Assays for telomerase activity

As part of its drug discovery program for inhibitors, Geron scientists developed an assay for telomerase activity, which was a modification of the method of Morin (Cell 59: 521, 1989).   As disclosed in WO 93/23572, one method for determining telomerase activity is “by measuring the rate of elongation of an appropriate repetitive sequence, having 2 or more, usually 3 or more, repeats of the telomere unit sequence, TTAGGG.”  In this assay, radiolabeled nucleotides are incorporated into an elongated oligonucleotide substrate containing the TTAGGG repeats; the reaction products are resolved by gel electrophoresis and visualized.  Because the telomerase enzyme stalls and can release the substrate after adding the first G in the repeat, the pattern obtained is a six nucleotide ladder of extended substrates.

Following on this initial assay, Geron scientists concocted a new assay, which is disclosed and claimed in US Patent 5,629,154.  As claimed, the new assay comprised:

  • preparing an extract from a cell sample;
  • mixing an aliquot of the extract with (i) a telomerase substrate that lacks a repeat sequence, and (ii) an oligonucleotide primer complementary to a telomeric repeat sequence; and
  • detecting a double-stranded DNA reaction product, one strand of which is an extended telomerase substrate containing repeat sequences and the other strand of which is extended primer.

The conditions for extension of the substrate and for extension of the primer are not specified in the claims and as such, the conditions are not limiting.  For example, the extension of the primer can be effected by a DNA polymerase, a template-dependent DNA ligase, or other suitable enzyme and the reaction product may be labeled by incorporation of radio-labeled dNTPs or by a 5’ label or by some other means.

A further improvement of this basic strategy at Geron resulted in the widely used TRAP (telomeric repeat amplification protocol) assay (Kim et al. 1994).  The claimed method of US 5,863,726 comprises:

  • incubating a cell sample with an exogenous telomerase substrate under conditions such that telomerase can catalyze extension of the substrate by addition of telomeric repeat sequences;
  • replicating (e.g., by amplification such as PCR) the extended substrate; and
  • correlating telomerase activity with presence of extended substrate and lack of activity with absence of extended substrate.

Although the claims don’t limit many of the elements, such as the nature of the substrate (e.g., lacking a telomeric repeat sequence), the method of replication, means for detecting the extended substrate, in practice most scientists perform TRAP using PCR and visualizing the amplified product by detection of radioactive-labeled nucleotides after the products are separated by electrophoresis.  As is typical of patents, dependent claims in the patent recite these specific elements as well as a variety of substitutes for the elements.   Other claims are directed to kits for detecting telomerase activity having (i) a telomerase substrate, and (ii) a primer that is complementary to a telomeric repeat sequence.

Further consolidating Geron’s position on assays for telomerase activity, US 5,804,380 claims using the TRAP assay for screening candidate modulators of telomerase activity (claim 1).  In addition, Geron claims a kit for detecting telomerase activity similar to the one in US ‘726 except that the substrate is labeled and instructions are included.  This kit claim actually falls within the scope of the claim in US ‘726, but is directed to what is probably a more likely format for commercial kits.

Geron has not left a lot of room for others in this area, as indicated by the dearth of other pending or issued patents.  CTRC Research Foundation in Texas however has a patent, US 5,856,096, that claims a ligation-style assay for telomerase activity.  In their method, activity is detected in a sample by:

  • incubating a sample with a telomerase substrate and dNTPs to form an extended substrate; then
  • adding two oligonucleotides to the extended substrate, in which the two oligonucleotides are adjacent to each other when annealed to the extended substrate;
  • adding a ligase that will ligate together the two oligonucleotides; and
  • detecting the ligated product.

Other claims are directed to use of assay to screen inhibitors of telomerase activity and to distinguish between processive and nonprocessive activities of telomerase.

Patent Data Title and relevant claims Family Data
US 5,629,154

Assignee:

Geron Corp.

Earliest priority:

12 Nov 1993

Filed:

28 Sep 1994

Granted:

13 May 1997

Expiry date:

13 May 2014

Telomerase activity assays

Claim 1:  A method for determining whether a cell sample contains telomerase activity, said method comprising the steps of: (a) preparing a cell extract from said cell sample; (b) placing an aliquot of said cell extract in a reaction mixture comprising a telomerase substrate lacking a telomeric repeat sequence and a buffer in which telomerase can catalyze extension of said telomerase substrate by addition of telomeric repeat sequences; (c) adding to said reaction mixture a primer comprising a sequence sufficiently complementary to a telomeric repeat to hybridize specifically thereto under conditions such that if an extended telomerase substrate is present in said reaction mixture, said primer will hybridize to said extended telomerase substrate and extend to form a complementary copy of said extended telomerase substrate, thereby forming duplex DNA molecules comprising an extended telomerase substrate bound to an extended primer; and (d) correlating presence of telomerase activity in said cell sample with presence of duplex DNA molecules comprising an extended telomerase substrate bound to an extended primer and absence of telomerase activity in said cell sample with absence of said duplex DNA molecules.

Patents:

AU 682082 B2

AU 688262 B2

CA 2173872 C

US 5,804,380

Assignee:

Geron Corp.

Earliest priority:

12 Nov 1993

Filed:

15 Apr 1996

Granted:

08 Sep 1998

Expiry date:

13 May 2014

Telomerase activity assays

Claim 19:  A kit for detecting telomerase activity, said kit comprising: (a) a labeled telomerase substrate; and (b) a primer comprising a sequence complementary to a telomeric repeat sequence; and (c) instructions.

See Appendix 2 for complete list of related family members.

Patents:

AU 723767 B2

CA 2173872  C

EP 0728207 B1

US 5,856,096

Assignee:

CTRC Research Foundation

Filed:

21 Sep 1995

Granted:

05 Jan 1999

Expiry date:

21 Sep 2015

Rapid and sensitive assays for detecting and distinguishing between processive and non-processive telomerase activities

Claim 1:  A method for detecting telomerase activity in a sample comprising the steps of: (i) obtaining said sample; (ii) contacting said sample with a telomerase primer and dNTPs under conditions permitting formation of a telomerase product; (iii) contacting the telomerase product of step (ii) with a first oligonucleotide and a second oligonucleotide under conditions permitting hybridization, wherein said first and said second oligonucleotides hybridize to said telomerase product such that no single-stranded region intervenes between said first and said second oligonucleotides to form a hybridized product; (iv) contacting the hybridized product and oligonucleotides with a ligase; and (v) detecting the ligated form of said first and said second oligonucleotides.

AU 7115496 A1

WO 97/11198 A1

US 5,863,726

Assignee:

Geron Corp.

Earliest priority:

12 Nov 1993

Filed:

12 Apr 1996

Granted:

26 Jan 1999

Expiry date:

13 May 2014

Telomerase activity assays

Claim 1:  A method for determining whether a cell sample contains telomerase activity, said method comprising the steps of: (a) collecting a cell sample; (b) incubating said cell sample in a reaction mixture comprising an exogenous telomerase substrate under conditions such that telomerase can catalyze extension of said telomerase substrate by addition of telomeric repeat sequences; (c) replicating said extended telomerase substrate; and (d) correlating presence of telomerase activity in said cell sample with presence of said extended telomerase substrate and absence of telomerase activity in said cell sample with absence of said extended telomerase substrate.

See Appendix 2 for complete list of family members.

Patents:

AU 723767 B2

CA 2173872 C