Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1998-12-23
2001-10-09
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S252300, C435S320100, C435S069100, C435S254110, C530S300000, C530S350000, C536S023200
Reexamination Certificate
active
06300110
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides methods and reagents for regulating telomere length and modulating telomerase activity in mammalian cells as well as for detecting, diagnosing, and treating related diseases and conditions in humans and other mammals. In an important embodiment, the invention provides oligonucleotide probes and primers, polynucleotide plasmids, peptides, proteins, antibodies, and enzymes relating to genes and gene products that regulate telomere length and telomerase activity in mammalian cells. The invention has diverse applications and provides important advances in the fields of molecular biology, chemistry, pharmacology, and medical therapeutic and diagnostic technology.
BACKGROUND OF THE INVENTION
The DNA at the ends of the telomeres of chromosomes in mammalian cells consists of double- and single-stranded nucleic acid composed of many tandem repeats of a simple nucleotide sequence referred to as the telomeric repeat sequence. Telomeres help maintain chromosome structure and function; the loss of telomeric DNA can activate the cellular processes that detect and control DNA damage and monitor and control cell proliferation and senescence. The maintenance of telomeres and the regulation of telomere length are vital cellular functions involved in transmitting genetic information from generation to generation, aging, the control of cell growth, and cancer. See Harley, 1991
, Mutation Research
256:271-282; and Blackburn, 1992
, Annu. Rev. Biocheni
. 61:113-129, each of which is incorporated herein by reference (note: references cited herein are provided for convenience; such citations are not to be construed as an admission of prior invention).
The multi-component telomerase ribonucleoprotein enzyme catalyzes the synthesis of the first strand of telomeric DNA synthesized during telomere elongation, using the RNA component of the enzyme as a template. Although the RNA component of human telomerase (hTR) and other mammalian telomerase enzymes has been identified, isolated, characterized, and described in the scientific literature, the protein components of the telomerase enzyme as well as most other cellular macromolecules involved in telomere maintenance and the regulation of telomere length and telomerase activity in mammalian cells have not. See Feng et al., 1995
, Science
269:1236-1241; PCT patent publication No. 96/01835; and pending U.S. patent application Ser. No. 08/521,634, filed Aug. 31, 1995, and Ser. No. 08/330,123, filed Oct. 27, 1994, each of which is incorporated herein by reference.
Many useful methods and reagents relating to telomere and telomerase biology have been described. See, e.g., U.S. Pat. No. 5,489,508; PCT patent publication Nos. 95/23572, 95/13381, 95/13382, and 95/13383; and U.S. patent application Ser. No. 08/632,662, filed Apr. 15, 1996, each of which is incorporated herein by reference. Significant improvements to and new opportunities for telomere- and telomerase-mediated therapies as well as related assays, screens, diagnostic methods, and reagents could be realized and obtained, however, if additional cellular macromolecules involved in mammalian telomere maintenance and the regulation of telomere length and telomerase activity could be identified, characterized, and made available in pure or isolatable form. In particular, the characterization of the nucleotide and corresponding amino acid sequences of such macromolecules could provide new and useful recombinant expression vectors and plasmids, as well as related reagents useful in medical therapeutic and diagnostic technology.
SUMMARY OF THE INVENTION
The present invention provides methods and reagents for regulating telomere length and modulating telomerase activity in mammalian cells as well as for detecting, diagnosing, and treating related diseases and conditions in humans and other mammals.
In one embodiment, the invention provides recombinant mammalian host cells containing:
(i) a recombinant or synthetic nucleic acid comprising at least about 10 to 15 to 25 to 100 or more contiguous nucleotides corresponding to an open reading frame sequence of a human gene TPC2 contained in a human DNA insert of an ~3.5 kb NotI-BstEII restriction fragment of plasmid pGRN109 (on deposit with the American Type Culture Collection under the accession number ATCC 97708); or
a synthetic or recombinant peptide or protein comprising at least about 6 to 10 to 15 to 25 to 100 or more contiguous amino acids corresponding to an amino acid sequence encoded by said open reading frame sequence; and
(ii) a recombinant or synthetic nucleic acid comprising at least about 10 to 15 to 25 to 100 or more contiguous nucleotides corresponding to an open reading frame sequence of a human gene TPC3 contained in a human DNA insert of an ~1.4 kb EcoRI-BamHI restriction fragment of plasmid pGRN92 (ATCC 97707); or
a synthetic or recombinant peptide or protein comprising at least about 6 to 10 to 15 to 25 to 100 or more contiguous amino acids corresponding to an amino acid sequence encoded by said open reading frame sequence of gene TPC3;
said TPC2 and TPC3 genes characterized in coding for proteins that regulate telomere length or modulate telomerase activity and are present in human or other mammalian cells that express telomerase activity.
Other mammalian host cells provided by the invention include those that comprise either or both TPC2- and TPC3-derived recombinant or synthetic nucleic acids, peptides, or proteins. Furthermore, the invention also provides such cells further modified to contain a synthetic or recombinant nucleic acid comprising at least about 10 to 15 to 25 to 100 or more contiguous nucleotides corresponding to a contiguous nucleotide sequence of human hTR located in an ~2.5 kb HindIII-SacI restriction fragment of pGRN33 (ATCC 75926).
The recombinant host cells of the invention have application in many useful methods also provided by the invention. For example, the invention provides recombinant host cells comprising novel expression vectors with expression control sequences operatively linked to nucleotide sequences encoding amino acids in a sequence substantially identical to the amino acid sequences encoded by the human TPC2 or TPC3 genes and, optionally, a recombinant hTR gene. These recombinant host cells are useful for producing recombinant human telomerase, for use in screens to identify agents that modulate telomerase activity or regulate telomere length, as well as for a variety of other purposes described more fully below. The recombinant host cells of the invention can also be incorporated into the germ line and/or somatic tissues of non-human transgenic mammals, as well as be administered to mammals for therapeutic purposes.
In another embodiment, the invention provides synthetic and recombinant oligonucleotides and nucleic acids in a variety of forms, i.e., isolatable, isolated, purified, or substantially pure, and for a variety of purposes, i.e., as probes or primers, as polynucleotide plasmids and vectors for introducing recombinant gene products that regulate telomere length or modulate telomerase activity in mammalian host cells, as restriction fragments for creating useful nucleic acids, and as reagents for therapeutic, diagnostic, and other applications. In particular, the invention provides recombinant or synthetic nucleic acids comprising at least about 10 to 15 to 25 to 100 or more contiguous nucleotides substantially identical or complementary in sequence to a contiguous nucleotide sequence located in either:
(i) an open reading frame sequence of a human gene TPC2 contained in a human DNA insert of an ~3.5 kb NotI-BstEII restriction fragment of plasmid pGRN109; or
(ii) an open reading frame sequence of a human gene TPC3 contained in a human DNA insert of an ~1.4 kb EcoRI-BamHI restriction fragment of plasmid pGRN92.
The novel oligonucleotide probes and primers of the invention typically comprise nucleotides in a sequence substantially identical or complementary to a sequence of nucleotides in a TPC2 or TPC3 gene or gene product to allow specific h
Adams Robert R.
Andrews William H.
Feng Junli
Villeponteau Bryant
Achutamurthy Ponnathapu
Earp David J.
Geron Corporation
Geron Corporation
Saidha Tekchand
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