein provide for efficient, large-scale production of pseudotyped virions without the need for multiple helper vectors.
  • the resultant pseudotyped virions can be used in numerous gene therapy applications.
  • the invention features a nucleic acid molecule including a first nucleotide sequence encoding an AAV Rep protein of a first serotype, a second nucleotide sequence encoding an AAV Cap protein of a second serotype, the second serotype being different from the first serotype, and a third nucleotide sequence encoding a transcription product having at least one Adenoviral helper function.
  • the nucleic acid molecule can be within a vector.
  • the AAV Rep protein can be an AAV serotype 2 protein.
  • the AAV serotype 2 Rep protein can be a Rep52 protein and/or a Rep78 protein. Both Rep52 and Rep78 proteins can be encoded by the first nucleotide sequence.
  • the AAV Cap protein can be an AAV serotype 1 protein and/or an AAV serotype 5 Cap protein.
  • the second nucleotide sequence encoding an AAV Cap protein can encode an AAV protein such as VP1, VP2, or VP3.
  • the second nucleotide sequence can encode all three AAV Cap proteins VP1, VP2, and VP3.
  • the transcription product having at least one Adenoviral helper function can be Adenovirus DNA binding protein, Adenovirus E4 protein, as well as Adenovirus virus associated RNA molecule.
  • the nucleic acid can be operably linked to at least one expression control sequence.
  • the first nucleotide sequence encoding an AAV Rep protein of a first serotype can be operably linked to a promoter. Examples of promoters include AAV p5 and AAV p19 promoters.
  • the second nucleotide sequence encoding an AAV Cap protein of a second serotype can also be operably linked to a promoter, such as an AAV p40 promoter.
  • the third nucleotide sequence encoding a transcription product having at least one Adenoviral helper function can further be operably linked to a promoter.
  • the nucleic acid molecule can further include a selectable marker such as a selectable marker that confers antibiotic resistance to a cell.
  • the invention features a cell including a nucleic acid molecule that includes a first nucleotide sequence encoding an AAV Rep protein of a first serotype, a second nucleotide sequence encoding an AAV Cap protein of a second serotype, the second serotype being different from the first serotype, and a third nucleotide sequence encoding a transcription product having at least one Adenoviral helper function.
  • the cell can be a mammalian cell.
  • the cell can further include a second nucleic acid that includes a polynucleotide (to be expressed) interposed between a first AAV inverted terminal repeat and a second AAV inverted terminal repeat.
  • the second nucleic acid can be within a vector.
  • the first and second AAV inverted terminal repeats can be AAV serotype 2 inverted terminal repeats.
  • the polynucleotide can encode a protein or a selectable marker such as green fluorescent protein.
  • the invention features a method of producing rAAV virions.
  • the method includes the steps of placing a cell having: 1) a nucleic acid molecule that includes a first nucleotide sequence encoding an AAV Rep protein of a first serotype, a second nucleotide sequence encoding an AAV Cap protein of a second serotype, the second serotype being different from the first serotype, and a third nucleotide sequence encoding a transcription product having at least one Adenoviral helper function and 2) a nucleic acid having a polynucleotide to be expressed interposed between a first AAV inverted terminal repeat and a second AAV inverted terminal repeat under conditions in which the first nucleic acid molecule is expressed, the second nucleic acid molecule is replicated, and rAAV virions are produced, and isolating the rAAV virions produced from the cell.
  • the cell can be a mammalian cell.
  • the step of placing the cell under conditions in which the first nucleic acid molecule is expressed and the second nucleic acid molecule is replicated includes placing the cell into a culture medium.
  • the step of isolating the rAAV virions produced from the cell includes separating the cell from the medium, lysing the cell to yield a cell lysate, and then isolating the rAAV virions from the cell lysate.
  • This step can also include subjecting the produced rAAV virions to an iodixanol step gradient and can further include subjecting the produced rAAV virions to ion exchange chromatography.
  • the produced rAAV virions can contain at least one AAV serotype 1 capsid protein or at least one AAV serotype 5 capsid protein.
  • gene is meant a nucleic acid molecule that codes for a particular protein, or in certain cases a functional or structural RNA molecule.
  • nucleic acid means a chain of two or more nucleotides such as RNA (ribonucleic acid) and DNA (deoxyribonucleic acid).
  • protein or “polypeptide” are used synonymously to mean any peptide-linked chain of amino acids, regardless of length or post-translational modification, e.g., glycosylation or phosphorylation.
  • nucleic acid molecule or polypeptide when referring to a nucleic acid molecule or polypeptide, the term “native” refers to a naturally-occurring (e.g., a wild-type; “WT”) nucleic acid or polypeptide.
  • WT wild-type
  • Rep protein is meant a polypeptide having at least one functional activity of a native AAV Rep protein (e.g., Rep 40, 52, 68, 78).
  • Cap protein is meant a polypeptide having at least one functional activity of a native AAV Cap protein (e.g., VP1, VP2, VP3).
  • a “functional activity” of a protein is any activity associated with the physiological function of the protein.
  • functional activities of Rep proteins include facilitating replication of DNA through recognition, binding and nicking of the AAV origin of DNA replication as well as DNA helicase activity.
  • Cap proteins include the ability to induce formation of a capsid, facilitate accumulation of single-stranded DNA, facilitate AAV DNA packaging into capsids (i.e., encapsidation), bind to cellular receptors, and facilitate entry of the virion into host cells.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors.”
  • a first nucleic acid sequence is “operably” linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked nucleic acid sequences are contiguous and, where necessary to join two protein coding regions, in reading frame.
  • expression control sequence refers to a nucleic acid that regulates the replication, transcription and translation of a coding sequence in a recipient cell.
  • expression control sequences include promoter sequences, polyadenylation (pA) signals, introns, transcription termination sequences, enhancers, upstream regulatory domains, origins of replication, and internal ribosome entry sites (“IRES”).
  • promoter is used herein to refer to a DNA regulatory sequence to which RNA polymerase binds, initiating transcription of a downstream (3′ direction) coding sequence.
  • proliferative typed a nucleic acid or genome derived from a first AAV serotype that is encapsidated or packaged by an AAV capsid containing at least one AAV Cap protein of a second serotype (i.e., one different from the first AAV serotype).
  • AAV inverted terminal repeats By “AAV inverted terminal repeats”, “AAV terminal repeats”, “ITRs”, and “TRs” are meant those sequences required in cis for replication and packaging of the AAV virion including any fragments or derivatives of an ITR which retain activity of a full-length or WT ITR.
  • rAAV vector and “recombinant AAV vector” refer to a recombinant nucleic acid derived from an AAV serotype, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, etc.
  • rAAV vectors can have one or more of the AAV WT genes deleted in whole or in part, preferably the rep and/or cap genes, but retain functional flanking ITR sequences.
  • a “recombinant AAV virion” or “rAAV virion” is defined herein as an infectious, replication-defective virus composed of an AAV protein shell encapsulating a heterologous nucleotide sequence that is flanked on both sides by AAV ITRs.
  • rAAV1 a rAAV virion having at least one AAV serotype 1 capsid protein.
  • rAAV5 a rAAV virion having at least one AAV serotype 5 capsid protein.
  • FIG. 1 is two plasmid maps (top:pXYZ1, bottom: pXYZ5).
  • FIG. 2 is a schematic illustration of purification schemes for rAAV1, 2, and 5 virions.
  • FIGS. 3A and B are chromatograms of rAAV virions purified by anion exchange and hydroxyapatite chromatography.
  • FIGS. 4A , B, and C are gels characterizing rAAV virion stocks.
  • A Silver-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel of rAAV1, 2, and 5 virion stocks (10 ul per lane). The titers of rAAV stocks are shown below each lane.
  • B Western blot analysis of rAAV1 and 2 virion stocks (10 ul per lane).
  • C Profile of anion exchange chromatography of an AAV5 virus (10 ul of each fraction per lane). Load is the iodixanol gradient purified material that was applied to the column; FT is the flow through, 1-5 are fractions eluted from the column. Monoclonal antibody B1 was used to detect AAV capsid proteins in Panels B and C.
  • FIGS. 5A and B are infectious center and dot blot assays.
  • the invention provides methods and compositions for producing pseudotyped AAV virions.
  • purified pseudotyped rAAV virions were produced in large quantities by introducing into host cells both (1) a first nucleic acid construct that contains AAV ITRs of a first AAV serotype, and encodes an exogenous nucleic acid (i.e., polynucleotide to be expressed in a cell infected with the virions produced); and (2) a second nucleic acid construct that encodes Ad transcription products having Ad helper function, Rep proteins of the first serotype, and Cap proteins of a second serotype.
  • the exogenous nucleic acid is located between two AAV ITRs that are the minimal cis-acting AAV sequences that direct replication and packaging of an AAV genome as well as an rAAV vector.
  • the second nucleic acid construct has sequences that encode (1) at least one AAV Cap protein of a first serotype, (2) at least one AAV Rep protein of a second serotype (i.e., serotype of the rAAV vector to be encapsidated), and (3) at least one transcription product having Ad helper function.
  • the first and second nucleic acids are introduced into host cells, which are then cultured under appropriate conditions to allow the host cells to replicate.
  • the portion of the first nucleic acid construct containing the two AAV ITRs and exogenous nucleic acid i.e., rAAV vector
  • the second nucleic acid construct is expressed, resulting in the production of transcription products having Ad helper function as well as Rep and Cap proteins.
  • Ad proteins such as E2A and E4, as well as Ad VA RNA provide helper functions that facilitate a productive AAV infection.
  • Rep proteins are essential for rAAV vector replication, while the Cap (e.g., VP1, VP2, VP3) proteins are structural proteins that are required for formation of the virion capsid.
  • the replicated rAAV vectors of a first serotype e.g., serotype 2
  • infectious rAAV virions i.e., an infectious virus particle containing an rAAV vector
  • cap proteins of a second serotype e.g., serotypes 1, 5
  • the first nucleic acid construct described above includes an exogenous nucleic acid and also contains other sequences that facilitates expression of the exogenous nucleic acid in a host cell.
  • An exogenous nucleic acid is a nucleic acid that is not native to AAV.
  • the exogenous nucleic acid is inserted into the construct in such a way that the nucleic acid is expressed.
  • the nucleic acid is placed within a construct (e.g., vector) at a particular location such that: (1) it is between two functional AAV ITRs of a particular serotype, (2) it is operatively linked with a promoter and (3) it is placed 5′ to a pA tail.
  • the exogenous nucleic acid can be any nucleic acid that is desired to be included in the rAAV to be produced so long as it does not exceed the number of nucleotides that can be encapsulated within a rAAV virion (i.e., approximately 5 kilobases).
  • Typical examples of such nucleic acids include those that encode a protein or an RNA. Proteins might, for example, be those that exert a therapeutic effect on a diseased cell (e.g., a human or non-human cell).
  • Genes that can be delivered by rAAV to exert a therapeutic effect include alpha-one antitrypsin, clotting factor IX, clotting factor VIII, clotting factor VII, dystrophin, ⁇ -, ⁇ -, ⁇ -, ⁇ -sarcoglycans, tyrosine hydroxylase, aromatic acid decarboxylase, GTP cyclohydrolasel, erythropoietin, aspartoacylase (ASPA), Nerve growth factor (NGF), lysosomal beta-glucuronidase (GUSB), insulin, alpha-synuclein, basic fibroblast growth factor (FGF-2), IGF1, alpha-galactosidase A (alpha-gal A), neurotrophin-3, Neuroglobin (Ngb), angoigenic proteins (vascular endothelial growth factor (VEGF165)), anti-angiogenic proteins, and any cytokines, including interferons (IFN- ⁇ , IFN- ⁇ , IFN
  • the second nucleic acid construct encodes transcription products having Ad helper function and AAV proteins that facilitate pseudotyping of an rAAV vector.
  • Such a nucleic acid construct encodes: 1) at least one AAV Cap protein of a first serotype, 2) at least one AAV Rep protein of a second serotype (i.e., serotype of the rAAV vector to be encapsidated), and 3) at least one transcription product having Ad helper function.
  • a nucleic acid encoding a Rep and/or Cap protein and transcription product having Ad helper function is inserted into the second nucleic acid construct in such a way that the nucleic acid is expressed.
  • the nucleic acid is placed within a construct (e.g., vector) at a particular location such that: (1) it is operatively linked with a promoter and (2) it is placed 5′ to a pA tail.
  • a nucleic acid encoding a Cap protein is any nucleic acid that encodes at least one functional Cap protein or functional derivative thereof.
  • the AAV cap gene encodes three capsid proteins: VP1, VP2 and VP3, and any one or combination of these three proteins may be expressed by a nucleic acid of the invention.
  • a nucleic acid encoding a Rep protein is any nucleic acid that encodes at least one functional Rep protein or functional derivative thereof. Any one or combination of the four AAV Rep proteins Rep40, Rep52, Rep68, and Rep78, may be expressed by a nucleic acid of the invention.
  • the rep and cap genes used in methods of the invention can be mutant or non-naturally occurring versions of AAV rep and cap genes.
  • nucleic acids encoding Rep and Cap proteins useful in the invention may be hybrid sequences containing portions of rep and cap genes from different serotypes.
  • rep and cap genes used in compositions and methods of the invention may include engineered as well as naturally-occurring rep and cap mutants.
  • a preferred rep gene according to the invention is a serotype 2 rep gene, while a preferred cap gene is a serotype 1 or 5 cap gene.
  • a nucleic acid encoding a transcription product having Ad helper function is any nucleic acid that encodes at least one protein or RNA molecule having Ad helper function.
  • Ad gene products that are known to provide Ad helper function include E1a, E1b, E2a, E4 (e.g., E4 orf6) and VA RNA.
  • Such nucleic acids can be mutant or non-naturally occurring versions of Ad nucleotide sequences. Mutants include those that are engineered as well as those that are naturally-occurring.
  • vectors for pseudotyping rAAV virions are constructed by combining the ORF coding for AAV Rep proteins of a first serotype and the ORF coding for capsid proteins of serotypes different from the first ( FIG. 1 ).
  • AAV helper plasmid such as pACG2R1C is constructed by substituting the AAV1 cap ORF for AAV2 cap ORF in pACG2 (Li et al., J. Virol. 71:5236-5243, 1997).
  • helper plasmid such as pACG2R5C
  • Rep2cap 1 and rep2cap5 helper sequences resulting from these constucts can be subcloned into an Ad helper plasmid, pXYZ, constructed from pAdEasy (Stratagene).
  • Ad helper plasmid pXYZ
  • several Ad genes penton, core protein, hexon, and Ad DNA polymerase
  • the left hand end of the Ad genome is removed to eliminate the possibility of generating infectious Ad and Ad structural proteins (some of which are cytotoxic).
  • the resultant plasmids pXYZ1 (26,256 bp) and pXYZ5 (26,147 bp) encode the AAV proteins and Ad transcription products required to pseudotype AAV2-ITR-containing nucleic acids into AAV1 and AAV5 capsids.
  • Both plasmids contain the Ad VA, E2A and E4 genes under the transcriptional control of their native promoters.
  • Both plasmid backbones also encode for ampicillin resistance.
  • rAAV vectors pseudotyped with AAV1 and AAV5 capsids can be generated using plasmids pACG2R1C and pACG2R5C, respectively, with plasmids pXX6 and pXYZ. See Xiao et al., J. Virol. 72:2224-2232, 1998.
  • the AAV and Ad transcription products can be expressed by more than one vector.
  • cells can be cotransfected with a first vector expressing the AAV genes and a second vector expressing transcription products.
  • the AAV proteins and Ad transcription products can be expressed by three different vectors. In this method, cells are transfected with the three vectors expressing the AAV proteins and Ad transcription products. In some applications, cells can be transfected with more than one vector expressing the AAV and Ad genes and a rAAV vector.
  • the exogenous nucleic acid and the nucleic acids encoding Rep, Cap and transcription products having Ad helper function are operably linked to one or more expression control sequences that facilitate gene expression in host cells.
  • operably linked nucleic acid sequences are contiguous and, where necessary to join two protein coding regions, in reading frame.
  • expression control sequences include promoters, insulators, response elements, introns, IRESs, silencers, enhancers, introns, initiation sites, termination signals, and pA tails.
  • any expression control sequence that facilitates gene expression in the host cell may be used.
  • control elements can include control sequences normally associated with the selected exogenous nucleic acid or nucleic acids encoding Rep and Cap. Alternatively, heterologous control sequences can be employed.
  • any of a number of promoters suitable for use in the selected host cell may be employed.
  • constitutive promoters of different strengths can be used to express the different AAV proteins.
  • Inducible promoters may also be used in compositions and methods of the invention.
  • the AAV p5 and p19 promoters are preferred.
  • Other promoters for use in the invention include both non-viral and viral promoters.
  • Non-viral promoters that may be used include ⁇ -actin and Factor IX promoters.
  • viral promoters examples include cytomegalovirus immediate early promoter (CMV), simian virus 40 (SV40) late promoter, Mouse Mammary Tumor Virus (MMTV) promoter (Grimm et al., Hum. Gene Ther. 9:2745-2760, 1998) and Ad E1A promoter.
  • CMV cytomegalovirus immediate early promoter
  • SV40 simian virus 40
  • MMTV Mouse Mammary Tumor Virus
  • vectors of the invention contain a selectable marker gene used to identify cells that contain the vector.
  • Suitable selectable marker genes for use in the invention include genes encoding enzymes that produce antibiotic resistance (e.g., those conferring resistance to ampicillin, penicillin, kanamycin, hygromycin, G418, or streptomycin), as well as those that encode enzymes that result in a colorimetric or fluorescent signal (e.g., green fluorescent protein, ⁇ -galactosidase).
  • the invention provides a cell containing a nucleic acid molecule having a nucleotide sequence encoding an AAV Rep protein of a first serotype, a nucleotide sequence encoding an AAV Cap protein of a second serotype, and a nucleotide sequence encoding a transcription product having at least one Ad helper function.
  • a cell according to the invention is any cell in which the nucleotide sequences can be expressed resulting in expression products (e.g., polypeptides, RNA molecules).
  • Cells of the invention may be non-mammalian cells (e.g., microorganisms, yeast cells, insect cells) or mammalian cells (e.g., human cells).
  • a cell according to the invention can further contain a second nucleic acid molecule containing a polynucleotide to be expressed interposed between two AAV ITRs.
  • both nucleic acid molecules are present within a vector (e.g., plasmid).
  • Preferred cells are those in which pseudotyped virions are formed based on the presence of the two nucleic acid molecules. Examples of useful cells for expressing nucleotide sequences resulting in the formation of pseudotyped rAAV virions include 293 (Graham et al., J. Gen. Virol. 36:59-72, 1977), HeLa (Bantel-Schaal et al., J. Virol. 73:939-947, 1984), and KB (Srivastava, A. Intervirology 27:138-147, 1987) cells.
  • the invention encompasses nucleotide sequences encoding transcription products (e.g., polypeptides, RNA) having at least one Ad helper function.
  • AAV is a helper-dependent virus, and as such, it requires co-infection with a helper virus such as Ad or cotransfection of helper virus DNA for a productive infection. See Ward and Berns, J. Virol., 70:4495, 1996.
  • Nucleotide sequences encoding transcription products having Ad helper function utilized in the present invention may be derived from any of a number of Ad serotypes that facilitate AAV infection. For example, sequences derived from Ad serotype 5 (Ad5) can be used.
  • nucleotide sequences encoding transcription products having Ad helper function reside in plasmids pXYZ1 and pXYZ5 for the generation of pseudotyped rAAV virions.
  • rAAV vectors and virions useful in the invention include those derived from a number of AAV serotypes, including 1, 2, 3, 4, 5, 6, and 7. Because of wide construct availability and extensive characterization, preferred rAAV vectors for use in the invention are those derived from serotype 2 (or mutants thereof). In methods of encapsidating rAAV2 vector contructs, use of serotype 2 Rep proteins is preferred. Because of tissue tropisms and purification methods described herein, preferred AAV Cap proteins are those derived from serotypes 1 and 5. Construction and use of AAV vectors and AAV proteins of different serotypes are discussed in Chao et al., Mol. Ther.
  • the invention also relates to the production of pseudotyped rAAV virions that have mutations within the virion capsid.
  • suitable AAV mutants may have ligand insertion mutations for the facilitation of targeting AAV to specific cell types.
  • the construction and characterization of AAV capsid mutants including insertion mutants, alanine screening mutants, and epitope tag mutants is described in Wu et al., (J. Virol. 74:8635-45, 2000).
  • Other rAAV virions that can be generated in methods of the invention include those capsid hybrids that are generated by molecular breeding of viruses as well as by exon shuffling. See Soong et al., Nat. Genet. 25:436-439, 2000; and Kolman and Stemmer Nat. Biotechnol. 19:423-428, 2001.
  • the nucleic acid molecules of the invention are useful in methods of producing pseudotyped rAAV virions.
  • Placing the cell in in vitro conditions includes placing the cell into a culture medium (e.g., DMEM supplemented with fetal bovine serum and antibiotics) in a humidified incubator (e.g., 5% CO 2 ) at a suitable temperature (e.g., 37°).
  • a culture medium e.g., DMEM supplemented with fetal bovine serum and antibiotics
  • a humidified incubator e.g., 5% CO 2
  • a suitable temperature e.g. 37°
  • the rAAV virions are subjected to a density gradient separation step, such as an iodixanol step gradient.
  • the virions can be further isolated (e.g., purified) by subjecting the virions to an additional purification step such as an ion exchange (e.g., anion exchange) chromatography step.
  • an ion exchange e.g., anion exchange
  • a cell used in the method is a mammalian cell (e.g., 293 cells).
  • the rAAV virions produced contain at least one AAV serotype 1 capsid protein.
  • the rAAV virions produced contain at least one AAV serotype 5 capsid protein.
  • the nucleic acids are introduced into the cells.
  • a number of known transfection techniques may be used. See, e.g., Graham et al., (Virology 52:456, 1973), Sambrook et al., supra, Chu et al., (Gene 13:197, 1981).
  • Particularly suitable transfection methods include calcium phosphate co-precipitation (Graham et al., Virol. 52:456-467, 1973), direct micro-injection into cultured cells (Capecchi, M. R.
  • the invention provides methods for purifying pseudotyped rAAV virions.
  • Methods of the invention involve applying a virus-containing sample to one or more purification steps, including density gradient separation and chromatography.
  • An example of a method for purifying rAAV virions includes several steps. First, a plurality of cells infected with rAAV virions is provided. From these infected cells, rAAV virions are collected. These virions are then subjected to a density gradient separation step such as one using an iodixanol gradient.
  • a typical iodixanol step gradient contains a 15% iodixanol step, a 25% iodixanol step, a 40% iodixanol step, and a 60% iodixanol step.
  • the iodixanol step can further include 1M NaCl.
  • the virion-containing iodixanol step is centrifuged, and the resultant virion-containing sample is collected from the iodixanol gradient step. This sample is then subjected to a chromatography step, such as an ion exchange or hydroxyapatite chromatography step.
  • Purification methods of the invention are particularly useful for purifying virions having capsids containing proteins from AAV serotypes 1 and 5 because these serotypes do not bind to heparin columns.
  • purification protocols are employed that use iodixanol density gradient centrifugation followed by anion exchange or hydroxyapatite chromatography.
  • Iodixanol is an iodinated density gradient media originally produced as an X-ray contrast compound for injection into humans.
  • iodixanol solutions can be made iso-osmotic at all densities. This property makes iodixanol an ideal media for analysis and downstream purification steps.
  • iodixanol has the capacity to separate free capsid proteins and empty capsids from vector genome-containing (full) capsids.
  • iodixanol is preferred in the invention, other suitable density gradient media might be substituted.
  • rAAV vectors are purified by column chromatography. Any chromatography method that allows purification of rAAV virions may be used.
  • ion exchange chromatography can be used. Ion exchange chromatography is a method that relies on charge interactions between the protein of interest and the ion exchange matrix, which is generally composed of resins, such as agarose, dextran, and cross-linked cellulose and agarose, that are covalently bound to a charged group. Charged groups are classified according to type (cationic and anionic) and strength (strong or weak).
  • Ion exchange chromatographic techniques generally take place in several steps: equilibration of the column to pH and ionic conditions ideal for target protein binding, reversible adsorption of the sample to the column through counterion displacement, introduction of elution conditions that change the buffer's pH or ionic strength in order to displace bound proteins, and elution of substances from the column in order of binding strength (weakly-bound proteins are eluted first).
  • Ion exchange chromatography is directly upgradable from a small-scale to a bulk-scale level.
  • Anionic exchange chromatography is a type of ionic exchange chromatography in which a negatively charged resin will bind proteins with a net positive charge.
  • anion-exchange resins examples include HiTrapQ by Pharmacia; MonoQ, MonoS, MiniQ, Source 15Q, 30Q, Q Sepharose, DEAE, and Q Sepharose High Performance by Amersham Biosciences (Piscataway, N.J.); WP PEI, WP DEAM, and WP QUAT by J. T. Baker (St.
  • Hydroxyapatite chromatography is another example of a suitable chromatography technique. Hydroxyapatite is a crystalline form of calcium phosphate. The mechanism of hydroxyapatite chromatography involves nonspecific interactions between negatively charged protein carboxyl groups and positively charged calcium ions on the resin, and positively charged protein amino groups and negatively charged phosphate ions on the resin.
  • hydroxyapatite resins examples include Bio-Gel HT and CHT ceramic resins by Bio-Rad (Hercules, Calif.); hydroxylapatite high resolution and hydroxylapatite fast flow by Calbiochem (San Diego, Calif.); HA Ultrogel by Ciphergen (Fremont, Calif.); and hydroxyapatite by Sigma-Aldrich (St. Louis, Mo.).
  • rAAV5 virions were purified using hydroxyapatite chromatography ( FIG. 3B ).
  • a preferred hydroxyapatite resin is ceramic hydroxyapatite by Bio-Rad, Hercules, Calif., as this is a stable, porous form of hydroxyapatite with an improved calcium:phoshpate ration, which overcomes low binding capacity due to excess phoshpate.
  • heparin-agarose chromatography is preferred ( FIG. 3A ). See, e.g, U.S. Pat. No. 6,146,874.
  • a combination of iodixanol step gradient followed by either affinity heparin (for purifying rAAV2), hydroxyapatite, or anion exchange chromatography (for purifying AAV1, 2 and 5) is used to facilitate the high-throughput of several viruses for direct comparison of transduction efficiency and specificity in animal models and cell culture. Scaled-up production of the viruses in tissue culture is facilitated by the use of cell factories, e.g., plastic trays with large culture surface areas (Nunc, Rochester, N.Y.). More importantly, purification of rAAV1, 2 and 5 virions on Q-Sepharose allows the comparison of virions purified using the same method.
  • the cell-factory based protocol results in virion stocks with titers of 1 ⁇ 10 12 ⁇ 1 ⁇ 10 13 vg/ml purified from 1 ⁇ 10 9 cells.
  • These chromatographic methods have the added benefit that they can be readily scaled up to purify virus from 1 ⁇ 10 10 cells.
  • the final yield of rAAV is approximately 1 ⁇ 5 ⁇ 10 11 IU or approximately 1 ⁇ 10 12 ⁇ 1 ⁇ 10 13 vector genomes, with P:I ratios that average 20 and rarely exceed 100.
  • Virions are also purified using chromatography in the absence of density gradient centrifugation.
  • lysates from infected cells can be directly subjected to chromatography for purification of rAAV virions.
  • chromatography For large-scale production methods of rAAV vectors involving chromatography, see Potter et al. (Methods Enzymol. 346:413-430, 2002).
  • AAV helper plasmids were constructed by combining the ORF coding for the AAV2 Rep proteins and the ORF coding for capsid proteins of serotypes 1 and 5.
  • the pACG2R1C helper plasmid was constructed by substituting the AAV1 cap ORF for AAV2 cap ORF in pACG2 (Li et al., J. Virol. 71:5236-5243, 1997) and a similar approach was applied to the pACG2R5C plasmid.
  • Rep2cap1 and rep2cap5 helper cassettes were then subcloned into an Ad helper plasmid, pXYZ, constructed from pAdEasy.
  • pXYZ To construct pXYZ, several Ad genes (penton, core protein, hexon, and Ad DNA polymerase) were disrupted and the left hand end of the Ad genome was removed to eliminate the possibility of generating infectious Ad and Ad structural proteins (some of which are cytotoxic).
  • the resultant plasmids pXYZ1 and pXYZ5 FIG. 1 ) were used to pseudotype AAV2-ITR-containing expression cassettes into AAV1 and AAV5 capsids, respectively.
  • Plasmid pAdEasy-1 (Stratagene, La Jolla, Calif.) was digested with SgfI and PmeI, the SgfI 3′-overhang was removed by treatment with T4 DNA-polymerase, and blunt ends were ligated to produce pAdEasyDel1. Upon digestion with ClaI and SalI, the 18.9 Kbp fragment was subcloned into pBlueScriptKS(-) to derive the pXYZ Ad helper plasmid.
  • pACG2R1C and pACG2R5C pseudotyping plasmids wtAAV1 DNA (Genbank Accession no. NC — 002077) and pAAV5-2 (Chiorini et al., J. Virol. 73:1309-1319, 1999) were used to amplify the ORFs coding for the capsid proteins of AAV1 and AAV5, respectively.
  • AAV1 cap ORF primers 5′GAGCAATAAATGATTTAAACCAGGTATG3′ (SEQ ID NO:1) and 5′GCTCTAGACCCGATGACGTAAGTCTTTTATCG3′ (SEQ ID NO:2) were used, and for the AAV5 cap ORF primers, 5′ GCCAATAAAGAACAGTAAATAATTTAAATAGTCATGTCTTTTGTTGATCACC3′ (SEQ ID NO:3) and 5′ GGTGATCAACAAAAGACATGACTATTTAAATTATTTACTGTTCTTTATTGGC3′ (SEQ ID NO:4) were used.
  • the hybrid plasmids pACG2R1C and pACG2R5C contain the ORF coding for the AAV2 Rep proteins, and the ORF coding for either AAV1 or AAV5 capsid proteins, respectively.
  • helper plasmids encode the AAV and Ad genes required to pseudotype AAV2 ITR-containing nucleic acids into AAV1 or AAV5 capsids.
  • rAAV vector constructs were assembled using the pTR-UF backbone (Klein et al., Exp. Neurol. 150:183-194, 1998; and Zolotukhin et al., J. Virol. 70:4646-4654, 1996), thereby containing ITRs from AAV2.
  • the mixture was incubated for 1 min at room temperature, at which time the formation of precipitate was stopped by diluting the mixture into 1100 ml of pre-warmed DMEM-complete.
  • the conditioned culture media was removed from the cells and the fresh precipitate-containing media was added immediately.
  • Cells were incubated at 37° C., 5% CO 2 for 60 hrs and the CaPO 4 precipitate was allowed to remain on the cells during this incubation period. At the end of the incubation the culture media was discarded, cells were washed with PBS, and harvested using PBS containing 5 mM EDTA.
  • the collected cells were centrifuged at 1000 ⁇ g for 10 minutes, resuspended in 60 ml Lysis Solution (150 mM NaCl, 50 mM Tris pH 8.4), combined, and stored at ⁇ 20° C. until purified.
  • Cells were lysed by 3 freeze/thaw cycles between dry ice-ethanol and 37° C. water baths. Other methods for lysing cells might also be used, e.g., microfluidization. Benzonase (Sigma, St. Louis, Mo.) was then added to the cell lysate (50 U/ml final concentration) and incubated for 30 min at 37° C. The crude lysate was clarified by centrifugation at 4000 ⁇ g for 20 minutes and the virus-containing supernatant was divided between four iodixanol gradients.
  • Benzonase Sigma, St. Louis, Mo.
  • Discontinuous iodixanol step gradients were formed in quick seal tubes (25 ⁇ 89 mm, Beckman, Fullerton, Calif.) by underlaying and displacing the less dense cell lysate (15 ml) with iodixanol prepared using a 60% (w/v) sterile solution of OptiPrep (Nycomed, Roskilde, Denmark) and PBS-MK buffer (1 ⁇ PBS containing 1 nM MgCl 2 and 2.5 mM KCl). Therefore, each gradient consisted of (from the bottom): 5 ml 60%, 5 ml 40%, 6 ml 25%, and 9 ml of 15% iodixanol; the 15% density step also contained 1 M NaCl.
  • Tubes were sealed and centrifuged in a Type 70 Ti rotor at 69,000 rpm (350,000 ⁇ g) for 1 hr at 18° C. Approximately 5 ml of the 60%-40% step interface was aspirated after side-puncturing each tube with a syringe equipped with an 18-gauge needle. The iodixanol band from each of the four gradients was combined; this could be frozen until column chromatography was performed.
  • the iodixanol gradient fraction was further purified and concentrated by column chromatography.
  • AAV2 virions a 3 ml heparin agarose Type I column (Sigma, St. Louis, Mo.) was equilibrated with 10 ml of PBS-MK buffer, then 10 ml of PBS-MK/1M NaCl, followed by 20 ml of PBS-MK buffer.
  • the virus-containing iodixanol fraction (20 ml) was loaded onto the column by gravity flow. The column was washed with 20 ml of PBS-MK buffer and eluted in 15 ml of PBS-MK/1M NaCl.
  • the AAV2 virions were purified using a 1 ml or 5 ml HiTrap Heparin column (Pharmacia) on an ATKA FPLC system (Pharmacia) run at 1 column volume per minute.
  • the virus was then concentrated and desalted in a Biomax 100K concentrator (Millipore, Bedford, Mass.) by three cycles of centrifugation. In each cycle the virus was concentrated to 1 ml following the addition of 10 ml of Lactated Ringer's or 1 ⁇ PBS. The virus was stored at ⁇ 80° in Lactated Ringer's or 1 ⁇ PBS.
  • a 5 ml HiTrap Q column (Pharmacia) was equilibrated at 5 ml/min with 5 column volumes (25 ml) of Buffer A (20 mM Tris, 15 mM NaCl, pH 8.5), then by 25 ml Buffer B (20 mM Tris, 500 mM NaCl, pH 8.5), followed by 25 ml of Buffer A using a Pharmacia ATKA FPLC system.
  • the 20 ml virus-containing iodixanol fraction was diluted 1:1 with Buffer A and applied to the column at a flow rate of 3-5 ml/min. After loading the sample, the column was washed with 10 column volumes (50 ml) of Buffer A. The virus was eluted with Buffer B and 2 ml fractions were collected.
  • rAAV5 virions For rAAV5 virions, a buffer exchange and concentration of the vector-containing iodixanol fraction was performed using a Millipore (Bedford, Mass.) BioMax 50 filter device and 50 mM Tris pH 7.5.
  • a Bio-scale Q5 (5 ml bed volume) CHT type I hydroxyapatite column (BioRad, Hercules, Calif.) was equilibrated with 5 ml Buffer C (20 mM potassium phosphate pH 7.5), then 7 ml Buffer D (500 mM Potassium phosphate pH 7.5), followed by 7 ml Buffer C at 1 ml/min using a BioRad (Hercules, Calif.) Biologic Duoflow system.
  • Virus was loaded onto the column at 1 ml/min and the column was washed with 7 ml Buffer C, and eluted with a 25 ml linear gradient of 0-100% Buffer D followed by 7 ml 100% Buffer D. The virus eluted with 0.2M K-phosphate.
  • Assays for infectious rAAV Stocks were assayed for infectious rAAV by the infectious center assay (ICA). In this assay, 96-well plates seeded with 2 ⁇ 10 4 C12 cells were infected 16 hours after seeding with 10-fold dilutions of rAAV and superinfected with WT Ad5 at a multiplicity of infection (MOI) of 10. Cells that had been infected by rAAV were then complemented for DNA replication and amplification of the rAAV genomes.
  • MOI multiplicity of infection
  • Cells were harvested and suspended in 5 ml of 1 ⁇ PBS, vacuum filtered onto nylon membranes (0.45 ⁇ m), and lysed with 0.5N NaOH/1.5M NaCl (this step also denatured and immobilized the DNA to the membrane) followed by neutralization with 1M Tris-HCl pH 7.0/2 ⁇ SSC (20 ⁇ SSC is 3M NaCl and 0.3M NaCitrate pH 7.0).
  • the immobilized DNA was probed for transgene DNA (i.e., exogenous DNA) and only those cells that had been productively infected with rAAV produced a spot.
  • the assay was accurate in the range of 10-200 spots (or infectious centers) per filter ( FIG. 5A ).
  • SCFA single cell fluorescence assay
  • Dot blot assay to determine the titer of rAAV physical particles and the particle to infectivity ratio.
  • the dot blot assay was used to determine the titer of rAAV virions that contained vector genomes ( FIG. 5B ).
  • Plasmid and unpackaged vector DNA was digested for 1 hour at 37° C. in a final volume of 200 ul containing SU of DNaseI (Roche, Basel, Switzerland), 10 mM Tris-HCl, pH 7.5, and 1 mM MgCl 2 .
  • Encapsidated rAAV vector genomes were liberated by adding an equal volume of 2 ⁇ proteinase K buffer (20 m M Tris-Cl, pH 8.0, 20 mM EDTA, pH 8.0, 1% SDS) followed by the addition of proteinase K (100 ug), and incubated at 37° C. for 1 hour.
  • the liberated vector DNA was phenol extracted and ethanol precipitated.
  • Precipitated DNA was dissolved in 40 ul of dH 2 O and diluted into 400 ul 0.4N NaOH/10 mM EDTA immediately prior to immobilization.
  • a two-fold dilution series of the plasmid DNA that was packaged was prepared in water and diluted into 400 ul 0.4N NaOH/10 mM EDTA immediately prior to immobilization.
  • Denatured vector DNA was immobilized onto a nylon membrane along with the plasmid standard curve using a dot blot apparatus (BioRad, Hercules, Calif.). The blots were probed for the transgene and exposed to film or Phosphorimager screen (Molecular Dynamics, Piscataway, N.J.).
  • the vector DNA signal was compared to the signal generated from the plasmid DNA standard curve, and extrapolated to determine a vector genome titer. A comparison of the vector genome titer to the ICA titer produced the P:I ratio.
  • AAV1 and AAV5 both lack significant binding to the heparin affinity resin used to purify rAAV2 virions
  • purification protocols were developed that use density gradient centrifugation followed by anion exchange or hydroxyapatite chromatography.
  • rAAV virions were purified by column chromatography. Three column resins were used: heparin-agarose, Q-sepharose, and hydroxyapatite.
  • AAV2 virions bound heparin-agarose ( FIGS. 6A and B), AAV5 virions bound hydroxyapatite, and AAV1, 2, and 5 virions bound Q-Sepaharose ( FIGS. 3A and 4 ).
  • rAAV2 virions eluted from heparin with 0.35M NaCl and rAAV5 virions eluted from hydroxyapatite with 0.2 M phosphate.
  • AAV1, 2, and 5 eluted from Q-Sepharose in 0.5 M NaCl.
  • virions produced were 99% pure with the three capsid proteins at the proper ratio of ⁇ 1:1:20 for VP1:VP2:VP3.
  • a combination of iodixanol step gradient followed by either affinity Heparin (for purifying rAAV2), hydroxyapatite (for purifying AAV5), or anion exchange chromatography (for purifying AAV1, 2 and 5) was used to facilitate the high throughput of several viruses for direct comparison of transduction efficiency and specificity in animal models and cell culture.
  • the infectious titer of rAAV was determined by measuring the ability of the virus to infect C12 cells expressing AAV2 rep and cap ORFs, unpackage, and replicate ( FIG. 5A ).
  • rep-cap expressing C12 cells were infected with serial dilutions of rAAV.
  • To score the infecting viral particle it was amplified through viral DNA replication, whereupon the number of viral genomes reached several thousand per cell. This amplification was achieved by co-infecting the cell with a saturating amount of Ad5 to initiate rep and cap gene expression required for AAV DNA replication.
  • the cells were then incubated for 40 hours, harvested, and transferred onto a nylon membrane and lysed.
  • the immobilized viral DNA was hybridized with a transgene-specific probe and the cells infected with rAAV particles were scored as black dots following autoradiography ( FIG. 5A ).
  • WT AAV may contaminate vector preparations, and rcAAV may be formed during the production of rAAV due to recombination between the rAAV genome and the AAV helper plasmid. Since expression of the AAV rep gene has been shown to affect transduction frequency (McLaughlin et al., J. Virol. 62:1963-1973, 1988; and Samulski et al., J. Virol. 63:3822-3828, 1989) and gene expression (Horer et al., J. Virol. 69:5485-5496, 1995; and Labow et al., J. Virol.
  • the dot blot assay was used to determine the titer of rAAV virions harboring vector genomes ( FIG. 5B ). This assay allowed direct comparisons of the potency of the different serotype virions administered to the same cell type.
  • the dot blot assay was performed on the same rAAV2-GFP stock as that of Example 2. The calculated vg titer was 8.2 ⁇ 10 12 vg/ml.
  • rAAV1-GFP and rAAV5-GFP virions purified by Q-sepharose chromatography and rAAV2 virions purified by heparin chromatography were used to transduce rat oval cells in culture ( FIG. 6 ).
  • the rAAV5-GFP transduced rat oval cells more efficiently than either rAAV2-GFP or rAAV1-GFP virions. Transduction of oval cells with rAAV vectors provides a therapeutic approach for treating liver disease or systemic protein deficiencies.
  • Glycogen storage disease type II mice (Raben et al., J. Biol. Chem. 273:19086-19092, 1998), which lack the lysosomal hyrolase acid ⁇ -glucosidase, were injected intramuscularly under 2,2,2-tribromoethanol (Avertin) anesthesia. Mice were administered 4 ⁇ 10 10 vector genomes of rAAV1-CMV-mGaa, expressing the murine Gaa cDNA.
  • Rat oval cells were isolated from male Fischer 344 rats (Petersen et al., Science 284:1168-1170, 1999; and Petersen et al., Hepatology 27:1030-1038, 1998). Briefly, a 2-acetylaminofluorene (2-AAF) tablet was inserted subcutaneously into the lower quadrant to suppress the hepatocyte proliferation. After 5-7 days a partial hepatectamy was performed to induce a severe hepatic injury. Seven days later the liver was perfused with a collagenase H solution. The oval cells were immediately sorted by fluorescence activated cell sorting (FACS) using a FITC-conjugated anti-rat Thy 1.1 antibody.
  • FACS fluorescence activated cell sorting
  • the purified oval cells were then plated onto sixteen well chamber slides and infected with the rAAV1, 2, and 5 viruses (10,000 vector genomes/cell) or mock infected. Nine days after infection the cells were visualized by either bright-field or fluorescent microscopy for the expression of GFP using a Zeiss Axiovert microscope.

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WO2003104413A3 (fr) 2004-07-08
US7094604B2 (en) 2006-08-22

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