Cech, Thomas R. (Potomac, MD, US)
Lingner, Joachim (Epalinges, CH)
Nakamura, Toru (San Diego, CA, US)
Chapman, Karen B. (Mill Valley, CA, US)
Morin, Gregg B. (Oakville, CA)
Harley, Calvin B. (Palo Alto, CA, US)
Andrews, William H. (Reno, NV, US)

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The Regents of the University of Colorado (Boulder, CO, US)

435/7.1

435/4, 435/7.23, 435/7.21, 435/7.2

G01N33/53; G01N33/567; G01N33/00; C12Q1/00

3817837		June, 1974	Tanenholtz et al.
3850752	PROCESS FOR THE DEMONSTRATION AND DETERMINATION OF LOW MOLECULAR COMPOUNDS AND OF PROTEINS CAPABLE OF BINDING THESE COMPOUNDS SPECIFICALLY	November, 1974	Schuurs et al.
3939350	Fluorescent immunoassay employing total reflection for activation	February, 1976	Kronick et al.
3996345	Fluorescence quenching with immunological pairs in immunoassays	December, 1976	Ullman et al.
4275149	Macromolecular environment control in specific receptor assays	June, 1981	Litman et al.
4277437	Kit for carrying out chemically induced fluorescence immunoassay	July, 1981	Maggio
4366241	Concentrating zone method in heterogeneous immunoassays	December, 1982	Tom et al.
4554101	Identification and preparation of epitopes on antigens and allergens on the basis of hydrophilicity	November, 1985	Hopp
4683195	Process for amplifying, detecting, and/or-cloning nucleic acid sequences	July, 1987	Mullis et al.
4683202	Process for amplifying nucleic acid sequences	July, 1987	Mullis
4816567	Recombinant immunoglobin preparations	March, 1989	Cabilly et al.
4965188	Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme	October, 1990	Mullis et al.
5001225	Monoclonal antibodies to a pan-malarial antigen	March, 1991	Taylor
5075227	Directional cloning	December, 1991	Hagen
5489508	Therapy and diagnosis of conditions related to telomere length and/or telomerase activity	February, 1996	West et al.
5583016	Mammalian telomerase	December, 1996	Villeponteau et al.
5597697	Screening assay for inhibitors and activators of RNA and DNA-dependent nucleic acid polymerases	January, 1997	Diamond
5639613	Methods for cancer diagnosis and prognosis	June, 1997	Shay et al.
5747317	Human telomerase RNA interacting protein gene	May, 1998	Cao
5770422	Human telomerase	June, 1998	Collins
5853719	Methods for treating cancers and pathogen infections using antigen-presenting cells loaded with RNA	December, 1998	Nair et al.
5917025	Human telomerase	June, 1999	Collins
5919656	Genes encoding telomerase protein 1	July, 1999	Harrington et al.
5919676	Adenoviral vector system comprising Cre-loxP recombination	July, 1999	Graham et al.
6093809	Telomerase	July, 2000	Cech et al.
6120764	Adenoviruses for control of gene expression	September, 2000	Graham et al.
6140087	Adenovirus vectors for gene therapy	October, 2000	Graham et al.
6166178	Telomerase catalytic subunit	December, 2000	Cech et al.
6258535	Mammalian telomerase	July, 2001	Villeponteau et al.
6261556	Purified telomerose	July, 2001	Weinrich et al.
6261836	Telomerase	July, 2001	Cech et al.
6306388	Methods for treating cancers and pathogen infections using antigen-presenting cells loaded with RNA	October, 2001	Nair et al.
6309867	Telomerase	October, 2001	Cech et al.
6337200	Human telomerase catalytic subunit variants	January, 2002	Morin
6387701	Method of identifying tumor antigens that elicit a T-cell response	May, 2002	Nair et al.
6440735	Dendritic cell vaccine containing telomerase reverse transcriptase for the treament of cancer	August, 2002	Gaeta
6444650	Antisense compositions for detecting and inhibiting telomerase reverse transcriptase	September, 2002	Cech et al.
6475789	Human telomerase catalytic subunit: diagnostic and therapeutic methods	November, 2002	Cech et al.
6517834	Purified telomerase	February, 2003	Weinrich et al.
6545133	Methods for purifying telomerase	April, 2003	Weinrich et al.
6608188	CRT-1 gene having reverse transcriptase motif	August, 2003	Tsuchiya et al.
6610839	Promoter for telomerase reverse transcriptase	August, 2003	Morin et al.
6617110	Cells immortalized with telomerase reverse transcriptase for use in drug screening	September, 2003	Cech et al.
6627619	Antisense compositions for detecting and inhibiting telomerase reverse transcriptase	September, 2003	Cech et al.
6767719	Mouse telomerase reverse transcriptase	July, 2004	Morin et al.
6777203	Telomerase promoter driving expression of therapeutic gene sequences	August, 2004	Morin et al.
6787133	Using purified telomerase to identify telomerase activators and inhibitors	September, 2004	Weinrich et al.
6808880	Method for detecting polynucleotides encoding telomerase	October, 2004	Cech et al.
6921664	Telomerase	July, 2005	Cech et al.
6927285	Genes for human telomerase reverse transcriptase and telomerase variants	August, 2005	Cech et al.
7005262	Methods for detecting nucleic acids encoding human telomerase reverse transcriptase	February, 2006	Cech et al.
7056513	Telomerase	June, 2006	Cech et al.
7091021	Inactive variants of the human telomerase catalytic subunit	August, 2006	Morin
7195911	Mammalian cells that have increased proliferative capacity	March, 2007	Cech et al.
7262174	Treatment for wounds	August, 2007	Jiang et al.
7262288	Nucleic acids encoding human telomerase reverse transcriptase and related homologs	August, 2007	Cech et al.
7285639	Antibody to telomerase reverse transcriptase	October, 2007	Cech et al.
20020164786	Novel telomerase	November, 2002	Cech et al.
20020187471	Novel telomerase	December, 2002	Cech et al.
20030009019	NOVEL TELOMERASE	January, 2003	Cech et al.
20030032075	Novel telomerase	February, 2003	Cech et al.
20030044953	Novel telomerase	March, 2003	Cech et al.
20030059787	Novel telomerase	March, 2003	Cech et al.
20030096344	Human telomerase catalytic subunit: diagnostic and therapeutic methods	May, 2003	Cech et al.
20030100093	Human telomerase catalytic subunit: diagnostic and therapeutic methods	May, 2003	Cech et al.
20040247613	Treating cancer using a telomerase vaccine	December, 2004	Cech et al.

CA2194393	January, 1996
CA2271718	May, 1998
GB2317891	April, 1998
JP9154575	June, 1997
WO/1982/001461	May, 1982			HUMAN HYBRIDOMAS,PRECURSORS AND PRODUCTS
WO/1984/003564	September, 1984			METHOD OF DETERMINING ANTIGENICALLY ACTIVE AMINO ACID SEQUENCES
WO/1993/023572	November, 1993			THERAPY AND DIAGNOSIS OF CONDITIONS RELATED TO TELOMERE LENGTH AND/OR TELOMERASE ACTIVITY
WO/1994/017210	August, 1994			AMPLIFICATION OF NUCLEIC ACID SEQUENCES
WO/1995/013382	May, 1995			THERAPY AND DIAGNOSIS OF CONDITIONS RELATED TO TELOMERE LENGTH AND/OR TELOMERASE ACTIVITY
WO/1996/001835	January, 1996			MAMMALIAN TELOMERASE
WO/1996/012811	May, 1996			YEAST TELOMERASE COMPONENTS AND METHODS USING THEM
WO/1996/019580	June, 1996			TELOMERASE PROTEIN COMPONENT
WO/1996/040868	December, 1996			ESSENTIAL OLIGONUCLEOTIDES OF VERTEBRATE TELOMERASE
WO/1997/038013	October, 1997			MODULATION OF MAMMALIAN TELOMERASE BY PEPTIDE NUCLEIC ACIDS
WO/1998/001542	January, 1998			HUMAN TELOMERASE
WO/1998/001543	January, 1998			HUMAN TELOMERASE GENE
WO/1998/007838	February, 1998			HIGHER ANIMAL TELOMERASE PROTEIN AND GENE ENCODING THE SAME
WO/1998/008938	March, 1998			PROTEINS HAVING TELOMERASE ACTIVITY
WO/1998/014592	April, 1998			TELOMERASE REVERSE TRANSCRIPTASE
WO/1998/014593	April, 1998			HUMAN TELOMERASE CATALYTIC SUBUNIT
WO/1998/021343	May, 1998			GENES ENCODING TELOMERASE PROTEINS
WO/1998/023759	June, 1998			IDENTIFICATION OF INHIBITORS THAT PREVENT ACCESS OF TELOMERASE TO CHROMOSOMAL TERMINUS
WO/1998/037181	August, 1998			TELOMERASE CATALYTIC SUBUNIT GENE AND ENCODED PROTEIN
WO/1998/045450	October, 1998			PURIFIED TELOMERASE
WO/1998/059040	December, 1998			HUMAN CATALYTIC TELOMERASE SUB-UNIT AND ITS DIAGNOSTIC AND THERAPEUTIC USE,
WO/1999/001580	January, 1999			COMPOSITIONS AND METHODS FOR DETECTION OF GENOMIC IMPRINTING DISORDERS
WO/1999/033998	July, 1999			REGULATORY DNA SEQUENCES OF THE HUMAN CATALYTIC TELOMERASE SUB-UNIT GENE, DIAGNOSTIC AND THERAPEUTIC USE THEREOF,
WO/1999/038964	August, 1999			PROMOTER REGIONS OF THE MOUSE AND HUMAN TELOMERASE RNA COMPONENT GENES
WO/1999/063945	December, 1999			VACCINATION STRATEGY TO PREVENT AND TREAT CANCERS
WO/2000/002581	January, 2000			ANTIGENIC PEPTIDES DERIVED FROM TELOMERASE
WO/2000/046355	August, 2000			TELOMERASE REVERSE TRANSCRIPTASE TRANSCRIPTIONAL REGULATORY SEQUENCES
WO/2000/061766	October, 2000			TELOMERASE-SPECIFIC CANCER VACCINE
WO/2000/073420	December, 2000			CREATION OF HUMAN TUMORIGENIC CELLS AND USES THEREFOR
WO/2001/060391	August, 2001			A UNIVERSAL VACCINE AND METHOD FOR TREATING CANCER EMPLOYING TELOMERASE REVERSE TRANSCRIPTASE
WO/2002/094213	November, 2002			POLYORGANOSILOXANE MICRO-EMULSION COMPOSITION AND RAW MATERIAL FOR COSMETICS
WO/2003/038047	May, 2003			HUMAN TELOMERASE REVERSE TRANSCRIPTASE AS A CLASS-II RESTRICTED TUMOR-ASSOCIATED ANTIGEN

Nair et al., J. Immunol 2000 165(12): 6949-6955.
Roitt et al (Immunology, 1993, Mosby, St. Louis, p. 6.4-6.5).
Roitt et al (Immunology, 1993, Mosby, St. Louis, p. 7.7-7.8).
Greenspan et al (Nature Biotechnology, 1999, 7:936-937).
Bowie et al (Science, 1990, 257:1306-1310).
Herbert et al (The Dictionary of Immunology, Academic Press, 3rd Edition, London, 1985, pp. 58-59).
Holmes (Exp. Opin.Invest. Drugs, 2001, 10(3):511-519).
Yi et al (Nucleic acids Research, 2001, 23:4818I-4825).
Krams et al (Am. J. Pathol., 2001, 159:1925-1932).
Hirashima (Int. Arch. Allergy Immunol., 2000, Suppl 1:6-9).
Benedict et al (J. Exp. Medicine, 2001, 193(1)89-99).
Jiang et al (JBC, 2003, 278(7) 4763-4769.
Burgess et al (J of Cell Bio. 111:2129-2138, 1990).
Lazar et al (Molecular and Cellular Biology, 1988, 8:1247-1252).
U.S. Appl. No. 10/877,124, Cech et al.
U.S. Appl. No. 10/877,146, Cech et al.
Anderson, W. French “Human Gene Therapy” Nature, Apr. 30, 1998, pp. 25-30, vol. 392, Supp.
Campbell, Keith & Wilmut, Ian “Totipotency or Multipotentiality of Cultured Cells: Applications and Progress” Theriogenology, Jan. 1997, pp. 63-72, vol. 47, Issue 1, Elsevier Science Inc.
Gearhart, John “New Potential for Human Embryonic Stem Cells” Science, Nov. 6, 1998; pp. 1061-1062, vol. 282, Issue 5391.
Hornsby, PJ et al. “Adrenocortical Cells Immortalized by Telomerase: Potential Use for Ex Vivo Gene Therapy” Journal of Anti-Aging Medicine, 2000, pp. 411-417, vol. 3, No. 4.
Ostler Elizabeth L. et al. “Telomerase and the Cellular Lifespan: Implications for the Aging Process” J. of Pediatric Endocrinology & Metabolism, 2000, pp. 1467-1476, vol. 13, Supplement 6, Freund Publishing House Ltd. London.
Thomson, James A. et al. “Embryonic Stem Cell Lines Derived from Human Blastocysts” Science, Nov. 6, 1998; pp. 1145-1147, vol. 282, Issue 5391.
Adams, Mark et al. “Initial Assessment of Human Gene Diversity and Expression Patterns Based Upon 83 Million Nucleotides of cDNA Sequence” The Genome Directory: Supplement to Neture Sep. 28, 1995, 1995, pp. 3-174, vol. 377, Issue 6547S.
Adamson, D. et al. “Significant Telomere Shortening in Childhood Leukemia”, Cancer Genet. Cytogenet, 1992; pp. 204-206, vol. 61.
Anderson and Young, “Quantitative Filter Hybridization” in Nucleic Acid Hybridization pp. 73-111 (1985).
Ausubel, F. et al., Current Protocols in Molecular Biology, 1996, vol. 1, Chapter 5, John Wiley & Sons, New York NY.
Autexier, C. & Greider, C. Telomerase and cancer: revisiting the telomere hypothesis; Trends in Biochemical Sciences, 1996, pp. 387-391, vol. 10, No. 21.
Autexier, C. et al., “Reconstitution of human telomerase activity and identification of a minimal functional region of the human telomerase RNA,” EMBO J, 1996, pp. 5928-5935, vol. 15.
Auxexier and Greider, “Functional reconstitution of wild-type and mutant Tetrahymena telomerase,” Genes Develop., 1994, pp. 563, vol. 8.
Avilion, A., “Characterization and expression of human telomerase,” Dissertation Abstracts International, 1996, pp. 5930-B, vol. 56, No. 11.
Bandyopadhyay et al. “The human melanocyte: a model system to study the complecity of cellular aging and transformation in non-fibroblastic cells”, Exp. Gerontol., 2001, pp. 1265, vol. 36.
Baringa, Marcia, “The Telomerase Picture Fills In”, Science, Apr. 25, 1997; pp. 528-529, vol. 276.
Berger and Kimmel (Eds), “Guide to Molecular Cloning Techniques”, Methods in Enzymology, 1987, vol. 152, Academic Press, San Diego CA.
Biessman et al., “Addition of Telomere-Associated HeT DNA Sequences “Heals” Broken Chromosome End s in Drosophila,” Cell, 1990, pp. 663, vol. 61.
Bitter, G. et al., “Expression and secretion vectors for yeast,” Methods in Enzymology, 1987, pp. 516-544, vol. 153, Academic Press, Inc.
Blackburn and Chiou, “Non-nucleosomal packaging of a tandemly repeated DNA sequence at termini of extrachromosomal DNA coding for rRNA in Tetrahymena,” Proc. Natl. Acad. Sci., 1981, pp. 2263, vol. 78.
Blackburn and Gall, “A tandernly repeated sequence at the termini of the extrachromosomal ribosomal RNA genes in Tetrahymena,” J. Mol. Biol., 1978, pp. 33, vol. 120.
Blackburn, “Telomerases,” Ann. Rev. Biochem., 1992, pp. 113-129, vol. 61.
Bodnar, A. et al., “Extension of Life-Span by Introduction of Telomerase into Normal Human Cells,” Science, 1998, pp. 349-352, vol. 279.
Bradford, “A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding,” Anal. Biochem., 1976. pp. 248-255, vol. 72.
Bramston et al. “The use of adenoviral vectors for gene therapy and gene transfer in vivo”, Curr. Opin. Biotechnol., 1995, p. 590, vol. 6.
Braunstein et al., “Transcriptional silencing in yeast is associated with reduced nucleosome acetylation,” Genes Develop., 1993, pp. 592-604, vol. 7.
Calvio et al., “Identification of hnRNP P2 as TLS/FUS using electrospray mass spectrometry,” RNA, 1995, pp. 724, vol. 1.
Caruthers et al., “New chemical methods for synthesizing polynucleotides,” Nucleic Acids Res. Symp. Ser., 1980, pp. 215-223.
Chan and Tye, “Organization of DNA sequences and replication origins at yeat telomeres,” Cell, 1983. p. 563, vol. 33.
Chiu, et al. “Replicative senescence and cell immortality: the role of telomeres and telomerase (44075)”, Proc. Soc. Exp. Bio. Med., 1997, pp. 99-106, vol. 214.
Chong, L. et al. “A Human Telomeric Protein”, Science, Dec. 1995, pp. 1663-1667, vol. 270.
Colbere-Garapin et al., “A new dominant hybrid selective marker for higher eukaryotic cells,” J. Mol. Biol., Jul. 1981, pp. 1-14, vol. 150, No. 1.
Cole et al., “The EBV-hybridoma technique and its application to human lung cancer,” Monoclonal Antibodies and Cancer Therapy, 1985, pp. 77-96, Alan R. Liss Inc., New York NY.
Collins et al., “Purification of Tetrahymena telomerase and cloning of genes encoding the two peotein components of the enzyme,” Cell, 1995, pp. 677-686, vol. 81.
Collins, Kathleen, “Structure and function of telomerase”, Current Opinion in Cell Biology, 1996, pp. 374-380, vol. 8.
Conrad et al. “RAP1 protein interacts with yeast telomers in vivo: Overproduction alters telomere structure and decreases chromosome stability,” Cell, 1990, pp. 739, vol. 63.
Coombs, Dictionary of Biotechnology, 1994, pp. 101-102, 283, 301, 332; Stockton Press, New York.
Cote et al., “Generation of human monoclonal antibodies reactive with cellular antigens,” Proc. Natl. Acad. Sci., 1983, pp. 2026, vol. 80.
Counter, C. et al. “Telomerase activity in human ovarian carcinoma”; Proc. Natl. Acad. Sci USA; Apr. 1994; vol. 91; pp. 2900-2904.
Counter, C. et al. “Telomerase Activity in Normal Leukocytes and in Hematologic Malignancies” Blood; May 1, 1995; pp. 2315-2320, vol. 85, No. 9.
Counter, C. et al. “Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity”, The EMBO Journal; 1992, pp. 1921-1929, vol. 11; No. 5, Oxford University Press.
Counter, C. et al. “The catalytic subunit of yeast telomerase,” Proc. Natl. Acad. Sci. U.S.A., Aug. 1997, 9202-9207, vol. 94.
Creighton, T. Proteins, Structures and Molecular Principles, 1983, WH Freeman and Co., New York, NY.
Dagarag et al. “Differential impairment of lytic and cytokine functions in senescent human immunodeficiency virus type 1 specific T lymphocytes”, J. Virology, 2003, p. 3077-3083, vol. 77, No. 5.
De Lange, T. et al. “Structure and Variability of Human Chromosome Ends”; Molecular and Cellular Biology; Feb. 1990; pp. 518-527, vol. 10, No. 2.
Dieffenbach and Dveksler (Eds.), PCR Primer, a Laboratory Manual, 1995, Cold Spring Harbor Press, Plainview NY.
Duplaa et al., “Quantitative analysis of polymerase chain reaction products using biotinylated dUTP Incorporation,” Anal. Biochem., 1993, p. 229, vol. 212.
Fang,G. et al., “Oxytricha telomere-binding protein: separable DNA-binding and dimerization domains of the α-subunit,” Genes and Development, 1993, pp. 870-882, vol. 7.
Feng et al., “The RNA Componant of Human Telomerase,” Science, 1995, pp. 1236-1241, vol. 269.
Flavell, R. & Mathias, R. “Prospects for transforming monocot crop plants”, Nature, Jan. 12, 1984, pp. 108-109, vol. 307.
Franco et al. “Clonal variation in phenotype and life span of human embryonic fibroblasts (MRC-5) transduced with the catalytic component of telomerase (hTERT)”, Exp. Cell Res., 2001, p. 14, vol. 268.
Freshney, Culture of Animal Cells, A Manual Basic Technique, pp. 3-4, Alan R. Liss, Inc. New York, 1983.
Genbank Accession No. A46242; Sep. 21, 1993.
GenBank Accession No. AA281296; Apr. 2, 1997.
Genbank Accession No. AA299878; Apr. 18, 1997.
Genbank Accession No. AA311750; Apr. 19, 1997.
Genbank Accession No. L38903; Jan. 30, 1995.
Genbank Accession No. Q06163; Nov. 1, 1995.
Genbank Accession No. S39696; Oct. 7, 1994.
Genbank Accession No. S53396; May 5, 1995.
GenBank Accession No. U95964; May 5, 1997.
GenBank Accession No. W70315; Jun. 19, 1996.
Genome Issue of Science, 1994, pp. 1981-2144, vol. 265.
Gilley, D. et al., “Altering specific telomerase RNA template residues affects active site function,” Genes Develop., 1995, pp. 2214-2226, vol. 9, Cold Spring Harbor Laboratory Press.
Glaser, P. et al. “Bacillus subtilis genome project: cloning and sequencing of the 97 kb region from 325° to 333°” Molecular Microbiology, 1993, pp. 371-384, vol. 10, No. 2.
Goodman, R. et al. “Gene Transfer in Crop Improvement”, Science, Apr. 3, 1987, pp. 48-54, vol. 236.
Gottschling, D. and Cech, T. “Chromatin Structure of the Molecular Ends of Oxytricha Mononuclear DNA: Phased Nucleosomes and a Telomeric Complex,” Cell, Sep. 1984, pp. 501-510, vol. 38.
Gottschling. D. and Zakian, V. “Telomere proteins: specific recognition and protection of the natural termini of Oxytricha macronuclear DNA,” Cell, 1986, pp. 195-205, vol. 47.
Gray, J.T. et al., “Cloning and expression of genes for the Oxytricha telomere-binding protein specific subunit interactions in the telomeric complex,” Cell, 1991, pp. 807-814, vol. 67.
Greenwood, S. et al., “Phylogenetic relationships within the class oligohymenophorea, phylum ciliophora, inferred from the complete small subunit rRNA gene sequences of Colpidium campylum, Glaucoma chattoni, and Opisthonecta henneguyi,” J. Mol. Evol., 1991, pp. 163-174, vol. 33.
Greider, C. “Telomerase is processive,” Mol. Cell. Biol., Sep. 1991, p. 4572-4580, vol. 11.
Greider, C. “Telomere Length Regulation,” Ann. Rev. Biochem., 1996, pp. 337-365, vol. 65, Cold Springs Harbor Laboratory Press, New York.
Greider, C. “Telomeres, Telomerase and Senescence”; BioEssays; 1990; pp. 363-369, vol. 12, No. 8.
Greider, C. and Blackburn, E. “A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis,” Nature, Jan. 1989, pp. 331-337, vol. 337.
Greider, C. and Blackburn, E. “Identification of a specific telomore terminal transferase activity in Tetrahymena extracts,” Cell, Dec. 1985, 405-413, vol. 43.
Hampton et al., Serological Methods a Laboratory Manual, 1990, APS Press, St Paul MN.
Harley, C. “Telomerase is not an oncogene”, Oncogene, 2002, p. 494, vol. 21.
Harley, C. “Telomere loss: Mitotic clock or genetic time bomb?” Mutation Research; 1991; pp. 271-282, vol. 256, Elsevier Science Publishers.
Harley, C. & Villeponteau, B. “Telomeres and telomerase in aging and cancer” Current Opinion in Genetics and Development; 1995, pp. 249-255, vol. 5.
Harley, C. et al. “Telomeres shorten during ageing of human fibroblasts”, Nature, May 31, 1990; pp. 458-460, vol. 345.
Harrington et al., “A Mammalian Telomerase-Associated Protein,” Science, Feb. 1997, pp. 973-977, vol. 275.
Harrington et al., “Human telomerase contains evolutionarily conserved catalytic and structural subunits,” Genes Dev., 1997, pp. 3109-3115, vol. 11.
Hartman and Mulligan, “Two dominant-acting selectable markers for gene transfer studies in mammalian cells,” Proc. Natl. Acad. Sci., 1988, pp. 8047, vol. 85.
Hastie, N. et al. “Telomere reduction in human colorectal carcinoma and with ageing”, Nature, Aug. 30, 1990, pp. 866-868, vol. 346.
He et al. “A simplified system for generating recombinant adenoviruses”, Proc. Natl. Acad. Sci., 1998, p. 2509, vol. 95.
Healy, K. C. “Telomere dynamics and telomerase activation in tumor progression: prospects for prognosis and therapy” Oncol. Res., 1995, pp. 121-130, vol. 7.
Henderson C. Cancer genetics gene regulates telomerase resulting in death of cancer cells; Gene Therapy Weekly; Sep. 11, 1995.
Henderson, C. and Blackburn, E.“An overhanging 3′ terminus is a conserved feature of temomeres,” Mol Cell. Biol., Jan. 1989, p. 345-348, vol. 9, No. 1.
Hiyama, E. et al. “Correlating telomerase activity levels with human neuroblastoma outcomes”; Nature Medicine; Mar. 3, 1995; pp. 249-255, vol. 1, No. 3.
Holzmann, K. et al. “Telomeric Associations and Loss of Telomeric DNA Repeats in Renal Tumors”, Genes, Chromosomes & Cancer, 1993, pp. 178-181, vol. 6.
Horn, T. et al., “Synthesis of oligonucleotides on cellulose. Part II: design and synthetic strategy to the synthesis of 22 oligodeoxynucleotides coding for gastric inhibitory polypeptide (GIP),” Nucleic Acids Res. Symp. Ser. No. 7, 1980, pp. 225-232.
Hudson et al., “An STS-based map of the human genome,” Science, 1995, p. 1945, vol. 270.
Huse, W. et al., “Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda,” Science, 1989, p. 1275-1281, vol. 246.
Jähne, A. et al. “Genetic Engineering of Cereal Crop Plants: A Review”, Euphytica, 1995, pp. 35-44, vol. 85, Kluwer Academic Publishers, Netherlands.
Jiang, Xu-Rong et al. “Telomerase expression in human somatic cells does not include changes associated with a transformed phenotype”, Nat. Genet., 1999, p. 111-114, vol. 21.
Johnson, P. et al., “Expression of Wild-Type p53 Is Not Compatible with Continued Growth of p53-Negative Tumor Cells”, Molecular and Cellular Biology, Jan. 1991, p. 1-11, vol. 11, No. 1.
Jolliffe, L.K., “Humanized antibodies: enhancing therapeutic utility through antibody engineering”, Int. Rev. Immunol., 1993, pp. 241-250, vol. 10.
Kilian, A. et al., “Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns in different cell types,” Hum. Mol. Genet., 1997, pp. 2011-2019, vol. 6, No. 12.
Kim, N. et al. “Specific Association of Human Telomerase Activity with Immortal Cells and Cancer”, Science, Dec. 23, 1994, pp. 2011-2014, vol. 266.
Kinsella et al. “Episomal vectors rapidly and stablyproduce high-titer recombinant retrovirus”, Hum. Gene Ther., 1996, p. 1405, vol. 7.
Kipling and Cooke, “Hypervariable ultra-long telomeres in mice,” Nature, 1990, p. 400, vol. 347.
Kiwaki et al. “Correction of ornithine transcarbamylase deficiency in adult spf(ash) mice and in OTC-deficient human hepatocytes with recombinant adenoviruses bearing the CAG promoter”, Hum. Gene Ther., 1996, p. 821, vol. 7.
Kiyono et al. “Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells” Nature, Nov. 1998, pp. 84-88, vol. 396, No. 6706.
Klingelhutz, A. et al. “Restoration of Telomeres in Human Papillomavirus- Immortalized Human Anogenital Epithelial Cells”; Molecular and Cellular Biology; Feb. 1994, pp. 961-969, vol. 14, No. 2.
Klobutcher, L. et al. “All gene-sized DNA molecules in four species of hypotrichs have the same terminal sequence and an unusual 3′ terminus,” Proc. Natl. Acad. Sci., 1981, p. 3015, vol. 78.
Koehler and Milstein, “Continuous cultures of fused cells secreting antibody of predefined specificity,” Nature, 1975, pp. 495-497, vol. 256.
Kozbor, D. & Roder, J. “The production of monoclonal antibodies from human lymphocytes,” Immunol. Today, 1983, p. 72, vol. 4.
Lamond, A. and Sproat, B. “Isloation and Characterization of Ribonucleoprotein Complexes”, RNA Processing vol. 1 A Practical Approach, 1994, pp. 103-140, Eds. Higgins & Hames, Oxford University Press, New York.
Lamond, A. et al. “Probing the structure and function of U2 snRNP with antisense oligonucleotides made of 2′-OMe RNA,” Cell, Jul. 1989, pp. 383-390, vol. 58.
Lendvay, T. et al., “Senescence mutants of Saccharomyces cerevisiae with a defect in telomere replication identify three additional EST genes,” Genetics, Dec. 1996, pp. 1399-1412, vol. 144.
Lewis, A. & Crowe, J.S. “Generation of humanized monoclonal antibodies by ‘best fit’ framework selection and recombinant polymerase chain reaction”, Year Immunol., 1993, pp. 110-118, vol. 7.
Lingner et al., “Telomerase RNAs of different ciliates have a common secondary structure and a permuted template,” Genes Develop., 1994, p. 1984, vol. 8.
Lingner et al., “Purification of telomerase from Euplotes adeiculatus: requirement of a primer 3′ overhang,” Proc. Natl. Acad. Sci., 1996, pp. 10712-10717, vol. 93.
Lingner et al., “Reverse transcriptase motifs in the catalytic subunit of telomerase,” Science, Apr. 25, 1997; pp. 561-567, vol. 276.
Lingner et al., “Telomerase and DNA End Replication: No Longer a Lagging Strand Problem?,” Science, 1995, p. 1533, vol. 269.
Linking telomerase and tumors; Genesis Report- DX; 1995; vol. 4, No. 6; Publisher Genesis Group Associates.
Lowy, I. et al. “Isloation of transforming DNA: Cloning the hamster aprt gene,” Cell, Dec. 1980, p. 817-823, vol. 22.
Lundblad, V. & Blackburn, E., Letter to the Editor entitled, “RNA-Dependent Polymerase Motifs in EST1: Tentative Identification of a Protein Component of an Essential Yeast Telomerase”, Cell, Feb. 23, 1990, pp. 529-530, vol. 60.
Lustig, A. & Petes, T. “Identification of yeast mutants with altered telomere structure,” Proc. Natl. Acad. Sci., Mar. 1986, pp. 1398-1402, vol. 83.
Lustig, Arthur J., “The identification of telomerase subunits: catalysing telomere research”, Trends in Cell Biology; Aug. 1997, pp. 299-302, vol. 7.
Maddox, D.E. et al. “Elevated serum levels in human pregnancy of a molecule immunochemically similar to eosinophil granule major basic protein,” J. Exp. Med., Oct. 1983, pp. 1211-1226, vol. 158.
Makarov, V. et al. “Nucleosomal Organization of Telomere-Specific Chromatin in Rat,” Cell, 1993, p. 775-787, vol. 73.
Malicki, J. et al. “A human HOX4B regulatory element provides head-specific expression in Drosophila embryos ”, Nature, Jul. 23, 1992, pp. 345-357, vol. 358.
McEachern, M. & Blackburn, E. “Runaway telomere elongation caused by telomerase RNA gene mutations,” Nature, Aug. 1995, pp. 403-409, vol. 376.
Melby, P. et al. “Quantitative measurement of human cytokine gene expression by polymerase chain reaction,” J. Immunol. Meth., 1993, pp. 235-244, vol. 159.
Merrifield, R.B. “Solid phase peptide synthesis. I. The synthesis of tetrapeptide,” J. Am. Chem. Soc., Jul. 1963, pp. 2149-2154, vol. 85.
Meyerson, M. et al., “hEST2, the Putative Human Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cells and during Immortalization,” Cell, Aug. 1997, pp. 785-795, vol. 90.
Microbix Biosystems, Inc.; Admax™ adenovirus creation kits. www.
Murasawa et al. “Constitutive human telomerase reverse transcriptase expression enhances regenerative properties of endothelial progenitor cells”, Circulation, 2002, pp. 1133-1139, vol. 106.
Murray, In McGraw Hill Yearbook of Science and Technology, 1992, pp. 191-196, McGraw Hill, New York.
Nakamura, T. et al., “Telomerase Catalytic Subunit Homologs from Fission Yeast and Human,” Science, Aug. 1997, pp. 955-959, vol. 277.
Nakayama et al. “TLP1: A Gene Encoding a Protein Component of Mammalian Telomerase Is a Novel Member of WD Repeats Family,” Cell, 1997, pp. 875-884, vol. 88.
Nakayama, J. et al. “Cloning of a Candidate cDNA Encoding a Proteinaceous Component of Mammalian Telomerase”, Molecular Biology Cell Abstracts Supp. 7, 1996, p. 857-884, 286a Section 1664.
Natarajan et al. “Major histocompatibility complex determinants select T-cell receptor alpha chain variable region dominance in a peptide-specific response.” Proc. Natl. Acad. Sci., Oct. 1992, pp. 8874-8878, vol. 19.
Nielsen et al., “Peptide nucleic acids (PNAs): Potential antisense and anti-gene agents,” Anticancer Drug Des., 1993, pp. 53-63, vol. 8.
O'Hare et al., Proc. Natl. Acad. Sciences; Jan. 16, 2001; pp. 646-651, vol. 98.
Oka, Y. et al. “Inverted terminal repeat sequence in the macronuclear DNA of Stylonychia pustulata,” Gene, 1980, pp. 301-306, vol. 10.
Olovnikov, A.M., “A Theory of Marginotomy: The incomplete copying of template margin in enzymic synthesis of polynucleotides and biological significance of the phenomenon,” J. Theor. Biol., 1973, p. 181, vol. 41.
Orlandi et al., “Cloning immunoglobulin variable domains for expression by the polymerase chain reaction,” Proc. Natl. Acad. Sci., May 1989, pp. 3833-3837, vol. 86.
Paszkowski, Jerzy et al. “Direct gene transfer to plants,” The EMBO Journal, 1984, pp. 2717-2722, vol. 3, No. 12.
Pear et al. “Production of high-titer helper-free retroviruses by transient transfection,” Proc. Natl. Acad. Sci., 1993, p. 8392, vol. 92, USA.
Potrykus, I. et al. “Direct gene transfer to cells of a graminaceous monocot”, Mol. Gene. Genet., 1985, pp. 183-188, vol. 199.
Prescott, D. “The DNA of Ciliated Protozoa,” Microbiol. Rev., Jun. 1994, pp. 233-267, vol. 58, No. 2.
Price, C.M. “Fluorescence in Situ Hybridization” Blood Reviews, 1993, p. 127, vol. 7.
Ramirez et al. “Putative telomere-independent mechanisms of replicative aging reflect inadequate growth conditions”, Genes Dev., 2001, p. 398, vol. 15.
Raymond, E., et al.; Agents that target telomerase and telomeres; Curr. Opin. Biotechnol.; 1996; 7:583-91.
Rhodes, C. et al. “Transformation of maize by electroporation of embryos,” Meth. Mol. Biol., 1995, pp. 121-131, vol. 55.
Rhyu, M.S. “Telomeres, telomerase, and immortality”; J. Natl. Cancer Inst.; Jun. 21, 1995; pp. 884-894, vol. 87, No. 12.
Roberge, J. et al., “A strategy for a convergent synthesis of N-linked glycopeptides on a solid support,” Science, Jul. 1995, pp. 202-204, vol. 269.
Romero, D. and Blackburn, E. “A conserved secondary structure for telomerase RNA,” Cell, Oct. 1991, pp. 343-353, vol. 67.
Rudolph et al. “Inhibition of experimental liver cirrhosis in mice by telomerase gene delivery”, Science, 2000, p. 1253, vol. 287.
Sadelain et al. “Generation of high-titer retroviral vector capable of expressing high levels of the human β-globulin gene”, Proc. Natl. Acad. Sci., 1995, p. 6728, vol. 92, USA.
Sambrook, J. et al., Chapter 16: “Expression of Cloned Genes in Cultured Mammalian Cells”, Molecular Cloning, A Laboratory Manual, 1989, Cold Spring Harbor Press, Plainview NY.
Sambrook, J. et al., Chapter 17: “Expression of Cloned Genes in Escherichia coli”, Molecular Cloning, A Laboratory Manual, 1989, Cold Spring Harbor Press, Plainview NY.
Sambrook, J. et al., Chapter 8: “Construction and Analysis of cDNA Libraries”, Molecular Cloning, A Laboratory Manual, 1989, Cold Spring Harbor Press, Plainview NY.
Sandell, L. et al., “Transcription of yeast telomere alleviates telomere position effect without affecting chromosome stability,” Proc. Natl. Acad. Sci., 1994, pp. 12061-12065, vol. 91.
Sanger, F. et al., “DNA sequencing with chain-terminating inhibitors,” Proc. Natl. Acad. Sci., 1977, pp. 5463-5467, vol. 74, No. 12.
Scharf, D. et al. “Heat stress promoters and transcription factors,” Result Probl. Cell Differ. 1994, pp. 125-162, vol. 20.
Schena et al. “Parallel human genome analysis: Microarray-based expression monitoring of 1000 genes”, Proc. Natl. Acad. Sci., Oct. 1996, pp. 10614-10619, vol. 93, USA.
Schwartz, H. et al. “Telomere Reduction in Giant Cell Tumor of Bone and with Aging”; Cancer Genet Cytogenet; 1993; pp. 132-138, vol. 71, Elsevier Science Publishing Co., Inc., New York, U.S.A.
Shampay, J. and Blackburn, E. “Generation of telomere-length heterogeneity in Saccharomyces cerevisiae,” Proc. Natl. Acad. Sci., Jan. 1988, pp. 534-538, vol. 85.
Sheen, F. and Levis, R. “Transposition of the LINE-like retrotransposon TART to Drosophila chromosome termini,” Proc. Natl. Acad. Sci., Dec. 1994, pp. 12510-12514, vol. 91.
Singer, M. & Gottschling, D. “TLC1: Template RNA Component of Saccharomyces cerevisiae Telomerase,” Science, Oct. 1994, pp. 404-409, vol. 266.
Singer, M. “Unusual Reverse Transcriptases”, Journal of Biological Chemistry; 1995, pp. 24623-23626, vol. 270, No. 42.
Smith, J. & Yeh, G. “Telomere Reduction in Endometrial Adenocarcinoma”; Am. J. Obstet, Gynecol.; Dec. 1992; pp. 1883-1887, vol. 167, No. 6.
Starling, J.A. et al., “Extensive telomere repeat arrays in mouse are hypervariable,” Nucleic Acids Res., 1990, pp. 6881-6888, vol. 18, No. 23.
Swanton, M. et al., “Arrangement of Coding and Non-coding Sequences in the DNA Molecules Coding for rRNAs in Oxytricha sp.,” Chromosoma, 1980, pp. 203-215, vol. 77.
Tait, J. et al. “Structure and Polymorphisms of the Human Annexin III (ANX3) Gene”, Genomics, 1993, pp. 79-86, vol. 18, No. 1.
Tani et al. “Transduction of LacZ gene into leukemia cells using viral vectors of retrovirus and adenovirus”, Leukemia 9 suppl. 1, 1995, p. S64.
Tommerup, H. et al., “Unusual chromatin in human telomeres,” Mol. Cell. Biol., Sep. 1994, p. 5777-5785, vol. 14, No. 9.
Trask, B. “Fluorescence in situ hybridization: application in cytogenetics and gene mapping,” (1991) Trends Genet., May 1991, pp. 149-154, vol. 7, No. 5.
Vaziri, H. & Benchimol, S. “Reconstitution in telomerase activity in normal human cells leads to elongation of telomeres and extended replicative life span”, Curr. Biol., 1998, p. 279-282, vol. 8, No. 5.
Verma et al., “Human Chromosomes: A Manual of Basic Techniques,” 1988, Pergamon Press, New York NY.
Watson, J.D. “Origin of Concatemeric T7 DNA,” Nature New Biol., Oct. 1972, pp. 197-201, vol. 239.
Weinrich, S. et al., “Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT,” Nat. Genet., Dec. 1997, pp. 498-502, vol. 17, No. 4.
Wellinger, R. et al., “Origin activation and formation of single-strand TG1-3 tails occur sequentially in late S phase on a Yeast linear plasmid,” Mol. Cell. Biol., Jul. 1993, pp. 4057-4065, vol. 13, No. 7.
Wellinger, R. et al., “Saccharomyces Telomeres Acquire Single-Strand TG1-3 Tails Late in S Phase,” Cell, 1993, pp. 51-60, vol. 72.
Whitehead Institute/MIT Center for Genome Research, Genetic Map of the Mouse, Database Release 10, Apr. 28, 1995.
Wigler, M. et al., “Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells,” Cell, 1997, pp. 223-232, vol. 11.
Wigler, M. et al., “Transformation of mammalian cells with an amplifiable dominant-acting gene,” Proc. Natl. Acad. Sci., Jun. 1980, pp. 3567-3570, vol. 77, No. 6.
Winter, G. & Harris, W. “Humanized Antibodies,”, Trends Pharmacol. Sci., May 1993, pp. 139-143, vol. 14.
Winter, G. and Milstein, C. “Man-made antibodies,” Nature, Jan. 1991, pp. 293-299, vol. 349.
Wirth, Urs et al; “Immediate-Early RNA 2.9 and Early RNA 2.6 of Bovine Herpesvirus 1 Are 3′ Coterminal and Encode of Putative Zinc Finger Transactivator Protein”; Journal of Virology; May 1992; pp. 2763-2772, vol. 66, No. 5.
Wright, J. et al., “Saccharomyces telomeres assume a non-nucleosomal chromatin structure,” Genes Develop., 1992, pp. 197-210, vol. 6.
Yu et al., “In vivo alteration of telomere sequences and senescence caused by mutated Tetrahymena telomerase RNAs,” Nature, Mar. 1990, p. 126-132, vol. 344.
Zahler and Prescott, “Telomere terminal transferase activity in the hypotrichous ciliate Oxytricha nova and a model for replication of the ends of linear DNA molecules,” Nucleic Acids Res., 1988, p. 6953, vol. 16.
Zakian, “Telomeres: Beginning to Understand the End,” Science, 1995, pp. 1601-1606, vol. 270.
Zaug, Arthur et al., “Catalysis of RNA Cleavage by a Ribozyme Derived from the Group I Intron of Anabaena Pre-tRNALeu,” Biochemistry, 1994, pp. 14935-14947, vol. 33.
Zaug, Arthur et al., “Method for determining RNA 3′ ends and application to human telomerase RNA”, Nucleic Acids Research, 1996, pp. 532-533, vol. 24, No. 3.
Yoshimi Okada & Joue Ikeda (editors), Japanese Molecular Biology Association edition, Development of Molecular Biology 13, “Expression of information and control in higher plant”, Feb. 10, 1990, Maruzen Corp. pp. 262-263.
U.S. Appl. No. 60/038,750, Counter et al.
U.S. Appl. No. 08/974,584, Cech et al.
U.S. Appl. No. 09/432,503, Cech et al.
U.S. Appl. No. 09/721,477, Cech et al.
Adaptive definition, www.answers.com/topic/adaptive, 1 page (2007).
Alberts, B. et al., Molecular Biology of the Cell, Newton Press Ltd., p. 326, Fig. 7-43 (Jul. 20, 1995).
Anderson, W. French, “Human Gene Therapy,” Nature 392(Supp.):25-30 (Apr. 30, 1998).
Ayyoub, M. et al., “Lack of tumor recognition by hTERT peptide 540-548-specific CD8+T Cells from melanoma patients reveals inefficient antigen processing,” Eur. J. Immunol. 31:2642-51 (2001).
Bachand, F. & Autexier, C., “Functional regions of human telomerase reverse transcriptase and human telomerase RNA required for telomerase activity and RNA- protein interactions,” Mol. Cell. Biol. 21:1888-97 (2001).
Beasley, E. et al., “Statistical refinement of primer design parameters,” PCR Applications, Innis et al., Eds., Academic Press, San Diego, pp. 55-71 (1999).
Bellone, M. et al., “In vitro priming of cytotoxic T lymphocytes against poorly immunogenic epitopes by engineered antigen-presenting cells,” Eur. J. Immunol. 24:2691-8 (1994).
Bellone, M. et al., “Rejection of a nonimmunogenic melanoma by vaccination with natural melanoma peptides on engineered antigen-presenting cells,” J. Immunol. 158:783-9 (1997).
Boczkowski, D. et al., “Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo,” J. Exp. Med. 184:465-72 (1996).
Bryan, T. et al., “A mutant of Tetrahymena telomerase reverse transcriptase with increased processivity,” J. Biol. Chem. 275:24199-207 (2000).
Bryan, T. et al., “Telomerase reverse transcriptase genes identified in Tetrahymena thermophilia and Oxytricha trifallax,” Proc. Natl. Acad. Sci. USA 95:8479-84 (1998).
Bryan, T. et al., “Telomerase RNA bound by protein motifs specific to telomerase reverse transcriptase,” Molec. Cell 6:493-99 (2000).
Colgin, L. et al., “The hTERTα splice variant is a dominant negative inhibitor of telomerase activity,” Neoplasia 2(5):426-32 (2000).
EMBL Database entry Greenberg et al., AF051911, XP002091313 (Apr. 6, 1998).
EMBL Database entry Martin-Rivera et al., AF073311, XP002091314 (Sep. 9, 1998).
Farmery, M. & Bulleid, N., “Major histocompatibility class I folding, assembly, and degradation: A paradigm for two-stage quality control in the endoplasmic reticulum,” Prog. Nucl. Acid Res. Mol. Biol. 67: 235-68 (2001).
Flower, D. “Towards in silico prediction of immunogenic epitopes,” Trends Immunol. 24(12):667-74 (2003).
Friedman, K. et al., “Essential functions of amino-terminal domains in the yeast telomerase catalytic subunit revealed by selection for variable mutants,” Genes Dev. 13(21):2863-74 (1999).
Frolkis, M. et al., “Dendritic cells reconstituted with human telomerase gene induce potent cytotoxic T-cell response against different types of tumors,” Cancer Gene Ther. 10:239-49 (2003).
Greenberg, R. et al., “Expression of mouse telomerase reverse transcriptase during development, differentiation, and proliferation,” Oncogene 16:1723-30 (1998).
Greener, M., “Telomerase: The search for a universal cancer vaccine,” Mol. Med. Today 6:257 (2000).
Greider, C., “Telomerase and senescence: The history, experiment, the future,” Curr. Biol. 8(5):R178-81 (1998).
Gura, T., “Antisense has growing pains,” Science 270:575-7 (1995).
Haering, C. et al., “Analysis of telomerase catalytic subunit mutants in vivo and in vitro in Schizosaccharomyces pombe,” Proc. Natl. Acad. Sci. USA 97:6367-72 (2000).
Hahn, W. et al., “Inhibition of telomerase limits the growth of human cancer cells,” Nature Med. 5:1164-70 (1999).
Harrington, L. et al., “Gel shift and UV cross-linking analysis of Tetrahymena telomerase,” J. Biol. Chem. 270(15):8893-901 (1995).
Heiser, A. et al., “Human dendritic cells transfected with renal tumor RNA stimulate polyclonal T-cell responses against antigens expressed by primary and metastatic tumors,” Cancer Res. 61:3388-93 (2001).
Heiser, A. et al., “Induction of polyclonal prostate cancer-specific CTL using dendritic cells transfected with amplified tumor RNA,” J. Immunol. 166:2953-60 (2001).
Hernåndez, J. et al., “Identification of a human telomerase reverse transcriptase peptide of low affinity for HLA A2.1 that induces cytotoxic T lymphocytes and mediates lysis of tumor cells,” Proc. Natl. Acad. Sci. USA 99(19):12275-80 (2002).
Huston, J. et al., “Protein engineering of antibody binding sites: Recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli,” Proc. Natl. Acad. Sci. USA 85:5879-83 (1988).
Johnstone, A. & Thorpe, R., Immunochemistry in Practice, 2nd Ed., Blackwell Scientific Publications, Oxford, pp. 30, 49-50 (1987).
Lai, C. et al., “RNA binding domain of telomerase reverse transcriptase,” Mol. Cell. Biol. 21:990-1000 (2001).
Lanfranchi, G. et al., “Identification of 4370 epxressed sequence tags from a 3′-end-specific cDNA library of human skeletal muscle by DNA sequencing and filter hybridization,” Genome Res. 6:35-42 (1996).
Langford, L. et al., “Telomerase activity in ordinary meningiomas predicts poor outcome,” Hum. Pathol. 28(4):416-20 (1997).
Leem et al., “The human telomerase gene: complete genomic sequence and analysis of tandem repeat polymorphisms in intronic regions,” Oncogene 21:769-77 (2002).
Li, H. et al., “Protein phosphatase 2A inhibits nuclear telomerase activity in human breast cancer cells,” J. Biol. Chem. 272:16729-32 (1997).
Martin-Rivera, L. et al., “Expression of mouse telomerase catalytic subunitin embryos and adult tissues,” Proc. Natl. Acad. Sci. USA 95:10471-6 (Sep. 1998).
Meyers, R., Ed., Molecular Biology and Biotechnology, A Comprehensive Desk Reference, Wiley-VCH, New York, p. 187 (1995).
Minev, B. et al., “Cytotoxic T cell immunity against telomerase reverse transcriptase in humans,” Proc. Natl. Acad. Sci. USA 97(9):4796-801 (2000).
Morin, G., “The implications of telomerase biochemistry for human disease,” Eur. J. Cancer 33(5):750-60 (1997).
Nair, S. et al., “Antigen-presenting cells pulsed with unfractionated tumor-derived peptides are potent tumor vaccines,” Eur. J. Immunol. 27:589-97 (1997).
Nair, S. et al., “Induction of cytotoxic t cell responses and tumor immunity against unrelated tumors using telomerase reverse transcriptase RNA transfected dendritic cells,” Nature Med. 6(8):1011-7 (2000).
NGO et al., The Protein Folding Problem and Tertiary Structure Predictor, Mertz et al., Eds., pp. 433, 492-495 (1994).
Ohyashiki, J. et al., “Quantitative relationship between functionally active telomerase and major telomerase components (hTERT and hTR) in acute leukaemia cells,” Brit. J. Cancer 92:1942-7 (2005).
Parker, K. et al., “Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains,” J. Immunol. 152(1):163-75 (1994). 1 page Abstract only.
Perez, H. et al., “Human formyl peptide receptor ligand binding domain(s). Studies using an improved mutagenesis/expression vector reveal a novel mechanism for the regulation of receptor occupancy,” J. Biol. Chem. 269(36):22485-7 (1994).
Ping, L. et al., “Dramatic increase of telomerase activity during dendritic cell differentiation and maturation,” J. Leukoc. Biol. 74:270-6 (2003).
Skolnick, J. & Fetrow, J., “From genes to protein structure and function: Novel applications of computational approaches in the genomic area,” Tibtech 18:34-9 (2000).
Solheim, J., “Class I MHC molecules: Assembly and antigen presentation,” Immunol. Rev. 172:11-19 (1999).
Stratagene Catalog, p. 39 (1988).
Su, Z. et al., “Immunological and clinical responses in metastatic renal cancer patients vaccinated with tumor RNA-transfered dendritic cells,” Cancer Res. 63:2127-33 (2003).
Vonderheide, R. et al., “Vaccination of cancer patients against telomerase induces functional antitumor CD8+ T lymphocytes,” Clin. Cancer Res. 10:828-39 (2004).
Xia, J. et al., “Identification of functionally important domains in the N-terminal region of telomerase reverse transcriptase,” Mol. Cell. Biol. 20:5196-207 (2000).
Zakharova, O. et al., “Structural constraints in the HIV-1 reverse transcriptase-primer/template complex for the initiation of DNA synthesis from primer tRNALys3,” Biochem. 37:13343-8 (1998).

Canella, Karen A.

Reddig, Peter J.

Townsend and Townsend and Crew LLP

This application is a continuation-in-part of U.S. application Ser. No. 09/843,676, filed Apr. 26, 2001, and a continuation-in part of U.S. application Ser. No. 10/044,692, filed Jan. 11, 2002, and a continuation of U.S. patent application Ser. No. 09/432,503, filed Nov. 2, 1999, which is a continuation of U.S. patent application Ser. No. 08/974,549 filed Nov. 19, 1997, U.S. Pat. No. 6,166,178, which is a continuation-in-part application of U.S. patent application Ser. No. 08/915,503, filed Aug. 14, 1997, abandoned, and a continuation-in-part application of U.S. patent application Ser. No. 08/912,951, filed Aug. 14, 1997, U.S. Pat. No. 6,475,789 and a continuation-in-part of application of U.S. patent application Ser. No. 08/911,312, filed Aug. 14, 1997, abandoned, all three of which are continuations-in-part of U.S. patent application Ser. No. 08/854,050, filed May 9, 1997, U.S. Pat. No. 6,261,836, which is a continuation-in-part of U.S. patent application Ser. No. 08/851,843, filed May 6, 1997, U.S. Pat. No. 6,093,809, which is a continuation-in-part of U.S. patent application Ser. No. 08/846,017, filed Apr. 25, 1997, abandoned, which is a continuation-in-part of U.S. application Ser. No. 08/844,419 filed Apr. 18, 1997, abandoned. This application also claims priority to Patent Convention Treaty Patent Application Serial No.: PCT/US97/17885 (published on Apr. 9, 1998 as WO 98/14593) and to Patent Convention Treaty Patent Application Serial No.: PCT/US97/17618 (published on Apr. 9, 1998 as WO 98/14592), both designating the U.S. and filed in the U.S. Receiving Office on Oct. 1, 1997. Each of the aforementioned applications is explicitly incorporated herein by reference in its entirety and for all purposes. This application also incorporates by reference copending U.S. patent application Ser. No. 08/974,584, filed Nov. 19, 1997, in its entirety and for all purposes.

What is claimed is:

1. A method of identifying a biological sample as containing human telomerase reverse transcriptase (hTRT) protein (SEQ ID NO:2), comprising: a) combining the biological sample with a monoclonal or recombinant antibody or antigen binding fragment thereof that specifically binds hTRT protein (SEQ ID NO:2), under conditions where the antibody or fragment forms a complex with said hTRT protein; b) detecting complex formed between said antibody or antigen binding fragment thereof and hTRT protein (SEQ ID NO:2); and c) identifying the sample as containing hTRT protein (SEQ ID NO:2) if said complex is detected.

2. The method of claim 1, which is an enzyme-linked immunosorbant assay method.

3. The method of claim 1, which is a radioimmunoassay method.

4. The method of claim 1, wherein the detecting comprises fluorescent activated cell sorting.

5. The method of claim 1, which is a Western blotting method.

6. The method of 1 wherein said monoclonal or recombinant antibody or antigen binding fragment thereof binds in the motif regions (SEQ. ID NO: 13).

7. The method of claim 1, wherein the antibody is detectably labeled.

8. The method of claim 1, wherein the antibody fragment is selected from the group consisting of Fv, Fab, Fab′, Fabc and F(ab′)₂ fragments.

9. The method of claim 1, wherein the antibody or antigen binding fragment specifically binds to a sequence selected from SEQ ID NO: 232, SEQ ID NO:233, SEQ ID NO:234, or SEQ ID NO:235.

10. A method of analyzing a biological sample for hTRT protein (SEQ ID NO:2), comprising: a) combining the biological sample with an antibody or antigen binding fragment thereof that specifically binds telomerase reverse transcriptase (hTRT) protein (SEQ ID NO:2), under conditions where the antibody or fragment forms a complex with said hTRT protein; b) detecting complex formed between said antibody or antigen binding fragment thereof and hTRT protein (SEQ ID NO:2); and c) identifying the sample as containing hTRT protein (SEQ ID NO:2) if said complex is detected.

11. The method of claim 10, which is an enzyme-linked immunosorbant assay method.

12. The method of claim 10, which is a radioimmunoassay method.

13. The method of claim 10, wherein the detecting comprises fluorescent activated cell sorting.

14. The method of claim 10, which is a Western blotting method.

15. The method of claim 10 wherein the hTRT protein is a naturally occurring gene product.

16. The method of claim 10, wherein the biological sample is a cell fraction.

17. The method of claim 10, wherein a monoclonal antibody is used.

18. The method of claim 10 wherein an antibody fragment selected from the group consisting of Fv, Fab, Fab′, Fabc and F(ab′)₂ fragments is used.

19. The method of claim 10, wherein the antibody is detectably labeled.

20. The method of claim 10, wherein the biological sample is a tissue biopsy or blood cell.

This invention was made with Government support under Grant No. GM28039, awarded by the National Institutes of Health. The Government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention is related to novel nucleic acids encoding the catalytic subunit of telomerase and related polypeptides. In particular, the present invention is directed to the catalytic subunit of human telomerase. The invention provides methods and compositions relating to medicine, molecular biology, chemistry, pharmacology, and medical diagnostic and prognostic technology.

BACKGROUND OF THE INVENTION

The following discussion is intended to introduce the field of the present invention to the reader. The citation of various references in this section is not to be construed as an admission of prior invention.

It has long been recognized that complete replication of the ends of eukaryotic chromosomes requires specialized cell components (Watson, 1972 , Nature New Biol., 239:197; Olovnikov, 1973, J. Theor. Biol., 41:181). Replication of a linear DNA strand by conventional DNA polymerases requires an RNA primer, and can proceed only 5′ to 3′. When the RNA bound at the extreme 5′ ends of eukaryotic chromosomal DNA strands is removed, a gap is introduced, leading to a progressive shortening of daughter strands with each round of replication. This shortening of telomeres, the protein-DNA structures physically located on the ends of chromosomes, is thought to account for the phenomenon of cellular senescence or aging (see, e.g., Goldstein, 1990, Science 249:1129; Martin et al., 1979, Lab. Invest. 23:86; Goldstein et al., 1969, Proc. Natl. Acad. Sci. USA 64:155; and Schneider and Mitsui, 1976, Proc. Natl. Acad. Sci. USA, 73:3584) of normal human somatic cells in vitro and in vivo.

The length and integrity of telomeres is thus related to entry of a cell into a senescent stage (i.e., loss of proliferative capacity). Moreover, the ability of a cell to maintain (or increase) telomere length may allow a cell to escape senescence, i.e., to become immortal.

The structure of telomeres and telomeric DNA has been investigated in numerous systems (see, e.g, Harley and Villeponteau, 1995, Curr. Opin. Genet. Dev. 5:249). In most organisms, telomeric DNA consists of a tandem array of very simple sequences; in humans and other vertebrates telomeric DNA consists of hundreds to thousands of tandem repeats of the sequence TTAGGG. Methods for determining and modulating telomere length in cells are described in PCT Publications WO 93/23572 and WO 96/41016.

The maintenance of telomeres is a function of a telomere-specific DNA polymerase known as telomerase. Telomerase is a ribonucleoprotein (RNP) that uses a portion of its RNA moiety as a template for telomere repeat DNA synthesis (Morin, 1997, Eur. J. Cancer 33:750; Yu et al., 1990 , Nature 344:126; Singer and Gottschling, 1994, Science 266:404; Autexier and Greider, 1994, Genes Develop., 8:563; Gilley et al., 1995, Genes Develop., 9:2214; McEachern and Blackburn, 1995 , Nature 367:403; Blackburn, 1992, Ann. Rev. Biochem., 61:113; Greider, 1996, Ann. Rev. Biochem., 65:337). The RNA components of human and other telomerases have been cloned and characterized (see, PCT Publication WO 96/01835 and Feng et al., 1995, Science 269:1236). However, the characterization of the protein components of telomerase has been difficult. In part, this is because it has proved difficult to purify the telomerase RNP, which is present in extremely low levels in cells in which it is expressed. For example, it has been estimated that human cells known to express high levels of telomerase activity may have only about one hundred molecules of the enzyme per cell.

Consistent with the relationship of telomeres and telomerase to the proliferative capacity of a cell (i.e., the ability of the cell to divide indefinitely), telomerase activity is detected in immortal cell lines and an extraordinarily diverse set of tumor tissues, but is not detected (i.e., was absent or below the assay threshold) in normal somatic cell cultures or normal tissues adjacent to a tumor (see, U.S. Pat. Nos. 5,629,154; 5,489,508; 5,648,215; and 5,639,613; see also, Morin, 1989 , Cell 59: 521; Shay and Bacchetti 1997, Eur. J. Cancer 33:787; Kim et al., 1994, Science 266:2011; Counter et al., 1992 , EMBO J. 11: 1921; Counter et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91, 2900; Counter et al., 1994, J. Virol. 68:3410). Moreover, a correlation between the level of telomerase activity in a tumor and the likely clinical outcome of the patient has been reported (e.g., U.S. Pat. No. 5,639,613, supra; Langford et al., 1997, Hum. Pathol. 28:416). Telomerase activity has also been detected in human germ cells, proliferating stem or progenitor cells, and activated lymphocytes. In somatic stem or progenitor cells, and in activated lymphocytes, telomerase activity is typically either very low or only transiently expressed (see, Chiu et al., 1996 , Stem Cells 14:239; Bodnar et al., 1996, Exp. Cell Res. 228:58; Taylor et al., 1996, J. Invest. Dermatology 106: 759).

Human telomerase is an ideal target for diagnosing and treating human diseases relating to cellular proliferation and senescence, such as cancer. Methods for diagnosing and treating cancer and other telomerase-related diseases in humans are described in U.S. Pat. Nos. 5,489,508, 5,639,613, and 5,645,986. Methods for predicting tumor progression by monitoring telomerase are described in U.S. Pat. No. 5,639,613. The discovery and characterization of the catalytic protein subunit of human telomerase would provide additional useful assays for telomerase and for disease diagnosis and therapy. Moreover, cloning and determination of the primary sequence of the catalytic protein subunit would allow more effective therapies for human cancers and other diseases related to cell proliferative capacity and senescence.

BRIEF SUMMARY OF THE INVENTION

The present invention provides an isolated, substantially pure, or recombinant protein preparation of a telomerase reverse transcriptase protein, or a variant thereof, or a fragment thereof. In one embodiment the protein is characterized as having a defined motif that has an amino acid sequence:

Trp-R ₁ -X ₇ -R ₁ -R ₁ -R ₂ -X-Phe-Phe-Tyr-X-Thr-Glu-X ₈₋₉ -R ₃ -R ₃ -Arg-R ₄ -X ₂ -Trp (SEQ ID NOS:11 and 12)

where X is any amino acid and a subscript refers to the number of consecutive residues, R ₁ is leucine or isoleucine, R ₂ is glutamine or arginine, R ₃ is phenylalanine or tyrosine, and R ₄ is lysine or histidine. In one embodiment the protein has a sequence of human TRT. In other embodiments, the invention relates to peptides and polypeptides sharing substantial sequence identity with a subsequence of such proteins.

In a related embodiment the invention provides an isolated, substantially pure or recombinant nucleic acid that encodes a telomerase reverse transcriptase protein. In one embodiment the nucleic acid encodes a protein comprising an amino acid sequence (SEQ ID NOS:11 and 12):

Trp-R ₁ -X ₇ -R ₁ -R ₁ -R ₂ -X-Phe-Phe-Tyr-X-Thr-Glu-X ₈₋₉ -R ₃ -R ₃ -Arg-R ₄ -X ₂ -Trp.

In another embodiment, the nucleic acid has a sequence that encodes the human TRT protein. In other embodiments, the invention relates to oligonucleotides and polynucleotides sharing substantial sequence identity or complementarity with a subsequence of such nucleic acids.

In one embodiment, the invention relates to human telomerase reverse transcriptase (hTRT) protein. Thus, in one embodiment, the invention provides an isolated, substantially pure, or recombinant protein preparation of an hTRT protein, or a variant thereof, or a fragment thereof. In one embodiment, the protein is characterized by having an amino acid sequence with at least about 75% or at least about 80% sequence identity to the hTRT protein of FIG. 17 (SEQ ID NO:2), or a variant thereof, or a fragment thereof. In a related aspect, the hTRT protein has the sequence of SEQ ID NO:2. In some embodiments, the protein has one or more telomerase activities, such as catalytic activity. In one embodiment, the hTRT protein fragment has at least 6 amino acid residues. In other embodiments, the hTRT protein fragment has at least 8, at least about 10, at least about 12, at least about 15 or at least about 20 contiguous amino acid residues of a naturally occurring hTRT polypeptide. In still other embodiments, the hTRT protein fragment has at least about 50 or at least about 100 amino acid residues.

The invention also provides a composition comprising an hTRT protein and an RNA. The RNA may be a telomerase RNA, such as a human telomerase RNA. In one embodiment, the hTRT protein and the human telomerase RNA (hTR) form a ribonucleoprotein complex with a telomerase activity.

In one embodiment, the invention provides isolated human telomerase comprising hTRT protein, such as a substantially pure human telomerase comprising hTRT protein and comprising hTR. In one embodiment, the telomerase is at least about 95% pure. The telomerase may be isolated from a cell, such as a recombinant host cell in or a cell that expresses telomerase activity.

In another aspect, the invention provides an isolated, synthetic, substantially pure, or recombinant polynucleotide comprising a nucleic acid sequence that encodes an hTRT protein. In one embodiment, the polynucleotide has a nucleotide sequence encoding an hTRT protein that has an amino acid sequence as set forth in FIG. 17 (SEQ ID NO:2) or a sequence that comprises one or more conservative amino acid (or codon) substitutions or one or more activity-altering amino acid (or codon) substitutions in said amino acid sequence. In a related aspect, the polynucleotide hybridizes under stringent conditions to a polynucleotide having the sequence as set forth in FIG. 16 (SEQ ID NO:1). In another related aspect, the nucleotide sequence of the polynucleotide has a smallest sum probability of less than about 0.5 when compared to a nucleotide sequence as set forth in FIG. 16 (SEQ ID NO:1) using BLAST algorithm with default parameters.

In another aspect, the invention provides a polynucleotide having a promoter sequence operably linked to the sequence encoding the hTRT protein. The promoter may be a promoter other than the naturally occurring hTRT promoter. In a related aspect, the invention provides an expression vector comprising the promoter of the hTRT.

The invention also provides an isolated, synthetic, substantially pure, or recombinant polynucleotide that is at least ten nucleotides in length and comprises a contiguous sequence of at least ten nucleotides that is identical or exactly complementary to a contiguous sequence in a naturally occurring hTRT gene or hTRT mRNA. In some embodiments the polynucleotide is an RNA, a DNA, or contains one or more non-naturally occurring, synthetic nucleotides. In one aspect, the polynucleotide is identical or exactly complementary to the contiguous sequence of at least ten contiguous nucleotides in a naturally occurring hTRT gene or hTRT mRNA. For example, the polynucleotide may be an antisense polynucleotide. In one embodiment, the antisense polynucleotide comprises at least about 20 nucleotides.

The invention further provides a method of preparing recombinant telomerase by contacting a recombinant hTRT protein with a telomerase RNA component under conditions such that said recombinant protein and said telomerase RNA component associate to form a telomerase enzyme capable of catalyzing the addition of nucleotides to a telomerase substrate. In one embodiment, the hTRT protein has a sequence as set forth in FIG. 17 (SEQ ID NO:2). The hTRT protein may be produced in an in vitro expression system and mixed with a telomerase RNA or, in another embodiment, the telomerase RNA can be co-expressed in the in vitro expression system. In one embodiment the telomerase RNA is hTR. In an alternative embodiment, the contacting occurs in a cell, such as a human cell. In one embodiment, the cell does not have telomerase activity prior to the contacting of the hTRT and the RNA, or the introduction, such as by transfection, of an hTRT polynucleotide.

In one embodiment, the telomerase RNA is expressed naturally by said cell.

The invention also provides a cell, such as a human, mouse, or yeast cell, containing the recombinant polynucleotides of the invention such as a polynucleotide with an hTRT protein coding sequence operably linked a promoter. In particular aspects, the cell is a vertebrate cell, such as a cell from a mammal, for example a human, and has an increased proliferative capacity relative to a cell that is otherwise identical but does not comprise the recombinant polynucleotide or has an increased telomerase activity level relative to a cell that is otherwise identical but does not comprise the recombinant polynucleotide. In some embodiments the cell is immortal.

In related embodiments, the invention provides organisms and cells comprising a polynucleotide encoding a human telomerase reverse transcriptase polypeptide, such as a transgenic non-human organism such as a yeast, plant, bacterium, or a non-human animal, for example, a mouse. The invention also provides for transgenic animals and cells from which an hTRT gene has been deleted (knocked-out) or mutated such that the gene does not express a naturally occurring hTRT gene product. Thus, in alternative embodiments, the transgenic non-human animal has a mutated telomerase gene, is an animal deficient in a telomerase activity, is an animal whose TRT deficiency is a result of a mutated gene encoding a TRT having a reduced level of a telomerase activity compared to a wild-type TRT and is an animal having a mutated TRT gene with one or more mutations, including missense mutations nonsense mutations, insertions, or deletions.

The invention also provides an isolated or recombinant antibody, or fragment thereof, that specifically binds to an hTRT protein. In one embodiment, the antibody binds with an affinity of at least about 10 ⁸ M ⁻¹ . The antibody may be monoclonal or may be a polyclonal composition, such as a polyclonal antisera. In a related aspect, the invention provides a cell capable of secreting the antibody, such as a hybridoma.

The invention also provides a method for determining whether a compound or treatment is a modulator of a telomerase reverse transcriptase activity or hTRT expression. The method involves detecting or monitoring a change in activity or expression in a cell, animal or composition comprising an hTRT protein or polynucleotide following administration of the compound or treatment. In one embodiment, the method includes the steps of providing a TRT composition, contacting the TRT with the test compound and measuring the activity of the TRT, where a change in TRT activity in the presence of the test compound is an indicator that the test compound modulates TRT activity. In certain embodiments, the composition is a cell, an organism, a transgenic organism or an in vitro system, such as an expression system, which contains a recombinant polynucleotide encoding an hTRT polypeptide. Thus, the hTRT of the method may be a product of in vitro expression. In various embodiments the detection of telomerase activity or expression may be by detecting a change in abundance of an hTRT gene product, monitoring incorporation of a nucleotide label into a substrate for telomerase, monitoring hybridization of a probe to an extended telomerase substrate, monitoring amplification of an extended telomerase substrate, monitoring telomere length of a cell exposed to the test compound, monitoring the loss of the ability of the telomerase to bind to a chromosome, or measuring the accumulation or loss of telomere structure.

In one aspect, the invention provides a method of detecting an hTRT gene product in a biological sample by contacting the biological sample with a probe that specifically binds the gene product, wherein the probe and the gene product form a complex, and detecting the complex, where the presence of the complex is correlated with the presence of the hTRT gene product in the biological sample. The gene product may be RNA, DNA or a polypeptide. Examples of probes that may be used for detection include, but are not limited to, nucleic acids and antibodies.

In one embodiment, the gene product is a nucleic acid which is detected by amplifying the gene and detecting the amplification product, where the presence of the complex or amplification product is correlated with the presence of the hTRT gene product in the biological sample.

In one embodiment, the biological sample is from a patient, such as a human patient. In another embodiment the biological sample includes at least one cell from an in vitro cell culture, such as a human cell culture.

The invention further provides a method of detecting the presence of at least one immortal or telomerase positive human cell in a biological sample comprising human cells by obtaining the biological sample comprising human cells; and detecting the presence in the sample of a cell having a high level of an hTRT gene product, where the presence of a cell having a high level of the hTRT gene product is correlated with the presence of immortal or telomerase positive cells in the biological sample.

The invention also provides a method for diagnosing a telomerase-related condition in a patient by obtaining a cell or tissue sample from the patient, determining the amount of an hTRT gene product in the cell or tissue; and comparing the amount of hTRT gene product in the cell or tissue with the amount in a healthy cell or tissue of the same type, where a different amount of hTRT gene product in the sample from the patient and the healthy cell or tissue is diagnostic of a telomerase-related condition. In one embodiment the telomerase-related condition is cancer and a greater amount of hTRT gene product is detected in the sample.

The invention further provides a method of diagnosing cancer in a patient by obtaining a biological sample from the patient, and detecting a hTRT gene product in the patient sample, where the detection of the hTRT gene product in the sample is correlated with a diagnosis of cancer.

The invention further provides a method of diagnosing cancer in a patient by obtaining a patient sample, determining the amount of hTRT gene product in the patient sample; and comparing the amount of hTRT gene product with a normal or control value, where an amount of the hTRT gene product in the patient that is greater than the normal or control value is diagnostic of cancer.

The invention also provides a method of diagnosing cancer in a patient, by obtaining a patient sample containing at least one cell; determining the amount of an hTRT gene product in a cell in the sample; and comparing the amount of hTRT gene product in the cell with a normal value for the cell, wherein an amount of the hTRT gene product greater than the normal value is diagnostic of cancer. In one embodiment, the sample is believed to contain at least one malignant cell.

The invention also provides a method for a prognosing a cancer patient by determining the amount of hTRT gene product in a cancer cell obtained from the patient; and comparing the amount of hTRT in the cancer cell with a prognostic value of hTRT consistent with a prognosis for the cancer; where an amount of hTRT in the sample that is at the prognostic value provides the particular prognosis.

The invention also provides a method for monitoring the ability of an anticancer treatment to reduce the proliferative capacity of cancer cells in a patient, by making a first measurement of the amount of an hTRT gene product in at least one cancer cell from the patient; making a second measurement of the level of the hTRT gene product in at least one cancer cell from the patient, wherein the anticancer treatment is administered to the patient before the second measurement; and comparing the first and second measurements, where a lower level of the hTRT gene product in the second measurement is correlated with the ability of an anticancer treatment to reduce the proliferative capacity of cancer cells in the patient.

The invention also provides kits for the detection of an hTRT gene or gene product. In one embodiment, the kit includes a container including a molecule selected from an hTRT nucleic acid or subsequence thereof, an hTRT polypeptide or subsequence thereof, and an anti-hTRT antibody.

The invention also provides methods of treating human diseases. In one embodiment, the invention provides a method for increasing the proliferative capacity of a vertebrate cell, such as a mammalian cell, by introducing a recombinant polynucleotide into the cell, wherein said polynucleotide comprises a sequence encoding an hTRT polypeptide. In one embodiment, the hTRT polypeptide has a sequence as shown in FIG. 17. In one embodiment, the sequence is operably linked to a promoter. In one embodiment, the hTRT has telomerase catalytic activity. In one embodiment, the cell is human, such as a cell in a human patient. In an alternative embodiment, the cell is cultured in vitro. In a related embodiment, the cell is introduced into a human patient.

The invention further provides a method for treating a human disease by introducing recombinant hTRT polynucleotide into at least one cell in a patient. In one embodiment, a gene therapy vector is used. In a related embodiment, the method further consists of introducing into the cell a polynucleotide comprising a sequence encoding hTR, for example, an hTR polynucleotide operably linked to a promoter.

The invention also provides a method for increasing the proliferative capacity of a vertebrate cell, said method comprising introducing into the cell an effective amount of hTRT polypeptide. In one embodiment the hTRT polypeptide has telomerase catalytic activity. The invention further provides cells and cell progeny with increased proliferative capacity.

The invention also provides a method for treating a condition associated with an elevated level of telomerase activity within a cell, comprising introducing into said cell a therapeutically effective amount of an inhibitor of said telomerase activity, wherein said inhibitor is an hTRT polypeptide or an hTRT polynucleotide. In one embodiment, the inhibitor is a polypeptide or polynucleotide comprising, e.g., at least a subsequence of a sequence shown in FIG. 16, 17 , or 20 . In additional embodiments, the polypeptide or polynucleotide inhibits a TRT activity, such as binding of endogenous TRT to telomerase RNA.

The invention also provides a vaccine comprising an hTRT polypeptide and an adjuvant. The invention also provides pharmacological compositions containing a pharmaceutically acceptable carrier and a molecule selected from: an hTRT polypeptide, a polynucleotide encoding an hTRT polypeptide, and an hTRT nucleic acid or subsequence thereof.

DESCRIPTION OF THE FIGURES

FIG. 1 shows highly conserved residues in TRT motifs from human (SEQ ID NO:13), S. pombe (tez1) (SEQ ID NO:14), S. cerevisiae (EST2) (SEQ ID NO:15) and Euplotes aediculatus (p123) (SEQ ID NO:16). Identical amino acids are indicated with an asterisk (*) [raised slightly], while the similar amino acid residues are indicated by a dot (•). Motif “0” in the figure is also called Motif T; Motif “3” is also called Motif A.

FIG. 2 shows the location of telomerase-specific and RT-specific sequence motifs of telomerase proteins and other reverse transcriptases. Locations of telomerase-specific motif T and conserved RT motifs 1, 2 and A-E are indicated by boxes. The open rectangle labeled HIV-1 RT delineates the portion of this protein shown in FIG. 3.

FIG. 3 shows the crystal structure of the p66 subunit of HIV-1 reverse transcriptase (Brookhaven code 1HNV). The view is from the back of the right hand to enable all motifs to be shown.

FIG. 4 shows multiple sequence alignment of telomerase RTs (Sp_Trt1p, S. pombe TRT (SEQ ID NOS:24-29) [also referred to herein as “tez1p”]; hTRT, human TRT (SEQ ID NOS:30-35); Ea _— 123, Euplotes p123 (SEQ ID NOS:36-41); Sc_Est2p, S. cerevisiae Est2p) (SEQ ID NOS:42-48) and members of other RT families (Sc_a1, cytochrome oxidase group II intron 1-encoded protein from S. cerevisiae mitochondria (SEQ ID NOS:51-56), Dm_TART, reverse transcriptase from Drosophila melanogaster TART non-LTR retrotransposable element) (SEQ ID NOS:57-63; HIV-1, human immunodeficiency virus reverse transcriptase (SEQ ID NOS:64-68)). TRT con (SEQ ID NOS:17-23) and RT con (SEQ ID NOS:49 and 50) represent consensus sequences for telomerase RTs and non-telomerase RTs. Amino acids are designated with an h, hydrophobic; p, polar; c, charged. Triangles show residues that are conserved among telomerase proteins but different in other RTs. The solid line below motif E highlights the primer grip region.

FIG. 5 shows expression of hTRT RNA in telomerase-negative mortal cell strains and telomerase-positive immortal cell lines as described in Example 2.

FIG. 6 shows a possible phylogenetic tree of telomerases and retroelements rooted with RNA-dependent RNA polymerases.

FIG. 7 shows a restriction map of lambda clone Gø5.

FIG. 8 shows a map of chromosome 5p with the location of the STS marker D5S678 (located near the hTRT gene) indicated.

FIG. 9 shows the construction of a hTRT promoter-reporter plasmid.

FIGS. 10A and 10B show coexpression in vitro of hTRT and hTR to produce catalytically active human telomerase.

FIG. 11, in two pages, shows an alignment of sequences from four TRT proteins from human (hTRT; SEQ ID NOS:72-79), S. pombe Trt1 (spTRT; SEQ ID NOS:80-87), Euplotes p123 (Ea _— 123; SEQ ID NOS:88-95), and S. cerevisiae EST2p TRT (Sc_Est2; SEQ ID NOS:96-104) and identifies motifs of interest. TRT con (SEQ ID NOS:69, 21, 70 and 71) shows a TRT consensus sequence. RT con (SEQ ID NOS:49 and 50) shows consensus residues for other reverse transcriptases. Consensus residues in upper case indicate absolute conservation in TRT proteins.

FIG. 12 shows a Topoisomerase II cleavage site (SEQ ID NO: 108) and NFkB binding site motifs (NFkB_CS1=SEQ ID NO:105; NFkB-MH-I.2=SEQ ID NO:106; NFkB_CS2=SEQ ID NO:107) in an hTRT intron, with the sequence shown corresponding to SEQ ID NO:7.

FIGS. 13A and 13B show the sequence of the DNA encoding the Euplotes 123 kDa telomerase protein subunit ( Euplotes TRT; SEQ ID NO:109).

FIG. 14 shows the amino acid sequence of the Euplotes 123 kDa telomerase protein subunit ( Euplotes TRT protein; SEQ ID NO:110).

FIGS. 15A-15F show the DNA (SEQ ID NO:111) and amino acid (SEQ ID NO:112) sequences of the S. pombe telomerase catalytic subunit ( S. pombe TRT).

FIG. 16, in two pages, shows the hTRT cDNA sequence, with the sequence shown corresponding to SEQ ID NO:1.

FIG. 17 shows the hTRT protein encoded by the cDNA of FIG. 16. The protein sequence shown corresponds to SEQ ID NO:2.

FIG. 18 shows the sequence of clone 712562, with the sequence shown corresponding to SEQ ID NO:3.

FIG. 19 shows a 259 residue protein encoded by clone 712562, with the sequence shown corresponding to SEQ ID NO:10.

FIGS. 20A-20E show the sequence of a nucleic acid with an open reading frame encoding a Δ182 variant polypeptide, with the sequence shown corresponding to SEQ ID NO:4. This Figure also shows the amino acid sequence of this Δ182 variant polypeptide, with the amino acid sequence shown corresponding to SEQ ID NO:5.

FIGS. 21A-21E show sequence from an hTRT genomic clone, with the sequence shown corresponding to SEQ ID NO:6. Consensus motifs and elements are indicated, including sequences characteristic of a topoisomerase II cleavage site, NFkB binding sites, an Alu sequence and other sequence elements.

FIG. 22 shows the effect of mutation of the TRT gene in yeast, as described in Example 1.

FIG. 23 shows the sequence of EST AA281296, corresponding to SEQ ID NO:8.

FIG. 24 shows the sequence of the 182 basepairs deleted in clone 712562, with the sequence shown corresponding to SEQ ID NO:9.

FIG. 25 shows the results of an assay for telomerase activity from BJ cells transfected with an expression vector encoding an hTRT protein (PGRN133) or a control plasmid (pBBS212) as described in Example 13.

FIG. 26 is a schematic diagram of the affinity purification of telomerase showing the binding and displacement elution steps.

FIG. 27 is a photograph of a Northern blot of telomerase preparations obtained during a purification protocol, as described in Example 1. Lane 1 contained 1.5 fmol telomerase RNA, lane 2 contained 4.6 fmol telomerase RNA, lane 3 contained 14 fmol telomerase RNA, lane 4 contained 41 fmol telomerase RNA, lane 5 contained nuclear extract (42 fmol telomerase), lane 6 contained Affi-Gel-heparin-purified telomerase (47 fmol telomerase), lane 7 contained affinity-purified telomerase (68 fmol), and lane 8 contained glycerol gradient-purified telomerase (35 fmol).

FIG. 28 shows telomerase activity through a purification protocol.

FIG. 29 is a photograph of a SDS-PAGE gel, showing the presence of an approximately 123 kDa polypeptide and an approximately 43 kDa doublet from Euplotes aediculatus.

FIG. 30 is a graph showing the sedimentation coefficient of Euplotes aediculatus telomerase.

FIG. 31 is a photograph of a polyacrylamide/urea gel with 36% formamide showing the substrate utilization of Euplotes telomerase.

FIG. 32 shows the putative alignments of telomerase RNA template (SEQ ID NO:113), and hairpin primers with telomerase RNA.

FIG. 33 is a photograph of lanes 25-30 of the gel shown in FIG. 31, shown at a lighter exposure level (G ₄ T ₄ G ₄ T ₄ =SEQ ID NO:114).

FIG. 34 shows the DNA sequence of the gene encoding the 43 kDa telomerase protein subunit from Euplotes (SEQ ID NO:115).

FIGS. 35A-35D show the DNA sequence (SEQ ID NO:115), as well as the amino acid sequences of all three open reading frames of the 43 kDa telomerase protein subunit from Euplotes (a=SEQ ID NOS:116-140; b=SEQ ID NOS:141-162; c=SEQ ID NOS:163-186).

FIGS. 36A and 36B show a sequence comparison between the 123 kDa telomerase protein subunit of Euplotes (SEQ ID NO:187) (upper sequence) and the 80 kDa polypeptide subunit of T. thermophila (SEQ ID NO:188) (lower sequence).

FIGS. 37A and 37B show a sequence comparison between the 123 kDa telomerase protein subunit of E. aediculatus (SEQ ID NO:189) (upper sequence) and the 95 kDa telomerase polypeptide of T. thermophila (SEQ ID NO:190) (lower sequence).

FIG. 38 shows the best-fit alignment between a portion of the “La-domain” of the 43 kDa telomerase protein subunit of E. aediculatus (SEQ ID NO:191) (upper sequence) and a portion of the 95 kDa polypeptide subunit of T. thermophila (SEQ ID NO:192) (lower sequence).

FIG. 39 shows the best-fit alignment between a portion of the “La-domain” of the 43 kDa telomerase protein subunit of E. aediculatus (SEQ ID NO:193) (upper sequence) and a portion of the 80 kDa polypeptide subunit of T. thermophila (SEQ ID NO:194) (lower sequence).

FIG. 40 shows the alignment and motifs of the polymerase domain of the 123 kDa telomerase protein subunit of E. aediculatus (SEQ ID NOS:38-41) and the polymerase domains of various reverse transcriptases including a cytochrome oxidase group II intron 1-encoded protein from S. cerevisiae mitochondria (al S.c. (group II)) (SEQ ID NOS:204, 205, 54, 206, and 56), Dong (LINE) (SEQ ID NOS:200-203), and yeast ESTp (L8543.12) (SEQ ID NOS:45, 46, 211 and 212), HIV-RT (SEQ ID NOS:207-210) and consensus (SEQ ID NOS:195-199).

FIG. 41 shows the alignment of a domain of the 43 kDa telomerase protein subunit (SEQ ID NO:213) with various La proteins (human La=SEQ ID NO:214; Xenopus LaA=SEQ ID NO:215; Drosophila La=SEQ ID NO:216; S.c. Lhplp=SEQ ID NO:217).

FIG. 42 shows the nucleotide sequence encoding the T. thermophila 80 kDa protein subunit.

FIG. 43 shows the amino acid sequence of the T. thermophila 80 kDa protein subunit (SEQ ID NO:219).

FIG. 44 shows the nucleotide sequence encoding the T. thermophila 95 kDa protein subunit (SEQ ID NO:220).

FIG. 45 shows the amino acid sequence of the T. thermophila 95 kDa protein subunit (SEQ ID NO:221).

FIG. 46 shows the amino acid sequence of L8543.12 (“Est2p”) (SEQ ID NO:222).

FIG. 47 shows the alignment of the amino acid sequence encoded by the Oxytricha PCR product (SEQ ID NO:223) with the Euplotes p123 sequence (SEQ ID NO:224).

FIG. 48 shows the DNA sequence of Est2 (SEQ ID NO:225).

FIG. 49 shows partial amino acid sequence from a cDNA clone encoding human telomerase peptide motifs (SEQ ID NO:13).

FIG. 50 shows partial DNA sequence of a cDNA clone encoding human telomerase peptide motifs (SEQ ID NO:8).

FIG. 51 shows the amino acid sequence of tez1, also called S. pombe trt (SEQ ID NO:112).

FIGS. 52A and 52B show the DNA sequence of tez1 (SEQ ID NO:111). Intronic and other non-coding regions are shown in lower case and exons (i.e., coding regions) are shown in upper case.

FIG. 53 shows the alignment of EST2p (SEQ ID NO:226), Euplotes (SEQ ID NO:227), and Tetrahymena SEQ ID NO:228) sequences, as well as consensus sequence (SEQ ID NOS:229-231).

FIG. 54 shows the sequences of peptides (SEQ ID NOS:232-237) useful for production of anti-hTRT antibodies.

FIGS. 55A and 55B present a schematic summary of the tez1 ⁺ sequencing experiments.

FIG. 56 shows two degenerate primers (SEQ ID NOS:238 and 241) used in PCR to identify the S. pombe homolog of the E. aediculatus p123 sequences (SEQ ID NOS:239 and 240).

FIG. 57 shows the four major bands produced in PCR using degenerate primers to identify the S. pombe homolog of the E. aediculatus p123 sequences (SEQ ID NOS:239 and 240).

FIGS. 58A and 58B show the alignment of the M2 PCR product (SEQ ID NO:243) with E. aediculatus p123 (SEQ ID NO:242), S. cerevisiae (SEQ ID NO:244), and Oxytricha (SEQ ID NO:223) telomerase protein sequences. Also shown are the actual genomic sequences (SEQ ID NOS:246 and 249) and the peptides encoded (SEQ ID NOS:245 and 250), degenerate primers Poly4 (SEQ ID NO:238) and Poly 1 (SEQ ID NO:244), and homologous regions of the M2 PCR product (SEQ ID NO:247) and its encoded peptide region (SEQ ID NO:248).

FIG. 59 is a schematic showing the 3′ RT PCR strategy for identifying the S. pombe homolog of the E. aediculatus p123.

FIG. 60 shows characteristics of the libraries used to screen for S. pombe telomerase protein sequences and shows the results of screening the libraries for S. pombe telomerase protein sequences.

FIG. 61 shows the positive results obtained with the HindIII-digested positive genomic clones containing S. pombe telomerase sequence.

FIG. 62 is a schematic showing the 5′ RT PCR strategy used to obtain a full length S. pombe TRT clone.

FIG. 63 shows the alignment of RT domains from telomerase catalytic subunits for S. pombe (S.p.) (SEQ ID NOS:251-255), S. cerevisiae (S.c.) (SEQ ID NOS:256-260) and E. aediculatus (E.a.) (SEQ ID NOS:261-265). Consensus sequences=SEQ ID NOS:49 and 50.

FIGS. 64A-64J show the alignment of the sequences from Euplotes (“Ea_p123”) (SEQ ID NO:110), S. cerevisiae (“Sc_Est2p”) (SEQ ID NO:222), and S. pombe (“SP_T1p1p”) (SEQ ID NO:112). In Panel A, the shaded areas indicate residues shared between two sequences. In Panel B, the shaded areas indicate residues shared between all three sequences.

FIG. 65 shows the disruption strategy used with the telomerase genes in S. pombe.

FIG. 66 shows the experimental results confirming disruption of tez1.

FIG. 67 shows the progressive shortening of telomeres in S. pombe due to tez1 disruption.

FIGS. 68A-68C show the DNA (SEQ ID NO:266) and amino acid (SEQ ID NO:267) of the ORF encoding an approximately 63 kDa telomerase protein encoded by the EcoRI-NotI insert of clone 712562.

FIG. 69 shows an alignment of reverse transcriptase motifs from various sources, E aediculatus p123 (SEQ ID NOS:268-273), S pombe tez1 (SEQ ID NOS:274-279), S. cerevisiae EST2 (SEQ ID NOS:280-285), and human Hs TCP1 (SEQ ID NOS:286-291), with various consensus residues and motif sequences (SEQ ID NOS:49 and 50) indicated.

FIG. 70 provides a restriction and function map of plasmid pGRN121.

FIGS. 71A and 71B show the results of preliminary nucleic acid sequencing analysis of a hTRT cDNA sequence (SEQ ID NO:292).

FIGS. 72A-72I show the preliminary nucleic acid sequence of hTRT (SEQ ID NO:292) and deduced ORF sequences in three reading frames (a=SEQ ID NOS:293-320; b=SEQ ID NOS:321-333; c=SEQ ID NOS:334-342).

FIG. 73 provides a restriction and function map of plasmid pGRN121.

FIGS. 74A-74F show refined nucleic acid sequence (SEQ ID NO:343) and deduced ORF sequences (SEQ ID NO:344) of hTRT.

FIG. 75 shows a restriction map of lambda clone 25-1.1.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

Telomerase is a ribonucleoprotein complex (RNP) comprising an RNA component and a catalytic protein component. The present invention relates to the cloning and characterization of the catalytic protein component of telomerase, hereinafter referred to as “TRT” (telomerase reverse transcriptase). TRT is so named because this protein acts as an RNA-dependent DNA polymerase (reverse transcriptase), using the telomerase RNA component (hereinafter, “TR”) to direct synthesis of telomere DNA repeat sequences. Moreover, TRT is evolutionarily related to other reverse transcriptases (see Example 12).

In one aspect, the present invention relates to the cloning and characterization of the catalytic protein component of human telomerase, hereinafter referred to as “hTRT.” Human TRT is of extraordinary interest and value because, as noted supra, telomerase activity in human (and other mammalian cells) correlates with cell proliferative capacity, cell immortality, and the development of a neoplastic phenotype. For example, telomerase activity, and, as demonstrated in Example 2, infra, levels of human TRT gene products and are elevated in immortal human cells (such as malignant tumor cells and immortal cell lines) relative to mortal cells (such as most human somatic cells).

The present invention further provides methods and compositions valuable for diagnosis, prognosis, and treatment of human diseases and disease conditions, as described in some detail infra. Also provided are methods and reagents useful for immortalizing cells (in vivo and ex vivo), producing transgenic animals with desirable characteristics, and numerous other uses, many of which are described infra. The invention also provides methods and reagents useful for preparing, cloning, or re-cloning TRT genes and proteins from ciliates, fungi, vertebrates, such as mammals, and other organisms.

As described in detail infra, TRT was initially characterized following purification of telomerase from the ciliate Euplotes aediculatus . Extensive purification of E. aediculatus telomerase, using RNA-affinity chromatography and other methods, yielded the protein Ap123”. Surprisingly, p123 is unrelated to proteins previously believed to constitute the protein subunits of the telomerase holoenzyme (i.e., the p80 and p95 proteins of Tetrahymena thermophila ). Analysis of the p123 DNA and protein sequences (Genbank Accession No. U95964; FIGS. 13 and 14) revealed reverse transcriptase (RT) motifs consistent with the role of p123 as the catalytic subunit of telomerase (see, e.g., FIGS. 1, 4 and 11 ). Moreover, p123 is related to a S. cerevisiae (yeast) protein, Est2p, which was known to play a role in maintenance of telomeres in S. cerevisiae (Genbank Accession No. S5396), but prior to the present invention was not recognized as encoding a telomerase catalytic subunit protein (see, e.g., Lendvay et al., 1996, Genetics, 144:1399).

In one aspect, the present invention provides reagents and methods for identifying and cloning novel TRTs using: nucleic acid probes and primers generated or derived from the TRT polynucleotides disclosed (e.g., for cloning TRT genes and cDNAs); antibodies that specifically recognize the motifs or motif sequences or other TRT epitopes (e.g., for expression cloning TRT genes or purification of TRT proteins); by screening computer databases; or other means. For example, as described in Example 1, PCR (polymerase chain reaction) amplification of S. pombe DNA was carried out with degenerate-sequence primers designed from the Euplotes p123 RT motifs B′ and C. Of four prominent products generated, one encoded a peptide sequence homologous to Euplotes p123 and S. cerevisiae Est2p. Using this PCR product as a probe, the complete sequence of the S. pombe TRT homologue was obtained by screening of S. pombe cDNA and genomic libraries and amplifying S. pombe RNA by reverse transcription and PCR(RT-PCR). The complete sequence of the S. pombe gene (“trt1”; GenBank Accession No. AF015783; FIG. 15) revealed that homology with p123 and Est2p was especially high in the reverse transcriptase motifs. S. pombe trt1 is also referred to as tez1.

Amplification using degenerate primers derived from the telomerase RT motifs was also used to obtain TRT gene sequences in Oxytricha trifallax and Tetrahymena thermophila , as described in Example 1.

The Euplotes p123, S. pombe trt1, and S. cerevisiae Est2p nucleic acid sequences of the invention were used in a search of a computerized database of human expressed sequence tags (ESTs) using the program BLAST (Altschul et al. al, 1990, J. Mol. Biol. 215:403). Searching this database with the Est2p sequence did not indicate a match, but searching with p123 and trt1 sequences identified a human EST (Genbank accession no. AA281296; see SEQ ID NO:8), as described in Example 1, putatively encoding a homologous protein. Complete sequencing of the cDNA clone containing the EST (hereinafter, “clone 712562”; see SEQ ID NO:3) showed that seven RT motifs were present. However, this clone did not encode a contiguous human TRT with all seven motifs, because motifs B′, C, D, and E were contained in a different open reading frame (ORF) than the more NH ₂ -terminal motifs. In addition, the distance between motifs A and B′ was substantially shorter than that of the three previously characterized TRTs. Clone 712562 was obtained from the I.M.A.G.E. Consortium; Lennon et al., 1996 , Genomics 33:151.

A cDNA clone, pGRN121, encoding a functional hTRT (see FIG. 16, SEQ ID NO:1) was isolated from a cDNA library derived from the human 293 cell line as described in Example 1. Comparing clone 712562 with pGRN121 showed that clone 712562 has a 182 base pair (see FIG. 24, SEQ ID NO:9) deletion between motifs A and B. The additional 182 base pairs present in pGRN121 place all of the TRT motifs in a single open reading frame, and increase the spacing between the motif A and motif B′ regions to a distance consistent with the other known TRTs. As is described infra in the Examples (e.g., Example 7), SEQ ID NO:1 encodes a catalytically active telomerase protein having the sequence of SEQ ID NO:2. The polypeptide of SEQ ID NO:2 has 1132 residues and a calculated molecular weight of about 127 kilodaltons (kD).

As is discussed infra, and described in Example 9, infra, TRT cDNAs possessing the 182 basepair deletion characteristic of the clone 712562 are detected following reverse transcription of RNA from telomerase-positive cells (e.g., testis and 293 cells). hTRT RNAs lacking this 182 base pair sequence are referred to generally as “Δ182 variants” and may represent one, two, or several species. Although the hTRT variants lacking the 182 basepair sequence found in the pGRN121 cDNA are unlikely to encode a fully active telomerase catalytic enzyme, they may play a role in telomerase regulation, as discussed infra, and/or have partial telomerase activity, such as telomere binding or hTR binding activity, as discussed infra.

Thus, in one aspect, the present invention provides an isolated polynucleotide with a sequence of a naturally occurring human TRT gene or mRNA including, but not limited to, a polynucleotide having the sequence as set forth in FIG. 16 (SEQ ID NO:1). In a related aspect, the invention provides a polynucleotide encoding an hTRT protein, fragment, variant or derivative. In another related aspect, the invention provides sense and antisense nucleic acids that bind to an hTRT gene or mRNA. The invention further provides hTRT proteins, whether synthesized or purified from natural sources, as well as antibodies and other agents that specifically bind an hTRT protein or a fragment thereof. The present invention also provides many novel methods, including methods that employ the aforementioned compositions, for example, by providing diagnostic and prognostic assays for human diseases, methods for developing therapeutics and methods of therapy, identification of telomerase-associated proteins, and methods for screening for agents capable of activating or inhibiting telomerase activity. Numerous other aspects and embodiments of the invention are provided infra.

One aspect of the invention is the use of a polynucleotide that is at least ten nucleotides to about 10 kb or more in length and comprises a contiguous sequence of at least ten nucleotides that is identical or exactly complementary to a contiguous sequence in a naturally occurring hTRT gene or hTRT mRNA in assaying or screening for an hTRT gene sequence or hTRT mRNA, or in preparing a recombinant host cell.

A further aspect of the invention is the use of an agent increasing expression of hTRT in the manufacture of a medicament for the treatment of a condition addressed by increasing proliferative capacity of a vertebrate cell, optionally the medicament being for inhibiting the effects of aging.

Yet a further aspect of the invention is the use of an inhibitor of telomerase activity in the manufacture of a medicament for the treatment of a condition associated with an elevated level of telomerase activity within a human cell.

The proteins, variants and fragments of the invention, and the encoding polynucleotides or fragments, are also each provided in a further aspect of this invention for use as a pharmaceutical.

The invention further includes the use of a protein, variant or fragment, or of a polynucleotide or fragment, in each case as defined herein, in the manufacture of a medicament, for example in the manufacture of a medicament for inhibiting an effect of aging or cancer.

Another aspect of the invention is a polynucleotide selected from:

(a) the DNA having a sequence as set forth in FIG. 16;

(b) a polynucleotide of at least 10 nucleotides which hybridizes to the foregoing DNA and which codes for an hTRT protein or variant or which hybridizes to a coding sequence for such a variant; and,

(c) DNA sequences which are degenerate as a result of the genetic code to the DNA sequences defined in (a) and (b) and which code for an hTRT polypeptide or variant.

In certain embodiments of the present invention, the hTRT polynucleotides are other than the 389 nucleotide polynucleotide of SEQ ID NO:8 and/or other than clone 712562, the plasmid containing an insert, the sequence of which insert is shown in FIG. 18 (SEQ ID NO:3).

The description below is organized by topic. Part II further describes amino acid motifs characteristic of TRT proteins, as well as TRT genes encoding proteins having such motifs. Parts III-VI describe, inter alia, nucleic acids, proteins, antibodies and purified compositions of the invention with particular focus on human TRT related compositions. Part VII describes, inter alia, methods and compositions of the invention useful for treatment of human disease. Part VI describes production and identification of immortalized human cell lines. Part IX describes, inter alia, uses of the nucleic acids, polynucleotides, and other compositions of the invention for diagnosis of human diseases. Part X describes, inter alia, methods and compositions of the invention useful for screening and identifying agents and treatments that modulate (e.g., inhibit or promote) telomerase activity or expression. Part XI describes, inter alia, transgenic animals (e.g., telomerase knockout animals and cells). Part XII is a glossary of terms used in Parts I-XI. Part XIII describes examples relating to specific embodiments of the invention. The organization of the description of the invention by topic and subtopic is to provide clarity, and not to be limiting in any way.

II. TRT Genes and Proteins

The present invention provides isolated and/or recombinant genes and proteins having a sequence of a telomerase catalytic subunit protein (i.e., telomerase reverse transcriptase), including, but not limited to, the naturally occurring forms of such genes and proteins in isolated or recombinant form. Typically, TRTs are large, basic, proteins having reverse transcriptase (RT) and telomerase-specific (T) amino acid motifs, as disclosed herein. Because these motifs are conserved across diverse organisms, TRT genes of numerous organisms may be obtained using the methods of the invention or identified using primers, nucleic acid probes, and antibodies of the invention, such as those specific for one or more of the motif sequences.

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