Human gene sequences

Not surprisingly, the co-assignees Geron Corp. and University of Colorado (current assignee of University Technology Corp., the former Colorado University organization that handled licensing for all CU campuses) dominate this area, in sheer numbers of patents as well as in scope.  A minimum of 16 U.S. patent applications claim priority from an initial filing in October 1996 (which did not contain any human-derived nucleotide sequences).  At least 10 of these applications have resulted in granted U.S. patents, some of which are directed to human telomerase and others to homologues from other species.  In addition, numerous patent applications (up to about 55) were filed in countries other than the United States.  Many of these have been granted.

Before launching into a discussion of the Geron / University of Colorado patents, a few comments are warranted on the likely disposition of a patent application filed by Whitehead Institute (Boston, MA).  A series of U.S. provisional patent applications were filed between February and October 1997, which were based on the work of the Robert Weinberg lab (Meyerson et al. 1997e).  A PCT application (WO 98/37181) was also filed, but never converted into national applications in the non-U.S. designated countries.  Because U.S. patent procedures were conducted in secret until 2000, nothing is known about the status of any U.S. patent applications.  Because no patent has issued to date, any U.S. applications most likely have either been abandoned or are in interference[1] with one or more Geron / UC patents.

Fig 5.  Brief history of Geron / University of Colorado selected patent application filings

Following a series of rapid succession of filings of patent applications on 18 April, 25 April, and 6 May 1997, containing incomplete sequences of human telomerase, on 9 May 1997, the Geron / University of Colorado groups filed a patent application with the full-length sequence.  US 6,261,836 B1 was granted on 17 July 2001 from this filing; its claims are directed to “human telomerase reverse transcriptase protein”.

With respect only to the disclosure related to human telomerase, the application details the cloning of hTERT.  Briefly, partial homologous sequences were identified in a BLAST search of publicly available EST sequences queried against the Euplotes and Schizosaccharomyces telomerase sequences.  A match was scored for an EST sequence derived from a partial cDNA clone (GenBank accession AA281296).  When the open reading frame of this clone was aligned to the query sequences, the presence of signature motifs strongly suggested that this was the long-sought after human telomerase.  Using standard techniques, additional 5’ sequence information was obtained as well as a lambda vector clone (l25-1.1) that had complementary sequence that was sub-cloned into a plasmid (pGRN121 – ATCC accession no. 209016).  The sequence of the insert revealed the entire open reading frame encoding the human telomerase protein.  Interestingly, pGRN121 contained an insertion of 182 nucleotides relative to the GenBank accession AA281296.  The open reading frame of pGRN121 encoded a protein of 1132 amino acids having a molecular weight of approximately 127,000 daltons.  SEQ ID No: 224 presents the nucleotide and amino acid sequences of hTERT.

The independent claims are directed to nucleic acid sequences encoding the hTERT protein, hTERT protein encoded by the sequences, cells comprising the nucleic acid sequence, and a method of preparing the telomerase complex using the claimed hTERT protein and a telomerase RNA component.

Claim 1 asserts a “synthetic or recombinant human telomerase reverse transcriptase” (hTERT)

  • protein;
  • variant of the protein;
    o        variant is encoded by a polynucleotide that hybridizes under stringent conditions to a complement of SEQ ID NO: 224 (hTERT); or
  • fragment of the protein;

AND

  • the protein, variant or fragment has telomerase catalytic activity when complexed with a telomerase RNA component.

Literally read, the claim terms, “hTERT protein”, “variant of the protein”, and “fragment of the protein” extend beyond the disclosed amino acid sequence.  The term “hTERT protein” has no limitation of specific sequence, as would have been expected.  Its only limitation is the requirement of catalytic activity (discussed below).  But what exactly falls within the scope of “hTERT protein”?

Reading the claim literally, any human TERT protein that has catalytic activity would be encompassed.  Any hTERT protein could arguably include polymorphisms, RNA splice variants, deletions, etc.  It’s unclear whether or not the modifier “human” means that the sequence has to be a natural sequence.  If the telomerase is not a natural sequence found in humans, then what makes a telomerase a “human” telomerase?  Another reason to believe that only natural sequences, e.g., the amino acid sequence of SEQ ID NO: 225 and polymorphisms, are encompassed is the recitation of “variant of the protein” as an alternative element.  If the term “protein” also included non-natural sequences such as those encoded by hybridizing polynucleotides, then there wouldn’t have been any need to recite “variants” in the claim.

Reading the claim in light of the specification, however, an argument can be made that the term only encompasses the specific amino acid sequence set forth in SEQ ID NO: 225, because that is the only amino acid sequence shown.  (The Federal Circuit Court applies a strict written description requirement to biotechnology inventions, which usually limits the claims to the precise sequences disclosed in the patent application.)   This interpretation however presumes that a court would find it so.  As it stands in 2006, though, most patent lawyers lament the lack of certainty of claim interpretation.

While by no means certain, the term hTERT protein probably means just SEQ ID NO: 225.  This conclusion is supported by the presumed meaning of “fragment thereof”, which refers to sequences found within SEQ ID NO: 225 – see below for more discussion.  If hTERT refers just to SEQ ID NO: 225, then polymorphisms of hTERT and RNA splice variants are excluded.

“Fragments” of hTERT are contemplated to be “of various lengths. In one embodiment, the portion of polypeptide comprises fragments of lengths greater than 10 amino acids. However, the present invention also contemplates polypeptide sequences of various lengths, the sequences of which are of which are included within SEQ ID NOS: …225, from 5 to 1100 amino acids (as appropriate, based on the length of (SEQ ID NOS: ….. 225).”  So, from this description, is the minimal length of a fragment 5 amino acids?  10 amino acids?  Or some other length?  Because of the requirement that the fragment have catalytic activity, the minimum length of a fragment is not terribly relevant.  To have catalytic activity, a fragment of hTERT would need to be substantially longer than 5 or 10 amino acids.  What we can infer from the description however is that the fragment sequence is found within SEQ ID NO: 225.  So, fragments of polymorphic hTERT or RNA splice variant hTERT do not appear to be encompassed by this element of the claim.

On the other hand, “variant of the protein” carries a definition in the claim itself – a protein encoded by a hybridizing nucleic acid, which hybridizes under “stringent conditions”.  According to the patent application, “stringency” typically occurs in a range from about 5° C to about 20° C to 25° C below Tm of the probe.  From the point of view of hybridization kinetics, this is not much of a limitation.  While it is often stated that the maximum divergence of sequences that hybridize at about 20-25° C below Tm is 25% mismatch, it is the stringency of the wash conditions that generally controls the amount of mismatch (or conversely, the amount of identity) observed.[2]  The common hybridization condition of 20-25° C below Tm is a consequence of experiments begun in the 1970s by Wetmur and Davidson, who determined that this temperature resulted in maximal hybridization efficiency, not necessarily maximal hybridization specificity.  Even though it is an erroneous belief, many, or most, scientists would consider the stated stringent hybridization conditions to yield nucleic acid molecules having at least 75-80% identity to the probing sequence.  The various RNA splice variants would hybridize under these conditions, as would many related sequences.  Because of the activity requirement, at least some of the splice variants would necessarily be excluded from the claimed telomerase molecules.

Any protein / variant / fragment of hTERT has a major limitation in that it must have “telomerase catalytic activity” when complexed with a telomerase RNA.  While this term is not explicitly defined, the most probable meaning is that the catalytic activity is using a “portion of its internal RNA moiety as a template for telomere repeat DNA synthesis” and more specifically, “extend[ing] the G strand of telomeric DNA.”  (col. 3, lines 17-30) Example 4 provides the method for assaying telomerase activity; no other assay appears to be described.  The assay is for extension of dGTP on an oligonucleotide substrate; assay conditions do not mention the inclusion of a telomerase RNA component, however.

US 6,921,664 has identical disclosure to US ‘836.  The claims however recite a recombinant expression vector comprising an encoding region for telomerase protein, a variant or a fragment.  As for the claims of US ‘836, the protein, variant or fragment must have telomerase catalytic activity when complexed with a telomerase RNA.  Similarly, the nucleic acid encoding telomerase hybridizes to the complement of SEQ ID NO: 224 under stringent conditions.

Because of how the claim is written, it appears that a nucleic acid sequence that incorporates enough codon differences (taking advantage of codon degeneracy) wouldn’t fall within the scope of this claim because it couldn’t hybridize to the complement of SEQ ID NO: 224.

US 6,927,285 has a disclosure written prior to establishing full-length telomerase sequence.  Only a partial sequence, replete with sequence errors, was presented.  To overcome that deficiency, Geron / University of Colorado made a biologic deposit of the plasmid pGRN121, which contained an insert encoding the full-length telomerase protein.  Under U.S. patent law a biological deposit can satisfy the enablement – and written description –  requirements.   The deposit was therefore a wise decision.

This patent has six independent claims to the telomerase sequence.  With reference to pGRN121, both claim 1 and claim 5 are directed to an isolated cDNA encoding hTERT, wherein the cDNA is contained in pGRN121 (claim 1) or wherein the cDNA hybridizes to the insert of pGRN121 (claim 5). The cDNA of Claim 2 has the restriction map of pGRN121.

Claims 3 and 6 claim an isolated nucleic acid encoding a “naturally occurring” hTERT or variant thereof (claim 3) and an isolated cDNA encoding hTERT (claim 6), wherein the nucleic acid or the cDNA hybridize to the incomplete sequence presented as SEQ ID NO: 173.  Claim 4 recites SEQ ID NO:173 with a 5’-Met codon.

Usually claims to a deposited sequence would be considered to be relatively narrow.  Essentially such claims are equivalent to a claim to SEQ ID NO: N.  In a twist however, claims in this patent recite hybridizing sequences, effectively broadening the scope.  The requirement exists though that the nucleic acids must encode human telomerase reverse transcriptase protein.  As discussed for other patents in this section, a plausible interpretation is that the claim encompasses the “wild-type” or full-length telomerase and possibly polymorphisms.

A continuation-in-part of US 6,261,836, the claims of US 6,475,789 are directed to mammalian cells that contain a recombinant nucleic acid sequence that encodes hTERT.  The disclosure of US ‘789 first describes the consensus motifs that are characteristic of TERT proteins and nucleic acids identified in species such as OxytrichiaSchizosaccharomycesEuplotes, and human.  Most of the remaining disclosure provides pages of detail about the human gene and protein, including how to clone the gene, recombinant expression of the protein, purification of the protein, and raising antibodies to the protein.  Multiple activities of the protein are discussed along with descriptions of assays for the activities.  The activities include reverse transcriptase activity, telomere binding, dNTP binding, and telomerase RNA binding.

Uses for the nucleic acids and proteins comprise the remainder of the disclosure.  The uses include treatment of cancer and other diseases and conditions, vaccine production, and increasing the proliferative capacity and production of immortalized cells and animals.  As well, diagnostic assays for the presence of telomerase are provided.  These assays are proposed for diagnosis and prognosis of cancer and other conditions and monitoring cells in culture, among other uses.

Although the disclosure is hefty, because of U.S. patent law, claims are limited to one invention – as determined by the U.S. Patent Office.[3]  In this case, the eight claims are directed to:

A mammalian cell that contains

·         a nucleic acid sequence that encodes TERT protein, variant or fragment having telomerase catalytic activity;

o        the TERT sequence hybridizes to the hTERT sequence (ID NO: 1) using stringent conditions.

The terms and hybridization conditions have all been discussed above for US ‘836.  Whereas the claims of US ‘836 claim a TERT protein (or variant or fragment), the claims here are directed to nucleic acid sequences.  Because of how the claim is written, it appears that a nucleic acid sequence that incorporates enough codon differences (taking advantage of codon degeneracy) wouldn’t fall within the scope of this claim because it couldn’t hybridize to SEQ ID NO: 1.

The title of US 6,444,650 is “Antisense compositions for detecting and inhibiting telomerase reverse transcriptase.”  This very slim patent is directed to antisense oligonucleotides that specifically anneal to hTERT nucleic acid molecules.  These include not only sequences encoding hTERT, but also any upstream, flanking, noncoding, and transcriptional control elements, hTERT pre-mRNA, mRNA, cDNA, and the like.  Antisense is considered to be at least 7 nt up to about 100 nt, but is often in the range from 10-50 nt.  The inventors prefer approximately 30 nt long antisense.

Like their other patent applications, “specific binding” or “specific hybridization” is that annealing to a target polynucleotide that occurs under stringent conditions.  In this patent, “stringent conditions” are also defined as from about 5 to about 20 to 25° C below Tm of the target sequence in 1 M NaCl.  (See discussion about this definition under US 6,261,836.)   Other definitions of terms are broader in this disclosure than in earlier disclosures – e.g., hTERT activity now refers to one or more of activities found in naturally-occurring full-length hTERT protein.

The lead claim recites antisense oligonucleotides that

·         hybridize to SEQ ID NO: 1 under stringent conditions; and

·         inhibits expression of hTERT mRNA.

Although the text of the patent discusses antisense oligonucleotides that anneal to other than hTERT coding sequences, the claim limits them to annealing to SEQ ID NO: 1.  This sequence contains approximately 50 bases of 5’-noncoding sequence, the entire hTERT coding sequence, and approximately 575 bases of 3-noncoding sequence.  According to the claim, the antisense could have a different complementary sequence than found in SEQ ID NO: 1 as long as the antisense anneals under the stated conditions.  As is well known, hybridization kinetics and duplex stability is not the same for oligonucleotides as it is for polynucleotides over about 600 bases long.  Base mismatches in short duplexes can significantly affect thermal stability.  With these considerations, this claim is unlikely to encompass widely divergent oligonucleotide sequences; a prediction of the likely boundaries is outside the scope of this landscape.

Rounding out the group of Geron patents, US 6,610,839 claims hTERT promoter sequences.  A human genomic DNA library was screened to identify a clone containing hTERT coding sequences.  One isolated clone (lGF5) contained approximately 13 kb of DNA upstream of the start site of the cDNA sequence.  Subfragments of this region were tested for promoter activity by linking them to a reporter gene sequence.  Claims were granted to the deposited lambda phage clone, hybridizing nucleic acid sequences, and specific sequences, along with 80% identical sequences.

Patent Data Title and relevant claims Family Data**
US 6,261,836

Assignee:

Geron Corp.

University Technology Corp.

Earliest priority:

01 Oct 1996

Filed:

09 May 1997

Granted:

17 Jul 2001

Expiry date:

01 Oct 2016 *

Telomerase

Claim 1:  A synthetic or recombinant human telomerase reverse transcriptase (hTRT) protein, or a variant thereof, or a fragment thereof, wherein said variant is encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide having a sequence complementary to SEQ ID NO: 224, and wherein said hTRT protein, variant, or fragment has telomerase catalytic activity when complexed with a telomerase RNA.

See Appendix 1 for complete list of family members.

Patents:

AU 734089

GB 2317891

EP 841396

Patent applications:

CA 2267664

EP 0954585

EP 1333094

US 6,444,650

Assignee:

Geron Corp.

University Technology Corp.

Earliest priority:

01 Oct 1997

Filed:

31 Mar 1998

Granted:

03 Sep 2002

Expiry date:

01 Oct 2017 *

Antisense compositions for detecting and inhibiting telomerase reverse tran-scriptase

Claim 1:  An isolated antisense oligo-nucleotide that hybridizes to a target DNA having the nucleotide sequence of SEQ. ID NO:1 at 5° C. to 25° C. below Tm in aqueous solution at 1 M NaCl; wherein Tm is the melting temperature of a complementary oligonucleotide hybridized to the target DNA in aqueous solution at 1 M NaCl, wherein the complementary oligonucleotide is exactly complementary to SEQ. ID NO:1 and the same length as the antisense oligonucleotide; and wherein hybridization of the antisense oligonucleotide to an mRNA encoding hTRT (SEQ. ID NO:1) inhibits expression of the mRNA.

See Appendix 1 for complete list of family members.

Patents:

EP 841396

Patent applications:

CA 2267664

EP 0954585

US 6,475,789

Assignee:

University Technology Corp.

Geron Corp.

Earliest priority:

01 Oct 1996

Filed:

14 Aug 1997

Granted:

05 Nov 2002

Expiry date:

01 Oct 2016 *

Human telomerase catalytic subunit: diagnostic and therapeutic methods

Claim 1:  A mammalian cell that contains a recombinant polynucleotide comprising a nucleic acid sequence that encodes a telomerase reverse transcriptase protein, variant, or fragment having telomerase catalytic activity when complexed with a telomerase RNA, wherein said recombinant polynucleotide hybridizes to a DNA having a sequence complementary to SEQ ID NO: 1 at 5° C. to 25° C. below Tm in aqueous solution at 1 M NaCl, wherein Tm is the melting temperature of a complementary polynucleotide hybridized to said DNA in aqueous solution at 1M NaCl, wherein the complementary polynucleotide is exactly complementary to SEQ ID NO: 1 and is the same length as the recombinant polynucleotide.

See Appendix 1 for complete list of family members.

Patents:

AU 734089

GB 2317891

EP 841396

Patent applications:

CA 2267664

EP 0954585

US 6,610,839

Assignee:

Geron Corp.

Earliest priority:

14 Aug 1997

Filed:

01 Oct 1997

Granted:

26 Aug 2003

Expiry date:

14 Aug 2017

Promoter for telomerase reverse transcriptase

Claim 1: An isolated nucleic acid comprising a promoter sequence that either: a) is contained in lambda phage GΦ5 deposited as ATCC Accession No. 98505; or b) hybridizes to the DNA of lambda phage GΦ5 at 5 to 25° C. below the melting temperature (Tm) of a double-stranded DNA having the sequence of lambda phage GΦ5 in aqueous solution at 1 M NaCl; wherein the promoter sequence promotes transcription in cells endogenously expressing human telomerase reverse transcriptase (hTRT).

See Appendix 1 for complete list of family members.

Patents:

GB 2321642

US 6,921,664

Assignee:

University of Colorado

Geron Corp.

Earliest priority:

18 Apr 1997

Filed:

18 Jan 2002

Granted:

26 Jul 2005

Expiry date:

18 Apr 2017 *

Telomerase

Claim 1:  A recombinant expression vector containing a polynucleotide that comprises an encoding region for a telomerase reverse transcriptase protein, variant, or fragment, wherein the protein, variant or fragment has telomerase catalytic activity when complexed with a telomerase RNA, and wherein a single-stranded DNA consisting of said encoding region hybridizes to a second single-stranded DNA at 5° C. to 25° C. below Tin aqueous solution at 1 M NaCl, wherein said second DNA is exactly complementary to SEQ. ID NO:224, and Tis the melting temperature under the same reaction conditions of double-stranded DNA having the sequence of SEQ. ID NO:224.

See Appendix 1 for complete list of family members.

Patents:

EP 841396

Patent applications:

CA 2267664

US 6,927,285

Assignee:

Geron Corp.

University Technology Corp.

Earliest priority:

18 Apr 1997

Filed:

12 Nov 1999

Granted:

09 Aug 2005

Expiry date:

18 Apr 2017 *

Genes for human telomerase reverse transcriptase and telomerase variants

Claim 1:  An isolated cDNA encoding human telomerase protein, wherein said cDNA is contained in plasmid pGRN121 having ATCC Deposit Accession No. 209016.

Claim 3:  An isolated nucleic acid encoding a naturally occurring human telomerase reverse transcriptase protein or variant thereof, wherein the polynucleotide hybridizes to a nucleic acid having the sequence in SEQ ID NO:173 at 5C to 25C below Tm in aqueous solution at 1M NaCl.

See Appendix 1 for complete list of family members.

Patents:

AU 734089

GB 2317891

EP 841396

Patent applications:

CA 2267664

EP 0954585

EP 1333094

** Patents and applications that are published in English and that have claims similar to the listed United States patent are specifically mentioned. The claims needed to be quite similar before it was included.  Even if not mentioned, a non-U.S. patent could have claims that encompass similar subject matter.  For example, a patent claiming a cell having an introduced nucleic acid encoding telomerase would encompass a cell containing an expression vector expressing telomerase, but is not listed as an “equivalent”.