Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
2000-11-09
2003-10-28
Falk, Anne-Marie (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S013000, C800S014000, C800S021000, C800S025000, C435S325000, C435S352000, C435S354000, C435S361000
Reexamination Certificate
active
06639123
ABSTRACT:
STATEMENT REGARDING FEDERALLY-SPONSORED R&D
Not Applicable
REFERENCE TO MICROFICHE APPENDIX
Not applicable.
FIELD OF THE INVENTION
The present invention relates to cells and non-human transgenic animals that have been engineered to be deficient in the gene encoding the melcanocortin-3 receptor protein (MC-3R). It is shown herein that MC-3R deficient transgenic animals have increased fat mass and reduced lean body mass, showing that the MC-3R protein is involved in the regulation of body fat and lean body mass. The MC-3R deficient transgenic animals of the present invention, including a MC-3R/MC-4R double knockout mouse, can be used to select for and test potential modulators (e.g., agonists or antagonists) of MC-3R, as well as dual modulators of MC-3R and MC-4R. It is shown herein that MC-3R serves a non redundant role, when compared to MC-4R, in the regulation of energy homeostasis. To this end, the present invention also relates to methods of screening for MC-3R modulators which effect body weight and associated methods of treating various disorders or diseases responsive to the action of one or more of the melanocortin receptors, including but not limited to obesity (by reducing appetite, increasing metabolic rate, reducing fat intake or reducing carbohydrate craving), diabetes mellitus (by enhancing glucose tolerance, decreasing insulin resistance), hypertension, hyperlipidemia, osteoarthritis, cancer, gall bladder disease, sleep apnea, depression, anxiety, compulsion, neuroses, insomnia/sleep disorder, substance abuse, pain, male and female sexual dysfunction (including impotence, loss of libido and erectile dysfunction), fever, inflammation, immunomodulation, rheumatoid arthritis, learning memory, modulation of cytokine release, skin tanning, acne and other skin disorders, neuroregeneration and neuroprotective and cognitive and memory enhancement including the treatment of Alzheimer's disease.
BACKGROUND OF THE INVENTION
Melanocortin receptors belong to the rhodopsin sub-family of G-protein coupled receptors (GPCR's). Five different subtypes are known. These melanocortin receptors bind and are activated by peptides such as &agr;-, &bgr;, or &ggr;-melanocyte stimulating hormones (&agr;-, &bgr;-, &ggr;-MSH) derived from the pro-opiomelanocortin (POMC) gene. A wide range of physiological functions are believed to be mediated by melanocortin peptides and their receptors.
Desarnaud et al. (1994
, Biochem J
. 299 (2): 367-372) disclose a cDNA clone encoding mouse MC-3R.
Roselli-Rehfuss et al. (1993
, Proc. Natl. Acad. Sci
90: 8856-8860) disclose a cDNA clone encoding rat MC-3R cDNA.
U. S. Pat. No. 5,622,860 (issued Apr. 22, 1997) and U.S. Pat. No. 5,703,220 (issued Dec. 30, 1997) to Yamada and Gantz, disclose DNA molecules which encode human MC-3R and human MC-4R, respectively (see also Gantz, et al., 1993
, J. Biol. Chem
. 268(11): 8246-8250).
The agouti mouse represents a naturally occurring obese rodent, with a late life onset of obesity which is not corticosterone dependent. The obesity in this model results from the ectopic expression of the 131 amino acid agouti protein. Agouti is normally only expressed in the skin where it controls hair color. The protein is a paracrine antagonist of the melanocortin-1 receptor (MC-1R), a G-protein coupled receptor of the hair follicle. MC-1R agonism, through its natural ligand, &agr;-MSH raises cAMP and the expression of the enzyme tyrosinase. Low levels of tyrosinase, which result from agouti antagonism of MC-1R, result in reduced conversion of the hair color pigment pheomelanin to eumelanin. As a result a light (agouti) rather than black hair color results. The obese phenotype of the agouti mouse was ascribed to the expression of agouti in the brain, where it antagonizes MC-3R and MC-4R receptors. This conclusion was corroborated by the generation of an MC-4R knockout mouse which recapitulates the obese phenotype of the agouti mutant mouse (see U.S. Pat. No. 5,932, 779, issued Aug. 3, 1999 to Lee et al.) In rodents, MC-4R has been implicated as a key regulator of feeding behavior which regulates body weight through studies with peptide agonists and antagonists (Fan et al., 1997
, Nature
385:165-168) and with a MC-4R knock-out mouse (Huszar et al., 1997
, Cell
88:131-141, see also U.S. Pat. No. 5,932,779, issued Aug. 3, 1999 to Lee et al).
It is desirable to discover new drugs for the treatment of body weight disorders which selectively modulate a melanocortin receptor within the host.
It is also desirable to identify additional receptor targets which are involved in regulating body weight.
The present invention also addresses and meets these needs by disclosing MC-3R-deficient animal cells and/or MC-3R/MC-4R deficient animal cells, related non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates which are also MC-3R-deficient or MC-3R/MC-4R deficient.
The present invention addresses and meets these needs by disclosing methods of screening for compounds.which effect body weight comprising the screening and selection of compounds which modulate the MC-3R.
SUMMARY OF THE INVENTION
The present invention relates to animal cells which are homozygous for an MC-3R deficiency due to a disruption in the gene(s) encoding MC-3R. To this end, the present invention also relates to non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates which are MC-3R deficient (MC-3R null) due to a disruption in the gene(s) encoding MC-3R.
The present invention further relates to animal cells, non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates which are heterozygous for a functional MC-3R gene native to that animal.
The present invention also relates in part to animal cells, non-human transgenic embryos and non-human transgenic littermates having a non-native gene encoding a MC-3R protein expressed either in the presence or absence of the native (wild type) MC-3R. Preferably, the non-native MC-3R gene is the human MC-3R gene.
The present invention also relates to transgenic embryos, non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates which are either homozygous, heterozygous or hemizygous for deletion of at least a portion of the MC-3R gene in combination with a homozygous, heterozygous or hemizygous deletion at separate alleles which in their wild type form encode at least one additional melanocortin receptor, especially a melanocortin receptor shown to be involved in body weight regulation, such as MC-4R. Therefore, aspects of the invention relate to transgenic embryos, non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates which are MC-3R
−/+
/MC-4R
−/−
; MC-3R
−/+
/MC-4R-4
−/+
; MC-3R
−/−
/MC-4R
−/+
, as well as hemizygous alternatives in reference to the two separate alleles. An especially preferred aspect of the present invention relates to MC-3R
−/−
/MC-4R
−/−
double knockout mice and related transgenic embryos, non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates.
The transgenic cells and animals of the present invention are useful in the study of the effect of modulators on the activity of the MC-3R gene and/or protein or the expression of the MC-3R gene and/or protein as concerning the regulation of body weight, including but not limited to disorders such as obesity, diabetes, cardiovascular disease, anorexia, cachexia, cancer, male and female sexual dysfunction, pain, memory, neuronal regeneration and neuropathy.
The present invention also relates to MC-3R-based assays to select for modulators of this receptor protein which affect regulation of body weight through the various known disorders associated with regulation of body weight, as described herein. For example, a MC-3R modulator may be used to treat these body weight disorders, such as a MC-3R agonist to treat obesity or a MC-3R antagonist to
Chen Airu S.
Chen Howard Y.
Van der Ploeg Leonardus H. T.
Falk Anne-Marie
Hand J. Mark
Merck & Co. , Inc.
Tribble Jack L.
Xu Yang
LandOfFree
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