WO2003104392A9 - Reactifs ameliores et procedes de production de parvovirus - Google Patents

Reactifs ameliores et procedes de production de parvovirus

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Publication number
WO2003104392A9
WO2003104392A9 PCT/US2002/038423 US0238423W WO03104392A9 WO 2003104392 A9 WO2003104392 A9 WO 2003104392A9 US 0238423 W US0238423 W US 0238423W WO 03104392 A9 WO03104392 A9 WO 03104392A9
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WO
WIPO (PCT)
Prior art keywords
parvovirus
aav
coding sequences
rep
polynucleotide
Prior art date
Application number
PCT/US2002/038423
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English (en)
Other versions
WO2003104392A3 (fr
WO2003104392A2 (fr
Inventor
Richard Jude Samulski
Joseph E Rabinowitz
Original Assignee
Univ North Carolina
Richard Jude Samulski
Joseph E Rabinowitz
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Filing date
Publication date
Application filed by Univ North Carolina, Richard Jude Samulski, Joseph E Rabinowitz filed Critical Univ North Carolina
Priority to AU2002367943A priority Critical patent/AU2002367943A1/en
Publication of WO2003104392A2 publication Critical patent/WO2003104392A2/fr
Publication of WO2003104392A9 publication Critical patent/WO2003104392A9/fr
Publication of WO2003104392A3 publication Critical patent/WO2003104392A3/fr

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    • 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
    • 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

Definitions

  • This invention relates to improved reagents and methods for producing virus, and more particularly relates to improved reagents and methods for producing parvovirus stocks.
  • the adeno-associated viruses are members of the family Parvoviridae and the genera Dependoviruses. Serotypes 1 through 4 were . originally identified as contaminates of adenovirus preparations (Carter and Laughlin (1984) in, The Parvoviruses p. 67-152 New York, N.Y.) whereas type 5 was isolated from a patient wart that was HPV positive. To date, seven molecular clones have been generated representing the serotypes of AAV (Bantel-Schaal et al. (1999) J. Virol. 73: 939, Chiorini et al. (1999) J. Virol. 73:1309, Muramatsu et al.
  • AAV type 2 As a vector has been facilitated by 30 years of studying its biology in vitro.
  • Recombinant AAV type 2 (rAAV2) has proven to be a suitable gene transfer vector in many different organisms (Monohan and Samulski (2000) Gene Ther. 7:24, Rabinowitz and Samulski (1998) Curr. Opin. Biotechnol. 9:470).
  • rAAV2 Recombinant AAV type 2
  • the present invention is based, in part, on the discovery of new transacting elements that mediate parvovirus replication and the production of new parvovirus particles.
  • the parvovirus replication proteins contain functional domains that are implicated in different aspects of viral propagation, i.e., genomic DNA replication and capsid assembly/packaging. Accordingly, based on an understanding of these elements, the present invention provides improved reagents (e.g., polynucleotides encoding parvovirus replication and/or capsid proteins, and vectors and cells comprising the same) for producing parvoviruses. Also provided are improved methods for producing hybrid parvoviruses (i.e., the viral terminal repeats and capsid are from different parvoviruses) using the inventive reagents.
  • the present invention provides a polynucleotide comprising parvovirus rep coding sequences and parvovirus cap coding sequences, the rep coding sequences encoding a DNA binding domain from a first parvovirus; the rep coding sequences further encoding a capsid interacting domain from a different parvovirus from the first parvovirus; and the cap coding sequence comprising sequences from the different parvovirus.
  • the invention provides a polynucleotide comprising adeno-associated virus (AAV) rep coding sequences and AAV cap coding sequences, the rep coding sequences having a 5' portion and a 3' portion; the 5' portion comprising rep coding sequences from a first AAV; the 3' portion comprising rep coding sequences from a different AAV from the first AAV; and the cap coding sequences comprising sequences from the different AAV.
  • AAV adeno-associated virus
  • a cell comprising parvovirus rep coding sequences and parvovirus cap coding sequences, the rep coding sequences encoding a DNA binding domain from a first parvovirus; the rep coding sequences further encoding a capsid interacting domain from a different parvovirus from the first parvovirus; the cap coding sequences comprising sequences from said different parvovirus; and the rep coding sequences being stably integrated into the genome of the cell.
  • the invention provides a cell comprising adeno- associated virus (AAV) rep coding sequences and AAV cap coding sequences, the rep coding sequences having a 5' portion and a 3' portion; the 5' portion comprising rep coding sequences from a first AAV; the 3' portion comprising rep coding sequences from a different AAV from the first AAV; the cap coding sequences comprising sequences from the different AAV; and the rep coding sequences being stably integrated into the genome of the cell.
  • AAV adeno- associated virus
  • a method of producing a recombinant hybrid parvovirus particle comprising providing to a cell permissive for parvovirus replication: (a) a recombinant parvovirus template comprising (i) a heterologous nucleotide sequence, and (ii) a parvovirus terminal repeat sequence; (b) parvovirus rep coding sequences and parvovirus cap coding sequences; the rep coding sequences encoding a DNA binding domain from a first parvovirus that interacts with the parvovirus terminal repeat to mediate replication of the recombinant parvovirus template; the rep coding sequences further encoding a capsid interacting domain from a different parvovirus from the first parvovirus; and the cap coding sequences comprising sequences from the different parvovirus; wherein the parvovirus terminal repeat sequence may be from the first parvovirus but not from the different parvovirus; under conditions sufficient for the replication and packaging of the recombinant parvovirus template; whereby recombinant hybrid
  • the present invention provides a method of producing a recombinant hybrid adeno-associated virus (rAAV) particle, comprising providing to a cell permissive for AAV replication: (a) a rAAV template comprising (i) a heterologous nucleotide sequence, and (ii) an AAV terminal repeat sequence; and (b) AAV rep coding sequences and AAV cap coding sequences; the rep coding sequences having a 5' portion and a 3' portion; the 5' portion comprising rep coding sequences from a first AAV that interacts with the AAV terminal repeat to mediate replication of the rAAV template; the 3' portion comprising rep coding sequences from a different AAV from the first AAV; and the cap coding sequences comprising sequences from the different AAV; wherein the AAV terminal repeat sequence may be from the first AAV but not from the different AAV; under conditions sufficient for the replication and packaging of the rAAV template; whereby infectious recomb
  • Figure 1 illustrates the construction and characterization of AAV serotype clones.
  • Panel A The capsid domain of each AAV serotype, generated by PCR, was cloned into the pBS+AAV2rep plasmid. The serotype specific capsid insertions (gray rectangles) are listed in order from type 1 to 5. Restriction sites are shown in the AAV2 diagram. Additionally, modifications containing the coding region of the carboxy termini of the Rep coding domain (gray striped) for each serotype were cloned into the constructs as needed.
  • Panel B An acrylamide gel of AAV serotype constructs pXR1 through 5 digested with BsflMI. White arrowheads point to common bands in the backbone and replication gene. A 50 bp DNA ladder (Amersham Pharmacia Biotech) flanks the lanes on which the constructs were loaded.
  • Figure 2 depicts Western analysis of lysates derived from serotype- specific transfections. Twenty-four hours post-transfection, 5 micrograms of total protein was loaded into each well. After transfer the blots were incubated with: Panel A) The anti-rep monoclonal antibody 1 F11 , (the sizes of the proteins listed on the right side); or Panel B) The anti-capsid monoclonal antibody B1 , with the capsid subunits listed on the right side. The serotype specific helpers used in the transfection are listed above each blot. Panel C) The B1 recognition site, as determined by Wobus et al. (2000) J. Virol. 74:9281 , is shown at the bottom of the figure. The amino acid sequence from this region for all five serotyes is shown. Asterisks indicate amino acids identical to AAV type 2.
  • Figure 3 illustrates a Hirt analysis of low molecular weight DNA isolated from 293 cells 24 hours after the three plasmid transfection with serotype specific plasmids. Equal amounts (2.5 micrograms) of undigested (lanes 1-5) and Dpn ⁇ digested DNA (lanes 7-11 ) from each serotype sample was loaded onto a 1% agarose gel (lane 6 DNA ladder). The resulting blot was probed with a 735 bp fragment of the GFP gene. Input DNA digested with Dpnl reveals the replicating monomer (M) and dimer transgene (D). The lower bands in lanes 7-11 are Dpn ⁇ digestion products of input plasmids.
  • Figure 4 shows the transduction efficiency of fractions collected from a heparin sepharose affinity column for rAAV serotypes 1 through 5.
  • the elution conditions were optimized originally for AAV2 (Zolotukhin et al. (1999) Gene Ther. 6:973).
  • Numbered fractions were collected in 0.5 ml volumes, W (waste) was collected in a single 10 ml fraction, and C (control) was an aliquot of the 1 ml volume of virus applied to the column.
  • Infections were performed in reference cell lines, with between 1/100 to 20 microliters of each fraction. Each bar represents the average of three separate infections, with the standard deviation indicated by the error bar.
  • Figure 5 depicts the transduction efficiency of rAAV serotype clones in CHO mutant and reference cell lines.
  • Cells (cell types are specified in the legend on right) were transduced with approximately 0.3 transducing units (TU)/cell, as determined relative to the reference cell lines (HeLa cells for AAV serotypes 1 , 2, 3 and 5, Cos1 cells for AAV4).
  • the transducing titers are given in TU/ ⁇ l for each serotype in each cell line.
  • the particle numbers used in this experiment were as follows: rAAV1 , 1.2 x 10 8 ; rAAV2, 5.6 x 10 8 ; rAAV3, 9.1 x 10 8 ; rAAV4, 2.3 x 10 8 ; and rAAV5, 5.4 x 10 8 .
  • Each bar represents the average of three separate infections, with the standard deviation indicated by the error bar.
  • Figure 6 illustrates GFP protein expression resulting from subretinal injections via in vivo fluorescence imaging.
  • Figure 7 shows an illustrative genomic DNA sequence for AAV-2; GenBank Accession No. NC 001401 ; SEQ ID NO: 19.
  • Figure 8 shows an illustrative genomic DNA sequence for AAV-1 ; GenBank Accession No. NC 002077; SEQ ID NO: 20.
  • Figures 9 shows an illustrative genomic DNA sequence for AAV-3A
  • Figure 10 shows an illustrative genomic DNA sequence for AAV-3B GenBank Accession No. NC 001863; SEQ ID NO: 22.
  • Figure 11 shows an illustrative genomic DNA sequence for AAV-4; GenBank Accession No. NC 001829; SEQ ID NO: 23.
  • Figure 12 shows an illustrative genomic DNA sequence for AAV-5 GenBank Accession No. NC Y18065; SEQ ID NO: 24.
  • Figure 13 shows an illustrative genomic DNA sequence for AAV-6; GenBank Accession No. NC 001862; SEQ ID NO: 25.
  • Figure 14 shows an illustrative genomic DNA sequence for AAV-7; GenBank Accession No. AF513851 ; SEQ ID NO: 26.
  • Figure 15 shows an illustrative genomic DNA sequence for AAV-8
  • Figure 16 shows an illustrative genomic DNA sequence for B19 parvovirus; GenBank Accession No. NC 000883; SEQ ID NO: 28.
  • Figure 17 shows an illustrative genomic DNA sequence for Minute Virus from Mouse (MVM); GenBank Accession No. NC 001510; SEQ ID NO: 29.
  • Figure 18 shows an illustrative genomic DNA sequence for goose parvovirus; GenBank Accession No. NC 001510; SEQ ID NO: 30.
  • Figure 19 shows an alignment of the amino acid sequence of exemplary Rep40. proteins from AAV1 (SEQ ID NO:31), AAV2 (SEQ ID NO:32), AAV3A (SEQ ID NO:33), AAV3B (SEQ ID NO:34), AAV4 (SEQ ID NO:35), AAV5 (SEQ ID NO:36), AAV6 (SEQ ID NO:37), AAV7 (SEQ ID NO:38) and AAV8 (SEQ ID NO:39), as well as a consensus sequence (SEQ ID NO:40). Dashes indicate gaps in the sequence and shading indicates positions of sequence homology.
  • Figure 20 shows an alignment of the amino acid sequence of exemplary Rep52 proteins from AAV1 (SEQ ID NO:41), AAV2 (SEQ ID NO:42), AAV3A (SEQ ID NO:43), AAV3B (SEQ ID NO:44), AAV4 (SEQ ID NO:45), AAV5 (SEQ ID NO:46), AAV6 (SEQ ID NO:47), AAV7 (SEQ ID NO:48) and AAV8 (SEQ ID NO:49), as well as a consensus sequence (SEQ ID NO:50).
  • Dashes indicate gaps in the sequence and shading indicates positions of sequence homology.
  • Figure 21 shows an alignment of the amino acid sequence of exemplary Rep68 proteins from AAV1 (SEQ ID NO:51), AAV2 (SEQ ID NO:52), AAV3A (SEQ ID NO:53), AAV3B (SEQ ID NO:54), AAV4 (SEQ ID NO:55), AAV5 (SEQ ID NO:56), AAV6 (SEQ ID NO:57), AAV7 (SEQ ID NO:58) and AAV8 (SEQ ID NO:59).
  • Dashes indicate gaps in the sequence and shading indicates positions of sequence homology.
  • Figure 22 shows an alignment of the amino acid sequence of exemplary Rep78 proteins from AAV1 (SEQ ID NO:60), AAV2 (SEQ ID NO:61), AAV3A (SEQ ID NO:62), AAV3B (SEQ ID NO:63), AAV4 (SEQ ID NO:64), AAV5 (SEQ ID NO:65), AAV6 (SEQ ID NO:66), AAV7 (SEQ ID NO:60), AAV1 (SEQ ID NO:60), AAV2 (SEQ ID NO:61), AAV3A (SEQ ID NO:62), AAV3B (SEQ ID NO:63), AAV4 (SEQ ID NO:64), AAV5 (SEQ ID NO:65), AAV6 (SEQ ID NO:66), AAV7 (SEQ ID NO:60), AAV2 (SEQ ID NO:61), AAV3A (SEQ ID NO:62), AAV3B (SEQ ID NO:63), AAV4 (SEQ ID NO:64), AAV5 (SEQ ID NO:65),
  • the present invention is based, in part, on the discovery of new transacting elements that mediate parvovirus replication and the production of new viral particles.
  • the parvovirus replication proteins contain functional domains that are implicated in different aspects of viral propagation, i.e., genomic DNA replication and capsid assembly/packaging. An°understanding of these elements has enabled improved reagents (e.g., packaging constructs and cells) and methods for producing stocks of parvovirus vectors.
  • Nucleotide sequences are presented herein by single strand only, in the 5' to 3' direction, from left to right, unless specifically indicated otherwise. Nucleotides and amino acids are represented herein in the manner recommended by the lUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three letter code, both in accordance with 37 CFR ⁇ 1.822 and established usage. See, e.g., Patentln User Manual, 99-102 (Nov. 1990) (U.S. Patent and Trademark Office).
  • Parvoviridae including autonomously-replicating parvoviruses and dependoviruses.
  • the autonomous parvoviruses include members of the genera Parvovirus, Erythrovirus, Densovirus, Iteravirus, and Contravirus.
  • Exemplary autonomous parvoviruses include, but are not limited to, minute virus of mouse, bovine parvovirus, canine parvovirus, chicken parvovirus, feline panleukopenia virus, feline parvovirus, goose parvovirus, H1 parvovirus, muscovy duck parvovirus, and B19 virus.
  • Other autonomous parvoviruses are known to those skilled in the art. See, e.g., BERNARD N.
  • the genus Dependovirus contains the adeno-associated viruses (AAV), including but not limited to, AAV type 1 , AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV. See, e.g., BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers).
  • AAV adeno-associated viruses
  • the parvovirus particles, capsids and genomes of the present invention are preferably from, but not limited to, AAV.
  • AAV AAV genomes
  • the genomic sequences of the various different serotypes of AAV and the autonomous parvoviruses, as well as the sequences of the terminal repeats (TRs), Rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank.
  • tropism refers to entry of the virus into the cell, optionally and preferably followed by expression (e.g., transcription and, optionally, translation) of sequences carried by the viral genome in the cell, e.g., for a recombinant virus, expression of the heterologous nucleotide sequences(s).
  • expression e.g., transcription and, optionally, translation
  • sequences carried by the viral genome in the cell e.g., for a recombinant virus, expression of the heterologous nucleotide sequences(s).
  • transcription of a heterologous nucleic acid sequence from the viral genome may not be initiated in the absence of trans-acting factors, e.g., for an inducible promoter or otherwise regulated nucleic acid sequence.
  • gene expression from the viral genome may be from a stably integrated provirus, from a non-integrated episome, as well as any other form in which the virus may take within the cell.
  • transduction or “infection” of a cell by a parvovirus or AAV means that the parvovirus/AAV enters the cell to establish an active (i.e., lytic) infection.
  • transduction of a cell by AAV means that the AAV enters the cell to establish a latent infection. See, e.g., BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter 69 (3d ed., Lippincott-Raven Publishers).
  • a "3' portion” of a polynucleotide indicates a segment of the polynucleotide that is downstream of another segment.
  • the term “3' portion” is not intended to indicate that the segment is necessarily at the 3' end of the polynucleotide, or even that it is necessarily in the 3' half of the polynucleotide, although it may be.
  • a "5' portion” of a polynucleotide indicates a segment of the polynucleotide that is upstream of another segment.
  • the term “5' portion” is not intended to indicate that the segment is necessarily at the 5' end of the polynucleotide, or even that it is necessarily in the 5' half of the polynucleotide, although it may be.
  • polypeptide encompasses both peptides and proteins, unless indicated otherwise.
  • a "polynucleotide” is a sequence of nucleotide bases, and may be RNA, DNA or DNA-RNA hybrid sequences (including both naturally occurring and non-naturally occurring nucleotide), but are preferably either single or double stranded DNA sequences.
  • an "isolated” polynucleotide e.g., an "isolated DNA” or an “isolated RNA” means a polynucleotide separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polynucleotide.
  • an "isolated" polypeptide means a polypeptide that is separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polypeptide.
  • a “therapeutic polypeptide” is a polypeptide that may alleviate or reduce symptoms that result from an absence or defect in a protein in a cell or subject.
  • a “therapeutic polypeptide” is one that otherwise confers a benefit to a subject, e.g., anti-cancer effects or improvement in transplant survivability.
  • heterologous nucleotide sequence will typically be a sequence that is not naturally occurring in the virus.
  • a heterologous nucleotide sequence may refer to a viral sequence that is placed into a non-naturally occurring environment (e.g., by association with a promoter with which it is not naturally associated in the virus).
  • vector or “gene delivery vector” may refer to a parvovirus (e.g., AAV) particle that functions as a gene delivery vehicle, and which comprises viral DNA (i.e., the vector genome) packaged within a parvovirus (e.g., AAV) capsid.
  • a parvovirus e.g., AAV
  • the term “vector” may be used to refer to the vector genome/vDNA alone.
  • a "recombinant parvovirus vector genome” is a parvovirus genome (i.e., vDNA) into which a heterologous (e.g., foreign) nucleotide sequence (e.g., transgene) has been inserted.
  • a “recombinant parvovirus particle” comprises a recombinant parvovirus vector genome packaged within a pa ⁇ /ovirus capsid.
  • rAAV vector genome is an AAV genome (i.e., vDNA) that comprises a heterologous nucleotide sequence.
  • rAAV vectors generally require only the 145 base terminal repeat(s) in cis to generate virus. All other viral sequences are dispensable and may be supplied in trans (Muzyczka, (1992) Curr. Topics Microbiol. Immunol. 158:97).
  • the rAAV vector genome will only retain the minimal terminal repeat (TR) sequence(s) so as to maximize the size of the transgene that can be efficiently packaged by the vector.
  • TR minimal terminal repeat
  • the structural and non-structural protein coding sequences may be provided in trans (e.g., from a vector, such as a plasmid, or by stably integrating the sequences into a packaging cell).
  • the rAAV vector genome comprises at least one AAV terminal repeat, more typically two AAV terminal repeats, which generally will be at the 5' and 3' ends of the heterologous nucleotide sequence(s).
  • template or "substrate” is used herein to refer to a polynucleotide sequence that may be replicated to produce the parvovirus viral DNA.
  • the template will typically be embedded within a larger nucleotide sequence or construct, including but not limited to a plasmid, naked DNA vector, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC) or a viral vector (e.g., adenovirus, herpesvirus, Epstein-Barr Virus, AAV, baculoviral, retroviral vectors, and the like).
  • the template may be stably incorporated into the chromosome of a packaging cell.
  • the methods and reagents herein may further be used to produce a duplexed parvovirus particle as described in international patent publication WO 01/92551 (the disclosure of which is incorporated herein by reference in its entirety).
  • a "rAAV particle” comprises a rAAV vector genome packaged within an AAV capsid.
  • a “parvovirus terminal repeat” may be from any parvovirus, including autonomous parvoviruses and AAV (all as defined above).
  • An “AAV terminal repeat” may be from any AAV with serotypes 1 , 2, 3, 4, 5, 6, 7 and 8 being preferred, and serotypes 1 , 2 and 5 being more preferred.
  • the term “terminal repeat” includes synthetic sequences that function as an AAV inverted terminal repeat, such as the "double-D sequence" as described in United States Patent No. 5,478,745 to Samulski et al., the disclosure of which is incorporated in its entirety herein by reference.
  • the AAV terminal repeats need not have a wild-type terminal repeat sequence (e.g., a wild-type sequence may be altered by insertion, deletion, truncation or missense mutations), as long as the terminal repeat mediates the desired functions, e.g., replication, virus packaging, integration, and/or provirus rescue, and the like.
  • parvovirus or AAV "rep coding sequences" indicate the nucleic acid sequences that encode the parvoviral or AAV non-structural proteins that mediate viral replication and the production of new virus particles.
  • the pa ⁇ /ovirus and AAV replication genes and proteins have been described in, e.g., BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-Raven Publishers).
  • the "rep coding sequences" need not encode all of the parvoviral or AAV Rep proteins.
  • the rep coding sequences do not need to encode all four AAV Rep proteins (Rep78, Rep 68, Rep52 and Rep40), in fact, it is believed that AAV5 only expresses the spliced Rep68 and Rep40 proteins.
  • the rep coding sequences encode at least those replication proteins that are necessary for viral genome replication and packaging into new virions.
  • the rep coding sequences will generally encode at least one large Rep protein (i.e., Rep78/68) and one small Rep protein (i.e., Rep52/40).
  • the rep coding sequences encode the AAV Rep78 protein and the AAV Rep52 and/or Rep40 proteins. In other embodiments, the rep coding sequences encode the Rep68 and the Rep52 and/or Rep40 proteins. In a still further embodiment, the rep coding sequences encode the Rep68 and Rep40 proteins.
  • the replication proteins be encoded by the same polynucleotide.
  • the NS-1 and NS-2 proteins (which are splice variants) may be expressed independently of one another.
  • the p19 promoter may be inactivated and the large Rep protein(s) expressed from one polynucleotide and the small Rep protein(s) expressed from a different polynucleotide.
  • the viral promoters may not be recognized by the cell, and it is therefore necessary to express the large and small Rep proteins from separate expression cassettes.
  • a "chimeric rep coding sequence” is a rep coding sequence comprising segments from two or more parvoviruses and encoding replication proteins, or portions thereof, from two or more parvoviruses.
  • the chimeric rep coding sequences will generally encode a DNA binding domain from one parvovirus and a capsid interacting domain from another parvovirus. These two different functional domains may be expressed as a single polypeptide or as separate polypeptides (e.g., due to different transcriptional and/or translational starts sites and/or differential splicing).
  • the rep coding sequences may encode a large Rep protein that is chimeric and a small Rep protein that is not. Neither protein may be chimeric if each protein is expressed from a separate polynucleotide.
  • the parvovirus or AAV "cap coding sequences” encode the structural proteins that form a functional parvovirus or AAV capsid (i.e., can package DNA and infect target cells).
  • the cap coding sequences will encode all of the parvovirus or AAV capsid subunits, but less than all of the capsid subunits may be encoded as long as a functional capsid is produced.
  • the cap coding sequences will be present on a single nucleic acid molecule.
  • the parvovirus particles of the invention are "hybrid" parvovirus particles in which the viral terminal repeats and viral capsid are from different parvoviruses.
  • Hybrid parvoviruses are described in more detail in international patent publication WO 00/28004 and Chao et al., (2000) Molecular Therapy 2:619 (the disclosures of which are incorporated herein in their entireties).
  • the viral terminal repeats and capsid are from different serotypes of AAV (i.e., a "hybrid AAV particle").
  • the parvovirus capsid may further be a "chimeric" capsid (e.g., containing sequences from different parvoviruses, preferably different AAV serotypes) or a "targeted" capsid (e.g., having a directed tropism) as described in international patent publication WO 00/28004.
  • a "DNA binding domain” is a replication protein or portion thereof that binds to and interacts with the parvovirus terminal repeats (e.g., AAV terminal repeats).
  • the DNA binding domain binds to the parvovirus terminal repeats and mediates replication of the AAV genome (e.g., by nicking the hairpin loop in the parvovirus terminal repeats by site-specific endonuclease cleavage at the terminal resolution site).
  • the AAV Rep78/68 proteins, the large Rep proteins from autonomous parvoviruses such as the MVM NS-1 protein, B19 NS-1 protein, the densovirus Junonia coenia NS-1 protein, and the goose parvovirus Rep1 protein (Smith et al., (1999) J. Virology 73:2930) are illustrative examples of proteins comprising "DNA binding domains".
  • a "capsid interacting domain” is a replication protein or portion thereof that interacts with the pa ⁇ /ovirus capsid to facilitate packaging of new virions (e.g., by facilitating particle assembly or packaging of the genomic DNA into assembled particles). While not wishing to be held to any particular theory of the invention, the capsid interacting domain may be associated with helicase activity.
  • the AAV Rep52/40 proteins, the small Rep proteins from autonomous parvoviruses such as the MVM NS-2 protein, B19 NS-2 protein, the densovirus Junonia coenia NS-2 protein, and the goose parvovirus Rep2 protein are illustrative examples of capsid interacting domains.
  • the large Rep proteins i.e., autonomous parvovirus NS-1 protein or AAV Rep78/68
  • which include the small Rep protein sequences also comprise capsid interacting domains.
  • the parvovirus replication proteins comprise a DNA binding domain that interacts with the parvovirus terminal repeats to initiate DNA replication and a capsid interacting domain that mediates new particle assembly (e.g., by mediating capsid assembly and/or packaging of the viral DNA into the capsid).
  • International patent publication WO 00/28004 discloses hybrid parvovirus particles in which viral genomes comprising terminal repeat(s) from one parvovirus are packaged within a capsid from a different parvovirus. This publication did not disclose that production of such hybrid parvoviruses may be improved or optimized by modifying the viral replication proteins.
  • the linear, single-stranded DNA genome of AAV encodes two open reading frames (rep and cap) flanked by 145 bp inverted terminal repeats (TR) (Srivastava et al., (1983) J. Virol. 45:555).
  • Replication of the AAV genome uses two viral components, the terminal repeat that serves as the origin of replication (Hauswirth et al., (1977) Virology 78:488; Straus et al., (1976) Proc. Natl. Acad. Sci. USA 73:742; Samulski et al., (1983) Cell 33:135; Senepathy et al., (1984) J. Mol. Biol.
  • the rep gene encodes four multifunctional proteins (Hermonat et al., (1984) J. Virology
  • Rep proteins transcribed from the p5 promoter (Rep78 and Rep68), are essentially identical except for unique carboxy termini generated from unspliced (Rep78) and spliced (Rep68) transcripts, respectively (Srivastava et al, (9183) J. Virol. 45:555).
  • Rep78 and Rep68 Two smaller rep proteins (Rep52, Rep40), transcribed from the p19 promoter are amino terminal truncations of Rep78 and Rep68, respectively.
  • Several biochemical activities of Rep78 and Rep68 have been implicated in the process of AAV replication. These include specific binding to the AAV terminal repeat (Ashktorab et al., (1989) J. Virology 63:3034; Im et al., 1989) J. Virology 63:3095; Snyder et al., (1993) J. Virology 67:6096) and site-specific endonuclease cleavage at the terminal resolution site (trs) (Im et al., (1990) J.
  • Rep78/68 also possess ATP dependent DNA-DNA helicase (Im et al., (1990) J. Virology 63:447; Im et al., (1992) J. Virology 66:1119) and DNA-RNA helicase as well as ATPase activities (Wonderling et al., (1995) J. Virology 69:3542).
  • Rep78/68 In addition to these activities associated with replication, Rep78/68 also regulate transcription from the viral promoters (Beaton et al., (1989) J. Virology 63:4450; Labow et al., (1986) J. Virology 60:251 ; Tratschin et al., (1986) Mol. Cellular Biol. 6:2884; Kyostio et al., (1994) J. Virology 68:2947; Pereira et al., (1997) J.
  • Virology 71 :1079) have been shown to mediate viral targeted integration (Xiao, W., (1996), "Characterization of cis and trans elements essential for the targeted integration of recombinant adeno-associated virus plasmid vectors", Ph.D. Dissertation, University of North Carolina-Chapel Hill; Balague et al., (1997) J. Virology 71:3299; LaMartina et al., (1998) J. Virology 72:7653; Pieroni et al., (1998) Virology 249:249).
  • the present investigations have determined that the parvovirus replication proteins comprise a capsid interacting domain(s) that is involved in capsid assembly and/or packaging. Accordingly, in a hybrid parvovirus production system, more efficient viral production may be achieved by providing a capsid interacting domain that is compatible with or optimized for the particular parvovirus capsid. Thus, for example, the present investigations have determined that the packaging of a recombinant AAV2 genome (i.e., with AAV2 terminal repeats) in an AAV5 capsid may be improved by providing AAV Rep proteins that comprise an AAV5, as opposed to an AAV2, capsid interacting domain.
  • Figures 19-22 comprise a capsid interacting domain.
  • the rep coding sequences encode the AAV Rep52 and/or Rep40 protein or the homologous regions of the large Rep proteins (i.e., the rep coding sequences comprise the sequences 3' of the AAV p19 promoter, with or without the intron region).
  • the rep coding sequences encode a functional portion (i.e., functions as a capsid interacting domain, as defined above) of the AAV small Rep proteins (e.g., a carboxy terminal portion).
  • the rep coding sequences encode the zinc finger domain, nuclear localization signal, dimerization domain(s), NTP binding pocket, the Rep splice domain (i.e., the intron domain), and/or the carboxyl terminal regions encoded by the rep sequences 3' of the p40 promoter.
  • the capsid interacting domain comprises approximately the carboxy terminal 50, 75, 100, 125, 150 or 200 amino acids of the Rep42 or Rep50 proteins.
  • the rep coding sequences comprise the coding sequences 3' of the p40 promoter.
  • the rep coding sequences comprise sequences encoding from amino acid 225 or 380 using AAV2 Rep78/68 numbering (or the analogous position of other AAV) through to the carboxyl end of the Rep78 or Rep68 protein (see, e.g., Chiorini et al., (1999) J. Virology 73:1309; Figures 19-22).
  • the rep coding sequences comprise sequences encoding the amino acids from the p19 promoter, p40 promoter or Accl site through to about amino acid 225 or amino acid 380 using AAV2 numbering of the Rep78 protein or the analogous position on the Rep78 proteins of other AAV.
  • the Rep coding sequences comprise sequences encoding the amino acid sequence from the p19 promoter, the p40 promoter, or the Accl site through to the beginning of the intron region (i.e., about amino acid 528 of AAV2 using Rep78/68 numbering, or the analogous position of other AAV).
  • the rep coding sequences comprise sequences encoding the amino acid sequence from about amino acid 225 or 380 using AAV2 numbering of the Rep78/68 proteins (or the analogous position on the Rep78/68 proteins of other AAV) through to beginning of the intron region.
  • the rep coding sequences encode a capsid interacting domain from an autonomous parvovirus.
  • the autonomous parvovirus replication proteins have structural and functional homology with the AAV Rep proteins (see, e.g., Yoon et al., (2001) J. Virology 75:3230; Smith et al. (1999) J. Virology 73:2930; Zadori et al., (1995) Virology 212: 562; BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers).
  • the MVM NS-2 protein, NS-2 protein of B19 and Rep2 protein of goose parvovirus may comprise a capsid interacting domain.
  • the capsid interacting domain is a functional portion (i.e., functions as a capsid interacting domain, as defined hereinabove) of the NS-2/Rep2 protein (i.e., the "small" replication protein) of an autonomous parvovirus (e.g., a carboxy terminal portion, a portion encoding zinc finger domains, a nuclear localization domain, an NTP binding pocket, a dimerization domain and/or a portion having helicase activity).
  • the capsid interacting domain comprises approximately the carboxy terminal 50, 75, 100, 125, 150 or 200 amino acids of the NS-2 protein.
  • the capsid interacting domain may be provided by the small Rep proteins and/or the homologous regions located in the carboxyl terminal portion of the large Rep proteins.
  • the rep coding sequences encoding the capsid interacting domain are selected so that the capsid interacting domain is compatible with the parvovirus capsid to be packaged.
  • the viral capsid and capsid interacting domain will typically be from the same parvovirus (e.g., AAV5 capsid and AAV5 capsid interacting domain or B19 capsid and B19 capsid interacting domain). Alternatively, there may be compatibility with respect to these elements among some of the parvoviruses.
  • the AAV1 and AAV2 capsid interacting domains may be compatible with both the AAV1 and AAV2 capsids.
  • the feline panleukemia virus and canine parvovirus elements may be able to interact with each other. Compatibility may be routinely determined by those skilled in the art by evaluation of sequence homology and by use of the techniques described herein.
  • One or more of the parvovirus replication proteins comprise a DNA binding domain(s) that interacts with the parvovirus terminal repeats. This functional element in the replication proteins interacts with the viral DNA to mediate replication thereof.
  • production of hybrid parvoviruses may be improved (e.g., higher titers achieved) by selecting a DNA binding domain that is compatible with or optimized for the parvovirus terminal repeat(s).
  • production titers may be improved by providing a DNA binding domain that is compatible with the AAV2 terminal repeat(s) and a capsid interacting domain that is compatible with the AAV5 capsid.
  • the large AAV Rep proteins (Rep78/68) comprise a DNA binding domain.
  • rep coding sequences are provided that encode one or both of the AAV large Rep proteins, where the Rep protein(s) is selected so as to be compatible with the AAV terminal repeats to be packaged.
  • the rep coding sequences may encode a functional portion of the AAV large Rep proteins (e.g., an amino-terminal portion).
  • the rep coding sequences encode the AAV large Rep protein specific amino acid sequences, i.e., the amino-terminal amino acid sequences that are not present in the AAV Rep52/40 proteins (i.e., encoded by the rep coding sequences 5' of the AAV p19 promoter) (see, e.g., Chiorini et al., (1999) J Virology 73:1309; Figures 19-22).
  • the rep coding sequences encode approximately the amino-terminal 50, 75, 100, 150, 190, 200, 210, 215, 220, 225, 230, 240, 250, or 270 amino acids of the AAV large Rep78/68 proteins.
  • the rep coding sequences encode approximately the amino-terminal 50, 75, 100, 150, 190, 200, 210, 215, 220, 225, 230, 240, 250, or 270 amino acids of the AAV2 large Rep78/68 proteins or the analogous sequences in other AAV serotypes (see, e.g., Chiorini et al., (1999) J. Virology 73:1309; Figures 7 - 22).
  • the autonomous parvovirus large replication proteins also have DNA binding domains.
  • the MVM and H1 NS-1 proteins, the goose parvovirus Rep1 protein, the B19 NS-1 protein, and the Junonia coenia densovirus NS-1 protein may comprise DNA binding domains that mediate replication of the viral DNA.
  • the DNA binding domain is a functional portion of an autonomous parvovirus NS-1/Rep1 protein (i.e., the "large" replication protein).
  • the functional portion may be an amino terminal portion of the large Rep protein.
  • the rep coding sequences encode approximately the amino terminal 50, 75, 100, 150, 190, 200, 210, 215, 220, 225, 230, 240, 250, or 270 amino acids of the autonomous parvovirus NS- 1/Rep1 protein (i.e., large Rep protein).
  • the replication protein DNA binding domain is selected so as to be compatible with the viral terminal repeats to be packaged.
  • the viral terminal repeat and DNA binding domain may be from the same parvovirus (e.g., AAV5 terminal repeats and AAV5 DNA binding domain or B19 terminal repeats and B19 DNA binding domain). Alternatively, there may be compatibility among some of the parvovirus DNA binding domains and the terminal repeats.
  • the AAV2 and AAV3 terminal repeats are highly homologous; thus, AAV2 terminal repeats can interact with DNA binding domains from AAV2 or AAV3 Rep protein, and vice versa.
  • AAV2 and AAV3 with AAV1 and AAV4, although higher titers may be achieved with the serotype-specific DNA binding domain and terminal repeat.
  • closely related autonomous parvoviruses such as feline panleukemia virus and canine pa ⁇ /ovirus, or MVM and H1 virus.
  • an AAV DNA binding domain will be used with an AAV terminal repeat
  • an autonomous parvovirus DNA binding domain will be used with autonomous parvovirus terminal repeats.
  • Compatibility between terminal repeats and DNA binding domains from different parvoviruses may be routinely determined by those skilled in the art by evaluation of sequence homology and by use of the methodologies described herein.
  • a chimeric large Rep protein comprises a DNA binding domain from one parvovirus and a capsid interacting domain from a different parvovirus.
  • the DNA binding domain interacts with the TRs and the capsid interacting domain interacts with the capsid.
  • these functions are provided by different Rep proteins; a large Rep protein comprises a DNA binding domain that interacts with the TRs, and a capsid interacting domain of a small Rep protein interfaces with the capsid.
  • the present invention provides polynucleotide sequences comprising chimeric parvovirus rep coding sequences (e.g., chimeric AAV rep coding sequences) and chimeric replication proteins encoded thereby.
  • the chimeric parvovirus rep coding sequences encode a DNA binding domain from one parvovirus (preferably, an AAV) and a capsid interacting domain from another parvovirus.
  • the inventive rep coding sequences may further be associated with the parvovirus cap coding sequences in a single polynucleotide.
  • the invention provides a polynucleotide comprising parvovirus rep coding sequences and parvovirus cap coding sequences, the rep coding sequences encoding a DNA binding domain from a first parvovirus (preferably, the first parvovirus is an AAV), where the rep coding sequences further encode a capsid interacting domain from a different parvovirus from the first parvovirus; and the cap coding sequences comprise sequences that encode a parvovirus capsid that is compatible with and interacts with the capsid interacting domain to facilitate capsid assembly and/or packaging (as described above).
  • This rep/cap polynucleotide sequence may be used to produce hybrid parvoviruses, i.e., a parvovirus having terminal repeats from one parvovirus and a capsid from another parvovirus, where the terminal repeats are compatible with the DNA binding domain encoded by the rep coding sequences and the capsid is compatible with the capsid interacting domain encoded by the rep coding sequences.
  • the invention provides a polynucleotide comprising parvovirus rep coding sequences and parvovirus cap coding sequences, the rep coding sequences encoding a DNA binding domain from a first parvovirus (preferably, the first parvovirus is an AAV), where the rep coding sequences further encode a capsid interacting domain from a different parvovirus from the first parvovirus; and the cap coding sequences comprising sequences from the different parvovirus.
  • a polynucleotide comprising parvovirus rep coding sequences and parvovirus cap coding sequences, the rep coding sequences encoding a DNA binding domain from a first parvovirus (preferably, the first parvovirus is an AAV), where the rep coding sequences further encode a capsid interacting domain from a different parvovirus from the first parvovirus; and the cap coding sequences comprising sequences from the different parvovirus.
  • the first parvovirus is an AAV.
  • the different parvovirus is an AAV.
  • both the first and different parvoviruses are AAV.
  • the rep coding sequences comprise a 5' and 3' portion, the 5' portion encoding the DNA binding domain from the first parvovirus; and the 3' portion encoding the capsid interacting domain from the different parvovirus.
  • the invention provides a polynucleotide comprising AAV rep coding sequences and AAV cap coding sequences, the rep coding sequences having a 5' and a 3' portion, the 5' portion comprising rep coding sequences from a first AAV and the 3' portion comprising rep coding sequences from a different AAV from the first AAV; and the cap coding sequences comprising sequences from the different AAV.
  • the capsid interacting domain and DNA binding domain encoded by the polynucleotides are as described above.
  • the capsid interacting domain is from an autonomous parvovirus (e.g., B19 virus, H1 virus, canine parvovirus, feline panleukemia virus, muskovy duck parvovirus, goose parvovirus, and MVM).
  • the capsid interacting domain is from an AAV (e.g., AAV1 , AAV2 or AAV5).
  • the DNA binding domain may be from an autonomous parvovirus (e.g., B19 virus, H1 virus, canine parvovirus, feline panleukemia virus, muskovy duck parvovirus, goose parvovirus, and MVM) or from an AAV (e.g., AAV1 , AAV2 or AAV5).
  • an autonomous parvovirus e.g., B19 virus, H1 virus, canine parvovirus, feline panleukemia virus, muskovy duck parvovirus, goose parvovirus, and MVM
  • AAV e.g., AAV1 , AAV2 or AAV5
  • both the rep coding sequences and cap coding sequences are AAV sequences (i.e., the AAV rep and cap sequences).
  • the rep coding sequences encode an AAV1 , AAV2, AAV4 or AAV5 DNA binding domain.
  • the rep coding sequences encode an AAV1 , AAV2 or AAV5 capsid interacting domain.
  • the cap coding sequences encode a capsid of the same serotype as the capsid interacting domain.
  • the rep coding sequences encode an AAV5 DNA binding domain and an AAV1 , AAV2, AAV3, AAV4, AAV6, AAV7 or AAV8 capsid interacting domain.
  • the cap coding sequences encode a capsid of the same serotype as the capsid interacting domain.
  • the rep coding sequences encode an AAV5 capsid interacting domain and an AAV1 , AAV2, AAV3, AAV4, AAV6, AAV7 or AAV8 DNA binding domain.
  • the cap coding sequences encode an AAV5 capsid.
  • the capsid interacting domain and capsid are from an autonomous parvovirus (e.g., B19 virus, goose parvovirus, H1 virus, MVM virus, and the like) and the DNA binding domain is from an AAV.
  • an autonomous parvovirus e.g., B19 virus, goose parvovirus, H1 virus, MVM virus, and the like
  • the DNA binding domain is from an AAV.
  • the rep coding sequences and cap coding sequences may be operatively associated with an expression control sequence, for example, a promoter.
  • the rep coding sequences and cap coding sequences are each operatively associated with separate expression control sequences (e.g., promoters). Promoters and other transcriptional and translational control elements are described in more detail hereinbelow in connection with the discussion of transgenes that may be encoded by a recombinant parvovirus genome.
  • the rep coding sequences are operatively associated with a parvovirus promoter, more preferably, the AAV p5 or p19 promoters.
  • the coding sequences for the large and small Rep proteins may be associated with separate transcriptional and/or translational control elements so that expression of these proteins may be differentially regulated.
  • rep coding sequences encoding the DNA binding domain and the capsid interacting domain may be operatively associated with different promoters.
  • the rep coding sequences encoding the capsid interacting domain are operatively associated with an AAV p19 promoter.
  • Chimeric AAV rep coding sequences may encode two, three or all four of the AAV Rep proteins. Typically, at least one of the large AAV Rep proteins and one of the small Rep proteins will be encoded. Further, it is not necessary that all of the Rep proteins encoded by the rep coding sequences be chimeric. For example, the large Rep protein may be chimeric and the small Rep protein need not be chimeric. As a further alternative, neither protein may be chimeric if each protein is expressed from a separate polynucleotide.
  • the present invention also encompasses vectors comprising the polynucleotides comprising the inventive chimeric rep coding sequences, optionally in conjunction with the cap coding sequences.
  • the vector may be any vector known in the art. Illustrative vectors include, but are not limited to, plasmids, naked DNA vectors, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), cosmids, and viral vectors.
  • any suitable viral vector may be employed, as known in the art, including single and double-stranded RNA and DNA viral vectors, with DNA being preferred.
  • Exemplary viral vectors may derived be from Poxviridae (e.g., pox virus or vaccinia virus), Papoviridae (e.g., BKV, JCV, or SV40), Adenoviridae (e.g., adenovirus), Herpesviridae (e.g., Herpes Simplex Virus), Hepadnaviridae (e.g., HBV), Retroviridae (e.g., HIV, SIV, MoMLV, RSV, HTLV), Picomaviridae (e.g., poliovirus, rhinovirus, coxsackieviruses, Caliciviridae, Togaviridae (e.g., alphaviruses, rubella) Flaviviridae (e.g., yellow fever virus), Parvoviridae
  • Preferred viral vectors include AAV, adenovirus, herpesvirus, EBV, baculovirus, and retroviral (e.g., lentiviral) vectors.
  • the present invention also encompasses cells containing the inventive nucleotide sequences, vectors and chimeric replication proteins described above.
  • the cell may be any cell known in the art, including bacterial, protozoan, yeast, fungal, plant and animal cells. Animal cells (e.g., insect and mammalian cells) are preferred. Mammalian cells (e.g., human) and insect cells are more preferred.
  • the cell contains both chimeric rep coding sequences and the appropriate cap coding sequences, but the cap coding sequences and rep coding sequences are on separate polynucleotide molecules. Further, the rep coding sequences and/or the cap coding sequences may be stably integrated into the cellular chromosomes. Cells for use in methods of virus production and methods of gene delivery are described in more detail hereinbelow.
  • the present invention also encompasses methods of producing hybrid parvovirus particles using the inventive polynucleotide sequences, vectors and cells.
  • methods known to those skilled in the art of producing AAV and parvovirus vectors may be used to produce hybrid parvovirus vectors using the inventive reagents disclosed herein (see, e.g., WO 00/28004, the disclosure of which is incorporated herein in its entirety by reference).
  • Hybrid parvovirus particles may be produced by introducing a rAAV template to be replicated and packaged into a permissive or packaging cell, as those terms are understood in the art (e.g., a "permissive” cell can be infected or transduced by the virus; a "packaging” cell is a stably transformed cell that expresses the replication and/or capsid proteins).
  • the invention provides a method of producing a recombinant parvovirus particle, comprising providing to a cell (a) a recombinant parvovirus template comprising (i) a heterologous nucleotide sequence, and (ii) at least one parvovirus terminal repeat sequence; and (b) chimeric parvovirus rep coding sequences and parvovirus cap coding sequences; the chimeric rep coding sequences encoding a DNA binding domain from a first parvovirus that interacts with the parvovirus terminal repeat to mediate replication of the recombinant parvovirus template; the rep coding sequences further encoding a capsid interacting domain from a different pa ⁇ /ovirus from the first parvovirus; and the cap coding sequences comprising sequences that encode a parvovirus capsid that is compatible with and interacts with the capsid interacting domain to facilitate capsid assembly and/or packaging.
  • the cap coding sequences are from a different parvovirus than the parvovirus terminal repeat
  • the recombinant parvovirus template and parvovirus rep coding sequences and parvovirus cap coding sequences are provided under conditions so that hybrid pa ⁇ /ovirus particles comprising the recombinant parvovirus template are produced in the cell.
  • the hybrid parvovirus particles will be infectious.
  • the invention provides a method of producing a recombinant parvovirus particle, comprising providing to a cell (a) a recombinant parvovirus template comprising (i) a heterologous nucleotide sequence, and (ii) at least one parvovirus terminal repeat sequence; and (b) chimeric parvovirus rep coding sequences and parvovirus cap coding sequences; the chimeric rep coding sequences encoding a DNA binding domain from a first parvovirus (preferably, an AAV) that interacts with the parvovirus terminal repeat to mediate replication of the recombinant parvovirus template; the rep coding sequences further encoding a capsid interacting domain from a different parvovirus from the first parvovirus; and the cap coding sequences comprising sequences from the different parvovirus.
  • a recombinant parvovirus template comprising (i) a heterologous nucleotide sequence, and (ii) at least one parvovirus terminal repeat sequence
  • the cap coding sequences are further from a different parvovirus than the parvovirus terminal repeat sequence, such that the resulting parvovirus particle is "hybrid".
  • the recombinant parvovirus template and parvovirus rep coding sequences and parvovirus cap coding sequences are provided under conditions so that infectious parvovirus particles comprising the rAAV template are produced in the cell.
  • the recombinant parvovirus template is a rAAV template and the parvovirus terminal repeat is an AAV terminal repeat sequence.
  • the cap coding sequences and the rep coding sequences encoding the capsid interacting domain are AAV sequences.
  • the recombinant parvovirus template (e.g., rAAV template) has two terminal repeat sequences at the 5' and 3' ends of the heterologous nucleotide sequence.
  • the parvovirus terminal repeat is from the first parvovirus (e.g., the same parvovirus as the DNA binding domain).
  • the chimeric rep coding sequences comprise a 5' portion and a 3' portion; the 5' portion encoding the DNA binding domain from the first parvovirus and the 3' portion encoding the capsid interacting domain from the different parvovirus.
  • the invention provides a method of producing a rAAV particle, comprising providing to a cell: (a) a rAAV template comprising (i) a heterologous nucleotide sequence, and (ii) at least one AAV terminal repeat sequence (preferably, two AAV two terminal repeat sequences at the 5' and 3' ends of the heterologous nucleotide sequence); and (b) chimeric AAV rep coding sequences and AAV cap coding sequences; the chimeric rep coding sequences having a 5' portion and a 3' portion; the 5' portion comprising rep coding sequences from a first AAV that interacts with the AAV terminal repeat to mediate replication of the rAAV template; the 3' portion comprising rep coding sequences from a different AAV from the first AAV; and the cap coding sequences comprising sequences from the different AAV.
  • the cap coding sequences are from a different AAV than the AAV terminal repeat sequence, such that the resulting AAV particle is
  • the rAAV template, AAV rep coding sequences and AAV cap coding sequences are provided under conditions so that rAAV particles comprising the rAAV template are produced in the cell.
  • the recombinant parvovirus template comprises a segment (e.g., a terminal repeat) that permits packaging within the parvovirus capsid.
  • the rep coding sequences and cap coding sequences are not associated with parvovirus terminal repeats to prevent packaging of these sequences into the parvovirus capsid.
  • Other methods of preventing the packaging of AAV rep/cap sequences are known in the art (see, e.g., U.S. Patent 6,329,181 to Xiao et al.).
  • the method further comprises the step of collecting the infectious recombinant parvovirus particles.
  • the method may further comprise the step of lysing the cell prior to collecting the recombinant parvovirus particles.
  • the parvovirus template comprises an AAV terminal repeat sequence (preferably two AAV terminal repeat sequences at the 5' and 3' ends of the vDNA). It is further preferred that the AAV terminal repeat(s) is from AAV1 , AAV2, AAV4 or AAV5. Alternatively, the parvovirus template comprises an autonomous parvovirus terminal repeat (e.g. B19).
  • the rep coding sequences encode an AAV1 , AAV2, AAV4 or AAV5 DNA binding domain.
  • the parvovirus terminal repeat is an AAV terminal repeat and is compatible with the DNA binding domain, and is preferably from the same AAV serotype as the DNA binding domain. In the case of a DNA binding domain from AAV5, the parvovirus terminal repeat will also preferably be from AAV5.
  • Any suitable permissive or packaging cell known in the art may be employed to produce parvovirus vectors utilizing the invention, including bacterial, protozoan, yeast, fungal, plant and animal cells. Mammalian and insect cells are preferred. Preferred insect cells are those that are infected by baculovirus, e.g., Sf9 cells. Also preferred are trans-complementing packaging cell lines that provide functions deleted from a replication-defective helper virus, e.g., 293 cells or other E1a trans-complementing cells.
  • a baculovirus system is used to produce parvovirus particles in mammalian (e.g., human) or insect cells.
  • the baculovirus may be used to provide the parvovirus template, rep coding sequences and/or cap coding sequences to the cell.
  • the inventive parvovirus rep coding sequences and cap coding sequences may be provided by any method known in the art. Current protocols typically provide the pa ⁇ /ovirus rep and cap genes on a single plasmid.
  • the parvovirus rep coding sequences and cap coding sequences need not be provided by a single construct, although it may be convenient to do so.
  • the parvovirus rep and/or cap sequences may be provided by any viral or non-viral vector.
  • the rep/cap sequences may be provided by a hybrid adenovirus, baculovirus or herpesvirus vector, as described in the previous section (e.g., inserted into the E1a or E3 regions of a deleted adenovirus vector).
  • EBV vectors may also be employed to express the parvovirus cap coding sequences and/or rep coding sequences.
  • One advantage of this method is that EBV vectors are episomal, yet will maintain a high copy number throughout successive cell divisions (i.e., are stably integrated into the cell as extra-chromosomal elements, designated as an "EBV based nuclear episome", see Margolski, (1992) Curr. Top. Microbiol. Immun. 158:67).
  • the rep coding sequences and/or cap coding sequences may be stably integrated into chromosome of the cell.
  • the AAV rep and cap sequences described above will not be flanked by the AAV packaging sequences (e.g., AAV terminal repeats), to prevent rescue and/or packaging of these sequences.
  • AAV packaging sequences e.g., AAV terminal repeats
  • Other methods of preventing the packaging of AAV rep/cap sequences are known in the art (see, e.g., U.S. Patent 6,329,181 to Xiao et al.)
  • the recombinant parvovirus template (preferably, a rAAV template) is as described herein, and may be provided to the cell using any method known in the art.
  • the recombinant parvovirus template may be supplied by a non-viral (e.g., plasmid) or viral vector (as described above).
  • the recombinant parvovirus template is supplied by a herpesvirus or adenovirus vector (e.g., inserted into the E1a or E3 regions of a deleted adenovirus).
  • a herpesvirus or adenovirus vector e.g., inserted into the E1a or E3 regions of a deleted adenovirus.
  • Palombo et al., (1998) J. Virology 72:5025 describe a baculovirus vector carrying a reporter gene flanked by the AAV terminal repeats.
  • EBV vectors may also be employed to deliver a rAAV template, as described above with respect to the rep/cap genes.
  • the AAV template is provided by a replicating rAAV virus.
  • an AAV provirus is stably integrated into the chromosome of the cell.
  • helper virus functions to complete the viral life cycle.
  • Both adenovirus and herpes simplex virus may serve as helper viruses for AAV. See, e.g., BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers).
  • exemplary helper viruses include, but are not limited to, Herpes simplex (HSV) varicella zoster, cytomegalovirus, and Epstein-Barr virus.
  • HSV Herpes simplex
  • MOI multiplicity of infection
  • duration of the infection will depend on the type of virus used and the packaging cell line employed. Any suitable helper vector may be employed.
  • helper virus sequences necessary for AAV replication are known in the art.
  • the helper functions are provided by the adenovirus early genes, more particularly, the E1a, E2a, E4orf6 and VA RNA adenovirus sequences.
  • these sequences will be provided by a helper adenovirus or herpesvirus (preferably, adenovirus) vector.
  • the adenovirus or herpesvirus sequences may be provided by another non-viral or viral vector, e.g., as a non-infectious adenovirus miniplasmid that carries all of the helper genes required for efficient AAV production as described by Ferrari et al., (1997) Nature Med.
  • the vector can be introduced into the packaging cell by any suitable method known in the art, as described above.
  • Herpesvirus may also be used as a helper virus in AAV packaging methods.
  • Hybrid herpesviruses encoding the AAV Rep protein(s) may advantageously facilitate more scalable AAV vector production schemes.
  • a hybrid herpes simplex virus type I (HSV-1 ) vector expressing the AAV-2 rep and cap genes has been described (Conway et al., (1999) Gene Therapy 6:986 and WO 00/17377, the disclosures of which are incorporated herein in their entireties).
  • the helper virus functions may be provided by a packaging cell with the helper genes embedded in the chromosome or maintained as a stable extrachromosomal element.
  • helper virus sequences cannot be packaged into AAV virions, e.g., are not flanked by AAV terminal repeats.
  • helper virus sequences e.g., adenovirus sequences
  • This helper construct may be a non-viral or viral construct, but is preferably a hybrid adenovirus or hybrid herpesvirus comprising the AAV rep/cap genes as described above.
  • the AAV rep/cap sequences and the adenovirus helper sequences are supplied by a single adenovirus helper vector.
  • This vector further contains the rAAV template.
  • the AAV rep/cap sequences and/or the rAAV template may be inserted into a deleted region (e.g., the E1a or E3 regions) of the adenovirus.
  • the AAV rep/cap sequences and the adenovirus helper sequences are supplied by a single adenovirus helper vector.
  • the rAAV template is provided as a plasmid template.
  • the AAV rep/cap sequences and adenovirus helper sequences are provided by a single adenovirus helper vector, and the rAAV template is integrated into the cell as a provirus.
  • the rAAV template is provided by an EBV vector that is maintained within the cell as an extrachromosomal element (e.g., as an EBV based nuclear episome).
  • the AAV rep/cap sequences and adenovirus helper sequences are provided by a single adenovirus helper.
  • the rAAV template is provided as a separate replicating viral vector.
  • the rAAV template may be provided by a rAAV particles or a second recombinant adenovirus particle.
  • the hybrid adenovirus vector typically comprises the adenovirus 5' and 3' cis sequences sufficient for adenovirus replication and packaging (i.e., the adenovirus terminal repeats and PAC sequence).
  • the AAV rep/cap sequences and, if present, the rAAV template are embedded in the adenovirus backbone and are flanked by the 5' and 3' cis sequences, so that these sequences may be packaged into adenovirus capsids.
  • the adenovirus helper sequences and the AAV rep/cap sequences are not flanked by the AAV packaging sequences (e.g., the AAV terminal repeats), so that these sequences are not packaged into the AAV virions.
  • the adenovirus is grown for a time sufficient to produce an adenovirus stock, optionally in the presence of a suitable helper virus or in a suitable packaging cell as known in the art (i.e., to complement any genes deleted or inactivated in the adenovirus vector).
  • AAV vector stocks free of contaminating helper virus may be obtained by any method known in the art.
  • AAV and helper virus may be readily differentiated based on size.
  • AAV may also be separated away from helper virus based on affinity for a heparin substrate (Zolotukhin et al. (1999) Gene Therapy 6:973).
  • deleted replication-defective helper viruses are used so that any contaminating helper virus is not replication competent.
  • an adenovirus helper lacking late gene expression may be employed, as only adenovirus early gene expression is required to mediate packaging of AAV virus.
  • Adenovirus mutants defective for late gene expression are known in the art (e.g., ts100K and ts149 adenovirus mutants).
  • the reagents and methods disclosed herein may be employed to produce high-titer stocks of the inventive parvovirus vectors, preferably at essentially wild-type titers.
  • the parvovirus stock has a titer of at least about 10 5 transducing units (tu)/ml, more preferably at least about 10 6 tu/ml, more preferably at least about 10 7 tu/ml, yet more preferably at least about 10 8 tu/ml, yet more preferably at least about 10 9 tu/ml, still yet more preferably at least about 10 10 tu/ml, still more preferably at least about 10 11 tu/ml, or more.
  • titer of at least about 10 5 transducing units (tu)/ml, more preferably at least about 10 6 tu/ml, more preferably at least about 10 7 tu/ml, yet more preferably at least about 10 8 tu/ml, yet more preferably at least about 10 9 tu/ml, still yet more preferably at least about 10 10 tu/ml, still more preferably at least about 10 11 tu/ml, or more.
  • the parvovirus stock preferably has a titer of at least about 1 , 5, 10, 20, 50, 100, 250, 500, 1000, 2500 tu/cell or more.
  • virus titers achieved with the inventive methods and chimeric rep coding sequences (or Rep proteins) may advantageously be greater than the titers achieved using a non-chimeric rep coding sequence as a control, e.g., at least about 2-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 250-fold, 500-fold or 1000-fold greater or more.
  • virus titers of a hybrid AAV particle comprising a recombinant AAV2 genome (i.e., with AAV2 TRs) packaged within a different AAV serotype capsid may be higher using a chimeric rep coding sequence of the invention that is compatible with both the AAV2 TRs and the capsid as compared with the titers achieved using a non-chimeric AAV2 rep coding sequence.
  • the parvovirus vectors produced according to the present invention are useful for the delivery of nucleic acids to cells in vitro, ex vivo, and in vivo.
  • the parvovirus vectors can be advantageously employed to deliver or transfer nucleic acids to animal, more preferably mammalian, cells.
  • Nucleic acids of interest include nucleic acids encoding polypeptides, preferably therapeutic (e.g., for medical or veterinary uses) or immunogenic (e.g., for vaccines) polypeptides.
  • the recombinant parvovirus genome will only retain the minimal terminal repeat (TR) sequence(s) so as to maximize the size of the transgene that can be efficiently packaged by the vector.
  • TR minimal terminal repeat
  • the structural and non-structural protein coding sequences may be provided in trans (e.g., from a vector, such as a plasmid, or by stably integrating the sequences into a packaging cell).
  • the recombinant parvovirus vector genome comprises at least one terminal repeat, more typically two terminal repeats, which generally flank the 5' and 3' ends of the heterologous nucleotide sequence(s).
  • nucleic acids of interest include nucleic acids encoding polypeptides, preferably therapeutic (e.g., for medical or veterinary uses) or immunogenic (e.g., for vaccines) polypeptides.
  • the recombinant vector genome is preferably approximately the size of the wild-type parvovirus genome (e.g., the AAV genome) corresponding to the parvovirus capsid into which it will be packaged (e.g., from about 80% to about 105% of wt) and comprises an appropriate packaging signal.
  • the AAV capsid disfavors packaging of vDNA that substantially deviate in size from the wt AAV genome.
  • the genome is preferably approximately 5.2 kb in size or less.
  • the genome is preferably greater than about 3.6, 3.8, 4.0, 4.2, or 4.4 kb in length and/or less than about 5.4, 5.2, 5.0 or 4.8 kb in length.
  • the heterologous nucleotide sequence(s) will typically be less than about 5 kb in length (more preferably less than about 4.8 kb, still more preferably less than about 4.4 kb in length, yet more preferably less than about 4.2 kb in length) to facilitate packaging of the duplexed template by the parvovirus (e.g., AAV) capsid.
  • the present invention may be further used to produce a parvovirus vector to deliver a therapeutic polypeptide.
  • Therapeutic polypeptides include, but are not limited to, cystic fibrosis transmembrane regulator protein (CFTR), dystrophin (including the protein product of dystrophin mini-genes, see, e.g, Vincent et al., (1993) Nature Genetics 5:130), utrophin (Tinsley et al., (1996) Nature 384:349), clotting factors (e.g., Factor XIII, Factor IX, Factor X, etc.), erythropoietin, angiostatin, endostatin, catalase, tyrosine hydroxylase, superoxide dismutase, leptin, the LDL receptor, lipoprotein lipase, omithine transcarbamylase, ⁇ -globin, ⁇ -globin, spectrin, ⁇ i-antitrypsin, adenosine
  • heterologous nucleotide sequences encode suicide gene products (e.g., thymidine kinase, cytosine deaminase, diphtheria toxin, and tumor necrosis factor), proteins conferring resistance to a drug used in cancer therapy, tumor suppressor gene products (e.g., p53, Rb, Wt-1 ), and any other polypeptide that has a therapeutic effect in a subject in need thereof.
  • suicide gene products e.g., thymidine kinase, cytosine deaminase, diphtheria toxin, and tumor necrosis factor
  • proteins conferring resistance to a drug used in cancer therapy e.g., tumor suppressor gene products (e.g., p53, Rb, Wt-1 ), and any other polypeptide that has a therapeutic effect in a subject in need thereof.
  • Heterologous nucleotide sequences encoding polypeptides include those encoding reporter polypeptides (e.g., an enzyme). Reporter polypeptides are known in the art and include, but are not limited to, Green Fluorescent Protein, ⁇ -galactosidase, alkaline phosphatase, and chloramphenicol acetyltransferase gene.
  • the nucleic acid of interest may encode an antisense nucleic acid, a ribozyme (e.g., as described in U.S. Patent No. 5,877,022), RNAs that effect spliceosome-mediated frans-splicing (see, Puttaraju et al., (1999) Nature Biotech. 17:246; U.S. Patent No. 6,013,487; U.S. Patent No. 6,083,702), interfering RNAs (RNAi) that mediate gene silencing (see, Sharp et al., (2000) Science 287:2431) or other non-translated RNAs, such as "guide” RNAs (Gorman et al., (1998) Proc. Nat. Acad. Sci. USA 95:4929; U.S. Patent No. 5,869,248 to Yuan et al.), and the like.
  • RNAi interfering RNAs
  • the parvovirus vector may also encode a heterologous nucleotide sequence that shares homology with and recombines with a locus on the host chromosome. This approach may be utilized to correct a genetic defect in the host cell.
  • the present invention may be used to produce a parvovirus vector to express an immunogenic polypeptide in a subject, e.g., for vaccination.
  • the nucleic acid may encode any immunogen of interest known in the art including, but are not limited to, immunogens from human immunodeficiency virus, influenza virus, gag proteins, tumor antigens, cancer antigens, bacterial antigens, viral antigens, and the like.
  • the use of parvoviruses as vaccines is known in the art (see, e.g., Miyamura et al., (1994) Proc. Nat. Acad. Sci USA 91 :8507; U.S. Patent No.
  • the antigen may be presented in the parvovirus capsid. Alternatively, the antigen may be expressed from a heterologous nucleic acid introduced into a recombinant vector genome. Any immunogen of interest may be provided by the parvovirus vector.
  • Immunogens of interest are well-known in the art and include, but are not limited to, immunogens from human immunodeficiency virus, influenza virus, gag proteins, tumor antigens, cancer antigens, bacterial antigens, viral antigens, and the like.
  • an immunogenic polypeptide, or immunogen may be any polypeptide suitable for protecting the subject against a disease, including but not limited to microbial, bacterial, protozoal, parasitic, and viral diseases.
  • the immunogen may be an orthomyxovirus immunogen (e.g., an influenza virus immunogen, such as the influenza virus hemagglutinin (HA) surface protein or the influenza virus nucleoprotein gene, or an equine influenza virus immunogen), or a lentivirus immunogen (e.g., an equine infectious anemia virus immunogen, a Simian Immunodeficiency Virus (SIV) immunogen, or a Human Immunodeficiency Virus (HIV) immunogen, such as the HIV or SIV envelope GP160 protein, the HIV or SIV matrix/capsid proteins, and the HIV or SIV gag, pol and env genes products).
  • an influenza virus immunogen such as the influenza virus hemagglutinin (HA) surface protein or the influenza virus nucle
  • the immunogen may also be an arenavirus immunogen (e.g., Lassa fever virus immunogen, such as the Lassa fever virus nucleocapsid protein gene and the Lassa fever envelope glycoprotein gene), a poxvirus immunogen (e.g., vaccinia, such as the vaccinia L1 or L8 genes), a flavivirus immunogen (e.g., a yellow fever virus immunogen or a Japanese encephalitis virus immunogen), a filovirus immunogen (e.g., an Ebola virus immunogen, or a Marburg virus immunogen, such as NP and GP genes), a bunyavirus immunogen (e.g., RVFV, CCHF, and SFS viruses), or a coronavirus immunogen (e.g., an infectious human coronavirus immunogen, such as the human coronavirus envelope glycoprotein gene, or a porcine transmissible gastroenteritis virus immunogen, or an avian infectious bronchitis virus immunogen).
  • the immunogen may further be a polio immunogen, herpes antigen (e.g., CMV, EBV, HSV immunogens) mumps immunogen, measles immunogen, rubella immunogen, diptheria toxin or other diptheria immunogen, pertussis antigen, hepatitis (e.g., hepatitis A or hepatitis B) immunogen, or any other vaccine immunogen known in the art.
  • herpes antigen e.g., CMV, EBV, HSV immunogens
  • mumps immunogen e.g., measles immunogen
  • rubella immunogen e.g., diptheria toxin or other diptheria immunogen
  • pertussis antigen e.g., hepatitis A or hepatitis B immunogen
  • hepatitis e.g., hepatitis A or hepatitis B
  • the immunogen may be any tumor or cancer cell antigen.
  • the tumor or cancer antigen is expressed on the surface of the cancer cell. Exemplary cancer and tumor cell antigens are described in S.A. Rosenberg, (1999) Immunity 10:281 ).
  • cancer and tumor antigens include, but are not limited to: BRCA1 gene product, BRCA2 gene product, gp100, tyrosinase, GAGE-1/2, BAGE, RAGE, NY-ESO-1 , CDK-4, ⁇ - catenin, MUM-1 , Caspase-8, KIAA0205, HPVE, SART-1 , PRAME, p15, melanoma tumor antigens (Kawakami et al., (1994) Proc. Natl. Acad. Sci. USA 91 :3515); Kawakami et al., (1994) J. Exp. Med., 180:347); Kawakami et al., (1994) Cancer Res.
  • MART-1 Coulie et al., (1991 ) J. Exp. Med. 180:35), gp100 (Wick et al., (1988) J. Cutan. Pathol. 4:201) and MAGE antigen, MAGE-1 , MAGE-2 and MAGE-3 (Van der Bruggen et al., (1991 ) Science, 254:1643); CEA, TRP-1 , TRP-2, P-15 and tyrosinase (Brichard et al., (1993) J. Exp. Med. 178:489); HER-2/neu gene product (U.S. Pat. No.
  • telomeres nuclear matrix proteins
  • prostatic acid phosphatase papilloma virus antigens
  • antigens associated with the following cancers melanomas, metastases, adenocarcinoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, colon cancer, non-Hodgkins lymphoma, Hodgkins lymphoma, leukemias, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer and others (see, e.g., Rosenberg, (1996) Ann. Rev. Med. 47:481-91 ).
  • heterologous nucleotide sequence may encode any polypeptide that is desirably produced in a cell in vitro, ex vivo, or in vivo.
  • the parvovirus vectors may be introduced into cultured cells and the expressed gene product isolated therefrom.
  • heterologous nucleotide sequence(s) of interest may be operably associated with appropriate control sequences.
  • the heterologous nucleic acid may be operably associated with expression control elements, such as transcription/translation control signals, origins of replication, polyadenylation signals, and internal ribosome entry sites (IRES), promoters, enhancers, and the like.
  • expression control elements such as transcription/translation control signals, origins of replication, polyadenylation signals, and internal ribosome entry sites (IRES), promoters, enhancers, and the like.
  • IRS internal ribosome entry sites
  • promoter/enhancer elements may be used depending on the level and tissue- specific expression desired.
  • the promoter/enhancer may be constitutive or inducible, depending on the pattern of expression desired.
  • the promoter/enhancer may be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional
  • Promoter/enhancer elements that are native to the target cell or subject to be treated are most preferred. Also preferred are promoters/enhancer elements that are native to the heterologous nucleic acid sequence.
  • the promoter/enhancer element is chosen so that it will function in the target cell(s) of interest. Mammalian promoter/enhancer elements are also preferred.
  • the promoter/enhance element may be constitutive or inducible. Inducible expression control elements are preferred in those applications in which it is desirable to provide regulation over expression of the heterologous nucleic acid sequence(s).
  • Inducible promoters/enhancer elements for gene delivery are preferably tissue-specific promoter/enhancer elements, and include muscle specific (including cardiac, skeletal and/or smooth muscle), neural tissue specific (including brain-specific), eye (including retina-specific and cornea-specific), liver specific, bone marrow specific, pancreatic specific, spleen specific, and lung specific promoter/enhancer elements.
  • Other inducible promoter/enhancer elements include hormone-inducible and metal-inducible elements.
  • Exemplary inducible promoters/enhancer elements include, but are not limited to, a Tet on/off element, a RU486-inducible promoter, an ecdysone-inducible promoter, a rapamycin-inducible promoter, and a metalothionein promoter.
  • heterologous nucleic acid sequence(s) will be transcribed and then translated in the target cells
  • specific initiation signals are generally required for efficient translation of inserted protein coding sequences.
  • exogenous translational control sequences which may include the ATG initiation codon and adjacent sequences, can be of a variety of origins, both natural and synthetic.
  • the parvovirus vectors produced according to the present invention also provide a means for delivering heterologous nucleotide sequences into a broad range of cells, including dividing and non-dividing cells.
  • the parvovirus vectors may be employed to deliver a nucleotide sequence of interest to a cell in vitro, e.g., to produce a polypeptide in vitro or for ex vivo gene therapy.
  • the vectors are additionally useful in a method of delivering a nucleotide sequence to a subject in need thereof, e.g., to express an immunogenic or therapeutic polypeptide. In this manner, the polypeptide may thus be produced in vivo in the subject.
  • the subject may be in need of the polypeptide because the subject has a deficiency of the polypeptide, or because the production of the polypeptide in the subject may impart some therapeutic effect, as a method of treatment or otherwise, and as explained further below.
  • the parvovirus vectors produced according to the present invention may be employed to deliver any foreign nucleic acid with a biological effect to treat or ameliorate the symptoms associated with any disorder related to gene expression.
  • Illustrative disease states include, but are not limited to: cystic fibrosis (and other diseases of the lung), hemophilia A, hemophilia B, thalassemia, anemia and other blood disorders, AIDs, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, epilepsy, and other neurological disorders, cancer, diabetes mellitus, muscular dystrophies (e.g., Duchenne, Becker), Gaucher's disease, Hurler's disease, adenosine deaminase deficiency, glycogen storage diseases and other metabolic defects, retinal degenerative diseases (and other diseases of the eye), diseases of solid organs (e.g., brain, liver, kidney, heart), and the like.
  • cystic fibrosis and other diseases of the lung
  • a gene transfer vector may be administered that encodes any therapeutic polypeptide.
  • Gene transfer has substantial potential use in understanding and providing therapy for disease states. There are a number of inherited diseases in which defective genes are known and have been cloned. In general, the above disease states fall into two classes: deficiency states, usually of enzymes, which are generally inherited in a recessive manner, and unbalanced states, which may involve regulatory or structural proteins, and which are typically inherited in a dominant manner. For deficiency state diseases, gene transfer could be used to bring a normal gene into affected tissues for replacement therapy, as well as to create animal models for the disease using antisense mutations.
  • parvovirus vectors produced according to the methods of the present invention permit the treatment of genetic diseases.
  • a disease state is treated by partially or wholly remedying the deficiency or imbalance that causes the disease or makes it more severe.
  • the use of site-specific recombination of nucleic sequences to cause mutations or to correct defects is also possible.
  • the parvovirus vectors produced according to the present invention may also be employed to provide an antisense nucleic acid to a cell in vitro or in vivo. Expression of the antisense nucleic acid in the target cell diminishes expression of a particular protein by the cell.
  • antisense nucleic acids may be administered to decrease expression of a particular protein in a subject in need thereof.
  • Antisense nucleic acids may also be administered to cells in vitro to regulate cell physiology, e.g., to optimize cell or tissue culture systems.
  • the parvovirus vector may encode any other non- translated RNA, as described in more detail hereinabove.
  • parvovirus vectors produced according to the instant invention find further use in diagnostic and screening methods, whereby a gene of interest is transiently or stably expressed in a cell culture system, or alternatively, a transgenic animal model. Delivery of Immunogenic Polypeptides.
  • parvovirus vectors produced according to the present invention may be used to produce an immune response in a subject.
  • a parvovirus vector comprising a nucleotide sequence encoding an immunogen may be administered to a subject, and an active immune response is mounted by the subject against the immunogen. Immunogens are as described hereinabove. Preferably, a protective immune response is elicited.
  • the parvovirus vector may be administered to a cell ex vivo and the altered cell is administered to the subject.
  • the heterologous nucleotide sequence is permitted to be introduced into the cell, and the cell is administered to the subject, where the heterologous nucleotide sequence encoding the immunogen is preferably expressed and induces an immune response in the subject against the immunogen.
  • the cell is an antigen presenting cell (e.g., a dendritic cell) or a cancer.
  • an “active immune response” or “active immunity” is characterized by “participation of host tissues and cells after an encounter with the immunogen. It involves differentiation and proliferation of immunocompetent cells in lymphoreticular tissues, which lead to synthesis of antibody or the development of cell-mediated reactivity, or both.” Herbert B. Herscowitz, Immunophysiology: Cell Function and Cellular Interactions in Antibody Formation, in IMMUNOLOGY: BASIC PROCESSES 117 (Joseph A. Bellanti ed., 1985). Alternatively stated, an active immune response is mounted by the host after exposure to immunogens by infection or by vaccination.
  • Active immunity can be contrasted with passive immunity, which is acquired through the "transfer of preformed substances (antibody, transfer factor, thymic graft, interleukin-2) from an actively immunized host to a non-immune host.”
  • a "protective" immune response or “protective” immunity as used herein indicates that the immune response confers some benefit to the subject in that it prevents or reduces the incidence of disease.
  • a protective immune response or protective immunity may be useful in the treatment of disease, in particular cancer or tumors (e.g., by causing regression of a cancer or tumor and/or by preventing metastasis and/or by preventing growth of metastatic nodules).
  • the protective effects may be complete or partial, as long as the benefits of the treatment outweigh any disadvantages thereof.
  • the pa ⁇ /ovirus vector carrying the heterologous nucleotide sequence is administered in an immunogenically effective amount, as described below.
  • the parvovirus vectors produced according to the present invention may also be administered for cancer immunotherapy by administration of a parvovirus vector expressing cancer cell antigens or any other immunogen that produces an immune response against a cancer cell.
  • an immune response may be produced against a cancer cell antigen in a subject by administering a parvovirus vector comprising a heterologous nucleotide sequence encoding the cancer cell antigen, for example to treat a patient with cancer.
  • the parvovirus vector may be administered to a subject in vivo or by using ex vivo methods, as described herein.
  • cancer encompasses tumor-forming cancers.
  • cancer cell antigen encompasses tumor antigens.
  • cancer has its understood meaning in the art, for example, an uncontrolled growth of tissue that has the potential to spread to distant sites of the body (i.e., metastasize).
  • exemplary cancers include, but are not limited to, leukemias, lymphomas, colon cancer, renal cancer, liver cancer, breast cancer, lung cancer, prostate cancer, ovarian cancer, melanoma, and the like. Preferred are methods of treating and preventing tumor-forming cancers.
  • Tumor is also understood in the art, for example, as an abnormal mass of undifferentiated cells within a multicellular organism. Tumors can be malignant or benign. Preferably, the methods disclosed herein are used to prevent and treat malignant tumors.
  • Cancer cell antigens according to the present invention have been described hereinabove.
  • treating cancer or “treatment of cancer” it is intended that the severity of the cancer is reduced or the cancer is at least partially eliminated.
  • these terms indicate that metastasis of the cancer is reduced or at least partially eliminated. It is further preferred that these terms indicate that growth of metastatic nodules (e.g., after surgical removal of a primary tumor) is reduced or at least partially eliminated.
  • prevention of cancer or “preventing cancer” it is intended that the methods at least partially eliminate or reduce the incidence or onset of cancer. Alternatively stated, the onset of cancer in the subject may be slowed, controlled, decreased in likelihood or probability, or delayed.
  • cells may be removed from a subject with cancer and contacted with parvovirus particles produced according to the instant invention.
  • the modified cell is then administered to the subject, whereby an immune response against the cancer cell antigen is elicited.
  • This method is particularly advantageously employed with immunocompromised subjects that cannot mount a sufficient immune response in vivo (i.e., cannot produce enhancing antibodies in sufficient quantities).
  • immunomodulatory cytokines e.g., ⁇ -interferon, ⁇ -interferon, ⁇ -interferon, ⁇ - interferon, ⁇ -interferon, interleukin-1 ⁇ , interleukin-1 ⁇ , interleukin-2, interleukin- 3, interleukin-4, interleukin 5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11 , interleukin 12, interleukin-13, interleukin-14, interleukin-18, B cell Growth factor, CD40 Ligand, tumor necrosis factor- ⁇ , tumor necrosis factor- ⁇ , monocyte chemoattractant protein- 1 , granulocyte-macrophage colony stimulating factor, and lymphotoxin).
  • immunomodulatory cytokines preferably, CTL inductive cytokines
  • Cytokines may be administered by any method known in the art. Exogenous cytokines may be administered to the subject, or alternatively, a nucleotide sequence encoding a cytokine may be delivered to the subject using a suitable vector, and the cytokine produced in vivo.
  • the parvovirus vector may be administered as part of a method of treating cancer by administering anti-cancer agents (e.g., cytokines, tumor suppressor gene products, as described above)
  • the parvovirus particle may be administered to a cell in vitro or to a subject in vivo or by using ex vivo methods, as described herein and known in the art.
  • Parvovirus vectors produced according to the present invention find use in both veterinary and medical applications. Suitable subjects for ex vivo gene delivery methods as described above include both avians and mammals, with mammals being preferred.
  • avian as used herein includes, but is not limited to, chickens, ducks, geese, quail, turkeys and
  • the term "mammal” as used herein includes, but is not limited to, humans, bovines, ovines, caprines, equines, felines, canines, lagomorphs, etc. Human subjects are most preferred. Human subjects include neonates, infants, juveniles, and adults.
  • the present invention provides a
  • composition comprising a virus particle of the invention in a pharmaceutically acceptable carrier and/or other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc.
  • the carrier will typically be a liquid.
  • the carrier may be either solid or liquid.
  • the carrier will be
  • water that contains the additives usual for injection solutions, such as stabilizing agents, salts or saline, and/or buffers.
  • a “physiologically acceptable carrier” is one that is not toxic
  • physiologically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free, phosphate buffered saline.
  • physiologically acceptable carriers include pharmaceutically acceptable carriers.
  • biologically or otherwise undesirable i.e., the material may be administered to a subject without causing any undesirable biological effects.
  • a pharmaceutical composition may be used, for example, in transfection of a cell ex vivo or in administering a viral particle or cell directly to a subject.
  • One aspect of the present invention is a method of transferring a nucleotide sequence to a cell in vitro.
  • the virus particles may be added to the cells at the appropriate multiplicity of infection according to standard transduction methods appropriate for the particular target cells. Titers of virus to administer can vary, depending upon the target cell type and number, and the particular virus vector, and can be determined by those of skill in the art without undue experimentation.
  • the cell(s) to be administered the parvovirus vector may be of any type, including but not limited to neural cells (including cells of the peripheral and central nervous systems, in particular, brain cells such as neurons and oligodendricytes), lung cells, cells of the eye (including retinal cells, retinal pigment epithelium, and corneal cells), epithelial cells (e.g., gut and respiratory epithelial cells), muscle cells, dendritic cells, pancreatic cells (including islet cells), hepatic cells, myocardial cells, bone cells (e.g., bone marrow stem cells), hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, germ cells, and the like.
  • neural cells including cells of the peripheral and central nervous systems, in particular, brain cells such as neurons and oligodendricytes
  • lung cells including retinal cells, retinal pigment epithelium, and corneal cells
  • epithelial cells e.
  • the cell may be any progenitor cell.
  • the cell can be a stem cell (e.g., neural stem cell, liver stem cell).
  • the cell may be a cancer or tumor cell.
  • the cells can be from any species of origin, as indicated above.
  • the parvovirus vectors may be administered to cells in vitro for the purpose of administering the modified cell to a subject.
  • the cells have been removed from a subject, the parvovirus vector is introduced therein, and the cells are then replaced back into the subject.
  • Methods of removing cells from subject for treatment ex vivo, followed by introduction back into the subject are known in the art (see, e.g., U.S. patent No. 5,399,346; the disclosure of which is incorporated herein in its entirety).
  • the recombinant parvovirus vector is introduced into cells from another subject, into cultured cells, or into cells from any other suitable source, and the cells are administered to a subject in need thereof.
  • Suitable cells for ex vivo gene therapy are as described above. Dosages of the cells to administer to a subject will vary upon the age, condition and species of the subject, the type of cell, the nucleic acid being expressed by the cell, the mode of administration, and the like. Typically, at least about 10 2 to about 10 8 , preferably about 10 3 to about 10 6 cells, will be administered per dose in a pharmaceutically acceptable carrier.
  • the cells transduced with the parvovirus vector are preferably administered to the subject in a therapeutically effective amount in combination with a pharmaceutical carrier.
  • a “therapeutically effective” amount as used herein is an amount that provides sufficient expression of the heterologous nucleotide sequence delivered by the vector to provide some improvement or benefit to the subject.
  • a “therapeutically effective” amount is an amount that will provide some alleviation, mitigation, or decrease in at least one clinical symptom in the subject.
  • cells that have been transduced with a parvovirus vector may be administered to elicit an immunogenic response against the delivered polypeptide (e.g., expressed as a transgene or in the capsid).
  • an immunogenic amount of the polypeptide in combination with a pharmaceutically acceptable carrier is administered.
  • An "immunogenic amount” is an amount of the expressed polypeptide that is sufficient to evoke an active immune response in the subject to which the pharmaceutical formulation is administered.
  • the dosage is sufficient to produce a protective immune response (as defined above).
  • the degree of protection conferred need not be complete or permanent, as long as the benefits of administering the immunogenic polypeptide outweigh any disadvantages thereof.
  • a further aspect of the invention is a method of treating subjects in vivo with the parvovirus particles.
  • Administration of the parvovirus particles produced according to the present invention to a human subject or an animal in need thereof can be by any. means known in the art for administering virus vectors.
  • the parvovirus vector is delivered in a therapeutically effective dose in a pharmaceutically acceptable carrier.
  • the parvovirus vectors of the invention may be administered to elicit an immunogenic response (e.g., as a vaccine).
  • vaccines of the present invention comprise an immunogenic amount of infectious virus particles as disclosed herein in combination with a pharmaceutically acceptable carrier.
  • the dosage is sufficient to produce a protective immune response (as defined above).
  • the degree of protection conferred need not be complete or permanent, as long as the benefits of administering the immunogenic polypeptide outweigh any disadvantages thereof.
  • Subjects and immunogens are as described above. Dosages of the parvovirus particles to be administered to a subject will depend upon the mode of administration, the disease or condition to be treated, the individual subject's condition, the particular virus vector, and the nucleic acid to be delivered, and can be determined in a routine manner.
  • Exemplary doses for achieving therapeutic effects are virus titers of at least about 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 3 , 10 14 , 10 15 transducing units or more, preferably about 10 8 - 10 13 transducing units, yet more preferably 10 12 transducing units.
  • more than one administration e.g., two, three, four or more administrations
  • more than one administration may be employed to achieve the desired level of gene expression.
  • Exemplary modes of administration include oral, rectal, transmucosal, topical, transdermal, in utero (or in ovo), inhalation, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, and intraarticular) administration, and the like, as well as direct tissue or organ injection, alternatively, intrathecal, direct intramuscular, intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • the parvovirus vector administered to the subject may transduce any permissive cell or tissue. Suitable cells for transduction by the parvovirus vectors are as described above.
  • the nucleotide sequence of interest is delivered to the liver of the subject. Administration to the liver may be achieved by any method known in the art, including, but not limited to intravenous administration, intraportal administration, intrabiliary administration, intra-arterial administration, and direct injection into the liver parenchyma.
  • the parvovirus particles are administered intramuscularly, more preferably by intramuscular injection or by local administration (as defined above). Delivery to the brain is also preferred. In other preferred embodiments, the parvovirus particles of the present invention are administered to the lungs.
  • the parvovirus vectors disclosed herein may be administered to the lungs of a subject by any suitable means, but are preferably administered by administering an aerosol suspension of respirable particles comprised of the parvovirus vectors, which the subject inhales.
  • the respirable particles may be liquid or solid.
  • Aerosols of liquid particles comprising the pa ⁇ /ovirus vectors may be produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is known to those of skill in the art. See, e.g., U.S. Patent No. 4,501 ,729. Aerosols of solid particles comprising the parvovirus vectors may likewise be produced with any solid particulate medicament aerosol generator, by techniques known in the pharmaceutical art.
  • the plasmid pBS+ (Stratagene) was used as the backbone for cloning AAV serotypes 1 to 5 capsid genes.
  • the AAV2 replication gene was subcloned into the plasmid pAAV2Cap (Rabinowitz et al. (1999), Virology, 265:274) by using a Xba ⁇ (blunt ended) Swa ⁇ digestion of pACG2 (Li et al. (1997) J. Virol. 71:5236) and the SmaUSwa ⁇ digestion of pAAV2Cap.
  • the new plasmid, pAAV2rep was digested with ⁇ /ael, blunt ended with mung bean nuclease, and digested with Swa ⁇ , removing the capsid genes and leaving only the replication gene from AAV2.
  • Viral sequences from AAV1 , -2, -3, -4, -5 were cloned from ATCC stocks. Primers were designed for each of these serotypes, such that the Swal was present before the coding region of Vp1 and a unique Not ⁇ site was present after the polyadenylation site.
  • the forward primers used were AAV1 and -2 (5'-AATCAGGTATGGCTGCCGAT-3' SEQ ID NO:1), AAV3 (5'-AAATCAGGTATGGCTGCTGAT-3' SEQ ID NO:2), AAV4 (5'- AAATCAGGTATGGCTGCTGACGGTTAC-3' SEQ ID NO:3), and AAV5 (5'- AAATCAGGTATGGCTTTTGTTGATCAC-3' SEQ ID NO:4).
  • the reverse primers used were AAV1 , -2, -3, and -4 (5'-AATCAGGTATGGCTGCCGAT-3' SEQ ID NO:1), AAV3 (5'-AAATCAGGTATGGCTGCTGAT-3' SEQ ID NO:2), AAV4 (5'- AAATCAGGTATGGCTGCTGACGGTTAC-3' SEQ ID NO:3), and AAV5 (5'- AAATCAGGTATGGCTTTTGTTGATCAC-3' SEQ ID NO:4).
  • the reverse primers used were AAV
  • Pfu polymerase (Stratagene) was used in the PCR to generate the serotype-specific clones with blunt ends. These serotype-specific capsid coding fragments were then cloned into pAAV2rep. A BsfNI digestion was used to confirm the presence and orientation of the positive clones (Fig. 1, panel B). In clones containing the serotype 3, 4, or 5 capsid gene, a portion of the serotype-specific rep gene was substituted for that of the AAV2 rep gene.
  • the AAV3 serotype clone was digested with >4ccl, as was the original plasmid (nucleotide 1424 to 4355 of the AAV3 sequence, Fig. 1 , panel A).
  • the AAV4 serotype clone was digested with Acc ⁇ and .gel, as was the original plasmid (nucleotides 1479 to 4488 of the AAV4 sequence, Fig. 1 , panel A).
  • the AAV5 serotype clone was digested with BamYW, as was the original plasmid (nucleotides 1071 to 2276 of the AAV5 sequence, Fig 1 , panel A).
  • clones were designated pXR1 , -2, -3, -4, and -5 respectively.
  • the vector plasmid pTR/CMV/GFP enhanced green fluorescent protein [EGFP] transgene
  • CMV cytomegalovirus
  • helper plasmid pXX6-80 containing adenovirus genes required for AAV production was a gift from Xiao Xiao, and the plasmid pCB-AAT was provided by Terry Flotte.
  • All cell lines (293 human embryonic kidney, HeLa, Cos 7, Cos 1 , CHO K1 and CHO K1 mutant p) were originally obtained from ATCC, and maintained in 5% C0 2 saturation at 37°C. 293T, HeLa, Cos1 and Cos7 cells were cultured in Dulbecco's Modified Eagle's medium (DM EM) with 10% fetal bovine serum (Sigma), and CHO K1 , where as CHO Kland were cultured in F12 Nutrient Mixture (HAM) with 10% fetal bovine serum.
  • DM EM Dulbecco's Modified Eagle's medium
  • HAM F12 Nutrient Mixture
  • Example 3 Production of Serotvpe-Specific Recombinant AAV Vectors Production of all recombinant AAV vectors used in these investigations utilized a three-plasmid transfection scheme outlined in Rabinowitz et al. (2002) J Virol. 76:(ln press) and Xiao et al. (1998), J. Virol. 72:2224.
  • Cells were transfected using Superfect (Qiagen) according to manufacturer's specifications, in which equimolar amounts of each plasmid — 75micrograms of pTR/CMV/GFP (vector), 150 micrograms of pXX6-80 (Ad helper), and 75 micrograms of serotype-specific plasmids pXR1 , -2, -3, -4, or -5 — was used. Cells were harvested 48 h post-transfection and recombinant virus was isolated as described in Rabinowitz et al. (1999), Virology, 265:274.
  • Recombinant viruses were purified with two rounds of cesium chloride isopycnic centrifugation. To each milliliter of viral supernatant, 0.59 g of CsCI was added, and 1.38 g of CsCI/ml was added to a final volume of 12 ml. The solution was centrifuged at 37,000 rpm for 36 to 48 h (Son/all Ultra 80 rotor). Peak fractions from the first cesium gradient were determined by infection of HeLa cells except for AAV4, which was tested on Cos1 cells. A second CsCI gradient was incorporated for further purity at 65,000 rpm for 4 h (Beckman NVT-65 rotor).
  • Peak fractions from the second cesium gradient were determined by dot blot hybridization described below, then subjected to heparin-Sepharose column chromatography as described in Rabinowitz et al. (2002) J Virol. 76;(ln press) and Rabinowitz et al. (1999), Virology, 265:274.
  • Example 4 SDS Polyacrylamide Gel Electrophoresis and Western Blots
  • Cell-free lysates were derived from transfected human embryonic kidney cells 293 as described in Rabinowitz et al. (1999), Virology, 265:274 and subject to Western blot analysis. Briefly, an aliquot was removed, to which PMSF at 100 Dg/ml (Sigma), pepstatin A at 1 Dg/ml (Sigma), and leupeptine at 2 Dg/ml (Sigma) were added. Protein concentrations were estimated with a BCA Protein Assay kit (Pierce). Equivalent amounts of protein (5 Dg/lane) from lysates of each serotype were fractionated on a 10% denaturing polyacrylamide gel and then transferred to Hybond ECL nitrocellulose membranes (Amersham Pharmacia Biotech).
  • DNA from AAV serotype transfected 293 cells was isolated 24 h post- transfection using a method described in Hirt (1967) J. Mol: Biol. 26:365. Approximately half of each sample was digested with Dpn ⁇ , and 2.5 micrograms of digested and undigested DNA was fractionated on a 0.8% agarose gel. After transferring the DNA onto GeneScreen Plus charged nylon membranes (NEN), the membranes were probed with a 735 basepair Not ⁇ fragment from pTR/CMV/GFP that carries the coding sequence for the packaged transgene (GFP). The positive signal was visualized by phosphorescence autoradiography using a Storm Phosphorlmager and quantitated using ImageQuant software (Molecular Dynamics).
  • Example 6 Heparin Column Chromatographv
  • a preset program (12 ml of buffer at 137 mM NaCI, 2 mM MgCI 2 , and 2 mM KCI run over the column at a flow rate 0.5 ml/min for the flowthrough and wash, followed by a waste wash of 10 ml of the same buffer at 1 ml/min) was implemented before elution.
  • the elution step used a continuous salt gradient from 137 mM NaCI to 1 M NaCI at a flow rate of 0.5 ml/min.
  • Transducing units were determined for each fraction by transducing either HeLa (AAV1 , -2, -3, and -5) or Cos1 cells (AAV4) in triplicate.
  • the titer of the input virus was also determined so that a percentage of the recovered TU to the total number could be calculated.
  • Dot blots were performed as described previously in Rabinowitz et al. (1999), Virology, 265:274. Two to 10 microliters from fractions off the cesium gradient, after dialysis, or off the heparin column were blotted onto GeneScreen Plus charged nylon membranes (NEN) using a dot blot manifold (Schleicher and Schuell) and the DNA was UV cross-linked to the membranes at 60 mJ with a Stratalinker 1800 UV crosslinker (Stratagene). The blots were probed for positive signal with the GFP DNA probe described in Example 4.
  • Example 9 ELISA Analysis of Factor IX and 1 -Antitrypsin NOD/Scid, BALB/c, and C57/BL mice (Jackson Laboratories) were maintained and treated in accordance with the guidelines of the Animal Care and Use Committee of University of North Carolina at Chapel Hill. Portal vein and muscular injection were performed and Canine factor IX antigen was detected by ELISA as previously described in Chao et al. (2000) Mol. Ther. 2:619. A modified double antibody sandwich enzyme-linked immunosorbent assay was used for measurement of alpha-1 -antitrypsin in biologic fluids as described in Michalski et al. (1985) J. Immunol. Methods 83:101.
  • Example 10 Subretinal Injection and in vivo Fluorescence Imaging
  • AAV serotype specific hybrid helper plasmids utilized a common AAV2 rep gene pACG2 described in Li et al. (1997) J. Virol. 71 :5236, and the respective capsid coding sequences from each of the 5 serotypes (Fig. 1 panel A).
  • the ACG mutation of the p5 start site was chosen because this mutation has been shown to improve vector production by reducing rep78/68 while increasing AAV cap expression (Li et al. (1997) J. Virol. 71:5236).
  • pACG2 rep sequences were cloned into pAAV2Cap (Stratagene pBS+ backbone previously described (Rolling et al. (1999) Hum. Gene Ther.
  • AAV2 ACG replication gene and the serotype specific capsid sequences were called pXR1-5.
  • BstN ⁇ digestion (Fig. 1 panel B) and DNA sequencing determined correct orientation and nucleotide sequence of the serotype specific capsid gene.
  • vector yields varied significantly among these constructs (Tablel).
  • additions of serotype specific non-capsid coding sequences (5' to the VP1 start site) reduced this variation (see Fig. 1 , Table 1).
  • helper proteins Functional activity of these helper proteins were determined by replication and encapsidation of rAAV type 2 GFP template.
  • serotypes 1 , 4, and 5 were observed to have yields within 4 fold of each other after numerous production runs (5x), with serotypes 2 and 3 demonstrating the highest yields (9.8 x 10 12 , 7.2 x 10 11 respectively, Table 1).
  • the serotype 1 helper produced the least amount of virus (1.27x10 11 and Table 1 ), consistent with the Hirt replication analysis.
  • Example 14 Heparin Column Binding Profiles of AAV Hybrid Serotypes
  • iodixanol gradient and/or heparin column binding has allowed for the rapid purification of rAAV type 2 (Auricchio et al. (2001 ) Hum. Gene Ther. 12:71 , Zolotukhin et al. (1999) Gene Ther. 6:973).
  • these methods allow for purification of vector with better ratio of transducing units to particle numbers. This purification scheme was applied to the hybrid serotypes and elution profiles determined by GFP transduction as described in Example 8
  • Recombinant AAV2 demonstrated elution profiles as previously published (Zolotukhin et al. (1999) Gene Ther. 6:973), with less than 1 % of starting material recovered in the flow-through and the majority of the TU being recovered in the elution step (Fig. 4, AAV2).
  • rAAV 3 displayed more efficient binding to the column and elution profiles near identical to type 2. These results indicate that the elution conditions used for rAAV2 purification can be applied to hybrid rAAV3.
  • Recombinant AAV1 and -5 displayed similar profiles to one another with 60% of type 1 and 80% of type 5 TU recovered in the flow-through (Fig. 4, AAV1 and 5). The virus eluted from the salt gradient represent only a small portion of the total applied to the column (Fig. 4, AAV1 and 5).
  • transgene were chicken D-actin promoter with the CMV enhancer-driven hAAT (both C57/BL and BALB/c mice were injected with 5 x 10 10 particles/mouse) and CMV immediate-early promoter-driven dog factor IX (dF9) (SCID mice were injected with 2 x 10 11 particles/mouse, and BALB/c mice were injected with 10 10 particles/mouse.
  • CMV enhancer-driven hAAT both C57/BL and BALB/c mice were injected with 5 x 10 10 particles/mouse
  • dF9 CMV immediate-early promoter-driven dog factor IX
  • SCID mice CMV immediate-early promoter-driven dog factor IX mice were injected with 2 x 10 11 particles/mouse
  • BALB/c mice were injected with 10 10 particles/mouse.
  • b For each group of animals, serotype, and route of injection, a minimum of three animals were used. Abbreviations for route of injection:
  • Two B19 helper plasmids carrying the B19 VP1 and VP2 coding sequences and a chimeric non-structural protein containing N-terminal elements from the AAV2 Rep 78/68 protein and C-terminal elements from the B19 NS-1 protein is constructed in a similar manner to the AAV hybrid helper plasmids described in Examples 1 and 9 by cloning the appropriate B19 viral sequences into the pXR2 construct described in Example 1. These include both the complete B19 capsid (VP1 and VP2) protein coding sequences, and the C-terminal portion of the B19 NS-1 protein.
  • the two helper plasmids are constructed by the ligation of the PCR amplification products of: the B19 sequences described in Shade et al. (1986) J. Virol. 58:921 , and the backbone of pXR2 containing the N-terminal portion of the AAV-2 rep coding sequence.
  • the chimeric Rep proteins that are coded for in these constructs contain: (1) the portion of the N-terminal amino acids of AAV Rep78/68 up to the p19 promoter, and (2) up to the p40 promoter, and the appropriate C- terminal amino acids from B19 NS-1.
  • the PCR primers that are used to prepare (1) are as follows: 5'-ATGGTAAACTGGTTGTGTGAAAAC-3' (SEQ ID NO:7) and 5'-GCAACAACATAATTTTTTAACCAC-3' (SEQ ID NO:8), which amplify B19 sequences as described in Shade et al. (1986) J. Virol. 58:921 , and 5'-GTACCTGGCTGAAGTTTTTGATCT-3' (SEQ ID NO:9) and 5'- GGCCGCTCGATAAGCTTTTGTTCC-3' (SEQ ID NO:10), which amplify the pXR2 backbone carrying the AAV2 rep coding region up to the P19 promoter.
  • the PCR primers used to prepare (2) are as follows: 5'- AGCACGAGTGGTGGTGAAAGCTCT-3' (SEQ ID NO:11) and 5'- GCAACAACATAATTTTTTAACCAC-3' (SEQ ID NO: 8), which amplify B19 sequences as described in Shade et al. (1986) J. Virol. 58:921 , and 5'- TGGCTGGCGAACTGACTCGCGCAC-3' (SEQ ID NO:12) 5'-
  • GGCCGCTCGATAAGCTTTTGTTCC-3' (SEQ ID NO:10), which amplify the pXR2 backbone carrying the AAV2 rep coding region up to the P40 promoter. In both cases, the resulting PCR products are then blunt end ligated together to generate the desired construct, and whose structure is confirmed by restriction digestion.
  • the resulting AAV helper plasmids are used in conjunction with the Ad helper plasmid pXX6-80 to package the rAAV-2 transgene pTRUF as described in Example 2.
  • the resulting virus preparations from this AAV helper construct are analyzed for particle yield as described in Example 11.

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Abstract

L'invention porte sur des polynucléotides comprenant les séquences de codage rep de parvovirus chimères (par exemple AAV). Lesdits polynucléotides codent facultativement pour les séquences de codage cap de parvovirus L'invention porte également sur des vecteurs et cellules comprenant les polynucléotides de l'invention, et sur des procédés améliorés de constitution de stocks de parvovirus hybrides utilisant les séquences de codage rep de parvovirus chimères de l'invention.
PCT/US2002/038423 2001-12-18 2002-12-02 Reactifs ameliores et procedes de production de parvovirus WO2003104392A2 (fr)

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US8241622B2 (en) 2001-07-13 2012-08-14 University Of Iowa Research Foundation Adeno-associated virus vectors with intravector heterologous terminal palindromic sequences
HU230406B1 (hu) 2001-11-13 2016-04-28 The Trustees Of The University Of Pennsylvania Eljárás adeno-asszociált vírus(AAV)szekvenciák detektálására és/vagy azonosítására és az azzal azonosított új szekvenciák izolálására
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MX2016011686A (es) 2014-03-09 2016-11-07 Univ Pennsylvania Composiciones utiles en el tratamiento de deficiencia de la ornitina transcarbamilasa (otc).
WO2017139381A1 (fr) 2016-02-08 2017-08-17 University Of Iowa Research Foundation Procédés pour produire des virus adéno-associés/bocavirus parvovirus chimériques
MA43735A (fr) 2016-03-07 2018-11-28 Univ Iowa Res Found Expression médiée par aav utilisant un promoteur et un activateur synthétiques
WO2018132747A1 (fr) 2017-01-13 2018-07-19 University Of Iowa Research Foundation Petit arn non codant pour bocaparvovirus et ses utilisations
EP3456822A1 (fr) * 2017-09-19 2019-03-20 CEVEC Pharmaceuticals GmbH Indunctible aav rep gènes
KR20200130337A (ko) * 2018-03-06 2020-11-18 유니버시티 오브 플로리다 리서치 파운데이션, 인코포레이티드 Aav 키메라
CN108465107B (zh) * 2018-06-05 2020-02-21 山东信得科技股份有限公司 一种鸭2型腺病毒和番鸭细小病毒病二联灭活疫苗
US10557149B1 (en) * 2019-07-15 2020-02-11 Vigene Biosciences, Inc. Recombinantly-modified adeno-associated virus helper vectors and their use to improve the packaging efficiency of recombinantly-modified adeno-associated virus
WO2023220035A1 (fr) * 2022-05-09 2023-11-16 Synteny Therapeutics, Inc. Compositions d'érythroparvovirus et méthodes de thérapie genique
WO2023220043A1 (fr) * 2022-05-09 2023-11-16 Synteny Therapeutics, Inc. Érythroparvovirus à génome modifié pour thérapie génique

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