Variants of telomerase

This section presents patents directed to variants (alternate forms) of wild-type human telomerase.  Variants come in many different flavors, including:  mutations, insertions, deletions, polymorphisms, and RNA splice variants.  The first set of patents presented in this section is about RNA splice variants.  Two groups initially described RNA splice variants:  CAMBIA[1] and Bayer.  In the United States, CAMBIA obtained two granted patents, while Bayer appears to have largely abandoned their effort to obtain protection for splice variants, possibly because their disclosure of splice variants was later in time than CAMBIA’s.  Both CAMBIA and Bayer have been granted Australian patents however.

US 6,846,662 and US 6,916,642, which are owned by CAMBIA, have identical disclosures.[2]  The disclosure presents a number of RNA splice variants of telomerase.  Specifically, seven alternative “introns/exons” were identified, which theoretically could yield 128 different RNAs.  Because of frame-shifts or stop codons introduced by some of the alternative “introns/exons”, the number of different telomerase variant polypeptides would be much smaller.  Now that the genomic structure of the telomerase gene is known, these alternative sequences can be mapped.

Strikingly, most of the alternative sequences do not correspond to entire exons or introns, and as such, the RNA splice variants disclosed would not have been predicted by the genomic sequence.

The claims of the two patents are directed to proteins (US ‘662) and to nucleic acids (US ‘642).  The protein patent claims telomerase proteins having:

  • specific sequences of Figure 11 (claim 1);
  • proteins that are 90% identical to the specific sequences and that have either catalytic activity or binds telomerase RNA – and is not wild-type telomerase (claim 4);
  • individual alternative “introns/exons” (claim 2);
  • a splice variant lacking 13 amino acids from residues 711-722 (inclusive) and proteins having 90% identical amino acids as well as same requirements of claim 4 (claim 3);
  • a sequence encoded by a nucleic acid having at least one of the disclosed alternative introns/exons excluding wild-type telomerase (claim 6).

The lead claim of the nucleic acid patent recites a molecule encoding a splice variant in which the variant has at least one of six alternative introns/exons, which would yield 64 different proteins.  Other claims are directed to specific DNA sequences encoding variant telomerases presented in Figure 11 and sequences encoding a protein having at least 95% identity to the variants.  Individual nucleic acid sequences of the alternative introns/exons are also claimed.  There are also claims to nucleic acid probes and amplification primers for detecting and replicating RNA splice variants.   Methods for establishing patterns of expression of the variants, e.g., for detection or prognosis of cancer, form many of the remaining claims.  While not claiming all RNA splice variants, these patents encompass nearly every splice variant identified to date.

Australian patent AU 748442 is related to the two CAMBIA U.S. patents and has essentially identical disclosure.  The claims in AU ‘442 are broader however.

The lead claim encompasses a “nucleic acid molecule encoding a splice variant” of wild-type telomerase.  Moreover, the wild-type telomerase reference protein is not limited to humans, but includes related telomerases that are encoded by nucleic acid molecules that hybridize under conditions of low stringency to the region containing reverse transcriptase motifs.  By limiting the hybridization region to the most conserved part of telomerase and setting the hybridization conditions to low stringency, the reference telomerases will likely include many vertebrate species.  Low stringency hybridization may include molecules with as little as 50% identity and possibly less.  Thus, the claim covers splice variants of telomerase from a variety of vertebrate species.

In contrast to the broad claims of CAMBIA’s Australian patent, the claims in the Bayer patents AU 742489 and AU 745420 recite specific DNA sequences of splice variants.  Although the earliest priority date of AU ‘420 is before the earliest priority date of AU ‘442, splice variants weren’t disclosed in the earliest priority document for AU ‘420.  Thus, CAMBIA filed disclosure of splice variants prior to Bayer.  The lead claim in AU ‘420 recites four different variants encoded by nucleic acid sequences that are:

  • deleted for 182 bp (b region);
  • deleted for 36 bp (a region);
  • deleted for both the 182 bp and 36 bp; or
  • deleted for about 1800 bp at the 5’ end and has an alternate 3’ end.

The first three variants were described in AU ‘442 and appear to be encompassed by its claims, or they are identical claims (and probably not valid as a result).  The fourth variant is a fragment, and whether it is also covered by the claims of AU ‘442 requirse a more precise inquiry into how Australian patent law would interpret the claim of AU ‘420.

The second Bayer patent – AU ‘489 – claims isolated intron sequences or fragments of these sequences which have a regulatory effect.  In the intron sequences, mainly in intron 2, the inventors have identified potential binding sequences for DNA-binding proteins – regulatory proteins.  The regulatory proteins include C/EBP, CRE.2, Sp1, GRE, CREB, c-Myc, CCAAT site, and Rb site.  In addition to the numerous candidate binding sites in intron 2, one potential Sp1 binding site was found in intron 1 and one potential c-Myc binding site was found in the 5’-untranslated region.

These introns, intron 1 and 2, have not been found in RNA splice variants of telomerase to date.  Moreover, each intron sequence that is found in splice variants corresponds only to a portion of the intron.  Unless the portion specifically has a regulatory effect, and none have been identified so far, then the claims in Bayer’s patent AU ‘498 do not cover individual alternate intron/exon sequences.

Patent Data Title and relevant claims Family Data
US 6,846,662

Assignee:

CAMBIA

Earliest priority:

01 Jul 1997

Filed:

11 Feb 2000

Granted:

25 Jan 2005

Expiry date:

01 Jul 2017

Vertebrate telomerase genes and proteins and uses thereof

Claim1:  An isolated protein, wherein the protein comprises one of SEQ ID Nos. 37, 39, 42, 44, 46, 48, 50, 56-58, 60-62, 64-66, 68-70, 72-74, 76-78, 80-82, 84-86.

AU 748442 B2

BR PI9810643 A

CA 2294782 AA

CN 1270634 A

EP 0917579 A1

EP 1571215 A2

JP 2002/514928 T2

NZ 501962 A

US 2005/176022 A1

US 6,916,642 B1

WO 99/01560 A1

US 6,916,642

Assignee:

CAMBIA

Earliest priority:

01 Jul 1997

Filed:

11 Feb 2000

Granted:

12 Jul 2005

Expiry date:

01 Jul 2017

Vertebrate telomerase genes and proteins and uses thereof

Claim 1: An isolated nucleic acid molecule encoding a splice variant of a gene sequence capable of being spliced to result in a reference human telomerase encoding SEQ ID No: 2, wherein the splice variant has at least one of the following insertions or deletions:

(a) an insertion of sequences X (comprising SEQ ID No: 32) at nucleotide 1766 of SEQ ID No: 1;

(b) an insertion of nucleic acid sequence encoding sequence 1 (SEQ ID No: 24) at nucleotide 1950 of SEQ ID No: 1;

(c) a deletion of nucleotides 2131 through 2166 of SEQ ID No: 1;

(d) a deletion of nucleotides 2287 through 2468 of SEQ ID No: 1;

(e) an insertion of sequence 2 comprising SEQ ID No: 29 at nucleotide 2843 of SEQ ID No: 1; and

(f) an insertion of nucleic acid sequence encoding sequence 3 (SEQ ID No: 31) at nucleotide 3157 of SEQ ID No: 1,

and wherein the splice variant does not encode SEQ ID No: 2.

AU 748442 B2

BR PI9810643 A

CA 2294782 AA

CN 1270634 A

EP 0917579 A1

EP 1571215 A2

JP 2002514928 T2

NZ 501962 A
US 2005/176022 AA

US 6,846,662 B1

WO 99/01560 A1

AU 742489

Assignee:

Bayer

Earliest priority:

24 Dec 1997

Filed:

22 Dec 1998

Granted:

01 Jan 2002

Expiry date:

22 Dec 2018

Regulatory DNA sequences of the human catalytic telomerase sub-unit gene, diagnostic and therapeutic use thereof

Claim 1: Isolated DNA characterized in that the sequences are intron sequences in accordance with SEQ ID NO 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and/or 20 or fragments of these sequences which have a regulatory effect.

CA 2316282 AA

DE 19757984 A1

EP 1040195 A2

JP 2003519462 T2

US 2005/032094 A1

WO 99/33998 A3

AU 745420

Assignee:

Bayer

Earliest priority:

20 Jun 1997

Filed:

09 Jun 1998

Granted:

21 Mar 2002

Expiry date:

09 Jun 2018

Human catalytic telomerase sub-unit and its diagnostic and therapeutic use

Claim 1:  Functional equivalents, variants and catalytically active fragments of the catalytically active human telomerase subunit in isolated or purified form, comprising the amino acid sequence depicted in Fig. 2, characterized in that they comprise an amino acid sequence encoded by

(1)           the DNA sequence depicted in Fig. 1 with a deletion of 182 bp in length extending from nucleotide 2345 to 2526.

(2)           the DNA sequence depicted in Fig. 1 with a deletion of 36 bp in length extending from nucleotide 2184 to 2219.

(3)           the DNA sequence depicted in Fig. 1 with a deletion of 36 bp in length extending from nucleotide 2184 to 2219 and a deletion of 182 bp in length extending from nucleotide 2345 to 2526 or.

(4)         The DNA sequence depicted in Fig. 14.

CA 2294646 AA

DE 19816496 A1

EP 0990037 A2

JP 2002508662 T2

WO 98/59040 A3

AU 748442

Assignee:

CAMBIA

Earliest priority:

01 Jul 1997

Filed:

01 Jul 1998

Granted:

06 Jun 2002

Expiry date:

01 Jul 2018

Vertebrate telomerase genes and proteins and uses thereof

Claim 1:  An isolated nucleic acid molecule comprising a sequence corresponding to a nucleic acid molecule encoding a splice variant of a reference sequence of a catalytic subunit of a vertebrate telomerase,

Wherein the reference sequence has the nucleic sequence presented in Figure 1 or the reference sequence hybridizes under conditions of low stringency to the complement of nucleic acid sequence encoding amino acids 5605-915 of Figure 1.

BR PI9810643 A

CA 2294782 AA

CN 1270634 A

EP 0917579 A1

EP 1571215 A2

JP 2002514928 T2

NZ 501962 A

US 2005/176022 A1

US 6,846,662 B1

US 6,916,642 B1

WO 9/901560 A1

The second set of patents in this section is directed to telomerases deleted for specific regions of sequence.  All of these patents were granted to Geron.  The claims in one are directed to specific deletions that do not affect the activity of telomerase, while the claims in the other recite deletions that abolish telomerase activity.  According to the patent, a variant telomerase has “activity” if it has at least 40% of the activity of a wild-type telomerase.  “Lack of activity” means that the variant has less than 1% of “wild-type” activity; “intermediate activity” is used to describe a telomerase with between 1% and 40% activity.

The inventors discovered that amino acid residues 192-323 or residues 415-450 can be deleted and the resulting telomerase retains catalytic activity.  Some decrease was observed of the binding of the telomerase RNA component to these variants.  More interesting however, are the variants that are deleted for one or more of residues 192-450, 637-660, 638-660, 748-766, 748-764 and 1055-1071.  These variants not only lack telomerase catalytic activity but appear to inhibit the activity of wild-type telomerase – “dominant negative variants”.  For reference, the region from residues approximately 620-902 contains the telomerase-specific and reverse transcriptase motifs, and the splice variant that is dominant negative – Da – is deleted for residues 708-722.

The claims of US 6,337,200 are relatively straightforward.  Claim 1 recites a polynucleotide encoding an hTERT variant, which is deleted for at least 10 amino acids of residues 192-323 or 415-450.  Moreover, the variant has catalytic activity.  Other independent claims are directed to using the variants to increase the proliferative capacity of a human cell in vitro by expressing the variant in the cell and to producing the variant protein by expression in a host cell or in a cell-free expression system.

The claims of US 7,091,021 are directed to polypeptide variants and methods of using the variants to inhibit telomerase catalytic activity.  The simplest claim (claim 3) for polypeptide variants recites a telomerase deleted for one or more of residues 326-415, 560-565, 637-660, 748-766, 930-934, 1055-1071, or 1084-1116.  An unusual aspect of this claim is the recitation that the deletions “consist[ing] essentially of” the residues already mentioned.  “Consisting essentially of” is a patent term that “limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention (MPEP 2111.03, 8th ed.; emphasis in original).  It is the understanding of the author that “consisting essentially of” is rarely, if ever, used in biotechnology claims, and that the meaning of this term for biotechnology claims is uncertain.

Claim 2 is relatively simple and is directed to a variant lacking telomerase activity, in which the variant is wild-type telomerase deleted for one or more of the following:

  • at least 10 consecutive amino acids between 326-415, 637-660, 748-766, 1055-1071, 1084-1116;
  • amino acids 560-565; 930-934.

Claim 1 is similar to claim 2 except that “wild-type telomerase” is replaced by “a polypeptide encoded by DNA that hybridizes to” the complement of the sequence encoding wild-type telomerase.  Hybridization conditions are the same as for other Geron patents and have been discussed in detail elsewhere in this landscape.  An additional requirement is that the variants inhibit telomerase enzyme activity in situ.  This requirement doesn’t impose an additional limitation relative to the other claims; it is intended to exclude polypeptides that have the recited deletions but are not dominant negative variants.

Patent Data Title and relevant claims Family Data
US 6,337,200

Assignee:

Geron Corp.

Earliest priority:

31 Mar 1998

Filed:

03 Aug 1998

Granted:

08 Jan 2002

Expiry date:

31 Mar 2018

Human telomerase catalytic subunit variants

Claim 1:  A polynucleotide encoding a variant of human telomerase reverse transcriptase (hTRT), said variant having processive catalytic activity and comprising a deletion of at least 10 amino acids from region 192-323 or 415-450 of SEQ. ID NO:2.

AU 3375299 A1

US 2002/102686 A1

US 2006/275267 A1

US 7,091,021 B2

WO 99/50386 A3

US 7,091,021

Assignee:

Geron Corp.

Earliest priority:

31 Mar 1998

Filed:

21 Nov 2002

Granted:

15 Aug 2006

Expiry date:

31 Mar 2018

Inactive variants of the human telomerase catalytic subunit

Claim 1:  A polypeptide encoded by DNA that hybridizes to the sequence complementary to SEQ. ID NO:1 at 5.degree. C. to 25.degree. C. below Tm in aqueous solution at 1 M NaCl, wherein Tm is the melting temperature of double-stranded DNA having the sequence of SEQ. ID NO:1 under the same reaction conditions; wherein said polypeptide has one or more of the following deletions: a) residues 560 565, b) residues 930 934, c) at least 10 consecutive amino acids from residues 326 415, d) at least 10 consecutive amino acids from residues 637 660, e) at least 10 consecutive amino acids from residues 748 766, f) at least 10 consecutive amino acids from residues 1055 1071, or g) at least 10 consecutive amino acids from residues 1084 1116 of SEQ. ID NO:2; and wherein said polypeptide inhibits telomerase enzyme activity when introduced into a cell expressing human telomerase reverse transcriptase (hTRT) (SEQ. ID NO:2).

Continuation of ‘200 and has the same family history

[1] The original assignees were Cambia Biosystems LLC and Peter MacCallum Cancer Research Institute.

[2] In the United States, claim sets are often subject to “restriction”, meaning that the Patent Office believes that the claims are drawn to multiple inventions.  Because only one invention can be claimed in a patent, the applicant chooses an invention for examination; the remaining inventions can be examined in separate applications.  The separate applications have identical disclosures and are called “divisional” applications.