Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-07-13
2004-08-24
Falk, Anne-Marie (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S325000, C435S320100, C536S024100, C800S013000
Reexamination Certificate
active
06780610
ABSTRACT:
1. INTRODUCTION
The present invention generally relates to promoters, enhancers and other regulatory elements of smooth muscle cells (“SMC”). The invention more particularly relates to methods for the targeted knockout, or over-expression, of genes of interest within smooth muscle cells. The invention further relates to methods of conferring smooth muscle cell specific gene expression in vivo.
2. BACKGROUND OF THE INVENTION
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 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, hypertension, stroke, asthma and multiple gastrointestinal, urogenitcl 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 smooth muscle-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 artherosclerotic 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 0. et al., 1986,
Hum. Pathol.,
17:875-880. Major modifications include decreased expression of smooth muscle 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 consequence of vascular disease, but rather are likely to contribute to progression of the disease.
A key to understanding SMC differentiation is to identify transcriptional mechanisms that control expression of genes that are selective or specific for differentiated SMC and that are required for its principal differentiated function, contraction. Currently, studies are ongoing in which the expression of the contractile proteins SM &agr;-actin (Shimizu R. T. et al., 1995,
J. Biol. Chem.,
270:7631-7643; Blank R. S. et al., 1992,
J. Biol. Chem.,
267:984-989) and SM myosin heavy chain (SM-MHC)(White S. L. et al., 1996,
J. Biol. Chem.,
271:15008-15017; Katoh Y. et al., 1994,
J. Biol. Chem.,
269:30538-30545; Wantanabe M. et al., 1996,
Circ. Res.,
78:978-989; Kallmeier R. C. et al., 1995,
J. Biol. Chem.,
270:30949-30957; Madsen C. S. et al., 1997,
J. Biol. Chem.,
272:6332-6340; Madsen C. S. et al., 1997,
J. Biol. Chem.,
272:29842-29851), as well as a variety of proteins implicated in control of contraction including SM22&agr; (Li L. et al., 1996,
J. Cell. Biol.,
132:849-859; Kim S. et al., 1997,
Mol. Cell. Biol.,
17:2266-2278), h
1
-calponin (Miano J. M. et al., 1996,
J. Biol. Chem.,
271:7095-7103), h-caldesmon (Yano H. et al., 1994,
Biochem. Biophys. Res. Commun.,
201:618-626), telokin (Herring B. P. et al., 1996,
Am. J. Physiol.,
270:C1656-C1665) and desmin (Bolmont C. et al., 1990,
J. Submicrosc. Cytol. Pathol.,
22: 117-122) are being examined. Of these gene products, only SM-MHC expression appears to be completely restricted to SMC lineages throughout development (Miano J. et al., 1994,
Circ. Res.,
75:803-812), whereas all others show at least transient expression in non-SMC tissues (Owens G. K., 1995,
Physiol. Rev.,
75:487-517). As such, it appears that the SM-MHC gene is unique with regard to its potential utility for identification of SMC-specific transcriptional regulatory pathways and mechanisms.
To date, four SM-MHC isoforms (SMC-1A, SMC-1B, SMC-2A and SMC-2B) have been identified (Nagai R. et al., 1989,
J. Biol. Chem.,
264:9734-9737; White S. et al., 1993,
Am. J. Physiol.,
264:C1252-C1258; Kelley C. A. et al., 1993,
J. Biol. Chem.,
268:12848-12854), all of which are derived from alternative splicing of a single gene (Miano J. et al. 1994,
Circ. Res.,
75:803-812; Babij P. et al., 1989,
J. Mol. Biol.,
210:673-679). Alterations in expression of SM-MHC isoforms have been extensively documented in SMC that have undergone phenotypic modulation either when placed in culture (Rovner A. S., 1986,
J. Biol. Chem.,
261:14740-14745; Kawamoto S. et al., 1987,
J. Biol. Chem.,
262:7282-7288), or in vascular lesions of both humans and several animal models of vascular disease (Aikawa M. et al., 1997,
Circulation,
96:82-90; Sartore S, et al., 1994,
J. Vasc. Res.,
31:61-81). Thus, the SM-MHC gene represents an excellent candidate gene for delineating transcriptional pathways important for both normal development and diseased states.
Transcriptional regulation of the SM-MHC gene has been analyzed extensively in cultured SMC and several functional cis-elements have been identified. White S. L. et al., 1996,
J. Biol. Chem.,
271:15008-15017; Katoh Y. et al., 1994,
J. Biol. Chem.,
269:30538-30545; Wantanabe M. et al., 1996,
Circ. Res.,
78:978-989; Kallmeier R. C. et al., 1995,
J. Biol. Chem.,
270:30949-30957; Madsen C. S. et al., 1997,
J. Biol. Chem.,
272:6332-6340; Madsen C. S. et al., 1997,
J. Biol. Chem.,
272:29842-29851. However, because differentiation of SMC is known to be dependent on many local environmental cues that cannot be completely reproduced in vitro, cultured SMC are known to be phenotypically modified as compared to their in vivo counterparts (Owens G. K., 1995,
Physiol. Rev.,
75:487-517; Chamley-Campbell J. H. et al., 1981,
Atherosclerosis,
40:347-357). As such, certain limitations may apply regarding the usefulness of cultured SMC in defining transcriptional programs that occur during normal SMC differentiation and maturation within the animal.
Prior to the instant invention, no genetic elements that are completely specific for SMC and which have been proven to confer smooth muscle specific gene expression in vivo in transgenic animals have been defined, isolated or identified. Furthermore, as discussed above, previously characterized smooth muscle cell gene promoters including those for SM 22&agr; and SM &agr;-actin show activity in both SMC and non-SMC, thus limiting their use for purposes requiring SMC-specific gene targeting.
The current invention provides the major advance of identifying molecular elements that confer SMC-specific transcription in vivo during normal development. More specifically, the instant invention utilizes transgenic mice to identify DNA sequences that are critical for SM-MHC expression. Thus, the instant invention provides, for the first time, the identification of sufficient
Madsen Cort
Owens Gary K.
Falk Anne-Marie
Setagon, Inc.
Sullivan Daniel M.
Townsend and Townsend / and Crew LLP
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