WO1999064569A1 - Methods and compositions for generating recombinant adeno-associated virus vectors - Google Patents

Abstract

The present invention describes a plasmid co-transfection system that reduces homologous recombination which leads to generation of the wild-type (wt) adeno-associated virus 2 (AAV) during recombinant vector production. More particularly, the present invention describes the involvement of 10 nucleotides in the AAV D-sequence in such recombination events. Methods and compositions for the use of recombinant AAV plasmids and helper vectors lacking homology in the D-sequence, and helper plasmids lacking the adenovirus ITRs for use in gene therapy are described.

Description

DESCRIPTION

METHODS AND COMPOSITIONS FOR GENERATING RECOMBINANT ADENO-ASSOCIATED VIRUS VECTORS

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of gene therapy. More particularly, it concerns the engineering, propagation and use of recombinant adeno- associated viral vectors in the delivery of exogenous genes to cells.

2. Description of Related Art

Gene therapy protocols involving recombinant viral vectors are gaining wide attention and have immense potential to become the future mode of molecular medicine. Of the different viral vectors attempted to mediate gene transfer, the retrovirus and adenovirus based vector systems have been extensively investigated over a decade. Recently, adeno-associated virus (AAV) has emerged as a potential alternative to the more commonly used retro viral and adenoviral vectors. While studies with retroviral and adenoviral mediated gene transfer raise concerns over potential oncogenic properties of the former, and immunogenic problems associated with the latter, AAV has not been associated with any such pathological indications.

In addition, AAV possesses several unique features that make it more desirable than the other vectors. Unlike retroviruses, AAV can infect non-dividing cells; wild-type AAV has been characterized by integration, in a site-specific manner, into chromosome 19 of human cells (Kotin and Berns, 1989; Kotin et al, 1990; Kotin et al, 1991; Samulski et al, 1991); and AAV also possesses anti-oncogenic properties (Ostrove et al, 1981; Berns and Giraud, 1996). Recombinant AAV genomes are constructed by molecularly cloning DNA sequences of interest between the AAV ITRs, eliminating the entire coding sequences of the wild-type AAV genome. The AAV vectors thus produced lack any of the coding sequences of wild-type AAV, yet retain the property of stable chromosomal integration and expression of the recombinant genes upon transduction both in vitro and in vivo (Berns, 1990; Berns and Bohensky, 1987; Bertran et al, 1996; Kearns et al, 1996; Ponnazhagan et al, 1997a).

The production of recombinant AAV utilizes a vector containing a transgene cassette flanked by the viral ITRs. Recombinant vectors are generated by co- transfecting the recombinant AAV plasmid and a helper plasmid into adenovirus- infected cells (Samulski et al, 1987, Samulski et al, 1989). The helper plasmid contains the AAV rep and cap genes which provide Rep and Cap proteins in trans, respectively, required for efficient rescue of the recombinant AAV genome from the recombinant plasmid, followed by replication and encapsidation into progeny virions. Rescue, replication and packaging of the AAV genome does not occur from the helper plasmid which lacks the AAV-ITR. However, several reports have documented that wild-type AAV (wt AAV) particles also are generated during recombinant AAV vector production (Hargrove et al, 1997; Koeberl et al, 1997; Kube et al, 1997; Muzyczka, 1992; Ponnazhagan et al, 1997; Snyder et al, 1997), but the underlying mechanism of generation of the wt AAV remains unknown. Whether the contaminating AAV is truly authentic wt AAV, or whether these particles originate following recombination between the recombinant AAV and the helper plasmids also remains unclear. What is clear is that this wt AAV is replication-competent and this significantly impacts upon the possible use of AAV in gene therapy.

From the discussion above, it is clear that AAV is in excellent alternative to retrovirus and adenovirus mediated gene therapy. However, populations of recombinant AAV appear to acquire wild-type characteristics that make them less desirable as gene therapy vectors. If the problem of the generation of wt AAV particles could be addressed, recombinant AAV vectors would be an ideal gene therapy vector.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for producing a plasmid co-transfection system that reduces the generation of wild-type (wt) adeno- associated virus 2 (AAV) that occurs through recombination events during recombinant vector production.

Thus, in one aspect, the present invention provides a method for producing adeno-associated virus (AAV) particles by providing a helper plasmid encoding rep and cap polypeptides; providing a recombinant AAV plasmid; and introducing both the helper plasmid and the AAV plasmid into a cell under conditions supporting replication, rescue and packaging of the recombinant AAV genomes wherein there is no distal D sequence homology between the helper plasmid and the AAV plasmid. In defined aspects of the present invention, the method may be used to provide clinical grade adeno-associated virus.

In particularly preferred embodiments, the recombinant AAV plasmid lacks distal D sequences. In other preferred embodiments, the helper plasmid is an adenovirus that lacks adenoviral ITRs. In more defined preferred embodiment, the helper plasmid is pAAVp5 or pSP-19. In specific embodiments, the AAV plasmid lacks some or all of the distal 10 nucleotides of the D sequences. In particularly preferred embodiments the AAV plasmid lacks the distal 10 nucleotides of the D sequences.

In other embodiment, the AAV plasmid may comprise an expression cassette.

In certain embodiments, the expression cassette may comprise a polynucleotide under the control of a promoter operable in eukaryotic cells. In particularly preferred embodiment, the promoter may be an inducible promoter. In more defined embodiments, the promoter may be CMV IE, SV40 IE, HSV tk, β-actin, human globin α, human globin β, human globin γ, RSV, B19p6, AAVp5, alpha-1 antitrypsm, PGK, tetracyclin, MMTV or albumin promoter. In other aspects of this embodiment, the expression cassette further may comprise a polyadenylation signal. The polyadenylation signal may be any polyadenylation signal known to those of skill in the art as being compatible with the expression constructs being used. In particularly preferred embodiments, the polyadenylation signal may be an AAV polyadenylation signal, an SV40 polyadenylation signal or a BGH polyadenylation signal.

In preferred embodiments, the polynucleotide encodes a polypeptide, an antisense construct or a ribozyme. In particularly preferred embodiments, the polypeptide may be a hormone, a tumor suppressor, an inhibitor of apoptosis, a toxin, a lymphokine, a growth factor, an enzyme, a DNA binding protein or a single-chain antibody.

In particular embodiments, the tumor suppressor may be selected from the group consisting of p53, pl6, p21, MMAC1, p73, zacl, C-CAM, BRCAI and Rb. In certain embodiments, the inducer of apoptosis may be selected from the group consisting of Bax, Bak, Bim, Bik, Bid, Bad Harakiri, Ad E1B and an ICE-CED3 protease. In those embodiments employing a lymphokines, the lymphokines may be selected from the group consisting of IL-2, IL-2, IL-3, IL-4, IL-5, BL-6, IL-7, IL-8, IL- 9, IL-10, ΓL-1 1, IL-12, ΓL-13, ΓL-14, ΓL-IS, TNF, GMCSF, β-interferon and γ- interferon. In those embodiments where the polypeptide is a receptor, the receptor may be selected from the group consisting of CFTR, EGFR, VEGFR, IL-2 receptor and the estrogen receptor. It is contemplated that the polynucleotide may be an oncogene, the polynucleotide being positioned in an antisense orientation with respect to the promoter. In embodiments in which an oncogene is employed, the oncogene may be selected from the group consisting of ras, myc, neu, raf, erb, src, fins, fun, trk, ret, gsp, hst, and abl. The polypeptide may be, for example, selected from the group consisting of amylin, luteinizing hormone, follicle stimulating hormone and chorionic gonadotrophin. In those embodiments where the polypeptide is a hormone, such a hormone may be selected from the group consisting of growth hormone, prolactin, placental lactogen, luteinizing hormone, follicle-stimulating hormone, chorionic gonadotropin, thyroid-stimulating hormone, leptin, adrenocorticotropin (ACTH), angiotensin I, angiotensin LI, β-endorphin, β-melanocyte stimulating hormone (β- MSH), cholecystokinin, endothelin I, galanin, gastric inhibitory peptide (GIP), glucagon, insulin, lipotropins, neurophysins and somatostatin.

In other embodiments, the polypeptide may be an enzyme. Particularly preferred enzymes may be selected from the group consisting of adenosine deaminase, galactosidase, glucosidase, lecithin: cholesterol acyltransferase (LCAT), factor LX, sphingolipase, lysosomal acid lipase, lipoprotein lipase, hepatic lipase, pancreatic lipase related protein, pancreatic lipase and uronidase.

In particular aspects of the present invention it is contemplated that the method further may comprise purifying the AAV particles. In still further embodiments, the method further may comprising formulating the AAV particles in a pharmaceutically acceptable buffer, diluent or excipient.

In defined embodiments, the cell may express an adenovirus polypeptide essential to adenoviral replication. In particular aspects, the adenovirus polypeptide may be an El polypeptide In other embodiments, the cell may express both El A and

E1B. In particularly preferred embodiments the cell is an embryonic kidney cell. In more defined embodiments, the cell is a 293 cell.

In particularly defined aspects of the present invention, the rep and cap polypeptides are derived from AAV. In alternative and preferred embodiments, the cap polypeptide is derived from parvovirus B19. In more defined embodiments, the cap polypeptide comprises the cap VP2 protein. In still further alternative embodiments, the cap polypeptide further may comprise the cap VP1 protein as well as the VP2 protein.

Also provided by the present invention is a method for reducing wild-type adeno-associated virus (AAV)-like particles in a recombinant AAV population comprising providing an AAV plasmid lacking distal D sequences; and introducing the AAV plasmid into a cell, along with a helper plasmid encoding rep and cap polypeptides, under conditions supporting replication.

In especially preferred embodiments, the rep and cap polypeptides are derived from AAV. In other embodiments, the cap polypeptide is derived from B19. In defined embodiments, the helper plasmid lacks adenoviral ITRs. In particularly defined embodiments the helper plasmid is an adenovirus that lacks adenoviral ITRs. In preferred embodiments, the method may further comprising purifying the recombinant AAV population.

The present invention further contemplates a population of adeno-associated virus (AAV) particles comprising recombinant AAV plasmids, the population containing less than 3% percent wild-type AAV-like particles. In other preferred embodiments, the population contains less than 2% wild-type AAV-like particles. In another alternative preferred embodiments, the population contains less than 1% wild- type AAV-like particles. In another alternative preferred embodiments, the population contains less than 0.5% wild-type AAV-like particles. In another alternative preferred embodiment, the population contains less than 0.25% wild-type AAV-like particles. In especially preferred embodiments, the population of adeno-associated virus plasmids is essentially free of wild-type AAV-like particles. In further preferred embodiments the AAV plasmids comprises an expression cassette. In especially preferred embodiments, the expression cassette comprises a polynucleotide under the control of a promoter operable in eukaryotic cells. In further embodiments, the expression cassette further comprises a polyadenylation signal. The polynucleotide may encode a polypeptide, an antisense construct or a ribozyme. Particularly preferred polypeptides include hormones, tumor suppressors, inhibitors of apoptosis, toxins, lymphokines, growth factors, enzymes, DNA binding proteins, single-chain antibodies and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1: Schematic representation of the recombinant AAV helper plasmids. The three viral promoters are denoted by arrows, and the viral rep and cap genes are represented by shaded and cross-hatched boxes, respectively. The Ad-ITRs are denoted by closed boxes, and the plasmid vector backbones are indicated by thin lines.

FIG. 2A and FIG. 2B: (FIG. 2A) Southern blot analyses for identification of replication-competent wt AAV-like genomes. Following 4 rounds of amplification, low Mr DNA samples were digested with the indicated restriction endonucleases and analyzed on Southern blots using the right-half of the AAV DNA (EcoRI-SacI DNA fragment) probe, m and d denote the monomeric and dimeric replicative forms of the AAV genome. (FIG. 2B) Schematic representation of the AAV DNA replicative intermediates. The restriction endonuclease restriction recognition sites for Sacl (S), Xbal (X), Ball (B) enzymes from the wt and the wt AAV-like DNA are indicated. The EcoRI (E)-Xbal (X) DNA probe specific for the right-half of the AAV genome from plasmid pSub201 is depicted as a thick line, and AAV-ITRs are denoted as closed boxes.

FIG. 3: Experimental strategy for cloning the wt AAV-like genomes from recombinant vector stocks. These particles generated during the recombinant vector production are amplified through 4 successive rounds of infection of adenovirus-infected 293 cells. Low Mr DNA is digested with Ball restriction endonuclease and DNA fragments are cloned in a pBluescript SK(+) plasmid vector. AAV sequence-positive clones are subjected to nucleotide sequencing using the T3 and the T7 primers.

FIG. 4: Nucleotide sequences of the left and the right junctions between

AAV ITRs derived from the recombinant AAV-lacZ vector and the AAV sequence derived from the helper plasmid (pAAVp5). The D-sequence, downstream from the Ball site (CCAA), is shown in outline, shadow font and the AAV sequences from the helper plasmid are shown in bold, italic font. The sequence shown in outline, italic font in the right junction in plasmids in group A represents a duplication of the same sequence from the left junction. The underlined nucleotide pairs indicate the recombination junctions. Nucleotide sequences shown in this figure are also reported as the following SEQ ID Listings: Form La, SEQ LD NO: l; Form Lb, SEQ ID NO:2; Form Lc, SEQ ID NO:3; Form Ra, SEQ ID NO:4; Form Rb, SEQ ID NO:5; Form Re, SEQ ID NO:6; Form Rd, SEQ LD NO:7.

FIG. 5: Summary of nucleotide sequences of the left and the right junctions in AAV ITRs from the recombinant AAV-lacZ vector. The sequence at the top represents the AAV D-sequence (outline, shadow font) downstream from the Ball site. The three left and four right end sequences in the wt AAV-like genomes are shown in the middle. The sequence at the bottom indicates the recombination sites in AAV ITRs. The underlined nucleotides form the junctions, and each asterisk represents the frequency of the recombination events. Nucleotide sequences shown in this figure are also reported as the following SEQ ID Listings: AAV-ITR, SEQ ID NO:8; La, SEQ ID NO:9; Lb, SEQ ID NO: 10; Lc, SEQ ID NO: 11; Ra, SEQ ID NO: 12; Rb, SEQ ID NO: 13; Re, SEQ ID NO: 14; Rd, SEQ ID NO: 15.

FIG. 6: Nucleotide sequences of the left and the right junctions between

AAV-ITRs from the recombinant vector pWP-8A, and the AAV sequence from the pAAV/Ad helper plasmid. The D-sequence is shown in outline, shadow font and the helper plasmid sequences are shown in bold, italic font. The underlined nucleotides indicate the recombination junctions, and the asterisks represent the recombination frequency. Nucleotide sequences shown in this figure are also reported as the following SEQ LD Listings: AAV-ITR in pWP-8A, SEQ ID NO: 16; Junctions at the left end in the wt AAV-like genome- a, SEQ LD NO: 17; b, SEQ LD NO: 18; c, SEQ ID NO: 19; d, SEQ ID NO:20; e, SEQ ID NO:21; f, SEQ ID NO:22; Junctions at the right end in the wt AAV-like genome- a, SEQ ID NO:23; b, SEQ LD NO:24; c, SEQ ID NO:25.

FIG. 7: Nucleotide sequences of the junction fragments involving the adenovirus ITRs. The adenovirus ITR sequence is shown in bold, italic font at the top. The sequence in the middle corresponds to the D-sequence downstream from the Ball site in the recombinant AAV plasmid pWP-8A. The recombination sites in the Ad5 ITR sequences are indicated by the underlined nucleotides. The asterisks represent the recombination frequency. Nucleotide sequences shown in this figure are also reported as the following SEQ ID Listings: Ad5-ITR, SEQ LD NO:26; AAV-ITR in pWP-8A, SEQ ID NO: 16; Recombination Sites in the Ad5-ITR, SEQ LD NO:27.

FIG. 8: Nucleotide sequence analyses of recombinant junctions in the left ITR of the wt AAV-like genomes. The AAV D-sequence, starting with the Ball site (nt 122) is shown in outline fonts, and the rest of the AAV DNA sequence is shown in bold font. +Ad5-ITR denotes the helper plasmid that contains the Ad5- ITRs (pAAV/Ad), and -Ad5-ITR denotes the helper plasmid that lacks the Ad5-ITRs (pSP-19) which were used as helper-plasmids to generate the recombinant AAV vector stocks. The underlined nucleotides represent the recombination sites, and the numbers indicate the observed frequency of recombination events in 7 clones for the former, and in 22 clones for the latter that were analyzed. The nucleotide sequence shown in this figure is also reported as SEQ ID NO:28.

FIG. 9: Schematic structures of pSub201 and pD-10 recombinant AAV vectors. The D-sequence is shown as a shaded box in plasmid pSub201. In plasmid pD-10, the distal 10 nucleotides in the D-sequence have been replaced by a substitute (S)-sequence described previously (Wang et al, 1997). The relevant restriction endonuclease sites (Xbal in pSub201, and EcoRV in pD-10) for cloning a gene of interest are also indicated. Nucleotide sequences shown in this figure are also reported as the following SEQ ID Listings: D-20 Sequence, SEQ ID NO:29; D-10/S-lO Sequence, SEQ ID NO:30.

FIG. 10A and FIG. 10B: Southern blot analyses of replication of the recombinant AAV-lacZ and the wt AAV-like genomes generated from recombinant plasmids pCMVp-lacZ (in pSub201) or pBK-2 (CMVp-lacZ in pD-10) with helper plasmids containing Ad5- ITRs (pAAV/Ad), or lacking it (pSP-19), respectively. Equivalent amounts of low Mr DNA isolated at 72 hrs post-transfection from adenovirus co-infected 293 cells were analyzed on Southern blots using either lacZ- specific (FIG. 10A), or AAV-specific (FIG. 10B) DNA probes. Autoradiography was performed for 48 hrs (for FIG. 10A), and for 4 days (for FIG. 10B). m and d denote the monomeric and dimeric viral replicative DNA intermediates, respectively.

FIG. 11: Southern blot analyses of replication of wt AAV-like genomes present in recombinant vCMVp-lacZ vector stocks generated from the recombinant

AAV plasmid containing the entire D-sequence (pCMVp-lacZ) and the helper plasmid containing Ad5 ITRs (pAAV/Ad) (lanes 2,5,8,11), or from the recombinant AAV plasmid containing deletions in the distal 10 nucleotides in the D-sequence (pBK-2) and the helper plasmid, pSP-19, lacking Ad5 ITRs (pSP-19) (lanes 3,6,9,12). Equivalent amounts of low Mr DNA isolated at 72 hrs post-infection from adenovirus co-infected 293 cells were obtained following the indicated rounds of amplification (l-4x), and analyzed on Southern blots using an AAV-specific DNA probe. In lanes 1,4,7, and 10, low Mr DNA from mock-infected 293 cells were analyzed, m and d denote the monomeric and dimeric replicative forms of the AAV genome, respectively.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Viral vectors are widely utilized for a variety of gene transfer endeavors. For example, retroviral vectors have been used for a number of years to transform cell lines in vitro for the purpose of expressing exogenous polypeptides. More recently, with advancements in genetic therapies, various other vectors including adeno viruses and herpes viruses, along with retroviruses, have been utilized to transfer therapeutic genes into the cells of patients.

A variety of problems remain with respect to the use of viral vectors, however. First, many vectors are toxic to cells, and some may even be oncogenic. Second, expression of even non-toxic viral gene products may lead to the development of an immune response in the patient, thereby limiting the efficacy of the treatment and possibly preventing repeat administrations. Third, many viruses have limited host ranges and some may only replicate in dividing cells. Fourth, diminished transgene expression over time may further limit the effectiveness of the vector. And fifth, it may be difficult to generate viral vectors with sufficient titers to make treatments possible. All of these limitations have, in one case or another, hampered efforts by researchers to implement gene therapy. While retroviral vectors and adenoviral vectors have been associated with a wide variety of pathological indications, adeno-associated viral (AAV) vectors are considered especially desirable for a number of reasons. In the first instance, AAV is not associated with any known pathological indications. Further, AAV can infect non-dividing cells (Kotin et al, 1990; Kotin et al, 1991; Samulski et al, 1991) and also possesses anti-oncogenic properties (Berns and Giraud, 1996). AAV vectors can be produced that lack any of the coding sequences of wild-type AAV, yet retain the property of stable chromosomal integration and expression of the recombinant genes upon transduction both in vitro and in vivo (Bertran et al, 1996; Kearns et al, 1996; Ponnazhagan et al, 1997a).

Although an attractive alternative to other viral vectors, the use AAV as a delivery vector has been limited. Several reports have documented that wild-type (wt) AAV particles are generated during recombinant AAV vector production (Hargrove et al, 1997; Koeberl et al, 1997; Kube et al, 1997; Muzyczka, 1992; Ponnazhagan et al, 1997; Snyder et al, 1997). The underlying mechanism of generation of the wt AAV in these recombinant techniques to date has been an unresolved. Whether the contaminating AAV is truly authentic wt AAV, or whether these particles originate following recombination between the recombinant AAV and the helper plasmids also was unclear.

In pursuit of answers to these questions, the inventors carried out systematic analyses of the wt AAV genomes molecularly cloned from different recombinant AAV vector stocks. The present invention shows that the contaminating AAV is not authentic wt AAV but, rather, wt "AAV-like" particle. These wt AAV-like particles are generated by non-homologous recombination between the recombinant AAV and the helper plasmids. Furthermore, it appears that adenovirus ITRs in helper plasmids contribute to generation of these particles. Upon further investigation, it was revealed that the recombinatorial sites in the AAV-ITRs are clustered around the distal 10 nucleotides in the D-sequence, and the removal of the adenovirus ITRs from the helper plasmid, and/or deletion of the distal 10 nucleotides in the D-sequence from the recombinant plasmid, leads to elimination of replication-competent wt AAV-like particles. The present invention is, therefore, directed to the production of recombinant AAV vectors for use in gene therapy. Methods and compositions for achieving these objectives are described in further detail herein below.

A. Adeno-associated Virus and its use in Gene Therapy a. Adeno-associated Virus

AAV utilizes a linear, single-stranded DNA of about 4700 base pairs. Inverted terminal repeats (ITRs) flank the genome. Two genes are present within the genome, giving rise to a number of distinct gene products. The first, the cap gene, produces three different virion proteins (VP), designated VP-1, VP-2 and VP-3. The second, the rep gene, encodes four non-structural proteins (NS). One or more of these rep gene products is responsible for transactivating AAV transcription. The sequence of AAV is provided in Srivastava et al, (1983).

The three promoters in AAV are designated by their location, in map units, in the genome. These are, from left to right, p5, pl9 and p40. Transcription gives rise to six transcripts, two initiated at each of three promoters, with one of each pair being spliced. The splice site, derived from map units 42-46, is the same for each transcript. The four non-structural proteins apparently are derived from the longer of the transcripts, and three virion proteins all arise from the smallest transcript.

AAV is not associated with any pathologic state in humans. Interestingly, AAV is dependent upon co-infection with a helper virus, for its optimal replication (Berns and Bohenzky, 1987; Berns and Giraud, 1996). Such helper viruses include herpes simplex virus I and II, cytomegalovirus, pseudorabies virus and, of course, adenovirus. The best characterized of the helpers is adenovirus, and many "early" functions for this virus have been shown to assist with AAV replication. Low level expression of AAV rep proteins is believed to hold AAV structural expression in check, and helper virus infection is thought to remove this block.

In the absence of a helper virus, the wt-AAV genome integrates into the host chromosome in a site-specific manner and establishes a latent infection (Kotin and Berns, 1989; Kotin et al, 1991; Kotin et al, 1990; Samulski et al, 1991). Three elements of the AAV genome are required for the viral replicative life cycle. The first is a pair of inverted terminal repeats (ITRs) which fold into hairpin structures such that the 3'-end serves as a primer for AAV DNA replication (Hauswirth and Berns, 1977; Lusby et al, 1980; Srivastava, 1987; Samulski et al, 1982; Samulski et al, 1983). ITRs are also required for AAV genome encapsidation and integration (Giraud et al, 1995; Samulski et al, 1983; Wang et al, 1995). The second is the rep gene which codes for four viral replication (Rep) proteins (Berns and Giraud, 1996; Berns, et al, 1988; Muzyczka, 1992). Rep proteins are also required for viral gene expression, DNA encapsidation and site-specific integration (Ashktorab and Srivastava, 1989; Berns, et al, 1988; Chejanovsky and Carter, 1989; Chiorini et al, 1996; Flotte et al, 1994; Im and Muzyczka, 1989; Im and Muzyczka, 1990; Im and Muzyczka, 1992; Kube et al, 1997; McCarty et al, 1994a; McCarty et al, 1994b, Samulski et al, 1982; Senapathy et al, 1984). And the third is the cap gene which encodes the viral capsid (Cap) proteins required for viral assembly (Samulski et al, 1982; Srivastava et al, 1983).

ITRs are the sole cis-acting sequences required for viral DNA replication, encapsidation and integration (Samulski et al, 1989). Based on the fact that AAV is a non-pathogenic human parvovirus that can infect both dividing and non-dividing cells (Flotte et al, 1994; Podsakoff et al, 1994), and that it can stably integrate into the host chromosome, recombinant AAV vectors have been developed as a potentially useful alternative to the more commonly used retroviral and adenoviral vectors for human gene therapy (Berns and Giraud, 1996; Flotte and Carter, 1995; Muzyczka, 1992; Samulski et al, 1989; Shaughnessy et al, 1996; Srivastava, 1994). The terminal repeats of the AAV vector of the present invention can be obtained by restriction endonuclease digestion of AAV or a plasmid such as psub201, which contains a modified AAV genome (Samulski et al, 1987), or by other methods known to the skilled artisan, including but not limited to chemical or enzymatic synthesis of the terminal repeats based upon the published sequence of AAV. The ordinarily skilled artisan can determine, by well-known methods such as deletion analysis, the minimum sequence or part of the AAV ITRs which is required to allow function, i.e., stable and site-specific integration. The ordinarily skilled artisan also can determine which minor modifications of the sequence can be tolerated while maintaining the ability of the terminal repeats to direct stable, site-specific integration.

The AAV-ITRs contain a domain, designated the D-sequence which is comprised of a stretch of 20 nucleotides that are not involved in the HP formation (Berns and Bohenzky, 1987; Berns and Giraud, 1996; Srivastava et al, 1983). The inventors have recently shown that the D sequence plays a crucial role in high- efficiency rescue, selective replication, and encapsidation of the AAV genome and that a host cell protein, designated the D sequence-binding protein (D-BP), specifically interacts with this sequence (Wang, et al, 1996).

Mutational analyses of the D sequences were performed to evaluate their precise role in viral DNA rescue, replication, and packaging. These studies revealed that 10 nucleotides proximal to the HP structure in each of the D sequences are necessary and sufficient to mediate high-efficiency rescue, replication, and encapsidation of the viral genome in vivo. In in vitro studies, the same 10 nucleotides were found to be required for specific interaction with D-BP, but viral Rep protein- mediated cleavage at the functional terminal resolution site is independent of these sequences. The inventors show that AAV replication and terminal resolution functions can be uncoupled and that the lack of efficient replication of AAV DNA may not be a consequence of impaired resolution of the viral ITRs. b. Adeno-associated Viral Mediated Gene Therapy

AAV -based vectors have proven to be safe and effective vehicles for gene delivery in vitro, and these vectors are now being developed and tested in pre-clinical and clinical stages for a wide range of applications in potential gene therapy, both ex vivo and in vivo. However, the inventors (Ponnazhagan et al, 1997b; 1997c) and others (Carter and Flotte, 1996 ; Chatterjee et al, 1995; Ferrari et al, 1996; Fisher et al, 1996; Flotte et al, 1993; Goodman et al, 1994; Kaplitt et al, 1994 and 1996;

Kessler et al, 1996; Koeberi et al, 1997; Mizukami et al, 1996; Xiao et al, 1996) have repeatedly observed wide variations in AAV transduction efficiency in different cells and tissues in vitro as well as in vivo.

It would seem reasonable to suggest that AAV transduction efficiency correlates with the number of the putative cell surface receptors. It was recently revealed that membrane-associated heparan sulfate proteoglycan serves as the viral receptor for AAV type 2 (Summerford and Samulski, 1998). However, it has become clear from the inventors' present studies that such a correlation most probably does not exist since 293 cells that express relatively fewer numbers of these putative receptors are transduced most efficiently, an observation consistent with previously published reports (Ferrari et al, 1996; Fisher et al, 1996).

AAV-mediated efficient gene transfer and expression in the lung has already led to clinical trials for the treatment of cystic fibrosis (Carter and Flotte, 1996; Flotte et al, 1993). Similarly, the prospects for treatment of muscular dystrophy by AAV- mediated gene delivery of the dystrophin gene to skeletal muscle, of Parkinson's disease by tyrosine hydroxylase gene delivery to the brain, of hemophilia B by Factor EX gene delivery to the liver, and potentially of myocardial infarction by vascular endothelial growth factor gene to the heart, appear promising since AAV-mediated transgene expression in these organs has recently been shown to be highly efficient (Fisher et al, 1996; Flotte et al, 1993; Kaplitt et al, 1994 and 1996; Koeberi et al, 1997; McCown et al., 1996; Ping et al, 1996; Xiao et al, 1996). Since the present invention shows that the contamination of a recombinant AAV viral stock arises from recombination events between the D-sequences of the AAV with the ITR of the helper virus, it is now possible to generate uncontaminated AAV for use in such gene therapies.

B. Helper Viruses a. Adenovirus

Adenovirus is a linear, double-stranded DNA virus with a genome of about 36 kB. Adenovirus can infect a wide range of host cells in a non-integrative fashion. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans. Both ends of the viral genome contain 100-200 base pair inverted terminal repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication. The El region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shut-off (Renan, 1990). The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP, (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and all the mRNA's issued from this promoter possess a 5'-tripartite leader (TPL) sequence which makes them preferred mRNA's for translation.

Racher et al, (1995) disclosed improved methods for culturing 293 cells and propagating adenovirus. In one format, natural cell aggregates are grown by inoculating individual cells into 1 liter siliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium. Following stirring at 40 rpm, the cell viability is estimated with trypan blue. In another format, Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/1) is employed as follows. A cell inoculum, resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250 ml Erlenmeyer flask and left stationary, with occasional agitation, for 1 to 4 h. The medium is then replaced with 50 ml of fresh medium and shaking initiated. For virus production, cells are allowed to grow to about 80% confluence, after which time the medium is replaced (to 25% of the final volume) and adenovirus added at an MOI of 0.05. Cultures are left stationary overnight, following which the volume is increased to 100% and shaking commenced for another 72 h.

The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.

As stated above, adenovirus is relatively easy to grow and manipulate and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g., 10⁹-10ⁿ plaque-forming units per ml, and they are highly infectious. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et al, 1963; Top et al, 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.

b. Herpes Viruses

Because herpes simplex virus (HSV) is neurotropic, it has generated considerable interest in treating nervous system disorders. Moreover, the ability of HSV to establish latent infections in non-dividing neuronal cells without integrating in to the host cell chromosome or otherwise altering the host cell's metabolism, along with the existence of a promoter that is active during latency makes HSV an attractive vector. And though much attention has focused on the neurotropic applications of HSV, this vector also can be exploited for other tissues given its wide host range.

HSV also is relatively easy to manipulate and can be grown to high titers. Thus, delivery is less of a problem, both in terms of volumes needed to attain sufficient MOI and in a lessened need for repeat dosings. For a review of HSV as a gene therapy vector, see Glorioso et al. (1995).

HSV, designated with subtypes 1 and 2, are enveloped viruses that are among the most common infectious agents encountered by humans, infecting millions of human subjects worldwide. The large, complex, double-stranded DNA genome encodes for dozens of different gene products, some of which derive from spliced transcripts. In addition to virion and envelope structural components, the virus encodes numerous other proteins including a protease, a ribonucleotides reductase, a DNA polymerase, a ssDNA binding protein, a helicase/primase, a DNA dependent ATPase, a dUTPase and others.

HSV genes form several groups whose expression is coordinately regulated and sequentially ordered in a cascade fashion (Honess and Roizman, 1974; Honess and Roizman 1975; Roizman and Sears, 1995). The expression of α genes, the first set of genes to be expressed after infection, is enhanced by the virion protein number 16, or α-transinducing factor (Post et al, 1981; Batterson and Roizman, 1983; Campbell, et al, 1983). The expression of β genes requires functional α gene products, most notably ICP4, which is encoded by the α4 gene (DeLuca et al, 1985). γ genes, a heterogeneous group of genes encoding largely virion structural proteins, require the onset of viral DNA synthesis for optimal expression (Holland et al, 1980). In line with the complexity of the genome, the life cycle of HSV is quite involved. In addition to the lytic cycle, which results in synthesis of virus particles and, eventually, cell death, the virus has the capability to enter a latent state in which the genome is maintained in neural ganglia until some as of yet undefined signal triggers a recurrence of the lytic cycle. Avirulent variants of HSV have been developed and are readily available for use in gene therapy contexts (U.S. Patent No. 5,672,344).

c. Others Although adenovirus is described herein as an exemplary helper virus, additional helper viruses that may be useful herein include cytomegalovirus and pseudorabies virus. These viruses are well known to those of skill in the art.

Pseudorabies virus is described in U. S. Patents 5,736,319; 5,047,237; 4,514,497, 5,242,829, 5,674,709 and 4,810,634, in as much as these documents provide descriptions of the components of a pseudorabies virus and for modifying and using such viruses, these documents are each incorporated herein by reference.

Cytomegalovirus (CMV) is another virus well known to those of skill in the art. CMV is a member of the of the family of herpesviridae. CMV particles have a diameter of 120 to 200 nm and consist of a core containing double-stranded DNA, an icosahedral capsid and a surrounding envelope. Electron microscopic features of

CMV are well documented and include virions morphologically indistinguishable from other herpes viruses. Viral replication occurs in the nucleus of the host cell and involves the expression of immediate early, early and late classes of genes. The viral envelope is formed as assembled nucleocapsids bud from the inner surface of the nuclear membrane. U.S. Patents 5,720,957; 4,058,598; 4,762,780; 5,168,062, each incorporated herein by reference, provide additional background regarding CMV and disclosure pertaining to methods and compositions employing CMV. C. AAV Recombinant Vectors for Therapeutic Applications

In accordance with the present invention, two vectors are provided that, when introduced into the same cell, produce an apathogenic, recombinant AAV useful in gene therapy application. The components of these vectors are described in further detail herein below.

a. D-Sequence

The AAV-ITRs discussed above also contain an additional domain, designated the D-sequence (SEQ ID NO:29), which is discussed in further detail herein below. The present invention shows that the D-sequence contain recombinatorial "hotspots." The inventors have shown that about the distal 10 nucleotides of the D-sequence are responsible for the wt AAV contamination that is seen when helper virus is used to produce recombinant AAV. Thus, the present invention provides methods and compositions for decreasing or circumventing this problem.

In preferred embodiments of the present invention, there is provided a recombinant AAV in which the 10 nucleotides proximal to the HP structure in each of the D sequences are intact, whilst about the 10 nucleotides distal to the HP structure have been deleted, removed, other otherwise made non-functional. Thus, in a particularly preferred embodiment, the D-sequence of the recombinant AAV may comprise about the 10 nucleotides proximal to the HP structure and none of the nucleotides of the distal 10 nucleotides of the D-sequence. As the inventors have found that the distal 10 nucleotides of the D-sequence contain recombinatorial hotspots, it is contemplated that any mechanism which prevents such recombination between the recombinant AAV plasmid and the helper ITR region will be useful in the present invention. Thus it may not be necessary to delete, mutate, or otherwise remove all 10 of the distal nucleotides of the D-sequence, rather, it would be sufficient to delete, mutate or otherwise alter those residues that are involved in these recombinatorial events. An exemplary mutated D-sequence is shown as SEQ ID NO:30. Therefore, the D-sequence of the recombinant AAV may comprise about the 10 nucleotides proximal to the HP structure and a portion of the distal 10 nucleotides of the D-sequence. The term "a portion" may refer to one, two, three, four, five, six, seven, eight or nine of the nucleotides of the distal 10 nucleotides of the D-sequence, in this respect, such a portion of the sequence would preferably be one that has a reduced capacity to undergo recombination with a helper ITR. In other embodiments, the D-sequence of the recombinant AAV may comprise about 10 nucleotides proximal to the HP structure and about 10 distal nucleotides of the D sequence in which about 10 nucleotides do not comprise the wild-type D-sequence but a sequence that is inhibited from undergoing the recombination with the helper virus ITR regions. Such an exemplary sequence is shown as SEQ ID NO:30. In particular embodiments, it may be that one or more of the distal 10 nucleotides is blocked, thereby preventing it from undergoing a recombination event. By "blocked" it is intended to mean that a particular nucleotide is modified such that it can not take part in a particular reaction. Agents and methods for modifying a nucleotide in such a way are well known to those of skill in the art.

Techniques for deleting or mutating the nucleotide residues at a particular point in a give nucleotide sequence are well known to those of skill in the art. Given that the present invention has shown that about the 10 nucleotides of the D-sequence distal to the HP structure are responsible for the recombinatorial events that lead to the generation of replication competent wild-type like AAV particle, it will be well within the skill of one in the art to mutate, delete or otherwise alter each and/or any of the 10 residues to identify the minimal motif that is responsible for this recombinatorial action. Once such a motif is identified it can be used to generate the recombinant adeno-associated vectors of the present invention. By way of example, Wang et al,

(1997) is incorporated herein by reference, as providing disclosure on the mutation of the D sequence of AAV (SEQ ID NO:30, FIG. 9, D-lO/S-10 Sequence). b. Adeno-Associated Recombinant Vector

The first vector is derived primarily from AAV and can carry a recombinant DNA construct comprising a heterologous gene to be delivered to a target cell. This vector contains the AAV ITR regions but preferably may lack the presence of some or all of the 10 nucleotides of the D-sequence distal to the HP structure of AAV.

Thus, in a preferred embodiment for the therapeutic applications of the present invention, the AAV recombinant vectors comprise a first and second AAV ITR, which flank at least a first promoter operably linked to a heterologous gene. The terminal repeats can comprise all or an active portion of the ITRs of AAV. By active, it is meant that sufficient portions of the ITR exists to permit replication and packaging of the vector. Further, the ITRs mediate stable integration of the DNA sequence into a specific site in a particular chromosome. The entire DNA sequence, including the ITRs, the promoter, and the heterologous gene, is integrated into the genome. Therefore, in preferred embodiments, the ITRs or portions thereof, also permit integration. As described herein above, in an especially preferred embodiments, some or all of the distal 10 nucleotides of the D-sequence of the AAV ITRs have been deleted.

c. Adeno-Associated Virus Helper Vectors

The second vector is derived from adeno-associated virus, containing the rep and cap gene to help the AAV recombinant vector replicate. This second vector serves to provide structural and replicative functions that facilitate the packaging of the first vector, since the results presented herein show that the adenovirus ITR regions are involved in recombinatorial events. The presence of Ad5 ITRs in the helper plasmid promotes recombination between the recombinant AAV and the helper plasmids. It appears that most of the recombination events are clustered in the distal 10 nucleotides in the D-sequence. Further, the inventors showed that removal of the adenovirus ITRs from the AAV helper plasmid, the removal of the distal 10 nucleotides in the D-sequence, or the removal of both, eliminate generation of replication-competent wt AAV-like particles during recombinant AAV vector production. Thus, in particularly preferred embodiments of the present invention, the adeno-associated virus helper vector used in the present invention may lack some or all of the adenovirus ITR region(s). Of course, it is understood that there may be particular hotspots in the adenovirus ITR that are involved in these recombination events, thus given the teachings of the present invention, it is well within the skill of one in the art to identify such hotspots. These recombinations may involve particular nucleotides that can be deleted or mutated so that the recombination event will not occur.

The remaining components of the adeno-associated virus helper plasmid supply the replication (rep) function of AAV and the capsid (cap) function. The rep gene codes for four viral replication (Rep) proteins (Berns and Giraud, 1996; Berns, et al, 1988; Muzyczka, 1992). Rep proteins are also required for viral gene expression, DNA encapsidation and site-specific integration (Ashktorab and Srivastava, 1989; Berns, et al, 1988; Chejanovsky and Carter, 1989; Chiorini et al, 1996; Flotte et al, 1994; Im and Muzyczka, 1989; Im and Muzyczka, 1990; Im and Muzyczka, 1992; Kube et al, 1997; McCarty et al, 1994a; McCarty et al, 1994b, Samulski et al, 1982; Senapathy et al, 1984). The cap gene encodes the viral capsid (Cap) proteins required for viral assembly (Samulski et al, 1982; Srivastava et al, 1983). Each of these genes may be contained in a single expression cassette. An exemplary construct utilizes a CMV IE promoter operably linked to a cap gene from AAV, and a p5 promoter of AAV operably linked to the rep gene. Where multigene constructs are utilized, internal ribosome entry sites (IRES) may increase the level and fidelity of expression of the downstream gene (discussed below). In certain embodiments, the cap gene may be from yet another virus such as, for example, the human parvovirus B19 cap gene.

The currently available helper plasmids used to generate recombinant AAV vector stocks suffer from the problem that they yield limited amounts of the viral capsid proteins since they are unable to undergo DNA replication. The inventors propose to exploit human origins of replication (Or/) by inserting these sequences into the helper plasmids in order to facilitate plasmid DNA replication. One such Ori has been identified and isolated from the human choline acetyltransferase (CHAT) gene (Boulikas et al. 1998). This 500-bp DNA fragment, when inserted into a recombinant plasmid containing the CMV promoter-driven Luc gene, results in episomal replication of the plasmid for more than 2 months (approximately 70 cell generations), and more than 1 ,000-fold increase in Luc activity compared with initially-transfected cells. Two scenarios of helper plasmids containing origins of replication will be generated. The first scenario is a single plasmid containing rep and cap genes with an origin of replication. The second scenario is the use of two helper plasmids, one plasmid simply containing the rep gene and the second plasmid containing the cap gene and an origin of replication on the plasmid.

The inventors anticipate that by disrupting the AAV rep-cap cassette, and inserting the 500-bp Ori sequence into the cap plasmid, there will be a significant increase in the capsid protein production, hopefully leading not only to increased viral titers but also further reduction in the contaminating wild-type AAV-like particles (Wang et al, 1998). An additional advantage of this approach is that the need for repeated transfections for each round of recombinant vector packaging will be obviated since these plasmids will be retained in episomal forms, and aliquots of transfected cells can be stored frozen and reused multiple times.

Another approach will be to utilize a helper adenovirus plasmid which obviates the need to use helper adenovirus altogether (Xiao et al, 1998). Such adeno- AAV helper plasmid have been described recently (Grimm et al, 1998). The Ori sequence will be inserted into such a helper adenovirus plasmid. In addition to the benefits of the Ori sequence described above, the relatively low transfection efficiency of this -20 kb plasmid (Grimm et al, 1998) can thus be compensated for by its replication following transfection. In another embodiment, the present invention contemplates the use of helper vectors packaged in viral particles. In particular, the recombinant AAV vectors can be engineered to contain sufficient s-acting signals for their packaging in adenoviral capsids. The requirements for adenoviral packaging are well-defined by the field and largely reside in the adenoviral left end ITR. The only other major concerns relate to the compatability of inserted sequences and the overall size of the vector, which is up to about 40 kB. This system would require provision of adenoviral replicative and packaging functions in trans, either by a cell line or by a helper virus. Essential functions from adenovirus, which would need to be provided, include El A, E1B, E2A, E2B, E4 and L1-L5. 293 cells are well known in the art and can provide El A and E1B functions.

Generally, any high level expression promoter will suffice to drive these genes. However, certain advantages may accrue through the use of the homologous (normal) promoter for these genes and/or viruses. For example, position effects in the 5' untranslated portion of a gene may be difficult to duplicate in synthetic constructs and may be responsible for dramatic variations in expression levels. Similarly, given the dependence of the present vectors on superinfecting helper viruses, it may prove advantageous to utilize the homologous promoter. As mentioned above, the B 19 p6 promoter may be very useful in the context of the present invention, and is a homologous promoter with respect to the B19 cap gene. Another homologous promoter is the AAV p5 promoter for the AAV rep gene.

D. Propagation of Vectors and Transformation of Host Cells a. Propagation of r AAV

The following is an exemplary description of the propagation of vectors of the present invention. Of course the conditions described are only exemplary and in light of the present disclosure it will be possible for one of ordinary skill in the art to modify these propagation conditions according to particular needs. Approximately 10 μg each of the recombinant AAV plasmid and the AAV-helper plasmids are co- transfected per confluent 100 mm dish of 293 cells. Transfected cells are also infected with 10 plaque-forming units (pfu) per cell of human adenovirus type 2 (Ad2). Crude cell lysates are prepared 65-72 hrs post-infection by three cycles of freezing and thawing followed by heat-inactivation of Ad2 at 56°C for 30 min. Clarified supernatants are digested with DNase I (100 U/ml) for 1 hr at 37°C and adjusted to a density of 1.40 g/ml by addition of CsCl and centrifuged at 35,000 rpm for 40 hrs at 20°C. Equilibrium density gradients are fractionated by collecting drops through a puncture in the bottom of the centrifuge tube. The densities of all fractions are determined from refractive index measurements. All fractions are dialyzed against lxSSC (0.15 M NaCl, 0.015 M sodium citrate) and analyzed for viral DNA by slot blot analysis. Viral titers of rAAV generally ranges from 10¹¹ to 10¹³ particles/ml, and for rB19 from 10⁸ to 10⁹ particles/ml.

Virions can be produced by cotransfection of the helper plasmid and the AAV plasmid, followed by infection with a helper virus such as adenovirus, herpes virus or vaccinia virus. Transfection may be accomplished using any standard gene transfer mechanism: calcium phosphate precipitation, lipofection, electroporation, microprojectile bombardment or other suitable means. Following transfection, host cells are infected with a helper virus, virions are isolated and the helper virus is inactivated (e.g., heated at 56°C for one hour). The resulting helper free stocks of virions are used to infect appropriate target cells.

b. Transformation with rAAV

Bone Marrow Cells are an example of host cells that may be transformed by rAAV. Equivalent numbers of primary human low-density bone marrow (LDBM) mononuclear cells and differentiated and undifferentiated CD34+ cells are either mock-infected or infected with the recombinant B19-lacZ or recombinant AAV-lacZ vectors at a particle to cell ratio of 200:1 and 100,000:1, respectively, at 37°C for 1 hr. The cells are then washed with sterile phosphate-buffered saline (PBS), and incubated in tissue culture media containing 20% FBS and antibiotics at 37°C in a 5% CO₂ incubator in the presence of cytokines (interleukin-3 (IL-3), interleukin-6 (IL-6) and stem cell factor (SCF)) for a period of 48 hrs. Analysis of expression of the transduced lacZ gene is performed using the FITC-conjugated substrate for β-Gal and the PE-conjugated substrates for CD34 and CD33 antigens or glycophorin. Briefly, cells are incubated with 300 μM chloroquine for 30 min. at 30°C, following which chloroquine is removed by centrifugation and cells are incubated further with 33 μM Imagreen C12FDG β-gal substrate for 30 min. at 37°C. Following centrifugation, the cells are resuspended in fresh culture medium and analyzed using a Beckton- Dickinson FACScanner.

Function of the hybrid vectors of the present invention, i.e., the ability to mediate transfer and expression of the heterologous gene in hematopoietic stem or progenitor cells, can be evaluated by monitoring the expression of the heterologous gene in transduced cells. Obviously, the assay for expression depends upon the nature of the heterologous gene. Expression can be monitored by a variety of methods including immunological, histochemical or activity assays. For example, Northern analysis can be used to assess transcription using appropriate DNA or RNA probes. If antibodies to the polypeptide encoded by the heterologous gene are available, Western blot analysis, immunohistochemistry or other immunological techniques can be used to assess the production of the polypeptide. Appropriate biochemical assays can also be used if the heterologous gene is an enzyme. For example, if the heterologous gene encodes antibiotic resistance, a determination of the resistance of infected cells to the antibiotic can be used to evaluate expression of the antibiotic resistance gene.

Site-specific integration can be assessed, for example, by Southern blot analysis. DNA is isolated from cells transduced by the vectors of the present invention, digested with a variety of restriction enzymes, and analyzed on Southern blots with an AAV-specific probe. A single band of hybridization evidences site- specific integration. Other methods known to the skilled artisan, such as polymerase chain reaction (PCR) analysis of chromosomal DNA can be used to assess stable integration.

E. Regulatory Elements Throughout this application, the term "expression construct" or "expression cassette" is meant to include any type of genetic construct containing a nucleic acid coding for gene products in which part or all of the nucleic acid encoding sequence is capable of being transcribed. The transcript may be translated into a protein, but it need not be. In certain embodiments, expression includes both transcription of a gene and translation of mRNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding genes of interest. The terms "expression cassette" and "expression construct" are used interchangeably herein throughout.

a. Promoters

In describing both the helper virus which contains cap and rep genes, and the recombinant AAV-derived plasmid, which contains the transgene of interest, it should be noted that genetic elements will be required to drive the transcription of genes therein. Thus, the nucleic acid encoding a gene product is placed under the transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrases "under transcriptional control" or "operably linked" mean that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.

The term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II.

Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the start site for

RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co- operatively or independently to activate transcription.

The particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of direction the expression of the nucleic acid in the targeted cell. Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include either a human or viral promoter.

In various embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, β-actin, rat insulin promoter and glyceraldehyde-3 -phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest. The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose. By employing a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized.

Selection of a promoter that is regulated in response to specific physiologic or synthetic signals can permit inducible expression of the gene product. For example in the case where expression of a transgene, or transgenes when a multicistronic vector is utilized, is toxic to the cells in which the vector is produced in, it may be desirable to prohibit or reduce expression of one or more of the transgenes. Examples of transgenes that may be toxic to the producer cell line are pro-apoptotic and cytokine genes. Several inducible promoter systems are available for production of viral vectors where the transgene product may be toxic.

The ecdysone system (Invitrogen, Carlsbad, CA) is one such system. This system is designed to allow regulated expression of a gene of interest in mammalian cells. It consists of a tightly regulated expression mechanism that allows virtually no basal level expression of the transgene, but over 200-fold inducibility. The system is based on the heterodimeric ecdysone receptor of Drosophila, and when ecdysone or an analog such as muristerone A binds to the receptor, the receptor activates a promoter to turn on expression of the downstream transgene high levels of mRNA transcripts are attained. In this system, both monomers of the heterodimeric receptor are constituitively expressed from one vector, whereas the ecdysone-responsive promoter which drives expression of the gene of interest is on another plasmid. Engineering of this type of system into the gene transfer vector of interest would therefore be useful. Cotransfection of plasmids containing the gene of interest and the receptor monomers in the producer cell line would then allow for the production of the gene transfer vector without expression of a potentially toxic transgene. At the appropriate time, expression of the transgene could be activated with ecdysone or muristeron A.

Another inducible system that would be useful is the Tet-Off™ or Tet-On™ system (Clontech, Palo Alto, CA) originally developed by Gossen and Bujard (Gossen and Bujard, 1992; Gossen et al, 1995). This system also allows high levels of gene expression to be regulated in response to tetracycline or tetracycline derivatives such as doxycycline. In the Tet-On™ system, gene expression is turned on in the presence of doxycycline, whereas in the Tet-Off™ system, gene expression is turned on in the absence of doxycycline. These systems are based on two regulatory elements derived from the tetracycline resistance operon of E. coli. The tetracycline operator sequence to which the tetracycline repressor binds, and the tetracycline repressor protein. The gene of interest is cloned into a plasmid behind a promoter that has tetracycline- responsive elements present in it. A second plasmid contains a regulatory element called the tetracycline-controlled transactivator, which is composed, in the Tet-Off™ system, of the VP16 domain from the herpes simplex virus and the wild-type tertracycline repressor. Thus in the absence of doxycycline, transcription is constituitively on. In the Tet-On™ system, the tetracycline repressor is not wild type and in the presence of doxycycline activates transcription. For gene therapy vector production, the Tet-Off™ system would be preferable so that the producer cells could be grown in the presence of tetracycline or doxycycline and prevent expression of a potentially toxic transgene, but when the vector is introduced to the patient, the gene expression would be constituitively on.

In some circumstances, it may be desirable to regulate expression of a transgene in a gene therapy vector. For example, different viral promoters with varying strengths of activity may be utilized depending on the level of expression desired. In mammalian cells, the CMV immediate early promoter if often used to provide strong transcriptional activation. Modified versions of the CMV promoter that are less potent have also been used when reduced levels of expression of the transgene are desired. When expression of a transgene in hematopoetic cells is desired, retroviral promoters such as the LTRs from MLV or MMTV are often used. Other viral promoters that may be used depending on the desired effect include SV40, RSV LTR, HrV-1 and HIV-2 LTR, adenovirus promoters such as from the El A, E2A, or MLP region, AAV LTR, cauliflower mosaic virus, HSV-TK, and avian sarcoma virus.

Similarly tissue specific promoters may be used to effect transcription in specific tissues or cells so as to reduce potential toxicity or undesirable effects to non- targeted tissues. For example, promoters such as the PSA, probasin, prostatic acid phosphatase or prostate-specific glandular kallikrein (hK2) may be used to target gene expression in the prostate. Similarly, the following promoters may be used to target gene expression in other tissues (Table 1).

Table 1. Tissue specific promoters

Tissue Promoter

Pancreas Insulin elastin amylase pdr-1 pdx-1 glucokinase

Liver albumin PEPCK

HBV enhancer alpha fetoprotein apolipoprotein C alpha- 1 antitrypsin vitellogenin, NF-AB

Transthyretin

Skeletal muscle myosin H chain muscle creatine kinase dystrophin calpain p94 skeletal alpha-actin fast troponin 1

Skin keratin K6 keratin Kl Lung CFTR human cytokeratin 18 (K18) pulmonary surfactant proteins A, B and C

CC-10

PI

Smooth muscle sm22 alpha

SM-alpha-actin Endothelium Endothelin-1

E-selectin von Willebrand factor

TIE (Korhonen et /., 1995)

KDR flk-1

Melanocytes Tyrosinase Adipose tissue Lipoprotein lipase (Zechner et al, 1988) adipsin (Spiegelman et al, 1989) acetyl-CoA carboxylase (Pape and Kim, 1989) glycerophosphate dehydrogenase (Dani et al, 1989) adipocyte P2 (Hunt et al, 1986)

Blood β -globin In certain indications, it may be desirable to activate transcription at specific times after administration of the gene therapy vector. This may be done with such promoters as those that are hormone or cytokine regulatable. For example in gene therapy applications where the indication is a gonadal tissue where specific steroids are produced or routed to, use of androgen or estrogen regulated promoters may be advantageous. Such promoters that are hormone regulatable include MMTV, MT-1, ecdysone and RuBisco. Other hormone regulated promoters such as those responsive to thyroid, pituitary and adrenal hormones are expected to be useful in the present invention. Cytokine and inflammatory protein responsive promoters that could be used include K and T Kininogen (Kageyama et al, 1987), c-fos, TNF-alpha, C- reactive protein (Arcone et al, 1988), haptoglobin (Oliviero et al, 1987), serum amyloid A2, C/EBP alpha, IL-1, IL-6 (Poli and Cortese, 1989), Complement C3 (Wilson et al, 1990), IL-8, alpha-1 acid glycoprotein (Prowse and Baumann, 1988), alpha-1 antitypsin, lipoprotein lipase (Zechner et al, 1988), angiotensinogen (Ron et al, 1991), fibrinogen, c-jun (inducible by phorbol esters, TNF-alpha, UV radiation, retinoic acid, and hydrogen peroxide), collagenase (induced by phorbol esters and retinoic acid), metallothionein (heavy metal and glucocorticoid inducible), Stromelysin (inducible by phorbol ester, interleukin-1 and EGF), alpha-2 macroglobulin and alpha-1 antichymotrypsin.

It is envisioned that cell cycle regulatable promoters may be useful in the present invention. For example, in a gene therapy vector, use of a strong CMV promoter to drive expression of a first gene such as pi 6 that arrests cells in the Gl phase could be followed by expression of a second gene such as p53 under the control of a promoter that is active in the Gl phase of the cell cycle, thus providing a "second hit" that would push the cell into apoptosis. Other promoters such as those of various cyclins, PCNA, galectin-3, E2F1, p53 and BRCA1 could be used. Tumor specific promoters such as osteocalcin, hypoxia-responsive element (HRE), MAGE-4, CEA, alpha-fetoprotein, GRP78/BiP and tyrosinase may also be used to regulate gene expression in tumor cells. Other promoters that could be used according to the present invention include Lac-regulatable, chemotherapy inducible (e.g. MDR), and heat (hyperthermia) inducible promoters, radiation-inducible (e.g., EGR (Joki et al, 1995)), Alpha-inhibin, RNA pol HI fRNA met and other amino acid promoters, UI snRNA (Bartlett et al, 1996), MC-1, PGK, β-actin and α-globin. Many other promoters that may be useful are listed in Walther and Stein (1996).

It is envisioned that any of the above promoters alone or in combination with another may be useful according to the present invention depending on the action desired. In addition, this list of promoters is should not be construed to be exhaustive or limiting, those of skill in the art will know of other promoters that may be used in conjunction with the promoters and methods disclosed herein.

b. Enhancers

Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.

Below is a list of viral promoters, cellular promoters/enhancers and inducible promoters/enhancers that could be used in combination with the nucleic acid encoding a gene of interest in an expression construct. Additionally, any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of the gene. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct. Enhancer/promoter elements contemplated for use with the present invention include but are not limited to Immunoglobulin Heavy Chain, Immunoglobulin Light, Chain T- Cell Receptor, HLA DQ α and DQ β, β-Interferon, Interleukin-2, Interleukin-2 Receptor, MHC Class π 5, MHC Class π HLA-DRα, β-Actin, Muscle Creatine Kinase, Prealbumin (Transthyretin), Elastase I, Metallothionein, CoUagenase, Albumin Gene, α- Fetoprotein, τ-Globin, β-Globin, e-fos, c-HA-ras, Insulin, Neural Cell Adhesion Molecule (NCAM), αl-Antitrypsin, H2B (TH2B) Histone, Mouse or Type I Collagen, Glucose-Regulated Proteins (GRP94 and GRP78), Rat Growth Hormone, Human Serum Amyloid A (SAA), Troponin I (TN I), Platelet-Derived Growth Factor, Duchenne Muscular Dystrophy, SV40, Polyoma, Retroviruses, Papilloma Virus, Hepatitis B Virus, Human Immunodeficiency Virus, Cytomegalovirus, Gibbon Ape Leukemia Virus. Certain Inducible promoter elements and their associated inducers are listed in Table 2 below.

TABLE 2

Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

Thus, transgene expression will be driven by a selected promoter. The promoter selection will depend on the polypeptide to be expressed, the target tissue and the purpose for expression. For example, if the protein is simply to be produced in vitro and purified, a high level promoter will be utilized. If the protein is toxic to the cells, it may be desirable to regulate the expression of the protein such that cell proliferation is maximized prior to polypeptide expression. If the protein's processing or secretion is dependent upon a particular stage in the host cell's cycle, it may be desirable to employ a promoter that is regulated in an appropriate, cell cycle dependent fashion.

For example, the B19 p6 promoter provides for expression specific to erythroid progenitor cells. The nucleotide sequence of B19 from nucleotide number 200 to nucleotide number 424, as numbered by Shade et al. (1986), contains the p6 promoter. A consensus promoter-like sequence TAT AT ATA is located at nucleotide 320 in B19 and, thus, transcription is likely to originate about 30 nucleotides downstream. It is known that B19 fragments containing these sequences direct expression that is specific for erythroid progenitor cells, and that deletion of B19 coding sequences downstream from the promoter prevents replication of B19.

One of ordinary skill in the art can determine the minimum sequence and modifications of the p6 promoter which provide cell-specific, non-cytotoxic expression. This can be determined by infecting erythroid and non-erythroid cells with vectors containing the p6 promoter and assessing expression of the heterologous gene. The promoter sequence can be derived by restriction endonuclease digestion of B 19 or a cloned B19 plasmid such as pYT103 and pYT107 (Cotmore et al. (1984)) or by any other methods known to the skilled artisan, including but not limited to chemical or enzymatic synthesis based upon the published sequence of B 19. Other cell-specific promoters can be obtained by analogous methods, and the specificity of these promoters is determined by assessing expression in the appropriate cell type.

c. Selectable Markers

In certain embodiments of the invention, a cell may be identified in vitro or in vivo by including a marker in the vector. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the vector and, hence, the gene of interest. Usually, the inclusion of a drug selection marker aids in cloning and in the selection of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. Alternatively, enzymes such as herpes simplex virus thymidine kinase or chloramphenicol acetyltransferase (CAT) may be employed. Further examples of selectable markers are well known to one of skill in the art.

d. IRES

In certain embodiments of the present invention, the use of internal ribosome binding sites (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5'-methylated Cap-dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements from two members of the picanovirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message. Any heterologous open reading frame can be linked to IRES elements. This includes genes for secreted proteins, multi-subunit proteins, encoded by independent genes, intracellular or membrane-bound proteins and selectable markers. In this way, expression of several proteins can be simultaneously engineered into a cell with a single construct and a single selectable marker.

F. Transgenes

Virtually any transgene may be utilized in the vectors described herein. In a preferred embodiment, the heterologous gene encodes a biologically functional protein, i.e., a polypeptide or protein which affects the cellular mechanism of a cell in which the biologically functional protein is expressed. For example, the biologically functional protein can be a protein which is essential for normal growth of the cell or for maintaining the health of a mammal. The biologically functional protein can also be a protein which improves the health of a mammal by either supplying a missing protein, by providing increased quantities of a protein which is underproduced in the mammal or by providing a protein which inhibits or counteracts an undesired molecule which may be present in the mammal. The biologically functional protein can also be a protein which is a useful protein for investigative studies for developing new gene therapies or for studying cellular mechanisms.

a. Secreted Proteins

The cDNA's encoding a number of useful human proteins are available for insertion into vectors of the present invention. Examples would include soluble CD4, Factor VLLI, Factor LX, von WiUebrand Factor, TPA, urokinase, hirudin, interferons α and β, TNF, GM-CSF, antibodies, albumin, transferin and nerve growth factors. Cytokines such as IL-1, IL-2, IL-3, LL-4, IL-5, JL-6, IL-7, EL-8, IL-9, IL-10, IL-11, IL- 12 also are contemplated.

Peptide hormones are grouped into three classes with specific examples given for each. These classes are defined by the complexity of their post-translational processing. Class I is represented by Growth Hormone, Prolactin and Parathyroid hormone. A more extensive list of human peptides that are included in Class I is given in Table 3. These require relatively limited proteolytic processing followed by storage and stimulated release from secretory granules. Class II is represented by Insulin and Glucagon. A more extensive list of human peptide hormones that are included in Class II are given in Table 4. These peptides require further proteolytic processing, with both endoproteases and carboxypeptidases processing of larger precursor molecules occurring in the secretory granules. Class HI is represented by Amylin, Glucagon-like Peptide I and Calcitonin. Again, a more extensive list of Class HI human peptide hormones is given in Table 5. In addition to the proteolytic processing found in the Class II peptides, amidation of the C-terminus is required for proper biological function. Examples of engineering expression of all three of these classes of peptide hormones in a neuroendocrine cell are found in this patent.

TABLE 3 - Class I Human Peptide Hormones

Growth Hormone Follicle-stimulating Hormone

Prolactin Chorionic Gonadotropin

Placental Lactogen Thyroid-stimulating Hormone Luteinizing Hormone Leptin

TABLE 4 - Human Peptide Hormones Processed by Endoproteases from Larger Precursors

Adrenocorticotropin (ACTH) Gastric Inhibitory Peptide (GIP)

Angiotensin I and LI Glucagon β-endorphin Insulin Cholecystokinin Lipotropins

Endothelin I Neurophysins

Galanin Somatostatin β-Melanocyte Stimulating Hormone (β-MSH) TABLE 5 - Amidated Human Peptide Hormones

Calcium Metabolism Peptides: Calcitonin

Calcitoήin Gene related Peptide (CGRP) β-Calcitonin Gene Related Peptide Hypercalcemia of Malignancy Factor (1-40) (PTH-rP) Parathyroid Hormone-related protein (107-139) (PTH-rP) Parathyroid Hormone-related protein ( 107- 111 ) (PTH-rP)

Gastrointestinal Peptides:

Cholecystokinin (27-33) (CCK)

Galanin Message Associated Peptide, Preprogalanin (65-105) Gastrin I

Gastrin Releasing Peptide

Glucagon-like Peptide (GLP-1)

Pancreastatin

Pancreatic Peptide Peptide YY

PHM

Secretin

Vasoactive Intestinal Peptide (VIP)

Pituitary Peptides:

Oxytocin

Vasopressin (AVP) Vasotocin

Enkephalins:

Enkephalinamide Metorphinamide (Adrenorphin)

Alpha Melanocyte Stimulating Hormone (alpha-MSH) Atrial Natriuretic Factor (5-28) (ANF)

Amylin

Amyloid P Component (SAP-1)

Corticotropin Releasing Hormone (CRH)

Growth Hormone Releasing Factor (GHRH) Luteinizing Hormone-Releasing Hormone (LHRH)

Neuropeptide Y

Substance K (Neurokinin A )

Substance P

Thyrotropin Releasing Hormone (TRH) b. Non-Secreted Proteins

Two general classes of non-secreted proteins can be defined. The first are proteins that, once expressed in cells, stay associated with the cells in a variety of destinations. These destinations include the cytoplasm, nucleus, mitochondria, endoplasmic reticulum, golgi, membrane of secretory granules and plasma membrane. Non-secreted proteins are both soluble and membrane associated. The second class of proteins are ones that are normally associated with the cell, but have been modified such that they are now secreted by the cell. Modifications would include site-directed mutagenesis or expression of truncations of engineered proteins resulting in their secretion as well as creating novel fusion proteins that result in secretion of a normally non-secreted protein.

The cDNA's encoding a number of therapeutically useful human proteins are available. These include cell surface receptors, transporters and channels such as GLUT2, CFTR, leptin receptor, sulfonylurea receptor, β-cell inward rectifying channels, etc. Other proteins include protein processing enzymes such as PC2 and PC3, and PAM, transcription factors such as 1PF1, and metabolic enzymes such as adenosine deaminase, phenylalanine hydroxylase, glucocerebrosidase.

Engineering mutated, truncated or fusion proteins also is contemplated.

Examples of each type of engineering resulting in secretion of a protein are given (Ferber et al, 1991; Mains et al, 1995). Reviews on the use of such proteins for studying the regulated secretion pathway are also cited (Burgess and Kelly, 1987; Chavez et al, 1994).

Tumor suppressors also may be employed in the vectors of the present invention, this category of transgenes includes p53, pl6, CCAM, RB, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-LI, zacl, p73, VHL, MMAC1, FCC and MCC. Additional inducers of apoptosis include those of the Bcl-2 family, Ad E1B and ICE- CED3 proteases, similarly could find use according to the present invention. As mentioned above, various enzyme genes are of interest according to the present invention. Such enzymes include cytosine deaminase, hypoxanthine-guanine phosphoribosyltransferase, galactose- 1 -phosphate uridyltransferase, phenylalanine hydroxylase, glucocerbrosidase, sphingomyelinase, α-L-iduronidase, glucose-6- phosphate dehydrogenase, HSV thymidine kinase and human thymidine kinase.

In yet another embodiment, the heterologous gene may include a single-chain antibody. Methods for the production of single-chain antibodies are well known to those of skill in the art. The skilled artisan is referred to U.S. Patent No. 5,359,046, (incorporated herein by reference) for such methods. A single chain antibody is created by fusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule.

Single-chain antibody variable fragments (Fvs) in which the C-terminus of one variable domain is tethered to the N-terminus of the other via a 15 to 25 amino acid peptide or linker, have been developed without significantly disrupting antigen binding or specificity of the binding (Bedzyk et al, 1990; Chaudhary et al, 1990). These Fvs lack the constant regions (Fc) present in the heavy and light chains of the native antibody.

Antibodies to a wide variety of molecules can be used in combination with the present invention, including antibodies against oncogenes, toxins, hormones, enzymes, viral or bacterial antigens, transcription factors, receptors and the like.

c. Antisense Constructs

In some cases, one may wish to block the function or expression of a particular polypeptide. Antisense constructs are one way of addressing this situation. Antisense methodology takes advantage of the fact that nucleic acids tend to pair with "complementary" sequences. By complementary, it is meant that polynucleotides are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Inclusion of less common bases such as inosine, 5- methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.

Targeting double-stranded (ds) DNA with polynucleotides leads to triple-helix formation; targeting RNA will lead to double-helix formation. Antisense polynucleotides, when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability. Antisense RNA constructs, or DNA encoding such antisense RNA's, may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject.

Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. It is contemplated that the most effective antisense constructs will include regions complementary to intron/exon splice junctions. Thus, it is proposed that a preferred embodiment includes an antisense construct with complementarity to regions within 50-200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used. One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected. As stated above, "complementary" or "antisense" means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions. Naturally, sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology also are contemplated. For example, an antisense construct which has limited regions of high homology, but also contains a non-homologous region (e.g., ribozyme; see below) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.

It may be advantageous to combine portions of genomic DNA with cDNA or synthetic sequences to generate specific constructs. For example, where an intron is desired in the ultimate construct, a genomic clone will need to be used. The cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.

d. Ribozyme Constructs

Although proteins traditionally have been used for catalysis of nucleic acids, another class of macromolecules has emerged as useful in this endeavor. Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cech, 1987; Gerlach et al, 1987; Forster and Symons, 1987). For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cech et al, 1981; Michel and Westhof, 1990; Reinhold- Hurek and Shub, 1992). This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.

Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cech et al, 1981). For example, U.S. Patent No. 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes. Thus, sequence-specific ribozyme-mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon et al, 1991 ; Sarver et al, 1990). Recently, it was reported that ribozymes elicited genetic changes in some cells lines to which they were applied; the altered genes included the oncogenes H-ras, c-fos and genes of HIV. Most of this work involved the modification of a target mRNA, based on a specific mutant codon that is cleaved by a specific ribozyme.

G. Methods of Therapy

The hybrid vectors of the present invention are useful for gene therapy. In particular, the vectors of the present invention can direct erythroid cell-specific expression of a desired gene, and thus are useful in the treatment of hemoglobinopathies. Examples of maladies to be treated include thalassemia, sickle- cell anemia, diabetes, and cancer. The heterologous gene, in this context, can be the normal counterpart of one that is abnormally produced or underproduced in the disease state, for example β-globin for the treatment of sickle-cell anemia, and α- globin, β-globin or γ-globin in the treatment of thalassemia. The heterologous gene also can encode antisense RNA as described hereinabove.

For example, α-globin is produced in excess over β-globin in β-thalassemia.

Accordingly, β-thalassemia can be treated in accordance with the present invention by gene therapy with a vector in which the heterologous gene encodes an antisense RNA. The antisense RNA is selected such that it binds to a target sequence of the α-globin mRNA to prevent translation of α-globin, or to a target sequence of the α-globin DNA such that binding prevents transcription of α-globin DNA.

In the treatment of cancer the heterologous gene can be a gene associated with tumor suppression, such as retinoblastoma gene, p53, pl6, p21 or the gene encoding tumor necrosis factor.

The use of the hybrid vectors of the present invention for the treatment of disease involves, in one embodiment, the transduction of hematopoeitic stems cells (HSC) or progenitor cells with the claimed vectors. Transduction is accomplished, following preparation of mature virions containing the AAV vectors, by infection of HSC or progenitor cells therewith. Transduced cells may be located in patients or transduced ex vivo and introduced or reintroduced into patients, e.g., by intravenous transfusion (Rosenberg, 1990).

In ex vivo embodiments, HSC or progenitor cells are provided by obtaining bone marrow cells from patients and optionally enriching the bone marrow cell population for HSC. HSC can be transduced by standard methods of transfection or infected with mature virions for about one to two hours at about 37°C. Stable integration of the viral genome is accomplished by incubation of HSC at about 37°C for about one week to about one month. The stable, site-specific integration and erythroid cell-specific expression is assessed as described above. After the transduced cells have been introduced into a patient, the presence of the heterologous gene product can be monitored or assessed by an appropriate assay for the gene product in the patient, for example in peripheral red blood cells or bone marrow of the patient when expression is erythroid cell-specific. As described above, the specific assay is dependent upon the nature of the heterologous gene product and can readily be determined by one skilled in the art.

In a particular embodiment, β-thalassemia represents a heterologous group of clinical syndromes that are inherited as mutated alleles of genes that encode the human β-globin chain. These mutations affect all aspects of β-globin gene expression including transcription, splicing, polyadenylation, translation, and protein stability. The hallmark of β-thalassemia is the marked reduction or total absence of synthesis of normal adult hemoglobin (HbA; α₂ β₂). Despite significant advances in the understanding of basic underlying molecular mechanisms of β-thalassemia, treatment is limited to regular red blood cell transfusions and iron-chelation therapy. Treatment by bone marrow transplantation has also been attempted (Thomas et al. 1982), but an effective cure has not been found.

Accordingly, the vectors of the present invention are useful in the treatment of β-thalassemia. An AAV-B19 vector is constructed in which the heterologous gene is the normal human β-globin gene, with the resulting AAV-B19- β-globin vector allowing parvovirus-mediated transfer, site-specific integration and erythroid cell- specific expression of the normal human beta -globin gene in human hematopoietic cells.

Abnormal beta-globin expression in beta-thalassemia may result in the overabundance of alpha-globin mRNA relative to beta-globin mRNA. The present invention can not only provide a normal beta-globin gene, as described hereinabove, but can further be utilized to down-regulate the production of excess alpha-globin by providing a vector with an antisense RNA as the heterologous gene.

Hence, the present invention contemplates gene therapy for β-thalassemia comprising transduction of hematopoietic stem or progenitor cells with a hybrid vector encoding normal β-globin chains, or simultaneous transduction with a vector encoding a normal β-globin chain and a vector encoding an RNA antisense to alpha - globin mRNA or DNA. Alternately, a construction with more than one B19 p6 promoter, as described hereinabove, permits coincident expression of β-globin and antisense α-globin. Accordingly, transduction with a single vector effects both the provision of a normal β-globin gene and the down-regulation of excess α-chains. More specifically, bone marrow cells are transfected with the subject vectors, and transduced cells are introduced, by intravenous transfusion, into a patient. The stable integration of the vector can be assessed by PCR or Southern blot analysis and the expression of the heterologous gene can be evaluated by assaying for the heterologous gene product in the patient's peripheral blood cells or bone marrow cells. As described previously, the particular assay depends upon the nature of the heterologous gene product.

Yet another aspect of the present invention provides a method for delivery of a pharmaceutical product, a protein or an antisense RNA in a mammal. Since the normal differentiation of these stem cells results in production of mature erythrocytes, the transduction of stem cells with the subject vector ultimately yields a population of circulating, enucleate vesicles containing the gene product. This method comprises transducing hematopoietic stem or progenitor cells with the hybrid vector of the present invention and introducing, by intravenous transfusion or injection, the transduced cells into a mammal.

Transduction can be accomplished by transfecting cells with the hybrid vector by standard methods or infecting cells with mature AAV virions containing the hybrid vector at about 37°C. for about one to two hours. Stable integration of the recombinant viral genome is accomplished by incubating cells at about 37°C. for about one week to about one month. Transduced cells are recognized by assaying for expression of the heterologous gene, as described hereinabove. In this embodiment, the pharmaceutical product is encoded by the heterologous gene of the hybrid vector, and can be any pharmaceutical product capable of being expressed by the hybrid vector. Such products include alpha, beta and gamma -globin, insulin, GM-CSF, M- CSF, G-CSF, EPO, TNF, MGF, interleukins, the gene product of the retinoblastoma gene, p53 or adenosine deaminase. The coding sequences of the respective genes are known (Lee et al. (1985; GM-CSF); Broderick et al, (1987; APRT); Tratschin et al (1985; Neo^r); Huang et al (1988; RB-1); Liebhaber et al (1980; α-globin); Lawn et al. (1980; β-globin); Enver et al. (1989; γ-globin)) and thus can be easily provided as described hereinabove.

Therefore, the present invention can provide production of constitutive levels of heterologous gene products inside membrane vesicles, specifically red blood cells, for in situ treatment of disease. Optionally, the hybrid vector can further comprise a sequence which encodes a signal peptide or other moiety which facilitates the secretion of the gene product from the erythroid cell. Such sequences are well-known to one of ordinary skill in the art (Michaelis et al. 1982) and can be inserted into the subject vectors between the promoter and coding region by methods described herein above. This method can be used to treat a variety of diseases and disorders and is not limited to the treatment of hemoglobinopathies, since the heterologous gene is constitutively expressed and can be released from the red blood cell by virtue of a secretory sequence, or released when red blood cells are lysed in the liver and spleen.

H. Pharmaceutical Compositions and Routes of Administration

Where clinical applications are contemplated, it will be necessary to prepare pharmaceutical compositions containing the viral vectors of the present invention in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.

One will generally desire to employ appropriate salts and buffers to render delivery vectors stable and allow for uptake by target cells. Buffers also will be employed when viral preparations are introduced into a patient. Aqueous compositions of the present invention comprise an effective amount of the vector to cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula. The phrase "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well know in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incoφorated into the compositions.

The active compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions.

The active compounds may also be administered parenterally or intraperitoneally. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial an antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. For oral administration the polypeptides of the present invention may be incoφorated with excipients and used in the form of non-ingestible mouthwashes and dentifrices. A mouthwash may be prepared incoφorating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incoφorated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate. The active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries. The active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.

The compositions of the present invention may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethyl amine, histidine, procaine and the like.

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

I. Examples

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1 Materials and Methods

Cells, viruses, and plasmids: The human embryonic kidney cell line, 293, was obtained from the American

Type Culture Collection (Rockville, MD), and the human nasopharyngeal carcinoma cell line, KB, was obtained from Asok C. Antony, Indiana University School of Medicine (Indianapolis, LN). Cells were maintained as monolayer cultures in Iscove's- modified Dulbecco's medium (LMDM) supplemented with 10% fetal bovine serum (FBS) and penicillin and streptomycin as previously described (Nahreini and Srivastava, 1989). The human adenovirus type 2 (Ad2) stock was obtained from Kenneth H. Fife, Indiana University School of Medicine (Indianapolis, IN), and propagated as previously described (Nahreini and Srivastava, 1992).

The AAV helper plasmid containing the AAV coding sequences flanked by the adenovirus 5 (Ad5) ITRs, pAAV/Ad (Samulski et al, 1989), was supplied by Richard J. Samulski, University of North Carolina (Chapel Hill, NC). An AAV helper plasmid lacking the Ad5 ITRs, pSP-19, has been described previously (Wang et al, 1996), and an additional AAV helper plasmid, pAAVp5, was constructed in which the AAVp5 promoter sequences were inserted downstream of the polyadenylation signal of AAV as follows. A 693-bp Ball-Sad DNA fragment containing the AAVp5 promoter sequences was isolated from plasmid pSub201 (Samulski et al, 1987), digested with Bbvl, and treated with the Klenow fragment of E. coli DNA polymerase I. A 203-bp (Ball)-(BbvI) fragment was recovered and digested with Xbal, and ligated at the 3'-end of the AAV genome in plasmid pSP-19 partially digested with Xbal and completely digested with Spel. Each of these three helper plasmids is shown schematically in FIG. 1.

Recombinant AAV plasmids, pCMVp-lacZ, containing the human cytomegalovirus (CMV) immediate-early promoter-driven b-galactosidase gene (Ponnazhagan et al, 1997; Ponnazhagan et al, 1996), and pWP-8A containing the genes for resistance to tetracycline and the heφesvirus thymidine kinase promoter- driven gene for resistance to neomycin (Nahreini et al, 1993), have also been described previously. The construction of a plasmid pD-10, which contains deletions in the distal 10 nucleotides in the D-sequence within the AAV ITRs, was described recently (Wang et al, 1997). The CMV promoter-driven lacZ gene sequences were also inserted in the pD-10 vector to generate a recombinant plasmid, pBK-2. Standard cloning techniques were used for constructing all recombinant plasmids (Sambrook et al, 1989). Packaging of recombinant AAV:

DNA transfections were performed by the calcium phosphate co-precipitation method essentially as previously described (Sambrook et al, 1989). Briefly, 15 μg each of the recombinant AAV plasmid (pCMVp-lacZ, pWP-8A, or pBK-2) and the AAV helper plasmid (pAAVp5, pAAV/Ad, or pSP-19) were used per 15-cm dish of 70% confluent 293 cells. Eight hours post-transfection, the medium was replaced with fresh medium containing 20 plaque-forming units (pfu) of Ad2. The cultures were incubated at 37°C in a CO2 incubator for 65-72 hrs, and cells were harvested. The cell pellets were subjected to three cycles of freezing and thawing. CsCl was added to a final density of 1.4 g/cm3 and centrifuged in a SW50.1 swinging-bucket rotor at 35,000 φm for 48 hrs at 20°C. Fractions with refractive indices of 1.371- 1.374 were pooled and dialyzed in lx phosphate-buffered-saline (PBS), followed by exhaustive digestion with DNasel. Clarified supernatants were heated at 56°C for 30 min to inactivate Ad2. Equivalent amounts were analyzed on quantitative DNA slot- blots using 32P-labeled DNA probes specific for wt AAV, lacZ, or neo sequences as previously described (Kube and Srivastava, 1997).

AAV DNA replication and amplification assays:

Approximately 70% confluent 293 cells in 10-cm dishes were co-infected with 10 moi of the recombinant AAV and 10 pfu of Ad2. Seventy two hrs post-infection, low Mr DNA samples were isolated by the procedure described by Hirt (1967).

Equivalent amounts of low Mr DNA, with or without prior digestion with restriction endonucleases, were analyzed on Southern blots by using 32P-labeled DNA probe specific for AAV coding sequences. To amplify the replication-competent wt AAV in the recombinant vector stocks, 293 cells were co-infected with 10 moi of recombinant

AAV and 10 pfu of Ad2 as described above. Seventy two hrs post-infection, cells were harvested and subjected to three cycles of freezing and thawing. Clarified culture supernatants were used to infect 293 cells with 10 pfu of Ad2 again. This process was repeated for a total of four rounds. Molecular cloning and sequencing of the wild-type AAV-like genomes:

After four rounds of amplification, the supernatants were used to infect 293 cells with 10 pfu of Ad2. Low Mr DNA samples were isolated 72 hrs post-infection, digested with Ball restriction endonuclease, and cloned into the EcoRV site of plasmid pBluescript II SK(+). Bacterial colonies containing plasmids with AAV sequences were selected by in situ colony hybridization using 32P-labeled AAV DNA as a probe. Nucleotide sequence analyses of each of the inserted full-length AAV genomes were carried out using T3 and T7 primers as previously described (Wang and Srivastava, 1997).

EXAMPLE 2

Contamination of the recombinant AAV vector stocks with wild-type" AAV-like" particles

Several groups have reported that recombinant AAV stocks contain varying levels of contaminating wild-type AAV particles (Hargrove et al, 1997; Koeberi et al, 1997; Kube et al, 1997; Muzyczka, 1992; Ponnazhagan et al, 1997; Snyder et al, 1997). There are two possible sources of this contamination. First, the helper adenovirus stocks may contain low-levels of wt AAV, which is unlikely to be the case. And second, the contaminating AAV particles, which are not truly authentic AAV, but are generated by recombination events involving the recombinant AAV and the helper plasmids.

Indeed, wt AAV genomes present in the recombinant vector stocks were not exactly like the wt AAV genome because of differences in the patterns of hybridization following digestion with various restriction endonucleases and Southern blot analyses. For example, when recombinant vCMVp-lacZ vector stocks, produced following co-transfection with pCMVp-lacZ and pAAVp5 plasmids and purified on CsCl gradients, were used to infect Ad2-infected 293 cells and low Mr DNA were analyzed using the AAV right-end EcoRI-Xbal DNA fragment as a probe, the results shown in FIG. 2A were obtained. It is clear that the replicated AAV genomes could be digested with restriction enzymes Sad, Xbal and Ball, but not with Clal. Based on these results, the following conclusions can be drawn. First, the recombinant AAV vector preparations are free of the authentic wt AAV contamination since the wt AAV genome contains only one Sad site and no Xbal sites. And second, the contaminating AAV genomes are derived from the helper plasmid since there are three Sad and two Xbal sites in the helper plasmid (FIG. 2B), and digestion with Sad or Xbal generated the expected sized DNA fragments. Thus, these contaminating AAV particles are wt "AAV-like" particles.

EXAMPLE 3 Strategies for molecular cloning and sequencing of the wt AAV-like genomes

In order to investigate the mechanism of generation of these wt AAV-like particles, it became necessary to characterize these genomes at the nucleotide sequence level. FIG. 3 illustrates how these wt AAV-like particles might be generated during recombinant AAV vector production as well as the strategy to characterize these wt AAV-like genomes.

Briefly, following co-transfection of the recombinant AAV plasmid containing a gene of interest and the AAV helper plasmid containing the viral rep and cap genes in Ad2-infected 293 cells, most virions contain the recombinant AAV genome; however, a small population of virions contain the wt AAV-like genome which is comprised of AAV ITRs (derived from the recombinant AAV plasmid), and the viral rep and cap genes (derived from the helper plasmid), all of which are required for AAV replication and encapsidation. The viral genomes were amplified following four rounds of amplification in Ad2-infected 293 cells. Low Mr DNA samples were digested with Ball restriction endonuclease, which cleaves within the AAV haiφin structure at nucleotide 121 in plasmid pSub201 (Samulski et al., 1987), and molecularly cloned and sequenced as described in Example 1. Ball digestion of these replication-competent AAV-like genomes would be expected to yield one fragment containing the AAV D-sequence and the putative recombination junction sites since previous studies have documented that the first 10 nucleotides in the D-sequence are required for high-efficiency replication and encapsidation of the AAV genome (Wang et al, 1995; Wang et al, 1996; Wang et al, 1997). Indeed, a single DNA fragment was detected on Southern blots following digestion with Ball (FIG. 2A).

A total of 24 recombinant plasmids were sequenced. Twenty two of 24 plasmids contained intact AAV genomes. These plasmids could be divided into six groups, A, B, C, D, E, and F, the left and the right junction sequences from which are presented in FIG. 4. The left junction in plasmids from group A contains the first 19 nucleotides of the D-sequence and the left end of the AAV genome. Based on the sequence, it is clear that this genome is not the authentic wt AAV since it lacks the portion between the D-sequence and the AAV p5 promoter. The right junction in plasmids from group A contains the same 19 nucleotides of the D-sequence, 30 additional nucleotides that match the left end of plasmids from group A, and the right end of the helper plasmid. Thus, these wt AAV-like particles contain all of the elements required for DNA replication such as the AAV ITR, and the rep and the cap genes. Furthermore, these data suggest that the wt AAV-like particles are generated by non-homologous recombination between the recombinant AAV plasmid and the helper plasmid.

The 30 nucleotides at the right end of plasmid A are derived from the left end of the helper plasmid which suggests that the recombination event first occurred at the left end of the genome. The sequence at the right end of plasmids from group A arose most probably from repair and/or recombination between the left and the right ends of the recombinant AAV genome and not from that between the recombinant AAV and the helper plasmid DNA. In plasmids from group B, both ends contain the entire D- sequence, but the recombination junctions between the AAV ITR derived from the recombinant plasmid and the AAV genome derived from the helper plasmid are completely different. Similarly, in plasmids from group C, the left and the right ends contain 17 and 19 nucleotides in the D-sequence, respectively, but the recombination junctions between the AAV ITR and the AAV genome are totally different. These results suggest that recombination events involving the left and the right ends are independent of each other.

In plasmids from group D, the left end is the same as the left end in plasmids from group A, but the right end is different from the right end in plasmids from group

A. In plasmid from group E, the left end is the same as the left end in plasmids from group A, but the right end is the same as the right end in plasmids from group C. In plasmid from group F, the nucleotide sequence of the left end is the same as that of the left end in plasmids from group C, and the right end is the same as the right end in plasmids from group B. The frequencies of these recombination events are presented in Table 6. It appears that the Form Ra ITR is repaired from the Form La ITR in approximately 9% of the clones. The plasmid in group E is derived from recombination between plasmids in groups A and C, and the plasmid in group F is derived from recombination between plasmids in groups C and B, which together constitute approximately 9% of the clones. However, in approximately 82% of the clones examined, the recombination event involving each ITR occurs independently.

Table 6: Summary of recombination junctions in the wt AAV-like genomes. Each of the nucleotide sequences obtained from 22 different plasmids containing the wt AAV-like genome were categorized into 6 distinct groups (A-F), and the left (L) and the right (R) recombination junction sequences were grouped into

4 forms (a-d). Table 6

Group Left and right Number of Frequency junction sequences clones/22 plasmids (%)

A La + Ra 4/22 18.2%

B Lb + Rb 6/22 27.3%

C Lc + Rc 8/22 36.4%

D La + Rd 2/22 9.1%

E La + Rc 1/22 4.5%

F Lc + Rb 1/22 4.5%

The recombination junctions are illustrated in FIG. 5 which indicates that most of the recombination events are clustered in the distal 10 nucleotides in the D- sequence. Interestingly, however, there was no clear pattern of recombination sites in the AAV helper plasmid DNA. When the left junction (Form La) in plasmids from group A is compared with the right junction (Form Re) in plasmids from group C (FIG. 4), the recombination sites in the ITRs are the same, but the rest of the sequences are different. Taken together, these data indicate that the genesis of wt AAV-like genomes is a consequence of multiple, independent, non-homologous yet non-random recombination events involving the recombinant AAV and the helper plasmids as well as recombinant AAV-like genomes.

EXAMPLE 4 The role of adenovirus inverted terminal repeats in recombination In order to corroborate that the distal 10 nucleotides in the D-sequence were indeed the 'hot-spots' for recombination, the wt AAV-like genomes amplified from a different recombinant AAV vector (vTc.Neo) generated by co-transfection of the recombinant AAV plasmid, pWP-8A, and a different AAV helper plasmid, pAAV/Ad, were analyzed as described above except that each of the ends was analyzed independently. The nucleotide sequences of 6 left ends and 3 right ends of these genomes are presented in FIG. 6. These data further provide strong evidence that most of the recombination events involve the distal 10 nucleotides in the D- sequence, and that the recombination is non-homologous. Interestingly, however, upon closer examination, it became evident that each of the junction sequences also contained the Ad5 ITR sequences. The nucleotides in the Ad5 ITR sequence involved in recombination events are highlighted in FIG. 7. When a helper plasmid that lacked the Ad5 ITRs was used to generate recombinant AAV vectors, nearly a five-fold reduction in the recombination frequency leading to generation of wt AAV-like particles was observed. These data are summarized in FIG. 8. Based on sequence analyses of 7 independent clones that were obtained with a helper plasmid that contained Ad5-ITRs, there were 5 sites of recombination that lead to generation of biologically active wt AAV-like particles during packaging of recombinant AAV compared with 22 additional clones generated with a helper plasmid that lacked the Ad5-ITRs in which there were 3 sites of recombination. Similar results were obtained when the extent of generation of wt AAV-like particles was determined by quantitative DNA slot blot analyses (Example 5). Taken together, these results demonstrate that the Ad5-TTRs play a role in illegitimate recombination during the generation of the wt AAV-like particles.

EXAMPLE 5 Strategies for elimination of the wt AAV-like particles From the foregoing, it stood to reason that removal of the distal 10 nucleotides in the D-sequence from the recombinant AAV vector, and removal of the Ad5 ITRs from the AAV helper plasmid might prove beneficial in substantially reducing, if not completely eliminating, the generation of the wt AAV-like particles during recombinant AAV vector production. This possibility was experimentally tested as follows.

The potential hot-spots of recombination were deleted in a recombinant AAV plasmid, pD-10 (FIG. 9), the construction of which has recently been reported (Wang et al, 1997). This vector was used to generate a recombinant AAV plasmid, pBK-2, containing the CMV promoter-driven lacZ gene. Four sets of recombinant vCMVp- lacZ vector stocks were generated either with pCMVp-lacZ (pD-20), or pBK-2 (pD- 10) with pAAV/Ad and pSP-19 (prep/cap), respectively, as helper plasmids. Quantitative DNA slot-blot analysis of each of the stocks revealed that contamination with the wt AAV-like genomes was highest in vectors generated from plasmids pCMVp-lacZ+pAAV/Ad, and lowest in vectors generated from plasmids pBK- 2+pSP-19. These results, summarized in Table 7, suggest that both adenovirus ITRs and the distal 10 nucleotides in the AAV-ITRs promote the generation of the wt AAV-like particles.

Table 7

The extent of contamination with the wt AAV-like physical particles in vector stocks generated from various combinations of recombinant and helper plasmids.

Recombinant Helper plasmid^b Physical titers ofcontaminating plasmid" wild -type AAV-like particles (%)^c

PCMVp-lacZ pcpAAV/Ad 2.0 x 10⁹ (4.0)

PCMVp-lacZ pSP-19 4.0 x 10⁸ (0.8)

PBK-2 pAAV/Ad 5.0 x 10⁸ (1.0)

PBK-2 pSP-19 7.0 x 10⁷ (0.1)

^aPlasmids pCMVp-lacZ and pBK-2 contain the same CMV promoter- driven lacZ gene in recombinant AAV vector backbone containing 20 and 10 nucleotides, respectively, in the D-sequences in the viral ITRs. ^bIn plasmid pAAV/Ad, the AAV coding sequences are flanked by adenovirus ITRs, but deleted in plasmid pSP-19.

Contaminating wt AAV-like genomes in 5xl0¹⁰ particles/ml of each of the recombinant vCMVp-lacZ vector stocks were detected and quantitated on DNA slot blots as previously described (Kube et al., 1997).

Equivalent amounts of each of the vector stocks were also used to infect Ad2- infected 293 cells under identical conditions. Low Mr DNA isolated 72 hrs post- infection were analyzed on Southern blots using DNA probes specific for lacZ or AAV sequences, respectively. These results are shown in FIG. 10. It is evident that the lacZ probe detected the characteristic monomeric and dimeric replicative forms of the recombinant AAV genomes, the hybridization intensities of which were roughly the same in all vector preparations (FIG. 10A). Interestingly, however, when a replicate Southern blot was probed with the AAV probe, no AAV DNA replicative intermediates could be detected in the recombinant AAV vector stocks prepared with plasmids pBK-2+pSP-19. The extent of generation of replication-competent wt AAV-like particles was most pronounced in recombinant vector stocks generated with plasmids pCMVp-lacZ+pAAV/Ad, followed by that with plasmids pCMVp- lacZ+pSP-19, and pBK-2+pAAV/Ad (FIG. 10B). Even when the vector stocks generated with plasmids pBK-2+pSP-19 were amplified through 4 successive rounds of amplification in Ad2-infected 293 cells, the AAV probe failed to detect any replication-competent wt AAV-like particles, whereas abundant hybridization signals were detected in vector stocks generated with plasmids pCMVp-lacZ+pAAV/Ad even after one round of amplification (FIG. 11). Although a low-level of wt AAV-like genomes was generated even in the absence of adenovirus ITRs and the distal 10 nucleotides in the AAV D-sequence (Table 7), these genomes were replication- incompetent (FIG. 10A and FIG. 10B).

Thus, these results corroborate the contention that removal of the distal 10 nucleotides in the D-sequence from the recombinant AAV vector, and removal of the adenovirus ITRs from the AAV helper plasmid are sufficient to eliminate generation of replication-competent wt AAV-like particles during recombinant AAV vector production.

* * *

All of the compositions and/or disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Claims

CLAIMS:

1. A method for producing adeno-associated virus (AAV) particles comprising:

(a) providing a helper plasmid encoding rep and cap polypeptides;

(b) providing a recombinant AAV plasmid; and

(c) introducing both said helper plasmid and said AAV plasmid into a cell

under conditions supporting replication, rescue and packaging of the

recombinant AAV genomes

wherein there is no distal D sequence homology between said helper plasmid and said AAV plasmid.

2. The method of claim 1, wherein said recombinant AAV plasmid lacks distal D sequences.

3. The method of claim 1, wherein said helper plasmid is an adenovirus that

lacks adenoviral ITRs.

4. The method of claim 3, wherein said helper plasmid is pAAVp5 or pSP-19.

5. The method of claim 1, wherein said AAV plasmid lacks the distal 10

nucleotides of the D sequences.

6. The method of claim 5, wherein said helper plasmid is an adenovirus that

lacks adenoviral ITRs.

7. The method of claim 1, wherein said AAV plasmid comprises an expression

cassette.

8. The method of claim 7, wherein said expression cassette comprises a

polynucleotide under the control of a promoter operable in eukaryotic cells.

9. The method of claim 8, wherein, said promoter is an inducible promoter.

10. The method of claim 8, wherein said promoter is CMV IE, SV40 IE, HSV tk,

╬▓-actin, human globin ╬▒, human globin ╬▓, human globin ╬│, RSV, B19p6, ╬▒-1

antitrypsin, PGK, tetracyclin, MMTV or albumin promoter.

11. The method of claim 8, wherein said expression cassette further comprises a

polyadenylation signal.

12. The method of claim 11 , wherein said polyadenylation signal is an AAV

polyadenylation signal, an SV40 polyadenylation signal or a BGH

polyadenylation signal.

13. The method of claim 8, wherein said polynucleotide encodes a polypeptide.

14. The method of claim 13, wherein polypeptide is a hormone, a tumor

suppressor, an inhibitor of apoptosis, a toxin, a lymphokine, a growth factor,

an enzyme, a DNA binding protein or a single-chain antibody.

15. The method of claim 8, wherein said polynucleotide encodes an antisense construct.

16. The method of claim 8, wherein said polynucleotide encodes a ribozyme.

17. The method of claim 1, further comprising purifying said AAV particles.

18. The method of claim 1, further comprising formulating said AAV particles in

a pharmaceutically acceptable buffer, diluent or excipient.

19. The method of claim 1, wherein said cell expresses an adenovirus polypeptide

essential to adenoviral replication.

20. The method of claim 19, wherein said polypeptide is an El polypeptide.

21. The method of claim 20, wherein both El A and E IB are expressed by said

cell.

22. The method of claim 21, wherein said cell is an embryonic kidney cell.

23. The method of claim 22, wherein said cell is a 293 cell.

24. The method of claim 1, wherein said rep and cap polypeptides are derived from AAV.

25. The method of claim 1, wherein said cap polypeptide is derived from parvovirus B19.

26. The method of claim 1, wherein said cap polypeptide comprises the cap VP2 protein.

27. The method of claim 26, wherein said cap polypeptide further comprises the

cap VP1 protein.

28. A method for reducing wild-type adeno-associated virus (AAV)-like particles

in a recombinant AAV population comprising:

(a) providing an AAV plasmid lacking distal D sequences; and

(b) introducing said AAV plasmid into a cell, along with a helper plasmid

encoding rep and cap polypeptides, under conditions supporting

replication.

29. The method of claim 28, wherein said rep and cap polypeptides are derived

from AAV.

30. The method of claim 28, wherein said cap polypeptide is derived from B 19.

31. The method of claim 28, wherein said helper plasmid is an adenovirus that

lacks adenoviral ITRs.

32. The method of claim 28, wherein said helper plasmid is pAAVp5 or pSP-19.

33. The method of claim 28, further comprising purifying said recombinant AAV population.

34. A population of adeno-associated virus (AAV) particles comprising recombinant AAV plasmids, said population containing less than 3% percent

wild-type AAV-like particles.

35. The population of claim 34, containing less than 2% wild-type AAV-like

particles.

36. The population of claim 34, containing less than 1% wild-type AAV-like

particles.

37. The population of claim 34, containing less than 0.5% wild-type AAV-like

particles.

38. The population of claim 34, containing less than 0.25% wild-type AAV-like

particles.

39. The population of claim 34, being essentially free of wild-type AAV-like

particles.

40. The population of claim 34, wherein said AAV plasmids comprises an expression cassette.

41. The population of claim 40, wherein said expression cassette comprises a

polynucleotide under the control of a promoter operable in eukaryotic cells.

42. The population of claim 40, wherein said expression cassette further comprises

a polyadenylation signal.

43. The population of claim 41, wherein said polynucleotide encodes a

polypeptide.

44. The population of claim 43, wherein polypeptide is a hormone, a tumor

suppressor, an inhibitor of apoptosis, a toxin, a lymphokine, a growth factor,

an enzyme, a DNA binding protein or a single-chain antibody.

45. The population of claim 41, wherein said polynucleotide encodes an antisense

construct or a ribozyme.