Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
2001-04-17
2004-11-30
Falk, Anne-Marie (Department: 1636)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C536S024100, C435S320100, C800S013000
Reexamination Certificate
active
06825035
ABSTRACT:
1. INTRODUCTION
The present invention relates to promoters, enhancers and other regulatory elements that direct expression within smooth muscle cells (“SMC”). In particular, it relates to compositions comprising nucleotide sequences from the 5′ regulatory region and the first intron, and transcriptionally active fragments thereof, that control expression of a smooth muscle &agr;-actin (“SM &agr;-A”). Specifically provided are expression vectors, host cells and transgenic animals wherein an SM &agr;-A regulatory region is capable of controlling expression of a heterologous gene, over-expressing an endogenous SMC gene or an inhibitor of a pathological process or knocking out expression of a specific gene believed to be important for an SM-related disease in SMC. The invention also relates to methods for using said vectors, cells and animals for screening candidate molecules for agonists and antagonists of disorders involving SMC.
The present invention further relates to compositions and methods for modulating expression of compounds within SMC. The invention further relates to screening compounds that modulate expression within SMC. Methods for using molecules and compounds identified by the screening assays for therapeutic treatments also are provided.
2. BACKGROUND OF THE INVENTION
2.1 Gene Therapy
Somatic cell gene therapy is a strategy in which a nucleic acid, typically in the form of DNA, is administered to alter the genetic repertoire of target cells for therapeutic purposes. Although research in experimental gene therapy is a relatively young field, major advances have been made during the last decade. (Arai, Y., et al., 1997, Orthopaedic Research Society, 22:341). The potential of somatic cell gene therapy to treat human diseases has caught the imagination of numerous scientists, mainly because of two recent technologic advancements. Firstly, there are now numerous viral and non-viral gene therapy vectors that can efficiently transfer and express genes in experimental animals in vivo. Secondly, increasing support for the human genome project will allow for the identity and sequence of the estimated 80,000 genes comprising the human genome in the very near future.
Gene therapy was originally conceived of as a specific gene replacement therapy for correction of heritable defects to deliver functionally active therapeutic genes into targeted cells. Initial efforts toward somatic gene therapy relied on indirect means of introducing genes into tissues, called ex vivo gene therapy, e.g., target cells are removed from the body, transfected or infected with vectors carrying recombinant genes and re-implanted into the body (“autologous cell transfer”). A variety of transfection techniques are currently available and used to transfer DNA in vitro into cells; including calcium phosphate-DNA precipitation, DEAE-Dextran transfection, electroporation, liposome mediated DNA transfer or transduction with recombinant viral vectors. Such ex vivo treatment protocols have been proposed to transfer DNA into a variety of different cell types including epithelial cells (U.S. Pat. No. 4,868,116; Morgan and Mulligan WO87/00201; Morgan et al., 1987, Science 237:1476-1479; Morgan and Mulligan, U.S. Pat. No. 4,980,286), endothelial cells (WO89/05345), hepatocytes (WO89/07136; Wolff et al., 1987, Proc. Natl. Acad. Sci. USA 84:3344-3348; Ledley et al., 1987 Proc. Natl. Acad. Sci. 84:5335-5339; Wilson and Mulligan, WO89/07136; Wilson et al., 1990, Proc. Natl. Acad. Sci. 87:8437-8441), fibroblasts (Palmer et al., 1987, Proc. Natl. Acad. Sci. USA 84:1055-1059; Anson et al., 1987, Mol. Biol. Med. 4:11-20; Rosenberg et al., 1988, Science 242:1575-1578; Naughton & Naughton, U.S. Pat. No. 4,963,489), lymphocytes (Anderson et al., U.S. Pat. No. 5,399,346; Blaese, R. M. et al., 1995, Science 270:475-480) and hematopoietic stem cells (Lim, B. et al. 1989, Proc. Natl. Acad. Sci. USA 86:8892-8896; Anderson et al., U.S. Pat. No. 5,399,346).
Direct in vivo gene transfer recently has been attempted with formulations of DNA trapped in liposomes (Ledley et al., 1987, J. Pediatrics 110:1), in proteoliposomes that contain viral envelope receptor proteins (Nicolau et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:1068) and DNA coupled to a polylysine-glycoprotein carrier complex. In addition, “gene guns” have been used for gene delivery into cells (Australian Patent No. 9068389). It even has been speculated that naked DNA, or DNA associated with liposomes, can be formulated in liquid carrier solutions for injection into interstitial spaces for transfer of DNA into cells (Felgner, WO90/11092).
Numerous clinical trials utilizing gene therapy techniques are underway for such diverse diseases as cystic fibrosis and cancer. The promise of this therapeutic approach for dramatically improving the practice of medicine has been supported widely, although there still are many hurdles that need to be passed before this technology can be used successfully in the clinical setting.
Perhaps, one of the greatest problems associated with currently devised gene therapies, whether ex vivo or in vivo, is the inability to control expression of a target gene and to limit expression of the target gene to the cell type or types needed to achieve a beneficial therapeutic effect.
2.2 Tissue Specific Expression within Smooth Muscle Cells
Smooth muscle cells, often termed the most primitive type of muscle cell because they most resemble non-muscle cells, are called “smooth” because they contain no striations, unlike skeletal and cardiac muscle cells. Smooth muscle cells aggregate to form smooth muscle (“SM”) which constitutes the contractile portion of the stomach, intestine and uterus, the walls of arteries, the ducts of secretory glands and many other regions in which slow and sustained contractions are needed.
Abnormal gene expression in SMC plays a major role in numerous diseases including, but not limited to, atherosclerosis, coronary artery disease, hypertension, stroke, asthma and multiple gastrointestinal, urogenital and reproductive disorders. These diseases are the leading causes of morbidity and mortality in Western Societies, and account for billions of dollars in health care costs in the United States alone each year.
In recent years, the understanding of muscle differentiation has been enhanced greatly with the identification of several key cis-elements and trans-factors that regulate expression of muscle-specific genes. Firulli A. B. et al., 1997,
Trends in Genetics,
13:364-369; Sartorelli V. et al., 1993,
Circ. Res.,
72:925-931. However, the elucidation of transcriptional pathways that govern muscle differentiation has been restricted primarily to skeletal and cardiac muscle. Currently, no transcription factors have yet been identified that direct SM-specific gene expression, or SMC myogenesis. Owens G. K., 1995,
Physiol. Rev.,
75:487-517. Unlike skeletal and cardiac myocytes, SMC do not undergo terminal differentiation. Furthermore, they exhibit a high degree of phenotypic plasticity, both in culture and in vivo. Owens G. K., 1995,
Physiol. Rev.,
75:487-517; Schwartz S. M. et al., 1990,
Physiol. Rev.,
70:1177-1209. Phenotypic plasticity is particularly striking when SMC located in the media of normal vessels are compared to SMC located in intimal lesions resulting from vascular injury or atherosclerotic disease. Schwartz S. M., 1990,
Physiol. Rev.,
70:1177-1209; Ross R., 1993,
Nature,
362:801-809; Kocher 0. et al., 1991,
Lab. Invest.,
65:459-470; Kocher O. et al., 1986,
Hum. Pathol.,
17:875-880. Major modifications include decreased expression of SM isoforms of contractile proteins, altered growth regulatory properties, increased matrix production, abnormal lipid metabolism and decreased contractility. Owens G. K., 1995,
Physiol. Rev.,
75:487-517. The process by which SMC undergo such changes is referred to as “phenotypic modulation”. Chamley-Campbell J. H. et al., 1981,
Atherosclerosis,
40:347-357. Importantly, these alterations in expression patterns of SMC protein cannot simply be viewed as a consequen
Blank Randall
Mack Christopher
Owens Gary K.
Falk Anne-Marie
Setagon, Inc.
Sullivan Daniel M.
Townsend and Townsend / and Crew LLP
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