Link:  Pharm/Biotech Resources

Title:  Methods and compositions for efficient gene transfer using transcomplementary vectors

United States Patent:  6,875,610

Issued:  April 5, 2005

Inventors:  Higginbotham; James N. (Ames, IA); Link; Charles J. (Des Moines, IA); Ramsey; William J. (Ames, IA)

Assignee:  Human Gene Therapy Research Institute (Des Moines, IA)

Appl. No.:  871183

Filed:  May 31, 2001

Abstract

The invention includes a viral vector method and composition comprising transcomplementary replication incompetent viral vectors, preferably adenoviral vectors, which are cotransformed to a recipient cell. The two vectors complement each other and thus allow viral replication, in a synergistic combination which enhances both gene delivery and gene expression of genetic sequences contained within the vector.

Description of the Invention

FIELD OF THE INVENTION

This invention relates generally to genetic engineering and more specifically to improvements in components and methods used in genetic engineering, namely vectors. Vectors produced by the teachings herein can be used in any of a number of molecular protocols including in vitro, ex vivo or in vivo modification of nucleotide sequences present in cells.

BACKGROUND OF THE INVENTION

The field of gene therapy has made significant gains in recent years. The combination of genetic defects being identified and gene target/delivery methods being developed has led to an explosion in the number of clinical gene therapy protocols. The central focus of gene therapy is to develop methods for introducing new genetic material into somatic cells. To date two general classes of gene transfer methods have evolved. The first is DNA-mediated gene transfer and involves direct administration of DNA to the patient in various formulations. These methods use genes as medicines in a manner much like conventional organic or protein compounds. DNA-mediated gene transfer however has proven quite difficult. Methodology such as micro-injection, lipofection, and receptor mediated endocytosis have usually resulted in lower gene transfer, and have usually established only transient residence of the novel gene in the targeted cell. Permanent incorporation of genes into cells occurs rarely after DNA-mediated gene transfer in cultured cells (less than 1x10⁵ cells) and has not been significantly observed in vivo. Thus DNA-mediated gene transfer may be inherently limited to the use of genes as medicines that are administered by conventional parenteral routes to provide a therapeutic effect over predictable period of time. Studies of a therapeutic gene product may be constituted by repetitively dosing the patient with degenerate material much like conventional pharmaceutical medicines.

Viral gene transfer on the other hand involves construction of synthetic virus particles (vectors) that lack pathogenic functions. The virus particles are incapable of replication and contain a therapeutic or diagnostic gene within the viral genome which is delivered to cells by the process of infection. To date the viral vector which has achieved the most success is the retroviral vector. The prototype for a retroviral mediated gene transfer is a retroviral vector derived from Moloney Murine Leukemia Virus. Retroviral vectors have several properties that make them useful for gene therapy. First is the ability to construct a "defective" virus particle that contains the therapeutic gene and is capable of infecting cells but lacks viral genes and expresses no viral gene products which helps to minimize host response to potential viral epitopes.

Retroviral vectors are capable of permanently integrating the genes they carry into the chromosomes of the target cell. Considerable experience in animal models and initial experience in clinical trials suggest that these vectors have a high margin of safety.

Vectors based on adenovirus have recently proven effective as vehicles for gene transfer in vitro and in vivo in several cell types. Adenoviral vectors are constructed using a deleted adenoviral genome that lacks either the e-3, e-4 or gene region and/or the e-1 gene region that is required for producing a replicating adenovirus particle. Recombinant genes are inserted into the site of the deleted gene region(s). Adenoviral particles are then produced in a cell line that is able to express e-1, e-4 or e-3 genes and thus capable of assembling a viral particle which contains only the recombinant viral genome with the therapeutic gene.

Adenoviral vectors differ from retroviral vectors in that they do not integrate their genes into the target cell chromosome. Adenoviral vectors will infect a wide variety of both dividing and non-dividing cells in vitro and in vivo with a high level of efficiency providing expression of their recombinant gene for a period of several weeks to months.

Current technology has enabled construction of adenoviral vectors that are incapable of proliferating however they are not completely "defective" and will express a series of viral gene products which can generate host immune response to the viral epitopes presented causing quick elimination of the already transient vector.

Adenovirus is a non-enveloped, nuclear DNA virus with a genome of about 36 kb, which has been well-characterized through studies in classical genetics and molecular biology (Horwitz, M. S., "Adenoviridae and Their Replication," in Virology, 2^nd ed., Fields et al., eds., Raven Press, New York 1990). The viral genes are classified into early (known as E1-E4) and late (known as L1-L5) transcriptional units, referring to the generation of two temporal classes of viral proteins. The demarcation between these events is viral DNA replication.

Recombinant adenoviruses have several advantages for use as gene transfer vectors, including tropism for both dividing and non-diving cells, minimal pathogenic potential, ability to replicate to high titer for preparation of vector stocks, and the potential to carry large inserts (Berkner, K. L., Curr. Top. Micro. Immunol. 158:39-66, 1992; Jolly, D., Cancer Gene Ther. 1:51-64, 1994).

The cloning capacity of an adenoviral vector is proportional to the size of the adenovirus genome present in the vector. For example, a cloning capacity of about 8 kb can be created from the deletion of certain regions of the virus genome dispensable for virus growth, e.g., E3, and the deletion of a genomic region such as E1 whose function may be restored in trans from 293 cells (Graham, F. L., J. Gen. Virol. 36:59-72, 1977) or A549 cells (Imler et al., Gene Ther. 3:75-84, 1996). Such E1-deleted vectors are rendered replication-defective. The upper limit of vector DNA capacity is about 105%-108% of the length of the wild-type genome. Further adenovirus genomic modifications are possible in vector design using cell lines which supply other viral gene products in trans, e.g., complementation of E2 (Zhou et al., J. Virol. 70:7030-7038, 1996), complementation of E4 (Krougliak et al, Hum. Gene Ther. 6:1575-1586, 1995). Maximum carrying capacity can be achieved using adenoviral vectors deleted for all viral coding sequences (Kochanek et al., Proc. Natl. Acad. Sci. USA 93:5731-5736, 1996; Fisher et al., Virology 217:11-22, 1996).

Transgenes that have been expressed to date by adenoviral vectors include p53 (Wills et al., Hum. Gene Ther. 5:1079-188, 1994); dystrophin (Vincent et al., Nature Genetics 5:130-134, 1993; erythropoietin (Descamps et al., Hum. Gene Ther. 5:979-985, 1994; omithine transcarbamylase (Stratford-Perricaudet et al., Hum. Gene Ther. 1:241-256, 1990; We et al., J. Biol. Chem. 271:3639-3646, 1996); adenosine deaminase (Mitani et al., Hum. Gene Ther. 5:941-948, 1994); interleukin-2 (Haddada et al., Hum. Gene Ther. 4:703-711, 1993); and .alpha.1-antitrypsin (Jaffe et al., Nature Genetics 1:372-378, 1992); thrombopoictin (Ohwada et al., Blood 88:778-784, 1996); and cytosine deaminase (Ohwada et al., Hum. Gene Ther. 7:1567-1576, 1996).

The tropism of adenoviruses for cells of the respiratory tract has particular relevance to the use of adenovirus in Gene Ther. for cystic fibrosis (CF), which is the most common autosomal recessive disease in Caucasians. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that disturb the cAMP-regulated C1.about. channel in airway epithelia result in pulmonary dysfunction (Zabner et al., Nature Genetics 6:75-83, 1994). Adenoviral vectors engineered to carry the CFTR gene have been developed (Rich et al., Hum. Gene Ther. 4:461-476, 1993) and studies have shown the ability of these vectors to deliver CFTR to nasal epithelia of CF patients (Zabner et al., Cell 75:207-216, 1993), the airway epithelia of cotton rats and primates (Zabner et al., Nature Genetics 6:L75-83, 1994), and the respiratory epithelium of CF patients (Crystal et al., Nature Genetics 8:42-51, 1994). Recent studies have shown that administering an adenoviral vector containing a DNA sequence encoding CFTR to airway epithelial cells of CF patients can restore a functioning chloride ion channel in the treated epithelial cells (Zabner et al., J. Clin. Invest. 97:1504-1511, 1996).

Modifications to the adenovirus genomic sequences contained in the recombinant vector have been attempted in order to decrease the host immune response (Yang et al., Nature Genetics 7:362-369, 1994; Lieber et al., J. Virol. 70:8944-8960, 1996; Gorziglia et al., J. Virol. 70:4173-4178; Kochanek et al., Proc. Natl. Acad. Sci. USA 93:5731-5736, 1996; Fisher et al., Virology 217:11-22, 1996).

In addition to deletions in the adenovirus El region, first-generation adenoviral vectors often contain modifications to the E3 region in order to increase the packaging capacity of the vectors and to reduce viral gene expression (Yang et al., J. Virol. 69:2004-2015, 1995; Zsengeller et al., Hum. Gene Ther. 6:457-467, 1995; Brody et al., Hum. Gene Ther. 5:821-836, 1994). However, the adenovirus E3 regions contains certain proteins which modulate the host's antiviral immune response. The E3 transcription unit encodes the 12.5K, 6.7K, gp19K, 11.6K, 10.4K, 14.5K and 14.7K proteins (Wold et al., Trends Microbiol. 2:437-443, 1994). The E3 14.7K, 14.5K, and 10.4K proteins are able to protect infected cells from TNF-induced cytolysis. The adenovirus E3 gpl9K protein can complex with MHC Class 1 antigens and retain them in the endoplasmic reticulum, which prevents cell surface presentation and killing of infected cells by cytotoxic T-lymphocytes (CTLs) (Wold et al., Trends Microbiol. 437-443, 1994), suggesting that its presence in a recombinant adenoviral vector may be beneficial. The E3 11.6K gene (Adenovirus death protein) is required for cell lysis and the release of adenovirus from infected cells (Tollefson et al., J. Virol. 70:2296-2306, 1996; Tollefson et al., Virology 220:152-162, 1996).

Earlier designs of adenoviral vectors in which the E3 region was modified have shown only transient expression of a transgene in the lungs of test animals (Yang et al., J. Virol. 69:2004-2015; Zsengeller et al., Hum Gene Ther. 6:457-467, 1995).

Modifications to the adenovirus E4 region have been introduced into adenoviral vectors in order to reduce viral gene expression and to further increase carrying capacity (Armentano et al., Hum. Gene Ther. 6:1343-1353, 1995). However, experiments in which adenoviral vectors were introduced into nude mice demonstrated that the context of the adenovirus E4 genomic region was a determinant in the persistence of expression, especially when the CMV promoter was used to control expression of the transgene (Kaplan et al., Hum. Gene Ther. 8:45-56, 1997; Armentano et al., J. Virol. 71:2408-2416, 1997).

The current state of adenoviral vector as well as viral vector based gene delivery requires the development of novel adenoviral vectors which demonstrate a capability for persistence and sustained expression of a transgene.

SUMMARY OF THE INVENTION

The following invention involves methods and strategies for improving efficiency of gene transfer of vectors. According to the invention, it has been discovered that a synergistic combination of cotranscomplementary replication defective vectors achieves gene transfer at increased levels as high as 13 fold.

The invention includes a viral vector method and composition comprising two transcomplementary replication incompetent adenoviral vectors which are transduced to a recipient cell. The two vectors complement each other in trans and thus allow viral replication, in a synergistic combination which enhances both gene delivery and gene expression of genetic sequences contain within the vector. The combination of the two vectors in vivo, demonstrated tumor reduction in mice as high as 100%. Any replication incompetent viral vector may be used according to the invention including but not limited to retroviral vectors, adenoviral vectors, adeno-associated viral vectors, lentivirus vectors (human and other including porcine), Herpes virus vectors, Epstein-Ban virus vectors, SV40 virus vectors, pox virus vectors, pseudotype virus vectors.

A first vector is engineered and/or introduced to be replication deficient. A second vector is also engineered and/or introduced which will complement in trans the first vector providing for viral replication after transduction in recipient cells.

The invention comprises a transformation composition comprising a mixture of the two or more vectors, methods of transformation using this combination, and genetic engineering protocols which take advantage of this high gene transfer and expression efficiency to transform recipient host cells with exogenous nucleotide sequences.

General transformation techniques including construction and use of vectors, are all known to those of skill in the art, are generally described herein, and are also described in the references disclosed and incorporated herein.

DETAILED DESCRIPTION OF THE INVENTION

The following is a nonlimiting description of the adenovirus genome which may assist in designing the strategies and vectors of the invention. The Adenovirus genome is functionally divided into 2 major non-contiguous overlapping regions, early and late, based on the time of transcription after infection. The early regions are defined as those that are transcribed before the onset of viral DNA synthesis. The switch from early to late gene expression takes place about 7 hours after infection. The terms early and late are not to be taken too literally as some early regions are still transcribed after DNA synthesis has begun.

There are 6 distinct early regions; E1a, E1b, E2a, E2b, E3, and E4, each (except for the E2a-b region) with individual promoters, and one late region, which is under the control of the major late promoter, with 5 well characterized coding units (L1-L5). There are also other minor intermediate and/or late transcriptional regions that are less well characterized, including the region encoding the viral-associated (VA) RNAs. Each early and late region appears to contain a cassette of genes coding for polypeptides with related functions. Each region is transcribed initially as a single RNA which is then spliced into the mature mRNAs. More than 30 different mature RNA transcripts have been identified in Ad2, one of the most studied serotypes.

Once the viral DNA is inside the nucleus, transcription is initiated from the viral E1a promoter. This is the only viral region that must be transcribed without the aid of viral encoded trans-activators. There are other regions that are also transcribed immediately after cell infection but to a lesser extent, suggesting that the E1 region is not the only region capable of being transcribed without viral-encoded transcription factors. The E1a region codes for more than six polypeptides. One of the polypeptides from this region, a 51 kd protein, transactivates transcription of the other early regions and amplifies viral gene expression. The E1b region codes for three polypeptides. The large E1b protein (55 kd), in association with the E4 34 kd protein, forms a nuclear complex and quickly halts cellular protein synthesis during lytic infections. This 55 kd polypeptide also interacts with p53 and directly inhibits its function. A 19 kd transactivating protein encoded by the E1B region is essential to transform primary cultures. The oncogenicity of Ads in new-born rodents requires the E1 region. Similarly, when the E1 region is transfected into primary cell cultures, cell transformation occurs. Only the E1a region gene product is needed to immortalize cell cultures.

The E2a and E2b regions code for proteins directly involved in replication, i.e., the viral DNA polymerase, the pre-terminal protein and DNA binding proteins. In the E3 region, the 9 predicted proteins are not required for Ad replication in cultured cells. Of the 6 identified proteins, 4 partially characterized ones are involved in counteracting the immune system; a 19 kd glycoprotein, gp19 k, prevents cytolysis by cytotoxic T lymphocytes (CTL); and a 14.7 kd and a 10.4 kd/14.5 kd complex prevent, by different methods, E1a induced tumor necrosis factor cytolysis. The E4 region appears to contain a cassette of genes whose products act to shutdown endogenous host gene expression and upregulate transcription from the E2 and late regions. Once viral DNA synthesis begins, the late genes, coding mainly for proteins involved in the structure and assembly of the virus particle, are expressed.

The invention comprises the use of transcomplementary replication incompetent vectors, preferably adenoviral vectors. The two variants are replication deficient individually (in cis) but replication competent when combined in cells via transcomplementation. The system of the invention results in increased vector spread when introduced to tumor or other recipient cells. Any viral vector can be used according to the invention so long as two or more replication incompetent vectors are provided which, in trans are replication competent. One or both of the vectors are engineered to contain a nucleotide sequence the expression of which is desired in a host cell. The nucleotide sequence can be any sequence such as for example a therapeutic gene might be a tumor suppressor gene or a suicide gene. In one embodiment an adenoviral E4 deleted vector and an E1A/B deleted vector are introduced to recipient cells.

The compositions and methods of increasing gene transfer efficiency of viral vectors of the invention can be engineered by any of a number of techniques known to those of skill in the art of can be purchased, as many replication incompetent adenoviral vectors are commercially available and may be engineered to contain the exogenous nucleotide sequence of choice. It is to be understood that based upon the teachings herein, other viral vectors may be used in the transcomplementation scheme of the invention and are intended to be within the scope of the invention. The following is a summary of techniques for construction and transformation of the compositions and methods of the invention.

Genetic Engineering Techniques for Construction and Delivery of Vectors

A therapeutic gene to be expressed can then be introduced into the vector of the invention. The foreign DNA can comprise an entire transcription unit or expression cassette, promoter-gene-poly A or the vector can be engineered to contain promoter/transcription termination sequences such that only the gene of interest need be inserted. These types of control sequences are known in the art and include promoters for transcription initiation, optionally with an operator along with ribosome binding site sequences. Examples of such systems include beta-lactase (penicillinase) and lactose promoter systems, (Chang et al., Nature, 1977, 198:1056); the Tryptophan (trp) promoter system (Goeddel, et al., Nucleic Acid Res., 1980, 8:4057) and the lambda derived Pl promoter and N-gene ribosome binding site (Shimatake et al., Nature 1981, 292:128). Other promoters such as cytomegalovirus promoter or Rous Sarcoma Virus can be used in combination with various ribosome elements such as SV40 poly A. The promoter can be any promoter known in the art including constitutive, (supra) inducible, (tetracycline-controlled transactivator (tTA)-responsive promoter (tet system, Paulus, W. et al., "Self-Contained, Tetracycline-Regulated Retroviral Vector System for Gene Delivery to Mammalian Cells", J of Virology, January 1996, Vol. 70, No. 1, pp. 62-67)),or tissue specific, (such as those cited in Costa, et. Al., European journal of Biochemistry, 258 "Transcriptional Regulation Of The Tissue-Type Plasminogen Activator Gene In Human Endothelial Cells: Identification Of Nuclear Factors That Recognize Functional Elements In The Tissue-Type Plasminogen Activator Gene Promoter" pgs, 123-131 (1998); Fleischmann, M., et. al., FEBS Letters 440 "Cardiac Specific Expression Of The Green Fluorescent Protein During Early Murine Embryonic Development" pgs. 370-376, (1998); Fassati, Ariberto, et. Al., Human Gene Therapy, (9:2459-2468) "Insertion Of Two Independent Enhancers In The Long Terminal Repeat Of A Self Inactivating Vector Results In High-Titer Retroviral Vectors With Tissue Specific Expression" (1998); Valerie, Jerome, et. Al. Human Gene Therapy 9:2653-2659, "Tissue Specific Cell Cycle Regulated Chimeric Transcription Factors For The Targeting Of Gene Expression To Tumor Cells, (1998); Takehito, Igarashi, et. Al., Human Gene Therapy 9:2691-2698, "A Novel Strategy Of Cell Targeting Based On Tissue-Specific Expression Of The Ecotropic Retrovirus Receptor Gene", 1998; Lidberg, Ulf et.al. The Journal of Biological Chemistry 273, No.47, "Transcriptional Regulation Of The Human Carboxyl Ester Lipase Gene In Exocrine Pancreas" 1998; Yu, Geng-Sheng et. Al., The Journal of Biological Chemistry 273 No. 49, "Co-Regulation Of Tissue-Specific Alternative Human Carnitine Palmitoyltransferase IB Gene Promoters By Fatty Acid Enzyme Substrate" (1998)). These types of sequences are well known in the art and are commercially available through several sources, ATCC, Pharmacia, Invitrogen, Stratagene, Promega.

In a preferred embodiment the expression vehicles or vectors of the invention comprising the expression system also comprise a selectable marker gene to select for transformants as well as a method for selecting those transformants for propagation of the construct in bacteria. Such selectable marker may contain an antibiotic resistance gene, such as those that confer resistance to ampicillin, kanamycin, tetracycline, or streptomycin and the like. These can include genes from prokaryotic or eukaryotic cells such as dihydrofolate reductase or multi-drug resistance I gene, hygromycin B resistance that provide for positive selection. Any type of positive selector marker can be used such as neomycin or Zeosyn and these types of selectors are generally known in the art. Several procedures for insertion and deletion of genes are known to those of skill in the art and are disclosed. For example in Maniantis, "Molecular Cloning", Cold Spring Harbor Press. See also Post et al., Cell, Vol. 24:555-565 (1981). An entire expression system must be provided for the selectable marker genes and the genes must be flanked on one end or the other with promoter regulatory region and on the other with transcription termination signal (polyadenylation cite). Any known promoter/transcription termination combination can be used with the selectable marker genes. For example SV40 promoter and SV40 poly A.

In a most preferred embodiment the vector comprises a specifically engineered multi-cloning site within which several unique restriction sites are created. Restriction enzymes and their cleavage sites are well known to those of skill in the art.

Any of a number of standard gene delivery transformation methods can be used with the viral vectors created according to the invention including lipid mediated transfection, receptor mediated transfection, calcium phosphate transfection, electroporation particle bombardment, naked-direct DNA injection, diethylaminoethyl (DEAE-dextran transfection).

In a preferred embodiment, the viral vector is a retroviral vector. Examples of retroviral vectors which may be employed include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus.

The vectors of the invention are useful as agents to mediate viral-mediated gene transfer into eukaryotic cells. The replication incompetent vectors are constructed such that the majority of sequences coding for the structural genes of the virus are deleted and replaced by the therapeutic gene(s) of interest. Most often, the structural genes are removed using genetic engineering techniques known in the art. This may include digestion with the appropriate restriction endonuclease or, in some instances, with Bal 31 exonuclease to generate fragments containing appropriate portions of the vector.

Then new genes may be incorporated into the proviral backbone in several general ways. The most straightforward constructions are ones in which the structural genes of the retrovirus are replaced by a single gene which then is transcribed under the control of the viral regulatory sequences. Vectors have also been constructed which can introduce more than one gene into target cells. Usually, in such vectors one gene is under the regulatory control of the viral LTR, while the second gene is expressed either off a spliced message or is under the regulation of its own, internal promoter.

In yet another embodiment the vector comprises a Herpes Simplex Virus plasmid vector. Herpes simplex virus type-1 (HSV-1) has been demonstrated as a potential useful gene delivery vector system for gene therapy, Glorioso, J. C., "Development of Herpes Simplex Virus Vectors for Gene Transfer to the Central Nervous System. Gene Therapeutics: Methods and Applications of Direct Gene Transfer", Jon A. Wolff, Editor, 1994 Birkhauser Boston, 281-302; Kennedy, P. G., "The Use of Herpes Simplex Virus Vectors for Gene Therapy in Neurological Diseases", Q J Med, November 1993, 86(11):697-702; Latchman, D. S., "Herpes Simplex Virus Vectors for Gene Therapy", Mol Biotechnol, October 1994, 2(2):179-95.

HSV-1 vectors have been used for transfer of genes to muscle. Huard, J., "Herpes Simplex Virus Type 1 Vector Mediated Gene Transfer to Muscle", Gene Therapy, 1995, 2, 385-392; and brain, Kaplitt, M. G., "Preproenkephalin Promoter Yields Region-Specific and Long-Term Expression in Adult Brain After Direct In Vivo Gene Transfer Via a Defective Herpes Simplex Viral Vector", Proc Natl Acad Sci USA, Sep. 13, 1994, 91(19):8979-83, and have been used for murine brain tumor treatment, Boviatsis, E. J., "Long-Term Survival of Rats Harboring Brain Neoplasms Treated With Ganciclovir and a Herpes Simplex Virus Vector That Retains an Intact Thymidine Kinase Gene", Cancer Res, Nov. 15, 1994, 54(22):5745-51; Mineta, T., "Treatment of Malignant Gliomas Using Ganciclovir-Hypersensitive, Ribonucleotide Reductase-Deficient Herpes Simplex Viral Mutant", Cancer Res, Aug. 1, 1994, 54(15):3963-6.

Replication incompetent mini-viral vectors have been developed for easier operation and their capacity for larger insertion (up to 140 kb), Geller, Al, "An Efficient Deletion Mutant Packaging System for Defective Herpes Simplex Virus Vectors: Potential Applications to Human Gene Therapy and Neuronal Physiology", Proc Natl Acad Sci USA, November 1990, 87(22):8950-4; Frenkel, N., "The Herpes Simplex Virus Amplicon: A Versatile Defective Virus Vector", Gene Therapy. 1. Supplement 1, 1994. Replication incompetent HSV amplicons have been constructed in the art, one example is the pHSVlac vector by Geller et al, Science, 241, September 1988, incorporated herein by reference. These HSV amplicons contain large deletions of the HSV genome to provide space for insertion of exogenous DNA. Typically they comprise the HSV-1 packaging site, the HSV-1 "ori S" replication site and the IE 4/5 promoter sequence.

The expression system delivery composition of the present invention can be used for any diagnostic or therapeutic genetic engineering protocol including in vitro, ex vivo, or in vivo expression of a desired nucleotide sequence. For example the expression vehicles of the invention can be used in any of a number of therapeutic treatment protocols in the treatment of cancer such as by the Herpes simplex virus, thymidine kinase gene transfer system Martuza RL et al., "Experimental therapy of human glioma by means of a genetically engineered virus mutant", Science, 1991; 252:854-856). Also in ex vivo gene therapy protocols such as bone marrow purging (Seth P., et al., "Adenovirus-mediated gene transfer to human breast tumor cells: an approach for cancer gene therapy and bone marrow purging", Cancer Res. 56(6):1346-1351 (1996; Andersen, N. S., et al., "Failure of immunologic purging in mantle cell lymphoma assessed by polymerase chain reaction detection of minimal residual disease", Blood, 90(10):4212-4221 (1997)) thus when the transformed cells are reintroduced to the patient they will generate a decreased immune response. These may also be used for diagnostic purposes as well.

To fully exploit the benefits of the methods and compositions described herein, the use of many general gene therapy improvements are contemplated and are intended to be within the scope of this invention. In this manner, improvements as higher viral titer production, selection of therapeutic gene and promotor enhancer elements will be utilized, and are intended to be within the scope of the invention. These improvements are seen as simply characterized through routine experimentation and are intended to be within the scope of this invention.

Claim 1 of 8 Claims

What is claimed is:

1. A method for increasing gene transfer to recipient cells comprising:

introducing to said recipient cell a first replication incompetent adenoviral vector having an E1 deletion, and a second replication incompetent adenoviral vector having an E4 deletion, wherein one or both of said vectors comprise a nucleotide sequence the expression of which is desired in said recipient cell, wherein said first and second adenoviral vectors are transcomplementary, so that upon cotransduction viral replication is enabled, wherein each vector is produced independently of each other in separate trans-complementing packing cell lines.

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