This application claims priority under 35 U. S. C. § 119 from provisional patent application Serial No. 60/136,482, filed May 28, 1999 which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION This invention pertains to methods for the efficient, large-scale, helper virus-free production of recombinant adenovirus vectors that are devoid of most if not all viral genes using novel baculovirus/adenovirus hybrid helper vectors.

BACKGROUND OF THE INVENTION Recombinant adenovirus vectors are a highly efficient means of transferring genes into a wide variety of cell types in vivo. Although the adenovirus has a natural tropism for lung epithelium, adenovirus vectors have quite a broad host cell range. In addition to lung and other epithelial-derived tissues (Gilardi et al. 1990) (Stratford-Perricaudet et al. 1990):

(Rosenfeld et al. 1991); (Quantin et al. 1992); (Rosenfeld et al. 1992); (Bajocchi et al.

1993); (Yang et al. 1993), recombinant adenovirus vectors have been successfully used to transduce normally non-dividing cell types including hepatocytes (Levrero et al. 1991); (Jaffe et al. 1992); (Herz and Gerard 1993); (Ishibashi et al. 1993); (Li et al. 1993) and neuronal and glial cells of the central nervous system (Akli et al. 1993); (Davidson et cul.

1993); (Le Gal La Salle et al. 1993). Especially high transduction efficiencies are observed in liver, where 100% of all mouse hepatocytes could be transduced following intraportal infusion of recombinant adenovirus vectors (Li et al. 1993).

At present, the adenovirus vector system has two limitations that have prevented its application in various forms of somatic gene therapy. The first is the transient expression of genes delivered by this means. Apart from muscle, where low levels of persistent gene expression have been reporte (Rosenfeld et al. 1992); (Stratford-Perricaudet et al. 1992); (Vincent et al. 1993), expression of genes delivered by recombinant adenovirus vectors is largely transient in other tissues. For example, although 100% of hepatocytes were successfully transduced one week after intraportal infusion of a recombinant adenovirus vector expressing B-galactosidase, only 0.5% to 10% of hepatocytes were still positive for B- galactosidase activity by 14 to 16 weeks post-infusion (Li et al. 1993). This decline in the proportion of transduced cells was correlated with a decrease in the amount of vector DNA that was present, indicating that the decline in positive cells was not due to inactivation of transcription from the vector. More recent studies have used recombinant adenovirus vectors to express potentially therapeutic genes in normal animals (Herz and Gerard 1993) or in animal models of various monogenic diseases (Ishibashi et al. 1993); (Fang et al. 1994); (Kay and Woo 1994); (Li and Davidson 1995). In these latter studies, partial (Li and Davidson 1995) or complete (Ishibashi et al. 1993); (Fang et al. 1994); (Kay and Woo 1994) correction of the disease phenotype was observed shortly after infusion of a recombinant adenovirus vector expressing the deficient gene product. However, in studies where persistence was examined, the phenotypic correction was greatly diminished or totally reversed within 21 to 28 days of treatment (Fang et al. 1994); (Kay and Woo 1994); (Li and

Davidson 1995). Again, this lack of persistence was due to loss of the vector DNA from the target cells (Petricoin et al. 1994); (Kay and Woo 1994); (Li and Davidson 1995).

Several studies have indicated that the transient expression of genes delivered by El- deleted adenovirus vectors is primarily a consequence of a cytotoxic T cell-mediated immune response mounted against the transduced cells (Engelhardt et al. 1994a; Engelhardt et al. 1994b); (Yang et al. 1996a) (Yang et al. 1994a). These observations are supported by the fact that only slight decreases in the level of transgene expression for at least six months after adenovirus-mediated gene transfer in immune-deficient nulnu (Yang et al. 1994a) or SCID mice (Barr et al. 1995); R. Eisensmith, unpublished observations). Further support for this hypothesis is provided by the observation that administration of the immunosuppressive agent cyclosporine A can significantly prolong transgene expression following the infusion of E1-deleted adenovirus vectors (Engelhardt et al. 1994a); (Engelhardt et al. 1994b); (Fang et al. 1995), as can the depletion of CD4+ cells by the anti-CD4 antibody GK1.5 (Yang and Wilson 1995); (Yang et al. 1996b) (Kolls et al. 1996). To date, however, none of these studies have shown the indefinite persistence of a complete phenotypic correction following adenovirus-mediated transfer of a therapeutic gene in an animal model of human disease.

The second limitation to these vectors is that transduced cells become targets for host immune system. These vectors are not always safe, and repeated administration of these vectors is blocked by a strong immune response in the host system.

Because cells transduced with El-deleted recombinant adenovirus vectors appear to be specifically targeted due to residual amounts of late viral gene expression (Yang etal.

1994a); (Yang et al. 1994b), several studies have examined whether additional modifications of these vectors to reduce late viral gene expression can significantly increase their persistence in vivo (Engelhardt et al. 1994a; Engelhardt et al. 1994b) ; (Yang et al. 1994b); (Fang et al. 1995), (Armentano et al. 1995), (Krougliak and Graham 1995); (Wang et al.

1995); (Zhou et al. 1996); (Yeh et al. 1996a); (Gao et al. 1996); (Gorziglia et al. 1996). As many of these modified vectors are unable to grow in 293 cells, several new cell lines also were developed, expressing in trans the adenovirus genes deleted from the vectors, such as

E4 (Krougliak and Graham 1995); (Wang et al. 1995); (Gao et al. 1996); (Yeh et al. 1996b), E2a (Zhou et al. 1996); (Gorziglia et al. 1996), terminal protein precursor (Schaack et al.

1995); (Langer and Schaack 1996) or Ad polymerase (Amalfitano et al. 1998). Although some studies indicated that these so-called"second generation"adenovirus vectors are less toxic and less immunogenic (Wang et al. 1995); (Gao et al. 1996); (Gorziglia et al. 1996); (Hu et al. 1999), there is as yet no conclusive evidence that such vectors are capable of indefinite (or at least prolonged) persistence in immunocompetent animals. Furthermore, in nearly every case, the introduction of additional deletions into the vector genome has significantly decreased the resulting titers, making the vectors more difficult to produce in quantities sufficient to support preclinical and clinical studies.

The ultimate form of adenovirus vector modification is the creation of a so-called "gutless","gutted"or amplicon vector. This is a vector containing only the cis elements necessary for replication and packaging, but lacking most if not all adenovirus genes. All of the amplicon vectors created thus far (Mitani et al. 1995); (Fisher et al. 1996); (Kochanek et al. 1996); (Lieber et al. 1996); (Parks et al. 1996); (Alemany et al. 1997); (Chen et al. 1997); (Parks and Graham 1997); (Morsy et al. 1998); (Schiedner et al. 1998) ; (Chen et al. 1999) share a number of disadvantages. Foremost among these is the use of helper viruses or plasmid co-transfection to provide the necessary virus proteins in trans. These helper- dependent vectors are produced by simultaneous delivery of both helper and vector genome to a host cell. Both the helper virus and the vector should co-replicate in the same host cells to produce a mixture of a helper virus and a vector. This co-replication allows multiplying the amount of templates for expression of viral genes and producing sufficient amount vector genomes to package in virus particles. Although some techniques have been developed allowing partial removal of the helper virus, its complete elimination has never been achieved. Since the helper virus is a first-generation recombinant adenovirus expressing a number of virus genes, its presence in the final vector preparation may cause the same adverse effects, which were observed in first-generation adenovirus vectors. Thus the complete removal will greatly improve the quality of the vector, making it safer and

more persistent.

What is needed in the art is an adenovirus vector system which overcomes the drawbacks of the above-mentioned prior systems.

SUMMARY OF THE INVENTION Disclosed herein is a system which overcomes the limitations of both low yield and helper virus contamination that are associated with the previous approaches described above.

This system utilizes a novel baculovirus (BV)/adenovirus (Ad) or BV/Ad/SV40 hybrid vectors to deliver with high efficiency into the producer cells all of the adenovirus helper functions required to generate adenovirus amplicon vectors.

In one embodiment, the present invention describes a recombinant baculovirus hybrid helper virus that is comprised of baculovirus sequences and second viral structural and late viral sequences, where the baculovirus sequences encode the proteins necessary for replication and packaging of gutless viral vectors.

In another embodiment, the present invention describes a method of producing gutless viral vectors substatially free from helper virus contamination comprised of the steps of transfecting mammalian cells with a baculovirus hybrid helper virus comprised of baculovirus sequences and second viral structural and late viral sequences and a gutless vector, transducing the mammalian cell, replicating and packaging the gutless vector, and recovering virions.

In another embodiment, the present invention describes a recombinant baculovirus BAC-B4 (VSVG) deposited with the American Type Culture Collection (ATCC, Manassas, Virginia) on May 19,1999 and receiving ATCC Accession No. PTA-88.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 (A-C) shows the nucleotide sequence in (A) the synthetic loxP site I, (B) synthetic loxP site II and (C) polyadenylation site used in the vectors of the present invention.

Fig. 2 (A and B) are schematic representations showing the production of plasmids pL-VK-lox-SPA (2A) and pL/VK-LSL-1 (2B).

Fig. 3 is a schematic representation showing the production of plasmid pBAC-112.

Fig. 4 is a schematic representation showing the production of plasmid pBAC-B2-2.

Fig. 5 is a schematic representations showing the production of plasmid pBAC-B3-2.

Fig. 6 is a schematic representation showing the production of plasmid pBAC-B4 (VSVG).

Fig. 7 is a schematic representation showing the production of the recombinant baculovirus of the present invention.

Fig. 8 is a schematic diagram showing the production of plasmid pAVec4-2.

Fig. 9 is is a schematic diagram showing the production of plasmid pAVec9-2.

Fig. 10 is a schematic diagram showing the production of plasmid pAVec5.

Fig. 11 is a schematic diagram showing the production of plasmid pAVec 13.

Fig. 12 is a schematic diagram showing the structures of plasmid pBAC-B4, pBAC- B9, and pBAC-B 10.

Fig. 13 is a picture of the genetic stability of pBAC-B9 after 4 passages in E. coli.

Fig. 14 is a picture of the PCR analysis of vector production in HepG2, Huh7, and A549 cells.

DETAILED DESCRIPTION OF THE INVENTION All patent applications, patents and literature references cited herein are hereby incorporated in this specification in their entirety.

The most recent type of adenovirus vector, named"gutless"has been proposed as a promising tool for gene therapy. Since these vectors are deleted of most if not all adenovirus genes, they require a helper adenovirus for their propagation. Therefore all gutless vector stocks are contaminated to some degree by the helper virus. This contamination limits the value of currently existing gutless vectors in gene therapy applications. The present invention provides a system that allows production of gutless vectors that are free of helper

virus. Unlike the conventional helper-dependent system, the helper-free system does not include a helper adenovirus, which eliminates the necessity for time-and labor-consuming purification of the vector, reduces the potential immunogenicity of the vector, and improves its safety.

The term"substantial reduction"or"substantially reduced"refers to complete absence of helper virus in the system or less than about 0.1 % helper virus, based on the total content of the helper-free system.

The term"antibiotic resistance gene"refers to DNA that confers cellular resistance to an antibiotic, liketetracycline.

The term"stuffer DNA"refers to DNA which does not encode for any protein or has regulatory function. One example of stuffer DNA is intron DNA. The stuffer DNA may be of any length to achieve optimal packaging size, which may range from 27.5to 37.5 kilobases.

The term"transducing"refers to the transferring of genes from the vector to the cell.

A system for the propagation of gutless vectors is described herein. In this system, the adenovirus genes essential for the replication and packaging of the gutless vector are delivered into producer cells by a new recombinant baculovirus-adenovirus hybrid. In constructing the baculovirus-based helper viruses, the ability of baculovirus to carry large inserts (>15 kb) and to transduce a variety of mammalian cells advantageously used.

Recombinant baculoviruses, variants BacAd-B3-2 and BacAd-B3-2 (VSVG) have been produced by the present invention. Each of them carried a Cre recombinase-excisable copy of the plasmid pBHGlO that contains a complete genome of Ad5 lacking the packaging signal and E1A and E3 genes. One of the recombinant baculoviruses, BacAd-B3-2 (VSVG) also contained a polyhedrin promoter-driven VSVG gene allowing pseudotyping of the baculovirus. Pseudotyping is the process of altering a virus's envelope proteins so that the virus can bind to receptor molecules other than its natural receptor, allowing it to infect cell types that are different from those that it normally infects. This process can produce a virus that is completely pseudotyped (i. e., its natural envelope protein is completely replaced by a

heterologous envelope protein) or only partially pseudotyped (i. e. a second envelope protein is added to the original one). One of the baculovirus/adenovirus hybrids described herein [BAC-B4 (VSVG)] is an example of a baculovirus that is partially pseudotyped, as it expresses both its natural envelope protein, gp64, and the glycoprotein G envelope of the vesicular stomatitis virus (VSVG). The total size of the DNA inserts was 36 kb in BacAd- B3-2 and 38 kb in BacAd-B3-2 (VSVG). Despite this large size the genetic structure of both recombinant baculoviruses was stable. Both viruses give high yields in Sf9 insect cells with the resulting titers in the medium of 5X108 pfu/ml before concentration. These recombinant baculoviruses were used to infect 293 cells (available from Clontech) expressing Cre- recombinase, followed by transfection with a lacZ-containing gutless adenovirus vector. It was expected that the Cre-excised pBHG 10 would simultaneously express all necessary adenovirus genes, and would replicate to a high copy number, facilitating the replication of the vector genome. The latter is packaged into adenovirus virions while the helper genome remains unpackable. Using this system, a gutless vector expressing B-galactosidase was rescued. The vector obtained was completely free of helper virus as confirmed by passaging the resulting vector in 293 cells. The VSVG-pseudotyped helper was slightly more efficient in rescue of the gutless vector, likely due to increased ability to infect the 293-Cre cells.

Thus, the baculovirus-based helper system allows for efficient rescue and production of helper-free gutless vectors. Since the vectors generated in this system are free of helper this system will be a very useful alternative to current methods for the production on gutless vector.

In addition, two other baculovirus-based hybrid viruses, BAC-B9 and BAC-B10 (Fig. 12) have been produced. Both have structures similar to BAC-B4 (VSVG) with the addition of adenovirus ElA and E1B regions (BacB9) or 5.7 kb stuffer sequence into the adenovirus E3 region (BacBlO). Although BAC-B9 contains the Ad5 E1 region, the E1A promoter and the entire packaging signal were removed and replaced by the RSV promoter.

Additionally, the E1 region is placed outside of the excising region and in an orientation that is opposite to normal.

The resulting recombinant baculoviruses, BAC-B9 and BAC-B 10, contained inserts of approximately 43 kb and 45 kb, respectively. Serial passaging followed by Southern blotting analysis of the restriction pattern of the viral DNA in each passage showed that no genetic changes affecting the inserted sequences were detected after four subsequent large- scale preparations of BAC-B9 (Fig. 13). Similar data were obtained for BAC-B 10 (data not shown).

The ability of BAC-B9 to complement replication of gutless adenovirus vectors in the non-complementing cell lines Huh7, HepG2 and A549 was evaluted. PCR analysis of DNA revealed the presence of gutless vector DNA in crude lysates of the co-infected cells (Fig. 14). Vector DNA in the cells was not detected when infected with crude lysates only (without addition of BacB9). These findings indicate that BAC-B9 allows rescue and propagation of gutless vectors in non-complementing cells. Thus, the helper-free system can be adapted for use in non-complementing cell lines.

The present invention provides for a method of producing adenovirus vectors, adeno- associated virus based vectors, herpes virus-based vectors, retrovirus and lentivirus vectors, and SV40-based vectors, all of which could be free of contaminating helper virus.

The adenovirus genes expressed from the vector of the present invention are derived from the plasmid pBHGlO (Microbix Biosystems, Inc., Toronto, ON), which is presently used to rescue E1-deleted adenovirus vectors in conjunction with the El- transcomplementing 293 cell line. To create the BV/Ad hybrid, a baculovirus vector was rescued in which pBHGlO was flanked by two loxP sites (Hoess and Abremski. 1984).

When this vector was introduced into 293 cells in the presence of Cre recombinase, the pBHGlO sequences are excised from the baculovirus genome, circularized, and replicated to high copy number. Efficient trans-complementation of the adenovirus amplicon vector then occurs, with packaging and release of the recombinant adenovirus amplicon vector from the cell. The pBHGlO-derived adenovirus sequences are prevented from being packaged into infectious virus particles through deletion of the packaging signal. In alternative embodiments of the baculovirus/adenovirus hybrid vector, packaging of the adenovirus

helper sequences are further prevented through the incorporation of additional sequences, so that the size of the adenovirus genome exceeds the packaging capacity of the adenovirus particle (>108% of the wild-type genome; Bett et al., 1993).

The minimal requirements for an adenovirus amplicon vector that is produced consists of a plasmid containing all of the cis elements necessary for replication and packaging (the adenoviral ITRs and the packaging signal), an expression cassette containing the therapeutic gene, inert"stuffer DNA"sequences to achieve a vector of appropriate size for optimal packaging into adenovirus particles, and as few genes, viral or otherwise, as are necessary for the induction of amplification and expression of the helper genome. Optimal packaging size ranges from 27.5 to 37.5 kilobases. The only gene that is absolutely essential in the vector in its initial embodiment is the pTP gene.

Recombinant adeno-associated virus (rAAV) vectors based on AAV type 2 can safely transduce various cell types and result in persistent gene expression in vivo. The genome of the wild-type AAV contains two inverted terminal repeats (ITRs) with a packaging signal and encodes for two family of proteins, called rep and cap. For efficient propagation of AAV, some adenovirus products, namely the E1, E4, DBP and VA RNA, are also required in trans. The rAAV devoid of all viral genes can be propagated if all adenovirus helper functions as well as AAV rep and cap are expressed in trans. Currently, for the production of the rAAV, the helper function are provided by co-transfection of plasmids encoding all helper genes or by using appropriate vectors expressing rep and cap.

An alternative embodiment is the production of recombinant baculovirus-based helper virus for AAV production. This virus can be generated to contain an AAV fragment encoding rep and cap genes, which can be obtained from plasmid pAV2 (ATCC No. 37216).

Additionally, the adenovirus E4 region, the DBP-expressing cassette and the adenovirus fragment containing the VA1-RNA gene are incorporated in the recombinant baculovirus genome. The recombinant baculovirus is generated using Bac-to-Bac technology (Gibco Life Technologies) or any other system for generating the recombinant baculoviruses. This virus can be used to produce the rAAV by one of the following ways:

a) E 1-complementing cell line (293, PerC6 or other) is transfected with the plasmid DNA containing an rAAV vector genome followed by the infection by the BAC. AAV/Ad hybrid virus, b) E1-complementing cell line (293, PerC6 or other) is co-infected with the helper virus-free rAAV vector (generated as described above or by any other method) and by the BAC. AAV/Ad hybrid virus, or c) El-complementing cell line (293, PerC6 or other) is first modified to contain an integrated copy of the rAAV vector genome, and such a modified cell line then will be infected by the BAC/AAV/Ad hybrid virus.

Alternatively, the adenovirus E1 region will also be incorporated into the baculovirus, so that the propagation of the rAAV can be performed in any AAV-permissive cell line such as HeLa, Vero or other (available from ATCC). A third alternative is that the recombinant baculovirus-based helper virus for AAV production is generated to contain at least one of the following: an AAV fragment encoding rep or rep and cap genes, the adenovirus E1 and E4 regions, the DBP-expressing cassette and the adenovirus fragment containing the VA1-RNA gene. The elements listed above and not incorporated into the BAC/AAV hybrid virus must be provided by a cell line, in which the propagation occurs.

Alternatively, AAV rep and cap genes in the recombinant Bac/AAV/Ad hybrid virus can be flanked by two sites for a specific recombinase (such as loxP sites for Cre-recombinase or FRT sites for Flp recombinase) and contain a eukaryotic replication elements (such as Epstein- Barr virus EBNA-1 gene and oriP sequence or any other viral or non-viral eukaryotic replication origins). This allows excision of the AAV genes from the baculovirus backbone in the presence of corresponding recombinase (Cre recombinase if loxP sites were used), that is provided either the producing cell line or expressed by the Bac/AAV hybrid virus, and replication of the excised sequences resulting in increase of the copy number of AAV genes in producing cells and in increase of vector yields.

To produce lentivirus vectors such as a vector based on feline immunodeficiency virus (FIV) (available through ATCC with Accession No. VR-233) using the baculovirus-based

helper, the genetic elements required for production of the FIV vectors are incorporated into a recombinant baculovirus using the Bac-to-Bac technology (Gibco Life Technologies) or any other system for generating the recombinant baculoviruses. The recombinant baculovirus helper virus contains the following genetic elements: a) FIV structural cassette expressing gag and pol genes and driven by strong mammalian promoter (for example, EF-la promoter (Invitrogen)), b) the envelop gene (VSV G or any other capable of incorporating into FIV particles), and c) a vector genome, consisting of 5'LTR modified maximize transcription in producing cells, 3'LTR and the transgene driven by the FIV LTR promoter or by an internal promoter, such as PGK.

To produce the lentivirus vector, the recombinant baculovirus containing all three of the mentioned elements is used to transduce mammalian cells (for example, 293 cells or HepG2 cells or many other). This transduction converts the transduced cells into lentivirus-producing cells, and the resulting lentivirus (FIV) vector will be accumulated in cultural medium.

If all three genetic elements mentioned above are not incorporated in one baculovirus genome, the remaining ones can be delivered to the producing cells by other means (other baculoviruses, DNA transfection, etc.) or be provided by the producing cell.

For large-scale production, the vector, with or without a Cre expression cassette, is transfected into VK-33 or 293-Cre cells, respectively. VK-33 cells are a derivative cell line obtained through the stable transformation of 293 cells (Microbix Biosystems, Inc., Toronto, ON or ATCC CRL-1573) with the plasmid pTet-on (Clontech Laboratories Inc., Palo Alto CA), which contains both the tetracycline transactivator protein gene and the neomycin resistance gene, and the plasmid pVK58, which was constructed by insertion of an E4-encoding PCR fragment derived from the wild-type Ad 5 genome (ATCC# VR-5) between the BglII and NheI sites of the plasmid pBI-EGFP (Clontech Laboratories Inc., Palo Alto CA). The 293-Cre cell line is described in Lieber et al., 1996. This cell line could be created through stable transformation of 293 cells (Microbix Biosystems, Inc., Toronto, ON or ATCC# CRL-1573)

with any one of the plasmids sold by Clontech Laboratories Inc. that contain a Cre expression cassette. The cells are then super-infected with the BV/Ad hybrid helper virus at a multiplicity of infection (moi) of 100-2000. Cre expression from the vector leads to the release of the Ad helper genome from the hybrid helper. When transcomplemented by the E1 gene products provided by the 293 cells, the helper genome replicates to high copy number and expresses large quantities of the Ad proteins required to package the adenovirus amplicon vector. If El is incorporated into the BV/Ad hybrid helper virus, then any cell type that is transducible by recombinant baculovirus vectors could serve as a suitable cell for the production of recombinant adenovirus amplicon vectors using this system. Non-limiting examples of such cells include A549 (ATCC# CCL-185) or HepG2 (ATCC# HB-8065).

This novel system has many advantages over existing systems for the production of high-capacity recombinant adenovirus vectors. The first is that the vectors produced in this system are substantially free of contaminating helper virus. As this helper virus could provoke immunological or other responses in the host that could jeopardize both the efficacy and safety of the adenovirus vector, this is a significant benefit of the system of the present invention. Most (if not all) current production systems lack this feature. The second advantage is that the recombinant BV/Ad hybrid helper virus can be utilized to produce adenovirus amplicon vectors in almost any cultured human cell type. By eliminating the requirement for El- transcomplementing 293 cells, contamination of the adenovirus amplicon vector by replication- competent adenovirus (RCA) can be avoided. In addition, by eliminating the need for a specific cell type, both monolayer and suspension cell culturing can be used for the production of the adenovirus amplicon vector, providing greater flexibility and the potential for further cost reductions relative to existing systems. A third advantage of this system is the fact that the recombinant BV/Ad hybrid helper virus can be propagated to high titers easily, cheaply and with a minimum of human labor in cultured insect cells, thereby greatly reducing production costs.

Non-limiting examples of suitable insect cells include Sf9 (ATCC# CRL-1711) or Sf21 (Invitrogen, Inc.). The production system also can be easily expanded, with a minimum of human labor, to quickly, easily and cheaply produce vectors on a scale required for human

clinical trials. This is certainly not the case with system most commonly employed, which requires highly time-consuming serial passaging of the vector in the presence of empirically- determined amounts of helper virus (Parks et al., 1996; Morsy et al., 1998; Schiedner et al., 1998). Another advantage of this production system is that it can be adapted to enable titration of adenovirus amplicon vectors. Current vector production systems can only estimate indirectly the amount of virus DNA that is in a given preparation, but cannot accurately gauge the proportion of this vector DNA that is present in fully-infectious virus particles.

The majority of human gene therapy trials currently in progress utilize recombinant El- deleted adenovirus vectors. While some of the features of the adenovirus amplicon vector system may not be necessary for some of these applications (long-term persistence may not be an absolute requirement for certain trials where cancer or cardiovascular diseases are the targets), other features of this vector system such as its increased capacity may result in its increased use in these types of trials. Furthermore, in applications where highly-efficient and persistent gene transfer is required, e. g. the treatment of genetic diseases, diabetes or other acquired diseases, this vector system is currently without equal.

The vectors of the present invention can be used in numerous applications such as to treat inoperable cancers, reduce cholesterol, and reverse atherosclerosis. The genes necessary for the treatment of most of these conditions are known, and preliminary data are available showing that transient correction of these phenotypes can be achieved through the delivery of these genes by transient, E 1-deleted adenovirus vectors. It is believed that long-term corrections of these disease phenotypes will be achieved when these same genes are delivered by adenovirus amplicon vectors, which have now been shown to stably persist for periods exceeding one year in immunocompetent, non-human primates.

In an alternative embodiment of the present invention, baculovirus BAC-B4 can be modified by incorporation of an expression cassette for El outside of the loxP-flanked helper adenovirus genome so that production of recombinant adenovirus vectors is no longer restricted to VK-33 cells, but can occur in any other Ad-permissive cell line, such as A549 (ATCC# CCL- 185) or HepG2 (ATCC# HB-8065), transformed to express the tetracycline transactivator

protein (Clontech Laboratories Inc., Palo Alto, CA). The advantage to this embodiment is that other types of cells may be even more highly transducible by recombinant baculovirus vectors than VK-33, allowing for increased production efficiency and hence higher titers.

In another alternative embodiment, the modified baculovirus described above can be further engineered to add an expression cassette for the tetracycline transactivating protein (Clontech Laboratories Inc., Palo Alto CA) outside of the loxP-flanked helper adenovirus genome so that production of recombinant adenovirus vectors is no longer restricted to cell lines transformed by this gene, but can occur in any cell line. The advantage to this embodiment is similar to that described above. This modification as well as two following modifications will completely eliminate the risk of generating of replication-competent adenoviruses.

The modified baculovirus described above can be further engineered to add an expression cassette for the tetracycline transactivating protein outside the loxP-flanked helper adenovirus genome so that production of recombinant adenovirus vectors is no longer restricted to cell lines transformed by this gene, but can occur in any cell line. The advantage to this embodiment is similar to that described above.

Alternatively, the Cre-recombinase expressing cassette can be controlled by a constitutive mammalian promoter that does not work in insect cells (such as the HCMV immediately early promoter) (Invitrogen Corporation, Carlsbad CA) which can be incorporated into the backbone of the baculovirus described above. This alteration will remove all restrictions from the cell line, so that any cell line that is sensitive to baculovirus infection and permissive to adenovirus infection such as A549 (ATCC# CCL-185) or HepG2 (ATCC# HB- 8065) can be used for the production of recombinant gutless adenovirus vectors that are free of contaminating helper virus.

This recombinant baculovirus can also be modified by replacing Ad5 sequences with homologous sequences from adenoviruses of different serotypes, such as hAd40 (ATCC# VR- 931), BAV-3 (ATCC# VR-639), CELO (ATCC# VR-432) or EDS-76 (ATCC# VR-921), that will allow the packaging of helper virus-free gutless vector genomes into virus particles with different immunological properties.

Baculovirus BAC-B4 can be modified by the introduction of the SV40 origin of replication together with the SV40 T-Ag gene controlled by a mammalian promoter into the excisable adenovirus sequence, so that after the Cre-mediated excision, the adenovirus helper genome will be able to replicate using either the SV40 or adenovirus origins of replication. This approach could further increase the number of copies of the helper genome per cell and improve the yields of the helper-free gutless adenovirus vector. For example, the 2842 bp BamHI-SphI fragment of the plasmid pBRSV (ATCC# 45019), containing the SV40 origin of replication and the T-antigen gene, could be subcloned into a unique Clal site of pBAC-B4 (VSVG), generating a new plasmid that would express the SV40 T-antigen, and thus, upon Cre-mediated excision from the baculovirus backbone, would be able to replicate episomally to high copy number using the SV40 replication origin.

Baculovirus BAC-B4 can be modified to insert a fragment of DNA containing the AAV rep and cap genes (Samulski et al., 1987), which are required for the production of adeno- associated virus (AAV)-based vectors. This modified virus will now contain all of the AAV and Ad genes necessary for the production of helper virus-free AAV-based vectors.

Recombinant baculovirus BAC-B4 (VSVG) was deposited with the American Type Culture Collection (ATCC, Manassas, Virginia) on May 19,1999 and received ATCC Accession No. PTA-88.

In general, nucleic acid manipulations used in practicing the present invention employ methods that are well known in the art, as disclosed in, e. g., Molecular Cloning, A Laboratory Manual (2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring Harbor) and Current Protocols in Molecular Biology (Eds. Ausubel, Brent, Kingston, More, Feidman, Smith and Stuhl, Greene Publ. Assoc., Wiley-Interscience, NY, NY, 1997).

Preferred disclosed vectors, restriction enzymes, ligases, nucleases, and the like are commercially available from numerous sources, such as Sigma Chemical Co. (St. Louis, MO), New England Biolabs, Inc. (Beverly, MA) and Life Technologies (Rockville MD).

The present invention is described below in specific examples which are intended to further describe the invention without limiting the scope thereof.

Example 1: Method for the construction of a novel baculovirus vector that, by efficiently delivering a replicating, packaging-deficient helper genome into cultured mammalian cells, can be used for the rescue, propagation and titration of a wide range of vectors derived from recombinant viruses, including but not limited to adenoviruses, adeno-associated viruses and lentiviruses.

While the baculovirus-based approach to the rescue, production and titration of virus- derived vectors described in this application could be applied to any number of viruses and cell lines, the following example documents the construction of a baculovirus/adenovirus hybrid vector and 293 cell lines. This hybrid can be used to produce high-titers of recombinant adenovirus amplicon vectors (i. e., high-capacity vectors that are free of most, if not all, viral genes) that are substantially free of contaminating helper virus.

The first step in the construction process was the design and synthesis of two loxP sites and a polyadenylation signal (Figure 1). The synthetic ologonucleotides containing two loxP sites and a polyadenylation signal were sequentially introduced into the cloning vector pLitmus29 (Figures 2A and 2B; New England BioLabs). The resulting plasmid, pVK-LSL-1 (Figure 2B) was modified by insertion of a reporter gene encoding the Zeo-GFP fusion protein, obtained from plasmid pTracer CMV (Invitrogen Corporation, Carlsbad CA), yielding pBAC- 1.2 (Figure 3). The Spel-Xbal fragment of this latter plasmid was inserted into the pFastBacl vector (GIBCO Life Technologies, Rockville MD), which is a component of Bac-to-Bac system designed for the quick generation of recombinant baculoviruses (Figure 4). The resulting plasmid, pBAC-B2-2, was modified further through the insertion of a 32-kb Xbal-Cal fragment derived from pBHG10 (Microbix Biosystems, Inc., Toronto, ON) to produce pBAC-B3-2 (Figure 5). pBHGl 0 contains all of the genes necessary for the replication and packaging of the adenovirus genome. It also contains two fused adenovirus ITRs, which allows for the efficient initiation of adenovirus DNA replication from this plasmid. Although pBHGl 0 provides all of the necessary trans-functions required for adenovirus production, it lacks the cis-elements that

are required for packaging of the adenovirus DNA into mature adenovirus virions (the packaging signal). In the final cloning step, the vesicular stomatitis virus (VSV) glycoprotein G gene, obtained by PCR amplification from the genome of VSV (ATCC# VR-158), was inserted into pBAC-B3-2 between the baculoviral polyhedrin promoter and an SV40-derived polyadenylation signal. This resulted in pBAC-B4 (VSVG).

The adenovirus sequences in this plasmid are flanked by two loxP sites, which are the substrate for Cre-recombinase-mediated site-specific recombination. The recombinant plasmid pBAC-B4 (VSVG) was then used to generate a recombinant baculovirus [BAC-B4 (VSVG); Figure 7]. This was done according to the standard procedures supplied with the commercially- available Bac-to-Bac system (GIBCO Life Technologies, Rockville MD). Briefly, pBAC-B4 (VSVG) DNA was used to transform E. coli DHlOBac cells (Life Technologies, Inc., Rockville MD). These cells contain a so-called"bacmid". This bacmid is a copy of the baculovirus genome into which a minimal E. coli replicon has been incorporated, permitting it to be maintained in this E. coli strain at low copy number. The bacmid also contains a Tn7 integration site. In addition to the bacmid, DHlOBac carries a helper plasmid encoding transposon trans-functions. Since the pBAC-B4 (VSVG) contains Tn7L and Tn7R sites (the left and the right termini of the transposon Tn7), the transposition that occurs in the DH1 OBac cells results in incorporation of the entire insert flanked with Tn7L and Tn7R from pBAC-B4 (VSVG) into the bacmid. This resulting recombinant bacmid is then isolated from the E. coli and used to transfect the insect cell line Sf9 (ATCC# CRL-1711). These cells are permissive for replication of recombinant baculoviruses, and were capable of producing the novel recombinant baculovirus/adenovirus hybrid BAC-B4 (VSVG) at titers of 1 x 109 plaque forming units (pfu) per ml of culture medium. This virus-containing medium was further concentrated 25-fold using a Centricon-80 Plus Centrifugal Filter Device (Amicon), increasing the titers of the hybrid to 4x10'° pfu/ml. pBacB4 was digested with Pacl, which cuts at a unique site located within the E3 deletion that is present in the fragment of the Ad genome contained in the Bac/Ad hybrid vector.

A 5.8 kb Avrll fragment obtained from the human PAH gene, was isolated and subcloned into

the Pacl site. The PAH gene fragment can be of no specific source or sequence composition.

The AvrII and PacI ends in these fragments were joined together using a synthetic Avrll Pacl adapter consisting of following two oligonucleotides: 5'-CTAGGCTCACTGACGCCATAAT- 3'and 5'-TATGGCGTCAGTGAGC-3'. The product of this ligation was the plasmid pBacB 10.

The plasmid pBacB 10 then was used to generate a baculovirus/adenovirus hybrid using the Bac- to-Bac technology (GIBCO Life Technologies) as previously described.

The E1 region (positions 499-3510) was subcloned in the EcoR V site of the cloning vector pZero-2 (Invitrogen) using PCR cloning with the following two primers: 5'- CTCTTGAGTGCCAGCGAGTAGAGTT-3'and 5'-CTCAATCTGTATCTTCATCGCTAG-3' to generate pZeroEl. The structural integrity of the cloned fragment was confirmed by sequencing. The expression vector pREP7 (Invitrogen) was modified by replacing the XbaI- Acc651 fragment containing the RSV promoter with a fragment containing the PGK promoter that was derived from the commercially available plasmid pMSCVneo (Clontech). The resulting plasmid was dubbed pREP-PGK. An XballSpeI fragment containing E1 was excised from pZero-E 1 and inserted into a unique Nhel site in pREP-PGK to generate the plasmid pPGK-E 1.

The whole PGK-driven expression cassette was then excised from pPGK-E1 by Sall digestion.

A Sall-ClaI adapter consisting of the two oligonucleotides 5'-TCGAATGACGGATCCAT-3' and 5'-CGATGGATCCGTCAT-3'was attached to the ends of the fragment. The modified fragment then was inserted into a unique ClaI site of pBAC-B ì U IL0 produce pBAC-B9. The recombinant baculovirus BAC-B9 was generated from pBAC-B9 using the same Bac-to-Bac technology (GIBCO Life Technologies) as previously described.

Example 2: Method for the construction of an adenovirus amplicon vector that can be rescued, propagated and titered in the presence of the novel baculovirus/adenovirus hybrid vector.

The adenovirus cis elements necessary for virus replication and packaging are located

at the termini of the adenovirus genome. The regions are referred to as inverted terminal repeats or ITRs. Plasmid pNC-1.2, containing two Ad left ITRs amplified from human Ad 5 in a head- to-head configuration, was digested with EcoRl and a fragment of human genomic DNA was inserted to produce the plasmid pAVec4-2 or pAVec9-2 (Figures 8 and 9). This insertion was performed to optimize the size of the resulting adenovirus vector for packaging into mature virions. pAVec4-2 was then digested with Sall and an expression cassette consisting of the bacterial lacZ gene driven by the human elongation factor la (EFla) promoter, excised from plasmid pAVec2, was inserted to form the plasmid pAVec5 (Figure 10). PAvec5 was then digested with Pmel and Ascl, and an expression cassette in which the Cre recombinase gene (Life Technologies, Inc., Rockville MD) was driven by the tetracycline-inducible TRE promoter (Clontech Laboratories Inc., Palo Alto CA), derived from plasmid pP2-6f, itself a derivative of pAElsplB (Microbix Biosystems, Inc., Toronto, ON), was inserted to form the final vector, pAVec 13 (Figure 10). When digested by Sall, this vector forms a linear molecule in which the two adenovirus ITRs flank a lacZ expression cassette, a tet inducible Cre expression cassette, and a 19 kb"stuffer"DNA fragment obtained from an intronic region of the human phenylalanine hydroxylase gene. The only bacterial plasmid sequences still remaining are the ampicillin resistance gene (AP) and the ColE1 origin of replication (ori). Because Cre expression from this vector is driven by the tet-inducible promoter system, which has an absolute requirement for the tet transactivator protein, Cre expression from this vector can occur only in cells that express this protein, such as VK-33.

Example 3: Methods for the rescue, propagation and titering of recombinant adenovirus vectors lacking all viral genes using the novel baculovirus/adenovirus hybrid vector as a helper.

DNA transfection and rescue of the gutless adenovirus vector.

Mammalian VK-33 cells (293 cells transformed with expression cassettes for the"tet- on"transactivator and tet-inducible GFP) were grown at 3 7 ° C in 60mm dishes to approximately

70-80% confluence. The growth media (90% MEM + 10% Fetal Bovine Serum + 1% Penicillin/Streptomycin/Fungizone; all obtained from GIBCO) was removed and the cells were washed with phosphate-buffered saline (PBS) or fresh serum-free MEM (GIBCO).

Approximately 2x10l° pfu (0.5 ml) of the concentrated recombinant baculovirus [BAC-B4 (VSVG)] suspension was added to 106 293 cells. Adsorption of the virus was performed by incubating the cells with the virus suspension for 2 hours at 37°C in the presence of 5% CO2.

The cells were rocked gently every 15 minutes during the infection period. After two hours of incubation, the baculovirus inoculum was removed and fresh MEM growth media was added.

The infected cells then were immediately transfected with the adenovirus vector DNA, which was prepared as follows. Purified DNA of the pAVecl3 plasmid was linearized by digestion with the restriction enzyme Swal and purified using the QIAquick Gel Extraction Kit (Qiagen Inc). This digestion released the adenovirus ITRs, making them functional for initiating adenovirus replication. The purified plasmid DNA was transfected into 293 cells using either a standard calcium phosphate procedure or the Effectene Transfection Reagent (Qiagen Inc.).

At 24 hours after transfection, the transfection media was removed and replaced by fresh media.

At seven days after the infection/transfection procedure, the cells were harvested and a crude lysate of the transfected cells was prepared.

Example 4: Propagation of rescued adenovirus vector.

Subconfluent (80%) VK-33 cells grown in 60-mm dishes were infected by 0.5 ml (2x10'° pfus) of the recombinant baculovirus and 0.1 ml of the crude lysate containing the adenovirus vector rescued from the cells transfected as described immediately above. At five days postinfection, a crude lysate of the infected cells was made by subjecting the cell pellet to three rounds of freezing and thawing. These lysates were stored at-20 or-70°C. The multiplicity of infection (moi) of the vector was less than 1"blue-forming unit" (bfu)/cell at the initial states of vector propagation, but maximum vector yields were obtained when cells were infected with the vector at mois ranging from 2-5. One"blue-forming unit"is the amount of

a ß-galactosidase-expressing adenovirus vector necessary to produce one 293 cell that is positive for p-galactosidase expression after transduction of the cells by the vector followed by X-gal staining.

Example 5: Titration of the adenovirus vector.

The crude lysates from the propagation procedure described above were clarified by centrifugation at 700 rpm for ten minutes at 4°C and filtered through 0.8 micron filter units.

0.1 ml of the crude extract was used for infection of 293 cells. As the vector expressed E. coli (3-galactosidase, cells infected by serial dilutions of the vector could be identified by staining with 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-gal) dissolved to a concentration of 0.5 mg/ml in phosphate-buffered saline (Life Technologies, Inc., Rockville MD). In this procedure, the cells were fixed by a five minute exposure to 0.5% glutaraldehyde diluted in PBS at 20 hours after infection, rinsed several time with PBS, and then stained by exposure to the X-gal solution for five hours at 37°C. The number of positively-stained cells multiplied by the dilution factor corresponded to the infections titer of the recombinant adenovirus vector produced by this method.

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