Production enhancement of gene transfer vector of adenovirus base

Abstract

To provide a method for propagating a gene transfer vector of a completely deleted adenovirus base which delivers a gene harmful or toxic to an eukaryotic cell which is packaged without a helper virus.SOLUTION: A method includes: (a) providing an adenovirus packaging cell strain; (b) transfecting a completely deleted adenovirus vector module including at least one or more DNA insert, which includes both adenovirus inverted terminal repeats, a package signal and a gene sequence encoding target protein but does not include an adenovirus structural gene, into a cell strain by a construct of the completely deleted adenovirus vector module; and (c) transfecting a subset of an adenovirus late gene including L1, L2, L3, L4, L5, E2A and E4 and a replication-deficient cyclic package expression plasmid having the package signal into the cell strain.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Embodiments disclosed herein relate to the production of fully deleted adenovirus-based vectors that deliver harmful or toxic genes to eukaryotic cells packaged without a helper adenovirus, and more particularly to their use in gene therapy for gene and protein expression, vaccination, and cell and tissue modification. [Background technology]

[0002] Among the most commonly used vectors for the delivery of genetic material into human cells are the adenoviruses. Adenoviruses have been isolated from a number of different species, with over 100 different serotypes reported. The overall organization of the adenovirus genome is conserved between serotypes, such that certain functions are similarly located. Adenoviruses of different serotypes have been fully sequenced, and their genome sequences are publicly available. Many adults have been exposed to human adenoviruses of serotype 5 (Ad5), which have formed the basis of many gene transfer vectors.

[0003] The Ad5 genome is a linear, non-segmented, double-stranded DNA of approximately 35 kbp (size varies from group to group) with a theoretical capacity to code for 30-40 genes. The Ad5 genome is flanked on both sides by inverted terminal repeats (LITR and RITR) that are essential for adenovirus replication. The infectious cycle of adenoviruses such as Ad5 is divided into early and late stages. In the early stage, the virus is uncoated and the genome is transported to the nucleus, after which the early gene regions (E), E1, E2, E3 and E4, become transcriptionally active. E1 contains two regions, called E1A and E1 B. The E1A region (sometimes called the immediate early region) encodes two major proteins involved in the modification of the host cell cycle and the activation of other viral transcriptional regions. The E1 B region encodes two major proteins, 19K and 55K, that prevent the induction of apoptosis due to the activity of the EA protein, through different pathways. In addition, the E1 B-55K protein is required at a later stage for selective viral mRNA transport and inhibition of host protein expression. E2 is also divided into the E2A and E2B regions which together code for three proteins: the DNA binding protein, the viral polymerase and the proterminal protein, all involved in the replication of the viral genome. The E3 region codes for several proteins that are not required for replication in vitro but that subvert host defense mechanisms against viral infection. The E4 region codes for at least six proteins involved in several different functions related to splicing and transport of viral mRNA, transport of host cell mRNA, viral and cellular transcription and transformation.

[0004] All late proteins required for viral capsid formation and viral genome packaging are generated from a major late transcription unit that becomes fully active after the onset of viral DNA replication. A complex process of alternative splicing and polyadenylation generates more than 15 mRNA species that share three separate leader sequences. The early proteins E1 B-55K and E4-Orf3 and Orf6 play a central role in regulating late mRNA processing and export from the nucleus.

[0005] Packaging of the newly formed adenoviral genome in preformed capsids is mediated by at least two adenoviral proteins, late 52 / 55k and intermediate protein 1Va2, through interaction with the viral packaging signal (Ψ) located at the left end of the Ad5 genome. A second intermediate protein, pIX, is part of the capsid and is known to stabilize hexon-hexon interactions. In addition, pIX has been described to transactivate TATA-containing promoters such as the EIA promoter and the major late promoter (MLP).

[0006] Adenovirus-based vectors and adenovirus packaging cell lines Adenovirus-based vectors have been used as a means to achieve high levels of gene transfer into various cell types, as vaccine delivery vehicles, for gene transfer into tissue transplants, for gene therapy, and as a means to express recombinant proteins in cell lines and tissues that are otherwise difficult to transfect with high efficiency. Current systems for packaging adenovirus-based vectors consist of a host cell and a source of adenovirus late genes. Currently known host cell lines, including 293, OBI and PERC.6 cells, express only the early (non-structural) adenovirus genes, not the late adenovirus (structural) genes required for packaging. The adenovirus late genes have previously been provided by the adenovirus vector itself in cis or by a helper adenovirus virus in trans. Adenovirus vectors that provide the genes required for their encapsidation themselves carry a minimally modified adenovirus genome, deleted primarily in genes for E1, and occasionally also in E3 and other adenovirus regions.

[0007] More recently, "gutless" adenoviral vectors have been developed, i.e., vectors lacking DNA sequences encoding all viral proteins. Gutless adenoviral vectors contain only the ends of the viral genome (LITR and RITR), a gene of interest, such as a therapeutic gene, and a normal packaging recognition signal (Ψ) that allows selective packaging of this genome. However, in order to propagate gutless adenoviral vectors, a helper adenovirus is required, which contains the adenoviral genes necessary for replication and virion assembly, as well as the LITR, RITR, and Ψ. This helper virus-dependent system allows the introduction of up to about 35 kb of foreign DNA, but the helper virus contaminates preparations of gutless adenoviral vectors using this approach. Contamination with replication-competent helper viruses poses serious problems for gene therapy, vaccine, and transplantation applications due to the replication-competent virus and due to the host immune response to the adenoviral genes in the helper virus. One approach to reduce helper contamination in this helper virus-dependent vector system has been to introduce a conditional gene defect in the package recognition signal (Ψ) to make its DNA less likely to be packaged into virions. The gutless adenovirus vector produced in such a system still has significant contamination by helper virus. The ability to produce gutless adenovirus gene transfer vectors without helper virus contamination eliminates helper virus contamination, resulting in reduced vector toxicity and extended gene expression in human subjects and animals.

[0008] Adenoviral genes, especially adenoviral late genes carried by minimally modified adenoviral vectors or adenoviral helper viruses, are believed to 1) contribute to the inflammatory response seen after adenoviral-mediated gene therapy, 2) reduce the immune response to the gene of interest in vaccine applications, 3) interfere with normal cell function, and 4) result in protein contamination in protein expression applications. Moreover, they occupy space in minimally modified adenoviral vectors that could be beneficially used to carry other genetic information. Although adenoviral vectors have made significant progress in the past decade, significant drawbacks still make their use difficult.

[0009] Adenoviral Vectors for Gene Therapy and Protein Expression Gene delivery or gene therapy is a promising method for the treatment of acquired and inherited diseases. An ever-growing array of aberrantly expressed genes associated with life-threatening human diseases are being cloned and identified. The ability to express such cloned genes in humans will ultimately allow the prevention and / or cure of many important human diseases, diseases for which current treatments are inadequate or non-existent.

[0010] Unfortunately, however, the gene therapy protocols described thus far suffer from a variety of problems, including, inter alia, the short-term gene expression from the vector and the inability to effectively re-administer the same vector a second time, both of which may be caused by the host immune response to antigens associated with the vector and its therapeutic payload. Tissues that incorporate the virus and / or therapeutic gene are initially attacked by the host cellular immune response mediated by CD8+ cytotoxic T cells as well as CD4+ helper T cells, which dramatically limits the persistence of gene expression from the vector. Furthermore, the host humoral immune response mediated by CD4+ T cells further limits the effectiveness of current gene therapy protocols by inhibiting successful re-administration of the same vector.

[0011] For example, after initial administration of an adenoviral vector, serotype-specific antibodies are generated against epitopes of the major viral capsid proteins, namely penton, hexon and fiber. Given that such capsid proteins are the means by which adenovirus attaches itself to and subsequently infects cells, such antibodies can block or "neutralize" reinfection of cells with the same serotype of adenovirus or adenoviral vector. In the context of gene therapy and vaccines, this may require the use of different serotypes of adenovirus to administer one or multiple subsequent doses of exogenous therapeutic DNA. Furthermore, when using minimally modified adenoviral vectors or adenoviral helper virus-contaminated adenoviral vector preparations, both therapeutic and viral gene products are expressed on the target cells. These antigens can be recognized by a cellular immune response that leads to the destruction of the transduced cells or tissues, and thus the beneficial effects of gene therapy and vaccination may be negated. As a result of these immune-related obstacles, the widespread use of minimally modified viral vectors has been hindered.

[0012] At least 53 different forms of human adenoviruses, as well as numerous animal adenoviruses, have been characterized. The main distinguishing factor between these viruses is the humoral immune (i.e., antibody) response to the capsid hexon protein (encoded by various alleles of the L3 gene). Indeed, most of the variation between the different hexon proteins occurs in three "hyper" variable regions, and the humoral immune response to adenoviruses is centered in these hypervariable regions. Other structures, such as the fiber protein on the adenovirus surface, can also be recognized by the humoral immune system. Thus, interference with the humoral immune response by the activity of minimally modified adenoviral vectors can be mitigated by switching the adenoviral serotype between each application. Late adenoviral genes are less variable, and therefore the T cell responses induced by minimally modified adenoviral vectors or adenoviral helper viruses cannot be circumvented by switching the adenoviral serotype of the vector.

[0013] The human population is exposed to natural adenovirus infections of certain adenovirus serotypes. These subjects therefore carry humoral and cellular immune response-directing genes expressed by these adenoviruses and adenovirus vectors based on these serotypes of adenovirus. Two advances have attempted to overcome the problem: the use of "gutless" (completely deleted) adenovirus vectors, and the use of adenovirus vectors based on rare or animal adenoviruses expressing rare or animal serotypes. The use of "gutless" adenovirus vectors removes adenovirus genes, such as L3, from the therapeutic vector, but the propagation of these "gutless" adenovirus vectors still requires the presence of a helper adenovirus carrying the adenovirus genes. These helper viruses are a significant contaminant in the preparation of "gutless" adenovirus vectors. The use of minimally modified adenoviral vectors based on rare or animal serotypes can avoid the problem of pre-existing humoral immunity and perhaps to a lesser extent pre-existing cellular immunity in subjects who have been previously exposed to adenovirus of a given serotype.Nevertheless, minimally modified adenoviral vectors express adenoviral genes, including highly immunogenic L3, and can induce strong humoral and cellular immune responses against these adenoviral genes.Therefore, repeated application of minimally modified adenoviral vectors of a given serotype is not possible.

[0014] Adenoviruses as vaccine vectors Adenovirus vectors are transitioning from tools for gene replacement therapy to bona fide vaccine delivery vehicles. They are attractive vaccine vectors because they induce both innate and adaptive immune responses in mammalian hosts. Adenovirus vectors have been tested for delivery as subunit vaccine systems for a number of infectious diseases, such as malaria, tuberculosis, Ebola, and HIV-1. In addition, they have been explored as vaccines against different tumor-associated antigens. So far, most vaccines vectored based on adenovirus have been constructed as minimally modified adenovirus vectors of human and animal serotypes.

[0015] The kinetics of adenovirus gene expression has made it difficult to design adenovirus packaging systems; expression of the adenovirus early transcriptional region (E1A) gene induces expression of the adenovirus late genes (structural, immunogenic genes), which kill the cell.

[0016] Thus, host cells that constitutively express adenovirus early genes cannot carry the "wild-type" adenovirus late cistron. Previous host cells for propagating adenovirus vectors are not bona fide "packaging" cells. Specifically, 293, QBI and PERC 6 cells express only the early (non-structural) adenovirus genes, not the adenovirus late genes required for packaging. The adenovirus late and early genes must be provided. They have been previously provided by a minimally modified adenovirus vector in cis or by a helper adenovirus in trans.

[0017] Adenoviral genes found in minimally modified adenoviral vectors or contaminating helper adenoviruses contribute to inflammatory and immune responses to adenoviral vector preparations, reduce immune responses to the gene of interest of adenovirus-based vaccines, interfere with normal cellular function, and contribute to contamination in adenovirus-based protein expression.

[0018] It may be beneficial to use strong and broadly active promoters, such as viral promoters, for the expression of the gene of interest. This may be necessary to ensure effective gene therapy or strong immunogenicity. However, gene transfer vectors may carry genes of interest that are harmful or toxic to the host cells used for packaging the adenoviral vector. Their expression may therefore interfere with efficient encapsidation of the adenoviral vector, both minimally modified and "gutted" adenoviral vectors. These genes may be toxic or harmful by design, for example to eliminate cancerous cells after transduction, or may constitute bacterial or viral genes that are harmful or toxic to the host cells at high concentrations but are able to elicit a protective immune response, such as the Ebola glycoprotein. It may therefore be necessary to downregulate the expression of the gene of interest during packaging. The described invention addresses this problem. The described invention provides systems and methods for producing minimally modified or "gutted" (completely deleted) adenoviral vectors without the involvement of adenoviral helper viruses carrying genes of interest whose functions are deleterious or toxic to the host cells used to encapsidate these vectors. Uses of such vectors are described. Summary of the Invention

[0019] The embodiments disclosed herein relate to the construction and production of fully deleted adenoviral vectors packaged with helper viruses. They also relate to the production of minimally modified and fully deleted adenoviral vectors carrying genes of interest with functions and activities that are harmful or toxic to the host cells used to produce or encapsidate these vectors. They relate to these vectors being used in gene therapy, vaccination, cancer therapy and immunosuppressive therapy.

[0020] According to the embodiment exemplified herein, a system is provided that includes: (a) an adenovirus host cell for packaging an adenovirus vector; (b) a completely deleted adenovirus vector module construct; (c) a packaging expression plasmid carrying a gene capable of encapsidating the adenovirus vector module into an adenovirus capsid; (d) an expression construct incorporated in the packaging expression plasmid that can inhibit the expression of a gene of interest on a separate expression vector or on the adenovirus vector module; and (e) alternatively, a set of short inhibitory RNA or DNA fragments that can bind to and inhibit the expression of a gene of interest on the adenovirus vector module. Either the adenovirus vector module, the minimally modified or the completely deleted adenovirus vector, is optionally co-transfected with the packaging expression plasmid, the expression construct, or the set of RNA or DNA fragments that can inhibit the expression of a gene of interest on the adenovirus vector module.

[0021] The packaging expression vector and expression construct that inhibits the gene of interest cannot itself be encapsidated. The adenoviral vector module itself cannot replicate. According to an embodiment exemplified herein, a method for the propagation of an adenoviral vector carrying a harmful or toxic gene comprises: (a) providing an adenoviral packaging cell; (b) transfecting an adenoviral vector module deleted of all adenoviral genes into a packaging cell line; (c) transfecting a packaging expression plasmid into the packaging cell, where the fully deleted adenoviral vector module and the packaging expression plasmid transfect the packaging cell, resulting in encapsidation of the fully deleted adenoviral vector module in the adenoviral capsid independent of the helper virus; and (d) transfecting an inhibitory expression plasmid into the packaging cell, encoding expression of an antisense RNA of the gene of interest found on the fully deleted adenoviral vector module, where the gene of interest on the fully deleted adenoviral vector module is encapsidated in the adenoviral capsid independent of the helper virus. (e) transfecting a packaging cell with the fully deleted adenoviral vector module, a packaging expression plasmid and an inhibitory expression plasmid to inhibit expression of a gene of interest during vector encapsidation and improve packaging of the complete adenoviral vector module into an adenoviral capsid; (f) transfecting a short inhibitory RNA or DNA fragment that binds to a gene of interest found on the fully deleted adenoviral vector module, whereby expression of the gene of interest on the fully deleted vector module is inhibited; and (g) transfecting a packaging cell with the fully deleted adenoviral vector module, a packaging expression plasmid and the short inhibitory RNA or DNA fragment to inhibit expression of a gene of interest during vector encapsidation and improve packaging of the complete adenoviral vector module into an adenoviral capsid.

[0022] According to an embodiment exemplified herein, a method for the propagation of an adenoviral vector carrying a harmful or toxic gene is provided, comprising: (a) providing an adenoviral packaging cell; (b) introducing a minimally modified adenoviral vector into the packaging cell; (c) transfecting the packaging cell with an inhibitory expression plasmid encoding expression of an antisense RNA of a gene of interest found on a fully deleted adenoviral vector module, whereby expression of the gene of interest on the fully deleted adenoviral vector module is inhibited; and (d) transfecting the fully deleted adenoviral vector module, the packaging expression plasmid, and the inhibitory expression plasmid into the packaging cell to produce an antisense RNA of the gene of interest. Disclosed are methods that include inhibiting expression of a gene of interest during encapsidation and improving packaging of a complete adenoviral vector module into an adenoviral capsid; (e) transfecting a short inhibitory RNA or DNA fragment that binds to a gene of interest found on a fully deleted adenoviral vector module, whereby expression of the gene of interest on the fully deleted vector module is inhibited; and (g) transfecting a packaging cell with the fully deleted adenoviral vector module, a packaging expression plasmid and the short inhibitory RNA or DNA fragment to inhibit expression of the gene of interest during vector encapsidation and improve packaging of the complete adenoviral vector module into an adenoviral capsid.

[0023] In one embodiment, target cells are transduced with encapsidated fully deleted adenoviral vectors for the treatment of a condition, disease or disorder. In one embodiment, the encapsidated fully deleted adenoviral vectors are injected into a human subject such that expression of a gene of interest exerts a therapeutic function or induces an immune response. In one embodiment, the encapsidated fully deleted adenoviral vectors are used to transduce target cells or target tissues to modify their activity or to modify the response of other cellular and tissue components to the target cells or target tissues.

[0024] In some embodiments, the gene transfer vectors of the present disclosure are useful in methods of treating cancer. In some embodiments, the gene transfer vectors of the present disclosure are useful in methods of treating skin disorders. In some embodiments, the gene transfer vectors of the present disclosure are useful in methods of treating vascular disease. In some embodiments, the gene transfer vectors of the present disclosure are useful in methods of treating cardiac disease. In some embodiments, the gene transfer vectors of the present disclosure are useful in methods of treating cardiac disease. In some embodiments, the gene transfer vectors of the present disclosure are useful in methods of treating autoimmune disease. In some embodiments, the gene transfer vectors of the present disclosure are useful in methods of treating parasitic infections. In some embodiments, the gene transfer vectors of the present disclosure are useful in methods of treating viral infections. In some embodiments, the gene transfer vectors of the present disclosure are useful in methods of treating bacterial infections.

[0025] In some embodiments, the gene transfer vectors of the present disclosure are useful in methods of treating yeast infections. In some embodiments, the gene transfer vectors of the present disclosure are useful in methods of treating neurological diseases. In some embodiments, the gene transfer vectors of the present disclosure are useful in methods of treating genetic diseases.

[0026] In some embodiments, the encapsidated adenoviral vectors produced by the methods of the present disclosure are used as gene delivery vectors for protein expression. In some embodiments, the encapsidated adenoviral vectors produced by the methods of the present disclosure are used in vaccine development and production. In some embodiments, the encapsidated adenoviral vectors produced by the methods of the present disclosure are used as gene delivery vectors for immunosuppressive therapy. In one embodiment, target cells are transduced with a previously produced encapsidated adenoviral vector.

[0027] Other objects and features will be in part apparent and in part pointed out hereinafter. [Brief description of the drawings]

[0028] [Figure 1] FIG. 1 is a schematic diagram showing one embodiment of a fully deleted adenoviral vector module design. [Diagram 2] FIG. 1 is a schematic diagram showing one embodiment of the design of the packaged expression plasmid with and without an expression cassette capable of inhibiting the expression of a gene of interest on an adenoviral vector module. [Diagram 3] FIG. 1 is a schematic diagram showing one embodiment of the design of a minimally modified adenoviral vector. [Figure 4] FIG. 1 is a schematic diagram showing one embodiment of the design of an inhibitory expression plasmid carrying one or more expression cassettes capable of inhibiting the expression of one or more genes of interest found on an adenoviral vector module. [Diagram 5] FIG. 1 is a schematic diagram showing one embodiment of a method for encapsidation by co-transfection of a fully deleted adenoviral vector module with a packaging expression plasmid. [Figure 6]FIG. 1 is a schematic diagram showing one embodiment of a method for encapsidation by co-transfection of a fully deleted adenoviral vector module with a packaging plasmid and an expression plasmid that can inhibit expression of a gene of interest on the adenoviral vector module. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The present disclosure provides, inter alia, a fully deleted adenovirus vector module, a packaging expression plasmid and an adenovirus packaging cell for propagating a fully deleted adenovirus-based gene transfer vector packaged without an adenovirus helper virus. The gene transfer vector as a minimally modified fully deleted vector is used in gene and protein expression, vaccine development, cell and tissue engineering and gene therapy for immunosuppression therapy. Any subtype, mixture of subtypes, or chimeric adenovirus can be used as a source of DNA for making adenovirus gene transfer vectors. In one embodiment, the source of DNA is from human serotype 5.

[0030] In the system disclosed herein, the fully deleted adenoviral vector is co-transfected with a packaging expression plasmid. An expression cassette capable of interfering with a gene of interest is found on the packaging expression vector or on a separate co-transfected expression vector. An alternative RNA or DNA fragment that inhibits the expression of a gene of interest on the adenoviral vector module is co-transfected. In another system disclosed herein, a minimally modified adenoviral vector is exposed in the packaging cell to an expression vector that expresses a gene that can interfere with a gene of interest found on the packaging expression vector, or to an RNA or DNA fragment that is inhibitory to the expression of a gene of interest on the adenoviral vector. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In general, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, and nucleic acid chemistry and hybridization described below are well known and commonly used in the art. Standard techniques are used for recombinant nucleic acid methods, polynucleotide synthesis, and microbial culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification steps are generally performed according to manufacturer's specifications. Techniques and procedures are generally performed according to conventional methods in the art and various general references provided throughout this document (see generally Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Laboratory Press, Cold Spring Harbor, NY, which is incorporated herein by reference). Units, prefixes, and symbols may be shown in their SI accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation and amino acid sequences are written left to right in amino to carboxyl orientation, respectively. Numeric ranges are inclusive of the numbers defining the range and include each integer within the defined range.Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their commonly accepted single-letter codes. Unless otherwise specified, software, electrical, and electronic terms used herein are as defined in The New IEEE Standard Dictionary of Electrical and Electronics Terms (5'h edition, 1993). As used throughout this disclosure, the following terms shall be understood to have the following meanings unless otherwise indicated, and are more fully defined by reference to the entire specification. The terms "adenovirus" and "adenovirus particle" as used herein include any and all viruses that can be classified as adenoviruses, including any adenovirus that infects humans or animals, including all groups, subgroups, and serotypes. Thus, as used herein, "adenovirus" and "adenovirus particle" refer to the virus itself or its derivatives, encompassing all serotypes and subtypes, and both naturally occurring and recombinant forms. In one embodiment, such adenovirus infects human cells.

[0031] Such adenoviruses may be wild type or modified in various ways known in the art or disclosed herein. Such modifications include modifications to the adenoviral genome packaged into the particle to generate infectious virus. Such modifications include deletions known in the art, such as deletions in one or more of the Ela, EI b, E2a, E2b, E3 or E4 coding regions. An "adenoviral packaging cell" is a cell capable of packaging an adenoviral genome or a modified genome to produce viral particles. It may provide a defective gene product or its equivalent. Thus, the packaging cell may provide a complementary function for a gene deleted in the adenoviral genome and may package the adenoviral genome into an adenoviral particle. The production of such particles requires that the genome is replicated and that proteins necessary to assemble infectious virus are produced. The particle may also require certain proteins necessary for the maturation of the viral particle. Such proteins may be provided by a minimally modified vector, a packaging expression plasmid, or by the packaging cell. Exemplary host cells that can be used to generate packaging cell lines according to the present invention include, but are not limited to, A549, Hela, MRC5, W138, CHO cells, Vera cells, human embryonic retina cells, human embryonic kidney cells, or any eukaryotic cell, so long as the host cell is permissive for the propagation of adenovirus. Some host cell lines include adipocytes, chondrocytes, epithelial cells, fibroblasts, glioblastomas, hepatocytes, keratinocytes, leukemia, lymphoblastoid cells, monocytes, macrophages, myoblasts, and neuronal cells. Other cell types include, but are not limited to, cells derived from primary cell cultures, such as human primary prostate cells, human embryonic retina cells, and human stem cells. Eukaryotic diploid and aneuploid cell lines are included within the scope of the present invention.The packaging cells must be capable of expressing the products of the adenoviral vector and / or the packaged expression plasmid, as well as the products of the inhibitory expression cassette and / or vector, at appropriate levels of their products in order to generate high titer stocks of the recombinant gene transfer vector.

[0032] By "antigen" is meant a molecule that contains one or more epitopes that stimulate the host's immune system to elicit a cellular antigen-specific immune response or a humoral antibody response. Antigens therefore include proteins, polypeptides, antigenic protein fragments, oligosaccharides, polysaccharides, and the like. Furthermore, antigens can be derived from any known virus, bacteria, parasite, plant, protozoan, or fungus, and can be whole organisms. The term also includes tumor antigens. Similarly, oligonucleotides or polynucleotides that express antigens, for example in DNA immunization applications, are included in the definition of antigen. Also included are synthetic antigens, such as polyepitopes, contiguous epitopes, and other recombinantly or synthetically derived antigens (Bergmann et al. (1993) Eur. J. Immunol. 23:2777-2781; Bergmann et al. (1996) J. Immunol. 157:32423249; Suhrbier, A. (1997) Immunol. and Cell Bioi. 75:402408; Gardner et al. (1998) 12th World AIDS Conference, Geneva, Switzerland, Jun. 28-Jul. 3, 1998).

[0033] A "coding sequence" or a sequence that "encodes" a selected polypeptide is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or "control elements"). The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A transcribed sequence may be located 3' to the coding sequence. Transcription and translation of coding sequences are typically regulated by "control elements" that include, but are not limited to, transcriptional promoters, transcriptional enhancer elements, Shine-Dalgarno sequences, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), sequences for optimizing translation initiation (located 5' to the coding sequence), and translation termination sequences.

[0034] The term "construct" refers to at least one of the fully deleted adenoviral vector modules of the invention, the packaged expression vectors of the invention, or the inhibitory expression cassette located on an expression plasmid. As used herein, the term "delete" or "deleted" refers to removal, erasure, or removal.

[0035] The term "E1 region" as used herein refers to a group of genes present in the adenoviral genome. These genes, including but not limited to E1A and E1B, are expressed in the early stages of viral replication and activate the expression of other viral genes. In one embodiment, the adenoviral packaging cell line of the present disclosure contains all the coding sequences that make up the E1 region.

[0036] In one embodiment, the adenovirus packaging cell line of the present disclosure comprises several coding sequences that constitute the E1 region (e.g., E1A or E1B). As used herein, the term "E1A" refers to all gene products of the adenovirus E1A region, including the expression products of two major RNAs, 13S and 12S. These are translated into polypeptides of 289 and 243 amino acids, respectively. These two proteins differ in 46 amino acids that are spliced ​​out from the 12S mRNA, as described in Chow et al. (1980) Cold Spring Harb Symp Quant Bioi. 44 Pt 1:40114, and Chow et al. (1979) J. Mol. Biol. 134(2):265 303, which are specifically incorporated herein by reference. For purposes of the present invention, the packaging cell line may express the 289 polypeptide, the 243 polypeptide, or both the 289 polypeptide and the 243 polypeptide. The term E1A is also used herein in reference to partial and mutant E1A coding sequences. As used herein, the term "E1 B" refers to all gene products of the adenovirus E1 B region, including the three major polypeptides of 19 kd and 55 kd. The E1 8 19 kd and 55 kd proteins are important in cell transformation. For purposes of the present invention, a packaging cell line may express the 19 Kd polypeptide, the 55 Kd polypeptide, or both the 19 Kd and 55 Kd polypeptides. The term E1 B is also used herein in reference to partial and mutant E1B coding sequences.

[0037] The term "E2" as used herein refers to a cistron with at least three ORFs, all of which are involved in DNA replication, including the polymerase. The adenoviral E2 late promoter is described, for example, in Swaminathan, S., and Thimmapaya, 8. (1995) Gurr. Top. Microbial. Immunol., 199, 177-194. In the adenoviral system, the E2 late promoter, together with the E2 early promoter, functions to control the adenoviral E2 region and / or genes E2A and E2B. In this case, the synthesis of E2 mRNA is first initiated from the E2 early promoter. The switch to the E2 late promoter occurs about 5-7 hours after infection of the cells.

[0038] The term "E3 region" as used herein refers to a group of genes present in the adenovirus genome and expressed in the early stage of the viral replication cycle. These genes express proteins that interact with the host immune system. They are not necessary for viral replication in vitro, and therefore can be deleted in adenovirus vectors.

[0039] The term "E4 region" as used herein refers to a group of genes present in the adenovirus genome next to the right ITR and expressed in the early stage of the viral replication cycle.The E4 region contains at least 7 ORFs.The products of the E4 region promote the expression and replication of viral genes, interact with host cell components, and are involved in lytic infection and tumorigenesis.

[0040] The term "expression" refers to the transcription and / or translation of an endogenous gene, transgene or coding region in a cell. As used herein, a "fully deleted adenovirus", "gutless", "gutted", "minimal", "fully deleted" or "pseudo" vector refers to a linear double-stranded DNA molecule with at least one DNA insert (all or a fragment of at least one gene of interest (GOI)) containing inverted terminal repeats (ITRs), a viral packaging signal (Ψ), and a gene sequence encoding a protein of interest, separated by approximately 26-37 kb. The gene sequence may be regulatable. Regulation of gene expression can be achieved by one of the following: 1) change in gene structure: site-specific recombinase (e.g., Cre based on the Cre-loxP system) can activate gene expression by removing the inserted sequence between the promoter and the gene; 2) change in transcription: by induction (covered) or by relief of inhibition; 3) change in mRNA stability by specific sequences or siRNA incorporated into the mRNA; 4) change in translation by sequences in the mRNA.

[0041] The fully deleted adenoviral vector module does not contain any viral coding genes. The fully deleted adenoviral vector module is also called "high capacity" adenoviral vector because it can accommodate up to 36 kilobases of "foreign" DNA. Because the vector capsid can efficiently package only DNA that is 75-105% the size of the entire adenoviral genome, and therapeutic expression cassettes do not usually total 36 kb, "stuffer" DNA must be used to complete the genome size for encapsidation. Early fully deleted adenoviral vectors require a helper adenovirus carrying the essential adenoviral coding regions, so they are called "helper-dependent" adenoviruses.

[0042] As used herein, the term "gene expression construct" refers to a promoter, at least a fragment of a gene of interest, and a polyadenylation signal sequence. The fully deleted adenoviral vector module of the present disclosure includes a gene expression construct. A "gene of interest" or "GOI" can be one that exerts its effect at the RNA or protein level. Examples of genes of interest include, but are not limited to, therapeutic genes, immunomodulatory genes, viral genes, bacterial genes, protein producing genes, inhibitory RNAs or proteins, genes that encode products that are toxic or harmful to eukaryotic cells or regulatory proteins. For example, proteins encoded by therapeutic genes can be used in the treatment of genetic diseases, such as the use of eDNA encoding the cystic fibrosis transmembrane conductance regulator in the treatment of cystic fibrosis.

[0043] Additionally, a therapeutic gene may exert its effect at the level of RNA, for example by encoding an antisense message or a ribozyme, an siRNA known in the art, an alternative RNA splice acceptor or donor, a protein that affects splicing or 3' processing (e.g., polyadenylation), or a protein that affects the expression level of another gene in a cell (i.e., where gene expression is broadly considered to include all steps from the initiation of transcription to the production of the processed protein), perhaps by mediating changes in the rate of mRNA accumulation, changes in mRNA transport, and / or changes in post-transcriptional regulation, among others.

[0044] As used herein, the phrase "gene therapy" refers to the introduction of genetic material of interest (e.g., DNA or RNA) into a host to treat or prevent an inherited or acquired disease or condition. The genetic material of interest encodes a product (e.g., a protein polypeptide, peptide, or functional RNA) whose production in vivo is desired. For example, the genetic material of interest can encode a hormone, receptor, enzyme, or (poly)peptide of therapeutic value. Examples of genetic material of interest include DNA encoding cystic fibrosis transmembrane regulator (CFTR), factor VIII, low density lipoprotein receptor, beta-galactosidase, alpha-galactosidase, beta-glucocerebrosidase, insulin, parathyroid hormone, and alpha-1-antitrypsin. Another form of "gene therapy" may involve the delivery of "toxic" genes to remove unwanted cells or cell populations from an organism, for example, for the treatment of malignant growth. Examples of such toxic genes include, but are not limited to, bacterial L-methionase, monoclonal antibodies that block important cellular pathways, prodrug converting enzymes, bacterial toxins (botulinum toxin, tetanus toxin, shiga toxin, diphtheria toxin, cholera toxin, diphtheria toxin, anthrax toxin LF, listeriolysin), and plant toxins (ricin).

[0045] By "gene delivery vector" is meant a composition comprising an encapsidated fully deleted adenovirus-based vector of the present disclosure packaged without helper adenovirus.

[0046] The term "helper-independent" as used herein refers to the process for generating encapsidated, fully deleted adenovirus-based vector modules of the present disclosure that do not require the presence of a helper virus for replication and encapsidation.

[0047] Adenoviruses include minimally modified "first generation" and "second generation" adenovirus vectors. First generation adenovirus vectors refer to adenoviruses in which exogenous DNA replaces the E1 region, or optionally the E3 region, or optionally both the E1 and E3 regions. Second generation adenovirus vectors refer to first generation adenovirus vectors that contain, in addition to the E1 and E3 regions, additional deletions in the E2, E4, and any other regions, or combinations thereof, of the adenovirus genome.

[0048] The term "helper virus" as used herein refers to a virus that is used to produce copies of helper-dependent virus vectors that do not have the ability to replicate by themselves.Helper virus is used to co-infect cells with gutless virus, and provides the enzymes required for the replication of gutless virus genome and the structural proteins required for the assembly of gutless virus capsid.

[0049] The term "inhibitory expression cassette" as used herein refers to a DNA construct consisting of a promoter and a polyadenylation site into which a gene sequence capable of inhibiting the expression of a gene of interest located on a minimally modified adenoviral vector or a completely deleted adenoviral vector module is inserted and expressed. This "inhibitory" gene may be homologous to the gene of interest or may be antisense in orientation to the gene of interest. The "inhibitory" gene may code for a product that interferes with the gene of interest RNA in a different manner or may code for a product that interferes with the protein encoded by the gene of interest.

[0050] The terms "inhibitory RNA fragment" and "inhibitory DNA fragment" as used herein refer to RNA and DNA polynucleotides that have the ability to bind to the RNA of a gene of interest and thus interfere with the expression of the gene of interest. These RNA and DNA fragments may be homologous to the gene of interest or may be antisense in orientation to the gene of interest. These RNA and DNA fragments may be homologous to the gene of interest and can cleave the RNA and / or DNA of the gene of interest.

[0051] By "immune response" is preferably meant an adaptive immune response, such as a cellular or humoral immune response. In the present context, an "immunomodulatory molecule" is a polypeptide molecule that modulates, i.e. increases or decreases, the cellular and / or humoral host immune response against a target cell in a general or antigen-specific manner, preferably a polypeptide molecule that decreases the host immune response.

[0052] The term "inverted terminal repeat" as used herein refers to DNA sequences located at the left and right ends of the adenoviral genome. These sequences are identical to each other but are arranged in opposite directions. The length of the adenoviral inverted terminal repeat varies from about 50 bp to about 170 bp depending on the adenovirus serotype. The inverted terminal repeat contains several different cis-acting elements necessary for viral growth, such as the core origin of viral DNA replication and an enhancer element for activation of the E1 region.

[0053] The terms "in vivo gene therapy" and "in vitro gene therapy" are intended to encompass all past, present and future variations and modifications of what is commonly known to those of skill in the art and referred to as "gene therapy," including ex vivo applications.

[0054] As used herein, the term "linear DNA" refers to a non-circularized DNA molecule.

[0055] As used herein, the term "native" refers to that found in nature, wild-type, inherently, or congenitally.

[0056] The term "nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and, unless otherwise limited, encompasses known analogues which have the essential properties of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptides, nucleic acids).

[0057] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. Generally, "operably linked" means that the DNA sequences being linked are contiguous. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0058] The terms "packaging expression plasmid" and "packaging construct" or "pPac" refer to an engineered plasmid construct of a circular double-stranded DNA molecule, which contains at least a subset of adenovirus late genes (e.g., LI, L2, L3, L4, L5, E2A, and E4) under the control of a promoter. pPac can be deleted from an adenovirus of one or two of the inverted terminal repeats (ITRs) and does not contain a packaging signal (T). pPac is "replication-deficient" in that the viral genome does not contain sufficient genetic information alone to allow it to replicate independently and produce infectious viral particles in a cell. Any subtype, mixture of subtypes, or chimeric adenovirus can be used as a source of DNA to create a fully deleted adenovirus vector module and pPac. pPac can be circular or linear in nature.

[0059] As used herein, the term "packaging signal" refers to a nucleotide sequence present in the viral genome and necessary for the incorporation of the viral genome into the viral capsid during viral assembly.

[0060] The adenovirus packaging signal is naturally located at the left end downstream of the left inverted terminal repeat, sometimes designated "Ψ".

[0061] The term "pathogen" is used broadly to refer to any source of molecules that induce an immune response. Thus, pathogens include, but are not limited to, virulent or attenuated viruses, bacteria, fungi, protozoa, parasites, cancer cells, and the like. Typically, the immune response is elicited by one or more peptides produced by these pathogens. As described in detail below, genomic DNA encoding antigenic peptides from these and other pathogens is used to generate an immune response that mimics the response to natural infection. Given the teachings herein, it will also be apparent that the method includes the use of genomic DNA obtained from two or more pathogens.

[0062] "Permissive" cells support viral replication.

[0063] The term "plasmid" as used herein refers to an extrachromosomal DNA molecule that is separate from chromosomal DNA and can replicate independently of the chromosomal DNA. It is often circular and double stranded.

[0064] The term "polylinker" is used for an artificially synthesized short stretch of DNA with several unique restriction sites that allow for easy insertion of any promoter or DNA segment. The term "heterologous" is used for any combination of DNA sequences that are not normally found closely related in nature.

[0065] The term "promoter" is intended to mean a regulatory region of DNA that promotes transcription of a particular gene. A promoter usually contains a TATA box that can direct RNA polymerase II to begin RNA synthesis at the appropriate transcription start site for a particular coding sequence. A promoter may further contain other recognition sequences, generally located upstream or 5' of the TATA box, called upstream promoter elements, that affect the rate of transcription initiation. A "constitutive promoter" refers to a promoter that allows continuous transcription of its associated gene in many cell types. An "inducible promoter system" refers to a system that uses regulators (including small molecules, such as tetracycline, peptide and steroid hormones, neurotransmitters, and environmental factors, such as heat and osmolality) to induce or silence genes. Such systems are "analogs" in the sense that their response is graded and dependent on the concentration of the regulator. Also, such systems are reversible by removal of the regulator. The activity of these promoters is induced by the presence or absence of biotic or abiotic factors. Inducible promoters are powerful tools in genetic engineering because the expression of genes operably linked to them can be turned on or off at specific stages of development of the organism or specific tissues.

[0066] The term "propagate" or "propagated" as used herein refers to reproducing, doubling, or increasing in number, amount, or extent by any process.

[0067] As used herein, the term "purified" refers to the process of purifying or rendering free of foreign, adventitious or undesirable elements.

[0068] As used herein, the term "regulatory sequence" (also called "regulatory region" or "regulatory element") refers to a promoter, enhancer, or other segment of DNA to which regulatory proteins, such as transcription factors, preferentially bind. They control gene expression, and thus protein expression.

[0069] The term "recombinase" as used herein refers to an enzyme that catalyzes genetic recombination. Recombinase enzymes catalyze the exchange of short pieces of DNA between two long strands of DNA, specifically the exchange of regions of homology between paired paternal and maternal chromosomes.

[0070] The term "restriction enzyme" (or "restriction endonuclease" refers to an enzyme that cuts double-stranded DNA. The term "restriction site" or "restriction recognition site" refers to a specific sequence of nucleotides recognized by a restriction enzyme as a site for cutting a DNA molecule. These sites are commonly, but not necessarily, palindromic (because restriction enzymes usually bind as homodimers), and a particular enzyme may cut anywhere between or near two nucleotides within its recognition site.

[0071] The term "replication" or "replicating" as used herein refers to making identical copies of an object, such as, but not limited to, a virus particle. The term "replication-deficient" as used herein refers to a characteristic of a virus that cannot replicate in a natural environment. A replication-deficient virus is a virus that has deleted one or more genes essential for its replication, such as, but not limited to, the E1 gene. Replication-deficient viruses can be propagated in the laboratory in cell lines that express the deleted genes.

[0072] The term "stuffer" as used herein refers to a DNA sequence that is inserted into another DNA sequence to increase its size. For example, a stuffer fragment can be inserted into the adenovirus genome to increase its size to about 36 kb. A stuffer fragment usually does not code for any protein and does not contain regulatory elements for gene expression, such as transcription enhancers or promoters.

[0073] As used herein, the term "target" or "targeted" refers to a biological entity, such as, but not limited to, a protein, cell, organ, or nucleic acid, whose activity can be modified by an external stimulus. Depending on the nature of the stimulus, there may be no direct change in the target or a conformational change in the target may be induced.

[0074] As used herein, a "target cell" may exist as a single entity or may be part of a larger collection of cells. Such a "larger collection of cells" may include, for example, a cell culture (either mixed or pure), a tissue (e.g., epithelial or other tissue), an organ (e.g., heart, lung, liver, gallbladder, bladder, eye or other organ), an organ system (e.g., circulatory system, respiratory system, gastrointestinal system, urinary system, nervous system, integumentary system or other organ system), or an organism (e.g., birds, mammals, particularly humans, etc.). Preferably, the targeted organs / tissues / cells are of the circulatory system (e.g., including but not limited to, heart, blood vessels, and blood), respiratory system (e.g., nose, pharynx, larynx, trachea, bronchi, bronchial system, lungs, etc.), gastrointestinal system (e.g., including mouth, pharynx, esophagus, stomach, intestines, salivary glands, pancreas, liver, gallbladder, etc.), urinary system (e.g., kidneys, ureters, bladder, urethra, etc.), nervous system (e.g., including but not limited to, brain and spinal cord, and special sensory organs such as eyes), and integumentary system (e.g., skin). Even more preferably, the cells are selected from the group consisting of heart, blood vessels, lung, liver, gallbladder, bladder, eye cells, and stem cells. In one embodiment, the target cells are hepatocytes, and a method is provided for vetovector-mediated transplantation of allogeneic hepatocytes in a subject. In one embodiment, the target cells are keratinocytes, and a method is provided for vetovector-mediated transplantation of allogeneic keratinocytes in a subject, e.g., engineered skin. In one embodiment, the target cell is a pancreatic islet. In one embodiment, the target cell is a cardiomyocyte. In one embodiment, the target cell is a kidney cell, and a method is provided for vetovector-mediated transplantation of an allogeneic kidney in a subject. In one embodiment, the target cell is a fibroblast, and a method is provided for vetovector-mediated transplantation of an allogeneic fibroblast in a subject, for example, engineered skin. In one embodiment, the target cell is a neural cell.

[0075] In one embodiment, the target cell is a glial cell.

[0076] The term "transfection" as used herein refers to the introduction of DNA into cellular DNA (e.g., the introduction of an isolated nucleic acid molecule or construct of the present disclosure). The adenoviral packaging cell lines disclosed herein can be transfected with at least one of the fully deleted adenoviral vector modules or pPac of the present disclosure. The adenoviral packaging cell lines disclosed herein can be transfected or transduced with the DNA or viral particles of minimally modified adenoviral vectors.

[0077] As used herein, the term "transduction" refers to the introduction of a gene into a cell's DNA, either as DNA or by a gene transfer vector of the present disclosure. The gene transfer vector of the present disclosure is capable of transducing a target cell.

[0078] The term "vector" refers to a nucleic acid that can be used to infect a host cell and insert a polynucleotide therein. A vector is often a replicon. An expression vector allows for transcription of a nucleic acid inserted therein. Some common vectors include, but are not limited to, plasmids, cosmids, viruses, phages, recombinant expression cassettes, and transposons. The term "vector" can also refer to an element that aids in the transfer of a gene from one location to another.

[0079] The term "viral DNA" as used herein refers to the sequence of DNA found in a virus particle. The term "viral genome" as used herein refers to the entirety of DNA found in a virus particle that contains all the elements necessary for viral replication. The genome is replicated and transmitted to viral progeny with each cycle of viral replication.

[0080] The term "virion" as used herein refers to a virus particle. Each virion consists of genetic material within a protective protein capsid.

[0081] The term "wild type" as used herein refers to the typical form of an organism, strain, gene, protein, nucleic acid, or characteristic occurring in nature. Wild type refers to the most common phenotype in a natural population.

[0082] The terms "wild-type" and "naturally occurring" are used interchangeably.

[0083] Completely deleted adenovirus vectors As used herein, a fully deleted adenoviral vector module refers to a linear DNA sequence that contains only the cis-acting adenoviral sequences (i.e., ITR, ￥II) necessary for viral replication and encapsidation of the complete adenoviral vector module into an adenoviral capsid, or a circular sequence from the linear DNA sequence that can be released. A fully deleted adenoviral vector only possesses the cis-acting sequences (i.e., ITR, W) necessary for viral genome replication and encapsidation. Some systems and methods for packaging fully deleted adenoviral vectors (fully deleted adenoviral vector modules) require their co-infection with an adenoviral "helper" virus, which can be a source of immunogenic adenoviral antigens. Methods have been proposed to remove contaminating adenoviral helper viruses from therapeutic adenoviral vector preparations. One example is flanking (flaxing) the packaging site Ψ of the adenoviral helper virus with lox sequences for Cre recombinase. In theory, passaging a fully deleted adenovirus vector and a "floxed" adenovirus helper virus would reduce contamination by excising the Ψ (packaging) sequence from the adenovirus helper virus, thereby preventing packaging of the adenovirus helper virus. In practice, this approach reduces adenovirus helper virus contamination by 10 3It was not possible to reduce it below a factor of 1.

[0084] Figure 1 is a schematic diagram showing one embodiment of the design of a fully deleted adenoviral vector module. This fully deleted adenoviral vector module contains the cis-acting adenoviral sequences (i.e., ITR, ψ) necessary for viral replication and encapsidation into the adenoviral capsid of the complete adenoviral vector module, together with a non-adenoviral stuffer sequence and a polylinker site, into which an expression cassette of a single or several genes of interest can be cloned. Additional restriction sites are found within the stuffer, into which additional expression cassettes of a single or several genes of interest can be cloned.

[0085] 2 is a schematic diagram showing one embodiment of the design of a packaging expression plasmid with and without an expression cassette that can inhibit the expression of a gene of interest on an adenoviral vector module. The packaging expression plasmid consists of a plasmid of double-stranded DNA composed of a subset of adenoviral late genes (e.g., LI, L2, L3, L4, L5, E2A, and E4) under the control of a promoter. It can be deleted from one or two of the adenovirus inverted terminal repeats (ITRs) and does not contain a packaging signal ("Ψ").

[0086] FIG. 3 is a schematic diagram showing one embodiment of a minimally modified adenoviral vector carrying a subset of adenoviral late genes (e.g., LI, L2, L3, L4, L5, E2A, and E4) under the control of a promoter, as well as two adenoviral inverted repeats (ITRs) and a packaging signal (Ψ).

[0087] Figure 4 is a schematic diagram showing one embodiment of the design of an inhibitory expression plasmid, which carries one or more expression cassettes capable of inhibiting the expression of one or more genes of interest found on the adenoviral vector module.

[0088] 5 is a schematic diagram showing one embodiment of a method for packaging a fully deleted adenoviral vector module by co-transfection with a packaging expression plasmid. Both DNA constructs are mixed in a specific molar ratio and co-transfected into packaging cells to replicate and encapsidate the fully deleted adenoviral vector module. The genetic program of this process is encoded on the packaging expression plasmid. The encapsidated fully deleted adenoviral vector is recovered from the packaging cells.

[0089] 6 is a schematic diagram showing one embodiment of a method for encapsidation by co-transfection of a fully deleted adenoviral vector module with a packaging plasmid and an expression plasmid that can inhibit the expression of a gene of interest on the adenoviral vector module. The DNA constructs are mixed in a specific molar ratio and co-transfected into packaging cells to replicate and encapsidate the fully deleted adenoviral vector module. The genetic program of this process is encoded on a packaging expression plasmid. The expression of one or more genes of interest is inhibited by a gene, such as the antisense of the antisense construct of the gene of interest, expressed on an inhibitory expression plasmid. The encapsidated fully deleted adenoviral vector is recovered from the packaging cells.

[0090] Expression of one or more genes of interest is inhibited by the presence of an RNA or DNA fragment, such as an antisense of the gene of interest antisense construct. The encapsidated, fully deleted adenoviral vector is recovered from the packaging cells.

[0091] Those skilled in the art will appreciate that suitable methods of administering the disclosed gene transfer vectors to human subjects or animals for therapeutic purposes, e.g., gene therapy, immunosuppressive therapy, tissue engineering, vaccination, etc. (see, e.g., Rosenfeld et al., Science, 252, 431 434 (1991); Jaffe et al., Clin. Res., 39(2), 302A (1991); Rosenfeld et al., Clin. Res., 39(2), 311A ​​(1991); Berkner, BioTechniques, 6, 616 629 (19SS) are available, and more than one route can be used to administer the gene transfer vector, although certain routes may provide a more rapid and effective response than alternative administrations, excretion rates, drug combinations, and the importance of the route. Pharmaceutically acceptable excipients are also well known to those skilled in the art and are readily available. The choice of excipient will be determined in part by the condition and the host to be treated.

[0092] In the context of the present invention, the dose administered to an animal, particularly a human, will vary depending on the nature of the therapeutic transgene and / or molecule of interest, the composition used, the method of administration, and the particular site, as well as the particular method used to administer the transgene vector. Thus, there are a wide variety of suitable formulations of the transgene vector of the present invention. The following formulations and methods are merely exemplary and in no way limiting. However, oral, injectable, and aerosol formulations are preferred. Formulations suitable for oral administration can consist of a) liquid solutions, (b) capsules, sachets, or tablets, (c) suspensions in a suitable liquid, and (d) suitable emulsions. In one embodiment, the transgene vector of the present invention, alone or in combination with other suitable components, can be made into an aerosol formulation to be administered by inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. The transgene vector of the present invention can also be formulated as a pharmaceutical for non-pressurized formulations, such as nebulizers or atomizers. Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may contain suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives.

[0093] The formulations can be provided in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) state requiring only the addition of a sterile liquid excipient for injection, such as water, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the type described above. In the context of the present invention, the dose administered to animals, particularly humans, will vary depending on the gene or other sequence of interest, the composition used, the method of administration, and the particular site and organ being treated. The dose should be sufficient to produce a desired response, such as a therapeutic or immune response, within a desired time frame. Thus, the gene transfer vector of the present disclosure can be administered by one or more of the following routes: oral administration, injection (such as direct injection), topical, inhalation, parenteral administration, mucosal administration, intramuscular administration, intravenous administration, subcutaneous administration, intraocular administration, or transdermal administration. In one embodiment, the encapsidated fully deleted adenoviral vector of the present disclosure is administered by injection. In one embodiment, the gene transfer vector of the present disclosure is administered topically. In one embodiment, the gene transfer vector of the present disclosure is administered by inhalation. In one embodiment, the gene transfer vector of the present disclosure is administered by one or more of parenteral, mucosal, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration means and is formulated for such administration. Typically, a physician will determine the actual dosage of the gene transfer vector that is most suitable for an individual subject, and the dosage will vary according to the age, weight and response of the particular patient and the severity of the condition. The specific dose level and frequency of administration for any particular patient may vary and will depend on a variety of factors, including the activity of the particular compound used, the metabolic stability and length of action of that compound, age, weight, general health, sex, diet, mode and time of administration, excretion rate, drug combination, the severity of the particular condition, and the host being treated. In the context of the present invention, the dosage administered to animals, particularly humans, will vary according to the nature of the therapeutic transgene and / or immunomodulatory molecule of interest, the composition used, the method of administration, and the particular site and organism being treated. However, preferably, a dosage equivalent to an effective amount of GDV is employed.An "effective amount" is an amount sufficient to produce a desired effect in a host, which can be monitored using several endpoints known to those skilled in the art. For example, one desired effect is the introduction of a nucleic acid into a host cell. For example, but not limited to, a therapeutic effect (e.g., alleviation of any symptoms associated with the disease, condition, disorder or symptom being treated), or an effect by evidence of the introduced gene or coding sequence or its expression in the host (e.g., using polymerase chain reaction, northern or hybridization, or transcription assay to detect nucleic acid in the host cell, or using analysis, antibody-mediated detection, or specialization assay to detect protein or polypeptide encoded by the introduced nucleic acid or affected in level or function due to such introduction).

[0094] These described methods are by no means comprehensive, and further methods suitable for particular applications will be apparent to those skilled in the art. In this regard, it should be noted that the host response to the introduction of a gene transfer vector can vary depending on the dose of virus administered, the site of delivery, and the genetic makeup of the gene transfer vector, as well as the transgene and the means of inhibiting the immune response.

[0095] In general, to ensure effective introduction of the gene transfer vector of the present invention, it is preferable to use about 1 to about 5,000 copies of the gene transfer vector of the present invention per cell contacted, based on the approximate number of cells to be contacted, taking into account a given route of administration, and more preferably about 3 to about 300 pfu entering each cell. However, this is merely a general guideline and in no way precludes the use of higher or lower amounts as may be warranted in a particular application, either in vitro or in vivo. Similarly, the amount of the means for inhibiting an immune response, when in the form of a composition containing a protein, must be sufficient to inhibit an immune response to the recombinant gene transfer vector containing the transgene or gene of interest. For example, the actual dose and schedule may vary depending on whether the composition is administered in combination with other pharmaceutical compositions, or depending on inter-individual differences in pharmacokinetics, pharmacodynamics, and metabolism. Similarly, the amount may vary in in vitro applications depending on the particular cell type targeted or the means by which the gene transfer vector is introduced. One of skill in the art can readily make any necessary adjustments according to the needs of a particular situation.

[0096] Delivery of the gene transfer vector can be accomplished in vitro, ex vivo, as in laboratory procedures to transfect or transduce cell lines, or in vivo or ex vivo, as in the treatment of certain disease conditions. Once the gene transfer vector is delivered to the cell, the nucleic acid encoding the desired oligonucleotide or polynucleotide sequence can be positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the construct can be stably integrated into the genome of the cell. This integration can be in a specific location and orientation via homologous recombination (gene replacement), or integrated recombinationally (gene replacement), or integrated at a random, non-specific location (gene augmentation). In further preferred embodiments, the nucleic acid can be stably maintained in the cell as a separate episomal segment of DNA. Such a nucleic acid segment or "episome" encodes sufficient sequences to allow maintenance and replication independent of or in synchrony with the host cell cycle. The immune modulating gene can encode all or a functional portion of a CD8 polypeptide.

[0097] Additional immunomodulatory genes encoding immunomodulatory molecules are contemplated, including, but not limited to, IL-10, TGF-beta, IL-2, IL-12, IL-15, IL-18, IL-4 and GMCSF.

[0098] Gene Therapy for Gene and Protein Expression Gene therapy generally involves the introduction of a gene of interest or a therapeutic gene, also known as a transgene, into cells, the expression of which leads to the amelioration or treatment of a genetic disorder. The therapeutic gene involved may be a gene encoding a protein, a structural or enzymatic RNA, an inhibitory product such as antisense RNA or DNA, or any other gene product. Expression is the production of such a gene product or the effect that occurs as a result of the production of such a gene product. Thus, enhanced expression includes greater production of any therapeutic gene or enhanced role of its product in determining the state of a cell, tissue, organ or organism.

[0099] In general, the present invention relates to GDVs for the transfer of selected genetic material of interest (e.g., DNA or RNA) into cells in vivo. The present invention also relates to methods of gene therapy using the disclosed gene transfer vectors, and to genetically engineered cells produced thereby. Diseases that may be treated by the present invention include, but are not limited to, hemophilia A (due to factor VIII), Parkinson's disease, congestive heart failure, and cystic fibrosis.

[0100] In one embodiment, the gene transfer vector of the present disclosure carrying at least a fragment of a gene of interest can infect the myocardium in vivo after intracardiac injection. In one embodiment, the gene transfer vector of the present disclosure carrying at least a fragment of the CFTR gene can be introduced in situ into the lungs of a cystic fibrosis patient by aerosol inhalation. In one embodiment, the gene therapy vector of the present disclosure carrying at least a fragment of the gene encoding factor VIII can be introduced in situ into the arm muscle of a hemophilia A patient. In one embodiment, the gene transfer vector of the present disclosure carrying at least a fragment of the ADA gene can be transduced ex vivo into a subpopulation of bone marrow cells, and the transduced bone marrow cells can then be transplanted into a patient suffering from adenosine deaminase (ADA) deficiency. The specific therapeutic genes encoded by the gene therapy vectors of the present disclosure are not limited and include those useful for various therapeutic and research purposes, as well as reporter genes and reporter gene systems and constructs useful for tracking transgene expression and the efficacy of adenoviral vector transduction. Thus, by way of example, the following are possible classes of genes whose expression may be enhanced by using the GDV of the present disclosure: developmental genes (e.g., adhesion molecules, cyclin kinase inhibitors, Wnt family members, Pax family members, winged helix family members, Hox family members, cytokines / lymphokines and their receptors, growth or differentiation factors and their receptors, neurotransmitters and their receptors), cancer genes (e.g., ABU, BLCI, BCL6, CBFAI, CBL, CSFIR, ERBA, ERBB, EBRB2, ETSI, ETSI, ETV6, FGR, FOX, FYN, HCR, HRAS, JUN, KRAS, LCK, LKR ... YN, MDM2, MLL, MYB, MYC, MYCU, MYCN, NRAS, PIMI, PML, RET, SRC, TAU, TCL3 and YES), tumor suppressor genes (e.g., APC, BRCAI, BRCA2, MADH4, MCC, NFI, NF2, R131, TP53 and WTI), enzymes (e.g., ACP desaturase and hydroxylase, ADP-glucose pyrophorylase, ATPase, alcohol dehydrogenase,Amylase, amyloglucosidase, catalase, cellulase, cyclooxygenase, decarboxylase, dextrinase, esterase, DNA and RNA polymerase, hyaluronan synthase, galactosidase, glucanase, glucose oxidase, GTPase, helicase, hemicellulase, hyaluronidase, integrase, invertase, isomerase, kinase, lactase, lipase, lipoxygenase, lyase, isozyme, pectinase, pectinesterase, peroxidase, phosphatase, phospholipase, phosphorylase, polygalacturonase, proteinase and peptidase, planase, recombinase, reverse transcriptase, topoisomerase, xylanase, reporter genes (e.g., green fluorescent protein and its many color variants, Luciferase, etc.) ferase, CAT reporter system, beta-galactosidase, etc.), blood derivatives, hormones, lymphokines (including interleukins), interferons, TNF, growth factors, neurotransmitters or their precursors or synthetic enzymes, trophic factors (e.g., BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NTS, etc.), apolipoproteins (e.g., ApoAI, ApoAIV, ApoE, etc.), dystrophin or minidystrophic, tumor suppressor genes (e.g., p53, Rb, RapiA, DCC, k-rev, etc.), genes encoding factors involved in coagulation (e.g., factor VII, factor VI, factor IX, etc.), suicide genes (thymidine kinase, etc.), cytosine deaminase, or all or part of natural or artificial immunoglobulins (Fab, ScFv, etc.). Other examples of therapeutic genes include fus, interferon alpha, interferon beta, interferon gamma, and ADP (adenovirus death protein). The therapeutic gene may also be an antisense gene or sequence that allows expression in the target cell to control the expression of a cellular gene or the transcription of a cellular mRNA. Such a sequence may, for example, be transcribed in the target cell into an RNA complementary to the cellular mRNA. The therapeutic gene may also be a gene that encodes an antigenic peptide capable of generating an immune response in humans. In this particular embodiment, the present disclosure relates to a therapeutic gene for the treatment of a variety of diseases, particularly in microorganisms,This allows the production of vaccines that can immunize humans against viruses and cancer. Various enzyme genes are also considered therapeutic genes. Particularly suitable genes for expression include genes that are believed to be expressed at lower than normal levels in target cells of the subject mammal. Examples of particularly useful gene products include, but are not limited to, carbamoyl synthetase I, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate lyase, and arginase. Other desirable gene products include fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha-antitrypsin, glucose-6-phosphatase, low density lipoprotein receptor, porphobilinogen deaminase, factor VIII, factor IX, cystathione.beta.-synthase, branched-chain keto acid decarboxylase, albumin, isovaleryi-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, a-glucosidase, pyruvate carboxylase, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase (also called asP-protein), H-protein, T-protein, Menkes copper-transporting ATPase, and Wilson copper-transporting ATPase. Other examples of gene products include, but are not limited to, cytosine deaminase, galactose-1-phosphate hypoxanthine-guanine phosphoribosyltransferase, uridyltransferase, phenylalanine hydroxylase, glucosidase, sphingomyelinase, aL-iduronidase, glucose-6-phosphate dehydrogenase, HSV thymidine kinase, and human thymidine kinase. Hormones are another group of genes that can be used in the adenovirus-derived vectors described herein. Growth hormone, prolactin, placental lactogen, luteinizing hormone, follicle-stimulating hormone, chorionic gonadotropin, thyroid-stimulating hormone, leptin, adrenocorticotropic hormone (ACTH), angiotensin I and II, endorphins,Melanocyte-stimulating hormone (MSH), cholecystokinin, endothelin, galanin, gastric inhibitory peptide (GIP), glucagon, insulin, lipotropin, neurophysin, somatostatin, calcitonin, calcitonin gene-related peptide (CGRP), calcitonin gene-related peptide, hypercalcemia of malignancy factor (1-40), parathyroid hormone-related protein (107-139) (PTH-rP), parathyroid hormone-related protein (107-111) (PTH-rP), glucagon-like peptide (GLP-1), pancreatic peptide, peptide YY, PHM, secretin , vasoactive intestinal peptide (VIP), oxytocin, vasopressin (AVP), vasotocin, enkephalinamide, methorphinamide, 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 contemplated for insertion into the GDV of the present disclosure include, but are not limited to, interleukins and cytokines (interleukin 1 (IL-1), IL-2, IL-3, IL4, IL-5, IL-6, IL-7, IL-S, IL-9, IL-10, IL-11, IL-12, GM-CSF, and G-CSF.

[0101] Diseases that can be treated by the present invention include, but are not limited to, common genetic diseases such as phenylketonuria (phenylalanine-L-monooxygenase), adenosine deaminase deficiency, cystic fibrosis (cystic fibrosis conductance regulator), Parkinson's disease, ornithine caramyltransferase deficiency (OTC), hemophilia (factor IX deficiency, factor VIII deficiency), Tay-Sachs disease (N-acetylhexosamimidase A), cystic fibrosis with replacement of the cystic fibrosis conductance regulator gene, and other lipid storage diseases. Also, the gene encoding erythropoietin (EPO) can be used.

[0102] Additionally, bacterial, plant and eukaryotic toxins are contemplated as transgenes, including, but not limited to, botulinum toxin, tetanus toxin, shiga toxin, diphtheria toxin, cholera toxin, diphtheria toxin, anthrax toxin LF, listeriolysin and ricin.

[0103] The fully deleted adenoviral vector of the present disclosure does not contain adenoviral early region and late genes. The gene transfer vector of the present disclosure can be used to treat hyperproliferative diseases such as rheumatoid arthritis or restenosis by the introduction of genes encoding angiogenesis inhibitors or cell cycle inhibitors. The introduction of prodrug activators such as the HSVTK gene can also be used to treat hyperproliferative diseases, including cancer. In one embodiment, the gene transfer vector of the present disclosure includes a therapeutic gene sequence and a CD8 gene sequence, which can prevent an immune response to the therapeutic gene sequence, as described below. Such uses include, but are not limited to, factor VIII deficiency (hemophilia A), where patients do not produce protein from the defective gene (null allele). In these patients, an immune response can be mounted against the product of the therapeutic gene, similar to the immune response that frequently occurs in hemophilia A patients treated by injection of factor VIII protein. Adenoviral vectors have been used to produce recombinant proteins useful in the treatment of disease. However, contamination of therapeutic recombinant proteins with adenoviral proteins and / or adenoviruses is a continuing challenge.Therefore, there is a need for a system that supports growth from adenoviruses, has a reduced complement of adenoviral genes, and / or is free of replication-competent or helper adenovirus contamination.The co-expression of polypeptides for multimeric proteins can be regulated by co-expression from an adenoviral vector.For example, the expression of equimolar amounts of immunoglobulin heavy chains and immunoglobulin light chains is promoted by co-expression from an adenoviral vector.According to the aspects exemplified herein, in one embodiment, the protein expression protocol comprises administering a gene transfer vector of the present disclosure.

[0104] Vaccine Development In one embodiment, the present invention relates to biotechnology and the development and production of vaccines. The present invention is particularly useful in the manufacture of vaccines to help protect vertebrates, particularly mammals, and especially humans, against viral and bacterial pathogens.

[0105] The vaccines of the invention can be prophylactic (e.g., to prevent or ameliorate the effects of future infection with any natural or "wild" pathogen) or therapeutic (e.g., vaccines against cancer). Vaccination is the most important route to combat viral infections. Although several antiviral agents are available, typically the effectiveness of these agents is limited. Administering antibodies against the virus can be a good way to combat viral infections once an individual is infected (passive immunization), typically human or humanized antibodies seem promising for combating some viral infections. However, the most effective and safest way to combat viral infections is and may possibly be prevention by active immunization. Active immunization is commonly called vaccination, where the vaccine contains at least one antigenic determinant of the virus, preferably several different antigenic determinants of at least one virus, for example by incorporating at least one viral polypeptide or a protein derived from the virus into the vaccine (subunit vaccines). Typically, the formats mentioned so far contain an adjuvant to enhance the immune response. This is also possible for vaccines based on whole viruses, for example in inactivated formats. A further possibility is the use of a live but attenuated form of the pathogenic virus. A further possibility is the use of wild-type viruses, for example where adult individuals are not at risk of infection, but infants are, and may be protected by maternal antibodies, etc. The production of vaccines is not always an easy procedure. In some cases, the production of the viral material is on eggs, which makes purification of the material difficult and requires extensive safety measures against contamination, etc. Production on bacteria and / or yeasts is sometimes, but not always, an alternative to eggs, but this also requires many purification and safety steps. Production in mammalian cells would be an alternative, but all mammalian cells used so far require, for example, the presence of serum and / or attachment to a solid support for growth. In the first case, again, purification and safety are issues, as well as the need for, for example, proteases to support the replication of some viruses. In the second case, high yields and ease of production are further issues.Vaccines are still lacking for many viral diseases of great public health importance.Killed virus vaccines can be dangerous and expensive to produce, and are often not immunogenic.The inclusion of sequences encoding viral proteins in adenoviral vectors can avoid these problems, but there are challenges in making such adenoviral vectors.For example, there is little space in adenoviral vectors, and the immune response to adenoviral vectors interferes with the immune response to vaccine proteins.

[0106] It is therefore an object of the present invention to provide a gene transfer vector capable of long-term maintenance in a growing number of different cells in the host's body, thereby providing stable expression of the desired antigen. Another object of the present invention is to provide a gene transfer vector that is maintained for a long period of time in the cells that initially received the vector and transferred it to their daughter cells after cell mitosis. Yet another object of the present invention is to provide a gene transfer vector that expresses one or more genes of interest, preferably only those genes necessary for long-term maintenance in the recipient cells, and thus lacks components that are toxic or cause disease symptoms in the recipient. A further object of the present invention is to provide a gene transfer vector that mimics an attenuated live virus vaccine, particularly in its ability to multiply in the body, does not induce significant signs of disease, and does not express undesirable proteins that may induce adverse reactions in hosts injected with the DNA vaccine. The vaccine of the present invention comprises the gene transfer vector of the present invention or a mixture of said vectors in a suitable pharmaceutical carrier. The vaccine of the present invention is formulated using standard methods of vaccine formulation to produce a vaccine that is administered by any conventional route of administration, i.e., intramuscular, intradermal, etc. In a particular embodiment, the gene transfer vector of the invention is used to treat and / or prevent infection (passive immunization), typically human or humanized antibodies seem promising for combating some viral infections. However, the most effective and safe way to combat viral infections is and may possibly be prevention by active immunization. Active immunization is commonly called vaccination, where the vaccine contains at least one antigenic determinant of a virus, preferably several different antigenic determinants of at least one virus, for example by incorporating at least one viral polypeptide or a protein derived from the virus into the vaccine (subunit vaccines). Typically, the formats mentioned so far contain an adjuvant to enhance the immune response. This is also possible for vaccines based on whole viruses, for example in inactivated formats. A further possibility is the use of a live but attenuated form of the pathogenic virus.A further possibility is the use of wild-type viruses, for example where adult individuals are not at risk of infection, but infants are, and may be protected by maternal antibodies, etc. Vaccine production is not always an easy procedure. In some cases, production of viral material is on eggs, which makes purification of the material difficult and requires extensive safety measures against contamination, etc. Production on bacteria and / or yeasts is sometimes, but not always, an alternative to eggs, but this also requires many purification and safety steps. Production in mammalian cells would be an alternative, but all mammalian cells used so far require, for example, the presence of serum and / or attachment to a solid support for growth. In the first case, again, purification and safety are issues, as well as the need for, for example, proteases to support the replication of some viruses. In the second case, high yields and ease of production are further issues. Vaccines are still lacking for many viral diseases of great public health importance. Killed virus vaccines can be dangerous and expensive to produce and are often not immunogenic. Including sequences encoding viral proteins in adenoviral vectors may avoid these problems, but there are challenges in making such adenoviral vectors. For example, there is little space in adenoviral vectors, and immune responses to adenoviral vectors interfere with immune responses to vaccine proteins. Therefore, the object of the present invention is to provide a gene transfer vector that can be maintained long-term in a large number of different cells in the host's body, thereby providing stable expression of a desired antigen. Another object of the present invention is to provide a gene transfer vector that is maintained long-term in the cell that originally received the vector and transferred it to daughter cells after cell division. Yet another object of the present invention is to provide a gene transfer vector that expresses one or more genes of interest, as well as preferably only the genes necessary for long-term maintenance in recipient cells, and thus lacks components that are toxic or cause disease symptoms in the recipient.A further object of the present invention is to provide a gene transfer vector that mimics an attenuated live virus vaccine, particularly in its ability to multiply in vivo, does not induce significant symptoms of disease, and does not express undesirable proteins that may induce adverse reactions in a host injected with the DNA vaccine. The vaccine of the present invention comprises a gene transfer vector of the present invention or a mixture of said vectors in a suitable pharmaceutical carrier. The vaccine of the present invention is formulated using standard methods of vaccine preparation to produce a vaccine that is administered by any conventional route of administration, i.e., intramuscular, intradermal, etc. In a particular embodiment, the gene transfer vector of the present invention is used to treat and / or prevent infectious diseases.

[0107] The gene transfer vector of the present disclosure can be used in vaccine development to protect individuals against disease by inducing immunity. One advantage of using the GDV of the present disclosure for vaccine development is that the recipient's immune response is not biased by Ad genes. In one embodiment, the gene transfer vector of the present disclosure encodes one or more proteins and / or RNA (antigens) from a virus important to human health or agriculture. In one embodiment, the vaccine is used to protect individuals against disease by inducing immunity. In one embodiment, multiple genes of interest can be included for a multivalent vaccine from the same or different pathogens. In certain embodiments, the gene transfer vector further comprises one or more expression cassettes of a DNA sequence of interest. In certain cases, the DNA sequence of interest is a DNA sequence of an infectious pathogen. In certain embodiments, the infectious pathogen is a virus.

[0108] In certain specific embodiments, the virus is selected from the group consisting of Human Immunodeficiency Virus (HIV), Herpes Simplex Virus (HSV), Hepatitis C Virus, Influenzae Virus, Rotavirus, Papilloma Virus, Lentivirus, Enterevirus, or combinations thereof. In certain embodiments, the DNA sequence of interest is a bacterial DNA sequence. In certain embodiments, the bacteria is selected from the group consisting of Chlamydia trachomatis, Mycobacterium tuberculosis, and Mycoplasma pneumonia. In certain embodiments, the DNA sequence of interest is a DNA sequence of a fungal pathogen. In certain embodiments, the DNA sequence is a DNA sequence of HIV origin.

[0109] In one embodiment, the vaccine is used to protect an individual against influenza virus. In one embodiment, the influenza virus is swine flu. In one embodiment, the influenza virus is avian flu. In one embodiment, the one or more proteins and / or RNA of the gene transfer vector of the present disclosure are selected from one of hemagglutinin (HA), neuraminidase (NA), nucleocapsid (NP), M1 (matrix protein), M2 (ion channel), NS 1, NS 2 40 (NEP), lipid bilayer, PBI, PB2, or PA. Representative viral and bacterial candidates for the vaccine of the present disclosure are listed below. The genes of these vaccines are shown in parentheses. Viruses - Orthomyxovirus Influenza A - hemagglutinin (HA) and neuraminidase (NA), nucleoprotein (NP), Mu M2, NSI, NS2(NEP), PA, PB / , PB1-F2 and PB2) Influenza B Influenza C Viruses - Herpes Virus Herpes simplex 1 (oral herpes), herpes simplex 2 (genital herpes) - polypeptides; viral glycoproteins (designated gB, gC, gD, gE, gG, gH, gi, gJ, gK, gL and gM) are known and one is predicted (gN); glycoprotein band 0. Epstein-Barr (mononucleosis, Burkitt's lymphoma, nasopharyngeal carcinoma) - Epstein-Barr nuclear antigen [EBNA] 1, 2, 3A, 38, 3C, LP and LMP; gp3501220, also known as gp340; Cytomegalovirus - G / ycoprotein B, 1 EI, pp 89, gB and pp 65 are the minimum requirements in a vaccine to induce neutralizing antibody and cytotoxic T lymphocyte (CTL) responses. Immunization with additional proteins, e.g. gH, gN for neutralizing antibodies and El, exon 4 and pp 150 for CTL responses, will enhance the protective immune response. Varicella zoster virus (varicella and varicella zoster)-recombinant proteins derived from gE, gI and gB genes Kaposi's sarcoma-associated herpesvirus 8 (Kaposi's sarcoma) Herpes 6 (A and 8) Herpes 7 Herpes 8-glycoprotein B (gB); Viruses - Papillomavirus: For all HPV L1 capsid proteins, EI, E2, E6, and E7 genes HPV (High risk for cervical carcinoma: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59; Probable risk: 26, 53, 66, 68, 73, 82) HPV (common warts: 2, 7) HPV (plantar warts: 1, 2, 4, 63) HPV (Fiat Warts: 3, 10) HPV (anogenital warts: 6, 11, 42, 43, 44, 55) Viruses - Family Reoviridae Rotavirus A (gastroenteritis) - VP2 and VP6 proteins Virus - Coronavirus Severe acute respiratory syndrome coronaviruses (SARS-CoV and MERS-CoV) are enveloped positive-stranded RNA viruses with a 30 kb genome, structural proteins spike (S), envelope (E), membrane (M) and nucleocapsid (N) spike or nucleocapsid proteins, S and N genes, respectively, that encode the replicase (Rep) - human coronavirus 229E spike and envelope genes Human coronavirus NL63 Viruses - Astrovirus (gastroenteritis) - Astrovirus 87 kDa structural polyprotein Viruses - Norovirus (gastroenteritis) - Viral capsid genes, VPI and VP2 Viruses - Family Fiaviviridae Dengue - Promembrane (prM) and envelope (E) genes Japanese encephalitis-prM, E and NSI genes; prM and the envelope (E) coding region of the JE virus. Kyasanur Forest disease Murray Valley encephalitis St. Louis encephalitis Tick-borne encephalitis West Nile encephalitis Hepatitis C - Hepatitis C virus glycoprotein E2; Hepatitis C glycoproteins EI and E2; HCV core gene Viruses - Family Picornaviridae - Enterovirus Human enterovirus A (21 types, including some coxsackie A viruses) Human enterovirus B (57 types including enterovirus and coxsackie B virus) Human enterovirus C (14 types, including some Coxsackie A viruses) Human enterovirus 0 (three types: EV-68, EV-70, and EV-94)-VPI gene; Viruses - Picornaviridae - Rhinovirus Human rhinovirus A (serotype 74) Human rhinovirus B (25 serotypes) Human rhinovirus C (serotype 7)-rhinovirus-derived VPI; a surface protein critically involved in infection of respiratory cells Viruses - Picornaviridae - Hepatovirus Hepatitis A Viruses - Togaviridae - Aiphavirus Sindbis virus Eastern equine encephalitis virus Western equine encephalitis virus Venezuelan equine encephalitis virus Ross River virus O'nyong'nyong virus Viruses - Togaviridae - Rubivirus Rubella virus Viruses - Togaviridae - Hepevirus (Hepevi rus) Hepatitis E virus-ORF2 protein; recombinant HEY capsid protein; vaccine peptide has a 26 amino acid extension from the N-terminus of another peptide E2 of the HEY capsid protein Viruses - Togaviridae - Bomaviridae Barna disease virus-BDV nucleoprotein (BDV-N) Viruses - Togaviridae - Filoviridae Ebola virus Marburgvirus Viruses - Togaviridae - Paramyxovirus Measles Sendai virus Human parainfluenza virus 1 and 3, Mumps virus Human parainfluenza viruses 2 and 4 Human respiratory syncytial virus Newcastle disease virus Viruses - Togaviridae Retroviruses HIV-gag: p18, p24, p55; pol: p31, p51, p66; env: p41, p120, p160 Hepatitis B virus HTLVI, II Viruses - Togaviridae - Rhabdoviruses Rabies Viruses - Togaviridae - Arenaviruses Ranta virus Korean hemorrhagic fever Lymphocytic choriomeningitis virus Junin Machupo Lassa Sabia Guanarito California encephalitis Congo-Crimean hemorrhagic fever Ritt valley fever Viruses - Parvovirus Human parvovirus (B19) bacteria Bartonella Brucella (including B. abortus, B. canis, and B. suis) Burkholderia (Pseudomonas) mallei, B. pseudomallei Coxiella burnetii Francisella tularensis Mycobacterium bovis (except BCG strains, see Appendix B-11-A, Risk Group 2 (RG2) - Chlamydia-containing bacterial agents), M. tuberculosis Pasteurella multocida type B "buffalo" and other virulent strains Rickettsia akari, R.australis, R.canada, R.conorii, R.prowazekii, R.rickettsii, R.siberica, R.tsutsugamushi, R.typhi(R.mooseri) Yersinia pestis

[0110] A method of modulating an immune response in an individual includes administering to the individual a gene transfer vector of the present disclosure, the gene transfer vector encoding at least one therapeutic gene (gene of interest, "GOI").

[0111] vector In addition to adenovirus-based gene transfer vectors, other vector systems are envisioned in which assembly is prevented by the production of a protein encoded by a transgene within the vector, such as, but not limited to, retrovirus vectors, lentivirus vectors, adeno-associated virus vectors, SV40-based vectors, vaccinia virus vectors, and Epstein-Barr virus vectors.

[0112] The present invention is described in the following examples, which are provided to aid in the understanding of the present invention and should not be construed as limiting in any way thereafter. The following examples are provided to provide those skilled in the art with a complete disclosure and description of how to make and use the described invention, and are not intended to limit the scope of what the inventors regard as the invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0113] Of course, it should be understood that the examples are merely illustrative of certain embodiments of the invention only, and do not constitute limitations on the scope of the invention as defined by the claims appended at the end of this specification.

[0114] Although the disclosure has been described in detail, it will be apparent that modifications and variations are possible without departing from the scope of the appended claims. EXAMPLES

[0115] The following non-limiting examples are provided to further illustrate the present disclosure.

[0116] Example 1 Host cells, such as human embryonic kidney cells, are seeded into tissue culture flasks in eukaryotic tissue culture medium. They are incubated with CO 2The cells are cultured for 3 days under atmospheric oxygen tension (5%). A mixture of the following DNA constructs is added in a fixed ratio along with a transfectant, such as but not limited to calcium phosphate: a) a linear fully deleted adenoviral vector module carrying the Ebola glycoprotein gene in an expression cassette driven from a CMV promoter, b) a packaging expression plasmid, c) an inhibitory expression plasmid carrying an antisense construct of the Ebola glycoprotein driven from a CMV promoter. After 3 days of culture, the host cells are harvested and lysed by freeze-thawing to release the encapsidated fully deleted adenoviral vector.

[0117] When introducing elements of the present disclosure or preferred embodiments thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0118] In view of the above, it will be seen that the several objects of the disclosure are achieved and other advantageous results attained.

[0119] Since various changes can be made in the above products and methods without departing from the scope of the disclosure, it is intended that all matter contained in the above description should be interpreted as illustrative and not in a limiting sense.

Claims

1. A method for propagating a fully deleted adenovirus-based gene transfer vector, comprising: (a) providing an adenovirus packaging cell line; (b) transfecting the cell line with a fully deleted adenovirus vector module, the fully deleted adenovirus vector module construct comprising at least one or more DNA inserts including both adenovirus inverted terminal repeats, a packaging signal, and one or more gene sequences encoding one or more proteins of interest, but no adenovirus structural genes; and (c) transfecting the cell line with a fully deleted adenovirus vector module.

1. A method for producing a fully deleted adenovirus-based gene transfer vector comprising: (a) transfecting a cell line with a replication-deficient circular packaging expression plasmid having a subset of adenovirus late genes including L1, L2, L3, L4, L5, E2A and E4 and a packaging signal, wherein the fully deleted adenovirus vector construct and the packaging construct are capable of transfecting an adenovirus packaging cell line to result in encapsidation of the fully deleted adenovirus-based gene transfer vector independent of a helper adenovirus vector; and (b) transfecting the cell line with an inhibitory expression vector carrying one or more expression cassettes encoding one or more antisense constructs of gene sequences encoding one or more proteins of interest on the fully deleted adenovirus vector module.

2. The method of claim 1 , wherein the fully deleted adenoviral vector module is packaged into an adenoviral capsid without the aid of an adenoviral helper virus.

3. The method of claim 1, wherein a completely deleted adenoviral vector module carrying one or more genes of interest having a harmful or toxic function to a host cell is packaged into the adenoviral capsid.

4. A method for propagating a fully deleted adenovirus-based gene transfer vector, comprising: (a) providing an adenovirus packaging cell line; (b) transfecting the cell line with a fully deleted adenovirus vector module, the fully deleted adenovirus vector module construct comprising at least one or more DNA inserts including both adenovirus inverted terminal repeats, a packaging signal, and one or more gene sequences encoding one or more proteins of interest, but no adenovirus structural genes; and (c) transfecting the cell line with a fully deleted adenovirus vector module, the fully deleted adenovirus vector module construct comprising at least one or more DNA inserts including both adenovirus inverted terminal repeats, a packaging signal, and one or more gene sequences encoding one or more proteins of interest, but no adenovirus structural genes.

1. Transfecting a cell line with a replication-deficient circular packaging expression plasmid having a subset of adenovirus late genes including L2, L3, L4, L5, E2A and E4 and a packaging signal, wherein the fully deleted adenovirus vector construct and packaging construct are capable of transfecting an adenovirus packaging cell line to result in encapsidation of the fully deleted adenovirus-based gene transfer vector independent of a helper adenovirus vector; and (d) transfecting the cell line with an RNA fragment antisense to one or more genes of interest on the fully deleted adenovirus vector module.

5. The method of claim 4, wherein the fully deleted adenoviral vector module is packaged into an adenoviral capsid without the aid of an adenoviral helper virus.

6. The method of claim 4, wherein the fully deleted adenoviral vector, one or more genes of interest having a harmful or toxic function to a host cell, are packaged into the adenoviral capsid.

7. A method for propagating a fully deleted adenovirus-based gene transfer vector, comprising: (a) providing an adenovirus packaging cell line; (b) transfecting the cell line with a fully deleted adenovirus vector module, the fully deleted adenovirus vector module construct comprising at least one or more DNA inserts including both adenovirus inverted terminal repeats, a packaging signal, and one or more gene sequences encoding one or more proteins of interest, but no adenovirus structural genes; and (c) transfecting the cell line with a fully deleted adenovirus vector module.

1. Transfecting a cell line with a replication-deficient circular packaging expression plasmid having a subset of adenovirus late genes including L2, L3, L4, L5, E2A and E4 and a packaging signal, wherein the fully deleted adenovirus vector construct and packaging construct are capable of transfecting an adenovirus packaging cell line to result in encapsidation of the fully deleted adenovirus-based gene transfer vector independent of a helper adenovirus vector; and (d) transfecting the cell line with a DNA fragment antisense to one or more genes of interest on the fully deleted adenovirus vector module.

8. The method of claim 7, wherein the fully deleted adenoviral vector module is packaged into an adenoviral capsid without the aid of an adenoviral helper virus.

9. The method of claim 7, wherein a completely deleted adenoviral vector module carrying one or more genes of interest having a harmful or toxic function to a host cell is packaged into the adenoviral capsid.

10. A method for propagating a fully deleted adenovirus-based gene transfer vector, comprising: (a) providing an adenovirus packaging cell line; (b) transducing the cell line with an encapsidated fully deleted adenovirus vector, the encapsidated fully deleted adenovirus vector construct comprising at least one or more DNA inserts including both adenovirus inverted terminal repeats, a packaging signal, and one or more gene sequences encoding one or more proteins of interest, but no adenovirus structural genes; and (c) transducing the L 1. A method for producing a cell line comprising: (a) transfecting a replication-deficient circular packaging expression plasmid having a subset of adenovirus late genes including L1, L2, L3, L4, L5, E2A and E4 and a packaging signal, wherein the fully deleted adenovirus vector construct and packaging construct are capable of transfecting an adenovirus packaging cell line to effect encapsidation of the fully deleted adenovirus-based gene transfer vector independent of a helper adenovirus vector; and (b) transfecting the cell line with an inhibitory expression vector carrying one or more expression cassettes encoding antisense constructs of gene sequences encoding one or more proteins of interest.

11. The method of claim 10, wherein the fully deleted adenoviral vector module is packaged into an adenoviral capsid without the aid of an adenoviral helper virus.

12. The method of claim 10, wherein a completely deleted adenoviral vector module carrying one or more genes of interest having a harmful or toxic function to a host cell is packaged into the adenoviral capsid.

13. A method for propagating a fully deleted adenovirus-based gene transfer vector, comprising: (a) providing an adenovirus packaging cell line; (b) transducing the cell line with an encapsidated fully deleted adenovirus vector, the encapsidated fully deleted adenovirus vector construct comprising at least one or more DNA inserts including both adenovirus inverted terminal repeats, a packaging signal, and one or more gene sequences encoding one or more proteins of interest, but no adenovirus structural genes; and (c) transducing the L 1. Transfecting a cell line with a replication-deficient circular packaging expression plasmid having a subset of adenovirus late genes including L2, L3, L4, L5, E2A and E4 and a packaging signal, wherein the fully deleted adenovirus vector construct and packaging construct are capable of transfecting an adenovirus packaging cell line to result in encapsidation of the fully deleted adenovirus-based gene transfer vector independent of a helper adenovirus vector; and (d) transfecting the cell line with fragments antisense to one or more genes of interest on the fully deleted adenovirus vector module.

14. 14. The method of claim 13, wherein the fully deleted adenoviral vector module is packaged into an adenoviral capsid without the aid of an adenoviral helper virus.

15. The method of claim 13, wherein the fully deleted adenoviral vector, one or more genes of interest having a harmful or toxic function to a host cell, are packaged into the adenoviral capsid.

16. A method for propagating a fully deleted adenovirus-based gene transfer vector, comprising: (a) providing an adenovirus packaging cell line; (b) transducing the cell line with an encapsidated fully deleted adenovirus vector module construct, the encapsidated fully deleted adenovirus vector module comprising at least one or more DNA inserts including both adenovirus inverted terminal repeats, a packaging signal, and one or more gene sequences encoding one or more proteins of interest, but no adenovirus structural genes; and (c) transducing the cell line with an encapsidated fully deleted adenovirus vector module construct comprising at least one or more DNA inserts including both adenovirus inverted terminal repeats, a packaging signal, and one or more gene sequences encoding one or more proteins of interest, but no adenovirus structural genes.

1. Transfecting a cell line with a replication-deficient circular packaging expression plasmid having a subset of adenovirus late genes including L2, L3, L4, L5, E2A and E4 and a packaging signal, wherein the fully deleted adenovirus vector construct and packaging construct are capable of transfecting an adenovirus packaging cell line to result in encapsidation of the fully deleted adenovirus-based gene transfer vector independent of a helper adenovirus vector; and (d) transfecting the cell line with a DNA fragment antisense to one or more genes of interest on the fully deleted adenovirus vector module.

17. 17. The method of claim 16, wherein the fully deleted adenoviral vector module is packaged into an adenoviral capsid without the aid of an adenoviral helper virus.

18. The method of claim 16, wherein a completely deleted adenoviral vector module carrying one or more genes of interest having a harmful or toxic function to a host cell is packaged into the adenoviral capsid.

19. 1. A method for propagating a minimally modified adenoviral gene transfer vector, comprising: (a) providing an adenoviral packaging cell line; (b) transfecting the cell line with a construct encoding a minimally modified adenoviral vector carrying two ITRs, a packaging signal, and a subset of adenoviral late genes, such as those including L1, L2, L3, L4, L5, E2A, and E4, and carrying one or more expression cassettes carrying one or more genes of interest; and (c) transfecting the cell line with an inhibitory expression vector carrying one or more expression cassettes encoding antisense constructs of gene sequences encoding one or more proteins of interest on the minimally modified adenoviral vector.

20. 20. The method of claim 19, wherein the minimally modified adenoviral vector is packaged into an adenoviral capsid without the aid of an adenoviral helper virus.

21. 20. The method of claim 19, wherein a minimally modified adenoviral vector carrying one or more genes of interest having a function harmful or toxic to a host cell is packaged into the adenoviral capsid.

22. 1. A method for propagating a minimally modified adenoviral transfer vector, comprising: (a) providing an adenoviral packaging cell line; (b) transfecting the cell line with a construct encoding a minimally modified adenoviral vector carrying two ITRs, a packaging signal, and a subset of adenoviral late genes, such as those including L1, L2, L3, L4, L5, E2A, and E4, and carrying one or more expression cassettes carrying one or more genes of interest; and (c) transfecting the cell line with an RNA fragment antisense to the one or more genes of interest on the minimally modified adenoviral vector.

23. 23. The method of claim 22, wherein the minimally modified adenoviral vector is packaged into an adenoviral capsid without the aid of an adenoviral helper virus.

24. 23. The method of claim 22, wherein a minimally modified adenoviral vector carrying one or more genes of interest having a harmful or toxic function to a host cell is packaged into the adenoviral capsid.

25. A method for propagating a minimally modified adenoviral gene transfer vector, comprising: (a) providing an adenoviral packaging cell line; (b) transfecting the cell line with a construct encoding a minimally modified adenoviral vector carrying two ITRs, a packaging signal, and a subset of adenoviral late genes, such as those including L1, L2, L3, L4, L5, E2A, and E4, and carrying one or more expression cassettes carrying one or more genes of interest; and (c) transfecting the cell line with a DNA fragment antisense to the one or more genes of interest on the minimally modified adenoviral vector.

26. 16. The method of claim 15, wherein the minimally modified adenoviral vector is packaged into an adenoviral capsid without the aid of an adenoviral helper virus.

27. The method of claim 15, wherein a minimally modified adenoviral vector carrying one or more genes of interest having a harmful or toxic function to a host cell is packaged into the adenoviral capsid.

28. A method for propagating a minimally modified adenoviral gene transfer vector, comprising: (a) providing an adenoviral packaging cell line; (b) transducing the cell line with a minimally modified adenoviral vector carrying two ITRs, a packaging signal, and a subset of adenoviral late genes, such as those including L1, L2, L3, L4, L5, E2A, and E4, and carrying one or more expression cassettes carrying one or more genes of interest; and (c) transfecting the cell line with an inhibitory expression vector carrying one or more expression cassettes encoding antisense constructs of gene sequences encoding one or more proteins of interest on the minimally modified adenoviral vector.

29. 29. The method of claim 28, wherein the minimally modified adenoviral vector is packaged into an adenoviral capsid without the aid of an adenoviral helper virus.

30. 29. The method of claim 28, wherein a minimally modified adenoviral vector carrying one or more genes of interest having a function harmful or toxic to a host cell is packaged into the adenoviral capsid.

31. 1. A method for propagating a minimally modified adenovirus-based gene transfer vector, comprising: (a) providing an adenovirus packaging cell line; (b) transducing the cell line with a minimally modified adenovirus vector carrying two ITRs, a packaging signal, and a subset of adenovirus late genes, such as those including L1, L2, L3, L4, L5, E2A, and E4, and carrying one or more expression cassettes carrying one or more genes of interest; and (c) transfecting the cell line with RNA fragments antisense to the one or more genes of interest on the minimally modified adenovirus vector.

32. 29. The method of claim 28, wherein the minimally modified adenoviral vector is packaged into an adenoviral capsid without the aid of an adenoviral helper virus.

33. 29. The method of claim 28, wherein a minimally modified adenoviral vector carrying one or more genes of interest having a function harmful or toxic to a host cell is packaged into the adenoviral capsid.

34. 1. A method for propagating a minimally modified adenovirus-based gene transfer vector, comprising: (a) providing an adenovirus packaging cell line; (b) transducing the cell line with a minimally modified adenovirus vector carrying two ITRs, a packaging signal, and a subset of adenovirus late genes, such as those including L1, L2, L3, L4, L5, E2A, and E4, and carrying one or more expression cassettes carrying one or more genes of interest; and (c) transfecting the cell line with a DNA fragment antisense to the one or more genes of interest on the minimally modified adenovirus vector.

35. 35. The method of claim 34, wherein the minimally modified adenoviral vector is packaged into an adenoviral capsid without the aid of an adenoviral helper virus.

36. The method of claim 34, wherein a minimally modified adenoviral vector carrying one or more genes of interest having a function harmful or toxic to a host cell is packaged into the adenoviral capsid.

37. A method for propagating a helper-dependent adenovirus-based gene transfer vector, comprising: (a) providing an adenovirus packaging cell line; (b) transfecting the cell line with a fully deleted adenovirus vector module, the fully deleted adenovirus vector module construct comprising at least one or more DNA inserts including both adenovirus inverted terminal repeats, a packaging signal, and one or more gene sequences encoding one or more proteins of interest, but no adenovirus structural genes; and (c) transfecting the cell line with a fully deleted adenovirus vector module.

1. Transfecting or transducing a cell line with a construct encoding an adenovirus helper virus having a subset of adenovirus late genes including L2, L3, L4, L5, E2A and E4, with or without a packaging signal, wherein the fully deleted adenovirus vector construct and packaging construct are capable of transfecting an adenovirus packaging cell line in encapsidation of the fully deleted adenovirus-based gene transfer vector independent of the helper adenovirus vector; and (d) transfecting the cell line with an inhibitory expression vector carrying one or more expression cassettes encoding one or more antisense constructs of gene sequences encoding one or more proteins of interest on the fully deleted adenovirus vector module.

38. 38. The method of claim 37, wherein the fully deleted adenoviral vector module is packaged into an adenoviral capsid without the aid of an adenoviral helper virus.

39. 38. The method of claim 37, wherein a completely deleted adenoviral vector module carrying one or more genes of interest having a harmful or toxic function to a host cell is packaged into the adenoviral capsid.

40. A method for propagating a helper-dependent adenovirus-based gene transfer vector, comprising: (a) providing an adenovirus packaging cell line; (b) transfecting the cell line with a fully deleted adenovirus vector module, the fully deleted adenovirus vector module construct comprising at least one or more DNA inserts including both adenovirus inverted terminal repeats, a packaging signal, and one or more gene sequences encoding one or more proteins of interest, but no adenovirus structural genes; and (c) transfecting the cell line with a fully deleted adenovirus vector module.

1. Transfecting or transducing a cell line with a construct encoding an adenovirus helper virus having a subset of adenovirus late genes including L2, L3, L4, L5, E2A and E4, with or without a packaging signal, wherein the fully deleted adenovirus vector construct and packaging construct can transfect an adenovirus packaging cell line to result in encapsidation of the fully deleted adenovirus-based gene transfer vector independent of the helper adenovirus vector; and (d) transfecting the cell line with an RNA fragment antisense to one or more genes of interest on the fully deleted adenovirus vector module.

41. 41. The method of claim 40, wherein the fully deleted adenoviral vector module is packaged into an adenoviral capsid without the aid of an adenoviral helper virus.

42. The method of claim 40, wherein a completely deleted adenoviral vector module carrying one or more genes of interest having a harmful or toxic function to a host cell is packaged into the adenoviral capsid.

43. A method for propagating a helper-dependent adenovirus-based gene transfer vector, comprising: (a) providing an adenovirus packaging cell line; (b) transfecting the cell line with a fully deleted adenovirus vector module, the fully deleted adenovirus vector module construct comprising at least one or more inserts including both adenovirus inverted terminal repeats, a packaging signal, and one or more gene sequences encoding one or more proteins of interest, but no adenovirus structural genes; and (c) transfecting the cell line with a fully deleted adenovirus vector module.

1. Transfecting or transducing a cell line with a construct encoding an adenovirus helper virus having a subset of adenovirus late genes including L2, L3, L4, L5, E2A and E4, with or without a packaging signal, wherein the fully deleted adenovirus vector construct and packaging construct can transfect an adenovirus packaging cell line to result in encapsidation of the fully deleted adenovirus-based gene transfer vector independent of the helper adenovirus vector; and (d) transfecting the cell line with a DNA fragment antisense to one or more genes of interest on the fully deleted adenovirus vector module.

44. 41. The method of claim 40, wherein the fully deleted adenoviral vector module is packaged into an adenoviral capsid without the aid of an adenoviral helper virus.

45. The method of claim 40, wherein a completely deleted adenoviral vector module carrying one or more genes of interest having a harmful or toxic function to a host cell is packaged into the adenoviral capsid.

46. A method for propagating a helper-dependent adenovirus-based gene transfer vector, comprising: (a) providing an adenovirus packaging cell line; (b) transducing the cell line with a packaged fully deleted adenovirus vector module construct, the packaged fully deleted adenovirus vector module comprising at least one or more DNA inserts including both adenovirus inverted terminal repeats, a packaging signal, and one or more gene sequences encoding one or more proteins of interest, but no adenovirus structural genes; and (c) transducing the cell line with a packaged fully deleted adenovirus vector module construct comprising at least one or more DNA inserts including both adenovirus inverted terminal repeats, a packaging signal, and one or more gene sequences encoding one or more proteins of interest, but no adenovirus structural genes.

1. Transfecting or transducing a cell line with a construct encoding an adenovirus helper virus having a subset of adenovirus late genes including L2, L3, L4, L5, E2A and E4, with or without a packaging signal, wherein the fully deleted adenovirus vector construct and packaging construct are capable of transfecting an adenovirus packaging cell line to result in encapsidation of the fully deleted adenovirus-based gene transfer vector independent of the helper adenovirus vector; and (d) transfecting the cell line with an inhibitory expression vector carrying one or more expression cassettes encoding one or more antisense constructs of gene sequences encoding one or more proteins of interest on the fully deleted adenovirus vector module.

47. 47. The method of claim 46, wherein the fully deleted adenoviral vector module is packaged into an adenoviral capsid without the aid of an adenoviral helper virus.

48. 47. The method of claim 46, wherein a completely deleted adenoviral vector module carrying one or more genes of interest having a harmful or toxic function to a host cell is packaged into the adenoviral capsid.

49. A method for propagating a helper-dependent adenovirus-based gene transfer vector, comprising: (a) providing an adenovirus packaging cell line; (b) transducing the cell line with a packaged fully deleted adenovirus vector module construct, the packaged fully deleted adenovirus vector module comprising at least one or more DNA inserts including both adenovirus inverted terminal repeats, a packaging signal, and one or more gene sequences encoding one or more proteins of interest, but no adenovirus structural genes; and (c) transducing the cell line with a packaged fully deleted adenovirus vector module construct comprising at least one or more DNA inserts including both adenovirus inverted terminal repeats, a packaging signal, and one or more gene sequences encoding one or more proteins of interest, but no adenovirus structural genes.

1. Transfecting or transducing a cell line with a construct encoding an adenovirus helper virus having a subset of adenovirus late genes including L2, L3, L4, L5, E2A and E4, with or without a packaging signal, wherein the fully deleted adenovirus vector construct and packaging construct can transfect an adenovirus packaging cell line to result in encapsidation of the fully deleted adenovirus-based gene transfer vector independent of the helper adenovirus vector; and (d) transfecting the cell line with an RNA fragment antisense to one or more genes of interest on the fully deleted adenovirus vector module.

50. 50. The method of claim 49, wherein the fully deleted adenoviral vector module is packaged into an adenoviral capsid without the aid of an adenoviral helper virus.

51. 50. The method of claim 49, wherein a completely deleted adenoviral vector module carrying one or more genes of interest having a harmful or toxic function to a host cell is packaged into the adenoviral capsid.

52. A method for propagating a helper-dependent adenovirus-based gene transfer vector, comprising: (a) providing an adenovirus packaging cell line; (b) transducing the cell line with a packaged fully deleted adenovirus vector module construct, the packaged fully deleted adenovirus vector module comprising at least one or more DNA inserts including both adenovirus inverted terminal repeats, a packaging signal, and one or more gene sequences encoding one or more proteins of interest, but no adenovirus structural genes; and (c) transducing the cell line with a packaged fully deleted adenovirus vector module construct comprising at least one or more DNA inserts including both adenovirus inverted terminal repeats, a packaging signal, and one or more gene sequences encoding one or more proteins of interest, but no adenovirus structural genes.

1. Transfecting a cell line with a replication-deficient circular packaging expression plasmid having a subset of adenovirus late genes including L2, L3, L4, L5, E2A and E4 and a packaging signal, wherein the fully deleted adenovirus vector construct and packaging construct are capable of transfecting an adenovirus packaging cell line to result in encapsidation of the fully deleted adenovirus-based gene transfer vector independent of a helper adenovirus vector; and (d) transfecting the cell line with a DNA fragment antisense to one or more genes of interest on the fully deleted adenovirus vector module.

53. 53. The method of claim 52, wherein the fully deleted adenoviral vector module is packaged into an adenoviral capsid without the aid of an adenoviral helper virus.

54. 53. The method of claim 52, wherein a completely deleted adenoviral vector module carrying one or more genes of interest having a harmful or toxic function to a host cell is packaged into the adenoviral capsid.

55. A method for propagating a fully deleted adenovirus-based gene transfer vector, comprising: (a) providing an adenovirus packaging cell line; (b) transfecting the cell line with a fully deleted adenovirus vector module, the fully deleted adenovirus vector module construct comprising at least one or more DNA inserts including both adenovirus inverted terminal repeats, a packaging signal, and one or more gene sequences encoding one or more proteins of interest, but no adenovirus structural genes; and (c) transfecting the cell line with a fully deleted adenovirus vector module. and transfecting a cell line with a replication-deficient circular packaging expression plasmid carrying one or more expression cassettes encoding one or more antisense constructs of gene sequences encoding one or more proteins of interest on a fully deleted adenoviral vector module, the expression plasmid having a subset of adenoviral late genes including L1, L2, L3, L4, L5, E2A and E4 and a packaging signal, wherein the fully deleted adenoviral vector construct and the packaging construct are capable of transfecting an adenoviral packaging cell line to result in encapsidation of the fully deleted adenoviral-based gene transfer vector independent of a helper adenoviral vector.

56. 56. The method of claim 55, wherein the completely deleted adenoviral vector module is packaged into an adenoviral capsid without the aid of an adenoviral helper virus.

57. 56. The method of claim 55, wherein a completely deleted adenoviral vector module carrying one or more genes of interest having a harmful or toxic function to a host cell is packaged into the adenoviral capsid.

58. A method for propagating a fully deleted adenovirus-based gene transfer vector, comprising: (a) providing an adenovirus packaging cell line; (b) transducing the cell line with an encapsidated fully deleted adenovirus vector, the encapsidated fully deleted adenovirus vector construct comprising at least one or more DNA inserts including both adenovirus inverted terminal repeats, a packaging signal, and one or more gene sequences encoding one or more proteins of interest, but no adenovirus structural genes; and (c) transducing the L and transfecting a cell line with a replication-deficient circular packaging expression plasmid carrying one or more expression cassettes encoding one or more antisense constructs of gene sequences encoding one or more proteins of interest on a fully deleted adenoviral vector module, the expression plasmid having a subset of adenoviral late genes including L1, L2, L3, L4, L5, E2A and E4 and a packaging signal, wherein the fully deleted adenoviral vector construct and the packaging construct are capable of transfecting an adenoviral packaging cell line to result in encapsidation of the fully deleted adenoviral-based gene transfer vector independent of a helper adenoviral vector.

59. 59. The method of claim 58, wherein the completely deleted adenoviral vector module is packaged into an adenoviral capsid without the aid of an adenoviral helper virus.

60. 59. The method of claim 58, wherein a completely deleted adenoviral vector module carrying one or more genes of interest having a harmful or toxic function to a host cell is packaged into the adenoviral capsid.

61. A method for propagating a helper-dependent adenovirus-based gene transfer vector, comprising: (a) providing an adenovirus packaging cell line; (b) transfecting the cell line with a fully deleted adenovirus vector module, the fully deleted adenovirus vector module construct comprising at least one or more DNA inserts including both adenovirus inverted terminal repeats, a packaging signal, and one or more gene sequences encoding one or more proteins of interest, but no adenovirus structural genes; and (c) transfecting the cell line with at least one or more adenovirus structural genes including L1, L2, L31, L41, L51, L61, L71, L81, L91, L101, L111, L121, L131, L141, L151, L161, L171, L181, L191, L201, L211, L221, L231, L241, L251, L261, L271, L281, L291, L301, L311, L321, L331, L341, L351, L361, L371, L381, L391, L401, L41, L42, L43, L44, L451, L461, L471, L481, L491, L501, L511, L512, L521, L531, L541, L551, L561, L571, L581, L591, L621, L631, L641, L651, L661, L671, L681, L691, L701, L71, L721, L731, L741, L751, L761, L771, L781, L791, L821, L and transfecting or transducing a cell line with a construct encoding an adenovirus helper virus carrying one or more expression cassettes encoding one or more antisense constructs of gene sequences encoding one or more proteins of interest on a fully deleted adenovirus vector module, with or without a packaging signal for a subset of adenovirus late genes including E2A1 and E41, wherein the fully deleted adenovirus vector construct and the packaging construct are capable of transfecting an adenovirus packaging cell line to provide for encapsidation of the fully deleted adenovirus-based gene transfer vector independent of the helper adenovirus vector.

62. 62. The method of claim 61, wherein the completely deleted adenoviral vector module is packaged into an adenoviral capsid without the aid of an adenoviral helper virus.

63. The method of claim 61, wherein a completely deleted adenoviral vector module carrying one or more genes of interest having a harmful or toxic function to a host cell is packaged into the adenoviral capsid.

64. A method for propagating a helper-dependent adenovirus-based gene transfer vector, comprising: (a) providing an adenovirus packaging cell line; (b) transducing the cell line with a packaged fully deleted adenovirus vector module construct, the packaged fully deleted adenovirus vector module comprising at least one or more DNA inserts including both adenovirus inverted terminal repeats, a packaging signal, and one or more gene sequences encoding one or more proteins of interest, but no adenovirus structural genes; and (c) transducing the cell line with an adenovirus post-transduction vector comprising L1, L2, L3, L4, L5, E2A and E4 with or without a packaging signal. and transfecting or transducing a cell line with a construct encoding an adenovirus helper virus carrying one or more expression cassettes encoding one or more antisense constructs of gene sequences encoding one or more proteins of interest on a fully deleted adenovirus vector module, the fully deleted adenovirus vector construct and packaging construct being capable of transfecting an adenovirus packaging cell line to result in encapsidation of the fully deleted adenovirus-based gene transfer vector independent of the helper adenovirus vector.

65. 65. The method of claim 64, wherein the completely deleted adenoviral vector module is packaged into an adenoviral capsid without the aid of an adenoviral helper virus.

66. 65. The method of claim 64, wherein a completely deleted adenoviral vector module carrying one or more genes of interest having a harmful or toxic function to a host cell is packaged into the adenoviral capsid.

67. A method for propagating a viral vector, comprising: (a) a producer cell; (b) delivering to the producer cell an engineered vector genome capable of providing genetic information for the propagation of the viral vector; and (c) delivering to the producer cell an inhibitory expression vector carrying one or more expression cassettes encoding one or more antisense constructs of a gene sequence encoding one or more proteins of interest encoded within the viral vector genome.

68. 68. The method of claim 67, wherein the viral vector belongs to the group of retrovirus-based or DNA virus-based vectors.

69. 68. The method of claim 67, wherein the genetic information for the propagation of the viral vector is divided into two or more DNA fragments.

70. 68. The method of claim 67, wherein the genetic information for the propagation of the viral vector is delivered as a fragment of RNA.

71. 1. A method for propagating a viral vector, comprising: (a) a producer cell; (b) delivering to the producer cell an engineered vector genome capable of providing genetic information for the propagation of the viral vector; and (c) delivering to the producer cell one or more inhibitory gene fragments of one or more antisense constructs of a gene sequence encoding one or more proteins of interest encoded within the viral vector genome.

72. 72. The method of claim 71, wherein the inhibitory gene fragment is a DNA expression vector.

73. 72. The method of claim 71, wherein the inhibitory gene fragment consists of DNA.

74. 72. The method of claim 71, wherein the inhibitory gene fragment consists of RNA.

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