WO1999027123A9 - Modified sv40 viral vectors - Google Patents

Abstract

The present invention provides SV40-based viral vectors for use in the delivery of genes to target cells. The unique combination of multiple viral systems provides for vectors with increased carrying capacity and extended host range when compared to normal SV40 vectors. Uses for the vectors include the expression of proteins in vitro and in vivo.

Description

DESCRIPTION

MODIFIED SV40 VIRAL VECTORS

BACKGROUND OF THE INVENTION

The government may own certain rights in this invention pursuant to grants from a Specialized Program of Research Excellence, no. P50-CA70907.

A. FIELD OF THE INVENTION

The present invention relates generally to viral vectors and their use as expression vectors for transforming human cells, both in vitro and in vivo. More specifically, the invention relates to modified SV40 expression constructs lacking coding sequences and their packaging into capsids that permit infection of human cells.

B. DESCRIPTION OF RELATED ART

SV40 is one of the most thoroughly studied mammalian viruses. Since the 1980's, it has been a popular viral vector for expressing foreign genes in mammalian cells (Hamer, 1980). More recently, the SV40 vector has been reevaluated for use in gene therapy and found capable of mediating persistent transgene expression in mice (Strayer, 1996; Strayer & Milano, 1996) Initially, SV40 vectors were constructed by replacing early or later gene regions with a transgene, after which recombinant virus vectors were propagated with a wild-type SV40 virus or a temperature sensitive mutant, acting as helper viruses. The resulting preparation therefore contained a mixture of recombinant and helper viruses at a ration of about 3:7 (Hamer, 1980).

With the advent of COS-7 cells, which are transformed with an origin- defective mutant of SV40 and capable of supporting the lytic cycle of SV40 vectors with early region deletions, helper-free propagation of recombinant SV40 vectors became possible. However, this system was limited by the size of the transgene that could be accommodated (<2.5 kB) and the effects of SV40 late gene expression on the host cell (toxicity) and the organism (immune response).

Adenoviral vectors, on the other hand, have become one of the leading vectors for gene transfer, particularly in gene therapy contexts. They have been studied rigorously in both in vitro and in vivo contexts because of the ability to generate high titer stocks, their high transduction efficiency and their ability to infect a variety of tissue types in different species. In addition, the availability of cell lines to complement defects in adenoviral replication functions provides for the use of replication defective mutants carrying, in the place of selected structural genes. recombinant inserts of interest. However, the complexity of the adenoviral genome, and the toxicity of these products, has made it difficult to generate cells lines that are capable of complementing more than one or two of the virally-encoded functions.

Thus, there remains a need for improved viral vectors for use in gene transfer. In particular, there is a need to provide vectors with improved capacity for foreign genetic material while, at the same time, reducing the amount of viral material that may results in host cell toxicity and/or immune response. These considerations must be balanced against the ability to generate high titer viral stocks in an convenient fashion.

SUMMARY OF THE INVENTION

Therefore, it is a goal of the present invention to provide improved compositions and methods for the transfer of genetic material into cells, both in vitro and in vivo.

In satisfying these goals, there is provided a method for producing an infectious SV40 viral vector comprising (a) providing an SV40 viral vector J

comprising an SV40 origin of replication and an expression region operably linked to a first promoter active in eukaryotic cells, wherein said vector lacks SV40 coding sequences; (b) providing a replication-deficient adenoviral helper virus having the late region of JC virus or BK virus under the control of a second promoter active in eukaryotic cells; (c) infecting host cells with said adenoviral helper virus; (d) transfecting said host cells with said vector; (e) culturing said host cells for a period of time sufficent to permit said vector to replicated and be packaged; and (f) harvesting said packaged vector in an infectious form. The first promoter may be a CMV IE or SV40 IE promoter. Similarly, the second promoter may be a CMV IE or SV40 IE promoter. The method of claim 1 , wherein said host cells are cultured for about 3 to about 5 days, or more specifically, for about 4 days. The host cells may be transfected using a method selected from the group consisting of calcium phosphate precipitation, electroporation, lipofection and protoplast fusion. The host cells may express a polyomavirus large T antigen, for example, like COS-7 cells. The adenoviral helper virus may lack El regions.

The expression region of the vector may comprise a nucleic acid coding for a gene, for example, a tumor suppressor, a cytokine, an enzyme, a hormone, a receptor, an inducer of apoptosis, an antisense construct, especially one derived from an oncogene. The oncogene may be ras, myc, neu, raf erb, src, fins, jun, trk, ret, gsp. hst, bcl or abl. The tumor suppressor may be p53, pl6, p21, MMAC1, p73, zacl , BRCAI or Rb. The cytokine may be IL-2, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL- 9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, TNF, GMCSF, β-interferon or γ- interferon. The enzyme may be cytosine deaminase, adenosine deaminase, β- glucuronidase, hypoxanthine guanine phosphoribosyl transferase, galactose-1- phosphate uridyltransferase, glucocerbrosidase, glucose-6-phosphatase, thymidine kinase or lysosomal glucosidase. The hormone may be growth hormone, nerve growth factor, insulin, adrenocorticotropic hormone, parathormone, follicle- stimulating hormone, luteinizing hormone, epidermal growth factor or thyroid stimulating hormone. The receptor may be CFTR, EGFR, VEGFR, IL-2 receptor or the estrogen receptor. The inducer of apoptosis may be Bax, Bak, Bcl-X_s, Bik, Bid, Bad, Harakiri, Ad El B or an ICE-CED3 protease.

In another embodiment, there is provided an SV40 viral vector comprising an SV40 origin of replication and an expression region operably linked to a promoter active in eukaryotic cells, wherein said vector lacks SV40 coding sequences. The promoter may be CMV IE or SV40 IE. The expression region may comprises a nucleic acid coding for a polypeptide, such as those set forth above.

In yet another embodiment, there is provided a replication-deficient adenoviral helper virus having the late region of JC virus or BK virus under the control of a promoter active in eukaryotic cells. The promoter may be CMV IE or SV40 IE. The helper virus may lack El regions.

In still another embodiment, there is provided a method for expressing a selected polypeptide in a human cell comprising (a) providing an infectious viral particle comprising a JC virus or BK virus-derived capsid and an SV40 viral vector comprising (i) an SV40 origin of replication, and (ii) an expression region comprising said a nucleic acid encoding said polypeptide operably connected to a first promoter active in eukaryotic cells, wherein said vector lacks SV40 coding sequences; (b) contacting said viral particle with said cell under conditions permitting transfer of said vector into said cell; and (c) culturing said cell under conditions permitting the expression of said polypeptide. The cell may be located in a human subject.

In still yet another embodiment, there is provided a method for treating a human disease comprising (a) providing an infectious viral particle comprising a JC virus- or BK virus-derived capsid and an SV40 viral vector comprising (i) an SV40 origin of replication, and (ii) an expression region comprising said a nucleic acid encoding said therapeutic polypeptide operably connected to a first promoter active in eukaryotic cells, wherein said vector lacks SV40 coding sequences; and (b) administering said viral particle to a human subject having said disease under conditions permitting transfer of said vector into cells of said human subject. The disease may be cancer, diabetes or cystic fibrosis. The promoter may be CMV IE or SV40 IE.

In yet another embodiment, there is provided a method for producing an infectious SV40 viral vector comprising (a) providing an SV40 viral vector comprising an S V40 origin of replication and an expression region operably linked to a first promoter active in eukaryotic cells, wherein said vector lacks SV40 coding sequences; (b) providing a helper cell expressing the late region of JC virus or BK virus under the control of a second promoter active in eukaryotic cells, along with polypeptides essential for the replication of said SV40 viral vector; (c) introducing into said host cells said SV40 vector; (d) culturing said host cells for a period of time sufficent to permit said vector to replicated and be packaged; and (e) harvesting said packaged vector in an infectious form.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

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: Schematics of two recombinant adenoviruses. each of which contains a SV40 capsid expression cassette. The expression cassette is inserted in the adenoviral El region, from left to right, in Ad/CMV-CAP and from right to left in Ad/SV-CAP. Both vectors have polyadenylation sequences from SV40 late genes.

FIG. 2: Time course of production of SV40-GFP vector. Titers were determined by TCID₅₀ assay in COS-7 cells. Values represent the mean (+/- standard error) of this duplicated experiment.

FIGS. 3 A and 3B: DNA analysis of vector preparations. FIG. 3 A - PCR analysis of CMV-GFP expression in COS-7 cells (lane 1); transfected with pEGFP- Nl (positive control) mock infected (lane 2); infected with Ad/CMV-CAP (lane 3); infected with Ad-SV-CAP (lane 4); infected with Ad/CMV-LacZ (lane 5). Cell lysates were treated with Dnase I, and the DNAs isolated were subsequently subjected to PCR to detect the presence of CMV-GFP. Lane M, 100 bp ladder. FIG. 3B - PCR analysis of DNAs isolated from COS-7 cells infected with Ad/CMV-CAP. (V) - transfected with pEGFP-Nl (+, positive control) to detect CMV-GFP. (2) SV40 large T; (3) Adenoviral E4 and (4) Adenoviral El. Lane M, 100 bp ladder; +, positive control; v, testing DNA.

FIG. 4: SV40 viral vector. PGFP-N1 is 4.7 kB in length, a = CMV IE promoter; b = Green Fluorescent Protein gene & multipurpose cloning site (XX); c = SV40 polyA; d = fl origin and ampicillin promoter; e = SV40 and SV40 early promoter; f = Kan^r/Neo^r; g = HSV tk polyA; h = pUC origin.

FIG. 5: Comparison of genome structures of JC virus. BK virus and SV40. FIG. 5 - Comparison of JC virus, BK virus and SV40 VP1 amino acid sequence.

FIGS. 6A and 6B: Capsid Gene/ Adenoviral Constructs. FIG. 6A - Adenovirus-BK virus capsid construct. FIG. 6B - Adenovirus-JC virus capsid construct.

RECTIFIED SHEET(RULE 91 ) PCT/ISA DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Gene therapy provides a promising new approach to treating genetically-based diseases, such as cancer. To be effective, such therapy must achieve a sufficiently high expression level in a sufficient number of cells; repeated administration of the vector containing the therapeutic gene also may be required. These requirements may be problematic for a number of reasons, however.

First, in order for the virus to replicate efficiently, all the normal virus replication functions must be available. If these functions remain associated with the viral vector itself, there will be limited space for the inclusion of the heterologous construct. In addition, the expression of viral genes from the vector in the host cell may result in toxicity to the host or an induction of the host's immune response, both of which may be problematic in the event that survival of the target cell is desired. Immune responses also may prove detrimental to repeated therapies.

On the other hand, removing viral functions from the vector may reduce the ability to grow stocks to high titers, especially in the case of viral functions that are essential to viral replication. Suitable helper cell lines have been developed for both adenovirus (293 cells) and SV40 (COS-7), but these systems suffer from the limited number of viral functions that are provided by the helper cell (293 cells) and from the possibility of recombination (COS-7), resulting in the production of wild-type virus. Nonetheless, these systems show examples of successful trα/M-complementation of defective viral vectors.

Thus, the challenge remains to engineer viral vectors to minimize the amount of viral material that must be included, thereby maximizing the amount of foreign material that can be inserted. At the same time, it is necessary that the engineered virus still propagate, at least in vitro, to high titers, thereby permitting its use in a therapeutic context. A. The Present Invention

The present invention involves the combination of different attributes from at least three different viruses. The first element is the viral vector itself. The vector is derived from simian virus 40 (SV40). This well studied viral vector is easy to manipulate and can be grown to high titers. However, its rather small genome does not permit the inclusion of larger exogenous genetic constructs. Thus, viral functions - as many as possible - should be engineered out of the virus. According to the present invention, all SV40 coding sequences are removed, leaving only an SV40 origin of replication.

In order to compensate for the functional defects in the SV40 vector, two systems are provided to assist or "help" the SV40 vector replicate and package. First, a helper cell line is provided which carries the functions necessary for SV40 to replicate. Primarily, this is a cell line transformed with a polyoma early region, including the large and small T antigens. Preferred is the SV40 early region. An example of a cell line which will provide the necessary function is the COS-7 cell line.

A second "helper" function is provided by a replication-defective adenovirus.

This vector carries the late functions of polyomavirus. In particular, these late functions include the viral virion proteins - VP1, VP2 and VP3 - and optionally the agnoprotein. However, because SV40 is not highly tropic for human cells, the late functions are derived from a polyoma virus that readily infects human cells. These include JC virus and BK virus virion proteins, which are highly homologous to SV40 and each other. FIG. 5.

The helper cell is infected with the helper adenovirus and transfected with the

SV40 vector. The helper cell provides the early, replicative functions for the vector, and the helper adenovirus provides the late, packaging function. After a sufficient

RECTIFIED SHEETfRULE 91) PCT/ISA culture time, the virus is harvested and may be used to infect suitable human target cells. Various details of these materials, this process and uses therefor are provided below.

In another embodiment, the "helping" functions of the helper adenovirus, i. e. , the structural genes VP1-3, also can be incorporated into the helper cell line, thereby obviating the need to provide and infect with the helper adenovirus. These genes, to the extent that they may be toxic to the helper cell, may be controlled by promoter which are inducible. Inducibility can be conferred by an external element, e.g., a drug, by the SV40 vector, or as an indicident of its introduction into the helper cells (e.g., cellular shock).

B. Viral Vectors i) SV40 Virus Simian virus 40 (SV40) was discovered in 1960 as a contaminant in polio vaccines prepared from rhesus monkey kidney cell cultures. It was found to cause tumors when injected into newborn hamsters. The genome is a double-stranded, circular DNA of about 5000 bases encoding large (708 AA) and small T antigens (174 AA), agnoprotein and the structural proteins VP1, VP2 and VP3. The respective size of these molecules is 362, 352 and 234 amino acids.

Little is known of the nature of the receptors for any polyoma virus. The virus is taken up by endocytosis and transported to the nucleus where uncoating takes place. Early mRNA's initiate viral replication and is necessary, along with DNA replication, for late gene expression. Near the origin of replication, promoters are located for early and late transcription. Twenty-one base pair repeats, located 40-103 nucleotides upstream of the initiation transcription site, are the main promoting element and are binding sites for Spl, while 72 base pair repeats act as enhancers.

Large T antigen, one of the early proteins, plays a critical role in replication and late gene expression and is modified in a number of ways, including N-terminal acetylation, phosphorylation, poly-ADP ribosylation, glycosylation and acylation. The other T antigen is produced by splicing of the large T transcript. The corresponding small T protein is not strictly required for infection, but it plays a role in the accumulation of viral DNA.

DNA replication is controlled, to an extent, by a genetically defined core region that includes the viral origin of replication. The SV40 element is about 66 bp in length and has subsequences of AT motifs, GC motifs and an inverted repeat of 14 bp on the early gene side. Large T antigen is required for initiation of DNA replication, and this protein has been shown to bind in the vicinity of the origin. It also has ATPase, adenylating and helicase activities.

After viral replication begins, late region expression initiates. The transcripts are overlapping and, in some respect, reflect different reading frames (VP1 and VP2/3). Late expression initiates is the same general region as early expression, but in the opposite direction. The virion proteins are synthesized in the cytoplasm and transported to the nucleus where they enter as a complex. Virion assembly also takes place in the nucleus, followed by lysis and release of the infectious virus particles.

It is contemplated that the present invention will encompass SV40 vectors lacking all coding sequences. The region from about 5165-5243 and about 0-325 contains all of the control elements necessary for replication and packaging of the vector and expression of any included genes. Thus, minimal SV40 vectors are derived from this region and contain at least a complete origin of replication.

Because large T antigen is believed to be involved in the expression of late genes, and no large T antigen is expressed in the target cell, it will be desired that the promoter driving the heterologous gene be a polyomavirus early promoter, or more preferably, a heterologous promoter. Thus, where heterologous control elements are utilized, the SV40 promoter and enhancer elements are dispensable. ii) Human Papovaviruses

JC virus and BK virus are the only known human polyomaviruses. JC virus was isolated in 1971 by inoculating human fetal brain cells with extracts of diseased brain tissue from patients with progressive multifocal leukoencephalopathy. BK virus was isolated in the same year from the urine of an immunosuppressed renal transplant recipient. The genomes of both these viruses show close homology with SV40. They both are oncogenic when inoculated subcutaneously or intracerebrally into newborn hamsters. BK virus results in brain tumors of ventricular surfaces, whereas JC virus inoculation is associated with tumors of neural origin.

BK virus can be propagated on human epithelial and fibroblast cells. It also grows in human embryonic kidney cells (HEK), diploid lung fibroblasts, urothelial cells and fetal brain cells. JC virus has a more restricted host range than BK virus. JC virus grows well in primary human fetal glial cells and also replicates in human fetal Schwann cells and fetal astrocytes. It also has been isolated from urothelial cells and has been adapted to grow in human amnion, adult brain and HEK cells.

The host range and tissue specificity of polyomaviruses are determined by the interaction of viral and cellular factors. These factors include attachment sites and factors that are involved in DNA replication and gene expression. However, it is envisioned that viral DNA replication may be assayed without difficulty by use of various standard assays, conducted in vitro, thereby determining which cell types will support replication. Strictly speaking, it may not be necessary to replicate the viral genome if the transgene expression, mediated by heterologous promoters, is strong enough. In any event, inclusion of the JC virus or BK virus VP1-3 genes will provide the targeting functions for this system.

iii) Adenovirus

Human adenoviruses are double-stranded DNA viruses with genome sizes of approximate 36 kB. As a model system for eukaryotic gene expression, adenoviruses have been widely studied and well characterized, which makes them an attractive system for development of adenovirus as a gene transfer system. This group of viruses is easy to grow and manipulate, and exhibit a broad host range in vitro and in vivo. In lytically infected cells, adenoviruses are capable of shutting off host protein synthesis, directing cellular machinery to synthesize large quantities of viral proteins, and producing copious amounts of virus.

The El region of the genome includes El A and E1B which encode proteins responsible for transcription regulation of the viral genome, as well as a few cellular genes. E2 expression, including E2A and E2B, allows synthesis of viral replicative functions, e.g. DNA-binding protein, DNA polymerase, and a terminal protein that primes replication. E3 gene products prevent cytolysis by cytotoxic T cells and tumor necrosis factor and appear to be important for viral propagation. Functions associated with the E4 proteins include DNA replication, late gene expression, and host cell shutoff The late gene products include most of the virion capsid proteins, and these are expressed only after most of the processing of a single primary transcript from the major late promoter has occurred. The major late promoter (MLP) exhibits high efficiency during the late phase of the infection (Stratford-Perricaudet and Perricaudet, 1991).

As only a small portion of the viral genome appears to be required in cis, adenovirus-derived vectors offer excellent potential for the substitution of large DNA fragments when used in connection with cell lines such as 293 cells. Ad5 -transformed human embryonic kidney cell lines (Graham et al, 1977) have been developed to provide the essential viral proteins in trans. The characteristics of adenoviruses rendered them good candidates for use in gene transfer both in vitro and in vivo (Grunhaus & Horwitz, 1992).

In general, adenovirus gene transfer systems are based upon recombinant, engineered adenovirus which is rendered replication-incompetent by deletion of a portion of its genome, such as El, and yet still retains its competency for infection.

Sequences encoding relatively large foreign proteins can be expressed when additional deletions are made in the adenovirus genome. For example, adenoviruses deleted in both El and E3 regions are capable of carrying up to 10 kB of foreign DNA and can be grown to high titers in 293 cells (Stratford-Perricaudet and Perricaudet, 1991).

According to the present invention, adenovirus will act as a helper for the SV40 vector, in particular, by providing polyomavirus structural proteins (VPl-3) necessary for capsid formation. These overlapping genes, selected from either BK virus or JC virus, are inserted into the adenoviral helper under the control of a suitable promoter. Preferably, the VPl-3 gene cassette is inserted in the El region of the adenovirus. This permits replication of this virus in 293 cells, which complement the El defect. Upon infection of a SV40 helper cell, which cannot provide the missing El functions, the VPl-3 genes are expressed from the helper virus but replication of the virus will not take place (or take place only at low levels).

Adenoviruses are known to be heat labile, whereas SV40 vectors are not. In the present invention, one concern is the carryover, in the SV40 vector stock, of contaminating adenoviral helper virus. These viruses may arise from particles that have not infected cells during the SV40 vector stock production. Alternatively, there may be some low level replication during the vector stock preparation that results in the production of new adenoviral helper virus. By subjecting the SV40 vector stock to heat, one can reduce, and possibly eliminate, contaminating adenoviral helper virus. Heating the sample for about 30 min. at a temperature between about 50°C and about 60°C, preferably about 56°C, is sufficient to reduce contaminating adenoviral helper virus to undetectable levels. This same treatment has no effect on the SV40 vector stock titer.

iv) Helper Cells

Three distinct helper cells are used according to the present invention. First, as mentioned above, the adenoviral helper vector likely will comprise an El replacement. Because the El region encodes products essential for viral replication (El A and E1B), these functions must be provided from another source - a helper cell line. The cell line also should be within the host range of the adenovirus. Typically, for complementing El defects, those of skill in the art use 293 cells, which contain adenoviral El sequences integrated into the genome. These cells are highly effective at providing trans-complementation of both El A and E1B viral deletions. Thus, this cell will be used as a preliminary step in the production of the adenoviral helper vector.

The second helper cell is required to supply functions that support the replication of the SV40 vector. Again, the SV40 coding regions have been replaced with transgene sequences and, therefore, a number of functions must be supplied in trans. In this particular instance, SV40 "early" functions are needed to facilitate the replication of the SV40 vector. Primarily, these functions derive from the SV40 T antigen. Thus, a cell line that, at a minimum, provides an SV40 large T antigen is required. In addition, one would naturally select a cell type that supports SV40 replication. An example of such a cell line is the COS-7 cell line.

The third helper cell contains not only the early functions provided by cells such as COS-7, but late functions that would otherwise be provided by the helper adenovirus. These late functions are the polyomavirus structural proteins (VPl-3) necessary for capsid formation. These overlapping genes, selected from either BK virus or JC virus, are inserted into the the cell line under the control of a suitable promoter. To the extent that these products are non-toxic, a constitutive promoter may be used. However, it may be preferred that expression of VPl-3 in the cell is regulated such that expression is limited to that time during which the SV40 vector is being packaged. Regulation of expression can be accomplished by various means. The method of choice will involve linking the VPl-3 to an inducible promoter, as discussed further below. v) Control Regions

In order for (i) the SV40 viral vector to effect expression of a transcript encoding a selected gene, (ii) the helper cell to transcribe the SV40 complementing genes, and (iii) the adenoviral helper virus to synthesize JC virus and BK virus capsid proteins, the polynucleotides encoding these genes will be under the transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the host cell, or introduced synthetic machinery, that is required to initiate the specific transcription of a gene. The phrase "under transcriptional control" means that the promoter is in the correct location in relation to the polynucleotide to control RNA polymerase initiation and expression of the polynucleotide.

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 30-1 10 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 cooperatively or independently to activate transcription.

The particular promoter that is employed to control the expression of a therapeutic gene is not believed to be critical, so long as it is capable of expressing the polynucleotide in the targeted cell. Thus, where a human cell is targeted, it is preferable to position the polynucleotide 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 preferred embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter and the Rous sarcoma virus long terminal repeat can be used to obtain high-level expression of the polynucleotide 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 polynucleotides is contemplated as well, provided that the levels of expression are sufficient to produce a growth inhibitory effect.

By employing a promoter with well-known properties, the level and pattern of expression of a polynucleotide following transfection can be optimized. For example, selection of a promoter which is active in specific cells, such as tyrosinase (melanoma), alpha- fetoprotein and albumin (liver tumors), CC10 (lung tumor) and prostate-specific antigen (prostate tumor) will permit tissue-specific expression of the therapeutic gene.

In a particular embodiment, it will be desired to employ an inducible promoter or promoter system to control expression of one or more genes included within helper cells of the present invention. Selection of a promoter that is regulated in response to specific signals permits inducible expression of the gene product. 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 constitutively 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 constitutively on. In the Tet-On™ system, the tetracycline repressor is not wild type and in the presence of doxycycline activates transcription. For gene transfer 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.

Another inducible promoter system is disclosed in WO 98/21350. The promoter region of adenovirus E4 was replaced with a synthetic promoter composed of a minimal TATA box and five consensus 17-mer GAL4-binding site elements (GAL4/TATA). Since most mammalian cells express no GAL4-like activity, a synthetic GAL4-responsive promoter containing GAL4-binding sites and a TATA box should have no or extremely low basal activity in the absence of a GAL4 transactivator, and high activity in its presence. GAL4 is a transcriptional activator derived from yeast, that when fused to a highly acidic portion of the herpes simplex virus protein VP16, is a very potent activator of transcription (Sadowski et al., 1988). Thus, genes that have GAL4 binding sites in their promoter regions are highly activated by the introduction of the GAL4-VP16 fusion protein. This technology could be applied to expression of any of the genes in the helper cell, particularly, VPl-3.

Table 1. below, shows various different inducible regulatory elements and the factors that cause their induction. TABLE 1

Enhancers were originally detected as genetic elements that increased transcription from a promoter located at a distant position on the same molecule of DNA. This ability to act over a large distance had little precedent in classic studies of prokaryotic transcriptional regulation. Subsequent work showed that regions of DNA with enhancer activity 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.

Additionally, any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could be used to drive expression of a particular construct. Use of a T3, T7 or SP6 cytoplasmic expression system is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacteriophage promoters if the appropriate bacteriophage polymerase is provided, either as part of the delivery complex or as an additional genetic expression vector.

According to the present invention, a number of different promoters are required. It is contemplated that these promoters may be the same or different, but the selection of particular promoters for particular uses may be advantageous. For example, it is envisioned that promoters in the helper cell lines be subject to the control of the viral vector they are to support or constitutive at a level that is sufficient to support viral replication.

Similarly, the viral constructs have unique roles to play and, hence, the selection of particular promoter to accomplish specific tasks is advantageous. For example, because the promoter controlling the VPl-3 genes in the adenoviral helper vector should be active at the appropriate juncture in the replicative cycle of SV40. Therefore, use of the endogenous BK or JC promoter, or the corresponding promoter from SV40, is preferred. In certain embodiments of the invention, the use of internal ribosome binding site (IRES) elements may prove advantageous in accordance with the present invention. These 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 picornavirus 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.

In addition, it may be desirable to include polyadenylation signals in the vectors. These signals serve to terminate transcription and to stabilize mRNA transcripts produced from the vectors. A preferred polyadenylation signal is an SV40 polyadenylation signal. vi) Genes

The present invention contemplates the use of a variety of different genes inserted into the SV40 vector. For example, genes encoding enzymes, hormones, cytokines, oncogenes, receptors, tumor suppressors, transcription factors, drug selectable markers, toxins and various antigens are contemplated as suitable genes for use according to the present invention. In addition, antisense constructs derived from oncogenes are other "genes" of interest according to the present invention.

p53 currently is recognized as a tumor suppressor gene. High levels of mutant p53 have been found in many cells transformed by chemical carcinogenesis, ultraviolet radiation, and several viruses. The p53 gene is a frequent target of mutational inactivation in a wide variety of human tumors and is already documented to be the most frequently-mutated gene in common human cancers. It is mutated in over 50% of human NSCLC (Hollstein et al, 1991) and in a wide spectrum of other tumors.

The p53 gene encodes a 393-amino acid phosphoprotein that can form complexes with host proteins such as large-T antigen and E1B. The protein is found in normal tissues and cells, but at concentrations which are minute by comparison with transformed cells or tumor tissue. Interestingly, wild-type p53 appears to be important in regulating cell growth and division. Overexpression of wild-type p53 has been shown in some cases to be anti-proliferative in human tumor cell lines. Thus, p53 can act as a negative regulator of cell growth (Weinberg, 1991) and may directly suppress uncontrolled cell growth or indirectly activate genes that suppress this growth. Thus, absence or inactivation of wild-type p53 may contribute to transformation. However, some studies indicate that the presence of mutant p53 may be necessary for full expression of the transforming potential of the gene.

Wild-type p53 is recognized as an important growth regulator in many cell types. Missense mutations are common for the p53 gene and are essential for the transforming ability of the oncogene. A single genetic change prompted by point mutations can create carcinogenic p53. Unlike other oncogenes, however, p53 point mutations are known to occur in at least 30 distinct codons, often creating dominant alleles that produce shifts in cell phenotype without a reduction to homozygosity. Additionally, many of these dominant negative alleles appear to be tolerated in the organism and passed on in the germ line. Various mutant alleles appear to range from minimally dysfunctional to strongly penetrant, dominant negative alleles (Weinberg, 1991).

Casey and colleagues have reported that transfection of DNA encoding wild- type p53 into two human breast cancer cell lines restores growth suppression control in such cells (Casey et al, 1991). A similar effect has also been demonstrated on transfection of wild-type, but not mutant, p53 into human lung cancer cell lines (Takahasi et al, 1992). p53 appears dominant over the mutant gene and will select against proliferation when transfected into cells with the mutant gene. Normal expression of the transfected p53 does not affect the growth of cells with endogenous p53. Thus, such constructs might be taken up by normal cells without adverse effects. It is thus proposed that the treatment of p53-associated cancers with wild type p53 will reduce the number of malignant cells or their growth rate.

The major transitions of the eukaryotic cell cycle are triggered by cyclin- dependent kinases, or CDK's. One CDK, cyclin-dependent kinase 4 (CDK4), regulates progression through the Gi. The activity of this enzyme may be to phosphorylate Rb at late Gi. The activity of CDK4 is controlled by an activating subunit, D-type cyclin, and by an inhibitory subunit, the pi 6 has been biochemically characterized as a protein that specifically binds to and inhibits CDK4, and thus may regulate Rb phosphorylation (Serrano et al, 1993; Serrano et al, 1995). Since the pl6 protein is a CDK4 inhibitor (Serrano, 1993), deletion of this gene may increase the activity of CDK4, resulting in hypeφhosphorylation of the Rb protein, pi 6 also is known to regulate the function of CDK6. pi 6 belongs to a newly described class of CDK-inhibitory proteins that also includes pi 6 , p21 , and p27 . The pi 6 gene maps to 9p21, a chromosome region frequently deleted in many tumor types. Homozygous deletions and mutations of the pi 6 gene are frequent in human tumor cell lines. This evidence suggests that the pi 6 gene is a tumor suppressor gene. This inteφretation has been challenged, however, by the observation that the frequency of the pi 6 gene alterations is much lower in primary uncultured tumors than in cultured cell lines (Caldas et al, 1994; Cheng et al, 1994; Hussussian et al, 1994; Kamb et al, 1994; Kamb et al, 1994; Mori et al, 1994; Okamoto et al, 1994; Nobori et al, 1995; Orlow et al, 1994; Arap et al, 1995). Restoration of wild-type pl6^INK4 function by transfection with a plasmid expression vector reduced colony formation by some human cancer cell lines (Okamoto, 1994; Arap, 1995).

C-CAM is expressed in virtually all epithelial cells (Odin and Obrink, 1987). C-CAM, with an apparent molecular weight of 105 kD, was originally isolated from the plasma membrane of the rat hepatocyte by its reaction with specific antibodies that neutralize cell aggregation (Obrink, 1991). Recent studies indicate that, structurally, C-CAM belongs to the immunoglobulin (Ig) superfamily and its sequence is highly homologous to carcinoembryonic antigen (CEA) (Lin and Guidotti, 1989). Using a baculovirus expression system, Cheung et al. (1993) demonstrated that the first Ig domain of C-CAM is critical for cell adhesive activity.

Cell adhesion molecules, or CAM's are known to be involved in a complex network of molecular interactions that regulate organ development and cell differentiation (Edelman, 1985). Recent data indicate that aberrant expression of CAM's maybe involved in the tumorigenesis of several neoplasms; for example, decreased expression of E-cadherin, which is predominantly expressed in epithelial cells, is associated with the progression of several kinds of neoplasms (Edelman and Crossin, 1991; Frixen et al, 1991; Bussemakers et al, 1992; Matsura et al, 1992; Umbas et al, 1992). Also, Giancotti and Ruoslahti (1990) demonstrated that increasing expression of ₅β] integrin by gene transfer can reduce tumorigenicity of Chinese hamster ovary cells in vivo. C-CAM now has been shown to suppress tumors growth in vitro and in vivo.

Other tumor suppressors that may be employed according to the present invention include RB, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zacl, p73,

VHL, MMAC1, FCC and MCC. Inducers of apoptosis, such as Bax, Bak, Bcl-X_s,

Bik, Bid, Harakiri, Ad E1B, Bad and ICE-CED3 proteases, similarly could find use according to the present invention.

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.

Hormones are another group of gene that may be used in the SV40 vectors described herein. Included are growth hormone, prolactin, placental lactogen, luteinizing hormone, follicle-stimulating hormone, chorionic gonadotropin, thyroid- stimulating hormone, leptin, adrenocorticotropin (ACTH), angiotensin I and II, β- endoφhin, β-melanocyte stimulating hormone (β-MSH), cholecystokinin, endothelin I, galanin, gastric inhibitory peptide (GIP), glucagon, insulin, lipotropins, neurophysins, somatostatin, calcitonin, calcitonin gene related peptide (CGRP), β- calcitonin gene related peptide, hypercalcemia of malignancy factor (1-40), parathyroid hormone-related protein (107-139) (PTH-rP), parathyroid hormone- related protein (107-11 1) (PTH-rP), glucagon-like peptide (GLP-1), pancreastatin, pancreatic peptide, peptide YY, PHM, secretin, vasoactive intestinal peptide (VIP), oxytocin, vasopressin (AVP), vasotocin, enkephalinamide, metoφhinamide, 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 and thyrotropin releasing hormone (TRH).

Other classes of genes that are contemplated to be inserted into the SV40 vectors of the present invention include interleukins and cytokines. Interleukin 1 (IL- 1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1 IL-12, GM-CSF and G- CSF.

Other therapeutics genes might include genes encoding antigens such as viral antigens, bacterial antigens, fungal antigens or parasitic antigens. Viruses include picornavirus, coronavirus, togavirus, flavirviru, rhabdovirus, paramyxo virus, orthomyxovirus, bunyavirus, arenvirus, reovirus, retrovirus, papovavirus, parvovirus, heφesvirus, poxvirus, hepadnavirus, and spongiform virus. Preferred viral targets include influenza, heφes simplex virus 1 and 2, measles, small pox, polio or HIV. Pathogens include trypanosomes, tapeworms, roundworms, helminths, . Also, tumor markers, such as fetal antigen or prostate specific antigen, may be targeted in this manner. Preferred examples include HIV env proteins and hepatitis B surface antigen. Administration of a vector according to the present invention for vaccination puφoses would require that the vector-associated antigens be sufficiently non- immunogenic to enable long term expression of the transgene, for which a strong immune response would be desired. Preferably, vaccination of an individual would only be required infrequently, such as yearly or biennially, and provide long term immunologic protection against the infectious agent.

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, (incoφorated 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 are contemplated, such as oncogenes, toxins, hormones, enzymes, viral or bacterial antigens, transcription factors or receptors.

Another class of molecules that could be expressed in target cells are antisense molecules. Antisense methodology takes advantage of the fact that nucleic acids tend to pair with "complementary" sequences, thereby inhibiting transcription and/or translation of the corresponding gene and gene product, respectively. 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-mefhylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing. Oncogenes such as ras, myc, neu, raf, erb, src, fins, jun, trk, ret, gsp, hst, bcl and abl also are suitable targets for antisense constructs.

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.

Another genetic construct to be transferred, according to the present invention, is a ribozyme. 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 Cook, 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 (Cook et al, 1981; Michel and Wesfhof, 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; Cook 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. C. Production Of Polypeptides //! Vitro

The ability to produce biologically active polypeptides is increasingly important to the pharmaceutical industry. Over the last decade, advances in biotechnology have led to the production of important proteins and factors from bacteria, yeast, insect cells and from mammalian cell culture. Mammalian cultures have advantages over cultures derived from the less advanced lifeforms in their ability to post-translationally process complex protein structures such as disulfide-dependent folding and glycosylation. Neuroendocrine cell types have added unique capacities of endoproteolytic cleaving, C-terminal amidation and regulated secretion. Indeed, mammalian cell culture is now the preferred source of a number of important proteins for use in human and animal medicine, especially those which are relatively large, complex or glycosylated. The present invention may be exploited for transferring genetic material into suitable mammalian host cells for the puφose of producing desirable polypeptides.

i) Anchorage-dependent versus non-anchorage-dependent cultures

Animal and human cells can be propagated in vitro in two modes: as non- anchorage dependent cells growing freely in suspension throughout the bulk of the culture; or as anchorage-dependent cells requiring attachment to a solid substrate for their propagation (/. e. , a monolayer type of cell growth).

Non-anchorage dependent or suspension cultures from continuous established cell lines are the most widely used means of large scale production of cells and cell products. Large scale suspension culture based on microbial (bacterial and yeast) fermentation technology has clear advantages for the manufacturing of mammalian cell products. The processes are relatively simple to operate and straightforward to scale up. Homogeneous conditions can be provided in the reactor which allows for precise monitoring and control of temperature, dissolved oxygen, and pH, and ensure that representative samples of the culture can be taken. However, suspension cultured cells cannot always be used in the production of biologicals. Suspension cultures are still considered to have tumorigenic potential and thus their use as substrates for production put limits on the use of the resulting products in human and veterinary applications (Petricciani, 1985; Larsson and Litwin, 1987). Viruses propagated in suspension cultures as opposed to anchorage-dependent cultures can sometimes cause rapid changes in viral markers, leading to reduced immunogenicity (Bahnemann, 1980). Finally, sometimes even recombinant cell lines can secrete considerably higher amounts of products when propagated as anchorage- dependent cultures as compared with the same cell line in suspension (Nilsson and Mosbach, 1987). For these reasons, different types of anchorage-dependent cells are used extensively in the production of different biological products.

ii) Reactors and processes for suspension

Large scale suspension culture of mammalian cultures in stirred tanks was undertaken. The instrumentation and controls for bioreactors adapted, along with the design of the fermentors, from related microbial applications. However, acknowledging the increased demand for contamination control in the slower growing mammalian cultures, improved aseptic designs were quickly implemented, improving dependability of these reactors. Instrumentation and controls are basically the same as found in other fermentors and include agitation, temperature, dissolved oxygen, and pH controls. More advanced probes and autoanalyzers for on-line and off-line measurements of turbidity (a function of particles present), capacitance (a function of viable cells present), glucose/lactate, carbonate/bicarbonate and carbon dioxide are available. Maximum cell densities obtainable in suspension cultures are relatively low at about 2-4 x 10 cells/ml of medium (which is less than 1 mg dry cell weight per ml), well below the numbers achieved in microbial fermentation.

Two suspension culture reactor designs are most widely used in the industry due to their simplicity and robustness of operation - the stirred reactor and the airlift reactor. The stirred reactor design has successfully been used on a scale of 8000 liter capacity for the production of interferon (Phillips et al, 1985; Mizrahi, 1983). Cells are grown in a stainless steel tank with a height-to-diameter ratio of 1 : 1 to 3 : 1. The culture is usually mixed with one or more agitators, based on bladed disks or marine propeller patterns. Agitator systems offering less shear forces than blades have been described. Agitation may be driven either directly or indirectly by magnetically coupled drives. Indirect drives reduce the risk of microbial contamination through seals on stirrer shafts.

The airlift reactor, also initially described for microbial fermentation and later adapted for mammalian culture, relies on a gas stream to both mix and oxygenate the culture. The gas stream enters a riser section of the reactor and drives circulation. Gas disengages at the culture surface, causing denser liquid free of gas bubbles to travel downward in the downcomer section of the reactor. The main advantage of this design is the simplicity and lack of need for mechanical mixing. Typically, the height-to-diameter ratio is 10:1. The airlift reactor scales up relatively easy, has good mass transfer of gasses and generates relatively low shear forces.

Most large-scale suspension cultures are operated as batch or fed-batch processes because they are the most straightforward to operate and scale up. However, continuous processes based on chemostat or perfusion principles are available.

A batch process is a closed system in which a typical growth profile is seen. A lag phase is followed by exponential, stationary and decline phases. In such a system, the environment is continuously changing as nutrients are depleted and metabolites accumulate. This makes analysis of factors influencing cell growth and productivity, and hence optimization of the process, a complex task. Productivity of a batch process may be increased by controlled feeding of key nutrients to prolong the growth cycle. Such a fed-batch process is still a closed system because cells, products and waste products are not removed.

In what is still a closed system, perfusion of fresh medium through the culture can be achieved by retaining the cells with a fine mesh spin filter and spinning to prevent clogging. Spin filter cultures can produce cell densities of approximately 5 x 10 cells/ml. A true open system and the simplest perfusion process is the chemostat in which there is an inflow of medium and an outflow of cells and products. Culture medium is fed to the reactor at a predetermined and constant rate which maintains the dilution rate of the culture at a value less than the maximum specific growth rate of the cells (to prevent washout of the cells mass from the reactor). Culture fluid containing cells and cell products and byproducts is removed at the same rate. These perfused systems are not in commercial use for production from mammalian cell culture.

iii) Non-perfused attachment systems

Traditionally, anchorage-dependent cell cultures are propagated on the bottom of small glass or plastic vessels. The restricted surface-to-volume ratio offered by classical and traditional techniques, suitable for the laboratory scale, has created a bottleneck in the production of cells and cell products on a large scale. In an attempt to provide systems that offer large accessible surfaces for cell growth in small culture volume, a number of techniques have been proposed: the roller bottle system, the stack plates propagator, the spiral film bottles, the hollow fiber system, the packed bed, the plate exchanger system, and the membrane tubing reel. Since these systems are non-homogeneous in their nature, and are sometimes based on multiple processes, they suffer from the following shortcomings - limited potential for scale-up, difficulties in taking cell samples, limited potential for measuring and controlling the system and difficulty in maintaining homogeneous environmental conditions throughout the culture.

Despite these drawbacks, a commonly used process of these systems is the roller bottle. Being little more than a large, differently shaped T-flask, simplicity of the system makes it very dependable and, hence, attractive. Fully automated robots are available that can handle thousands of roller bottles per day, thus eliminating the risk of contamination and inconsistency associated with the otherwise required intense human handling. With frequent media changes, roller bottle cultures can achieve cell densities of close to 0.5 x 10 cells/cm^" (corresponding to 10 cells/bottle or 10 cells/ml of culture media).

iv) Cultures on microcarriers In an effort to overcome the shortcomings of the traditional anchorage- dependent culture processes, van Wezel (1967) developed the concept of the microcarrier culturing systems. In this system, cells are propagated on the surface of small solid particles suspended in the growth medium by slow agitation. Cells attach to the microcarriers and grow gradually to confluency of the microcarrier surface. In fact, this large scale culture system upgrades the attachment dependent culture from a single disc process to a unit process in which both monolayer and suspension culture have been brought together. Thus, combining the necessary surface for a the cells grow with the advantages of the homogeneous suspension culture increases production.

The advantages of microcarrier cultures over most other anchorage-dependent, large-scale cultivation methods are several fold. First, microcarrier cultures offer a high surface-to-volume ratio (variable by changing the carrier concentration) which leads to high cell density yields and a potential for obtaining highly concentrated cell η products. Cell yields are up to 1-2 x 10 cells/ml when cultures are propagated in a perfused reactor mode. Second, cells can be propagated in one unit process vessels instead of using many small low-productivity vessels (i.e., flasks or dishes). This results in far better utilization and a considerable saving of culture medium. Moreover, propagation in a single reactor leads to reduction in need for facility space and in the number of handling steps required per cell, thus reducing labor cost and risk of contamination. Third, the well-mixed and homogeneous microcarrier suspension culture makes it possible to monitor and control environmental conditions (e.g., pH, p0₂, and concentration of medium components), thus leading to more reproducible cell propagation and product recovery. Fourth, it is possible to take a representative sample for microscopic observation, chemical testing, or enumeration. Fifth, since microcarriers settle out of suspension easily, use of a fed-batch process or harvesting of cells can be done relatively easily. Sixth, the mode of the anchorage-dependent culture propagation on the microcarriers makes it possible to use this system for other cellular manipulations, such as cell transfer without the use of proteolytic enzymes, cocultivation of cells, transplantation into animals, and perfusion of the culture using decanters, columns, fluidized beds, or hollow fibers for microcarrier retainment. Seventh, microcarrier cultures are relatively easily scaled up using conventional equipment used for cultivation of microbial and animal cells in suspension.

v) Microencapsulation of mammalian cells One method which has shown to be particularly useful for culturing mammalian cells is microencapsulation. The mammalian cells are retained inside a semipermeable hydrogel membrane. A porous membrane is formed around the cells permitting the exchange of nutrients, gases, and metabolic products with the bulk medium surrounding the capsule. Several methods have been developed that are gentle, rapid and non-toxic and where the resulting membrane is sufficiently porous and strong to sustain the growing cell mass throughout the term of the culture. These methods are all based on soluble alginate gelled by droplet contact with a calcium- containing solution. Lim (1982) describes cells concentrated in an approximately 1% solution of sodium alginate which are forced through a small orifice, forming droplets, and breaking free into an approximately 1% calcium chloride solution. The droplets are then cast in a layer of polyamino acid that ionically bonds to the surface alginate. Finally the alginate is reliquefied by treating the droplet in a chelating agent to remove the calcium ions. Other methods use cells in a calcium solution to be dropped into a alginate solution, thus creating a hollow alginate sphere. A similar approach involves cells in a chitosan solution dropped into alginate, also creating hollow spheres.

Microencapsulated cells are easily propagated in stirred tank reactors and, with beads sizes in the range of 150-1500 μm in diameter, are easily retained in a perfused reactor using a fine-meshed screen. The ratio of capsule volume to total media volume can kept from as dense as 1 :2 to 1 :10. With intracapsular cell densities of up to 10 the effective cell density in the culture is 1-5 x 10 .

The advantages of microencapsulation over other processes include the protection from the deleterious effects of shear stresses which occur from sparging and agitation, the ability to easily retain beads for the puφose of using perfused systems, scale up is relatively straightforward and the ability to use the beads for implantation.

vi) Perfused attachment systems

Perfusion refers to continuous flow at a steady rate, through or over a population of cells (of a physiological nutrient solution). It implies the retention of the cells within the culture unit as opposed to continuous-flow culture which washes the cells out with the withdrawn media (e.g., chemostat). The idea of perfusion has been known since the beginning of the century, and has been applied to keep small pieces of tissue viable for extended microscopic observation. The technique was initiated to mimic the cells milieu in vivo where cells are continuously supplied with blood, lymph, or other body fluids. Without perfusion, cells in culture go through alternating phases of being fed and starved, thus limiting full expression of their growth and metabolic potential. The current use of perfused culture is in response to the challenge of growing cells at high densities (i.e., 0.1-5 x 10 cells/ml). In order to increase densities beyond 2-4 x 10 cells/ml (or 2 x 10 cells/cm ), the medium has to be constantly replaced with a fresh supply in order to make up for nutritional deficiencies and to remove toxic products. Perfusion allows for a far better control of the culture environment (pH, pO₂, nutrient levels, etc.) and is a means of significantly increasing the utilization of the surface area within a culture for cell attachment.

Microcarrier and microencapsulated cultures are readily adapted to perfused reactors but, as noted above, these culture methods lack the capacity to meet the demand for cell densities above 10 cells/ml. Such densities will provide for the advantage of high product titer in the medium (facilitating downstream processing), a smaller culture system (lowering facility needs), and a better medium utilization (yielding savings in serum and other expensive additives). Supporting cells at high density requires extremely efficient perfusion techniques to prevent the development of non-homogeneity. This means the use of highly sophisticated procedures and apparati and has, until recently, been confined to a relatively small scale.

vii) CelliGen™ bioreactor system

The development of a perfused packed-bed reactor using a bed matrix of a non-woven fabric has provided a means for maintaining a perfusion culture at densities exceeding 10 cells/ml of the bed volume (CelliGen™, New Brunswick Scientific, Edison, NJ; Wang et al, 1992; Wang et al, 1993; Wang et al, 1994). Briefly described, this reactor comprises an improved reactor for culturing of both anchorage- and non-anchorage-dependent cells. The reactor is designed as a packed bed with a means to provide internal recirculation. Preferably, a fiber matrix carrier is placed in a basket within the reactor vessel. A top and bottom portion of the basket has holes, allowing the medium to flow through the basket. A specially designed impeller provides recirculation of the medium through the space occupied by the fiber matrix for assuring a uniform supply of nutrient and the removal of wastes. This simultaneously assures that a negligible amount of the total cell mass is suspended in the medium. The combination of the basket and the recirculation also provides a bubble-free flow of oxygenated medium through the fiber matrix. The fiber matrix is a non- woven fabric having a "pore" diameter of from 10 μm to 100 μm, providing for a high internal volume with pore volumes corresponding to 1 to 20 times the volumes of individual cells.

In comparison to other culturing systems, this approach offers several significant advantages. With a fiber matrix carrier, the cells are protected against mechanical stress from agitation and foaming. The free medium flow through the basket provides the cells with optimum regulated levels of oxygen, pH, and nutrients. Products can be continuously removed from the culture and the harvested products are free of cells and produced in low-protein medium which facilitates subsequent purification steps. Also, the unique design of this reactor system offers an easier way to scale up the reactor. Currently, sizes up to 30 liter are available. One hundred liter and 300 liter versions are in development and theoretical calculations support up to a 1000 liter reactor. This technology is explained in detail in WO 94/17178 (August 4, 1994, Freedman et al), which is hereby incoφorated by reference in its entirety.

A number of culturing parameters, used in conjunction the CelliGen™ system, have been demonstrated to play a role in increased production. For example, the CelliGen™ Plus reactor system, including the use of non- woven polyester fiber matrix (preferably, Fibra-Cel™) and centrifugal lift impeller (preferably, Fibra-Cel™) are system components that give improved yields. Also, several media formulations have been employed with improved performance. For example, use of serum free medium is preferred, as is the use of cholesterol rich lipid extract (0.01% to 0.10%, volume to volume), ascorbic acid (from between about 0.001 to 0.100 mM), glutamate (rather than 2 mM glutamine) at 2 to 20 mM, preferably 4 mM, alpha ketoglutarate (rather than 2 mM glutamine) at 2 to 20 mM, preferably 4 mM, and the absence of growth promoting factors.

viii) CellCube™ bioreactor system The Cellcube™ (Corning-Costar) module provides a large styrenic surface area for the immobilization and growth of substrate attached cells. It is an integrally encapsulated sterile single-use device that has a series of parallel culture plates joined to create thin, sealed laminar flow spaces between adjacent plates. The Cellcube™ module has inlet and outlet ports that are diagonally opposite each other and help distribute the flow of media to the parallel plates. The medium is constantly recirculated from the module through an oxygenator and back to the cube. The external oxygenator provides a bubble free stream of oxygenated medium and allows for the additional control of the pH of the medium. With concurrent addition of fresh medium, medium with secreted product and wastes can be harvested continuously, retaining the cell population in the cube. During the first few days of growth the culture is generally satisfied by the media contained within the system after initial seeding. The amount of time between the initial seeding and the start of the media perfusion is dependent on the density of cells in the seeding inoculum and the cell growth rate. The measurement of nutrient concentration in the circulating media is a good indicator of the status of the culture. When establishing a procedure it may be necessary to monitor the nutrients composition at a variety of different perfusion rates to determine the most economical and productive operating parameters.

Cells within the system reach a higher density of solution (cells/ml) than in traditional culture systems. Many typically used basal media are designed to support 1-2 x 10 cells/ml/day. A typical CellCube™ run with an 21 000 cm^" surface, contains approximately 1.2 liters of media within the module. The final cell density can exceeds 2.5 x

cells/ml in the culture vessel. At confluence, depending on the cell line used, media required can vary anywhere form 4-16 module volumes per day.

The advantage of the CellCube™ system is that it to a large extent replicates the conditions the cells experience in T flask culture. This allows for very linear scale up of any culture that is successfully grown in flask culture without severe loss in per- cell performance.

ix) Purification of Proteins

Protein purification techniques are well known to those of skill in the art. These techniques tend to involve the fractionation of the cellular milieu to separated the amylin form other components of the mixture. Having separated amylin from the other plasma components the amylin sample may be purified using chromatographic and electrophoretic techniques to achieve complete purification. Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.

It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE (Capaldi et al, Biochem.

Biophys. Res. Comm., 76:425, 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary.

High Performance Liquid Chromatography (HPLC) is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain and adequate flow rate. Separation can be accomplished in a matter of minutes, or a most an hour. Moreover, only a very small volume of the sample is needed because the particles are so small and close- packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample.

Gel chromatography, or molecular sieve chromatography, is a special type of partition chromatography that is based on molecular size. The theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size. As long as the material of which the particles are made does not adsorb the molecules, the sole factor determining rate of flow is the size. Hence, molecules are eluted from the column in decreasing size, so long as the shape is relatively constant. Gel chromatography is unsuφassed for separating molecules of different size because separation is independent of all other factors such as pH, ionic strength, temperature, etc. There also is virtually no adsoφtion, less zone spreading and the elution volume is related in a simple matter to molecular weight. Affinity Chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction. The column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (alter pH, ionic strength, temperature, etc.).

A particular type of affinity chromatography useful in the purification of carbohydrate containing compounds is lectin affinity chromatography. Lectins are a class of substances that bind to a variety of polysaccharides and glycoproteins.

Lectins are usually coupled to agarose by cyanogen bromide. Conconavalin A coupled to Sepharose was the first material of this sort to be used and has been widely used in the isolation of polysaccharides and glycoproteins other lectins that have been include lentil lectin, wheat germ agglutinin which has been useful in the purification of N-acetyl glucosaminyl residues and Helix pomatia lectin. Lectins themselves are purified using affinity chromatography with carbohydrate ligands. Lactose has been used to purify lectins from castor bean and peanuts; maltose has been useful in extracting lectins from lentils and jack bean; N-acetyl-D galactosamine is used for purifying lectins from soybean; N-acetyl glucosaminyl binds to lectins from wheat germ; D-galactosamine has been used in obtaining lectins from clams and L-fucose will bind to lectins from lotus.

The matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability. The ligand should be coupled in such a way as to not affect its binding properties. The ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand. One of the most common forms of affinity chromatography is immunoaffinity chromatography. The generation of antibodies that would be suitable for use in accord with the present invention is discussed below. D. Pharmaceuticals And Methods Of Treating Disease

In another embodiment of the present invention, there are provided methods for the treatment of various disease states. Treatment methods will involve treating an individual with an effective amount of a viral particle, as described above, containing a therapeutic gene of interest. An effective amount is described, generally, as that amount sufficient to detectably and repeatedly to ameliorate, reduce, minimize or limit the extent of a disease or its symptoms. More rigorous definitions may apply, including elimination, eradication or cure of disease.

Administration of the therapeutic virus particle to a patient will follow general protocols for the administration of chemotherapeutics, taking into account the toxicity, if any, of the vector. It is anticipated that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the described gene therapy.

Where clinical application of a gene therapy is contemplated, it will be necessary to prepare the complex as a pharmaceutical composition appropriate for the intended application. Generally this will entail preparing a pharmaceutical composition that is essentially free of pyrogens, as well as any other impurities that could be harmful to humans or animals. One also will generally desire to employ appropriate salts and buffers to render the complex stable and allow for complex uptake by target cells.

Aqueous compositions of the present invention comprise an effective amount of the compound, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions can also be referred to as inocula. The phrases "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absoφtion 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 also can be incoφorated into the compositions.

The compositions of the present invention may include classic pharmaceutical preparations. Dispersions also can 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.

i) Disease States

A wide variety of disease states may be treated with compositions according to the present invention. In essence, any disease that can be treated by provision of a protein or nucleic acid is amenable to this approach. Disease states include a variety of genetic abnormalities such as diabetes, cancer, cystic fibrosis and various other diseases that could be treated by increasing or decreasing expression of a protein in a target cell.

Depending on the particular disease to be treated, administration of therapeutic 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. Topical administration would be particularly advantageous for treatment of skin cancers. Alternatively, administration will be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients. In certain embodiments, ex vivo therapies also are contemplated. Ex vivo therapies involve the removal, from a patient, of target cells. The cells are treated outside the patient's body and then returned. One example of ex vivo therapy would involve a variation of autologous bone marrow transplant. Many times, ABMT fails because some cancer cells are present in the withdrawn bone marrow, and return of the bone marrow to the treated patient results in repopulation of the patient with cancer cells. In one embodiment, however, the withdrawn bone marrow cells could be treated while outside the patient with an viral particle that targets and kills the cancer cell. Once the bone marrow cells are "purged," they can be reintroduced into the patient.

The treatments may include various "unit doses." Unit dose is defined as containing a predetermined-quantity of the therapeutic composition calculated to produce the desired responses in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, and the particular route and formulation, are within the skill of those in the clinical arts. Also of import is the subject to be treated, in particular, the state of the subject and the protection desired. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. Unit dose of the present invention may conveniently may be described in terms of plaque forming units (pfu) of the viral construct. Unit doses range from 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10ⁿ, 10¹², 10¹³ pfu and higher.

Preferably, patients will have adequate bone marrow function (defined as a peripheral absolute granulocyte count of > 2,000 / mm and a platelet count of 100,000 / mm^J), adequate liver function (bilirubin < 1.5 mg / dl) and adequate renal function (creatinine < 1.5 mg / dl). ii) Cancer

One of the preferred embodiments of the present invention involves the use of viral vectors to deliver therapeutic genes to cancer cells. Target cancer cells include cancers of the lung, brain, prostate, kidney, liver, ovary, breast, skin, stomach, esophagus, head & neck, testicles, colon, cervix, lymphatic system and blood. Of particular interest are non-small cell lung carcinomas including squamous cell carcinomas, adenocarcinomas and large cell undifferentiated carcinomas.

According to the present invention, one may treat the cancer by directly injection a tumor with the viral vector. Alternatively, the tumor may be infused or perfused with the vector using any suitable delivery vehicle. Local or regional administration, with respect to the tumor, also is contemplated. Finally, systemic administration may be performed. Continuous administration also may be applied where appropriate, for example, where a tumor is excised and the tumor bed is treated to eliminate residual, microscopic disease. Delivery via syringe or catherization is preferred. Such continuous perfusion may take place for a period from about 1-2 hours, to about 2-6 hours, to about 6-12 hours, to about 12-24 hours, to about 1-2 days, to about 1 -2 weeks or longer following the initiation of treatment. Generally, the dose of the therapeutic composition via continuous perfusion will be equivalent to that given by a single or multiple injections, adjusted over a period of time during which the perfusion occurs.

For tumors of > 4 cm, the volume to be administered will be about 4-10 ml (preferably 10 ml), while for tumors of < 4 cm, a volume of about 1-3 ml will be used (preferably 3 ml). Multiple injections delivered as single dose comprise about 0.1 to about 0.5 ml volumes. The viral particles may advantageously be contacted by administering multiple injections to the tumor, spaced at approximately 1 cm intervals.

In certain embodiments, the tumor being treated may not, at least initially, be resectable. Treatments with therapeutic viral constructs may increase the resectability of the tumor due to shrinkage at the margins or by elimination of certain particularly invasive portions. Following treatments, resection may be possible. Additional viral treatments subsequent to resection will serve to eliminate microscopic residual disease at the tumor site.

A typical course of treatment, for a primary tumor or a post-excision tumor bed, will involve multiple doses. Typical primary tumor treatment involves a 6 dose application over a two week period. The two week regimen may be repeated one, two, three, four, five, six or more times. During a course of treatment, the need to complete the planned dosings may be reevaluated.

Cancer therapies also include a variety of combination therapies with both chemical and radiation based treatments. Combination chemotherapies include, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin. daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, taxol, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate.

Combination radiation therapies may be x- and γ-irradiation. Dosage ranges for x-irradiation range from daily doses of 2000 to 6000 roentgens for prolonged periods of time (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosages for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by neoplastic cells.

Various combinations may be employed, gene therapy is "A" and the radio- or chemotherapeutic agent is "B":

A/B/A B/A/B B/B/A A/A/B A B/B B/A/A A/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B A/B/A B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A B/A A A A B/A

The terms "contacted" and "exposed," when applied to a cell, are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. To achieve cell killing or stasis, both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.

The therapeutic compositions of the present invention are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified. A typical composition for such puφose comprises a pharmaceutically acceptable carrier. For instance, the composition may contain 10 mg, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well known parameters.

Additional formulations are suitable for oral administration. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. When the route is topical, the form may be a cream, ointment, salve or spray.

E. 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: PACKAGING SYSTEM FOR SV40 VECTORS LACKING VIRAL CODING SEQUENCES

I. Materials and Methods

Cell culture

293 and COS-7 cells were obtained from American Type Culture Collection (Rockville, MD) and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/1 glucose, 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin.

Construction of recombinant adenovirus SV40 DNA was purchased from Sigma (St. Louis, MO). pADl/CMV was constructed by replacing the RSV-LTR in pADL.l/RSV (Fang et al, 1994) with the CMV early promoter. pADl/CMV-CAP, which contains SV40 late genes driven by the CMV promoter, was constructed by inserting a 2.5 kB fragment from the SV40 viral genome (bp 273-2771) at the Ee RV site of pADl/CMV. pADl/SV-CAP was constructed by inserting a 3.27 kB Taql- and 5c/I-digested fragment from SV40 at the Claϊ/BamUl site of pXCJL 1 (gift of Dr. F. Graham, McMaster University, Canada). Recombinant adenoviruses Ad/CMV-CAP and Ad/SV-CAP were constructed by cotransfecting 293 cells with a 35 kB CM fragment from dl324 and pADl/CMV- CAP (for Ad/CMV-CAP) or pADl/SV-CAP (for Ad/SV-CAP). The recombinants were identified by restriction digestion of viral genomes with BamHl.

Preparation of recombinant SV40 Vector

For use in preparing recombinant SV40 vectors, plasmid pGFP-Nl, which contains the SV40 replication origin and a green fluorescent protein (GFP)-expressing cassette driven by CMV, was obtained from Clontech (Palo Alto, CA). In brief, COS- 7 cells were seeded at 1 x 10 /10-cm dish and then infected with adenoviral vector at MOI 500 1 h prior to transfection. Plasmid DNA was transfected into cultured cells by calcium phosphate methods (Graham and Van Der Eb, 1973). Cells were then trypsinized and suspended in 1 ml medium. Cell suspensions were frozen and thawed three times to release the virus. Then, cell debris was removed after centrifugation at 13,000 φm for 5 min.

Titration of recombinant SV40 Virus

Titers of infectious particles were determined by an end-point titer assay (median tissue culture infective dose [TCID₅₀]). In brief, COS-7 cells were plated onto a 96-well microtiter plate at 10 cells/100 ml/well. Viral stocks were serially diluted with DMEM containing 10% bovine calf serum and then transferred in quadruplicate to COS-7-seeded plates at 100 μl/well. After culture for 2 days, the plates were examined under a fluorescent microscope and scored for the presence of GFP. Titers were determined using the liteφrint Analysis program (Lynn, 1992).

DNA assay

Aliquots of viral stock (0.37 ml each) were digested with 100 μg DNase I for

20 min to remove cellular DNA. Each mixture was then digested with 100 μg proteinase K in 1% SDS, 20 mM EDTA at 55°C for 2 h. After extraction with phenol, viral DNA was precipitated by ethanol. The final product was dissolved in 30 μl H₂0. The DNA was then subjected to polymerase chain reaction (PCR™) as described previously (Fang et al, 1994) using the following PCR™ primers: (Eider et al. 1981) 5'-acgcaaatgggcggtag-3' and 5'-cgctgaacttgtggccg-3' for CMV and GFP; (Hamer, 1980) 5'-gacactctatgcctgtg-3' and 5'-gagcagtggtggaatgc-3' for SV40 large T antigen gene; (Strayer and Milano, 1996) 5'-ccttgtaccggaggtgatc-3' and 5'-cacactctatcacccactg-3' for the adenovirus E1A region; (Strayer, 1996) 5'- ggaaatatgactacgtc-3' and 5'-aagtccacgcctacatg-3' for the adenovirus E4 region.

II. Results Recombinant adenovirus expressing SV40 capsid

Two recombinant adenoviruses, Ad/CMV-CAP and Ad/SV-CAP, each containing an SV40-capsid-expressing cassette, were constructed (FIG. 1). Ad/CMV- CAP contains SV40 late genes driven by the human CMV immediate-early (CMV-IE) gene promoter, one of the strongest promoters in a variety of cells (Guo et al , 1996). Because the SV40 late gene-expressing cassette in Ad/SV-CAP is the same as in the SV40 virus, the splicing of the late mRNAs and the ratio of the late proteins in Ad/SV-CAP-infected cells remained the same as in SV40-infected cells. The recombinants were identified by DNA assays. A single plaque from each construct was expanded and titrated on 293 cells.

Packaging ofSV40 vector

Plasmid pGFP-Nl was used to test whether the recombinant adenoviruses containing an SV40 late gene-expressing cassette were able to package plasmids having an SV40 replication origin. pGFP-N 1 is about nine-tenths the size of the SV40 genome and contains both the SV40 replication origin and a GFP-expressing cassette driven by the CMV promoter (FIG. 4). There are no SV40 coding sequences in the plasmid. To package pGFP-Nl in SV40 capsids, COS-7 cells were infected with recombinant adenovirus at an MOI of 500 1 h prior to transfection with pEGFP- Nl . Preliminary studies showed that over 70% of COS-7 cells were transduced by adenovirus at MOI of 500. The cells were then transfected with pEGFP-Nl, and the medium was changed 5 h after transfection. Four days after transfection, the cells were harvested and cell lysates were titrated on COS-7 cells for the presence of GFP- expressing vector (Table 2). No detectable GFP-expressing vector was found when Ad/SV-CAP or control virus was used to package pGFP-Nl . In contrast, GFP- expressing vectors were typically 10 -lOVlO cells when packaged with Ad/CMV- CAP.

TABLE 2

Titration of SV40 Vectors Packaged with Recombinant Adenoviruses

Adenovirus Titer of SV40 vectors

Ad/CMV-CAP 3.1 x 10⁵

Ad/SV-CAP ND

Ad/CMV-LacZ ND

ND-Ad/RSV-Luc ND

ND: not detectable

To determine the time course of the packaging, COS-7 cells were harvested over time after infection with Ad/CMV-CAP and transfection with pEGFP-Nl. Cell lysate were then titrated using TCID₅₀ as the end point. GFP-expressing vector was detected 1 day after transfection, and its level peaked at day 4 (FIG. 2). In subsequent studies, the cells were harvested at 4 days after infection and transfection.

Detection of helper virus in vector preparations by PCR™ and plaque assay The SV40 early gene, late gene and replication origin are contained in COS-7 cells, Ad/CMV-CAP, and pEGFP-Nl , respectively. Thus, the chances of generating wild-type SV40 virus by recombination should be very low. Furthermore, El-deleted recombinant adenovirus is believed to be replication defective and so should not be packaged. To test for the presence of SV40-GFP vector, wild-type SV40 and recombinant adenovirus in vector preparations, viral DNA was isolated after digestion of cell lysates with DNase I. Viral DNA was then subjected to PCR™ with primers specific for CMV-GFP, adenoviral El, adenoviral E4 and SV40 large T genes. The presence of CMV-GFP and adenoviral E4 were readily detected by PCR™; however, adenoviral El and SV40 large T were not detected at all (FIG. 3). To determine the amount of recombinant adenovirus in the vector preparations, cell lysates were titrated by plaque assay on 293 cells and COS-7 cells. While no plaques were titrated on COS-7 cells, about 1.9 x 10 plaque-forming units were titrated on 293 cells. Thus, no contamination by wild-type SV40 was detected by either PCR™ or plaque assay; however, the helper recombinant adenovirus was present at low titers in the vector preparations. This result is consistent with the inventors' previous observation that the El -deleted recombinant adenovirus still replicate at low levels when cells were infected at high MOI (Fang et al, 1997).

Heat inactivation of helper virus

SV40 virus is known to be relatively resistant to heat inactivation, while adenovirus is known to be heat labile. To test whether the helper recombinant adenovirus can be heat inactivated without affecting the titer of SV40 vector, vector preparations were incubated at 56°C for 30 min and subsequently titrated on 293 cells by plaque assay and on COS-7 cells by TCID₅₀ assay. A non-heat-inactivated vector preparation was used as a positive control and a mock-infected preparation as a negative control. While the titer for GFP-expressing vector remained unchanged after heat inactivation, the level of plaque-forming units for adenovirus dropped from 2 x 10 to undetectable.

III. Discussion The inventors have demonstrated that a plasmid containing the SV40 replication origin can be packaged to SV40 vector. Moreover, several pieces of data strongly imply that transduction of pEGFP-Nl after packaging with Ad/CMV-CAP is SV40-mediated. First, packaging with control virus did not produce vectors that could transduce pEGFP-Nl. Second, PCR™ assays showed no pEGFP-Nl DNA in cell lysates containing plasmid packaged with control virus, whereas pEGFP-Nl DNA was readily detected in cell lysates containing plasmid packaged with Ad/CMV-CAP. Third, heat inactivation diminished the titer of infectious adenovirus to undetectable levels but had no effect on the titer of GFP-expressing vector. Together, these results ruled out protein- or adenovirus-mediated transduction of GFP.

SV40 virus has long been considered nonpathogenic in humans. Indeed, the contamination of early preparations of polio vaccine by wild-type SV40 virus produced no significant side effects (Fraumeni Jr., 1963; Lewis Jr., 1973). However, the recent finding of the SV40 genome in some tumor samples has raised the alarm of possible SV40 pathogenesis in humans (Lednicky et al. 1995; Carbone et al, 1996). Nevertheless, the complete removal of viral coding sequences from SV40 will generate a vector similar to and presumably just as safe as retroviral or adeno- associated viral vectors (AAV). Moreover, unlike retroviral vector, SV40 vector can easily be concentrated to high titer, and unlike AAV, SV40 is double-stranded and will not require helpers for transgene expression.

Current limitations on the use of SV40 vectors include low transduction efficiencies in human cells. Though SV40 can reportedly transduce human hematopoietic and peripheral blood cells (Oppenheim et al, 1986; Strayer et al, 1997), the inventors have found that the transduction efficiency of SV40 vector in human cells is low and that no detectable gene transduction occurs in human lung carcinoma cell lines HI 299 and A549 after infection of both lines with the SV40-GFP vector at MOI 1 and 5. The inventors have seen, however, few GFP-transduced cells among K562 cells infected with the SV40-GFP vector at MOI 1. Thus, changes in the SV40 capsid may be required in order to expand the host range of SV40 vectors.

EXAMPLE 2: CONSTRUCTS INCORPORATING JC AND BK VIRUS CAPSID PROTEIN INTO AN ADENOVIRAL HELPER VIRUS

I. Materials And Methods

Construction Of Adenoviral/Bk Helper Virus

An adenoviral helper virus, containing the VI -3 coding regions from BK virus, is constructed as follows. First, the CMV promoter is inserted into pADl as described above, creating pADl/CMV. Second, an approximately 2.7 kB Hpall-Xbal fragment is generated from BK virus genomic DNA by double digestion with the appropriate enzymes. The fragment, which contains the coding regions for VPl-3, is blunted and cloned into the EcoRV site of pAD 1 /CMV, creating pAD 1 /CMV/BK 123. FIG. 6A. The cassette generated above then is cotransfected with a 35 kB Clal fragment from dl324, as described above, creating the recombinant adenoviral helper Ad5/CMV/BK123.

Construction Of Adenoviral/ Jc Helper Virus

An adenoviral helper virus, containing the VI -3 coding regions from JC virus, is constructed as follows. First, the CMV promoter is inserted into pADl as described above, creating pADl/CMV. Second, the JC virus VP1 gene is obtained by PCR using the primers 5'-GAAGGATCCGAAGATGGCCC-3' and 5'- GGGATCCTTACAGCAT-3'. These primers introduce BamHl sites into the ends of the PCR product. The product is cloned into the BamHl site of pADl/CMV, creating pADl/CMV-1. If the construct is reversed (i.e., antisense), with respect to the CMV promoter, a restriction with BamHl and subsequent religation will generate 50% transformants having the correct orientation. And third, the VP2/VP3 coding regions are obtained by cutting viral DNA with Accl and EcoRI and isolating an approximately 1 kB fragment. This fragment is blunted and cloned into the EcoRV of pADl/CMV-1 , creating pADl /CMV- 1-2/3. This plasmid contains the VPl-3 genes of JC virus in a single cassette. FIG. 6B. The cassette generated above then is cotransfected with a 35 kB Clal fragment from dl324, also as described above, creating the recombinant adenoviral helper Ad5/CMV/JC123.

Replication OfSv40 Viral Vector Supported By Ad5/Cmv/Bkl 23 And Ad5/Cmv/Jcl23 Helper Virus

To package pEGFP-Nl in SV40 capsids, COS-7 cells are infected with either

Ad5/CMV/BK123 or Ad5/CMV/JC123 at an MOI of 500 1 h prior to transfection. with pEGFP-Nl . The cells are then transfected with pEGFP-Nl, and the medium is changed 5 h after transfection. Four days after transfection, the cells are harvested and cell lysates are titrated on COS-7 cells for the presence of GFP-expressing vector. Contaminating adenovirus may be eliminated by heat inactivation at 56°C.

Infection Of Tarset Cells With Ad5/Cmv/Bkl23 And Ad5/Cmv/Jcl23 Helper Virus

Putative target cells for testing the performance of these vectors include tumors of neural origin including meduloblastomas, neuroblastomas, meningiomas and gliomas. Particular cell lines include ATCC CRL-8621, T98G, U251 mG, U373 MG and A-172 (all p53 deficienct), U-87 MG, EFC-2 and D54 MG (all wild-type p53). Other cell lines include those of renal or pancreatic origin, such as HEK cells or the cell lines ASPC-1, BXPC-3, Capan-1 , CFPAC-1 , MiaPaca-2 and Panc-1. Diploid lung fibroblasts (WI38), fetal brain cells and osteosarcomas (SaoS2) also could be employed.

REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incoφorated herein by reference:

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Claims

CLAIMS:

1. A method for producing an infectious SV40 viral vector comprising:

a) providing an SV40 viral vector comprising an SV40 origin of replication and an expression region operably linked to a first promoter active in eukaryotic cells, wherein said vector lacks SV40 coding sequences; b) providing a replication-deficient adenoviral helper virus having the late region of JC virus or BK virus under the control of a second promoter active in eukaryotic cells; c) infecting host cells with said adenoviral helper virus; d) transfecting said host cells with said vector; e) culturing said host cells for a period of time sufficent to permit said vector to replicated and be packaged; and f) harvesting said packaged vector in an infectious form.

2. The method of claim 1, wherein said first promoter is a CMV IE or SV40 IE promoter.

3. The method of claim 1, wherein said second promoter is a CMV IE or SV40 IE promoter.

4. The method of claim 1 , wherein said host cells are cultured for about 3 to about 5 days.

5. The method of claim 4, wherein said host cells are cultured for about 4 days.

6. The method of claim 1 , wherein said host cells are transfected using a method selected from the group consisting of calcium phosphate precipitation, electroporation, lipofection and protoplast fusion.

7. The method of claim 1 , wherein said expression region comprises a nucleic acid coding for a gene.

8. The method of claim 7, wherein said gene is a tumor suppressor.

9. The method of claim 7, wherein said gene is a cytokine.

10. The method of claim 7, wherein said gene is an enzyme.

11. The method of claim 7, wherein said gene is a hormone.

12. The method of claim 7, wherein said gene is a receptor.

13. The method of claim 7, wherein said gene is an inducer of apoptosis.

14. The method of claim 1, wherein said expression region comprises an antisense construct.

15. The method of claim 14, wherein said antisense construct is derived from an oncogene.

16. The method of claim 15, wherein said oncogene is selected from the group consisting ras, myc, neu, raf, erb, src, fins, jun, trk. ret, gsp, hst, bcl and abl.

17. The method of claim 8, wherein said tumor suppressor is selected from the group consisting of p53, pi 6, p21, MMAC1 , p73, zacl , BRCAI and Rb.

18. The method of claim 9, wherein said cytokine is selected from the group consisting of IL-2, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1, IL-12, IL-13, IL-14, IL-15, TNF, GMCSF, β-interferon and γ-interferon.

19. The method of claim 10, wherein said enzyme is selected from the group consisting of cytosine deaminase, adenosine deaminase, β-glucuronidase, hypoxanthine guanine phosphoribosyl transferase, galactose-1 -phosphate uridyltransferase, glucocerbrosidase, glucose-6-phosphatase, thymidine kinase and lysosomal glucosidase.

20. The method of claim 11, wherein said hormone is selected from the group consisting of growth hormone, nerve growth factor, insulin, adrenocorticotropic hormone, parathormone, follicle-stimulating hormone, luteinizing hormone, epidermal growth factor and thyroid stimulating hormone.

21. The method of claim 12, wherein said receptor is selected from the group consisting of CFTR, EGFR, VEGFR, IL-2 receptor and the estrogen receptor.

22. The method of claim 13, wherein said inducer of apoptosis is selected from the group consisting of Bax, Bak, Bcl-X_s, Bik, Bid, Bad, Harakiri, Ad E1B and an ICE- CED3 protease.

23. The method of claim 1, wherein said host cells express a polyomavirus large T antigen.

24. The method of claim 23, wherein said host cells are COS-7 cells.

25. The method of claim 1, wherein said adenoviral helper virus lacks El regions.

26. An SV40 viral vector comprising an SV40 origin of replication and an expression region operably linked to a promoter active in eukaryotic cells, wherein said vector lacks SV40 coding sequences.

27. The vector of claim 26, wherein said promoter is CMV IE or SV40 IE.

28. The vector of claim 26, wherein said expression region comprises a nucleic acid coding for a polypeptide.

29. The method of claim 28, wherein said gene is selected from the group consisting of a tumor suppressor, a cytokine., an enzyme, a hormone, an inducer of apoptosis and a receptor.

30. The method of claim 26, wherein said expression region comprises an antisense construct.

31. The method of claim 30, wherein said antisense construct is derived from an oncogene.

32. A replication-deficient adenoviral helper virus having the late region of JC virus or BK virus under the control of a promoter active in eukaryotic cells. The helper virus of claim 32, wherein said promoter is CMV IE or SV40 IE.

34. The helper virus of claim 32, wherein said virus lacks El regions.

35. A helper cell, the genome of which comprises (a) one or more genes encoding polypeptides that are sufficient to support replication of the vector of claim 26 and (b) genes encoding the VPl-3 genes of JC virus.

36. A helper cell, the genome of which comprises (a) one or more genes encoding polypeptides that are sufficient to support replication of the vector of claim 26 and (b) genes encoding the VPl-3 genes of BK virus.

35. A method for expressing a selected polypeptide in a human cell comprising:

a) providing an infectious viral particle comprising a JC virus or BK virus-derived capsid and an SV40 viral vector comprising (i) an SV40 origin of replication, and

(ii) an expression region comprising said a nucleic acid encoding said polypeptide operably connected to a first promoter active in eukaryotic cells, wherein said vector lacks SV40 coding sequences; b) contacting said viral particle with said cell under conditions permitting transfer of said vector into said cell; and c) culturing said cell under conditions permitting the expression of said polypeptide.

36. The method of claim 37, wherein said cell is in a human subject.

37. A pharmaceutical preparation formulated for use in a method for treating a human disease comprising:

a) providing an infectious viral particle comprising a JC virus- or BK virus-derived capsid and an SV40 viral vector comprising

(i) an SV40 origin of replication, and

(ii) an expression region comprising said a nucleic acid encoding said therapeutic polypeptide operably connected to a first promoter active in eukaryotic cells, wherein said vector lacks S V40 coding sequences; and b) administering said viral particle to a human subject having said disease under conditions permitting transfer of said vector into cells of said human subject.

38. The method of claim 39, wherein said disease is selected from the group consisting of cancer, diabetes and cystic fibrosis.

39. The method of claim 39, wherein said promoter is CMV IE or SV40 IE.

42. A method for producing an infectious SV40 viral vector comprising:

a) providing an SV40 viral vector comprising an SV40 origin of replication and an expression region operably linked to a first promoter active in eukaryotic cells, wherein said vector lacks SV40 coding sequences; b) providing a helper cell expressing the late region of JC virus or BK virus under the control of a second promoter active in eukaryotic cells, along with polypeptides essential for the replication of said SV40 viral vector; c) introducing into said host cells said SV40 vector; d) culturing said host cells for a period of time sufficent to permit said vector to replicated and be packaged; and e) harvesting said packaged vector in an infectious form.