Antisense modulation of talin expression

Details Antisense modulation of talin expression Antisense modulation of talin expression

2000-10-30

2002-04-16

Wang, Andrew (Department: 1635)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of regulating cell metabolism or physiology

C435S006120, C435S325000, C435S366000, C435S091100, C536S023100, C536S024500, C536S025300, C514S04400A

Reexamination Certificate

active

06372492

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of Talin. In particular, this invention relates to antisense compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding human Talin. Such oligonucleotides have been shown to modulate the expression of Talin.
BACKGROUND OF THE INVENTION
Cell adhesive contacts are critical for the development and maintenance of multicellular organisms. These contacts are mediated by cell adhesion molecules (CAMs), a versatile class of compounds expressed on the cell surface. Cells adhere to one another and to extracellular substrates through the concerted action of a variety of CAMs, which act as both receptors and ligands on opposing cells.
These contacts are also critical for the bidirectional transduction of signals to the interior of the cell and, consequently, to the modulation of biochemical pathways. Integrins, one group of transmembrane CAMs, are heterodimeric cation-dependent glycoproteins and have been found in all tissues examined. They are comprised of a large extracellular domain, a transmembrane domain and a smaller cytoplasmic domain. It is the extracellular domain of the integrin that acts as a receptor for various matrix proteins, while the cytoplasmic domain has been shown to interact with actin filaments of the cytoskeleton and with cytoplasmic proteins such as talin, paxillin, filamin and focal adhesion kinase (FAK) (Critchley et al.,
Biochem. Soc. Symp
., 1999, 65, 79-99).
Talin is a cytoplasmic protein that is concentrated at points of cell adhesion, membrane ruffles, the leading lamellae of the cell periphery and aligned with microfilament bundles, which acts to link cytoskeletal proteins to the integrins, thereby linking the extracellular matrix to other cells. Talin is a large dimeric protein and is post-translationally modified through phosphorylation by protein kinase C-delta (Litchfield and Ball,
Biochem. Biophys. Res. Commun
., 1986, 134, 1276-1283; Watters et al.,
Exp. Cell. Res
., 1996, 229, 327-335) as well as by proteolytic cleavage events making it a central component of many signaling cascades.
Talin interacts with several cytoskeletal proteins including actin filaments, where it functions as a crosslinker, with these interactions being very sensitive to ionic strength, temperature and pH (Goldmann et al.,
Eur. J. Biochem
., 1999, 260, 439-445; Schmidt et al.,
Arch. Biochem. Biophys
., 1999, 366, 139-150; Zhang et al.,
Biochem. Biophys. Res. Commun
., 1996, 218, 530-537). Talin also interacts with myosin (Lin et al.,
Biochem. Biophys. Res. Commun
., 1998, 249, 656-659) and vinculin (Burridge and Mangeat,
Nature
, 1984, 308, 744-746; Goldmann et al.,
J. Muscle Res. Cell Motil
., 1996, 17, 1-5), as well as the plasma membrane fibronectin receptor (Horwitz et al.,
Nature
, 1986, 320, 531-533) and platelet integrins (Knezevic et al.,
J. Biol. Chem
., 1996, 271, 16416-16421). Collectively, these studies strongly implicate talin in cell adhesion regulation and cell morphology. Interestingly, when the talin protein is added to liposomes in solution, holes form in the liposomes and become increasingly larger with increasing concentrations of added talin. This ‘opening up’ of the liposome continues until the liposome is transformed to a stable bilayer (Saitoh et al.,
Proc. Natl. Acad. Sci. U.S.A
., 1998, 95, 1026-1031).
In tissues, talin has been localized to myotendinous junctions (sites of skeletal muscle adherence to tendons), where expression was shown to be regulated by mechanical loading (Frenette and Tidball,
Am. J. Physiol.,
1998, 275, C818-825; Tidball et al.,
J. Cell. Biol
., 1986, 103, 1465-1472). Recently, it was further demonstrated that, in cardiomyocytes, talin participates in the transmission of contractile force to the extracellular matrix (Imanaka-Yoshida et al.,
Cell Motil. Cytoskeleton
, 1999, 42, 1-11). These studies imply that the integrity and concentration of the talin protein could play a major role in determining muscle strength and cardiac function.
In prostate tissues, talin is highly expressed in epithelial cells, with lower levels in the stroma. The expression of talin in these cells is down-regulated by androgens, a phenomenon known to contribute to the development of prostate cancer (Betts et al.,
FEBS Lett
., 1998, 434, 66-70).
Albiges-Rizo et al. have demonstrated that down-regulation of talin alters cell adhesion by slowing cell spreading of HeLa cells. In these studies, HeLa cells were transfected with a 5′ region of the talin gene, in antisense orientation, under the control of an inducible promoter. It was shown that a reduction of talin expression impaired the folding and processing of integrins as well as reducing focal contacts (Albiges-Rizo et al.,
J. Cell. Sci
., 1995, 108, 3317-3329).
Talin, originally called P235 in platelets, is an abundant protein in platelets and constitutes greater than 3% of the total protein (Beckerle et al.,
J. Cell. Biochem
., 1986, 30, 259-270; Collier and Wang,
FEBS Lett
., 1982, 143, 205-210; Collier and Wang,
J. Biol. Chem
., 1982, 257, 6937-6943). The distribution of talin in resting and activated human platelets undergoes an activation-dependent change in its subcellular location. In resting platelets, which are nonadhesive, talin is uniformly distributed throughout the cytoplasm. In contrast, in activated platelets, talin is concentrated at the cytoplasmic face of the plasma membrane (Beckerle et al.,
J. Cell. Biol
., 1989, 109, 3333-3346). The regulated redistribution of talin raises the possibility that talin plays a role in platelet adhesion (Beckerle and Yeh,
Cell. Motil. Cytoskeleton
, 1990, 16, 7-13).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of talin and strategies aimed at modulating talin expression and/or function have involved the use of antibodies and molecules that block protein kinase C activity. Priddle et al.,
using gene replacement vectors, isolated mouse embryonic stem cells in which both copies of the talin gene were disrupted. These cells, when undifferentiated, were unable to spread, showed reduced adhesion and expressed low levels of beta integrin. However, upon differentiation, these cells demonstrate talin-independent wild-type characteristics (Priddle et al.,
J. Cell. Biol
., 1998, 142, 1121-1133).
Using monoclonal antibodies raised against the platelet form of talin, Bolton et al. have mapped the epitopes along the talin protein that are recognized by these antibodies and shown that microinjection of antibodies to two separate regions of talin inhibit cell motility (Bolton et al.,
Cell. Motil. Cytoskeleton
, 1997, 36, 363-376). Implementing the same antibody strategy, others have shown that the talin dimer is comprised of two dumbbell-shaped antiparallel subunits (Isenberg and Goldmann,
FEBS Lett
., 1998, 426, 165-170).
However, because these strategies are untested as therapeutic protocols, there remains a long-felt need for additional agents capable of effectively inhibiting talin function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of talin expression. The present invention provides compositions and methods for modulating talin expression using said antisense technology.
SUMMARY OF THE INVENTION
The present invention is directed to antisense compounds, particularly oligonucleotides, which are targeted to a nucleic acid encoding Talin, and which modulate the expression of Talin. Pharmaceutical and other compositions comprising the antisense compounds of the invention are also provided. Further provided are methods of modulating the expression of Talin in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention. Further provided are methods of treating an animal, particula

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