WO2003104392A2

WO2003104392A2 - Improved reagents and methods for producing parvoviruses

Info

Publication number: WO2003104392A2
Application number: PCT/US2002/038423
Authority: WO
Inventors: Richard Jude Samulski; Joseph E. Rabinowitz
Original assignee: University Of North Carolina At Chapel Hill
Priority date: 2001-12-18
Filing date: 2002-12-02
Publication date: 2003-12-18
Also published as: AU2002367943A8; WO2003104392A9; AU2002367943A1; WO2003104392A3

Abstract

The present invention provides polynucleotides comprising chimeric parvovirus (e.g., AAV) rep coding sequences. The polynucleotides optionally encode parvovirus cap coding sequences. Also provided are vectors and cells comprising the inventive polynucleotides. Further provided are improved methods of making hybrid parvovirus stocks using the chimeric rep coding sequences of the invention.

Description

Improved Reagents and Methods for Producing Parvoviruses

Related Application Information

This application claims the benefit of United States Provisional Application Serial No. 60/341 ,919, Filed 18 December 2001 ; the disclosure of which is incorporated herein by reference in its entirety.

Statement of Federal Support

This invention was made, in part, with government support under grant numbers DK54419, HL51818 and GM59290 from the National Institutes of Health. The United States government has certain rights to this invention.

Field of the Invention

This invention relates to improved reagents and methods for producing virus, and more particularly relates to improved reagents and methods for producing parvovirus stocks.

Background of the Invention

The adeno-associated viruses (AAV) 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. (1996) Virology 221:208, Rutledge et al. (1998) J. Virol. 72:309, Srivastava et al. (1983) J.Virol. 45:555, Xiao et al. (1999) J. Virol. 73:3994). These clones have provided valuable reagents for studying the molecular biology of serotype specific infection. Transduction of these viruses naturally results in latent infections, with completion of the life cycle generally requiring helper functions not associated with AAV viral gene products. As a result, all of these serotypes are classified as non-pathogenic and are believed to share a safety profile similar to the more extensively studied AAV type 2 (Carter and Laughlin (1984) in, The Parvoviruses p. 67- 152 New York, N.Y.).

The extensive development of 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). As the number of applications evaluating gene transfer increases in vitro and in vivo, limitations to efficient rAAV type 2 transduction have become apparent (Bartlett et al. (2000) J. Virol. 74:2777, Davidson et al. (2000) Proc. Natl. Acad. Sci. USA 97:3428, Hansen et al. (2001) J Virol. 75:4080, Samulski et al. (1999) in, Adeno-associated viral vectors Cold Spring Harbor, N.Y., Walters et al. (2000) J. Virol 74:535, Xiao et al. (1999) J. Virol. 73:3994, Zabner et al. (2000) J. Virol. 74:3852). The natural tropism of any virus, including rAAV type 2, is a fundamental limitation to efficient gene transfer. With the identification of the AAV type 2 receptor, the requirements for efficient entry in target cells have become a critical topic of study (Summerford and Samulski (1998) J. Virol. 72:1438). Efforts have been made to overcome these restrictions by broadening the host range using either bispecific antibodies to the virion shell (Bartlett et al. (1999) Nat. Biotechnol. 17:181 ) or through capsid insertional mutagenesis (International patent publication WO 00/28004; Rabinowitz et al. (1999) Virology 265:274; Girod et al. (1999) Nat. Med. 5:1052, Wu et al. (2000) J.Virol. 74:8635). While these efforts are beginning to bear fruit, utilizing the other serotypes of AAV may yet provide additional resources for making safe and efficient gene transfer vectors. To this end, a number of studies have begun to show the utility of serotype specific vectors in vitro and in vivo (International patent publication WO 00/28004; Chao et al. (2000) Mol. Ther. 2:619, Chiorini et al. (3999) J. Virol. 73:1309, Chiorini et al. (1998) Mol. Cell. Biol. 18:5921 , Davidson et al. (2000) Proc. Natl. Acad. Sci. USA 97:3428, Hildinger et al. (2001 ) J. Virol. 75:6199, Xiao et al. (1999) J. Virol. 783:3994, Zabner et al. (2000) J. Virol. 74:3852). In general, each of these studies uncovered broader cell type specificity with increased gene transfer in vivo.

International patent publication WO 00/28004 describes "hybrid" parvoviruses that result from cross-packaging of a recombinant parvovirus genome in a capsid from a different parvovirus. There is a need in the art to further characterize the virus-specific vector components (e.g., the cis-acting terminal repeats [TRs] and the trans-acting virion shell and replication proteins) to produce a new generation of optimized hybrid parvovirus vectors and reagents, and methods for producing the same.

Summary of the Invention

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.

Accordingly, as one aspect, 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. As another aspect, 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.

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. As a further aspect, 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.

Also provided is 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 parvovirus particles comprising the parvovirus capsid encoded by the cap coding sequences and packaging the recombinant parvovirus template are produced in the cell.

As still a further aspect, 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 recombinant hybrid AAV particles comprising the AAV capsid encoded by the cap coding sequences and packaging the rAAV template are produced in the cell.

These and other aspects of the invention are set forth in more detail in the description of the invention below.

Brief Description of the Drawings 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 Bst \. 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 1F11 , (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 Dpnl 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 (WI) 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/μl for each serotype in each cell line. The particle numbers used in this experiment (per microliter) were as follows: rAAV1 , 1.2 x 10⁸; rAAV2, 5.6 x 10⁸; rAAV3, 9.1 x 10⁸; rAAV4, 2.3 x 10⁸; and rAAV5, 5.4 x 10⁸. 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. Subretinal injections of rAAV were performed via a transcleral transchoroidal approach on wild-type Wistar rats as previously described (Rolling et al. (1999) Hum. Gene Ther. 10:641). Briefly, the sclera and the choroid were punctured, a 33-gauge needle was then inserted in a tangential direction under an operating microscope. Three microliters of each of the five hybrid rAAV serotypes (5x10¹⁰ particles/ml) was delivered into the subretinal space (n = 3, for each serotype). A new method using fundus photography has been developed and performed in order to control the accuracy and reproducibility of subretinal injections (Rolling et al. in preparation). GFP protein expression in live rats was monitored by fluorescent retinal imaging using a Canon UVI retinal camera connected to a digital imaging system and Lhedioph Win software at 12, 26 and 46 days post-injection.

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;

GenBank Accession No. NC 001729; SEQ ID NO: 21.

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

GenBank Accession No. AF513852; SEQ ID NO: 27.

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:67) and AAV8 (SEQ ID NO:68), as well as a consensus sequence (SEQ ID NO:69). Dashes indicate gaps in the sequence and shading indicates positions of sequence homology.

Detailed Description of the Invention

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.

The present invention will now be described with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in

the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

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). Except as otherwise indicated, standard methods known to those skilled in the art may be used for the construction of recombinant parvovirus and rAAV constructs, packaging vectors expressing the parvovirus rep and/or cap sequences, and transiently and stably transfected packaging cells. Such techniques are known to those skilled in the art. See, e.g., SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed. (Cold Spring Harbor, NY, 1989); F. M. AUSUBEL ef al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

The following terms are used in the description herein and the appended claims: The term "parvovirus" as used herein encompasses the family

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. FIELDS et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). 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).

The parvovirus particles, capsids and genomes of the present invention are preferably from, but not limited to, AAV. 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. See, e.g., GenBank Accession Numbers NC 002077, NC 001401 , NC 001729, NC 001863, NC 001829, NC 001862, NC 000883, NC 001701 , NC 001510, AF063497, U89790, AF043303, AF028705, AF028704, J02275, J01901 , J02275, X01457, AF288061 , AH009962, AY028226, AY028223, NC 001358, NC 001540, AF513851 , AF513852; the disclosures of which are incorporated herein in their entirety. See also, e.g., Srivistava et al., (1983) J. Virology 45:555; Chiorini et al., (1998) J. Virology 71:6823; Chiorini et al., (1999) J. Virology 73:1309; Bantel-Schaal et al., (1999) J. Virology 73:939; Xiao et al., (1999) J. Virology 73:3994; Muramatsu et al., (1996) Virology 221:208; Shade et al., (1986) J. Virol. 58:921 ; Gao et al., (2002) Proc. Nat. Acad. Sci. USA 99:11854; international patent publications WO 00/28061, WO 99/61601 , WO 98/11244; U.S. Patent No. 6,156,303; the disclosures of which are incorporated herein in their entirety. See also, Figures 7 - 22. An early description of the AAV1 , AAV2 and AAV3 terminal repeat sequences is provided by Xiao, X., (1996), "Characterization of Adeno-associated virus (AAV) DNA replication and integration," Ph.D. Dissertation, University of Pittsburgh, Pittsburgh, PA (incorporated herein it its entirety). The term "tropism" as used herein 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). Those skilled in the art will appreciate that 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. In the case of AAV, 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.

As used herein, "transduction" or "infection" of a cell by a parvovirus or AAV means that the pa rvo virus/AAV enters the cell to establish an active (i.e., lytic) infection. As used herein, "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).

The terms "5" portion" and "3' portion" are relative terms to define a spatial relationship between two or more elements. Thus, for example, 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. Likewise, 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.

As used herein, the term "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. As used herein, 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.

Likewise, an "isolated" polypeptide means a polypeptide that is separated or substantially free from at least some ofthe 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. Alternatively, a "therapeutic polypeptide" is one that otherwise confers a benefit to a subject, e.g., anti-cancer effects or improvement in transplant survivability.

A "heterologous nucleotide sequence" will typically be a sequence that is not naturally occurring in the virus. Alternatively, 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).

As used herein, the term "vector" pr "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. Alternatively, in some contexts, the term "vector" may be used to refer to the vector genome/vDNA alone.

As used herein, 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 parvovirus capsid.

Likewise, a "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). Typically, 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. 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).

The term "template" or "substrate" is used herein to refer to a polynucleotide sequence that may be replicated to produce the parvovirus viral DNA. For the purpose of vector production, 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). Alternatively, 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. As used herein, 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 parvovirus 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. For example, with respect to AAV, 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. Preferably, 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). In particular embodiments, 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.

Those skilled in the art will further appreciate that it is not necessary that the replication proteins be encoded by the same polynucleotide. For example, for MVM, the NS-1 and NS-2 proteins (which are splice variants) may be expressed independently of one another. Likewise, for AAV, 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. Typically, however, it will be more convenient to express the replication proteins from a single construct. In some systems, the viral promoters (e.g., AAV p19 promoter) may not be recognized by the cell, and it is therefore necessary to express the large and small Rep proteins from separate expression cassettes. In other instance, it may be desirable to express the large Rep and small Rep proteins separately, i.e., under the control of separate transcriptional and/or translational control elements. For example, it may be desirable to control expression of the large Rep proteins, so as to decrease the ratio of large to small Rep proteins. In the case of insect cells, it may be advantageous to down-regulate expression of the large Rep proteins (e.g., Rep78/68) to avoid toxicity to the cells (see, e.g., Urabe et al., (2002) Human Gene Therapy 13:1935).

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. According to the present invention, 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).

It is not necessary that all, or even any, of the proteins encoded by the chimeric rep coding sequences be chimeric proteins. For example, in the case of AAV, 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.

As used herein, 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). Typically, 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. Typically, but not necessarily, the cap coding sequences will be present on a single nucleic acid molecule.

The capsid structure of autonomous parvoviruses and AAV are described in more detail in BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-Raven Publishers).

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). Preferably, 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 parvovirus 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. Likewise, 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.

Chimeric Parvovirus Rep Coding Sequences.

The inventors have identified novel trans-acting elements that may be used to achieve more efficient (e.g., higher titer) production of hybrid parvovirus stocks. In particular, 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 (Rabinowitz et al.) 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. 179:1) and the rep gene products (Senepathy et al., (1984) J. Mol. Biol. 179:1, Hermonat et al., (1984) J. Virology 51 :329; Tratschin et al., (1984) J. Virology 51 :611 ). The rep gene encodes four multifunctional proteins (Hermonat et al., (1984) J. Virology

51 :329; Tratschin et al., (1984) J. Virology 51 :611 ; Mendelson et al., (1986) J Virology 60:823; Trempe et al., (1987) Virology 161:18) that are expressed from two promoters at map units 5 (p5) and 19 (p19). The larger 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). 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. Virology 63:447; Im et al., (1992) J. Virology 66:1119; Snyder et al., (1990) Cell 60:105; Snyder et al., (1990) J. Virology 64:6204). 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). 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), and 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). Mutant studies of the AAV Rep proteins have indicated that the activities of Rep can be divided into partially distinct functional domains that are spread throughout the protein (Chejanovsky et al., (1989) Virology 173:120; McCarty et al., (1992) J. Virology 66:4050; Yang et al., (1992) J. Virology 66;6058; Owens et al., (1993) J. Virology 67:997; Weitzman et al., (1996) J. Virology 70:2440; Walker et al., (1997) J. Virology 7 V.2122; Walker et al., (1997) J. Virology 71 :6996; Davis et al., (1999) J. Virology 73:2084; Urabe et al., (1999) J. Virology 73:2682). These include regions required for binding to the terminal repeat; a putative NTP-binding/ATPase domain, nuclear localization domain, dimerization domain, and residues putatively required for nicking and helicase functions (see, e.g., Chiorini et al., (1999) J. Virology 73:1309). Several mutations within the NTP-binding/ATPase domain that lacked trs endonuclease and viral replication were also defective for trans-activation functions suggesting a need for further mutant analysis (McCarty et al., (1992) J. Virology 66:4050). Likewise, the replication genes and proteins of a number of autonomous parvoviruses have been described. For example, minute virus of mouse (MVM) and H1 virus each produce two non-structural proteins, NS-1 and NS-2, which share similar activities to the AAV Rep proteins (see, e.g., BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-Raven Publishers). See also, Ding et al., (2002) J. Virology 76:338, characterizing the Junonia coenia densovirus NS-1 protein.

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.

The small AAV Rep proteins (Rep52 and Rep40) and the homologous regions that are found in the carboxyl portion of the large AAV Rep proteins (Rep78 and Rep68; see, e.g., Chiorini et al., (1999) J. Virology 73:1309,

Figures 19-22) comprise a capsid interacting domain. In embodiments of the invention, 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). Alternatively, 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). In other particular embodiments, 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. In still further embodiments, the capsid interacting domain comprises approximately the carboxy terminal 50, 75, 100, 125, 150 or 200 amino acids of the Rep42 or Rep50 proteins. In other embodiments, the rep coding sequences comprise the coding sequences 3' of the p40 promoter. Alternatively, 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). In still other embodiments, 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. In further embodiments, 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). In yet other illustrative embodiments, 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. In other embodiments of the invention, 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). For example, the MVM NS-2 protein, NS-2 protein of B19 and Rep2 protein of goose parvovirus may comprise a capsid interacting domain. In embodiments of the invention, 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). In particular embodiments, 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. For example, the AAV1 and AAV2 capsid interacting domains may be compatible with both the AAV1 and AAV2 capsids. Likewise, 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. Thus, 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). In the example of an AAV5 capsid with a rAAV2 genome, 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. Thus, in embodiments of the invention, 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. Alternatively, the rep coding sequences may encode a functional portion of the AAV large Rep proteins (e.g., an amino-terminal portion). In other embodiments of the invention, 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). As a further alternative, 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. In other alternative embodiments, 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. As illustrative examples, 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. In some embodiments, the DNA binding domain is a functional portion of an autonomous parvovirus NS-1/Rep1 protein (i.e., the "large" replication protein). For example, the functional portion may be an amino terminal portion of the large Rep protein. In embodiments of the invention, 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). In embodiments of the invention, 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. For example, 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. There may also be some compatibility of AAV2 and AAV3 with AAV1 and AAV4, although higher titers may be achieved with the serotype-specific DNA binding domain and terminal repeat. In general, it is preferred to use an AAV5 DNA binding domain to package AAV5 terminal repeats. Likewise, there may be compatibility between closely related autonomous parvoviruses, such as feline panleukemia virus and canine parvovirus, or MVM and H1 virus. Generally, an AAV DNA binding domain will be used with an AAV terminal repeat, and 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.

Accordingly, the present investigations have elucidated important functional domains in the parvovirus replication machinery. On the basis of this understanding, improved reagents and methods may be employed for more efficient production of hybrid parvoviruses.

While not wishing to be bound by any particular theory of the invention, in one model, 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. In an alternative model, 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. As one aspect, 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. In one embodiment, 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.

In another embodiment, 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.

According to the previous embodiments, it is preferred that the first parvovirus is an AAV. In other embodiments, the different parvovirus is an AAV. In still further embodiments, both the first and different parvoviruses are AAV.

In particular embodiments of the invention, 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.

In a further embodiment, 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.

According to the foregoing embodiments, the capsid interacting domain and DNA binding domain encoded by the polynucleotides are as described above. In embodiments of the invention, 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). In other embodiments, the capsid interacting domain is from an AAV (e.g., AAV1 , AAV2 or AAV5). Likewise, 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).

In certain embodiments, both the rep coding sequences and cap coding sequences are AAV sequences (i.e., the AAV rep and cap sequences).

In particular embodiments of the invention, the rep coding sequences encode an AAV1 , AAV2, AAV4 or AAV5 DNA binding domain. In other preferred embodiments, the rep coding sequences encode an AAV1 , AAV2 or AAV5 capsid interacting domain. According to this embodiment, it is further preferred that the cap coding sequences encode a capsid of the same serotype as the capsid interacting domain.

In other embodiments, the rep coding sequences encode an AAV5 DNA binding domain and an AAV1 , AAV2, AAV3, AAV4, AAV6, AAV7 or AAV8 capsid interacting domain. According to this embodiment, it is also preferred that the cap coding sequences encode a capsid of the same serotype as the capsid interacting domain.

In other embodiments, the rep coding sequences encode an AAV5 capsid interacting domain and an AAV1 , AAV2, AAV3, AAV4, AAV6, AAV7 or AAV8 DNA binding domain. According to this embodiment, it is preferred that the cap coding sequences encode an AAV5 capsid.

In other embodiments of the invention, 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.

The rep coding sequences and cap coding sequences may be operatively associated with an expression control sequence, for example, a promoter. In embodiments of the invention, 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. Preferably, the rep coding sequences are operatively associated with a parvovirus promoter, more preferably, the AAV p5 or p19 promoters.

As described above, it may be desirable to associate the coding sequences for the large and small Rep proteins with separate transcriptional and/or translational control elements so that expression of these proteins may be differentially regulated. For example, it may be advantageous to down- regulate expression of the large Rep proteins (e.g., Rep78/68) as compared with the small Rep proteins (e.g., Rep52/40).

Those skilled in the art will appreciate that the rep coding sequences encoding the DNA binding domain and the capsid interacting domain may be operatively associated with different promoters. In particular embodiments, 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), Picornaviridae (e.g., poliovirus, rhinovirus, coxsackieviruses, Caliciviridae, Togavi idae (e.g., alphaviruses, rubella) Flaviviridae (e.g., yellow fever virus), Parvoviridae (e.g., AAV), Coronaviridae (e.g., HDV, TGEV, IBV, MHV, BCV), Rhabdoviridae, Filoviridae, Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, and respiratory syncytial virus), Orthomyxoviridae (e.g., influenza virus), Bunyaviridae, Arenaviridae, hepatitis delta virus, Astroviruses, Epstein Barr Virus (EBV) and non-mammalian viruses, such as baculoviruses.

Preferred viral vectors include AAV, adenovirus, herpesvims, 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. In some embodiments, 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.

Methods of Parvovirus Production.

The present invention also encompasses methods of producing hybrid parvovirus particles using the inventive polynucleotide sequences, vectors and cells. In general, 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). In one embodiment, 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 parvovirus 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 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 hybrid parvovirus particles comprising the recombinant parvovirus template are produced in the cell. Preferably, the hybrid parvovirus particles will be infectious.

In another embodiment, 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. 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. According to the previous embodiments, it is preferred that the recombinant parvovirus template is a rAAV template and the parvovirus terminal repeat is an AAV terminal repeat sequence. In other embodiments, it is preferred that the cap coding sequences and the rep coding sequences encoding the capsid interacting domain are AAV sequences. It is further preferred that the recombinant parvovirus template (e.g., rAAV template) has two terminal repeat sequences at the 5' and 3' ends of the heterologous nucleotide sequence. In other embodiments, the parvovirus terminal repeat is from the first parvovirus (e.g., the same parvovirus as the DNA binding domain). In particular embodiments, 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.

In a further embodiment, 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 "hybrid."

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. Preferably, the recombinant parvovirus template comprises a segment (e.g., a terminal repeat) that permits packaging within the parvovirus capsid. Conversely, it is preferred that 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.).

In embodiments of the invention, 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.

Particular combinations of the capsid interacting domain and DNA binding domain are as described in the previous section. It is further preferred that 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).

In particular embodiments of the invention, the rep coding sequences encode an AAV1 , AAV2, AAV4 or AAV5 DNA binding domain. According to this embodiment, 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.

In embodiments of the invention, a baculovirus system is used to produce parvovirus particles in mammalian (e.g., human) or insect cells. For example, 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 parvovirus 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. For example, 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). As a further alternative, the rep coding sequences and/or cap coding sequences may be stably integrated into chromosome of the cell. Typically, and preferably, 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. 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. For example, the recombinant parvovirus template may be supplied by a non-viral (e.g., plasmid) or viral vector (as described above). In particular preferred embodiments, 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). As another illustration, 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.

In another preferred embodiment, the AAV template is provided by a replicating rAAV virus. In still other embodiments, an AAV provirus is stably integrated into the chromosome of the cell.

Production of new AAV particles typically involves 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. The multiplicity of infection (MOI) and the 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. In general, the helper functions are provided by the adenovirus early genes, more particularly, the E1a, E2a, E4orf6 and VA RNA adenovirus sequences. Typically, these sequences will be provided by a helper adenovirus or herpesvirus (preferably, adenovirus) vector. Alternatively, 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. 3:1295, Xiao et al., (1998) J. Virology 72:2224, and U.S. Patent Nos. 6,040,183 and 6,093,570. The vector can be introduced into the packaging cell by any suitable method known in the art, as described above.

Other methods of producing rAAV stocks have been described, including but not limited to, methods that split the rep and cap genes onto separate expression cassettes to prevent the generation of replication- competent AAV (see, e.g., Allen et al., (1997) J. Virol. 71 :6816), methods employing packaging cell lines (see, e.g., Gao et al., (1998) Human Gene Therapy 9:2353; Inoue et al., (1998) J. Virol. 72:7024; U.S. Patent No. 5,837,484; WO 98/27207; U.S. Patent No. 5,658,785; WO 96/17947), and other helper virus free systems (see, e.g., U.S. Patent No. 5,945,335 to Colosi).

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). Further, 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. It is preferred that these helper virus sequences cannot be packaged into AAV virions, e.g., are not flanked by AAV terminal repeats. Those skilled in the art will appreciate that it may be advantageous to provide the AAV rep coding sequences and cap coding sequences and the helper virus sequences (e.g., adenovirus sequences) on a single helper construct. 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.

In one particular embodiment, 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 E1 a or E3 regions) of the adenovirus.

In a further embodiment, 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.

In another embodiment, 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. Alternatively, 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). In a further exemplary embodiment, 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. For example, the rAAV template may be provided by a rAAV particles or a second recombinant adenovirus particle.

According to the foregoing methods, 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. As described above, it is preferred that 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.

Generally, 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. For example, 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). Preferably, deleted replication-defective helper viruses are used so that any contaminating helper virus is not replication competent. As a further alternative, 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. It is also preferred that the parvovirus stock has a titer of at least about 10⁵ transducing units (tu)/ml, more preferably at least about 10⁶ tu/ml, more preferably at least about 10⁷tu/ml, yet more preferably at least about 10⁸ tu/ml, yet more preferably at least about 10⁹ tu/ml, still yet more preferably at least about 10¹⁰ tu/ml, still more preferably at least about 10¹¹ tu/ml, or more.

Alternatively stated, 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.

The 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. For example, as demonstrated in the working examples, 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.

Recombinant Parvovirus Vectors.

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. In particular, 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. Typically, 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. 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).

Any heterologous nucleotide sequence(s) (as defined above) may be delivered in a parvovirus particle produced according to the present invention. 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. In the case of AAV, it is well-known in the art that the AAV capsid disfavors packaging of vDNA that substantially deviate in size from the wt AAV genome. In the case of an AAV capsid, the genome is preferably approximately 5.2 kb in size or less. In other embodiments, 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. Alternatively stated, 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, β-globin, α-globin, spectrin, αi-antitrypsin, adenosine deaminase, hypoxanthine guanine phosphoribosyl transferase, β- glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, branched-chain keto acid dehydrogenase, RP65 protein, cytokines (e.g., α- interferon, β-interferon, interferon-γ, interleukin-2, interleukin-4, granulocyte- macrophage colony stimulating factor, lymphotoxin, and the like), peptide growth factors and hormones (e.g., somatotropin, insulin, insulin-like growth factors 1 and 2, platelet derived growth factor, epidermal growth factor, fibroblast growth factor, nerve growth factor, neurotrophic factor -3 and -4, brain-derived neurotrophic factor, glial derived growth factor, transforming growth factor -α and -β, and the like), receptors (e.g., the tumor necrosis growth factor receptor), monoclonal antibodies (including single chain monoclonal antibodies; an exemplary Mab is the herceptin Mab). Other illustrative 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.

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, β-galactosidase, alkaline phosphatase, and chloramphenicol acetyltransferase gene.

Alternatively, 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 -rans-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.

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. 5,916,563 to Young e- a/., 5,905,040 to Mazzara et al., U.S. Patent No. 5,882,652, U.S. Patent No. 5,863,541 to Samulski et al.; the disclosures of which are incorporated herein in their entirety by reference). 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. For example, 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). 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.

Alternatively, the immunogen may be any tumor or cancer cell antigen. Preferably, 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 ). Other illustrative 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, β- 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. 54:3124), including 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. 4,968,603), CA 125, LK26, FB5 (endosialin), TAG 72, AFP, CA19-9, NSE, DU-PAN-2, CA50, SPan-1 , CA72-4, HCG, STN (sialyl Tn antigen), c- erbB-2 proteins, PSA, L-CanAg, estrogen receptor, milk fat globulin, p53 tumor suppressor protein (Levine, (1993) Ann. Rev. Biochem. 62:623); mucin antigens (international patent publication WO 90/05142); telomerases; nuclear matrix proteins; prostatic acid phosphatase; papilloma virus antigens; and 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 ).

Alternatively, the heterologous nucleotide sequence may encode any polypeptide that is desirably produced in a cell in vitro, ex vivo, or in vivo. For example, the parvovirus vectors may be introduced into cultured cells and the expressed gene product isolated therefrom.

It will be understood by those skilled in the art that the heterologous nucleotide sequence(s) of interest may be operably associated with appropriate control sequences. For example, 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. Those skilled in the art will appreciate that a variety of 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 initiation region is introduced.

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.

In embodiments wherein which the 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. These 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.

Gene Transfer Technology.

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.

In general, 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.

Alternatively, 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. For unbalanced disease states, gene transfer could be used to create a disease state in a model system, which could then be used in efforts to counteract the disease state. Thus parvovirus vectors produced according to the methods of the present invention permit the treatment of genetic diseases. As used herein, 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. Accordingly, 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. Alternatively, the parvovirus vector may encode any other non- translated RNA, as described in more detail hereinabove.

Finally, the 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.

As a further aspect, parvovirus vectors produced according to the present invention may be used to produce an immune response in a subject. According to this embodiment, 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. Alternatively, 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. Preferably, 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." Id. 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. Alternatively, 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.

According to the foregoing methods of inducing an immune response in a subject, it is preferred that the parvovirus 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. To illustrate, 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.

As used herein, the term "cancer" encompasses tumor-forming cancers. Likewise, the term "cancerous tissue" encompasses tumors. A "cancer cell antigen" encompasses tumor antigens. The term "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.

The term "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. By the terms "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. Preferably, 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. By the terms "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.

In particular embodiments, 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). It is known in the art that immune responses may be enhanced by immunomodulatory cytokines (e.g., α-interferon, β-interferon, γ-interferon, ω- interferon, τ-interferon, interleukin-1α, interleukin-1 β, 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-α, tumor necrosis factor-β, monocyte chemoattractant protein- 1 , granulocyte-macrophage colony stimulating factor, and lymphotoxin). Accordingly, immunomodulatory cytokines (preferably, CTL inductive cytokines) may be administered to a subject in conjunction with the parvovirus vectors.

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. In particular embodiments, 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.

Subjects, Pharmaceutical Formulations, and Modes of Administration.

5 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. The term "avian" as used herein includes, but is not limited to, chickens, ducks, geese, quail, turkeys and

10 pheasants. 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.

In particular embodiments, the present invention provides a

15 pharmaceutical composition comprising a virus particle of the invention in a pharmaceutically acceptable carrier and/or other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc. For injection, the carrier will typically be a liquid. For other methods of administration, the carrier may be either solid or liquid. For inhalation administration, the carrier will be

20 respirable, and will preferably be in solid or liquid particulate form. As an injection medium, it is preferred to use water that contains the additives usual for injection solutions, such as stabilizing agents, salts or saline, and/or buffers.

In general, a "physiologically acceptable carrier" is one that is not toxic

25 or unduly detrimental to cells. Exemplary physiologically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free, phosphate buffered saline. Physiologically acceptable carriers include pharmaceutically acceptable carriers.

By "pharmaceutically acceptable" it is meant a material that is not

30. biologically or otherwise undesirable, i.e., the material may be administered to a subject without causing any undesirable biological effects. Thus, such 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. Preferably, at least about 10³ infectious units, more preferably at least about 10⁵ infectious units, are administered to the cell. 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. Alternatively, the cell may be any progenitor cell. As a further alternative, the cell can be a stem cell (e.g., neural stem cell, liver stem cell). As still a further alternative, the cell may be a cancer or tumor cell. Moreover, 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. In particular embodiments, 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). Alternatively, 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 10² to about 10⁸, preferably about 10³ to about 10⁶ 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. Alternatively stated, a "therapeutically effective" amount is an amount that will provide some alleviation, mitigation, or decrease in at least one clinical symptom in the subject. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

In some embodiments, 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). Typically, a quantity of cells expressing 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. Preferably, 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. Preferably, 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). Typically, vaccines of the present invention comprise an immunogenic amount of infectious virus particles as disclosed herein in combination with a pharmaceutically acceptable carrier. Preferably, 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 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10³, 10¹⁴, 10¹⁵ transducing units or more, preferably about 10⁸- 10¹³ transducing units, yet more preferably 10¹² transducing units.

In particular embodiments, more than one administration (e.g., two, three, four or more administrations) 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. Alternatively, one may administer the virus in a local rather than systemic manner, for example, in a depot or sustained-release formulation. 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. In particularly preferred embodiments of the invention, 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.

In other preferred embodiments, 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 parvovirus 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.

Having described the present invention, the same will be explained in greater detail in the following examples, which are included herein for illustration purposes only, and which are not intended to be limiting to the invention.

Example 1 Construction of Plasmids

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 Xbal (blunt ended) Swa\ digestion of pACG2 (Li et al. (1997) J. Virol. 71 :5236) and the Sma\/Swa\ digestion of pAAV2Cap. The new plasmid, pAAV2rep, was digested with Λ/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 Svval 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'-

GCGGCCGCGAGACCAAAGTTCAACTGA-3' SEQ ID NO:5) and AAV5 (5'- CGGCCGCAAGAGGCAGTATTTTACTGA-3' SEQ ID NO:6). 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 Accl, as was the original plasmid (nucleotide 1424 to 4355 of the AAV3 sequence, Fig. 1 , panel A). The AAV4 serotype clone was digested with Accl and >4gel, as was the original plasmid (nucleotides 1479 to 4488 of the AAV4 sequence, Fig. 1 , panel A). The AAV5 serotype clone was digested with BamHI, as was the original plasmid (nucleotides 1071 to 2276 of the AAV5 sequence, Fig 1 , panel A). These clones were designated pXR1 , -2, -3, -4, and -5 respectively. An additional AAV5 serotype clone and the sixth construct shown in Fig. 1 , panel A, was constructed by digestion of the serotype specific clone with Ncol and BamHI (nucleotides 670 to 1076 of the AAV5 sequence), as well as the original plasmid. The vector plasmid pTR/CMV/GFP (enhanced green fluorescent protein [EGFP] transgene) contains AAV2 terminal repeats flanking a cytomegalovirus (CMV) immediate- early promoter driven transgene (Halbert et al. (2000) J. Virol. 74:1524). The 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.

Example 2 Cell Culture

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% CO₂ saturation at 37°C. 293T, HeLa, Cos1 and Cos7 cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) 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.

Example 3 Production of Serotype-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 CsCl 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 (Sorvall 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 CsCl 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). Primary monoclonal antibodies 1 F11 , (Hunter and Samulski (1992) J. Virol. 66:317), and B1 (a gift from Jurgen Kleinschmidt) were used to detect AAV Rep and capsid proteins, respectively, as previously described by using SuperSignal West Pico (Pierce) for the detection of the secondary antibody conjugate.

Example 5

Hirt Low Molecular Weight DNA Analysis from AAV Serotype Transfections

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 Dpnl, 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 Notl 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 Chromatography

After recombinant virus from each of the five serotypes was purified by two rounds of CsCl isopycnic centrifugation and dialyzed against three changes of 1X PBS (Gibco-BRL) plus 10% sorbitol, an equal volume of virus was injected into a 1 ml HiTrap heparin column (Amersham Pharmacia Biotech) by using Amersham Pharmacia Biotech AKTA fast-performance liquid chromatography. A preset program (12 ml of buffer at 137 mM NaCl, 2 mM MgCI₂, 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 NaCl to 1 M NaCl at a flow rate of 0.5 ml/min. For the flowthrough, wash, and elution steps, 0.5 ml fractions were collected, with the waste step collected in a single fraction. Transducing units (TU)/microliter 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.

Example 7

Dot Blot Analysis

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 8

In vitro Transduction Assay

Cell lines were infected with rAAV serotypes by using a volume of virus equivalent to 10⁵ TU. For each datum point at least three experiments were performed, and the average of these experiments is presented. The titers of the AAV serotypes 1 , 2, 3, and 5 were originally determined in HeLa cells, and that of AAV4 was determined in Cos1 cells. Between 1 x 10⁵ and 1.75 x 10⁵ cells were plated into 24 well plates 24 h prior to infection. The volume of each serotype equivalent to 10⁵ TU was added to 100 microliters of DMEM that included adenovirus, and this was added to each well dropwise. After 24 h the number of GFP-positive cells was counted. Eight fields/well were counted, and the average number of GFP-positive cells/well was determined; this number was multiplied by the dilution in order to obtain the number of TU/microliter.

Example 9 ELISA Analysis of Factor IX and α1-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

Subretinal injections of rAAV were performed via a transcleral transchoroidal approach on wild-type Wistar rats, as previously described in Rolling et al. (1999) Hum. Gene Ther. 10:641. Briefly, the sclera and the choroid were punctured, a 33-gauge needle was then inserted in a tangential direction under an operating microscope. Three microliters of each of the five hybrid rAAV serotypes (5x10¹⁰ particles/ml) was delivered into the subretinal space (n = 3, for each serotype). A new method using fundus photography has been developed and performed in order to control the accuracy and reproducibility of subretinal injections (Rolling et al. in preparation). GFP protein expression in live rats was monitored by fluorescent retinal imaging using a Canon UVI retinal camera connected to a digital imaging system and Lhedioph Win software. Retinas were examined at 12, 26, and 46 days post- injection.

Example 11

AAV Hybrid Helper Plasmids

The generation of 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. 10:641 ), and the capsid gene was removed, by a Swal/Nsil digestion. This intermediate plasmid was used for cloning each of the serotype specific capsid coding sequences (see Fig. 1 panel A and methods for details). The capsid genes of each serotype were PCR amplified from ATCC viral stocks, and the product cloned into this Swal blunted ended Nsil- digested intermediate. The new hybrid plasmids containing the common

AAV2 ACG replication gene and the serotype specific capsid sequences were called pXR1-5. BstNl digestion (Fig. 1 panel B) and DNA sequencing determined correct orientation and nucleotide sequence of the serotype specific capsid gene. When a number of independent isolates for each seroytpe specific helper were compared to traditional type 2 helpers, vector yields varied significantly among these constructs (Tablel). Surprisingly, additions of serotype specific non-capsid coding sequences (5' to the VP1 start site) reduced this variation (see Fig. 1 , Table 1). Additional analysis indicates that a Rep-specific capsid interacting domain is required for efficient serotype-specific encapsidation (Rolling et al., manuscript in preparation). Since these modified serotype specific helper plasmids (as described in Fig. 1 ) produced vector yields within the range of type 2 (Table 1 ), they were further analyzed in detail for AAV Rep and capsid production as well as ability to replicate AAV type 2 transgenes as described below. TABLE 1. Comparison of AAV helper constructs containing AAV2-onIy replication gene of portions o the serotype specific replication gene

AAV-2 only replication gene AAV-2/serotype chimeric Rep

Serotype Particles/ml (n) Particles/ml (n) rAAV1 1.27 x 10¹¹ (10) not done rAAV2 9.79 x 10¹² (2) not applicable rAAV3 9.00 x 10⁸ (1 ) 7.16 x 10¹¹ (3) rAAV4 2.04 x 10¹⁰ (4) 2.63 x 10¹¹ (2) rAAV5 7.64 x 10⁹ (1 ) 4.60 x 10¹¹ (5) rAAV5 to Ncol site 0 0 rAAV5 with AAV5 ITR 0 3.70 x 10⁷ (3)

Example 12 AAV Hybrid Helper Functions

A series of experiments were carried out on XR 1-5 helper plasmids to determine levels of type 2 Rep and serotype-specific capsid protein expression after transfection in 293 cells. Western blot analysis was used to detect the AAV rep gene products 78/68, 52/40 (Fig. 2 panel A) and the three capsid sub-units Vp1 , 2, and 3 (Fig. 2 panel B) at 24 h post-transfection. A comparison of AAV2 Rep proteins in the context of different serotype helper plasmids was carried out by using monoclonal antibody 1 F 1 which recognizes each of the four AAV2 replication proteins (Hunter et al. (1992) J. Virol. 66:317). Previous studies demonstrated that the p5 mutation in the context of the helper plasmid pACG-2 down regulated the expression of Rep 78/68, without affecting Rep 52/40 (Li et al. (1997) J. Virol. 71 :5236). The results, shown in Fig. 2 panel A, demonstrate that in each of the serotype specific helper constructs all 4 Rep proteins were made at levels equivalent to those described for the original AAV2 helper construct pACG-2 (Li et al. (1997) J. Virol. 71 :5236). The B1 monoclonal antibody (Wistuba et al. (1997) J. Virol. 71 :1341 ), which recognizes the amino acid recognition sequence IGTRYLTR (SEQ ID NO:13) in AAV type 2 structural proteins (Wobus et al. (2000) J. Virol. 74:9281 ), was used to identify the serotype specific capsid subunits. This motif is conserved in all serotypes except AAV4 (Fig. 2 panel C), which is evident by the lack of positive signal after Western analysis (Fig. 2 panel B, lane 4). For the other helper plasmids, all three capsid proteins were detected (Fig. 2 panel B). Although the data provided is a representative example, higher amounts of the structural proteins were consistently observed from serotype 1 compared with the other helper constructs (repeated 10X). Taken together, the results for replication and capsid protein expression for the five helper plasmids is within the range of AAV helper plasmids currently available for production (Haberman et al. (1999) in, Current Protocols in Neuroscience, vol. 1, New York, N.Y.).

Example 13 Replication and Cross-Packaging of Type 2 Vectors

Functional activity of these helper proteins were determined by replication and encapsidation of rAAV type 2 GFP template. The five serotype helper plasmids, the rAAV type 2 transgene pTRUFR, and the adenovirus helper plasmid (XX6-80) were triple transfected into 293T cells, and Hirt analysis carried out 24 hr post-transfection. Dpnl digestion followed by

Southern blot analysis using GFP gene specific probe determined the ratio of input plasmid to newly replicated vector DNA. All serotype specific helpers replicated the vector template (Fig. 3 Dpnl digested lanes). Although nearly equivalent amounts of input DNA were loaded in all lanes of the undigested samples, the highest level of transgene replication was observed from XR2 (Fig. 3 lane 2). Repeated replication analysis demonstrated that all other helpers were essentially equal with XR1 generating 40% of XR2 levels (Fig. 3 lanes 3-5 and 1 respectively). Packaging efficiencies were determined by dot blot hybridization (Table 1). All helper constructs generated high vector yields within 10¹¹ to 10¹² particles/ml. In general, 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 10¹², 7.2 x 10¹¹ respectively, Table 1 ). The serotype 1 helper produced the least amount of virus (1.27x10¹¹ and Table 1 ), consistent with the Hirt replication analysis.

Example 14 Heparin Column Binding Profiles of AAV Hybrid Serotypes The use of 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). In, addition, 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).

The elution profile of rAAV4 was unique. Although the majority of the TU applied to the column was recovered in the elution steps, a significant fraction of the TU was recovered in the flow-through (Fig. 4, AAV4). These results suggest that if the salt conditions were altered virus might be recovered in the elution step, or that another ion exchange column might improve the recovery of AAV serotype 4. Example 15 Hybrid Vector Transduction on Rodent, Monkey, and Human Cell Lines

Various cell lines were infected with the hybrid vectors in an effort to determine transducing titer in vitro (Fig. 5). Parental and mutant cell lines defected in heparan sulfate (HS) proteoglycan biosynthesis were analyzed for serotype specific vector transduction since previous studies have demonstrated a role for this cell surface protein in AAV type 2 infection. For rAAVI , a decrease in transduction on CHO pgs D cells that are heparin sulfate deficient was not observed. In fact, transduction efficiency for rAAVI was near identical for all cell lines tested (Fig. 5, AAV1). The lack of specific binding to heparin columns and ability to transduce mutant HS cell lines indicate that type 1 entry is distinct from type 2 and has yet to be identified. The transduction efficiency for rAAV2 and 3 were similar to what has been previously published (Handa et al. (2000) J. Gen. Virol. 81 :2077, Summerford and Samulski (1998) J. Virol. 72:1438), with each showing a dependence on cell surface heparin sulfate (Fig. 5, AAV2 and 3) and efficient binding to HS columns (Fig. 4, AAV2 and 3). Hybrid AAV type 5 transduction paralleled that of AAV type 1 for all cell lines tested. After the completion of these studies, evidence that identifies sialic acid as a cell surface molecule involved in AAV type 5 transduction has been described in Kaludov et al. (2001 ) J. Virol. 75:6884. The presence of sialic acid on the battery of cell lines tested here was not determined although efficient transduction was observed. Hybrid vector type 4 also transduced all cell lines tested. However the level of transduction never approached that observed on monkey derived cell lines (Fig. 5, AAV4). The restriction in the in vitro host range that has been observed with both the hybrid and traditional type 4 vectors indicates efficient transduction require a monkey specific factor. Overall, these results indicate that unlike AAV2 and -3, AAV1 , -4 and -5 do not require heparin sulfate for infection. These observations are in agreement with other studies evaluating the transduction requirements of non AAV type 2 serotypes (Chiorini et al. (1999) J. Virol. 73:1309, Chiorini et al. (1997) J. Virol. 71 :6823, Xiao et al. (1999) J. Virol. 73:3994), and demonstrate the influence of the serotype specific virion shell to that of the type 2 vector sequences in transduction. Example 16 Hierarchy of Gene Expression for AAV Hybrid Vectors in vivo

The efficiency of gene expression of both therapeutic and marker transgenes were tested when delivered by each of the five hybrid vectors in vivo. The expression of human alpha-1-antitrypsin (hAAT) and factor IX was tested in outbred and inbred mice, as well as immunodeficient mouse lines (Table 2). The hAAT expression was also examined with respect to the route of injection (i.e. portal vein, intramuscular, and intravenous) (Table 2). ELISA was used to monitored expression of the therapeutic transgenes which consistently demonstrated that AAV type 1 was superior to the other hybrid vectors tested. This observation was true for all strains of mice routes of administration or transgenes tested (Table 2). However, small differences in the efficiency of expression for the remaining serotypes with respect to each variable was observed. For example, the expression of hAAT from the 5 hybrid vectors demonstrated AAV type 4 as least efficient, a feature ascribed to AAV type 2 when factor IX was tested.

TABLE 2. Animal experiments with different serotypes of rAAV

Gene^a Mouse strain Score with Serotype⁰ (route of injection)¹³ AAV1 AAV2 AAV3 AAV4 AAV5 hAAT C57BL (i.m.) +++++ +++ ++ ++ ++++

C57BL (p.v.) +++++ ++ +++ +++ ++++

C57BL (i.v.) +++++ ++ +++ +++ ++++

BALB/c (i.v.) +++++ ++++ ++ ++ ++++

dF9 Scid (i.m.) +++++ + ++++ ++++ ++++

BALB/c (i.m.) +++++ + ++ ++ +++

^a The transgene were chicken D-actin promoter with the CMV enhancer-driven hAAT (both C57/BL and BALB/c mice were injected with 5 x 10¹⁰ particles/mouse) and CMV immediate-early promoter-driven dog factor IX (dF9) (SCID mice were injected with 2 x 10¹¹ particles/mouse, and BALB/c mice were injected with 10¹⁰ particles/mouse. For each group of animals, serotype, and route of injection, a minimum of three animals were used. Abbreviations for route of injection: i.m., intramuscular injection; p.v., portal vein injection; i.v., intravenous injection. ^c Scores for protein expression ranged from the maximum level of protein observed for each set of animals (+++++) to the lowest level of expression in the group (+). Example 17 Evaluation of AAV Serotypes Gene Transfer Efficiency in the Rat Retina

Previous studies have shown that AAV type 5 transduction is more efficient in brain when compared to type 4 or 2 (Davidson et al. (2000) Proc. Natl. Acad. Sci. USA 97:3428). In our study, a type 1 vector appeared superior to all others when tested in non-neuronal targets. The hybrid vector analysis was extended to neuronal targets using rat retina as a substrate and green florescent protein (GFP) as a reporter. Each of the five serotype vectors was determined for efficient in vivo transduction using fundus color photography after subretinal injections. At twelve days post-injection, GFP expression could be detected in rAAV5, -4 and -1 injected animals, with type 5 and 4 hybrids displaying the most intense GFP signal. No signal was detected in animals injected with rAAV2 or -3 at this concentration and time point (Fig. 6). At 26 days post-injection, GFP expression increased proportionally for rAAV5, -4 and -1 , with types 2 and -3 eventually displaying a small but positive signal (Fig. 6, 26 days). Finally, this trend continued for the duration of the experiment (i.e., 48 days; Fig. 6).

Based on these observations, a different hierarchy of serotype specific transgene expression was observed in retina when compared with non- neuronal tissues. In this setting serotypes 5 and 4 were superior followed by types 1 , 2 and 3 (Fig. 6). Low to moderate levels of expression from AAV type 1 was in sharp contrast with data obtained in non-neuronal tissue. Taken together, the expression of therapeutic and marker genes from the five hybrid serotypes demonstrates that AAV1 is the superior vehicle for non-neuronal delivery, while in the retina and in the brain (Davidson et al. (2000) Proc. Natl. Acad. Sci. USA 97:3428) AAV5 produces the highest levels of marker gene expression. All in all, these data demonstrate the importance of determining serotype specific transduction in vivo when considering AAV as the delivery system of choice. Example 18

AAV-Serotype Chimeric Rep

Reguirements for Cross-Packaging of Type 2 Vectors

As described above, the packaging efficiencies of type 2 rAAV vectors utilizing serotype chimeric Rep proteins were determined by dot blot hybridization and depicted in Table 1. These results indicate that chimeric Rep proteins with a serotype specific amino acids from the C-terminus are sufficient to increase the cross packaging efficiency of rAAV2 vectors 10 - 1000-fold when compared with cross packaging with type 2 Rep proteins alone. Further, chimeric Rep proteins that possess the first 243 amino acids of the N-terminus of type 2 Rep protein are capable of efficient cross packaging of rAAV2 vectors, whereas those chimeric Rep proteins that possess only the first 103 amino acids from the N-terminus of type 2 Rep protein are not capable of efficient packaging of rAAV2 vectors.

Example 19

Chimeric AAV-B19 Rep Proteins and

Packaging of Type 2 Vectors in B19 Capsids

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.

The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

THAT WHICH IS CLAIMED IS:

1. A polynucleotide comprising parvovirus rep coding sequences and parvovirus cap coding sequences, said rep coding sequences encoding a DNA binding domain from a first parvovirus; said rep coding sequences further encoding a capsid interacting domain from a different parvovirus from said first parvovirus; and said cap coding sequence comprising sequences from said different parvovirus.

2. The polynucleotide of Claim 1 , said rep coding sequences comprising a 5' portion and a 3' portion; said 5' portion encoding said DNA binding domain from said first parvovirus; said 3' portion encoding said capsid interacting domain from said different parvovirus.

3. The polynucleotide of Claim 1 or Claim 2, wherein said first parvovirus is an autonomous parvovirus.

4. The polynucleotide of Claim 1 or Claim 2, wherein said first parvovirus is an AAV.

5. The polynucleotide of Claim 4, wherein said first parvovirus is selected from the group consisting of AAV1 , AAV2, AAV3, AAV4, AAVδm AAV6, AAV7 and AAV8.

6. The polynucleotide of Claim 5, wherein said first parvovirus is

AAV2 or AAV5.

7. The polynucleotide of any of Claims 4-6, wherein said DNA binding domain comprises about the first 220 amino acids of the large Rep proteins from said first parvovirus.

8. The polynucleotide of any of Claims 4-6, wherein said DNA binding domain comprises the Rep 78/68-specific amino acid sequences from said first parvovirus.

9. The polynucleotide of any of Claims 1 -8, wherein said different parvovirus is an autonomous parvovirus.

10. The polynucleotide of Claim 9, wherein said different parvovirus is selected from the group consisting of B19 virus, canine parvovirus, feline panleukemia virus, muskovy duck parvovirus, goose parvovirus, minute virus of mouse, and H1 virus.

11. The polynucleotide of Claim 9 or Claim 10, wherein said capsid interacting domain comprises an autonomous parvovirus small Rep protein or a functional portion thereof.

12. The polynucleotide of Claim 11 , wherein said capsid interacting domain comprises a functional carboxy-terminal portion of said autonomous parvovirus small Rep protein.

13. The polynucleotide of any of Claims 1 -8, wherein said different parvovirus is an AAV.

14. The polynucleotide of Claim 13, wherein said capsid interacting domain comprises the AAV Rep52 or Rep40 amino acid sequences or a functional portion thereof.

15. The polynucleotide of Claim 13 or Claim 14, wherein: said first parvovirus is selected from the group consisting of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8; and said different parvovirus is selected from the group consisting of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8.

16. The polynucleotide of Claim 15, wherein said different parvovirus is selected from the group consisting of AAV1 , AAV4 and AAV5.

17. The polynucleotide of any of Claims 1 -16, wherein said polynucleotide is a DNA sequence.

18. The polynucleotide of any of Claims 1 -17, wherein said rep coding sequences encoding said capsid interacting domain are operatively associated with a promoter such that said capsid interacting domain can be expressed independently of said DNA binding domain.

19. The polynucleotide of Claim 19, wherein said promoter is the

AAV p19 promoter.

20. A polynucleotide comprising adeno-associated virus (AAV) rep coding sequences and AAV cap coding sequences, said rep coding sequences having a 5' portion and a 3' portion; said 5' portion comprising rep coding sequences from a first AAV; said 3' portion comprising rep coding sequences from a different AAV from said first AAV; and said cap coding sequences comprising sequences from said different AAV.

21. The polynucleotide of Claim 20, wherein said first AAV is selected from the group consisting of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8.

22. The polynucleotide of Claim 20 or Claim 21 , wherein said first AAV is AAV2.

23. The polynucleotide of Claim 20 or Claim 21 , wherein said first AAV is AAV5.

24. The polynucleotide of any of Claims 20-23, wherein said 5' portion comprises the rep coding sequences 5' of the AAV p19 promoter.

25. The polynucleotide of any of Claims 20-23 wherein said 5' portion encodes at least the first 220 amino acids of the Rep 78/68 proteins.

26. The polynucleotide of any of Claims 20-25, wherein said 3' portion encodes the Rep52 or Rep40 proteins or a functional portion thereof.

27. The polynucleotide of any of Claims 20-26, wherein: said first AAV is selected from the group consisting of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8; and said different AAV is selected from the group consisting of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8.

28. The polynucleotide of Claim 27, wherein said different AAV is selected from the group consisting of AAV1 , AAV4 and AAV5.

29. The polynucleotide of Claim 27, wherein said first AAV is AAV2 and said different AAV is AAV5.

30. The polynucleotide of Claim 27, wherein said first AAV is AAV5 and said different AAV is AAV2.

31. The polynucleotide of any of Claims 20-30, wherein said polynucleotide is a DNA sequence.

32. The polynucleotide of any of Claims 20-31 , wherein said 3' portion is operatively associated with a promoter such that said 3' portion can be expressed independently of said 5' portion.

33. The polynucleotide of Claim 32, wherein said promoter is the AAV p19 promoter.

34. A vector comprising the polynucleotide of any of Claims 1 -33.

35. The vector of Claim 34, wherein said vector is a plasmid vector.

36. The vector of Claim 34, wherein said vector is a viral vector.

37. The vector of Claim 36, wherein said vector is selected from the group consisting of an adenovirus vector, a baculovirus vector, an AAV vector, a herpesvirus vector, and an Epstein-Barr virus vector.

38. A cell comprising the polynucleotide of any of Claims 1-33.

39. The cell of Claim 38, wherein said polynucleotide is stably integrated into the genome of said cell.

40. A cell comprising the vector of any of Claims 34-39.

41. A cell comprising parvovirus rep coding sequences and parvovirus cap coding sequences, said rep coding sequences encoding a DNA binding domain from a first parvovirus; said rep coding sequences further encoding a capsid interacting domain from a different parvovirus from said first parvovirus; said cap coding sequences comprising sequences from said different parvovirus; and said rep coding sequences being stably integrated into the genome of said cell.

42. The cell of Claim 41 , wherein said cap coding sequences are stably integrated into the genome of said cell.

43. A cell comprising adeno-associated virus (AAV) rep coding sequences and AAV cap coding sequences, said rep coding sequences having a 5' portion and a 3' portion; said 5' portion comprising rep coding sequences from a first AAV; said 3' portion comprising rep coding sequences from a different AAV from said first AAV; said cap coding sequences comprising sequences from said different AAV; and said rep coding sequences being stably integrated into the genome of said cell.

44. The cell of Claim 43, wherein said cap coding sequences are stably integrated into the genome of said cell.

45. 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 parvovirus particles comprising the parvovirus capsid encoded by the cap coding sequences and packaging the recombinant parvovirus template are produced in the cell.

46. The method of Claim 45, further comprising the step of collecting the recombinant parvovirus particles.

47. The method of Claim 46, further comprising the step of lysing the cell prior to collecting the recombinant parvovirus particles.

48. The method of any of Claims 45-47, wherein the rep coding sequences and cap coding sequences cannot be packaged into the recombinant parvovirus particles.

49. The method of any of Claims 45-48, wherein the recombinant parvovirus template comprises the heterologous nucleotide sequence flanked by 5' and 3' parvovirus terminal repeats.

50. The method of any of Claims 45-49, wherein the rep coding sequences and/or cap coding sequences are provided by a plasmid.

51. The method of any of Claims 45-50, wherein the rep coding sequences and/or cap coding sequences are provided by a viral vector.

52. The method of Claim 51 , wherein the viral vector is selected from the group consisting of an adenovirus vector, herpesvirus vector, Epstein-Barr virus vector, and baculovirus vector.

53. The method of any of Claims 45-52, wherein the rep coding sequences are stably integrated into the cell.

54. The method of any of Claims 45-53, wherein the cap coding sequences are stably integrated into the cell.

55. The method of any of Claims 45-54, wherein the recombinant parvovirus template is provided by a plasmid or a viral vector or is stably integrated into the cell as a provirus.

56. The method of any of Claims 45-55, wherein the rep coding sequences comprise a 5' portion and a 3' portion; the 5' portion encoding the DNA binding domain from the first parvovirus; the 3' portion encoding the capsid interacting domain from the different parvovirus.

57. The method of any of Claims 45-56, wherein the parvovirus terminal repeat sequence is an autonomous parvovirus terminal repeat sequence and the first parvovirus is an autonomous parvovirus.

58. The method of Claim 57, wherein the terminal repeat sequence' is selected from the group consisting of a B19 virus, canine parvovirus, feline panleukemia virus, muskovy duck parvovirus, goose parvovirus, minute virus of mouse, and H1 virus terminal repeat sequence; and the first parvovirus is selected from the group consisting of B19 virus, canine parvovirus, feline panleukemia virus, muskovy duck parvovirus, goose parvovirus, minute virus of mouse, and H1 virus.

59. The method of Claim 57 or Claim 58, wherein the parvovirus terminal repeat sequence is from the first parvovirus.

60. The method of any of Claims 45-56, wherein the parvovirus terminal repeat sequence is an adeno-associated virus (AAV) terminal repeat sequence and the first parvovirus is an AAV.

61. The method of Claim 60, wherein the AAV terminal repeat sequence is selected from the group consisting of an AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8 terminal repeat sequence.

62. The method of Claim 60 or Claim 61 , wherein the first parvovirus is selected from the group consisting of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8.

63. The method of any of Claims 60-62, wherein the AAV terminal repeat sequence is from the first AAV.

64. The method of any of Claims 45-63, wherein the different parvovirus is an autonomous parvovirus.

65. The method of Claim 64, wherein the different parvovirus is selected from the group consisting of B19 virus, canine parvovirus, feline panleukemia virus, muskovy duck parvovirus, goose parvovirus, minute virus of mouse, and H1 virus.

66. The method of any of Claims 45-65, wherein the different parvovirus is an AAV.

67. The method of Claim 66, wherein: the first parvovirus is selected from the group consisting of

AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8; and the different parvovirus is selected from the group consisting of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8.

68. The method of Claim 66 or Claim 67, wherein: the parvovirus terminal repeat sequence is selected from the group consisting of a AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8 terminal repeat sequence; and the different parvovirus is selected from the group consisting of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8.

69. The method of any of Claims 66-68, wherein the different parvovirus is selected from the group consisting of AAV1 , AAV4 and AAV5.

70. The method of any of Claims 45-69, wherein the rep coding sequences encoding the capsid interacting domain are operatively associated with a promoter such that the capsid interacting domain may be expressed independently of the DNA binding domain.

71. The method of Claim 70, wherein the promoter is the AAV p19 promoter.

72. 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 recombinant hybrid AAV particles comprising the AAV capsid encoded by the cap coding sequences and packaging the rAAV template are produced in the cell.

73. The method of Claim 72, further comprising the step of collecting the infectious rAAV particles.

74. The method of Claim 73, further comprising the step of lysing the cell prior to collecting the infectious rAAV particles.

75. The method of any of Claims 72-74, further comprising providing helper virus sequences which encode the helper virus functions essential for a productive AAV infection, wherein the helper virus sequences cannot be packaged into the rAAV particles.

76. The method of any of Claims 72-75, wherein the rep coding sequences and cap coding sequences cannot be packaged into the rAAV particles.

77. The method of any of Claims 72-76, wherein the AAV template comprises the heterologous nucleotide sequence flanked by 5' and 3' AAV inverted terminal repeats.

78. The method of any of Claims 72-77, wherein the rep coding sequences and/or cap coding sequences are provided by a plasmid.

79. The method of any of Claims 72-78, wherein the rep coding sequences and/or cap coding sequences are provided by a viral vector.

80. The method of Claim 79, wherein the viral vector is selected from the group consisting of an adenovirus vector, herpesvirus vector, Epstein-Barr virus vector, and baculovirus vector.

81. The method of any of Claims 72-80, wherein the rep coding sequences are stably integrated into the cell.

82. The method of any of Claims 72-81 , wherein the cap coding sequences are stably integrated into the cell.

83. The method of any of Claims 72-82, wherein the rAAV template is provided by a plasmid or a viral vector or is stably integrated into the cell as a provirus.

84. The method of any of Claims 72-83, wherein the first AAV is selected from the group consisting of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8.

85. The method of any of Claims 72-84, wherein the AAV terminal repeat sequence is selected from the group consisting of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8 terminal repeat sequence.

86. The method of Claim 84 or Claim 85, wherein the first AAV is AAV2 or AAV5.

87. The method of any of Claims 84-86, wherein the AAV terminal repeat sequence is an AAV2 or AAV5 terminal repeat sequence.

88. The method of any of Claims 70-87, wherein the AAV terminal repeat sequence is from the first AAV.

89. The method of any of Claims 72-88, wherein: the first AAV is selected from the group consisting of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8; and the different AAV is selected from the group consisting of AAV1 ,

AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8.

90. The method of any of Claims 72-89, wherein: the AAV terminal repeat sequence is selected from the group consisting of an AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and

AAV8 terminal repeat sequence; and the different AAV is selected from the group consisting of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8.

91. The method of any of Claims 72-90, wherein the different AAV is selected from the group consisting of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8.

92. The method of Claim 91 , wherein the first AAV is AAV2 and the different AAV is AAV5.

93. The method of Claim 91 , wherein the first AAV is AAV5 and the different AAV is AAV2.

94. The method of any of Claims 72-93, wherein the 3' portion is operatively associated with a promoter such that the 3' portion can be expressed independently of the 5' portion.

95. The method of Claim 94, wherein the promoter is the AAV p19 promoter.