Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Transferases
Reexamination Certificate
1999-10-18
2001-07-17
Achutamurthy, Ponnathapu (Department: 1652)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Transferases
C435S194000, C435S252300, C435S320100, C435S091300, C536S023200, C530S412000, C530S413000
Reexamination Certificate
active
06261556
ABSTRACT:
BACKGROUND OF THE INVENTION
The American population is aging. The fastest growing segment of the population is persons over 85 years of age, who are expected to number over 30 million by the year 2040. This demographic surge is creating significant needs for drugs for the treatment of age-related diseases and has led to increased interest in the aging process and diseases associated with it, including cancer.
Organisms age, in part, because their cells have a finite capacity to continue dividing. As they reach that limit, cells become senescent. Cell senescence has been traced to the ends of a cell's chromosomes, the telomeres. With each cell division, the telomeres lose some DNA and become shorter. At some point this shortening becomes critical. Cells sense this and arrest chromosome replication to avoid further loss. Hence, the cell is no longer able to divide.
Not all cells become senescent. Single-cell organisms and certain mammalian cells have no fixed cell division limit. Investigators have discovered that many of these cells contain a ribonucleoprotein enzyme called telomerase. Telomerase replaces the DNA that is usually lost from the telomeres during cell division. (E. H. Blackburn, (1992)
Annu. Rev. Biochem.
61:113-29.) Consequently, the telomeres never shorten past the critical length and the cells never reach senescence.
Particularly interesting, investigators have found that the cells of many human cancers have telomerase. (C. B. Harley,
Mutation Research
1991 256:271-282.) This helps explain why cancer cells continue dividing without becoming senescent. It also suggests a potent weapon in the battle against cancer: If telomerase activity in cancer cells can be inhibited, the cancer cells are expected to reach senescence and cease dividing.
Developing methods to regulate telomerase activity requires sources of purified telomerase and, in particular, purified human telomerase. Purified telomerase would be useful in developing and testing assays for measuring telomerase activity, for example, to evaluate the assay and for use as a standard in the assay. Assays for telomerase are useful in characterizing cancer cells or pre-cancer cells, because most cancer cells express telomerase. Purified telomerase would be more useful than crude telomerase preparations to identify and test regulators, inhibitors or activators of telomerase activity in in vitro assays. Moreover, purified telomerase would facilitate a thorough biochemical analysis of the enzyme's mechanism, which may provide insight for development of mechanism-based regulators. Purified telomerase also would be useful in the preparation of antibodies against telomerase. Such antibodies would in turn be especially useful as reagents to purify human telomerase and may be useful in cancer diagnosis or prognosis. Purified telomerase also will help provide amino acid sequence information useful in cloning the various components of the ribonucleoprotein.
While there is a need for purified telomerase, the purification of the human enzyme has posed technical challenges. Telomerase is a rare ribonucleoprotein expressed in human cells only in very low abundance. It has been estimated that human cells known to express the highest levels of telomerase activity may have only about one hundred molecules of the enzyme per cell. The fact that telomerase is a complex, multi-component structure further impedes its purification. Human cells also possess comparatively very high levels of non-telomerase ribonucleoproteins. These other ribonucleoproteins might have chromatographic purification properties similar to the telomerase ribonucleoprotein, which makes purification of telomerase from human cells difficult. Thus, there is a need for purified telomerase and purified human telomerase, in particular.
SUMMARY OF THE INVENTION
Human telomerase has been purified to over 60,000-fold purity over cytoplasmic crude cell preparations. Two polypeptides that co-purify with fractions containing telomerase activity are present in the purified fractions in approximate stoichiometric amounts with the RNA component of human telomerase have been isolated. One polypeptide has amino and sequences consistent with nucleolin. The other polypeptide has amino acid sequences consistent with elongation factor 2 homolog.
In one aspect, this invention provides methods of purifying telomerase. The steps included in the method depend on the level of purification one desires. A method to purify telomerase from an impure composition containing organic biomolecules, for example, a nuclear extract of 293 cells, to at least 60,000-fold compared to crude extract (about 4% relative purity) involves:
(1) contacting the telomerase with a first matrix that binds molecules bearing a negative charge, for example, POROS® 50 HQ, separating telomerase from other organic biomolecules that do not bind to the matrix and collecting the telomerase;
(2) contacting the telomerase with a matrix that binds molecules bearing a positive charge, for example POROS® Heparin 20 HE-1, separating telomerase from other organic biomolecules that do not bind to the matrix and collecting the telomerase;
(3) contacting the telomerase with a second matrix that binds molecules bearing a negative charge, for example, SOURCE 15Q®, separating telomerase from other organic biomolecules that do not bind to the matrix and collecting the telomerase;
(4) contacting the telomerase with an affinity agent having specific affinity for telomerase, for example an oligonucleotide having the sequence 5′-cgttcctctt cctgcggcct-3′ (Oligo 14ab) (SEQ ID NO:7), separating telomerase from other organic biomolecules that do not bind to the affinity agent and collecting the telomerase; and
(5) separating the telomerase from other organic biomolecules according to molecular size, shape, or buoyant density, for example separating molecules according to size on a TosoHaas TSK-gel*G5000PW
XL
sizing column and collecting the telomerase.
Telomerase can be isolated to at least 500,000-fold purity by adding a second affinity step using anti-nucleolin antibodies or anti-TMG antibodies.
Telomerase can be purified to substantial purity (e.g., at least 1,000,000-fold) by further isolating it by gel electrophoresis.
The purification protocol also can include the step of contacting the telomerase with an intermediate-selectivity matrix, separating telomerase from other organic biomolecules that do not bind to the intermediate-selectivity matrix and collecting the telomerase, preferably before the affinity step.
Telomerase can be isolated to different levels of purity by altering, changing the sequence of, or eliminating any of the steps in the purification protocol. However, any protocol will include contacting the telomerase with an affinity agent. Contacting the telomerase with at least one matrix that binds molecules bearing a negative charge or a positive charge is the next preferred step or steps to include in the protocol.
The invention also provides purified telomerase and, more particularly, telomerase having at least 2000-fold, at least 3000-fold, at least 20,000-fold, at least 60,000-fold, at least 100,000-fold, at least 500,000-fold or at least 1,000,000-fold increased relative purity compared to crude cell extracts of 293 cells. The telomerase can be animal, mammalian and, more particularly, human. The invention also provides telomerase made by the purification steps above, or recombinantly.
In another aspect, this invention provides a recombinant polynucleotide comprising a nucleotide sequence that encodes a polypeptide having at least 5 consecutive amino acids of a protein component of human telomerase. In one embodiment the recombinant polynucleotide further comprises an expression control sequence operatively linked with the nucleotide sequence.
In another aspect, this invention provides a polynucleotide probe or primer that specifically hybridizes with a polynucleotide encoding a protein component of human telomerase.
In another aspect, this invention provides a recombinant cell comprising a
Atkinson, III Edward M.
Kealey James T.
Lichtsteiner Serge P.
Pruzan Ronald A.
Vasserot Alain P.
Achutamurthy Ponnathapu
Earp David J.
Geron Corporation
Geron Corporation
Saidha Tekchand
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