Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-06-10
2001-09-04
Clark, Deborah J. R. (Department: 1633)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S325000, C435S320100
Reexamination Certificate
active
06284882
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Technical Field
The subject invention relates to a promoter which regulates expression of the myostatin gene as well as methods of inhibiting this promoter and compositions used for such inhibition. In particular, inhibitors of the promoter prevent the expression of the myostatin gene and thus prevent muscle wasting.
2. Background Information
Myostatin, or growth/differentiation factor 8 (GDF-8), belongs to the transforming growth factor-&bgr; (TGF-&bgr;) superfamily (McPherron et al.,
Nature
387:83-90 (1997)). The human myostatin gene has been cloned (Nestor et al.
Proc. Natl. Acad. Sci.
95:14938-43 (1998)), and it has been reported that myostatin immunoreactivity is detectable in human skeletal muscle in both type 1 and type 2 fibers. With respect to function, myostatin may play a role in negatively regulating the growth and development of skeletal muscle (Nestor et al., supra).
The first evidence that myostatin may play a key role in negatively regulating muscle development came from a study with myostatin knock-out mice (McPherron et al.,
Nature
387:83-90 (1997)). In the myostatin null mice, the animals were rather normal except that they were significantly larger than wild-type mice and had a large and widespread increase in skeletal muscle mass. Furthermore, it was also determined that two breeds of cattle, characterized by increased muscle mass, have mutations in the myostatin coding sequence (McPherron et al.,
Proc. Natl. Acad. Sci.
94:12457-61 (1997)). Additionally, it should be noted that the serum and intramuscular concentrations of immunoreactive myostatin are increased in HIV-infected men with muscle wasting compared with healthy men, and correlate inversely with the fat-free mass index. These data support the hypothesis that myostatin is a negative regulator of skeletal muscle growth in adult men and contributes to muscle wasting in HIV-infected men (Nestor et al., supra).
In view of the above findings, a need exists for a manner of regulating myostatin expression, particularly in individuals who experience muscle wasting as a result of a condition or disease state such as, for example, aging, Autoimmune Deficiency Syndrome (AIDS), Multiple Sclerosis, and cancer. The present invention provides methods and compositions which may be utilized to help individuals with such muscle wasting conditions and provides further insight into the regulation of myostatin gene expression.
All U.S. patents and publications referred to herein are hereby incorporated in their entirety by reference.
SUMMARY OF THE INVENTION
The present invention encompasses an isolated nucleic acid sequence represented by
FIG. 2
(SEQ ID NO:1).
Additionally, the present invention encompasses a vector comprising the above-described nucleic acid sequence and a nucleic acid sequence encoding a reporter molecule. The nucleic acid sequence encoding the reporter molecule is operably linked to the nucleic acid sequence represented by FIG.
2
. The reporter molecule may be selected from the group consisting of, for example, luciferase, &bgr;-galactosidase and Chloramphenicol Acetyltransferase (CAT). Preferably, the reporter molecule is luciferase. The present invention also includes a host cell comprising the above-described vector.
Additionally, the present invention includes a purified antibody produced in response to immunization with myostatin as well as a composition comprising this purified antibody.
Furthermore, the present invention also includes a method of identifying a composition which inhibits activation of the myostatin promoter. This method comprises the steps of: a) constructing a vector comprising a nucleic acid sequence represented by
FIG. 2
(SEQ ID NO:1) and a nucleic acid sequence encoding a reporter molecule, the nucleic acid sequence encoding the reporter molecule being operably linked to the nucleic acid sequence encoding the sequence represented by
FIG. 2
; b) introducing the vector into a host cell for a time and under conditions suitable for expression of myostatin; c) exposing the host cell to a composition which may inhibit activation of the myostatin promoter and a substrate specific for the reporter molecule; and d) measuring the signal generated by reaction of the reporter molecule and the substrate in comparison to that produced by a control host cell, a smaller signal by the host cell of (c) indicating that the composition will inhibit activation of the myostatin promoter.]
Also, the present invention includes a method of identifying a composition which inhibits expression of myostatin comprising the steps of: a) adding an antibody selected from the group consisting of a monoclonal or a polyclonal antibody produced against myostatin to a solid phase; b) adding known concentrations of myostatin or a cell sample comprising myostatin exposed to the test composition, to the solid phase, in order to form a first complex between the antibody and the known concentrations of myostatin or myostatin in said cell sample; c) adding a second antibody to the first complex, selected from the group consisting of a monoclonal antibody or a polyclonal antibody produced against myostatin for a time and under conditions sufficient for formation of a second complex between the first complex and the second antibody; d) contacting the second complex with an indicator reagent which comprises a signal generating compound attached to an antibody against said antibody of said second complex, for a time and under conditions sufficient for formation of a third complex; e) detecting the presence of a measurable signal, absence of the signal indicating that the composition inhibits expression of myostatin and presence of the signal indicating that the composition does not inhibit expression of myostatin.
Moreover, the present invention also includes a method of identifying a composition which inhibits expression of myostatin comprising the steps of: a) coating a fixed amount of myostatin on a solid phase; b) adding known concentrations of myostatin or a cell sample comprising myostatin exposed to the composition; c) contacting an antibody selected from the group consisting of a monoclonal antibody or a polyclonal antibody produced against myostatin to the myostatin in (a) and (b) for a time and under conditions sufficient to form a first complex, wherein myostatin of (a) competes with the myostatin of (b), in a competition for the antibody; d) contacting the complex with an indicator reagent which comprises a signal generating compound attached to an antibody against the antibody of the first complex, for a time and under conditions sufficient to form a second complex; and e) detecting a measurable signal, a higher signal as compared to a control, indicating the composition inhibits myostatin expression.
Additionally, the present invention includes a method of identifying a composition, in a mixture of compositions, which prevents myostatin from binding to a myostatin receptor comprising the steps of: a) mixing purified myostatin with the mixture of compositions; b) passing the resulting mixture of step (a) through a filter having pores of a size such that a composition which is complexed to myostatin does not pass through the filter; and c) determining the structure of a complexed composition, thereby identifying a composition which prevents myostatin from binding to a myostatin receptor.
Also, the present invention encompasses a method of identifying a composition which prevents myostatin from binding to a myostatin receptor comprising the steps of: a) radiolabeling recombinant myostatin; b) incubating the radiolabeled myostatin with cells or membranes comprising a myostatin receptor; c) contacting the incubated mixture of step (b) with a composition of interest; d) separating radiolabeled myostatin bound to cells or membranes from unbound myostatin; and e) determining the amount of radioactivity in bound myostatin, compared to a control, a lower level of radioactivity in bound myostatin compared to said control indicating a composition which inhibits myos
Wang Jiahong
Wu-Wong Jinshyun R.
Abbott Laboratories
Becker Cheryl L.
Clark Deborah J. R.
Drabik Christopher E.
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