Use of exogenous &bgr;-adrenergic receptor and...

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Reexamination Certificate

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C424S093200, C435S320100, C435S455000, C435S458000

Reexamination Certificate

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06436908

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to gene therapy for treating chronic heart failure or other cardiac diseases associated with decreased myocardial &bgr;-adrenergic receptors and reduced myocardial function. More particularly, this invention relates to manipulation of genes and gene products that affect &bgr;-adrenergic receptors in myocardium in order to enhance myocardial function. The invention also relates to receptors, methods or systems for drug screening, and transgenic animals suitable for investigation of therapies for treatment of heart failure and other cardiac conditions.
BACKGROUND INFORMATION
G-protein-coupled receptors, such as the adrenergic receptors, elevate cellular levels of second messengers like cyclic-adenosine monophosphate (cAMP) and diacylglycerol thereby regulating and coordinating cellular metabolism and function. In the heart, &bgr;-adrenergic receptors (&bgr;-ARs), responsive to the sympathetic neurotransmitter norepinephrine and the adrenal medullary hormone epinephrine, stimulate adenylyl cyclase, raising myocardial cAMP and increasing cardiac contractility. Elevated circulating catecholamines and myocardial &bgr;-AR stimulation represent critical mechanisms for augmenting cardiac function during stress.
As is true for most G protein-coupled receptors, prolonged agonist exposure of &bgr;-ARs leads to a rapid decrease in responsiveness. Agonist-dependent desensitization can be initiated by phosphorylation of activated receptors by members of the G protein-coupled receptor kinase (GRK) family (W. P. Hausdorff et al.,
FASEB J.
4, 2881 (1990); J. Inglese et al.,
J. Biol. Chem.
268, 23735 (1993)). Phosphorylated receptors then interact with arresting proteins like &bgr;-arrestin to which they bind thereby sterically interdicting further coupling to G proteins (W. P. Hausdorff et al., 1990; J. Inglese et al., 1993). The &bgr;-adrenergic receptor kinase-1 (&bgr;ARK1) is a GRK which has been shown to specifically phosphorylate activated &bgr;2-ARs in vitro and which is hypothesized to phosphorylate &bgr;
2
-ARs in vivo leading to uncoupling and desensitization (W. P. Hausdorff et al., 1990; J. Inglese et al., 1993; M. J. Lohse et al.,
Proc. Natl. Acad. Sci. U.S.A.
86, 3011 (1989); M. J. Lohse et al.,
J. Biol. Chem.
265, 3202 (1990); S. Pippig et al.,
J. Biol. Chem.
268, 3201 (1993)).
The action of &bgr;ARK1 on the &bgr;
1
-AR has not yet been documented. &bgr;ARK1 is specifically targeted to activated receptors in the plasma membrane by a translocation event mediated via a specific protein-protein interaction between the carboxyl terminus of the kinase and the &bgr;&ggr; subunits of activated and dissociated G proteins (J. Pitcher et al.,
Science
257, 1264 (1992); W. J. Koch et al.,
J. Biol. Chem.
268, 8256 (1993)).
In chronic congestive heart failure, an illness affecting more than four million Americans, there is dramatic impairment of the myocardial &bgr;-AR system. Failing human ventricular myocardium contains 50% fewer &bgr;-ARs and shows parallel decreases in agonist-stimulated adenylyl cyclase activity and even greater decreases in agonist-mediated inotropy (M. R. Bristow et al.,
N. Engl. J. Med.
307, 205 (1982), M. R. Bristow et al.,
J. Mol. Cell. Cardiol.
17 (Suppl. 2), I12 (1990)). In addition, increases in inhibitory G-protein and G-protein receptor kinases (e.g. &bgr;-adrenergic receptor kinase) in heart failure may further impair receptor-mediated inotropy (T. Eschenhagen et al.,
Circulation Research
70, 688 (1992) and M. Ungerer et al.,
Circulation
v7, 454 (1993)). Therapeutic interventions, involving the administration of agonists to stimulate the &bgr;-AR/adenylyl cyclase systems have an inherently limited efficacy given the reduction in receptor targets in the diseased myocardium.
An additional possible contributor to the decreased myocardial &bgr;-AR responsiveness seen in chronic failing human hearts is that levels of &bgr;ARK1 are elevated (M. Ungerer et al.,
Circulation
87, 454 (1993); M. Ungerer et al.,
Circ. Res.
74, 206 (1994)). Thus, &bgr;-AR impairment in heart failure may have several underlying causes.
The field of transgenic technology has achieved significant advances in techniques for in vivo gene transfer in recent years (T. Ragot et al.,
Nature
361, 647 (1993); M. A. Rosenfeld et al.,
Cell
68, 143 (1992); R. J. Guzman, et al.,
Circulation Research
73, 1202 (1993)).
While several transgenic mice have been reported which express, for example, the c-myc proto-oncogene or SV-40 T-antigen affecting cardiac growth (J. L. Swain et al.,
Cell
50, 719 (1987); L. J. Field,
Science
239, 1029 (1988); E. B. Katz et al.,
Am. J. Physiol.
262, H1867 (1992)), to date there have been no reports concerning the ability of a transgene to affect myocardial contractility.
SUMMARY OF THE INVENTION
The present invention provides novel strategies for improving cardiac function, for example by overexpressing the &bgr;-AR in the myocardium, or by inhibiting the activity of &bgr;ARK. It utilizes molecular, in vitro and in vivo methodologies to assess the biochemical and physiological consequences of transgenic overexpression of the human &bgr;
2
-AR in the heart. In addition, the invention utilizes in vitro and in vivo methodologies to assess the consequences of overexpression or suppression of &bgr;ARK in the heart.
In general, the invention features gene therapy for disease states where specific receptor-mediated functions are lost or altered. In particular, defects in the &bgr;-adrenergic receptors (B-AR) and in inotropic responsiveness in heart failure are a therapeutic target.
The invention provides a method for the delivery of a gene for &bgr;-AR, e.g., &bgr;
2
-AR, and a “minigene” encoding a &bgr;ARK inhibitor for delivery to diseased heart tissue. The invention also provides transgenic mice with cardiac specific overexpression of &bgr;-AR or &bgr;ARK, and mice with cardiac specific expression of a &bgr;ARK inhibitor. In addition, the invention provides a means for screening drugs that may be useful in the treatment of heart disease.
According to the invention, gene transfer to heart tissue may be accomplished by in vivo methods of gene transfer such as those involving the use of recombinant replication deficient adenovirus. Procedures include gene transfer into cardiac muscle are described in the literature, for example in Kass-Eisler, A. et al.,
Proc. Natl. Acad. Sci. U.S.A.,
90: 11498-11502 (1993); Stratford-Perricaudet, L. D. et al.,
J. Clin. Invest.
90: 626-630 (1992); and Guzman, R. J. et al.,
Circ. Res.
73: 1201-1207 (1993).
The gene for the human &bgr;
2
-AR has been cloned (Kobilka, B. K. et al.,
Proc. Natl. Acad. Sci. U.S.A.
84:46-50) and in the present invention, the &bgr;-AR gene, e.g., &bgr;
2
-AR or other subtypes, is simply any nucleic acid sequence which codes for a &bgr;-AR, said receptor having the ability to couple to adenylyl cyclase. Thus variations in the actual sequence of the gene can be tolerated provided that the &bgr;-AR can be expressed and is able to couple to adenylyl cyclase.
A &bgr;-AR gene construct can be obtained through conventional recombinant DNA techniques.
It is an object of the present invention to provide transgenic mice whose germ cells and somatic cells contain overexpressed human &bgr;-AR in the heart, transgenic mice whose germ cells and somatic cells contain genes for cardiac overexpression of &bgr;ARK, and transgenic mice whose germ cells and somatic cells contain a gene for cardiac expression of a &bgr;ARK inhibitor. The transgenic mice of the invention will usually have expressed levels of the gene products in myocardial tissue that are at least 50% greater, and preferably at least 100% greater, than levels than would normally occur in mice. In addition, the transgenic mice's myocardial function (e.g. heart rate or contractility) can be increased or decreased by about 10% and preferably by about 20% as desired.
Transgenic mice in the present invention were created by microinjection of the desired gene construct in

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