Human telomerase catalytic subunit variants

Details Human telomerase catalytic subunit variants Human telomerase catalytic subunit variants

1998-08-03

2002-01-08

Crouch, Deborah (Department: 1632)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

C435S069100, C435S070100, C435S320100, C435S325000, C435S440000, C435S455000, C514S04400A, C530S350000, C536S023100, C536S023500

Reexamination Certificate

active

06337200

ABSTRACT:

FIELD OF THE INVENTION
The present invention is related to the catalytic protein 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 should not 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 polymerase 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 of normal human somatic cells in vitro and in vivo. 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 telomeric DNA synthesis (Morin, 1997,
Eur. J. Cancer
33:750). The length and integrity of telomeres and the telomerase expression status of a cell is thus related to entry of a cell into a senescent stage (i.e., loss of proliferative capacity), or the ability of a cell to escape senescence, i.e., to become immortal.
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). Thus, human telomerase is an ideal target for diagnosing and treating human diseases relating to cellular proliferation and senescence, such as cancer, or for increasing the proliferative capacity of a cell.
BRIEF SUMMARY OF THE INVENTION
In one aspect, the invention provides an isolated or recombinant hTRT polypeptide that has telomerase catalytic activity. In one embodiment, the hTRT polypeptide has a deletion of at least 25 residues in the regions corresponding to residues 192-323, 200-323, 192-271, 200-271, 222-240, 415-450, 192-323 and 415-450, or 192-271 and 415-450 of hTRT. In a related embodiment, residues 192-323, 200-323, 192-271, 200-271, 222-240, 415-450, 192-323 and 415-450, or 192-271 and 415-450 of hTRT are deleted. The invention also provides a polynucleotide comprising a nucleotide sequence encoding these hTRT polypeptides. In some embodiments, the polynucleotide includes a promoter sequence operably linked to the nucleotide sequence encoding the hTRT polypeptide.
The invention also provides a method of preparing recombinant telomerase by contacting a recombinant hTRT polypeptide containing a deletion as described supra with a telomerase RNA component under conditions such that the recombinant protein and the telomerase RNA component associate to form a telomerase enzyme capable of catalyzing the addition of nucleotides to a telomerase substrate. The hTRT polypeptide may be produced in an in vitro expression system and/or may be purified before the contacting step. In some embodiments, the contacting occurs in a cell.
The invention further provides a method for increasing the proliferative capacity of a vertebrate cell by introducing into a cell the recombinant hTRT polynucleotide encoding an hTRT deletion variant described supra. In a related embodiment, the invention provides a cell, such as a human cell or other mammalian cell, comprising a nucleotide sequence that encodes the hTRT deletion variant polypeptide. The invention provides such cells that have an increased proliferative capacity relative to a cell that is otherwise identical but does not comprise the recombinant polynucleotide.
In a different aspect of the invention, an isolated or recombinant hTRT polypeptide that has a deletion of amino acid residues 192-450, 560-565, 637-660, 638-660, 748-766, 748-764, or 1055-1071, where the residue numbering is with reference to the hTRT polypeptide having the sequence provided in
FIG. 1
, is provided. In a related aspect, the invention provides an isolated, recombinant, or substantially purified polynucleotide encoding this polypeptide, which in some embodiments includes a promoter sequence operably linked to the nucleotide sequence encoding the hTRT polypeptide.
The invention also provides a method of reducing telomerase activity in a cell by introducing the polynucleotide described supra (i.e., having a deletion of deletion of amino acid residues 192-450, 560-565, 637-660, 638-660, 748-766, 748-764, or 1055-1071) into a cell under conditions in which it is expressed.
In a related embodiment, the hTRT polypeptide has one or more mutations other than, or in addition to, a deletion of at least 25 residues in the regions corresponding to residues 192-323, 200-323, 192-271, 200-271, 222-240, 415-450, 192-323 and 415-450, or 192-271 and 415-450 of hTRT.


REFERENCES:
Bodnar et al., “Extension of Life-Span by Introduction of Telomerase into Normal Human Cells”Science 279:349-352.
Harrington et al., 1997, “Human telomerase contains evolutionarily conserved catalytic and structural subunits”Genes Dev. 11:3109-3115.
Kilian et al., 1997, “Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns in different cell types”Hum. Mol. Genet. 6:2011-2019.
Meyerson et al., 1997, “hEST2, the Putative Human Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cells and during Immortalization”Cell 90:785-795.
Nakamura et al., 1997, “Telomerase Catalytic Subunit Homologs from Fission Yeast and Human”Science 277:955.
Weinrich et al., 1997, “Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT”Nat. Genet. 1997 Dec. 1:17(4):498-502.

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