MXPA99004499A - Tissue specific expression of retinoblastoma protein - Google Patents

Abstract

Fusions of the transcription factor E2F and the retinoblastoma protein RB are provided, along with methods of treatment of hyperproliferative diseases.

Description

EXPRESSION OF RETINOBLASTOMA PROTEIN SPECIFIC TO TISSUE BACKGROUND OF THE INVENTION Both the retinoblastoma (RB) gene and the E2F transcription factor play a critical role in the control of cell growth (for a review see Adams, P. Kaelin,. Cancer Biology (Seminars in cancer biology) 6: 99-108 (1995)). The RB locus is frequently inactivated in several of the human tumor cells. The reintroduction of a wild-type RB gene (e.g., Bookstein et al., Science 247: 712-715 (1990)) or an RB protein (pRB) (e.g., Antelman et al, Oncogene 10: 697- 704 (1995)) in Rbneg / Rbmut cells can suppress growth in culture and tumorigenicity in vivo. While E2F serves to activate the transcription of S-phase genes, its activity is stopped by RB. RB stops the cells by blocking the G-phase output in S phase (for example, Do dy et al., Cell 73: 499-511 (1993)) but the precise path of the arrest remains unclear. Even when E2F forms complexes with RB, the formation of complexes is more efficient if a protein related to E2F, and DP-1 is present. E2F-1 and DP-1 form stable heterodimers that bind to DNA (eg, Qui et al; Genes and Dey, 6-: 953-964 (1992)). The DP-1- E2F complexes serve to cooperatively activate the transcription of E2F-dependent genes. Said transcription can be repressed by pRB in the same way as the transcription activated by E2F-1 or DP-1. The transcriptional repression of genes by RB in some cases can be achieved by fixing pRB on a promoter. For example, fusions, GAL4-pRB bind on the GAL4 DNA binding domain and repress transcription from elements p53, Sp-1 or AP-1 (Adnane, et al., J. Biol. Chem. 270: 8837-8843 (1995); einytraub, et al., Nature 358: 259-261 (1995)). Sellers, et al. (Proc. Natl. Acad. Sci. 92: 11544-11548 (1995)) present amino acid residues 1-368 of E2F with amino acids 379-792 or 379-928 of RB. Chag, et al. (Science 267: 518-521 (1995)) reported the use of an adenovirus RB construct defective for replication in reducing neointima formation in two animal models of restenosis, a hyperproliferative disorder. SUMMARY OF THE INVENTION The present invention offers the surprising result that a fusion of an E2F polypeptide with an RB polypeptide is more efficient to repress the transcription of the E2F promoter than RB alone, and that such fusions may cause the arrest of the E2F promoter cycle. the cell in several types of cells. Such fusions can therefore satisfy the urgent need for the therapy of hyperproliferative disorders, including cancer. One aspect of the present invention is a polypeptide comprising a fusion of a transcription factor, the transcription group comprising a DNA binding domain, and a retinoblastoma (RB) polypeptide, the RB polypeptide comprises a deletion domain. of growth. Another aspect of the present invention is DNA encoding said fusion polypeptide. The DNA can be inserted into an adenovirus vector. In some embodiments of the invention, the transcription factor is E2F. The cyclin A binding domain of E2F can be removed or non-functional. The E2F comprises amino acid residues from about 95 to about 194 or from about 95 to about 286 in some embodiments. The retinoblastoma polypeptide can be wild-type RB, RB56, or a variant or fragment thereof. In certain embodiments, the retinoblastoma polypeptide comprises amino acid residues from about 379 to about 928. Preferred amino acid substitutions of the RB polypeptide include residues 2, 608, 788, 807, and 811. Another aspect of the present invention is a expression vector comprising a DNA encoding a polypeptide, the polypeptide comprises a fusion of a transcription factor, the transcription factor comprises a DNA binding domain and a retinoblastoma (RB) polypeptide, the Rb polypeptide comprises a growth suppression domain. In certain embodiments, a tissue-specific promoter is operatively linked to the DNA encoding the fusion polypeptide. The tissue-specific promoter may be an alpha-smooth muscle actin promoter. Another aspect of the present invention is a method for the treatment of hyperproliferative disorders comprising administering to a patient a therapeutically effective dose of an E2F-RB fusion polypeptide. The hyperproliferative disorder can be cancer. In certain modalities, the hyperproliferative disorder is restenosis. The fusion polypeptide and the nucleic acid encoding the fusion polypeptide can be used to coat devices used for angioplasty. BRIEF DESCRIPTION OF THE DRAWINGS Figure IA shows the predicted amino acid sequence of E2F. Figure IB shows the nucleotide sequence of the transcription factor E2F. Figure 2a shows the nucleotide sequence of pBR in accordance with that presented by Lee, et al, (Nature 329: 642-645 (1987)). Figure 2B shows the predicted amino acid sequence of pRB. Figure 3 is a diagrammatic representation of pCTM. Figure 4 shows the nucleotide sequence of the plasmid pCTM. Figure 5 is a diagrammatic representation of pCTMI. Figure 6 shows the nucleotide sequence of pCTMI. Figure 7 is a diagrammatic representation of the pCTMIE plasmid. Figure 8 shows the nucleotide sequence of pCTMIE.

Figure 9 is a diagram showing fusion constructs E2F-RB used in the examples. All the constructs of E2F started at amino acid 95 and lacked part of the cyclin A binding domain. E2F-437 contained the DNA binding domain (black), the heterodimerization domain (white), and the transactivation domain (dotted). E2F-194 contained only the DNA binding domain. E2F-286 contained the DNA binding domain and the DP-1 heterodimerization domain. To generate E2F-194-RB56-5s and E2F-286-rb56-5s, the E2F constructs were fused in frame over codon 379 of RB. C706F is a mutation point of deactivation. Figure 10 is a diagram showing the transcriptional repression by fusion constructs E2F-RB.

Figure 11 (A-D) shows the expression of E2F-RB fusion protein in mammalian cell lines. The extracts were prepared from cells used in E2-CAT reporter assays or in FACS assays and were analyzed with an anti-RB monoclonal antibody. In panel A, results appear from C33A cells transfected with (3) RB56-H209, (4) wild-type RB56, (5) RB56-5S, (6) E2F86-5S, (7) E2F194- 5S, (8) E2F194, (9) E2F286, (10) E2F437. Segment (1) is a protein standard RB56. Segment (2) is a fictitious transaction. In panel B, the results appear for transfection of Saos-2 cells with (1) RB56, (2.3) E2F194-5S, and (4.5) E2F286-5s. In panel C, results for the transfection of 5637 cells with (2.3) RB56 wild-type, (4.5) RB56-5S; (6.7) E2F194 -5s; (7.8) E2F286-5s. Segment (1) is a protein standard of RB56. In panel D, the results appear for transfected NIH-3T3, 83) RB56, (4) E2F286-5S, (5) E2F194-5S. Segment (1) is a standard of RB56; segment (2) is a standard of RB110. Figure 12 shows flow cytometry histogram analysis of NIH-3T3 cells expressing RB. Figure 13, panel A, shows a comparison of the effects of a CMV-driven recombinant adenovirus (ACN56) with two isolates of an E2F-p56 fusion construct activated by human smooth muscle alpha actin consisting of amino acids 95 to 286 of E2F bound directly and in frame with p56 (amino acids 379-928 of RB cDNA), versus a control virus (ACN) in a 3 H-thymidine uptake assay in the A7R5 rat smooth muscle cell line. Panel (B) shows the effects of the same constructs on the AlO rat smooth muscle cell line. Figure 17 shows a comparison of the effects of the viruses described in Figure 13 on non-muscle cells. Panel (A) shows the results in the MDA MB468 breast carcinoma cell line. Panel (B) shows the results in the non-small cell lung cell carcinoma line H358. Figure 15, upper panel, shows the ability of relative infection by different cell line adenoviruses in accordance with what is judged by the level of beta-galactosity staining (beta-gal) after infection with equal amounts with a beta-gal which expresses a recombinant adenovirus driven by a CMV promoter. H358 is a non-small lung cell carcinoma cell line, MB468 is a line of breast carcinoma cells; A7R5 and AlO are smooth muscle cell lines. The lower portion of the figure shows the relative levels of p56 protein expressed in the same cells when infected with the recombinant ACN56 adenovirus, where the p56 cDNA is driven by the non-tissue-specific CMV promoter. Figure 16 shows the relative levels of protein in cells infected with the E2F-p56 fusion construct driven by the smooth muscle active alpha promoter (ASN286-56) .UN indicates uninfected; 50, 100, 250 and 500 refer to the multiplicities of infections (MOI). Figure 17 is a bar graph showing the ratio of intima per average area (as a measurement of the inhibition of neointima formation) from cross sections (n = 9) of rat carotid arteries that were damaged and treated with recombinant adenoviruses that express either beta-gal, RB (ACNRB) or p56 (ACN56), all under the control of the CMV promoter. Figure 18 is a series of 3 photographs showing restenosis in a rat angioplasty model. The panel on the left shows data of a normal animal; the central panel shows data of an injured animal and then treated with a recombinant virus expressing beta-gal; the panel on the right shows data of a wounded animal and then treated with a recombinant adenovirus expressing p56 (ACN56). Figure 19 shows tissue specificity of the alpha-smooth muscle actin promoter in accordance with that demonstrated by its selective ability to express the beta-gal transgene in muscle cells but not in non-muscle cells. The panels on the left compare the expression of beta-gal in the MB468 breast cell carcinoma line infected with either an MOI = as well with a beta-gal driven by CMV (ACNBGAL) versus an MOI = 100 with the construct of smooth muscle promoter (ASNBGAL). The panels on the right show the beta-gal expression of the A7R5 rat smooth muscle cell line infected with either M0I = 1 of ACNBGAL or MOI = 50 of ASNBGAL. The expression from ASNBGAL is observed in the muscle cell line, but it is absent in the non-muscle cell line, despite the highest degree of infectivity of the cells. Figure 20 shows the ability of a recombinant adenovirus expressing RB to transduce rat carotid arteries. Arteries treated with recombinant adenoviruses (IX 109 pfu) were harvested two days after balloon injury and infection. Cross sections were fixed and an antibody specific for RB was used in order to detect the presence of RB protein in the tissue. The control virus used was ACN. Staining with RB protein was evident in the sample treated with ACNRB, specifically with more important extensions. Figure 21 shows a comparison of the effects of a p56 recombinant adenovirus driven by CMV (ACN56E4) versus an E2F-p56 fusion construct driven by human smooth muscle alpha-actin promoter (ASN286-56) versus a control adenoviral construct which contains either CMV or smooth muscle alpha-actin promoters without a downstream transgene (isolated ACNÉ 3 or ASBE3-2 illustrated, respectively). The trials were the absorption of H-thymidine in either a smooth muscle cell line (A7R5) or a non-smooth muscle cell line (MDA-MB468, breast carcinoma) the results showed muscle tissue specificity using the alpha-actin promoter of smooth muscle and a specific inhibition by both p56 and E2F-p56 transgenes in relation to their respective controls. DESCRIPTION OF THE PREFERRED MODE The present invention offers RB fusion constructs including fusion polypeptides and vectors that encode them, and methods for the use of such constructs in the treatment of hyperproliferative diseases. In certain preferred embodiments of the invention, a RB polypeptide is fused to an E2F polypeptide. Any E2F species, typically E2F-1, -2, -3, -3, or -5 (see, for example, Wu et al., Mol. Cell. Biol. 15: 2536-2546 (1995); 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)), more typically E2F-1. Typically, the EF2 polypeptide comprises at least the E2F DNA binding domain, and may optionally include the cyclin A binding domain, the heterodimerization domain, and / or the transactivation domain. Preferably, the cyclin A binding domain is not functional. The nucleotide and amino acid sequence of E2F referred to herein are those of Genbank HUME2F, shown in Figure IA and Figure IB. The nucleic acid, preferably DNA, encoding an EF2 polypeptide of this type is fused in a reading frame on a RB polypeptide. The RB polypeptide can be any RB polypeptide, including conservative amino acid variants, allelic variants, amino acid substitution, amino acid removal, or insertion mutants, or fragments thereof. Preferably, the growth suppression domain, ie, amino acid residues 379-928, of the RB polypeptide is functional (Hierbert, et al., MCB 13: 3384-3391 (1993)).; Quin, et al. Genes and Dev. 6: 953-964 (1992)). In some embodiments, wild type p RB110 is employed. More preferably, a truncated version of RB, RB56 is employed. RB56 comprises amino acid residues 379-928 of pRBUO (Hiebert, et al., MCB 13: 3384-3391 (1993); Quin, et al., Genes and Dev. 6: 953-964 (1992)). In certain embodiments, amino acid variants of RB are employed at positions 2, 608, 612, 788, 807, or 811, either singly or in combination. The RB56-5s variants comprise wild-type RB56 having alanine substitutions at 608, 612, 788, 807 and 811. The numbering of the RB amino acids and nucleotides according to the RB sequence presented by Lee, et al. (Nature 329: 642-645 81987)), is hereby incorporated by reference in its entirety for all purposes, (Figure 2). The nucleic acids encoding the polypeptides of the present invention may be DNA or RNA. The term "nucleic acid sequence encoding" refers to a nucleic acid that directs the expression of a specific protein or peptide. The nucleic acid sequences include both the strand sequence of DNA that is transcribed into RNA and the sequence of RNA that is translated into protein. The nucleic acid sequences include both the full length nucleic acid sequences and the full length sequences derived from the full length protein. It is further understood that the sequence includes the degenerate codons of the native sequence or sequences that can be introduced to provide codon preference in a specific host cell. The term "vector" as used herein refers to viral expression systems, self-replicating self-replicating circular DNA (plasmid), and includes both expression plasmids and non-expression plasmids. When a recombinant micro organism or a cell culture is described as the host of an "expression vector", this includes both extra-chromosomal circular DNA and DNA that has been incorporated into the host chromosome or host chromosomes. When a vector is maintained by a host cell, the vector can be stably replicated by the cells during mitosis in the form of an autonomous structure, or is incorporated into the genome of the host. A vector containing multiple positionally and sequentially oriented genetic elements, i.e., operably linked with other necessary elements such as the nucleic acid in the vector encoding the constructs of the present invention can be transcribed, and if necessary, transfected into transfected cells . The term "gene" as used herein is intended to refer to a nucleic acid sequence encoding a polypeptide. This definition includes several sequence polymorphisms, mutations, and / or sequence variants, where such operations do not affect the function of the gene product. The term "gene" is intended to include not only the coding sequence but also regulatory regions such as promoters, enhancers, and termination regions. The term also includes all introns and other DNA sequences spliced from the mRNA transcript, together with variants resulting from alternative splice sites. The term "plasmid" refers to an autonomous circular DNA molecule capable of replication in a cell, and includes both types of expression and non-expression. When a recombinant micro organism or a cell culture is described as a host of an "expression plasmid" this includes both extrachromosomal circular RNA molecules and DNA that has been incorporated into the host chromosome or host chromosomes. When a plasmid is maintained by a host cell, the plasmid is either stably replicated by the cells during mitosis as an autonomous structure or is incorporated within of the host genome. The term "recombinant protein" or "recombinantly produced protein" refers to a peptide or a protein produced using non-native cells that do not have an endogenous copy of 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 will not be found in association with proteins and other subcellular components normally associated with a cell that produces the protein. The terms "protein" and "polypeptide" are used interchangeably here.

In general, a construct of the present invention is provided in an expression vector comprising the following elements sequentially linked at appropriate distances for functional expression. A tissue-specific promoter, an initiation site for transcription, a 3"untranslated region, a 5 'leader sequence of mRNA, a nucleic acid sequence encoding a polypeptide of the invention and a polyadenylation signal. as "operable link." Sequences of enhancers and other sequences that aid expression and / or secretion may also be included in the expression vector.Additional genes, such as those encoding drug resistance may be included 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 chloramfenicol acetyl transferase In the present invention, the tissue-specific expression of the RB constructs of the This invention is preferably achieved by the use of a promoter which is preferably used by a tissue of interest. Example of tissue-specific promoters include a promoter for creatine kinase, which has been used to direct the expression of dystrophin cDNA in muscle and cardiac tissue (Cox, et al., Nature 364: 725-729 (1993)) and immunoglobulin heavy or light chain promoters for the expression of suicide genes in B cells (Maxwell, et al.

Res. 51: 4299-4304 (1991). A specific regulatory region for endothelial cells has also been characterized (Jahroudi, et al., Mol. Cell Biol. 14: 999-1008 (1994)).

Retroviral amphotrophic vectors have been constructed which carry a herpes simplex virus thymidine kinase gene under the control of albumin promoter or alpha-fetoprotein promoter (Huber, et al., Proc. Natl. Acad. Sci. USA 88: 8039 -8043 (1991)) to target cells of liver lineage and hepatoma cells, respectively. Such tissue-specific promoters can be used in retroviral vectors (Hartzoglou, et al., J. Biol. Chem. 265: 17285-17293 (1990)) and adenovirus vectors (Fried an, 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, a preferred promoter for tissue-specific expression of exogenous genes is the human smooth muscle alpha-actin promoter. Reddy, et al. (J, Cell Biology 265: 1683-1687 (1990)) presented the isolation and nucleotide sequence of this promoter, while Nakano, et al. (Gene 99: 285-289 (1991)) presented the transcriptional regulation elements at the 5 'end and the first introns regions of the human smooth muscle alpha-actin gene (aortic type). Petropoulos, et al. (J. Virol. 66: 3391-3397 (1992)) presented a comparison of bactgerian chloramphenicol transferase (CAT) expression operably linked to chicken skeletal muscle alpha-actin promoter or cytoplasmic beta-actin promoter. These constructs were provided in a retroviral vector and were used to infect chicken eggs. Exemplary tissue-specific expression elements for liver include, but are not limited to, the HMG-CoA reductase promoter (Luskey, Mol. Cell, Biol. 7 (5): 1881-1893 (1987)); regulatory element 1 sterol (SRE-1; Smith et al., J. Biol. Chem. 265 (4): 2306-2310 (1990); phosphoenol pyruvate carboxykinase (PEPCK) promoter (Eisenberger et al., Mol. Cell. Biol. 21 (3) ): 1396-1403 (1992)), human C-reactive protein promoter (CRP) (Li et al., J. Biol. Chem. 265 (7): 4136-4142 (1990)), human glucokinase promoter (Tanizawa et al., Endocrinology 6 (7): 1070-81 (1992); promoter of cholesterol 7-alpha hydroylase (CYP-7) (Lee et al.

J. Biol. Chem. 269 (29): 14681-9 (1994)); beta-galactosidase alpha-2,6-sialyltransferase promoter (Svensson et al., J. Biol. Chem. 265 (34): 20863-8 ( 1990), promoter of insulin-like growth factor enlce protein (IGFBP-1) (Babajo et al Biochem Biophys, Res. Comm. 196 81): 480-6 (1993)); Aldolase B promoter (Bingle et al., Biochem J. 294 (Pt2): 473-9 (1993)); human transferrin promoter (Mendelzon et al., Nucí Acids, Res. 18 (19): 5717-21 (1990), type I collagen promoter (Houglu et al., J. Clin.

Invest. 94 (2): 808-14 (1994)). Exemplary tissue-specific expression elements for the prostate include, but are not limited to, the prostatic acid phosphatase (PAP) promoter (Bañas et al., Biochim Biophys. Acta. 1217 (2): 188-94 (1994); prostatic secretory protein promoter of 94 (PSP 94) (Nolet et al., Biochim Biophys, ACTA 1098 (2): 247-9 (1991)); prostate-specific antigen complex promoter (Casper et al., J. Steroid Biochem, Mol. 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) Exemplary tissue-specific expression elements for gastric tissue include, but are not limited to, the alpha subunit promoter of H + / K + -ATPase (Tanura et al., FEBS Letters 298 (2-3): 137-41 ( 1992).) Exemplary tissue-specific expression elements for the pancreas include, but are not limited to, the pancreatitis-associated protein promoter (PAP) (Dusetti et al., J. Bio. I. Chem. 268 (19): 14470-5 (1993)); the transcriptional elastase 1 enhancer (Kruse et al., Genes and Development 7 (5): 774-86 (1993)); the pancreas-specific amylase and elastase enhancer promoter (Wu et al., Mol.Cell. Biol. 11 (9): 4423-30 (1991); Keller et al., Genes &Dev. 4 (8): 1316- 21 (1990)); promoter of the pancreatic cholesterol esterase gene (Fontaine et al, Biochemistry 30 (28): 7008-14 (1991)). Exemplary tissue-specific expression elements for the endometrium include, but are not limited to, the uteroglobin promoter (Helftenbein et al., Annal, NY Acad. Sci. 622: 69-79 (1991)). Exemplary tissue-specific expression elements for adrenal cells include, but are not limited to, the cholesterol side chain dissociation (SCC) promoter (Rice et al., H. Biol. Chem. 265: 11713-20 (1990). Exemplary tissue-specific expression elements for the general nervous system include, but are not limited to, the gamma-gamma enolase promoter (neuron-specific enolase, NSE) (Forss-Petter et al., Neuron 5 (2): 187-97). (1990) Exemplary tissue-specific expression elements for the brain include, but are not limited to, the neurofilament heavy chain promoter (NF-H) (Schwartz et al., J. Biol. Chem. 269 (18): 13444- 50 81994)).

Exemplary tissue-specific expression elements for lymphocytes include, but are not limited to, the human CGL-1 / granzyme B promoter (Hanson et al., J. Biol. Chem. 266 (36): 24433-8 (1991)).; terminal deoxytransferase (TdT), lambda 5 promoter, VpreB, and lck (lymphocyte specific for tyrosine protein kinase p561ck) (Lo et al., Mol. Cell, Biol. 11 (10): 5229-43 (1991)); the promoter of human CD2 and its transcription enhancer 3"(Lake et al., EMBO J: 9 (10): 3129-36 (1990)), and the activation promoter specific for human T cells and NK (NKG5) (Houchins et al., Immunogenetics 37 (2): 102-7 (1993).) Exemplary tissue-specific expression elements for the colon include, but are not limited to, the pp60c-src promoter of tyrosine kinase (Tala onti et al., J. Clin. Invest 9l (l): 53-60 81993)), organ-specific neoantigens (OSNs), 40kDa molecular weight promoter (p40) 8Ilantzis et al., Microbiol. Immunol., 37 (2): 119-28 (1993) ); P-antigen promoter specific for the colon (Sharkey et al., Cancer 73 (3 supp.) 864-77 (1994)) Exemplary elements of tissue-specific expression for breast cells, include, but are not limited to, the human afa-lactalbumin promoter (Thean et al., British J. Cancer.61 (5): 773-5 (1990).) Other elements that help the specificity of expression in a tissue of interest may include leader secretory sequences, enhancers, nuclear localization signals, endosmolytic peptides, etc. Preferably, these elements are derived from the tissue of interest to assist in specificity.Techniques for the manipulation of nucleic acid sequences Nucleic acid of the present invention such as subcloning of nucleic acid sequences encoding polypeptides into expression vectors, labeling probes, DNA hybridization and the like are described generally in Sambrook et al., Molecular Cloning || Alaboratory Manual ( 2nd Edition), Volume 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989), which is incorporated herein by reference. such as "Sambrook et al." Once the DNA encoding a sequence of interest is isolated, and once said DNA is cloned, the encoded proteins can be expressed in several recombinantly manipulated cells. It is expected that persons skilled in the art know of various expression systems available for the expression of encoding DNA. No attempt is made to describe in detail the various known methods for the expression of proteins in prokaryotes or eukaryotes. As a brief summary, the expression of natural or synthetic nucleic acids encoding a sequence of interest is typically achieved by linking the DNA or cDNA to a promoter (either constitutive or inducible), followed by incorporation into an expression vector. The vectors may be suitable for replication and integration in prokaryotic or eukaryotic. Typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of polynucleotide sequence of interest. To obtain high-level expression of a cloned gene, it is desirable to construct expression plasmids that contain, at a minimum, a strong promoter to direct transcription, a ribosome binding site for the start of translation, and a transcription terminator. / translation. Expression vectors can comprise generic expression cassettes containing at least one terminator sequence independently, sequences that allow replication of the plasmid in eukaryotes and prokaryotes, i.e., linkage vectors, and selection markers for both prokaryotic and systemic systems eukaryotic See Sambrook et al. The E2F-RB fusion constructs of the present invention can be introduced into the tissue of interest in vivo or ex vivo by various methods. In some embodiments of the invention, the nucleic acid, preferably DNA, is produced in cells by methods such as micro injection, calcium phosphate precipitation, liposome fusion or biolistic. In additional embodiments, the DNA is directly absorbed by the tissue of interest. In other embodiments, the constructs are packaged in a viral vector system to facilitate introduction into cells. »Viral vector systems useful in the practice of the present invention include adenovirus, herpesvirus, adeno-associated virus, mouse miniature virus 8MVM), HIV, sindbis virus, and retroviruses such as Rous sarcoma virus, and MoMLV. Typically, the constructs of the present invention are inserted into vectors of this type to allow packaging of the E2F-RB expression construct, typically with accompanying viral DNA, infection of a sensitive host cell, and expression of the E2F-RB gene. A particularly useful vector is the adenovirus vector presented in Wills, et al. Human Gene Therapy 5: 1079-1088 (1994). In other embodiments of the present invention, the recombinant DNA constructs of the present invention are conjugated with a cell receptor ligand for facilitated absorption (e.g., imagination of the coated pits and internalization of the endosome) through a DNA binding portion (Wu et al., J. Biol. Chem. 263: 14621-14624 (1988); WO 92/06180). For example, the DNA constructs of the present invention can be linked through a polylysine portion to asialo-oromucido, which is a ligand for the asialoglycoprotein receptor of hepatocytes. Similarly, the viral envelopes used to package the constructs of the present invention can be modified by the addition of receptor ligands or antibodies specific for a receptor in order to allow receptor-mediated endocytosis in specific cells (eg, WO). 93/20221; WO 93/14188; WO 94/06923). In some embodiments of the invention, the DNA constructs of the present invention are linked to viral proteins, such as, for example, adenovirus particles, in order to facilitate endosytosis (Curiel, et al., Proc. Natl. Acad. Sci. USA 88: 8850-8854 (1991)). In other embodiments, the molecular conjugates of the present invention may include microtubule inhibitors (WO 94/06922); synthetic peptides that mimic the hemagglutinin of influenza virus (Plank, et al., J. Biol. Chem. 269: 12918-12924 (1994)); and nuclear localization signals such as SV40 T antigen (Wo 93/19768). In some embodiments of the invention, the RB polypeptides of the present 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" includes but is not limited to cells that have autonomic capacity and growth, ie, cells that exist and reproduce independently of normal regulatory mechanisms. Hyperproliferative diseases can be categorized as pathological, that is, they deviate from normal cells, which are characterized by a constitutive disease or they can be categorized as non-pathological, that is, deviation from the norm but not associated with a diseased state. Pathological hyperproliferative cells are characteristic of the following diseases: restenosis, diabetic retinopathy, thyroid hyperplasia, severe disease, psoriasis, benign prostatic hypertrophy, Li-Fraumeni syndrome, including breast cancer, sarcomas and other neoplasms, bladder cancer , colon cancer, lung cancer, several leukemias and lymphomas. Examples of nonpathological hyperproliferative cells are found, for example, in mammary duct epithelial cells during the development of lactation and also in cells associated with wound repair. The pathological hyperproliferative cells characteristically exhibit a loss of contact inhibition and a decrease in their ability to selectively adhere, which implies an additional decomposition of the intercellular communication. These changes include the stimulus to divide and the ability to secrete proteolytic enzymes.

The constructs of the present invention are useful in the therapy of various cancers and other conditions wherein the administration of RB is beneficial, including, but not limited to, peripheral vascular diseases as well as diabetic retinopathy. Even though any tissue can be targeted for certain tissue-specific expression elements, such as, for example, a promoter, the tissue-specific administration of an RB construct for hyperproliferative disorders, such as, for example, restenosis can be identified, for which A smooth muscle actin promoter is of particular interest. The compositions of the present invention are formulated for administration in a manner known per se in the art acceptable for administration to a mammal, preferably a human. In certain embodiments of the invention, the compositions of the present invention can be administered directly into a tissue through injection or into a blood vessel that feeds the tissue of interest. In additional embodiments of the invention, the compositions of the present invention are administered "locoregionally", i.e., intravesically, intralesionally, and / or topically. In other embodiments of the invention, the compositions of the present invention are administered systemically by injection, inhalation, suppositories, transdermal administration, etc. In additional embodiments of the invention, the compositions are administered through catheters or through other devices to allow access to a remote tissue of interest, for example an internal organ. The compositions of the present invention can also be administered in prolonged release type devices, implants, or encapsulated formulations to allow a slow or sustained release of the compositions. The invention offers compositions for administration, comprising a solution of the compositions of the invention dissolved or suspended in an acceptable vehicle, preferably an aqueous vehicle. Various aqueous vehicles can be employed, for example, water, regulated water, 0.8% saline, 0.3% glycine, hyaluronic acid, and the like. These compositions can be sterilized by conventional well-known sterilization techniques or can be sterilely filtered. . The resulting aqueous solutions can be packaged for use as they are or they can be lyophilized, the lyophilized preparation being combined with a sterile solution before administration. The compositions may contain pharmaceutically acceptable excipients in accordance with what is required by the approximate physiological conditions, for example pH adjustment and regulating agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, lactate sodium, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. The concentration of the compositions of the present invention in the pharmaceutical formulations can vary widely, ie, from less than about 0.1%, usually from at least about 2% to 20% to 50% or more by weight, and are selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. The compositions of the present invention can also be administered through liposomes. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, sheet layers and the like. In these preparations, the composition of the present invention to be delivered is incorporated as part of a liposome, either alone or in combination with a molecule that binds to a desired target, such as for example antibody, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired composition of the present invention can be administered systemically, or they can be targeted to a tissue of interest, where the liposomes then deliver the selected therapeutic / immunogenic peptide compositions.

Liposomes for use in the present invention are formed from lipids that form standard vesicles, which generally include negatively charged or neutral phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by taking into account, for example, the size of the liposome, the weakness with acids and the stability of the liposomes in the bloodstream. Several methods are available for liposome preparation, in accordance with what is described, for example in Szoka et al. Ann. Rev. Biophys. Bioeng. 9: 467 (1980), U.S. U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, which are incorporated herein by reference. A liposome suspension containing a composition of the present invention can be administered intravenously, locally, topically, etc., in a dose that varies in accordance with, inter alia, the method of administration, the composition of the invention administered, and the state of the disease that is being treated. In the case of solid compositions, conventional non-toxic solid carriers can be employed, which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and similar. For oral administration, a pharmaceutically acceptable non-toxic composition is formed by the incorporation of any of the excipients normally employed, such as, for example, the vehicles appearing in the above list, and generally from 10 to 95% of active ingredient, i.e., a or various compositions of the invention and more preferably in a concentration of 25% to 75%. For aerosol administration, the compositions of the present invention are preferably delivered in a finely divided form together with a surfactant and an impeller. Typical percentages of compositions of the invention are from 0.01% to 20% by weight, preferably from 1% to 10%. The surfactant must, of course, be non-toxic, and preferably soluble in the impeller. Representative examples of such agents are esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as for example caproic acid, octanoic acid, lauric acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, oleic acid and oleic acid with a polyhydric alcohol. aliphatic or its cyclic anhydride. Mixed esters such as mixed or natural glycerides can be used. The surfactant may constitute from 0.1% to 20% by weight of the composition, preferably from 0.25 to 5%. The rest of the composition is a usual driver. A vehicle can also be included, as desired, for example lecithin for intranasal administration. The constructs of the present invention can be further administered in a depot-type system, an encapsulated form, or an implant by well-known techniques. Similarly, the constructs can be administered through a pump to a tissue of interest. In some embodiments of the invention, the compositions of the present invention are administered ex vivo to cells or tissues explanted from a patient, and then returned to the patient. Examples of the ex vivo administration of the gene therapy construct include Arteaga 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 (l): 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 (l): 402-6 (1996). In some embodiments of the invention, the constructs of the present invention are administered to a cardiac artery after balloon angioplasty to avoid or reduce the severity of restenosis. The constructs of the present invention can be used to coat the device used for angioplasty (see, for example, Willart, et al Circulation 89: 2190-2197 81994); Frech, et al. Circulation 90: 2402-2413 (1995)). In additional embodiments, the fusion polypeptides of the present invention can be used in the same manner.

The following examples are provided to illustrate the present invention and not to limit it. EXAMPLES Example I Fusions E2F-RB A. Introduction In this example, expression plasmids encoding different segments of E2F fused to the RB56 polypeptide were constructed. RB56 is a subfragment of full length RB that contains the "bag" domains necessary for growth suppression (Hiebert, et al., MCB 13: 3384-3391 (1993); Qui, et al., Genes and Dev. 6: 953-964 (1992)). E2F194 contains the amino acids of E2F 95-194. This fragment contains only one E2F DNA binding domain. E2F286 contains the DNA binding domain and the DP-1 heterodimerization domain. Both fragments of E2F lack the N-terminal cyclin A-kinase binding domain, which seems to regulate downward the activity of E2F DNA binding (Krek et al., Cell 83: 1149-1158 (1995); et al., Cell 78: 161-172 (1994)). B. Construction of vectors Plasmid pCTM contains a CMV promoter, a tripartite adenovirus leader flanked by T7 and SP6 promoters, and a multiple cloning site with a bovine growth hormone polyadenylation site (BGH) and a polyadenylation site SV-40 downstream. A diagrammatic representation of pCTM is provided in Figure 3. The DNA sequence for pCTM is given in Figure 4. PCTMI was constructed from pCTM by digestion of pCTM with Xho I and Not I and subcloning an XhoI fragment -Not I of 180 base pairs from a pCMV-beta-gal vector (Clonetech). A diagrammatic representation of pCTMI is provided in Figure 5. The DNA sequence is provided in Figure 6. pCTMIE was constructed by amplifying the SV40 enhancer from an SV40 viral DNA in a polymerase chain reaction. The amplified product was digested with BglIII and inserted into pCMTI digested with BamHl and ligated in the presence of BamHl. The plasmid is represented diagrammatically in Figure 7. The DNA sequence is provided in Figure 8. pCTM-RB was prepared in the following manner. A 3.2 KB Xba I-Cla I fragment of pERRBc (Huang et al., Nature 350: 160-162 (1991)) containing the full-length human RB cDNA was ligated onto pCTM digested with Xba I-Cla I. prepared pCTM-RB56 by ligating the digested pCTM into a 1.7 KB Xba 1-Cla I fragment containing the coding sequence for RB56. PCTMI-RB, pCTMIE-RB, pCTMI-RB56 (amino acids 381-928) were all constructed by the same methods. C. RB-E2F fusion constructs Figure 9 shows the fusion constructs used in these studies. These E2F constructs started at amino acid 95 and lacked part of the cyclin A binding domain. E2F437 contained the DNA binding domain (black), the heterodimerization domain (blank) and the transactivation domain (dotted). E2F194 it contained only the DNA binding domain. E2F286 contained the DNA binding domain and the heterodimerization domain of DP-1. RB56-5s refers to a variant of RB that have their alanine institutions in amino acid residues 606, 612, 788, 807 and 811. In E2F194-RB56-5S and E2F286-RB56-5S, the E2F fragments were fused in frame over codon 379 of RB-5s. RB56-C706F contained a dot mutation inactivation (Kaye et al., Proc. Natl. Acad. Sci. U.S.A. 87: 6922-6926 (1990)). PCMV-E2F194 and pCMV-E2F437 were constructed in the following manner, a DNA coding for amino acids 95-194 of E2F (which contains the DNA binding domain) or amino acids 95-437 was amplified in a chain reaction of Polymerase, was digested with HindII, and ligated into pCMV-RB56 vectors digested with Smal / HindII. PCMVE2F286 was constructed by digestion of pCMV-E2F437 with AflII, treating the ends with DNA pol I (Klenow fragment) and ligand again in the presence of AflII. The ligation of the flattened end created a retention codon at position 287. pCMV-E2F286-5s ligand pE2F437 digested by AflII (flattened) / HindIII was constructed on a salt fragment I (flattened) -HindIII containing the coding sequence of RB56 -5S. PCTMIE-E2F194-5s and pCTMIE-E2F286-RB5s ligand pCTMIE (4.2 NB) digested by ExoRI-EcoRV were constructed with HindIII (flattened) fragments -EcoRI from pCMV-E2F194-RB5s or pCMV-E2F286-RB5s. D. Promoter repression To measure the effect of the E2F-RB fusion proteins, a C33A cervical carcinoma cell line (ATCC # HTB-31) with equivalent amounts of E2F194-RB56 or E2F RB56 was transfected with an E2-CAT reporter plasmid (see, eg, Weintraub et al., Nature 358: 259 -261 (1992)). In the C33A assay, 250,000 C33A cells were seeded in each of the wells of 6-well tissue culture dishes and allowed to adhere overnight. Each of the plasmids pCMV-RB56, pCMV-E2F RB56, or pCMV-E2F (calcium phosphate method, MBS transfection set, Stratagene) were co-transfected with 5 μg of reporter construct indicated E2-CAT or SVCAT and 2.5 μg of beta-gal plasmid (pCMV-beta, Clontech) per well in duplicate wells. The cells were harvested 72 hours after transfection and the extracts were prepared.

In the 5637 assay, 250,000 5637 cells were seeded in accordance with the above described. 1 μg each of RB or fusion plasmid E2F-RB, SV-CAT reporter plasmid or E2-CAT, and pCMV-beta-galactosidase were co-transfected using the lipofectin reagent (BR1, Bethesda, Maryland) in accordance with the manufacturer's instructions. CAT assays were performed employing either 20μL (C33A) or 50μL (5637) of cell extract (Gorman et al., Mol.Cell. Biol. 2: 1044 (1982)). TLCs were analyzed in a Phosphoimager SF (Molecular Dynamics). The CAT activities were normalized for transfection efficiency in accordance with the beta-galactosidase activities of each extract. The beta-galactosidase activities of the extracts were tested in accordance with that described by Rosenthal et al (Meth., Enzym, 152: 704 (1987)). The results of these studies were the following. Transfection of the E2-CAT reporter alone or in the presence of the non-functional control mutant RB56-H209 provided a relatively high CAT activity. Co-transfection of wild-type RB56 or of variable RB56-5s resulted in a 10 to 12-fold repression of CAT activity, indicating that RB56 or RB56-5s are both capable of efficiently repressing transcription-dependent transcription. E2F. E2F194-RB5s and E2F286-RB5s repressed the transcription approximately 50 times. The repression of the transcription required that the RB56 and E2F components of the fusion proteins according to the expression of E2F194 and E2F286 do not mediate repression of transcription. No repression of SV40-CAT transcription occurred with the E2F-RB constructs thus demonstrating the specificity of transcriptional repression by E2FRB for the E2 promoter. These results are shown diagrammatically in Figure 10. E. Cell cycle suspension The ability of the E2F-Rb fusion polypeptides to cause Gl suspension in Saos-2 (RB - / - cells) was investigated (ATCC # HTB- 85) and C33A cells. Previous studies have shown that repression of RB-mediated E2 promoter and Gl suspension are related in Saos-2 cells but dissociated in C33A (Rbmut) cells (Xu, et al., PNAS 92: 1357-1361 (1992)) . The cells were washed in PBS and fixed in 1 ml of 70% ethanol at a temperature of -20 ° C for 30 minutes. The cells were collected by centrifugation and resuspended in 0.5 ml of 2% serum containing 10 μg / ml of RNase A and incubated for 30 minutes at a temperature of 37 ° C in 0.5 ml of PBS containing propidium iodide (100μg / liter). ml) and added to each sample, mixed and the cells were mixed again from a FACS tube strainer. The FACS analysis was carried out in a FACS-Scan (Becton-Dockenson) using doublet discrimination. 5000-10000 CD20 + events were analyzed. The percentage of cells in G0 / G1, S and G2 / M was determined using a programmatic modeling Modfit). The results of this experiment were the following full-length RBllO and the truncated version RB56, but not the control mutant RB-H209, caused Gl suspension in Saos-2 cells (table 1). Similarly, RB56-5s, E2F-194-RB56-5S and E2F286-RB56-5S were all able to stop cells in G0 / G1. Transfection of the DNA binding domain, E2F194, does not block entry into S phase in Saos-2 as previously described for rodent cells (Dobrowolski, et al., Oncogene 9: 2605-2612 (1994)). In contrast, the fusion proteins, RB11, RB56, and E2F-RB were not able to stop the C33A cell lines, indicating that the repression of transcription observed in these cells does not result in a suspension of Gl. The ability of E2F-RB fusion proteins to suspend 5637 cells was also investigated (Table 2). RB56 and RB56-5s efficiently suspended cells in G0 / G1 (approximately 90% of cells in G0-G1), while E2F194-RB56-5s and E2F286-RB56-5s are slightly less efficient (approximately 80% of cells in Gl / Gl) to promote the arrest of G0 / G1. Without being limited by any theory, the less efficient suspension of Saos-2 and 5637 cells by the E2F-RB fusion proteins seems to be due to lower levels of constant state protein produced in these cells (Figure 11, panels b and c). Table 1: Regulation of the cell cycle by RB and E2F-RB fusion proteins in Rbneg cells% of cells 0020"*" G0 / G1 G2 / M phase S 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: Suppression of bladder cell growth 5637 by RB and fusion proteins E2F-RB% of cells G0 / G1 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 E2F194-5S 80.1 1.31 0 E2F-286-5S 79.21 8.1 0 F. Activity of fusion proteins in background of functional RB The activity of fusion proteins E2F-RB in cellular background containing functional RB was determined after the NIH-3T3 cells were transfected with RB56 or well E2F-RB56 fusion proteins and labeled with an anti-RB monoclonal antibody 3C8 (Wen et al., J. Immuno.Meth.169: 231-240 (1994)). A FACS analysis of the RB expression cells was carried out. The results appear in Figure 12. The population without compound (g) shows the characteristic distribution of the cell cycle for the NIH-3T3 cells (60% GO, 28% S, 10% G2 / M). In contrast, in cells transfected with RB56 (a, b) or in E2F-RB fusion proteins (c-f), more than 90% of the RB expression cells were stopped in G0 / G1. These data demonstrate that the ability of RB and E2F-RB56 fusion proteins to suspend G0 / G1 cells is not limited to tumor cells negative for RB. The relative levels of protein expressed in the transfected NIH-3T3 cells was also investigated. RBllO was not expressed efficiently in these cells. Thus, these data demonstrate that E2F-Rb fusion proteins are more efficient transcription repressors than pRB or RB56, alone, and that RB can repress transcription by remaining bound on E2F instead of directly blocking the transactivation domain of E2F. These data support the use of E2F-Rb fusion proteins as RB agonists in both RB + cells and negative RB cells or RB mutants. EXAMPLE II Tissue specific expression of E2F-RB fusions A. Recombinant adenovirus construction: In this experiment, recombinant adenovirus comprises an RB polypeptide under the control of a smooth muscle alpha actin promoter or CMV were generated. The smooth muscle alpha-actin promoter (bases-670 to +5, reddy et al. "Structure of the Human Smooth Muscle Alpha-Actin Gene" J. Biol Chem. 265: 1683-1687 (1990), Nakano, et al. «Transcriptional Regulatory Elements In The 5 'Upstreamand First Intron Regions of the Human Smooth Muscle (aortic type) alpha-Actin-Encoding Gene» Gene 99: 285-289 (1991) was isolated by polymerase chain reaction from a genomic library with restriction sites Xho I and Avr II in 5'and Xba I, Cia I and Hind III in 3 'aggregates for cloning purposes The fragment was subcloned as an Xho I fragment, Hind III in a plasmid for sequencing with the object of verifying the base composition A fusion construct 286-56 containing the DNA and the heterodimerization domain of E2F-1 (bases 95-286) joined in p56 (amino acids 379-929 of full length RB) was subcloned ) as a fragment Xba I, Co. I directly downstream of the alpha-actin promoter of mu smooth muscle, and this expression cassette was digested and cloned into the plasmid pAd / lTR / IX-co or an Xba I fragment into Avrll, Co. I to create the plasmid pASN286-56. This plasmid consisted of the inverted terminal repeat of type adenovirus (ITR), packing signals and enhancer of Ela followed by the promoter of alpha-actin of human smooth muscle and a cassette 286-56, and then the Ad 2 sequence 4021 -10462 (containing the poly A Elb / protein IX signal) on a pBR322 background. A recombinant adenovirus was produced by quantum procedures. The plasmid pASN285-56 was linearized with Ngo MI and cotransfected in 293 cells with the large rAd34 fragment digested by Co. I that had deletions in the E3 and E4 regions of adenovirus type 5. Ad34 was a derivative of type 5 with a 1.9 KB removal in the initial region 3 which resulted from the removal of the Xba I restriction fragment extending from the coordinates of Ad5 28593 to 30470 and a 1.4 KB removal of the initial region 4 that results from a Taq 1 fragment of E4 (coordinates 33055-35573) replaced with a cDNA containing E4 or 0RF6 and 6/7. The recombinant adenovirus produced by the homologous recombination was isolated and identified by restriction digestion analysis and further purified by limiting dilution. Additional recombinant control adenoviruses are described elsewhere and include the ACN control virus (CMV promoter), Wills, et al. «Gene Therapy For Hepatocellular Carcinoma: Chemosensitivity Conferred By Adenovirus-Mediated Transfer of HSV-1 Thymidine Kinase Gene» Cancer Gene Therapy 2: 191-197 (1995)), and ACN56 (RB expressed under the control of a CMV promoter) . The ACN56 was prepared as follows. A plasmid containing p56 cAND was constructed by replacing the p53 cDNA from plasmid ACNP53 (Wills et al, Human Gene Therapy 5: 1079-1088 (1994)) with a 1.7 KB Xba I-BamHI fragment isolated from the pET 9 a-Rb56 plasmid (Antelman et al., Oncogene 10: 697-704 (1995)) containing p56 cDNA. The resulting plasmid contained amino acids 381-928 of p56, inverted terminal repeat Ad5, viral packaging signals and Ela enhancer, followed by the immediate initial promoter of human cytomegalovirus (CMV) and the tripartite leader Ad 2 cDNA to boost the expression of p56. The cDNA of p56 was followed by the 4021-10462 sequence of Ad 2 on a pBR322 background. This plasmid was linearized with EcoRI and cotransfected with the large fragment of DL327 digested by bsp 106 (E3 removed; Timmappaaya et al. Cell 31: 543-551 (1982)) or h5ile4 (E4 removed, Hemstrom et al., J. Virol. 62: 3258-3264 (1988)). The recombinant viruses were further purified by limiting dilution. B. Cell Poliferation In this experiment, cell lines in culture were infected with RB constructs of recombinant adenoviruses to determine the relative expression of the RB polypeptide and the effect on cell proliferation. For H358 cells (ATCC # Crl 5807) and for MDA-MB468 cells (ATCC #HTB 132, breast adenocarcinoma), 5,000 cells / well were placed on a plate in normal growth medium in a 96 well microtiter plate ( Costar) and incubation was allowed overnight at a temperature of 37 ° C, with C02 at 7%. The viruses were serially diluted in growth medium and used to infect cells at the indicated doses for 48 hours. At this point, 3H-thymidine (Amersham, 0.5μCi / well) was added and the cells were incubated at a temperature of 37 ° for 3 additional hours before harvest. Both A7r5 cells (ATCC CRL1444, rat smooth muscle) and AlO cells (ATCC CRL 1476, rat smooth muscle) were seeded at 3,000 cells / well either in DME + 0.5% FCS or DME + 20% of FCS, respectively. The virus was serially diluted in the culture medium and used to infect the cells in the doses indicated in the figures. The infection and labeling procedure were the same for AlO cells as for H358 and MDA-MB468 cells except that 2 micro Ci / well marker was used. The A7r5 cells were not infected with the virus until 48 hours after seeding. 48 hours after infection, the serum concentration was cultured in 10% FCS and 2μCi / well 3H-thymidine was added and the incubation continued for an additional 3 hours before harvest. All cells were harvested by aspirating the well medium, trypsinizing the cells, and harvesting using a 96 well GF / C filter with a Packard Top cell harvester. The results appear in the graph as the mean percentage (+/- standard deviation) of control proliferation treated with mean versus virus dose in figures 13 and 14. Thus, Figure 13 shows a comparison of the effects of the P56 constructs. of adenovirus on AlO muscle cells and A7r5 cells. The p56 virus driven by CMV (ACN 56) inhibited the growth of AlO in approximately the same manner as the E2F fusion constructs driven by the actin promoter (ASN586-56 # 25,26). In Figure 14, the effects of the adenovirus constructs on the inhibition of the breast cancer cell line, MDAta468 MDA and a non-small lung carcinoma cell line, H358 are shown. In these experiments. E2F-p56 driven by the actin promoter was ineffective while p56 driven by the CMV promoter was effective in inhibiting the growth of non-smooth muscle cells. To determine if the non-smooth muscle cells were more infectious with adenoviruses than the smooth muscle cell lines employed, the four cell lines, H358, MB468, A7R5, and AlO were infected at an MOI of 5 with an adenovirus expressing beta-galactosidase (AcbetaGL, Wills, et al, Human Gene Therapy 5: 1079-1088 (1994)) and the degree of beta-gal staining was examined. As shown in Figure 15 (above) the non-smooth muscle cell lines were signifficantly more infectious than the smooth muscle cell lines. In an additional test, the cells were infected at higher multiplicities of infection (50, 100, 250, 500) with ACN56 and the amount of p56 present in the infected cells was detected by autoradiography. As can be seen in Figure 15 (below), the non-muscle cell lines presented significantly higher numbers of p56 present, since as a result of their greater infectivity, the infected cells had a higher viral load and consequently more copies of the p56 annealing driven by the non-tissue-specific CMV promoter. In a further experiment, the specificity of the smooth muscle promoter of actin for smooth muscle tissue was determined. In this experiment, expression levels were measured in cells infected with beta-gal constructs driven by different promoters. As can be seen in figure 19, despite the low infectivity of smooth muscle cells, expression was only evident in cells using the smooth muscle alpha-actin promoter.

Figure 21 presents a comparison of the effects of a p56 recombinant adenovirus driven by CMV (ACN56E4) versus an E2F-p56 fusion construct driven by human smooth muscle alpha-actin promoter (ASN286-56) versus an adenoviral control construct which contains either the smooth muscle alpha actin promoters or CMV without a current to low transgene (isolated ACNE3 or ASBE3-2, respectively). The trials were of 3 H-thymidine uptake in either a smooth muscle cell line (A7R5) or the non-muscle cell line (MDA-MB468, breast carcinoma). The results showed muscle tissue specificity using the alpha-actin smooth muscle promoter and a specific inhibition of p56 or E2F-p56 transgenes in relation to their respective controls. C. Inhibition of Restenosis The balloon injury model was based on the model described by Clowes et al. (Clowes, Lab. Invest 49: 327-333 (1983)). Male Sprague-Dawley rats weighing 400 to 500 grams but anesthetized with an intraperitoneal injection of sodium pentobarbital (45 mg / kg Abbot Laboratories, North Chicago, Illinois). The bifurcation in the left common carotid artery was exposed through a midline incision and the left, internal and external communal carotid arteries were temporally ligated.

A 2F enbolectomel catheter (Baxter Edwards Healthcare Corp., Irvine, CA) was introduced into the external carotid and said catheter advanced toward the distal ligation of the common carotid. The balloon was inflated with a saline solution and pulled to the arteriotomy site three times in order to produce a distention lesion, deendotelialization. The catheter was then removed. Adenovirus (1 x 109 pfu of Ad-RB (ACNRb) or Ad-p56 (ACN56) was injected in a volume of 10 μL diluted to lOOμl with 15% (w / v) of Poloxamer 407 (BASF, Parsippany, NJ) or Ad-Beta-Gal (1 x 109 pfu, diluted as above) through a cannula was inserted just near the bifurcation of the carotid in a temporarily isolated segment of the artery.The adenovirus solution was incubated for 20 minutes after which the viral infusion was removed and the cannula was removed.The artery of the proximal external carotid was then ligated and the blood flow to the common carotid artery was restored by releasing the ligatures.The experimental protocol was approved by the Institutional Animal Care and Use Committee (Institutional Committee on Animal Care and Use) and complied with the "Guide for the Care and Use of Laboratory Animáis" (Guide for the care and use of laboratory animals) (Publication NIH number 86- 23, revised 1985) Rats were sacrificed at 14 days of treatment with an intraperitoneal injection of pentobarbital (100 mg / kg). The segment initially damaged by the balloon in the left common carotid artery, from the proximal edge of the omohyoid muscle to the bifurcation of the carotid, was perfused with a saline solution and the surrounding tissues were removed. The tissue was fixed in 100% methanol until it became embedded in paraffin. Several sections of 4μm were cut from each tissue sample. One section of each sample was marked with hematoxylin and eosin and another with a Richardson combination of conventional elastic trichromatic staining for light microscopy analysis. The histological images of the cross sections of the arterial sections stained with hematoxylin and eosin or elastic trichrome were projected on a digitized board (Summagraphics) and the medial and luminal intimal areas were measured by quantitative morphometric analysis using a computerized sketch program (MACMEASURE, version 1.9, National Institute of Mental Health) (National Institute of Mental Health). The results were expressed as the mean +/- S.E.M. The differences between groups were analyzed using an unpaired two-tailed Student's t-test. Statistical significance was considered when the probability of a null effect was less than 0.05. The results appear in figures 17 and 18. In figure 17, the relative inhibition of neointima formation is shown graphically, demonstrating the ability of p56 and RB to inhibit neointima formation. Figure 18 provides photographic evidence of the dramatic reduction of neointima in the presence of p56. The carotid arteries treated with adenovirus were harvested from rats two days after the injury caused by the balloon and from the infections. The tissue was fixed in formalin regulated with phosphate until its integration in paraffin. The tissue was cut into 4μm cross sections and the wax was removed through xylene and graded alcohols. Endogenous peroxylase was quenched with 1% hydrogen peroxide for 30 minutes. The recovery of antigen in lOmM sodium citrate buffer, pH 6.0, was carried out at a temperature of 95 ° C for 10 minutes. A monoclonal anti-RB antibody (AB-5, Oncogene Sciences, Uniondale, New York) lOμg / ml was applied in PBS in a humid chamber at a temperature of 4 ° C for 24 hours. A secondary antibody from the Unitect Mouse Immunohistochemistry kit (Oncogene Sciences, Uniondale, New York) was applied in accordance with the manufacturer's instructions. Antibody complexes were visualized using 3,3'-diaminobenzidene (DAB, Vector Laboratories, Burlingame, CA). The plates were counter-labeled with hematoxylin and mounted. The results appear in figure 20.

All references mentioned herein are incorporated by reference in their entirety for all purposes.

Claims (1)

1. CLAIMS A nucleic acid encoding a fusion polypeptide, the polypeptide comprises 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 polypeptide of fusion does not have the functional cyclin A-kinase linkage domain of the E2F transcription factor. The nucleic acid of claim 1, wherein the nucleic acid is inserted into an adenovirus vector. An expression vector comprising DNA encoding a fusion polypeptide, the polypeptide comprises a DNA binding domain fusion of an E2F transcription factor and a functional growth suppression domain of a retinoblastoma (RB) polypeptide, where the fusion polypeptide does not have the functional cyclin A-kinase binding domain of the E2F transcription factor. The vector of claim 3, comprising a tissue-specific promoter operably linked to the DNA encoding the fusion. The vector of claim 4, wherein the tissue-specific promoter is a smooth muscle actin promoter. The expression vector of claim 3, wherein the vector is a viral vector. 7. The expression vector of claim 6, wherein the vector is an adenovirus vector. 8. The vector of claim 7, wherein the adenovirus vector is deficient for replications. 9. The vector of claim 3, wherein the expression vector is a plasmid. 10. The vector of claim 5, wherein the actin promoter is an alpha-actin promoter. ll. A nucleic acid sequence encoding a fusion polypeptide, the polypeptide comprises a fusion of about amino acids 95-194 of E2F (SEQ ID No. 1) and about amino acids 379-928 of RB (SEQ ID No. 4) . 12. The nucleic acid of claim 11, further comprising a vector. 13. The nucleic acid sequence of claim 12, wherein the vector is a viral vector. 14. The nucleic acid sequence of claim 13, wherein the viral vector is an adenoviral vector. 5. The nucleic acid sequence of claim 14, wherein the adenoviral vector is deficient in replication. 6. The nucleic acid sequence of claim 11, wherein the nucleic acid sequence further comprises a tissue-specific promoter, wherein the fusion polypeptide is expressed under the control of a tissue-specific promoter. The nucleic acid sequence of claim 16, wherein the tissue-specific promoter is a smooth muscle actin promoter. The nucleic acid sequence of claim 17, wherein the smooth muscle promoter is an alpha-actin promoter.