WO2001057228A1

WO2001057228A1 - Methods for treatment of restenosis using adenoviral vectors and transgene products

Info

Publication number: WO2001057228A1
Application number: PCT/US2001/002046
Authority: WO
Inventors: Abraham Scaria
Original assignee: Genzyme Corporation
Priority date: 2000-02-02
Filing date: 2001-01-22
Publication date: 2001-08-09
Also published as: AU2001234508A1

Abstract

The invention relates to a method for treatment of restenosis and other vascular proliferative disorders, using an adenoviral vector encoding a cytotoxic or cytostatic molecule, along with a cell-preserving molecule in a host cell, thereby allowing persistent transgene expression in the host. The method further relates to adenoviral vectors and/or transgene products useful for the treatment of restenosis. In a preferred embodiment, the vectors comprise the gene encoding a Fas ligand, and a gene which modulates the apoptotic effects of said Fas ligand, such as p35.

Description

METHODS FOR TREATMENT OF RESTENOSIS USING ADENOVIRAL VECTORS AND TRANSGENE PRODUCTS

This application is a continuation-in-part of the U.S. patent application serial number

09/496,248, filed on February 2, 2000.

FIELD OF THE INVENTION

The present invention relates to the use of adenoviral vectors and transgenes for the treatment of restenosis and other vascular proliferation disorders or diseases, such as vein graft failure. The methods and compositions for the present invention are particularly useful for allowing the persistent expression of a transgene with cytotoxic or cytostatic effects, such as Fas ligand, in a host cell, with reduced autocellular response, such as apoptosis of the cells transfected with Fas ligand and increased persistence of expression of the adenoviral construct within the cells. This increased persistence and reduced autocellular response allows extended transgene expression in the host, while minimizing cytotoxic or cytostatic effects upon the cells expressing the transgene.

BACKGROUND OF INVENTION The ability to deliver a transgene to a target cell or tissue and have it expressed therein to produce a desired phenotypic effect depends on the development of gene transfer vehicles that can safely and efficiently deliver an exogenous nucleic acid (transgene) to the recipient cell. To this end, most efforts have focused on the use of virus-derived vectors in order to exploit the natural ability of a virus to deliver its genetic content to a target cell. Early gene transfer strategies focused on retroviral vectors which have been the vectors of choice to deliver transgene coding for a biologically active molecules for gene therapy because of their ability to integrate into the cellular genome. However, retroviral vectors, have several disadvantages which have become evident. These include their tropism for dividing cells only, the possibility of insertional mutagenesis in the host genome upon integration of the vector nucleic acid into the host cell genome, decreased expression of the transgene over time, rapid inactivation of retro viruses by the serum complement system, and the possibility of generating replication-competent retroviruses (Jolly, Cancer Gene Therapy 1 :51-64, 1994; Hodgson, Bio/Technology 13:222-225, 1995). Ndenovirus, a nuclear double-stranded DΝA virus with a genome of about 36 kb, has been well-characterized through studies in classical genetics and molecular biology (Horwitz, M.S., "Adenoviπdae and Their Replication," in Virology, 2nd edition, Fields et al , eds., Raven Press, New York, 1990) The adenovirus genome is classified into early (known as E1-E4) and late (known as L1-L5) transcriptional units, referring to the generation of two temporal classes of viral proteins The demarcation between these events is viral DNA replication

The cloning capacity of an adenoviral vector is proportional to the size of the adenovirus genome present in the vector. For example, a cloning capacity of about 8 kb can be created from the deletion of certain regions of the virus genome dispensable for virus growth, e g , E3, and the deletion of a genomic region such as El whose function may be restored in trans from 293 cells (Graham, J. Gen Virol. 36 59-72, 1977) or A549 cells (Imler et al., Gene Therapy 3-75-84, 1996). Such E 1 -deleted vectors are rendered replication-defective The upper limit of vector DNA capacity for optimal carrying capacity is about 105%- 108% of the length of the wild-type genome. Further adenovirus genomic modifications are possible in vector design using cell lines which supply other viral gene products in trans, e g , complementation of E2a (Zhou et al., J

Virol. 70-7030-7038, 1996), complementation of E4 (Kroughak et al., Hum. Gene Ther. 6:1575- 1586, 1995, Wang et al., Gene Ther. 2-775-783, 1995), or complementation of protein IX (Caravokyπ et al , J Virol 69-6627-6633, 1995; Kroughak et al., supra).

Adenovirus-deπved vectors have several advantages, including tropism for both dividing and non-dividmg cells, lessened pathogenic potential, ability to replicate to high titer for preparation of vector stocks, and the potential to carry large DNA inserts (Berkner, Curr Top Micro. Immunol. 158:39-66, 1992; Jolly, Cancer Gene Therapy 1 -51-64, 1994). The cloning capacity of an adenoviral vector is a factor of the deletion of certain regions of the virus genome dispensable for virus growth (e g., E3) or deletions of regions whose function is restored in trans from a packaging cell line (e g , El, with complementation of El functions by 293 cells

(Graham, supra). The upper limit for optimal packaging may be extended to about 105%- 108% of wild-type adenoviral genome length for increased carrying capacity m the vector

Genes that have been expressed to date by adenoviral vectors include p53 (Wills et al., Human Gene Therapy 5: 1079-188, 1994); dystrophm (Vincent et al., Nature Genetics 5 130-134, 1993); erythropoietin (Descamps et al., Human Gene Therapy 5 979-985, 1994), ornithine transcarbamylase (Stratford-Pemcaudet et al., Human Gene Therapy 1:241-256, 1990; We et al , J. Biol. Chem 271 -3639-3646, 1996); adenosine deam ase (Mitani et al , Human Gene Therapy 5:941-948, 1994); interleukin-2 (Haddada et al., Human Gene Therapy 4:703-711, 1993); al- antitrypsin (Jaffe et al., Nature Genetics 1 :372-378, 1992); thrombopoietin (Ohwada et al, Blood 88:778-784, 1996); cytosine deaminase (Ohwada et al, Hum. Gene Ther. 7:1567-1576, 1996); human alpha-galactosidase A (PCT No. PCT/US98/22886), human beta-galactosidase (Arthur et al, Cancer Gene Therapy 4: 17-25, 1997); interleukin-7 (Arthur et al, supra); a Heφes Simplex Virus thymidine kinase gene (U.S. Patent No. 5,763,415) and cystic fibrosis transmembrane conductance regulator (CFTR) (U.S. Patent Nos. 5,670,488 and 5,882,877; Zabner et al., J. Clin. Invest. 97: 1504-151 1, 1996).

As discussed above, El -deleted replication-defective adenoviral (Ad) vectors are attractive vehicles for gene transfer to host cells because of their ability to transduce a wide variety of dividing and non-dividing cells in vivo (Stratford-Perricaudet et al., Hum. Gene Ther. 1:241-256 (1990); Rosenfeld et al., Cell 68: 143-155 (1992); Zabner et al., Cell 75:207-216 (1993); Crystal et al., Nat. Genetics 8:42-51 (1994); Zabner et al., Nat. Genetics 6:75-83 (1994)). Such vectors have been used for transfer of the gene encoding normal human cystic fibrosis transmembrane conductance regulator (CFTR) into airway epithelial cells of experimental animals (e.g. mice, cotton rats, monkeys) and to airway epithelium of individuals with cystic fibrosis (CF) (Rosenfeld et al., supra); Zabner et al., CeU 75:207-216 (1993); Crystal et al., supra; Zabner et al., Nat. Genetics 6:75-83 (1994)). Such vectors have transiently produced normal chloride ion channel function in CF patient airway epithelial cells. Second generation adenoviral vectors have been developed, such as gutless Ad vectors, termed pseudoadenoviral vectors or PAV, which are adenoviral vectors derived from the genome of an adenovirus containing minimal cts-acting nucleotide sequences required for the replication and packaging of the vector genome and which can contain one or more transgenes (See, U.S. Patent No. 5,882,877 which covers PAV vectors and methods for producing PAV, incoφorated herein by reference). Such PAV vectors, which can accommodate up to 36 kb of foreign nucleic acid, are advantageous because the carrying capacity of the vector is optimized, while the potential for host immune responses to the vector or the generation of replication-competent viruses is reduced. PAV vectors contain the 5' inverted terminal repeat (ITR) and the 3' ITR nucleotide sequences that contain the origin of replication, and the cis-acting nucleotide sequence required for packaging of the PAV genome, and can accommodate one or more transgenes with appropriate regulatory elements.

Still another strategy used to improve the overall properties of adenoviral vector proteins as well as transgene product is the induction of peripheral T-cell tolerance to both the adenoviral vector and the transgene products which, in many cases, may be a neoantigen in the patient. It is known that activation-induced cell death in T cells, in which apoptosis of the T cells is mediated by upregulation of fas and fas ligand (fasL), is responsible for down regulation of the T-cell response and T cell homeostasis (Watanabe-Fukunaga et al., Nature 314-317, 1992; Zhou et al., J. Exp. Med. 176: 1063-1072, 1992; Nagata, Adv. Immunol. 57: 129-144, 1994, and Dhein et al., Nature 373:438-441, 1995). Recently, it has been shown that adenovirus mediated fasL expression in balloon-injured rat carotid artery, even in animals pre-immunized with adenoviral vector, resulted in effective inhibition of neointima formation as well as protection of the Ad/fasL infected cells from immune destruction (Sata et al., Proc. Natl. Acad. USA 95: 1213-

1217, 1998). The mammalian fas/CD95 ligand (fasL) cell surface protein induces apoptosis of T cells responding to foreign antigens in transplantation (Griffith et al., Science 270: 1189-1192, 1995).

The p35 gene of baculovirus encodes a protein which blocks apoptosis of baculovirus- infected cells (Clem et al, Science 254:1388-1389, 1991; Hershberger et al., J. Virol. 68:3467- 3477, 1994; Bump et al., Science 269:1885-1888, 1995; Xue et al, Nature 377: 248-251, (1995)).

Recently, it has been disclosed that pretreatment of mice with an antigen-presenting line expressing Ad/fasL can induce Ad-specific T cell tolerance (WO98/52615, WO98/51340 and Zhang et al., Nature BioTechnology 16:1045-1049). Upon subsequent intravenous administration of an Ad/lacZ vector, persistent expression of the lacZ gene was achieved in the liver for 50 days. However, there are two major drawbacks to this procedure. The first is that the method used for expression of the fasL gene in the antigen-presenting cell line involves coinfection with two different adenoviral vectors, AdLoxpfasL (Zhang et al., J. Virol. 72:2483- 2490, 1998) and AxCANCre (Kanegae et al, Nucl. Acids Res. 23:3816-3821, 1995). In this method, AxCANCre expresses the Cre recombinase and is required to turn on expression of the fasL gene from the AdLoxpfasL vector. Thus, an extra component besides the vector which contains the fas gene is required for its effective delivery. Ad vectors constitutively expressing fasL cannot be grown to high titers, because fasL induces apoptosis of the 293 cells used to propagate the Ad vectors (Larregina et al., Gene Ther. 5:563-568, 1998). Secondly, the antigen- presenting cell line used was derived from a fas-mutant B6-lpr/lpr mouse, since fasL expression can kill normal cells expressing the fas receptor (Muruve et al., Hum. Gene Ther. 8:955-963, 1997; Kang et al., Nature. Med. 3:738-743, 1997 and Larregina et al., supra). Therefore, the expression of fasL would be extremely difficult to accomplish in cells derived from normal individuals.

SUMMARY OF THE INVENTION Thus, the present invention relates to recombinant adenoviral vectors and methods for treating a patient with a vascular proliferation disorder, such as restenosis or vein graft failure. The methods of the invention comprise administering to such a patient a composition comprising a recombinant adenoviral vector. The recombinant adenoviral vector preferably comprises an adenovirus genome from which at least the adenovirus El region has been deleted. Additional adenoviral early genes may also be deleted, and at least one heterologous nucleic acid encoding a cytotoxic or cytostatic molecule is inserted into said deletion. The cytotoxic or cytostatic molecule may be any molecule which will inhibit the growth, proliferation and/or development of smooth muscle cells. In preferred embodiments, the cytotoxic or cytostatic molecule is Fas ligand, β-adrenergic receptor kinase [β-ARK], the carboxy terminus of β-ARK [β-ARKct], P21, PI 6, P27, thymidine kinase [TK], or fusions of the above. The vectors of the present invention further comprise a nucleic acid encoding a cell-preserving molecule. The cell-preserving molecule may be any molecule which is able to protect the cell, or to inhibit or mediate the cytotoxic or cytostatic molecule's autocellular effects. Preferred cell-preserving molecules include P35, bcl-2 and bcl-xL. In preferred embodiments, the present invention comprises a method for treating a patient with restenosis or other vascular proliferative disorder, said method comprising administering to said patient a composition comprising a recombinant adenoviral vector, said recombinant adenoviral vector comprising an adenovirus genome from which at least the adenovirus El region has been deleted, wherein at least one heterologous nucleic acid encoding a cytostatic or cytotoxic molecule is inserted into said deletion, and wherein said vector further comprises a nucleic acid encoding a cell-preserving molecule. In a particular preferred embodiment, the cytotoxic or cytostatic molecule is FasL, and the cell-preserving molecule is p35.

The invention also relates to a method for treatment of vascular proliferative disorders, such as restenosis and vein graft disorder, using an adenoviral vector and/or transgene product in a host or host cell, thereby allowing persistent transgene expression in the host. In preferred embodiments, the adenoviral vectors encode a molecule which induces apoptosis, such as FasL, along with a gene which modulates the apoptotic effects of FasL and/or reduces the autocellular response of a host to cytotoxic polypeptides encoded by a transgene without (or with minimized) adverse autocellular responses to promote the efficacy of the cytotoxic or cytostatic effect of the transgene within the host.

The method further relates to adenoviral vectors and/or transgene products useful for the treatment of restenosis. In preferred embodiments, the vectors comprise the gene encoding a cytotoxic or cytostatic molecule, along with a gene encoding a cell-preserving molecule. In a particular preferred embodiment, the cytotoxic or cytostatic molecule is FasL, and the cell- preserving molecule is one which regulates the apoptotic effects of FasL. Such genes may preferably be inserted in the place of one or more deleted adenoviral genes. In a preferred embodiment, the gene encoding the cytotoxic or cytostatic molecule is inserted into a deletion of the El adenoviral gene, and the gene encoding the cell-preserving molecule is inserted into a deletion of the E3 adenoviral gene.

These vectors can provide increased persistence in the individual to whom they are administered, thereby reducing the need for multiple readministration, as well as reduced immunologic response upon administration or readministration. The invention also relates to methods for the use of such vectors in delivering genes to cells of an individual for expression therein.

The present invention also provides for the treatment of a host suffering from a vascular proliferative disorder with an intravenous or implanted administration of a composition of cells which have been transfected with an Ad vector comprising a nucleic acid encoding fasL, as well as an inhibitor of apoptosis, such as p35. The cells may be autologous or heterologous, but are preferably autologous cells from the patient. Preferred cell types for this embodiment include endothelial cells, smooth muscle cells and myoblasts. When the transfected cells are administered to the area of vascular defect or disease, expression of fasL on the surface of the transfected cells induces apoptosis of the surrounding cells through the fas/fasL apoptosis pathway, while the expression of the apoptosis inhibitor within the transfected cells protects the transfected cells from apoptosis. The present invention further provides for the transfection of cells in vivo by intradermal injection of an Ad vector comprising a transgene coding for a cytotoxic or cytostatic molecule and a gene encoding a cell-preserving molecule such as p35. The transfected cells thus administered will induce apoptosis of the surrounding cells through the fas/fasL apoptosis pathway, while the expression of the apoptosis inhibitor within the transfected cells protects the transfected cells from apoptosis for persistent expression. Thus, in certain embodiments, the present invention comprises methods for treating a patient with a vascular proliferative disorder comprising administering to said patient a composition comprising a recombinant adenoviral vector, said recombinant adenoviral vector comprising an adenovirus genome from which at least the adenovirus El region has been deleted, wherein at least one heterologous nucleic acid encoding a cytotoxic or cytostatic molecule and at least one heterologous nucleic acid encoding a cell-preserving molecule are both inserted into said deletion. In such methods, the preferred cytotoxic or cytostatic molecule may be selected from the group consisting of Fas ligand, β-adrenergic receptor kinase, the carboxy terminus of β-adrenergic receptor kinase, P21, PI 6, P27, thymidine kinase, and fusions of the above. The cell-preserving molecule is preferably selected from the group consisting of p35, BCL-2 and BCL-xl. In one preferred embodiment, the cytotoxic molecule is Fas ligand and the cell preserving molecule is p35. By way of example, the vascular proliferative disorder may be restenosis or vein graft failure.

In alternative embodiments, the present invention comprises methods for treating a patient with a vascular proliferative disorder comprising administering to said patient a composition comprising a recombinant adenoviral vector, said recombinant adenoviral vector comprising an adenovirus genome from which at least the adenovirus El region and part of the E3 region have been deleted, wherein at least one heterologous nucleic acid encoding a cytotoxic or cytostatic molecule is inserted into the El deletion and at least one heterologous nucleic acid encoding a cell-preserving molecule is inserted into the E3 deletion. Again, the cytotoxic or cytostatic molecule is preferably selected from the group consisting of Fas ligand, β-adrenergic receptor kinase, the carboxy terminus of β-adrenergic receptor kinase, P21, PI 6, P27, thymidine kinase, and fusions of the above, and the cell-preserving molecule is preferably selected from the group consisting of p35, BCL-2 and BCL-xl. By way of example, the vascular proliferative disorder may be restenosis or vein graft failure.

The present invention further includes methods for treating a patient with restenosis, said method comprising administering to said patient a composition comprising a recombinant adenoviral vector, said recombinant adenoviral vector comprising an adenovirus genome from which at least the adenovirus El region has been deleted, wherein at least one heterologous nucleic acid encoding a Fas ligand molecule is inserted into said deletion, and wherein said vector further comprises a nucleic acid encoding a p35 molecule.

Alternatively, the invention comprises methods for treating a patient with restenosis, said method comprising administering to said patient a composition comprising a recombinant adenoviral vector, said recombinant adenoviral vector comprising an adenovirus genome from which at least the adenovirus El region and a portion of the E3 region have been deleted, wherein at least one heterologous nucleic acid encoding a Fas ligand molecule is inserted into said El deletion, and wherein at least one heterologous nucleic acid encoding a p35 molecule is inserted into said E3 deletion.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention may be further understood with reference to the attached drawings, of which,

Figure 1 shows an Ad2 vector encoding fasL and p35.

Figure 2 shows the results of the LDH assay.

Figure 3 shows a DeAd vector genome.

Figure 4 shows a map of the plasmid pCMV. Figure 5 shows a map of the plasmid pAd/E4+/E3Δ2.9.

Figure 6 shows an Ad2 vector encoding a transgene, fasL and p35.

Figure 7 shows the effectiveness of pAd2/fasL/p35 in reducing restenosis in injured arteries.

DETAILED DESCRIPTION OF THE INVENTION

Studies from several laboratories have suggested that administration of El -deleted adenoviral (Ad) gene transfer vectors result only in transient transgene expression in immunocompetent animals. We have previously demonstrated that El-deleted Ad vectors can lead to persistent transgene expression in the liver and lung of immunocompetent mice, despite the presence of adenovirus-specific CTLs. It has now been found that El -deleted Ad vectors expressing a cytotoxic or cytostatic gene can be administered to smoothe muscle cells or successfully administered to patients with vascular proliferative disorders, such as restenosis or vein graft failure, in order to improve or remedy the vascular proliferative disorder, for example by inhibiting restenosis. These same vectors may give only transient transgene expression due to cytotoxic or cytostatic effects of the transgene. For example, cells which express FasL may be subject to increased apoptotic death induced by the FasL expressed within. In order to decrease or evade the cytotoxic or cytostatic effects of such Ad vector and/or transgene product we are using two approaches (a) further deletion of nonessential viral genes and (b) inclusion of genes encoding cell-preserving molecules. In the preferred embodiment of the present invention, Ad vectors are constructed that express the p35 gene from baculovirus. The p35 protein from baculovirus inhibits apoptosis by inhibiting several different caspases. In vitro studies demonstrate that Ad/p35 vectors inhibit both TNF and FasL mediated apoptosis of several human, monkey and mouse cell lines. Preliminary studies in mice suggest that the Ad/p35 vector is capable of reducing the in vivo liver toxicity associated with administration of high doses of Ad vector to the liver.

The present invention is directed to improved methods of treating vascular proliferative disorders by administering Ad vectors encoding a cytotoxic or cytostatic molecule, while diminishing or inhibiting adverse cytotoxic or cytostatic responses in host cells which express an Ad vector comprising a transgene coding for a cytotoxic or cytostatic molecule. The nucleic acid encoding a cytotoxic or cytostatic molecule and a cell-preserving molecule may be cloned into any recombinant adenoviral vector suitable for the delivery of a desired nucleic acid to a recipient cell. In preferred aspects of the invention, the adenoviral (Ad) vector is derived from adenovirus serotype 2 (Ad 2) and has a substantially deleted El and E3 region. Other adenovirus serotypes can also be used as backbones for the adenoviral vector including, ter alia, Ad 5, Ad 6, Ad 7, Ad 9, Ad 12, Ad 15, Ad 17, Ad 19, Ad 20, Ad 22, Ad 26, Ad 27, Ad 28, Ad 30 and Ad 39. From these enumerated adenovirus serotypes, Ad 2, Ad 5, Ad 6, Ad 7 and Ad 17 are preferred.

In one embodiment of the invention, the adenoviral vector comprises an adenovirus genome in which the El region and a 1.6 kb portion in the E3 region of the adenovirus genome from nucleotides 29292-30840 are deleted as disclosed in U.S. Application Ser. No. 08/839,553, incoφorated herein by reference. In a specific embodiment of the invention, the adenoviral vector is Ad2/CMV/E3Δ 1.6.

In other aspects of the invention, the vector is a partially-deleted adenoviral (DeAd) vector in which a majority of the adenovirus early genes (E1-E4) required for virus replication have been deleted from the vector. The DeAd vector is described in co-pending provisional application Ser. Nos. 60/083,841 filed May 1, 1998 and 60/118,118 filed February 1, 1999 and their corresponding international application No. PCT/US99/09590 filed April 30, 1999, incoφorated herein by reference. The deleted adenovirus genes, with the possible exception of E3, which is not required for replication, are inserted into a producer cell chromosome under the control of a conditional promoter in order to facilitate vector production. A preferred producer cell is the human 293 cell line which was established by transfecting human embryonic kidney cells with fragments of the Ad5 genome and selecting for a transformed phenotype (Graham et al., J. Gen Virol. 36:59-72, 1997). In addition, the A549 cell line and the KB cell line may also be used (all of which are available from ATCC).

In further aspects of the invention, the adenoviral vectors are pseudoadenoviral vectors (PAV) which are derived from the genome of an adenovirus which contain minimal cis-acting nucleotide sequences required for the replication and packaging of the vector genome and which can contain one or more transgenes (See, U.S. Patent No. 5,882,877 which covers PAV vectors and methods for producing PAV, incoφorated herein by reference). Such PAV vectors can accommodate up to 36 kb of foreign nucleic acid.

Additionally, the heterologous nucleic acid encoding cytotoxic or cytostatic molecules, e.g., FasL or 3-ARK, and the nucleic acids encoding the cell-preserving molecule, e.g., p35, may be operably linked to expression control sequences, e.g., a promoter that directs expression of the transgene or the cytotoxic or cytostatic molecule. As used herein, the phrase "operably linked" refers to the functional relationship of a polynucleotide/transgene with regulatory and effector sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences. For example, operative linkage of a nucleic acid to a promoter refers to the physical and functional relationship between the polynucleotide and the promoter, such that transcription of DNA is initiated from the promoter by an RNA polymerase that specifically recognizes and binds to the promoter, and wherein the promoter directs the transcription of RNA from the polynucleotide.

Promoter regions include specific sequences that are sufficient for RNA polymerase recognition, binding and transcription initiation. Additionally, promoter regions include sequences that modulate the recognition, binding and transcription initiation activity of RNA polymerase. Such sequences may be cis acting or may be responsive to trans acting factors. Depending upon the nature of the regulation, promoters may be constitutive or regulated. Examples of promoters are SP6, T4, T7, SV40 early promoter, cytomegalovirus (CMV) promoter and CMV-derived promoters, mouse mammary tumor virus (MMTV) steroid-inducible promoter, Moloney murine leukemia virus (MMLV) promoter, Rous sarcoma virus long terminal repeat (RSV-LTR) viral promoters, phosphoglycerate kinase (PGK) promoter and the like. Alternatively, the promoter may be an endogenous adenovirus promoter, for example the El a promoter or the Ad2 major late promoter (MLP) The promoter may also be capable of driving persistent transgene expression such as the CMV derived promoter element described by Armentano et al. in international patent application PCT/US99/00915 incoφorated herein by reference Similarly those of ordinary skill in the art can construct adenoviral vectors utilizing endogenous or heterologous polyA adenylation signals, e g , the SV40, BGH, adenoviral or native polyA signal

Where the vector contains multiple heterologous nucleic acid molecules, the nucleic acid molecules may be expressed from different promoters in order to optimize specific expression of each type of gene For example, the transgene expressing the cytotoxic or cytostatic molecule may be placed under the control of a constitutive promoter, and the transgene encoding the cell- preserving molecule may be placed under an inducible promoter Alternatively, multiple copies of the same promoter may be used for the expression of multiple nucleic acids or a single promoter may be operatively linked to heterologous nucleic acids placed adjacently to achieve simultaneous expression. Polynucleotides useful in the present invention include the p35 gene of baculovirus which encodes a protein that blocks apoptosis of baculovirus-infected cells (Clem et al., Science 254-1388-1389, 1991; Hershberger et al., J. Virol. 68-3467-3477, 1994; Bump et al., Science 269.1885-1888, 1995; Xue et al., Nature 377- 248-251, 1995) and the mammalian fas/CD95 ligand (fasL) gene which encodes a cell surface protein that induces apoptosis of T cells responding to foreign antigens in transplantation (Griffith et al., Science 270:1189-1192, 1995), the apoptosis related BCL-2 and BAX genes (Delgado et al., Neuropathology and Applied Neurobiology, 25-400-407 (1999); Zhu et al, Circulation 110(18 Suppl. -19-110 (Nov. 2, 1999); Rincheval et al, FEBS Letters 460-203-206, Oct. 29, 1999)); B-ARK and B-ARKct (Fulton et al., Surgical Forum, 47:341-345 (1996); Eckhart et al., Circulation, 98(17 SUPPL.) 1736 (1998), Choi et al., FASEB, 11( 3 )-A280 (1997)). Also useful as the cytotoxic or cytostatic genes in the present invention are pl6, p21 and p27 (Fujiwara et al., Experimental Hematology, 27- 1004-09 (1999), Nuovo et al., PNAS-USA, 96: 12754-12759 (1999); Wang et al., Circulation, 110:1700 (1999); Sato et al., Circulation, 110(18SUPP):I701 (1999); Kaul et al., Molecular and Cellular Biochemistry, 200: 183-186 (1999)). The disclosure of all of the above publications is hereby incoφorated by reference.

By way of example, in order to insert the polynucleotide/transgene into the vector, the polynucleotides/transgene and vector nucleic can be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined together with a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the termini of the restricted polynucleotide. These synthetic linkers contain nucleic acid sequences that correspond to a particular restriction site in the vector nucleic acid. Additionally, an oligonucleotide containing a termination codon and an appropriate restriction site can be ligated for insertion into a vector containing, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColEl for proper episomal replication; versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Other means are well known and available in the art.

In one preferred embodiment of the present invention, (as illustrated in Example 1), nucleic acid molecules encoding both cytotoxic or cytostatic molecule and a cell-preserving molecule is inserted into the El -deleted region of the adenoviral vector (Fig. 1 and Fig 4). Alternatively, a nucleic acid encoding an cytotoxic or cytostatic molecule is inserted into the El - deleted region of the adenoviral vector and a nucleic acid encoding a cell-preserving molecule is inserted into the E3-deleted region of the adenoviral vector (Fig. 2, Fig. 3, Fig. 5 and Fig. 6). In a preferred embodiment of the invention, a nucleic acid encoding p35 is inserted into the E3- deleted region of the adenoviral vector and a nucleic acid encoding a fasL is inserted into the El - deleted region of the adenoviral vector (Fig. 7) as described in a co-pending U.S. provisional patent application Ser. No. 60/130,415, filed April 21, 1999, incoφorated herein by reference. The present invention includes a method of using the vectors of the present invention for the treatment of vascular proliferative disorders or diseases, including restenosis and vein graft failure, by expressing a cytotoxic or cytostatic molecule using an adenoviral vector in suitable cells, such as smoothe muscle cells of an individual experiencing such vascular proliferative disorder. The adenoviral vector preferably comprises an adenovirus genome comprising a deletion of the El region and a deletion in a second region, wherein a first nucleic acid molecule is inserted into one deleted region and a second nucleic acid molecule is inserted into the other deleted region and wherein the vector is taken up by the cells. The first nucleic acid molecule of which encodes a cytotoxic or cytostatic molecule, including, inter alia, fasL, P21, PI 6, P27 [and fusions thereof] and p35. The second nucleic acid molecule encodes a cell-preserving molecule, such as p35.

Ad vectors of the present invention may introduced to the host by any route of administration, e.g. , intravenous, intranasal, intramuscular, etc. The transgene coding for a biologically active cytotoxic or cytostatic molecule can then be expressed in the absence of or with minimized autocyto toxic or autocytostatic response within the transfected host cells. The absent or reduced autocellular response allows for persistent expression of the transgene coding for a biologically active cytotoxic or cytostatic molecule with a minimized or eliminated host autocellular response. Alternatively, the Ad vector comprising a nucleic acid encoding the fasL gene and the p35 gene may be used to transfect cells ex vivo by intravenous or implanted administration, or in vivo by intradermal injection of the Ad vector or any other suitable route of administration known in the art. The transfected cells will be applied to the area of vascular disease or disorder where they can interact with the surrounding smoothe muscle cells. The adenoviral vector- transfected cells may be introduced by any route of administration, e.g., intravenous, intranasal, intramuscular, etc., which is suitable for reaching the area of vascular injury. As stated above, the transfected cells may be autologous or heterologous, and may be endothelial, smooth muscle cells, myoblasts or other cells suitable for transfection and administration.

Other routes of administration for the Ad vectors comprising the transgene coding for a biologically active molecule include conventional and physiologically acceptable routes such as direct delivery to target cells, organs or tissues, intranasal, intravenous, intramuscular, subcutaneous, intradermal, oral and other parenteral routes of administration. The Ad vectors may also be administered via inhalation of liquid or dry powder aerosols (e.g. as disclosed in U.S. provisional patent application Ser. No. 60/110,899, filed December 4, 1998, incoφorated herein by reference).

Dosage of an Ad vector which is to be administered to an individual is determined with reference to various parameters, including the condition to be treated, the age, weight and clinical status of the individual and particular molecular defect requiring the provision of a biologically active protein. The dosage is preferably chosen so that administration causes a specific phenotypic result, as measured by molecular assays or clinical markers described above. Dosages of an Ad vector of the invention which can be used for example in providing a transgene contained in a vector to an individual for persistent expression of a biologically active protein encoded by the transgene and to achieve a specific phenotypic result range from approximately 10⁸ infectious units (I.U.) to 10" I.U. for humans.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. "Dosage unit form" as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated, each unit containing a predetermined quantity of active ingredient calculated to produce the specific phenotypic result in association with the required physiological carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly depend on the unique characteristics of the adenoviral vector used in the formulation and the limitations inherent in the art of compounding. The principle active ingredient (the adenoviral vector) is compounded for convenient and effective administration with the physiologically acceptable carrier in dosage unit form as discussed above.

In addition, the Ad vectors of the present invention can be complexed with a cationic amphiphile, such as a cationic lipid, polyL-lysine (PLL), and diethylaminoethyldextran (DEAE- dextran) to provide increased efficiency of viral infection of target cells (See, e.g., WO98/22144, incoφorated herein by reference). Representative cationic lipids include those disclosed, for example, in U.S. Patent Nos. 5,238,185 and 5,767,099, with preferred lipids being GL-67 (N⁴- spermine cholesteryl carbamate), GL-53 (N⁴-spermine cholesteryl carbamate), and GL-89 (1- (N⁴-spermine)-2,3-dilaurylglycerol carbamate). Ad vectors complexed with DEAE dextran are particularly preferred. In addition, to further reduce the immune response which occurs from repeat administration of viral vectors, adenovirus and other viral vectors may be polymer-modified, e.g., complexed with polyethylene glycol (PEG), to reduce viral immunogenicity and allow for repeat administration of the vector (See, e.g., WO/98/44143, incoφorated herein by reference). Also, Ad vectors complexed with a cationic molecule, preferably DEAE, and a polyalkylene glycol polymer, e.g. PEG, as disclosed in U.S. provisional patent application Serial No. 60/097,653, filed August 24, 1998, are contemplated herein. The disclosures of this application are specifically incoφorated by reference herein.

Transfer of the transgene to the target cells by the Ad vectors of the invention can be evaluated by measuring the level of the transgene product in the target cell and correlating a phenotypic alteration associated with transgene expression. The level of transgene product in the target cell directly correlates with the efficiency of transfer of the transgene by the Ad vectors. Any method known in the art can be used to measure transgene product levels, such as ELISA, radioimmunoassay, assays using an fluorescent and chemiluminescent enzyme substrates. Expression of the transgene can be monitored by a variety of methods known in the art including, inter alia, immunological, histochemical and activity assays. Immunological procedures useful for in vitro detection of the transgene product in a sample include immunoassays that employ a detectable antibody. Such immunoassays include, for example, ELISA, Pandex microfluorimetric assay, agglutination assays, flow cytometry, serum diagnostic assays and immunohistochemical staining procedures which are well known in the art. An antibody can be made detectable by various means well known in the art. For example, a detectable marker can be directly or indirectly attached to the antibody. Useful markers include, for example, radionuclides, enzymes, fluorogens, chromogens and chemiluminescent labels.

For in vivo imaging methods, a detectable antibody can be administered to a subject and the binding of the antibody to the transgene product can be detected by imaging techniques well known in the art. Suitable imaging agents are known and include, for example, gamma-emitting radionuclides such as "'In, ^99mTc, ⁵¹Cr and the like, as well as paramagnetic metal ions, which are described in U.S. Patent No. 4,647,447. The radionuclides permit the imaging of tissues by gamma scintillation photometry, positron emission tomography, single photon emission computed topography and gamma camera whole body imaging, while paramagnetic metal ions permit visualization by magnetic resonance imaging. Relevant animals in which the transgene expression system may be assayed include, but are not limited to, mice, rats, monkeys and rabbits. Suitable mouse strains in which the transgene expression system may be tested include, but are not limited to, C3H, C57B1/6 (wild- type and nude) and Balb/c (available from Taconic Farms, Germantown, New York).

In order to determine the persistence of Ad vectors in the host, one skilled in the art can assay for the presence of these vectors by any means which identifies the transgene (and its expression), for example, by assaying for transgene or nucleic acid encoding the cytostatic or cytotoxic molecule mRNA level by RT-PCR, Northern blot or S 1 analysis, or by assaying for transgene protein expression by Western blot, immunoprecipitation, or radioimmunoassay. Alternatively, the presence of the Ad vector or the desired transgene DNA sequences per se in a host can be determined by any technique that identifies DNA sequences, including Southern blot or slot blot analysis, or other methods 'known to those skilled in the art. Where the vector contains a marker gene, e.g., lacZ coding for E. coli, β-galactosidase, the presence of the vector may be determined by these same assays or a specific functional assay that screens for the marker protein (e.g., X-gal). The persistence of a vector of the invention in the host can also be determined from the continued observation of a phenotypic alteration conferred by the administration of the Ad vector containing the transgene, e.g., the improvement or stabilization of vascular proliferative function following administration of a vector containing the gene encoding cytotoxic or cytostatic molecule to an individual with a vascular proliferative disorder. The practice of the invention employs, unless otherwise indicated, conventional techniques of recombinant DNA technology, protein chemistry, microbiology and virology which are within the skill of those in the art. Such techniques are explained fully in the literature. See, e.g., Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, Inc., New York, 1995.

The invention is further illustrated by reference to the following examples:

EXAMPLES

Example 1: Ad vectors expressing baculovirus protein p35

Construction ofAd/p35 vectors

The baculovirus p35 gene is cloned by PCR from baculovirus genomic DNA and is inserted into an expression cassette plasmid pCMV (see Figure 2) using the Xhol and Notl restriction sites (Figure 3), so that the gene of interest is cloned behind the CMV promoter and uses the SV40 polyA sequence. The entire expression cassette is then cut out using the unique Ryrll sites at either end of the cassette and cloned into the E3Δ2.9 deletion in an adenoviral plasmid, pAd/E4+/E3Δ2.9 (Figure 4) which contains the right end of Ad2. This plasmid is then cotransfected with digested DΝA from Ad2/CFTR5 so that homologous recombination occurs between the plasmid and the vector to yield vector Ad/CFTR/CMVp35 which can express CFTR and can also express baculovirus p35 from the CMV promoter.

Example 2: E1/E3 Deleted Ad Vectors Containing a Cvtotoxic/Cytostatic Gene and Cell- Preserving Gene. The Ad vectors are all El deleted (Ad2 nucleotides 358-3328 deleted) and E3 deleted

(Ad2 nucleotides 27971-30937 deleted). The transgene encoding the cytotoxic or cytostatic molecule (e.g., FasL, etc.) is inserted in the El -deleted region and cell-preserving molecule (e.g., p35, etc.) is inserted in the E3-deleted region. Both genes are driven by the CMV promoter. See Fig. 1 through Fig. 6. All vectors contain wild-type E2 and E4 regions but may also have an E4 deletion.

A cytotoxic/cytostatic transgene (e.g. Fas-L) expression cassette containing a transgene operably linked to a promoter (e.g. CMV or CMV-derived promoters) followed by a polyA signal, is cloned into an Ad2 -based vector with most of the El region deleted and retains wild- type E2 and E3 regions with open reading frame 6 (ORF6) of E4. The transgene expression cassette replaces the El region of the Ad2 vector. This vector, which encodes the cytotoxic or cytostatic molecule is termed Ad2/CT. See by analogy, Ad2/CFTR-2 and Ad2/CFTR-5 of Scaria et al., 1998, J. Virol. 72:7302-7309 and U.S. provisional patent application Ser. No. 60/130,415, incoφorated herein by reference.

The gene encoding the cell-preserving molecule (e.g., p35) is cloned by PCR and is inserted into an expression cassette plasmid pCMV (see Figure 4) using Xhol and Notl restriction sites, so that the cell-preserving molecule gene is cloned behind the CMV promoter and uses the SV40 polyA sequence. The entire expression cassette is then cut out using the unique Rsrll sites at either end of the cassette and cloned into the E3Δ2.9 deletion in an adenoviral plasmid, pAd/E4+/E3Δ2.9 (see Figure 5) which contains the right end of Ad2. This plasmid is then co- transfected with digested DΝA from Ad2/CT so that homologous recombination occurs between the plasmid and the vector to yield adenovirus [called "Ad/CT/CP"] which can express the cytotoxic/cytostatic molecule and the cell-preserving molecule from their respective promoters.

Example 3: Ad Vector Containing FasL and p35

The mouse fasL (fasL) cDΝA is cloned by PCR from a mouse testis quick-clone cDΝA library (Clonetech Laboratories) and is inserted into the expression plasmid, pCMV using the Xhol and Notl restriction sites (Figure 4), so that the cDΝA is cloned behind the CMV promoter and uses the SV40 polyA site. The entire expression cassette is then cut out using the unique Rsrll sites at either end of the cassette and cloned into the Rsrll site in the El deleted (Ad2 nucleotides 358 to 3328 deleted) pre- adenoviral plasmid called pAdEl-R which contains the left end of Ad2 including the protein IX gene and E2B sequences. This plasmid is then cotransfected with restricted DΝA from an Ad2/CFTR/p35 (such as the vector described above) vector so that homologous recombination occurs between the plasmid and the Ad vector to yield Ad/fasL/p35 which expresses fasL from the El region and p35 from the E3 region, both genes being driven by the CMV promoter (Fig. 4).

Alternatively, the transgene is inserted, as described above in Example 1, into the El- deleted region and nucleic acids encoding fasL and p35 are inserted into the E3-deleted region as a single expression cassette (as described above in Example 1 for a single molecule) such that all three, the transgene, fasL and p35, are contained within the same Ad vector (see Figure 1).

The Ad/fasL/p35 or Ad/fasL vector is produced in high titers of 10" I.U./ml in regular 293 cells used to produce El deleted Ad vectors.

To determine if the Ad/fasL/p35 or Ad/fasL vector kills infected cells, a cell killing assay is performed on CV-1 cells using an LDH kit purchased from Promega. The results are shown in Figure 2 and indicate that the Ad/fasL/p35 vector does not kill infected cells like the control Ad/fasL vector.

Example 4: DeAd Vectors

The present invention also encompasses the use of partially deleted adenoviral (DeAd) vectors into which the cytotoxic or cytostatic transgene and nucleic acid encoding the cell- preserving molecule (e.g., p35) is inserted and is explained more fully in regard to construction of a DeAd vector from an adenovirus of serotype 2 (Ad2). The Ad 2 DeAd genome is modified using conventional molecular cloning methods (See, e.g. Ausubel, F.M. et al., eds., 1987-1996, Current Protocols in Molecular Biology, John Wiley & Sons, Inc. New York; Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Glover, D.M. (ed.), DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II, 1985, incoφorated herein by reference) to achieve a vector with the characteristics as shown in Fig 1. Nucleotides between 358 and 6038 (numbering and sequence of Ad2 available from GenBank) of the adenovirus genome are deleted to remove the El coding region, the pIX gene, the pIVa2 gene, and the MLP promoter. The E2A coding region is deleted by removal of nucleotides 22666 to 23960; this removes the first ATG of the E2A protein encoding sequence, as well as a large portion of the protein coding region, without affecting genes encoded by the opposite strand of the virus. The E3 region is deleted by removal of nucleotides 27971 to 30937; this removes all E3 coding regions. The E4 region is deleted by removal of nucleotides 32815 to 35977; this removes all E4 coding regions. Through these deletions, the transgene packaging size of the vector is increased to approximately 12kb. The DeAd vector genome is further modified by positioning the dimerizer controlled promoter, the ecdysone controlled promoter, or the tetracycline doxycycline controlled promoter (Fontana et al, J. Immunol. 143:3230. 1995) in place of the MLP, i.e., just upstream from position 6038

Example 5: Construction of a DeAd Vector Containing Nucleic Acid Molecules Encoding a Cytotoxic or Cytostatic Molecule(s) and a Cell-Preserving Molecule, and Transfer of the Vector to Recipient Cells

A DeAd vector comprising the a nucleic acid(s) encoding for a cytotoxic or cytostatic molecule such as fasL, 3-ARK, 3-ARK-ct, or TK, and a cell-preserving molecule such as baculovirus p35 to cells of an individual with a vascular proliferative disorder, such as restenosis, is constructed by cloning the relevant nucleic acid (transgene encoding FasL, 3-ARK , /3-ARKct, or TK) operably linked to a promoter, such as the CMV promoter, CMV-derived promoter, the PGK promoter, α-1 antitrypsin promoter, the K19 promoter, or other promoter suitable for expression of the nucleic acid, preferably the CMV promoter, into the region downstream of position 358 in the DeAd vector or any other suitable cloning site, as disclosed in the international application PCT/US99/09590 filed April 30, 1999, incoφorated herein by reference, using conventional cloning techniques (Ausubel et al., eds., 1987-1996, Current Protocols in Molecular Biology, John Wiley & Sons, Inc. New York; Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Glover (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II; or as described in U.S. Patent No. 5,670,488, Armentano et al, J. Virol. 71: 2408-2416, 1997, Rich et al., Hum. Gene Ther. 4: 461-476, 1993, all of which are incoφorated herein by reference.

DeAd vectors comprising a nucleic acids encoding a cytotoxic/cytostatic molecule and a cell-preserving molecule are propagated in any of the producer cell lines, as disclosed in the international application PCT/US99/09590 filed April 30, 1999, incoφorated herein by reference, released from the cells by suitable techniques, such as cell lysis and purified by CsCl gradient centrifugation as described in Zabner et al., Nature Genetics 6:75-83, 1994, incoφorated herein by reference. The DeAd cytotox/cell-preserving vector is administered via DCs to pretreat a host or may be administered to a host without pretreatment as described above in Example 3. For in vivo administration, the DeAd/cytotox/cell-preserv vector may be administered by aerosol or other topical administration method to airway epithelia cells of a suitable animal (e.g. cotton rats, primates) or to individuals with cystic fibrosis (see, e.g. U.S. Patent No. 5,670,488 and Zabner et al., J. Clin. Invst. 97: 1504-1511, 1996, incoφorated herein by reference).

Expression of the transgene in host cells transfected with vectors of the present invention, and in treated animals and individuals may be detected by any means known to those of skill in the art, including detection of RNA transcripts and protein production. Phenotypic alterations correlating with expression of the relevant transgene may also be assessed. Example 5: Use of Adenoviral Vectors For the Treatment of Restenosis

Adenoviral vectors expressing fas ligand (Ad fasL) as described above have been reported to be very potent inhibitors of neointimal hypeφlasia occuring during restenosis. However, it has been extremely difficult to prepare high titer stocks of Ad/fasL because mammalian cell lines which are typically used for adenoviral vector production, such as 293 cells, express the fas receptor on the cell surface. These cells are therefore bound by fas ligand, and hence are killed prematurely by the Ad/fasL vector. In order to address this problem, we have engineered into the Ad/fasL vector the cDNA for p35, which is an inhibitor of apoptosis from baculovirus, as described in Example 2 above. The Ad/fasL/p35 vector grows to high titers of 10" IU/ml in 293 cells, compared to only 10⁹ IU/ml obtained from Ad/fasL vectors. Even though the Ad/fasL/p35 vector protects the transduced cell from apoptosis due to intracellular expression of p35, it remains able to kill adjacent, non-infected cells, such as vascular smooth muscle cells, monocytes and T cells. The efficacy of Ad/fasL/p35 was examined in a balloon- injured rat carotid artery model. The data in Figure 7 demonstrate that at 2 weeks post injury and treatment with adenoviral vectors, there was significantly reduced lesion size in the Ad/fasL/p35 treated arteries. These results were highly unexpected.

The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various embodiments and modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Various references and publications are cited within this specification, and the disclosures of all of which are hereby incoφorated herein by reference in their entireties.

Claims

WHAT IS CLAIMED

1. A method for treating a patient with a vascular proliferative disorder, said method comprising administering to said patient a composition comprising a recombinant adenoviral vector, said recombinant adenoviral vector comprising an adenovirus genome from which at least the adenovirus El region has been deleted, wherein at least one heterologous nucleic acid encoding a cytotoxic or cytostatic molecule and at least one heterologous nucleic acid encoding a cell-preserving molecule are both inserted into said deletion.

2. The method of claim 1, wherein the cytotoxic or cytostatic molecule is selected from the group consisting of Fas ligand, β-adrenergic receptor kinase, the carboxy terminus of β- adrenergic receptor kinase, P21, PI 6, P27, thymidine kinase, and fusions of the above.

3. The method of claim 2, wherein the cell-preserving molecule is selected from the group consisting of p35, BCL-2 and BCL-xl.

4. The method of claim 1, wherein the cytotoxic molecule is Fas ligand and the cell preserving molecule is p35.

5. The method of claim 1, wherein said vascular proliferative disorder is restenosis.

6. The method of claim 1, wherein said vascular proliferative disorder is vein graft failure.

7. A method for treating a patient with a vascular proliferative disorder, said method comprising administering to said patient a composition comprising a recombinant adenoviral vector, said recombinant adenoviral vector comprising an adenovirus genome from which at least the adenovirus El region and part of the E3 region have been deleted, wherein at least one heterologous nucleic acid encoding a cytotoxic or cytostatic molecule is inserted into the El deletion and at least one heterologous nucleic acid encoding a cell-preserving molecule is inserted into the E3 deletion.

8. The method of claim 7, wherein the cytotoxic or cytostatic molecule is selected from the group consisting of Fas ligand, β-adrenergic receptor kinase, the carboxy terminus of β- adrenergic receptor kinase, P21, PI 6, P27, thymidine kinase, and fusions of the above.

9. The method of claim 8, wherein the cell-preserving molecule is selected from the group consisting of p35, BCL-2 and BCL-xl.

10. The method of claim 7, wherein the cytotoxic molecule is Fas ligand and the cell preserving molecule is p35.

11. The method of claim 7, wherein said vascular proliferative disorder is restenosis.

12. The method of claim 7, wherein said vascular proliferative disorder is vein graft failure.

13. A method for treating a patient with restenosis, said method comprising administering to said patient a composition comprising a recombinant adenoviral vector, said recombinant adenoviral vector comprising an adenovirus genome from which at least the adenovirus El region has been deleted, wherein at least one heterologous nucleic acid encoding a Fas ligand molecule is inserted into said deletion, and wherein said vector further comprises a nucleic acid encoding a p35 molecule.

14. A method for treating a patient with restenosis, said method comprising administering to said patient a composition comprising a recombinant adenoviral vector, said recombinant adenoviral vector comprising an adenovirus genome from which at least the adenovirus El region and a portion of the E3 region have been deleted, wherein at least one heterologous nucleic acid encoding a Fas ligand molecule is inserted into said El deletion, and wherein at least one heterologous nucleic acid encoding a p35 molecule is inserted into said E3 deletion.

15. Use of a recombinant adenoviral vector, said recombinant adenoviral comprising an adenovirus genome from which at least the adenovirus El region has been deleted, wherein at least one heterologous nucleic acid encoding a cytotoxic or cytostatic molecule and at least one heterologous nucleic acid encoding a cell-preserving molecule are both inserted into said deletion for the preparation of a pharmaceutical composition for the treatment of a vascular proliferative disorder.