Tissue specific expression of retinoblastoma protein

Description

Both the retinoblastoma gene (RB) and the transcription factor E2F play critical roles in the regulation of cell growth [for a review, see Adams, P. & Kaelin, W., Seminars in Cancer Biology 6, 99-108 (1995)]. The RB locus is frequently inactivated in various human tumor cells. Reintroduction of the wild-type RB gene [e.g., Bookstein et al., Science 247, 712-715 (1990)] or RB protein (pRB) [e.g., Antelman et al., Oncogene 10, 697-704 (1995)] into RBneg / RBmut cells can suppress culture growth and in vivo tumorigenicity (their ability to form tumors). Since E2F is used to activate transcription of S-phase genes, its activity can be targeted by RB. RB arrests cells by blocking entry from G to S phase [e.g. Dowdy et al., Cell 73, 499-511 (1993)], but the precise mechanism of arrest remains unclear. Although E2F can form a complex with RB, the complex formation is more efficient in the presence of a protein related to E2F, DP-1. E2F-1 and DP-1 form a stable heterodimer that can bind to DNA [e.g., Qin et al., Genes and Dev. 6, 953-964 (1992)]. DP-1-E2F complexes serve to cooperatively activate transcription of E2F-dependent genes. Such transcription can be repressed by pRB in the same way as transcription activated by E2F-1 or DP-1. Transcriptional repression of genes by RB can in some cases be achieved by linking pRB to a promoter. For example, GAL4-pRB fusions bind to GAL4 DNA binding domains and repress transcription at p53, Sp-1, or AP-1 elements [Adnane et al., J. Biol. Chem. 270, 8837-8843 (1995); Weintraub et al., Nature 358, 259-261 (1995)]. Sellers et al. [Proc. Natl. Acad. Sci. 92, 11544-11548 (1995)] have described the fusion of amino acid residues 1-368 of E2F with residues 379-792 or 379-928 of RB. Chang et al. [Science 267, 518-521 (1995)] described the use of a replication-defective adenovirus RB construct to reduce neointima formation in two animal models of restenosis (a hyperproliferative disorder). The present invention is based on the surprising finding that fusions of an E2F polypeptide and an RB polypeptide are more effective in repressing E2F promoter-driven transcription than RB alone, and that these fusions are capable of inducing cell cycle arrest in a variety of cell types. Such fusions are urgently needed in the therapy of hyperproliferative disorders, such as cancer. In one aspect, the invention provides a polypeptide comprising a fusion of a transcription factor (the transcription factor comprising a DNA binding domain) and a retinoblastoma polypeptide (RB polypeptide) (the retinoblastoma polypeptide comprising a growth suppression domain). In another aspect, the invention provides DNA encoding such a fusion polypeptide. The DNA may be inserted into an adenoviral vector. In some embodiments of the invention, the transcription factor is E2F. The cyclin A binding domain of E2F may be deleted or may be non-functional. E2F may comprise about amino acid residues 95-194 or, in some preferred embodiments of the invention, about amino acid residues 95-286. The retinoblastoma polypeptide may be wild-type RB, RB56, or a variant or fragment thereof. In some preferred embodiments of the invention, the retinoblastoma polypeptide comprises amino acid residues from about 379 to about 928. Preferred amino acid substitutions are at positions 2, 608, 788, 807, and 811 in the RB polypeptide. In another aspect, the invention provides an expression vector comprising DNA encoding a polypeptide comprising a fusion of a transcription factor (the transcription factor comprising a DNA binding domain) and a retinoblastoma polypeptide (RB polypeptide) (the retinoblastoma polypeptide comprising a growth suppressor domain). In some preferred embodiments of the invention, a tissue-specific promoter is operably linked to the DNA encoding the fusion polypeptide. The tissue-specific promoter may be an alpha-actin promoter derived from smooth muscle. The E2F-RB fusion polypeptides of the invention can be used to treat hyperproliferative disorders. The hyperproliferative disorder can be cancer. In some preferred embodiments of the invention, the hyperproliferative disorder is restenosis. The fusion polypeptide or nucleic acid encoding the fusion polypeptide can also be used to coat devices used in vascular surgery (angioplasty). Figure 1A depicts the predicted amino acid sequence of E2F. Figure 1B shows the nucleotide sequence of the E2F transcription factor. Figure 2A shows the nucleotide sequence of pRB, as reported by Lee et al. (Nature 329, 642-645 (1987)). Figure 2B shows the deduced amino acid sequence of pRB. Figure 3 shows a graphical representation of the pCTM plasmid. Figure 4 shows the nucleotide sequence of plasmid pCTM. Figure 5 shows a graphical representation of the pCTMI plasmid. Figure 6 shows the nucleotide sequence of plasmid pCTMI. Figure 7 shows a graphical representation of the plasmid pCTM1E. Figure 8 shows the nucleotide sequence of plasmid pCTM1E. Figure 9 shows the fusion constructs used in the experiments. All E2F constructs start at amino acid position 95 and lack parts of the cyclin A-binding domain. E2F-437 contains a DNA-binding domain (black), a heterodimerization domain (white), and a transactivation domain (white). HU 226 662 contains Β1 (shaded). E2F-194 contains only a DNA-binding domain. E2F-286 contains a DNA-binding domain and a DP-1 heterodimerization domain. To generate E2F194-RB56-5S and E2F286-RB56-5S, the E2F constructs were fused in one step to codon 379 of RB-5s. C706F is an inactivating point mutation. Figure 10 depicts transcriptional repression of E2F-RB fusion constructs. Figure 11 (AD) depicts the expression of E2F-RB fusion proteins in mammalian cell lines. Extracts were prepared from the cells and used in E2-CAT reporter assays or FACS assays and analyzed with anti-RB monoclonal antibody. In row A, the results obtained from transfection of C33A cells with (3) RB56-H209, (4) wild-type RB56, (5) RB56-5s, (6) E2F286-5s, (7) E2F194-5s, (8) E2F194, (9) E2F286, (10) E2F437 are shown. Lane (1) is the RB56 protein standard. Lane (2) is a mock transfection. In lane B, results from transfection of Saos-2 cells with (1) RB56, (2,3) E2F194-5s, and (4,5) E2F286-5s are shown. In lane C, results from transfection of 5637 cells with (2,3) wild-type RB56, (4,5) RB56-5s, (6,7) E2F194-5s, (7,8) E2F286-5s are shown. Lane (1) is the RB56 protein standard. In lane D, results from transfection of NIH-3T3 with (3) RB56, (4) E2F286-5s, (5) E2F194-5s are shown.Band (1) is RB56 standard; band (2) is RB110 standard. Figure 12 depicts histograms of flow cytometric analyses of NIH-3T3 cells expressing RB. Figure 13A shows a comparison of the effects of a CMV-driven recombinant adenovirus (ACN56) with two isolates of an E2F-p56 fusion construct driven by the human smooth muscle alpha-actin promoter, containing amino acids 95-286 of E2F directly linked in one phase to p56 (amino acids 379-928 of RB cDNA), versus a control virus (ACN), in a 3H-thymidine uptake assay in the rat smooth muscle cell line A7R5. Figure (B) shows the effects of the same constructs in the rat smooth muscle cell line A10. Figure 14 shows the effects of the viruses described in Figure 13 in non-muscle cells. Figure (A) shows the results obtained in the breast carcinoma cell line MDA-MB468. Figure (B) shows the results obtained in the non-small cell lung carcinoma cell line H358. Figure 15 (top) shows the adenovirus infectivity of various cell lines, as determined by β-galactosidase (βgal) staining, following infection with the same amount of recombinant adenovirus expressing β-gal under the control of the CMV promoter. H358 is a non-small cell lung carcinoma cell line; MB468 is a breast carcinoma cell line; A7R5 and A10 are smooth muscle cell lines. The bottom of the figure shows the relative amount of p56 protein expressed in the same cell lines when infected with a recombinant adenovirus designated ACN56, in which the p56 cDNA is driven by a non-tissue-specific CMV promoter. Figure 16 shows the relative amounts of protein expressed in cells infected with an E2F-p56 fusion construct (ASN286-56) driven by the smooth muscle alpha-actin promoter. UN means uninfected; 50, 100, 250 and 500 mean the multiplicity of infection (MOI). The bar graph in Figure 17 depicts the intima / media area ratio (measured as inhibition of neointima formation) in cross-sections of rat carotid arteries (n=9) that were injured and treated with recombinant adenoviruses expressing β-gal, RB (ACNRB) or p56 (ACN56), all expression driven by a CMV promoter. Figure 18 shows three photographs depicting restenosis in a rat angioplasty model. The left image is from a normal animal; the middle image is from an animal injured and treated with a recombinant virus capable of expressing β-gal; and the right image is from an animal injured and treated with a recombinant adenovirus capable of expressing p56 (ACN56). Figure 19 shows the specificity of the smooth muscle-derived actin promoter, as it can selectively express the β-gal transgene in muscle cells but not in non-muscle cells. The left part of the figure compares β-gal expression in the breast carcinoma cell line MB468, infected with 1 multiplicity of infection (MOI) of CMV-driven β-gal (ACNBGAL) versus 100 MOI of the smooth muscle-derived promoter-driven construct (ASNBGAL). The right part of the figure shows β-gal expression in the rat smooth muscle cell line A7R5, infected with 1 multiplicity of infection (MOI) of CMV-driven β-gal (ACNBGAL). HU 226 662 β1 multiplicity of infection (MOI) ACNBGAL or 50 multiplicity of infection (MOI) ASNBGAL. ASNBGAL expression is seen in smooth muscle cell lines but not in non-muscle cell lines, despite the high infectivity of the cells. Figure 20 shows the extent to which a recombinant adenovirus expressing RB can transduce rat coronary arteries. Arteries treated with recombinant adenovirus (1*10 pfu) were cultured for two days after balloon injury and infection. Transverse sections were fixed and an RB-specific antibody was used to detect the presence of RB protein in the tissue. ACN was used as a control virus. RB protein staining was evident in the ACNRB-treated sample, especially at higher magnifications. Figure A21 shows a comparison of the effects of a CMV-driven p56 recombinant adenovirus (ACN56E4) with an E2F-p56 fusion construct driven by the human smooth muscle alpha-actin promoter (ASN286-56) and control adenovirus constructs containing the CMV or smooth muscle actin promoter without a 3'-direction (direction of synthesis) transgene (isolates ACNE3 or ASBE3-2 are shown, respectively). The assays are based on 1H-thymidine incorporation in a rat smooth muscle cell line (A7R5) or a non-muscle cell line (MDA-MB468, breast carcinoma). The results demonstrate muscle tissue specificity when using the smooth muscle alpha-actin promoter and specific inhibition with both the p56 and E2F-p56 transgenes compared to their respective controls. The invention provides RB-E2F fusion constructs, including fusion polypeptides and nucleic acids encoding them and expression vectors, and methods for using such constructs in the treatment of hyperproliferative diseases. Any E2F can be used, typically E2F-1, -2, -3, -4 or -5 [see, for example, Wu et al., Mol. Cell. Biol. 15, 2536-2546 (1995); Ivey-Hoyle et al., Mol. Cell. Biol. 13, 7802 (1993); Vairo et al., Genes and Dev. 9, 869 (1995); Beijersbergen et al., Genes and Dev. 8, 2680 (1994); Ginsberg et al., Genes and Dev. 8, 2665 (1994); Buck et al., Oncogene 11, 31 (1995)], typically E2F-1. Typically, the E2F polypeptide comprises at least the DNA binding domain of E2F, and optionally may comprise the heterodimerization domain and / or the transactivation domain. The cyclin A binding domain is absent or non-functional. The nucleotide and amino acid sequences of E2F described herein are from Genbank HUME2F and are shown in Figs. 1B and 1A.Such an E2F polypeptide-encoding nucleic acid, preferably DNA, is fused in-frame to a nucleic acid encoding an RB polypeptide. The RB polypeptide may be any RB polypeptide, for example, conservative amino acid variants, allelic variants, mutants containing substituted, deleted or inserted amino acids, or fragments thereof. The growth suppression domain (i.e., amino acid residues 379-928) of the RB polypeptide is preferably functional [Hiebert et al., MCB 13, 3384-3391 (1993); Qin et al., Genes and Dev. 6, 953-964 (1992)]. In some preferred embodiments of the invention, wild-type pRB110 is used. More preferably, truncated versions of RB, RB56, are used. RB56 is a 379-928. [Hiebert et al., MCB 13, 3384-3391 (1993); Qin et al., Genes and Dev. 6, 953-964 (1992)]. According to some preferred embodiments of the invention, RB 2.,. Amino acid variations at positions 608, 612, 788, 807 or 811, either singly or in combination, are used. The RB56-5s variant is a wild-type RB56 variant at positions 608, It has alanine substitutions at positions 612, 788, 807 and 811. The amino acids and nucleic acids of RB are numbered according to the RB sequence reported by Lee et al. [Nature 329, 642-645 (1987)], which is incorporated herein by reference in its entirety for all purposes (Figure 2). The nucleic acid encoding the polypeptide of the invention may be DNA or RNA. The term "coding nucleic acid sequence" means a nucleic acid that directs the expression of a specific protein or peptide. The nucleic acid sequence may be a sequence consisting of a DNA strand that is transcribed into RNA or an RNA sequence that is translated into protein. The nucleic acid sequences may be full-length nucleic acid sequences as well as partial sequences derived from a full-length protein. It is further understood that the sequence may include degenerate codons of the native sequence or sequences that may be introduced to provide codon preference in a specific host cell. The term "vector" is intended to include viral expression systems, autonomously replicating circular DNA (plasmids), and expression and non-expression plasmids. When a recombinant microorganism or cell culture is described as a host for an "expression vector", this includes both extrachromosomal circular DNA and DNA integrated into the host chromosome(s). When a vector is maintained in a host cell, the vector can be stably replicated by the cells during mitosis as an autonomous structure or can be integrated into the host genome. A vector contains multiple genetic elements, arranged in a positional and sequential manner, i.e., operably linked to other necessary elements, such that the nucleic acid encoding the fusion polypeptide in the vector can be transcribed and, if necessary, translated in transfected cells. The term “gene” as used herein refers to a nucleic acid sequence encoding a polypeptide. This definition is ma4 HU 226 662 Β1 includes various sequence polymorphisms, mutations and / or sequence variants in which such changes do not affect the function of the gene product. The term “gene” is intended not only to refer to coding sequences, but also to regulatory regions such as promoters, enhancers and termination regions. The term also includes all introns and other DNA sequences that are generated from the mRNA transcript during splicing, together with variants that result from alternative splicing. The term "plasmid" refers to an autonomous, circular DNA molecule that is capable of replication in a cell and can be of the expression or non-expression type. When a recombinant microorganism or cell culture is described as a host for an "expression plasmid", this refers to both this extrachromosomal circular DNA molecule and the DNA integrated into the chromosome(s) of the host. When a plasmid is maintained in a host cell, the plasmid can be stably replicated by the cells during mitosis as an autonomous structure or can be integrated into the host genome. A “recombinant protein” or “recombinantly produced protein” refers to a peptide or protein produced by the use of non-native cells that do not have an endogenous copy of the DNA capable of expressing the protein. The cells produce the protein because they have been genetically altered by the introduction of the appropriate nucleic acid sequence. The recombinant protein is not found associated with proteins or other subcellular components normally associated with the cell producing the protein. The terms “protein” and “polypeptide” are interchangeable. The constructs of the invention are generally produced in an expression vector containing the following elements, sequentially linked at an appropriate distance for functional expression: a tissue-specific promoter, a transcription initiation site, a 3'-terminal untranslated region, a 5'-terminal mRNA leader sequence, a nucleic acid sequence encoding a polypeptide of the invention, and a polyadenylation signal. Such a linkage is referred to as "operably linked." Enhancer sequences and other sequences that promote expression and / or secretion may also be included in an expression vector. It may also include additional genes, for example, those encoding drug resistance, to allow selection or screening for the presence of the recombinant vector. Such additional genes may include, for example, genes encoding neomycin resistance, multidrug resistance, thymidine kinase, beta-galactosidase, dihydrofolate reductase (DHFR), and chloramphenicol acetyltransferase. In the present invention, a promoter that is primarily active in the tissue of interest is preferably used for tissue-specific expression of the RB constructs of the present invention. Tissue-specific promoters include, for example, the creatine kinase promoter, which is used to drive expression of dystrophin cDNA in muscle and heart tissue [Cox et al., Nature 364, 725-729 (1993)]; immunoglobulin heavy or light chain promoters for expression of suicide genes in B cells [Maxwell et al., Cancer Res. 51, 4299-4304 (1991)]. Endothelial cell-specific regulatory regions have also been characterized [Jahroudi et al., Mol. Cell. Biol. 14, 999-1008 (1994)]. Amphoteric retroviral vectors have been constructed containing the thymidine kinase gene from herpes simplex virus under the control of either albumin or alpha-fetoprotein promoters [Huber et al., Proc. Natl. Acad. Sci. USA 88, 8039-8043 (1991)] to target liver cell lines and hepatoma cells, respectively. Such tissue-specific promoters have been incorporated into retroviral vectors [Hartzoglou et al., J. Biol. Chem. 265, 17285-17293 (1990)] and adenoviral vectors [Friedman et al., Mol. Cell. Biol. 6, 3791-3797 (1986); Wills et al., Cancer Gene Therapy 3, 191-197 (1995)] and still retain their tissue specificity. In the present invention, the preferred promoter for tissue-specific expression of exogenous genes is the smooth muscle alpha-actin promoter. Reddy et al. [J. Cell. Biology 265, 1683-1687 (1990)] reported the isolation and nucleotide sequence of the promoter, while Nakano et al. [Gene 99, 285-289 (1991)] reported the 5'-terminal transcriptional regulatory elements and the first intron regions of the human smooth muscle (aortic type) alpha-actin gene. Petropoulos et al. (1992) J. Virol. 66:3391-3397 compared the expression of bacterial chloramphenicol synthase (CAT) under the control of a chicken skeletal muscle alpha-actin promoter and a cytoplasmic beta-actin promoter. These constructs were inserted into a retroviral vector and used to infect chicken eggs. Tissue-specific expression elements for liver, such as HMG-CoA reductase promoter [Luskey, Mol. Cell. Biol. 7 (5), 1881-1893 (1987)]; sterol regulatory element 1 [SRE-1; Smith et al., J. Biol. Chem. 265 (4), 2306-2310 (1990)]; phosphoenolpyruvate carboxykinase (PEPCK) promoter [Eisenberger et al., Mol. Cell BioL. 12 (3), 1396-1403 (1992)]; human C-reactive protein (CRP) promoter [Li et al., J. Biol. Chem. 265 (7), 4136-4142 (1990)]; human glucokinase promoter [Tanizawa et al., Mol. Endocrinology 6 (7), 1070-81 (1992)]; cholesterol 7-alpha-hydroxylase (CYP-7) promoter [Lee et al., J. Biol. Chem. 269 (20), 14681-9 (1994)]; beta-galactosidase alpha-2,6-sialyltransferase promoter [Svensson et al., J. Biol. Chem. 265 (34), 20863-8 (1990)]; insulin-like growth factor binding protein (IGFBP-1) promoter [Babajko et al., Biochem. Biophys. Res. Comm. 196 (1), 480-6 (1993)]; aldolase B promoter [Bingle et al., Biochem. J. 294 (Pt2), 473-9 (1993)]; human transferrin promoter [Mendelzon et al., Nucl.Acids Res. 18 (19), 5717-21 (1990)]; type I collagen promoter [Houglum et al., J. Clin. Invest. 94 (2), 808-14 (1994)]. Tissue-specific expression elements for prostate, e.g. prostate-derived acid phosphatase HU 226 662 β1 (PAP) promoter [Bánás et al., Biochim. Biophys. Acta 1217 (2), 188-94 (1994)]; prostate-derived secretory protein 94 (PSP 94) promoter [Nolet et al., Biochim. Biophys. Acta 1098 (2), 247-9 (1991)]; prostate-specific antigen complex promoter [Casper et al., J. Steroid Biochem. Mól. Biol. 47 (1-6), 127-35 (1993)]; human glandular kallikrein gene promoter (hgt-1) [Lilja et al., World J. Urology 11 (4), 188-91 (1993)]. Tissue-specific expression elements for gastric tissue include the promoter for the alpha subunit of human H+ / K+-ATPase [Tanúra et al., FEBS Letters 298 (2-3), 137-41 (1992)]. Tissue-specific expression elements for the pancreas include the promoter for pancreas-associated protein (PAP) [Dusetti et al., J. Biol. Chem. 268 (19), 14470-5 (1993)]; the elastase-1 transcription enhancer [Kruse et al., Genes and Development 7 (5), 774-86 (1993)]; the promoter for pancreas-specific amylase and elastase enhancer [Wu et al., Mol. Cell. Biol. 11 (9), 4423-30 (1991); Keller et al., Genes & Dev. 4 (8), 1316-21 (1990)]; the promoter for the pancreatic cholesterol esterase gene [Fontaine et al., Biochemistry 30 (28), 7008-14 (1991)]. Tissue-specific expression elements for endometrium include, for example, the uteroglobin promoter [Helftenbein et al., Ann. NY Acad. Sci. 622, 69-79 (1991)]. Tissue-specific expression elements for adrenal cells include, for example, the promoter for the cholesterol side chain cleavage (SCC) enzyme [Rice et al., J. Biol. Chem. 265, 11713-20 (1990)]. Tissue-specific expression elements for the general nervous system include, for example, the promoter for gamma-enolase (neuron-specific enolase, NSE) [Forss-Petter et al., Neuron 5(2), 187-97 (1990)]. Tissue-specific expression elements for the brain include, for example, the neurofilament heavy chain (NF-H) promoter [Schwartz et al., J. Biol. Chem. 269 (18), 13444-50 (1994)]. Tissue-specific expression elements for lymphocytes include the human CGL-1 / granzyme-B promoter [Hanson et al., J. Biol. Chem. 266 (36), 24433-8 (1991)]; the promoters for terminal deoxytransferase (TdT), lambda-5, VpreB and lek (lymphocyte-specific tyrosine protein kinase p561ck) [Lo et al., Mol. Cell. Biol. 11 (10), 5229-43 (1991)]; the human CD2 promoter and its 3'-terminal transcriptional enhancer [Laké et al., EMBO J. 9 (10), 3129-36 (1990)] and the human NK and T-cell-specific activation (NKG5) promoter [Houchins et al., Immunogenetics 37 (2), 102-7 (1993)]. Tissue-specific expression elements for colon include the pp60c-src tyrosine kinase promoter [Talamonti et al., J. Clin. Invest. 91 (1), 53-60 (1993)]; organ-specific neoantigens (OSNs), mw 40 kDa (p40) promoter [llantzis et al., Microbiol. Immunol. 37 (2), 119-28 (1993)]; colon-specific antigen-P promoter [Sharkey et al., Cancer 73 (3 supp.), 864-77 (1994)]. Tissue-specific expression elements for breast cells include the human alpha-lactalbumin promoter [Thean et al., British J. Cancer 61 (5), 773-5 (1990)]. Other elements to promote specificity of expression may be present in a tissue of interest, such as secretory leader sequences, enhancers, nuclear localization signals, endosmolytic peptides, etc. These elements are preferably derived from the tissue of interest where they promote specificity. Techniques for nucleic acid manipulation of nucleic acid sequences of the invention include, for example, subcloning of nucleic acid sequences encoding polypeptides into expression vectors, labeling of probes, DNA hybridization, and the like, which are generally described in Sambrook et al., "Molecular Cloning - A Laboratory Manual" (2nd ed.), vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989), which is incorporated herein by reference. This manual is hereinafter referred to as "Sambrook et al. Once DNA encoding a sequence of interest has been isolated and cloned, the encoded proteins can be expressed in a variety of recombinantly produced cells. It is expected that those skilled in the art will be familiar with a variety of expression systems for expressing proteins encoded by DNA. We will not attempt to describe in detail the various known methods for expressing proteins in prokaryotes or eukaryotes. Briefly, expression of natural or synthetic nucleic acids encoding a sequence of interest is typically achieved by operably linking the DNA or cDNA to a promoter (which may be constitutive or inducible) and then inserting it into an expression vector. Vectors may be capable of replication and integration in prokaryotes or eukaryotes. Typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters suitable for expression of the polynucleotide sequence of interest. For the purpose of high-level expression of the cloned gene, it is desirable to construct expression plasmids that contain, at a minimum, a strong promoter to control transcription, a ribosome binding site for translation initiation, and a transcription / translation terminator.Expression vectors may also contain non-specific expression cassettes that contain at least one independent terminator sequence, sequences that allow plasmid replication in both eukaryotes and prokaryotes (i.e. shuttle vectors), and selectable markers that can be used in both prokaryotic and eukaryotic systems. See Sambrook et al. The E2F-RB fusion constructs of the invention can be delivered to the tissue of interest in vivo or ex vivo using a variety of methods. In some preferred embodiments of the invention, the method for delivering the vector into cells can be microinjection, calcium phosphate precipitation, liposome fusion, or biolistic methods. In further preferred embodiments of the invention, the DNA is directly taken up by the tissue of interest. In other preferred embodiments of the invention, the constructs are delivered via a viral vector6 HU 226 662 Packaged in a Β1 system to facilitate delivery into cells. Vital vector systems useful in the present invention include, for example, adenovirus, herpesvirus, adeno-associated virus, mouse minute virus (MVM), HIV, sindbisvirus, and retroviruses such as Rous sarcoma virus and MoMLV. The constructs of the present invention are typically inserted into such vectors to allow packaging of the E2F-RB expressing construct, typically accompanied by vital DNA, infection of a susceptible host cell, and expression of the E2F-RB gene. A particularly preferred vector is the adenovirus vector described by Wills et al. [Journal of Gene Therapy 5, 1079-1088 (1994)]. In another preferred embodiment of the invention, the recombinant DNA constructs of the invention are conjugated to a cellular receptor ligand to promote uptake (e.g., invagination of coated cisternae and endosome internalization) via a DNA binding group [Wu et al., J. Biol. Chem. 263, 14621-14624 (1988); WO 92 / 06180]. For example, the DNA constructs of the invention can be linked to asialooromucide, a ligand for asialo-glycoprotein receptors of hepatocytes, via a polylysine group. Similarly, the viral envelopes used to package the constructs of the invention can be modified by adding receptor ligands or receptor-specific antibodies to enable receptor-mediated endocytosis into specific cells (e.g., WO 93 / 20221, WO 93 / 14188; WO 94 / 06923). In some preferred embodiments of the invention, the DNA constructs of the invention are linked to viral proteins, such as adenovirus particles, to facilitate endocytosis [Curiel et al., Proc. Natl. Acad. Sci. USA 88, 8850-8854 (1991)]. In other preferred embodiments of the invention, the molecular conjugates of the invention can include microtubule inhibitors (WO / 9406922); synthetic peptides mimicking influenza virus hemagglutinin [Piánk et al., J. Biol. Chem. 269, 12918-12924 (1994)]; and nuclear localization signals, such as SV40 T-antigen (WO 93 / 19768). In some preferred embodiments of the invention, the fusion polypeptides of the invention are administered directly to a patient in need of treatment. "A therapeutically effective dose is a dose of polypeptide sufficient to prevent or reduce the severity of a hyperproliferative disorder. As used herein, the term "hyperproliferative cells" refers, for example, to cells that have the ability to grow autonomously, i.e., to exist and reproduce themselves independently of normal regulatory mechanisms. Hyperproliferative diseases can be categorized as pathological disorders, i.e., as a condition that differs from normal cells and is characteristic of a given disease, or as non-pathological disorders, i.e., as a condition that differs from normal cells but is not associated with a disease state.Pathological hyperproliferative cells are characteristic of the following disease states: restenosis, diabetic retinopathy, thyroid hyperplasia, Grave's disease, psoriasis, benign prostatic hypertrophy, Li-Fraumeni syndrome, e.g. breast cancer, sarcomas and other neoplasias, bladder cancer, colon cancer, lung cancer, various leukemias and lymphomas. Non-pathological hyperproliferative cells can be found, for example, in mammary duct epithelial cells during the development of milk production and in cells associated with wound healing. Pathological hyperproliferative cells typically lose contact inhibition and their selective adhesion capacity decreases, which results in further impairment of intracellular communication. These changes include stimulation of division and secretion of proteolytic enzymes. The constructs of the invention are useful in the therapy of various cancers and other conditions in which administration of RB is beneficial, such as peripheral vascular disease and diabetic retinopathy. While any tissue for which a tissue-specific expression element, such as a promoter, can be identified can be targeted, tissue-specific administration of an RB construct is of particular interest in hyperproliferative disorders, such as restenosis, in which case the smooth muscle-derived actin promoter is advantageously used. The compositions of the invention are formulated into a form acceptable for administration to a mammal, preferably a human, by methods known to those skilled in the art. In some preferred embodiments of the invention, the compositions of the invention may be administered directly into tissue by injection or into a blood vessel supplying the tissue in question. In further preferred embodiments of the invention, the compositions of the invention are administered "locoregionally", i.e. intravesically, intralesionally and / or topically. In other preferred embodiments of the invention, the compositions of the invention are administered systemically by injection, inhalation, suppository, transdermal delivery, etc. In further preferred embodiments of the invention, the compositions are administered via a catheter or by a device that allows access to difficult-to-reach tissues, such as internal organs.The compositions of the invention may also be administered using a larger kit, in implants or in encapsulated forms that allow for slow or sustained release of the compositions. The invention provides compositions for administration comprising the compositions of the invention in solution form, dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, such as water, buffered water, 0.8% sodium chloride solution, 0.3% glycine, hyaluronic acid, and the like. These compositions may be sterilized using conventional, well-known sterilization techniques, or may be sterile filtered. The aqueous solutions thus obtained may be packaged for use as is, or lyophilized, the lyophilized preparation being stored in a sterile environment prior to administration. HU 226 662 Β1 must be dissolved with water. The compositions may contain pharmaceutically acceptable excipients necessary to approximate physiological conditions, such as pH adjusting and buffering agents, ionic strength adjusting agents, wetting agents and the like, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitol monolaurate, triethanolamine oleate, etc. The concentration of the compositions of the invention in the dosage forms may vary widely, i.e. from less than 0.1%, usually at least about 2%, up to 20-50%, or even more (w / w%), and is selected primarily based on the volume, viscosity, etc. of the liquid, in accordance with the particular route of administration chosen. The compositions of the invention may also be administered with liposomes. Liposomes may be emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. In these preparations, the compositions of the invention to be delivered are part of a liposome, alone or in combination with a molecule capable of binding to the desired target, such as an antibody or other therapeutic or immunogenic compositions. The liposomes thus loaded or coupled with the desired composition of the invention may be delivered systemically or may be directed to the tissue of interest, where the liposomes then deliver the selected therapeutic / immunogenic peptide compositions. Liposomes used in the present invention are prepared from standard vesicle-forming lipids, which generally comprise neutral or negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids generally takes into account, for example, the size of the liposome, its sensitivity to acid, and the stability of the liposomes in the bloodstream. A variety of methods are available for preparing liposomes, for example, Szoka et al., Ann. Rev. Biophys. Bioeng. 9, 467 (1980), USP 4,235,871, 4,501,728, 4,837,028 and 5,019,369, which are incorporated herein by reference. The liposome suspension containing the composition of the invention may be administered intravenously, locally, topically, etc. at a dosage that may vary depending on, among other things, the route of administration, the composition of the invention to be delivered, and the disease state to be treated. In solid formulations, non-toxic solid carriers may be used, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate and the like. A pharmaceutically acceptable non-toxic formulation for oral administration is prepared by incorporating any of the normally used excipients, such as those listed above, and generally containing from 10 to 95% of the active ingredient, which is one or more of the compositions of the invention, and more preferably from 25 to 75% concentration. For administration by aerosol, the compositions of the invention are preferably formulated in the final form with a surfactant and a propellant. Typical percentages of the compositions of the invention are 0.01% to 20% (w / w), preferably 1% to 10%. The surfactant should of course be non-toxic and should preferably be soluble in the propellant. Such agents may be, for example, esters or partial esters of fatty acids having 6 to 22 carbon atoms, such as caproic acid, octanoic acid, lauric acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, olesteric acid or oleic acid with an aliphatic polyhydroxy alcohol or cyclic anhydride. Mixed esters, such as mixed or natural glycerides, may also be used. The amount of surfactant is 0.1% to 20% (w / w) of the composition, preferably 0.25-5%. The formulation is balanced in the usual manner with the propellant. If desired, it may also contain a carrier, for example lecithin for intranasal administration. The compositions of the invention may also be administered in kit-type systems, encapsulated form, or in implants, techniques well known to those skilled in the art. Similarly, the constructs may be delivered to the tissue of interest via a pump. In some preferred embodiments of the invention, the compositions of the invention can be administered ex vivo into cells or tissues removed from a patient and then returned to the patient. For ex vivo administration of gene therapy constructs, see, for example, Artega et al., Cancer Research 56 (5), 1098-1103 (1996); Nolta et al., Proc. Natl. Acad. Sci. USA 93 (6), 2414-9 (1996); Koc et al., Seminars in Oncology 23 (1), 46-65 (1996); Raper et al., Annals of Surgery 223 (2), 116-26 (1996); Dalesandro et al., J. Thorac. Cardi. Surg. 11 (2), 416-22 (1996); and Makarov et al., Proc. Natl. Acad. Sci. USA 93 (1), 402-6(1996). In some preferred embodiments of the invention, the constructs of the invention are administered to a carotid artery after balloon angioplasty to prevent or reduce the severity of restenosis. The compositions of the invention can be used as coatings for devices used in angioplasty [see, for example, Willart et al., Circulation 89, 2190-2197 (1994); French et al., Circulation 90, 2402-2413 (1995)]. In other preferred embodiments of the invention, the fusion polypeptides of the invention can be used in a similar manner. The following examples are intended to illustrate the solution according to the invention, without however limiting our claim to what has been described. Example 1 E2F-RB fusions A. Introduction In this experiment, we construct expression plasmids encoding different E2F segments fused to the RB56 polypeptide. RB56 is a subfragment of full-length RB that contains the “pocket” domains required for growth suppression [Hiebert HU 226 662 Β1 et al., MCB 13, 3384-3391 (1993); Qin et al., Genes and Dev. 6, 953-964 (1992)]. E2F194 contains amino acids 95-194 of E2F. This fragment contains only the DNA binding site of E2F. E2F286 contains the DNA binding domain and the DP-1 heterodimerization domain. Both E2F fragments lack the N-terminal cyclin-A kinase binding domain, which appears to regulate the DNA binding activity of E2F [Krek et al., Cell 83, 1149-1158 (1995); Krek et al., Cell 78, 161-172(1994)]. B. Generation of vectors The pCTM plasmid contains a CMV promoter, a three-part adenovirus leader sequence flanked by T7 and SP6 promoters, and a multiple cloning site with a polyadenylation site derived from bovine growth hormone (BGH) and an SV-40 polyadenylation site in the direction of synthesis. A schematic representation of pCTM is shown in Figure 3. The DNA sequence of pCTM is shown in Figure 4. pCTMI was prepared from pCTM by digesting pCTM with XhoI and NotI enzymes and subcloning a 180 bp XhoI / NotI intron fragment from the pCMV-β-gal vector (Clontech). pCTMI is schematically shown in Figure 5. The DNA sequence is shown in Figure 6. pCTMIE is prepared by amplifying the SV40 enhancer from SV40 viral DNA by polymerase chain reaction. The amplified product is digested with BglII and inserted into BamHI-digested pCMTI and ligated in the presence of BamHI. The plasmid is schematically depicted in Figure 7. The DNA sequence is shown in Figure 8. pCTM-RB was prepared as follows. The 3.2 kb XbaI / ClAI fragment of pETRBc containing the full-length human RB cDNA [Huang et al., Nature 350, 160-162 (1991)] was ligated into pCTM digested with XbaI / ClAI enzymes. pCTM-RB56 was prepared by ligating the digested pCTM to the 1.7 kb XbaI / ClAI fragment containing the RB56 coding sequence. pCTMI-RB, pCTMIE-RB, pCTMI-RB56 (amino acids 381-928) and pCTMIE-RB56 (amino acids 321-928) were prepared by the same method. C. E2F-RB fusion constructs Figure 9 shows the fusion constructs used in the experiments. These E2F constructs start at amino acid position 95 and lack parts of the cyclin A-binding domain. E2F437 contains the DNA-binding domain (black), heterodimerization domain (white), and transactivation domain (shaded). E2F194 contains only the DNA-binding domain. E2F286 contains the DNA-binding domain and the DP-1 heterodimerization domain. RB56-5S represents an RB variant with alanine substitutions at amino acids 606, 612, 788, 807, and 811. In E2F194-RB56-5s and E2F286-RB56-5s, the E2F fragments were fused in one step to codon 379 of RB-5s. The RB56-C706F contains an inactivating point mutation [Kaye et al., Proc. Natl. Acad. Sci. USA 87, 6922-6926 (1990)]. pCMV-E2F194 and pCMV-E2F437 were prepared as follows. DNA encoding amino acids 95-194 (containing the DNA binding domain) or amino acids 95-437 of E2F was amplified by polymerase chain reaction, digested with HindII, and ligated into SmaI / HindIII-digested pCMV-RB56 vectors. pCMV-E2F286 was prepared by digesting pCMV-E2F437 with AflII, treating the ends with DNA pol-1 (Klenow fragment), and religating in the presence of AflII. Blunt-end ligation creates a stop codon at position 287. pCMV-E2F286-5s was prepared by ligating AflII(blunt) / HindIII-digested pE2F437 to a SalI(blunt) / HindIII fragment containing the RB56-5s coding sequence. pCTMIE-E2F194-5s and pCTMIE-E2F286-RB5s were prepared by ligating EcoRI / EcoRV-digested pCTMIE (4.2 kb) to HindIII(blunt) / EcoRI fragments from pCMV-E2F194-RB5s or pCMV-E2F286-RB5s. D. Promoter repression To measure the effects of E2F-RB fusion proteins, a cervical carcinoma cell line (ATCC# HTB-31) is transfected with equivalent amounts of E2F194-RB56 or E2F-RB56 plus E2-CAT reporter plasmid [see, e.g., Weintraub et al., Nature 358, 259-261 (1992)]. In the C33A assay, 250,000 C33A cells were plated in 6-well tissue culture plates and allowed to adhere overnight. 5 pg of pCMV-RB56, pCMV-E2F-RB56, or pCMV-E2F plasmid was cotransfected with 5 pg of E2-CAT or SVCAT-tagged reporter construct and 2.5 pg of β-gal plasmid (pCMV-β, Clontech) per well (calcium phosphate method, MBS transfection kit, Stratagene) in duplicate. Cells were cultured for 72 hours after transfection and extracts were prepared. In the 5637 assay, 250,000 5637 cells were plated as described above, cotransfected with 1 pg each of RB or E2F-RB fusion plasmid, E2-CAT or SV-CAT reporter plasmid, and pCMV-β-galactosidase using Lipofectin reagent (BRL, Bethesda, Maryland) according to the manufacturer's instructions. The CAT assay was performed using 20 µl (C33A) or 50 µl (5637) of cell-free extract [Gorman et al., Mol. Cell. Biol. 2, 1044 (1982)]. The TLCs were analyzed on a Phosphoimager SF (Molecular Dynamics). The CAT activities were normalized to transfection efficiency according to the β-galactosidase activity of each extract. The β-galactosidase activities of the extracts were assayed according to the assay procedure described by Rosenthal et al. [Meth. Enzym. 152, 704 (1987)]. The results of the studies are as follows. Transfection with the E2-CAT reporter alone or in combination HU 226 662 Β1 in the presence of a functional, control RB56-H209 mutant resulted in relatively high CAT activity. Cotransfection with wild-type RB56 or the RB56-5s variant resulted in a 10- to 12-fold repression of CAT activity, indicating that either RB56 or RB56-5s can effectively repress E2F-dependent transcription. E2F194-RB5s and E2F286-RB5s repressed transcription by approximately 50-fold. Both RB56 and the E2F components of the fusion proteins are required for transcriptional repression, whereas expression of E2F194 and E2F286 does not mediate transcriptional repression. SV40-CAT transcription was not repressed by the E2F-RB constructs, supporting the specificity of transcriptional repression by E2FRB to the E2 promoter. These results are schematically shown in Figure 10. E. Cell cycle recording E2F-RB fusion polypeptides are tested for their ability to induce G1 arrest in Saos-2 (RB- / - cells) (ATCC No. HTB-85) and C33A cells. Previous experiments have shown that RB-mediated E2 promoter repression and G1 arrest are coupled in Saos-2 cells but are dissociated in C33A cells (RBmut) [Xu et al., PNAS 92, 1357-1361 (1992)]. Cells are washed in PBS and fixed in 1 ml of -20°C, 70% ethanol for 30 minutes. Cells are centrifuged and resuspended in 0.5 ml of 2% serum containing 10 pg / ml RNaseA and incubated for 30 minutes at 37°C. 0.5 ml PBS containing propidium iodide (100 pg / ml) was added to each sample, mixed, and the cells were filtered through a FACS-tube filter (FACS needle capstrainer). FACS analyses were performed on a FACS-Scan (BectonDickinson) using doublet discrimination. 5000-10,000 CD20+ events were analyzed.The percentage of cells in Go / Gr, S and G2 / M is determined using “Modfit modeling” software. The results of this experiment are described below. Both full-length RB110 and the truncated RB56 version caused, but the control mutant RB-H209 did not cause, Gr arrest in Saos cells (Table 1). Similarly, RB56-5s, E2F-194-RB56-5S and E2F286-RB56-5s were all able to arrest cells in G0 / G1. Transfection of the DNA-binding domain, E2F194, did not block entry into S phase in Saos-2, as previously described for cells from rodents [Dobrowolski et al., Oncogene 9, 2605-2612 (1994)]. In contrast, RB110, RB56 and E2F-RB fusion proteins were unable to anchor C33A cell lines, indicating that the transcriptional repression observed in these cells does not translate into Gr anchoring. We observed that E2F-RB fusion proteins were also able to arrest 5637 cells (Table 2). Both RB56 and RB56-5s were able to arrest cells in G0 / Gi efficiently (approximately 90% of cells were in Go / Gr), whereas E2F194-RB56-5s and E2F286-RB56-5s were somewhat less efficient (approximately 80% of cells were in Go / Gr) in promoting Go / Gr arrest. Without wishing to be bound by any theory, it appears that the less efficient arrest of both Saos-2 and 5637 cells by E2F-RB fusion proteins is due to the lower steady-state protein levels in these cells (Figure 11, panels b and c). Table 1 Cell cycle regulation by RB and E2F-RB fusion proteins in RBneg cells Cells (%) CD20+ Gq / Gi g2 / m S-phase H209 52.1 27.1 20.8 p56RB 78.8 14.2 7.0 p110RB 70.9 14.3 14.8 p56RB-5s 84.8 13.2 2.0 p56RB-p5 81.3 11.5 7.3 E2F-194-5S 77.8 14.9 7.3 E2F-286-5S 72.2 15.0 12.8 E2F-194 49.9 28.0 22.1 Table 2 Growth suppression of 5637-bladder cells by RB and E2F-RB fusion proteins 5637 / CD20+ Cells (%) Gq / Gt S G2M CD20 59.7 16.9 20.6 RB56-C706F 57.4 16.3 24.3 RB56WT 90.7 4.12 4.88 RB56-5S 89.91 3.51 6.1 E2F-194-5S 80.1 1.31 0 E2F-286-5S 79.21 8.1 0 F. Functional activity of fusion proteins In RB background The activity of E2F-RB fusion proteins was then determined in a cellular background containing functional RB. NIH-3T3 cells were transfected with RB56 or E2F-RB56 fusions and stained with the anti-RB monoclonal antibody 3C8 [Wen et al., J. Immunol. Meth. 169, 231-240 (1994)]. FACS analysis of RB-expressing cells was performed. The results are shown in Figure 12. The “non-gated” population (g) shows a typical cell cycle distribution for NIH-3T3 cells (60% G0, 28% S, 10% G2 / M). In contrast, in cells transfected with RB56 (a, b) or E2F-RB fusion proteins (c—f), more than 90% of RB-expressing cells were arrested in G0 / Gi. These data indicate that the ability of RB and E2F-RB56 fusions to arrest cells in G0 / Gr is not limited to RB-negative tumor cells. Transfected NIH-3T310 We also examined the relative amount of protein expressed in HU 226 662 Β1 cells. RB110 was not efficiently expressed in these cells. Thus, these data indicate that E2F-RB fusion proteins are more potent transcriptional repressors than p56 or RB56 alone, and that RB is more likely to repress transcription in the E2F-bound state than by directly blocking the E2F transactivation domain. These data support the potential use of E2F-RB fusions as RB antagonists in RB(+) cells and in RB-negative or RB-mutant cells. Example 2 Tissue-specific expression of E2F-RB fusions A. Production of recombinant adenovirus In this experiment, recombinant adenoviruses containing RB polypeptide are produced under the control of the CMV or smooth muscle-derived alpha-actin promoter. The smooth muscle α-actin promoter [bases from -670 to +5, Reddy et al., “Structure of the Human Smooth Muscle α-Actin Gene”, J. Biol. Chem. 265, 1683-1687 (1990); Nakano et al., “Transcriptional Regulatory Elements In The 5' Upstream and First Intron Regions of The Human Smooth Muscle (aortic type) α-Actin-Encoding Gene”, Gene 99, 285-289 (1991)] was isolated from a genomic library by PCR with the addition of 5'-terminal XhoI and AvrII sites and 3'-terminal XbaI, ClaI and HindIII sites for cloning purposes. The fragment was subcloned into a plasmid as an XhoI / HindIII fragment for sequencing to confirm the base composition. A fusion construct containing the DNA-binding and heterodimerization domain of E2F-1 (bases 95-286) fused to p56 (bases 379-928 of full-length RB) (bases 286-56) was used.) is subcloned as an XbaI / ClA1 fragment to the smooth muscle α-actin promoter upstream of the expression cassette, and this expression cassette is digested and cloned into the pAd / ITR / IX(-) plasmid as an XbaI-AvrII and ClA1 fragment to yield plasmid pASN286-56. This plasmid consists of an adenovirus type 5 inverted terminal repeat (ITR), packaging signals, and Ela enhancer, followed by the human smooth muscle α-actin promoter and 286-56 cassette, followed by the Ad2 sequence (4021-10462.) (containing an E1b / protein IX polyA signal) in a pBR322 background. Recombinant adenovirus is prepared using standard procedures. Plasmid pASN286-56 was linearized with NgoMI and cotransfected into 293 cells with a large fragment of ClaI-digested rAd34, which contains deletions in both the E3 and E4 regions of adenovirus type 5. Ad34 contains a 1.9 kb deletion in the early 3.region (derived from a deletion of an XbaI restriction fragment, at positions 28593-30470 of Ad5) and a 1.4 kb deletion in early region 4 (derived from the E4 Taq 1 fragment, at positions 33055-35573), which are replaced by cDNA containing E4-ORF6 and -6 / 7. Recombinant adenovirus produced by homologous recombination is isolated and identified by restriction digest analysis and further purified by limiting dilution. Other recombinant adenoviruses used as controls have been described, such as the ACN control virus [CMV promoter, Wills et al., “Gene Therapy for Hepatocellular Carcinoma: Chemosensitivity Conferred By Adenovirus-Mediated Transfer of The HSV-1 Thymidine Kinase Gene”, Cancer Gene Therapy 2, 191-197 (1995)] and ACN56 [RB expressed under the CMV promoter], ACN56 was prepared as follows. A plasmid containing p56cDNA was prepared by replacing the p53cDNA in the ACNP53 plasmid [Wills et al., Human Gene Therapy 5, 1079-1088 (1994)] with a 1.7 kb XbaI / BamHI fragment isolated from the pET9a-Rb56 plasmid [Antelman et al., Oncogene 10, 697-704 (1995)] containing p56cDNA. The resulting plasmid was p56 381-928. amino acids, Ad5 inverted terminal repeat sequence, viral packaging signals and Ela enhancer, followed by a human cytomegalovirus (CMV) immediate early promoter and Ad2 tripartite leader cDNA sequence to control p56 expression. The p56 cDNA is followed by Ad2 sequence (4021-10462.) in a pBR322 background. This plasmid is linearized with EcoRI and cotransfected with a large fragment of bsp106-digested DL327 [E3-deleted; Thimmappaaya et al., Cell 31, 543-551 (1982)] or h5ile4 [E4-deleted; Hemstrom et al., J. Virol. 62, 3258-3264 (1988)].Recombinant viruses are further purified by limited dilution. B. Cellular proliferation In this experiment, cell lines in culture are infected with recombinant adenoviral RB constructs to determine the relative expression of the RB polypeptide and the effect on cell proliferation. For H358 cells (ATCC No. Crl 5807) and MDA-MB468 cells (ATCC No. HTB 132, breast adenocarcinoma cells), 5000 cells were plated per well in normal growth medium in 96-well microtiter plates (Costar) and incubated overnight at 37°C in 7% carbon dioxide. Viruses were serially diluted in growth medium and used to infect cells at the indicated dose for 48 hours. 3H-thymidine (Amersham, 0.5 pCi / well) was then added and the cells were incubated at 37°C for an additional 3 hours until culture was complete. A7r5 cells (ATCC CRL1444, derived from rat smooth muscle) and A10 cells (ATCC CRL 1476, derived from rat smooth muscle) were plated at a density of 3000 cells / well in DME containing 0.5% FCS (A7r5 cells) or DME containing 20% ​​FCS (A10 cells). The virus was serially diluted in the plating medium and used to infect the cells at the doses indicated in the figures.The infection and labeling procedure is the same for A10 cells as for H358 and MDA-MB468 cells, except that. HU 226 662 Β1 μθ labeling was used per culture well. A7r5 cells were not infected with virus until 48 hours after plating. 48 hours after infection, serum concentration was increased to 10% FCS and 2 pCi / well of 3H-thymidine was added, and cells were incubated at 37°C for an additional 3 hours until culture was complete. The medium was aspirated from all cells in the culture well, the cells were trypsinized, and collected using a 96-well GF / C filter and a Packard Top cell counter. The results are shown in Figures 13 and 14 as the mean percentage (+ / - SD) of control proliferation induced by medium treatment as a function of virus dose. Thus, Figure 13 compares the effects of p56-adenovirus constructs on A10 and A7R5 smooth muscle cells. The CMV-driven p56 virus (CAN 56) inhibited A10 growth to approximately the same extent as the actin promoter-driven E2F fusion constructs (ASN586-56 #25,26). Figure 14 shows the effects of the adenovirus constructs on the breast cancer cell line MDA Μβ468 and the non-small cell lung carcinoma cell line H358. In these experiments, the actin promoter-driven E2F-p56 was ineffective, while the CMV promoter-driven p56 was effective in inhibiting the growth of non-smooth muscle cells. To determine whether non-smooth muscle cells are more susceptible to adenovirus infection than smooth muscle cell lines, four cell lines (H358, MB468, A7R5, and A10) were infected with 5 multiplicity of infection (MOI) of adenovirus capable of expressing β-galactosidase [AC6GL; Wills et al., Human Gene Therapy 5, 1079-1088 (1994)] and the extent of β-gal staining was examined. As shown in Figure 15 (top), non-smooth muscle cell lines were significantly more susceptible to infection than smooth muscle cell lines. In a further test, cells were infected with ACN56 at various multiplicity of infection (50, 100, 250, 500) and the amount of p56 in the infected cells was detected by autoradiography. As in the 15th.As shown in Figure 1 (bottom), non-smooth muscle cell lines contained significantly more p56, as their greater susceptibility to infection meant that infected cells contained more virus and therefore more copies of the p56 template driven by a non-tissue-specific CMV promoter. In a further experiment, we will determine the specificity of the smooth muscle-derived actin promoter for smooth muscle tissue. In this experiment, we will measure the level of β-gal expression in cells infected with β-gal constructs driven by different promoters. As can be seen in Figure 19, despite their lower susceptibility to infection, expression in smooth muscle cells was only evident in these cells using the smooth muscle-derived alpha-actin promoter. Figure 21 shows a comparison of the effects of a CMV-driven p56 recombinant adenovirus (ACN56E4) with those of an E2F-p56 fusion construct driven by the human smooth muscle alpha-actin promoter (ASN286-56) and a control adenovirus construct containing either the CMV or smooth muscle alpha-actin promoter without an upstream transgene (ACNE3 or ASBE3-2 isolates, respectively). The assay measures 3H-thymidine uptake in a smooth muscle cell line (A7R5) or a non-smooth muscle cell line (MDA-MBA468, breast carcinoma). The results demonstrate muscle tissue specificity when the smooth muscle alpha-actin promoter is used, and specific inhibition for both the p56 and E2F-p56 transgenes, relative to their respective controls. C. Inhibition of restenosis The balloon injury model is based on that described by Clowes et al. [Clowes, Lab. Invest. 49, 327-333 (1983)]. Male Sprague-Dawley rats weighing 400-500 g are anesthetized by intraperitoneal injection of sodium pentobarbital (45 mg / kg, Abbot Laboratories, North Chicago, Illinois). The left common carotid artery is accessed through a midline incision, and the left common internal and external carotid arteries are temporarily ligated. A 2F embolectomy catheter (Baxter Edwards Healthcare Corp., Irvine, CA) is inserted into the external carotid artery and advanced to the distal (distal) ligation of the common carotid artery. The balloon is filled with saline and pulled towards the site of the angiotomy, creating a three-fold inflated, deendothelialized lesion, then the catheter is withdrawn. 10 μl of adenovirus [1*109pfu Ad-RB (ACNRb) or Ad-p56 (ACN56)] diluted to 100 μΙ with 15% (wt / vol) Poloxamer 407 (BASF, Parsippany, NJ) is injected.) or Ad-6-Gal (1*109pfu, diluted as described above) through a puncture needle inserted into a temporarily isolated segment of the artery just adjacent to the carotid artery bifurcation. The adenovirus solution is incubated for 20 minutes, then the viral infusion is stopped and the puncture needle is removed. The proximal carotid artery is then ligated, and blood flow is restored in the common carotid artery by removing the ligature. The experimental procedure was approved by the Institutional Animal Care and Use Committee and is in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23, revised 1985). Rats were sacrificed 14 days after treatment by intraperitoneal injection of pentobarbital (100 mg / kg). The original balloon-injured segment of the left common carotid artery from the proximal edge of the omohyoid muscle to the carotid artery bifurcation was soaked in sodium chloride solution and excised free of surrounding tissue. The tissue was fixed in 100% methanol until paraffin embedding. Several 4 μm sections were cut from each tissue sample. One section from each sample was stained with hematoxylin and eosin, and another was stained with Richardson's combination elastic-tricolor stain for conventional light microscopic analysis. Histological images of cross-sections of arterial sections stained with hematoxylin and eosin or elastic tricolor dye are projected onto a digitizing screen (Summagraphics), and the intimal, medial HU 226 662 β1 and luminal areas were measured by quantitative, morphometric analysis using a computer-aided drawing program (MACMEASURE, version 1.9, National Institute of Mental Health). Results are averaged ±SEM. Differences between groups are analyzed using an unpaired, slow-tailed Student's t-test. Statistical significance is determined if the probability of a null effect is <0.05. The results are presented in Figures 17 and 18. Figure 17 graphically depicts the relative inhibition of neointima formation, demonstrating that p56 and RB are able to inhibit neointima formation. The photographs shown in Figure 18 show a dramatic reduction in neointima in the presence of p56. Adenovirus-treated carotid arteries were harvested from rats 2 days after balloon injury and infection. Tissues were fixed in phosphate-buffered formalin until paraffin embedding. Tissues were cut into 4-μm transverse sections and degreased in xylene and pure alcohols. Endogenous peroxidase was quenched with 1% hydrogen peroxide for 30 min. Antigen was retrieved in 10 mM sodium citrate buffer, pH 6.0, at 95°C for 10 min. Monoclonal anti-RB antibody was used at 10 pg / ml in PBS in a humid chamber at 4°C for 24 h. The secondary antibody was the Unitect Mouse Immunochemistry Kit (Oncogene Sciences, Uniondale, New York) according to the manufacturer's instructions. Antibody complexes were visualized with 3,3'-diaminobenzidine (DAB, Vector Laboratories, Burlingame, CA). Sections were thinly counterstained with hematoxylin and mounted on slides. The results are shown in Figure 20. The references cited are incorporated herein by reference in their entirety.

Claims

CLAIMS 1. A nucleic acid encoding a fusion polypeptide comprising a fusion of a DNA binding domain of an E2F transcription factor and a functional growth suppression domain of a retinoblastoma (RB) polypeptide, wherein the fusion polypeptide lacks a functional cyclin A kinase binding domain of the E2F transcription factor.

2. The nucleic acid of claim 1, inserted into an adenoviral vector.

3. An expression vector comprising DNA encoding a fusion polypeptide comprising a fusion of a DNA binding domain of an E2F transcription factor and a functional growth suppression domain of a retinoblastoma (RB) polypeptide, wherein the fusion polypeptide lacks a functional cyclin A kinase binding domain of the E2F transcription factor.

4. The expression vector of claim 3, comprising a tissue-specific promoter operably linked to the DNA encoding the fusion.

5. The expression vector of claim 4, wherein the tissue-specific promoter is a smooth muscle actin promoter.

6. The expression vector of claim 3, which is a viral vector.

7. The expression vector of claim 6, which is an adenoviral vector.

8. The expression vector of claim 7, which is a replication-deficient adenovirus vector.

9. The expression vector of claim 3, which is a plasmid.

10. The expression vector of claim 5, wherein the actin promoter is an alpha-actin promoter.

11. A nucleic acid sequence encoding a fusion polypeptide comprising a fusion of amino acid residues 95-194 of E2F (Figure 1A) and amino acid residues 379-928 of RB (Figure 2B).

12. A vector comprising the nucleic acid sequence of claim 11.

13. The vector of claim 12, which is a viral vector.

14. The vector of claim 13, which is an adenoviral vector.

15. The vector of claim 12, which is a replication-deficient adenoviral vector.

16. The nucleic acid sequence of claim 11, further comprising a tissue-specific promoter, wherein the fusion polypeptide is expressed under the control of the tissue-specific promoter.

17. The nucleic acid sequence of claim 16, wherein the tissue-specific promoter is a smooth muscle actin promoter.

18. The nucleic acid sequence of claim 17, wherein the smooth muscle actin promoter is an alpha-actin promoter.