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
2000-02-22
2001-08-28
Kunz, Gary L. (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S070100, C435S071100, C435S071200, C435S325000, C435S471000, C435S252300, C435S320100, C536S023500
Reexamination Certificate
active
06280973
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to opioid receptors from mammalian species and the genes corresponding to such receptors. Specifically, the invention relates to the isolation, cloning and sequencing of complementary DNA (cDNA) copies of messenger RNA (mRNA) encoding a novel mammalian opioid receptor gene. The invention also relates to the construction of recombinant expression constructs comprising cDNA of this novel opioid receptor gene, said recombinant expression constructs being capable of expressing opioid receptor protein in cultures of transformed prokaryotic and eukaryotic cells. Production of the receptor protein in such cultures is also provided. The invention relates to the use of such cultures of such transformed cells to produce homogeneous compositions of the novel opioid receptor protein. The invention also provides cultures of such cells producing this opioid receptor protein for the characterization of novel and useful drugs. Antibodies against and epitopes of this novel opioid receptor protein are also provided by the invention.
2. Background of the Invention
The use (and abuse) of opiates, archetypally opium and morphine, have been known since antiquity (reviewed in Brownstein, 1993, Proc. Natl. Acad. Sci. USA 90: 5391-5393). Since the nineteenth century, chemical characterization and synthesis of a number of morphine analogues have been achieved in an effort to discover a compound with the analgesic effects of morphine that lacks or is substantially attenuated in its addictive potential. These efforts have proven fruitless to date.
The biology behind the reasons why morphine and morphine-like compounds display both analgesic and addictive properties was first elucidated by the discovery of endogenous morphine-like compounds termed enkephalins (see DiChara & North, 1992, Trends in Phamacol. Sci. 13: 185-193 for review). Accompanying this finding of an endogenous opiate was the biochemical evidence for a family of related but distinct opiate receptors, each of which displays a unique pharmacological profile of response to opiate agonists and antagonists (see McKnight & Rees, 1991, Neurotransmissions 7: 1-6 for review). To date, four distinct opiate receptors have been described by their pharmacological profiles and anatomical distribution; these comprise the &mgr;, &dgr;, &kgr; and &sgr; receptors (the &sgr; receptor has been determined to be a non-opioid receptor with cross-reactivity to some opioid agonists).
Thus, mammalian opioid receptors are known in the art, and some of these proteins have been isolated biochemically and their corresponding genes have been recently cloned using genetic engineering means.
Kieffer et al., 1992, Proc. Natl. Acad. Sci. USA 89: 12048-12052 disclosed the isolation of a cDNA copy of the mouse &dgr;-opioid receptor by expression cloning.
Evans et al., 1992, Science 258: 1952-1955 disclose the isolation of a cDNA copy of the mouse &dgr;-opioid receptor by expression cloning.
Chen et al., 1993, Molec. Pharmacol. 44: 8-12 disclose the isolation of a cDNA copy of the rat &mgr;-opioid receptor.
Yasuda et al., 1993, Proc. Natl. Acad. Sci. USA 90: 6736-6740 disclose the isolation of a cDNA copy of each of the mouse &kgr;- and &dgr;-opioid receptor.
Bzdega et al., 1993, Proc. Natl. Acad. Sci. USA 90: 9305-9309 disclose the isolation and chromosomal location of the &dgr;-opioid receptor in the mouse.
In 1991, U.S. pharmaceutical companies spent an estimated $7.9 billion on research and development devoted to identifying new therapeutic agents (Pharmaceutical Manufacturer's Association). The magnitude of this amount is due, in part, to the fact that hundreds, if not thousands, of chemical compounds must be tested in order to identify a single effective therapeutic agent that does not engender unacceptable levels of undesirable or deleterious side effects. There is an increasing need for economical methods of testing large number of chemical compounds to quickly identify those compounds that are likely to be effective in treating disease.
This is of particular importance for psychoactive and psychotropic drugs, due to their pharmacological importance and their potential to greatly benefit or greatly harm human patients treated with such drugs. At present, few such economical systems exist. Conventional screening methods require the use of animal brain slices in binding assays as a first step. This is suboptimal for a number of reasons, including interference in the binding assay by non-specific binding of heterologous (i.e., non-receptor) cell surface proteins expressed by brain cells in such slices; differential binding by cells other than neuronal cells present in the brain slice, such as glial cells or blood cells; and the possibility that putative drug binding behavior in animal brain cells will differ from the binding behavior in human brain cells in subtle but critical ways. The ability to synthesize human opioid receptor molecules in vitro would provide an efficient and economical means for rational drug design and rapid screening of potentially useful compounds. For these and other reasons, development in vitro screening methods for psychotropic drugs has numerous advantages and is a major research goal in the pharmaceutical industry.
SUMMARY OF THE INVENTION
The present invention relates to the cloning, expression and functional characterization of a mammalian methadone-specific opioid receptor (MSOR) gene. The invention comprises nucleic acids having a nucleotide sequence of a novel mammalian MSOR gene. The nucleic acids provided by the invention comprise a complementary DNA (cDNA) copy of the corresponding mRNA transcribed in vitro from the MSOR genes of the invention. Also provided are the deduced amino acid sequence of the cognate protein of the cDNA provided by the invention.
This invention provides nucleic acids, nucleic acid hybridization probes, recombinant eukaryotic expression constructs capable of expressing the MSOR receptors of the invention in cultures of transformed cells, such cultures of transformed eukaryotic cells that synthesize the MSOR receptors of the invention, homogeneous compositions of the MSOR receptor protein, and antibodies against and epitopes of the MSOR receptor protein of the invention. Methods for characterizing these receptor proteins and methods for using these proteins in the development of agents having pharmacological uses related to these receptors are also provided by the invention.
In a first aspect, the invention provides a nucleic acid having a nucleotide sequence encoding a mammalian methadone-specific opioid receptor. In a preferred embodiment, the nucleic acid encodes the rat MSOR receptor. In this embodiment of the invention, the nucleotide sequence includes 1452 nucleotides of the rat MSOR cDNA comprising 1101 nucleotides of coding sequence, 181 nucleotides of 5′ untranslated sequence and 170 nucleotides of 3′ untranslated sequence. In this embodiment of the invention, the nucleotide sequence of the MSOR receptor consists essentially of the nucleotide sequence depicted in
FIGS. 1A through 1C
(SEQ ID No:3). The use of the term “consisting essentially of” herein is meant to encompass the disclosed sequence and includes allelic variations of this nucleotide sequence, either naturally occurring or the product of in vitro chemical or genetic modification. Each such variant will be understood to have essentially the same nucleotide sequence as the nucleotide sequence of the corresponding MSOR disclosed herein.
The corresponding MSOR protein molecule, having the deduced amino acid sequence consisting essentially of the sequence shown in
FIGS. 1A through 1C
(SEQ ID No.:4), is also claimed as an aspect of the invention. The use of the term “consisting essentially of” herein is as described above. Similarly, the MSOR protein molecule, having the deduced amino acid sequence consisting essentially of the sequence shown in
FIGS. 1A through 1C
(SEQ ID No.:4), is also claimed as an aspect of the invention. MSOR protein molecul
Bunzow James R.
Civelli Olivier
Grandy David K.
Kunz Gary L.
Landsman Robert S.
McDonnell & Boehnen Hulbert & Berghoff
Oregon Health & Science University
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