WO1996040868A1 - Oligonucleotides essentiels de la telomerase de vertebre - Google Patents

Oligonucleotides essentiels de la telomerase de vertebre Download PDF

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WO1996040868A1
WO1996040868A1 PCT/US1996/009517 US9609517W WO9640868A1 WO 1996040868 A1 WO1996040868 A1 WO 1996040868A1 US 9609517 W US9609517 W US 9609517W WO 9640868 A1 WO9640868 A1 WO 9640868A1
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telomerase
rna
dna
htr
activity
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PCT/US1996/009517
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Carol Greider
Chantal Autexier
Ronald Pruzan
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Cold Spring Harbor Laboratory
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Priority to JP9501821A priority Critical patent/JPH11507818A/ja
Priority to AU61022/96A priority patent/AU701903B2/en
Priority to EP96918335A priority patent/EP0832190A1/fr
Publication of WO1996040868A1 publication Critical patent/WO1996040868A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Telomerase is an enzyme essential for telomere length maintenance. Conventional DNA polymerases cannot complete the replication of chromosome ends and, without a mechanism to overcome this problem, chromosomes are predicted to shorten with each round of cell division. Watson, J.D. (1972) Nature New Biol . 239 : 197-201 . Telomerase is a specialized telomere specific polymerase comprised of RNA and protein components, which elongate chromosomes through de novo nucleotide sequence addition.
  • telomere length A steady state equilibrium of telomere length is established in immortal single cell eukaryotes and is regulated by a number of different genes. Greider (1994) Current Opinion in Genetics and Dev. 4:203-211.
  • telomere length maintenance does not occur in primary human somatic cells and when they are passaged in culture, telomere length decreases in these cells.
  • Primary human cells have a limited lifespan in culture and telomere shortening correlates well with loss of replicative capacity. Harley, et al . (1990) Nature 345:458-460; Allsopp, et al . (1992) Proc . Natl . Acad. Sci . USA, USA 89:10114-10118.
  • Telomere shortening in tissues in vivo has been demonstrated for fibroblasts, leukocytes, and endothelial cells. Germ cell telomeres do not shorten with age, suggesting the germline is protected from telomere loss .
  • telomere shortening may be due to the inability of conventional polymerase to replicate chromosome ends.
  • Kim, et al . (1994) Science 266.- 2Q11-201S were not able to detect telomerase in a large number of primary cell lines and primary human tissues. In contrast to normal human cells, cancer cells from tissue culture and those taken directly from tumors contain detectable telomerase activity.
  • telomere and DNA oligonucleotides comprising truncated segments of the gene encoding telomerase RNA component, such as human telomerase RNA component, which are essential for telomerase activity (e.g., human telomerase activity) in cells and tissues.
  • telomerase RNA component such as human telomerase RNA component
  • DNA and RNA oligonucleotides sharing the biochemical and biological function of these essential oligonucleotides and differing only in alteration, substitution and/or deletion of one or more nucleotides
  • oligonucleotides which do not affect the activity of the enzyme.
  • These oligonucleotides can be derived from other vertebrates, especially mammals. Oligonucleotides which hybridize to the above-described DNA or RNA sequences are also included within the scope of this invention.
  • the truncated vertebrate telomerase of this invention is an essential oligonucleotide (RNA) and telomerase protein.
  • the protein component can be comprised of more than one subunit.
  • the RNA is encoded by DNA selected from nucleotides 44-204 of hTR as shown in Figure 6 (SEQ ID NO:l) .
  • Other essential oligonucleotides include sequences encompassing nucleotides 1-203, 1-273, 1-418, or DNA encompassing nucleotides 44-204 and sequential deoxyribonucleotides but shorter than nucleotides 1-445 (SEQ ID NO:2) of hTR as shown in Figure 6.
  • RNA component activity is required for functional RNA component activity.
  • RNA component activity is required for functional RNA component activity.
  • RNA component activity is required for telomerase activity and, thus, for maintenance of telomeric length in chromosomes.
  • truncated telomerase in which the RNA component is shorter than the complete RNA component of human telomerase has been produced and shown to have enzymatic activity.
  • telomere protein components can be produced by combining telomerase protein components with oligonucleotides prepared by recombinant methods, oligonucleotides which are isolated from sources in which they occur in nature or oligonucleotides which are synthetically produced. Similar types of truncated telomerases can be constructed by combining truncated oligonucleotides from other vertebrate telomerase RNA components with telomerase protein.
  • This invention also provides recombinant vertebrate telomerase in which the components are telomerase protein and the entire RNA component or a truncated RNA component, such as those encoded by nucleotides 44-204 of hTR as shown in Figure 6 (SEQ ID NO:l) or other essential oligonucleotides including sequences encompassing nucleotides 1-203, 1-273, 1-418, or DNA encompassing nucleotides 44-204 and sequential deoxyribonucleotides but less than 1-445 of hTR as shown in Figure 6, or the 30 nucleotide sequence (nucleotides 170-199 of SEQ ID NO:l) which is required for functional RNA component activity.
  • the components are telomerase protein and the entire RNA component or a truncated RNA component, such as those encoded by nucleotides 44-204 of hTR as shown in Figure 6 (SEQ ID NO:l) or other essential oligonucleotides including sequence
  • the telomerase protein can be synthesized, produced recombinantly or obtained from sources in which it occurs in nature.
  • the oligonucleotides of this invention can be used by themselves or combined with the protein of vertebrate telomerase for use in diagnostic or therapeutic methods and in assays for telomerase. Oligonucleotides that encompass the essential region of vertebrate telomerase are especially useful to block the function of telomerase by, for example, forming triple helices with DNA encoding RNA. components, preventing transcription.
  • essential oligonucleotides may serve as probes or primers to detect the presence of telomerase in cells and tissues.
  • probes or primers can be used diagnostically to determine the presence and amount of telomerase in cell, tissue or fluid samples obtained from an individual.
  • oligonucleotides of this invention as well as the vertebrate telomerases described above can be used to treat disorders arising from the presence of normal or abnormal telomerase or to provide telomerase wherever it could be beneficial.
  • Oligonucleotides in a sense or antisense orientation can prevent or inhibit telomerase activity by binding to essential regions of the RNA component or to telomerase protein.
  • Sense or antisense sequences can be delivered with or without telomerase protein by methods of gene therapy (such as infection or transfection) , as can plasmid or expression vectors encompassing recombinant DNA encoding vertebrate telomerase.
  • telomeres consisting of oligonucleotides, telomerase, or truncated telomerase, alone or combined with a suitable carrier, diluent or salt are also included in this invention. These compounds can be therapeutically applied to stimulate or modify the effects of telomerase in order to treat conditions, disorders or diseases arising from the lack of or abnormal telomerase activity. Examples of such uses include initiation or restoration of telomerase activity to counteract senescence or to prevent immortalization, and prevention or inhibition of telomerase activity in immortalized cells such as tumor cells or parasites.
  • telomere activity of invading eukaryotic parasites or tumors can also be detected and quantified. Therefore, the present invention provides a diagnostic tool through which inhibitors of telomerase activity can be tested and developed, and by which diseases such as cancer, or infections, such as yeast or protozoan diseases, can be diagnosed.
  • Figure 1 shows that the reconstitution of human telomerase activity after MNase treatment is specific to hTR.
  • Figure 2 shows the activity of telomerase reconstituted with telomerase RNA mutations and assayed in the absence or presence of dATP.
  • Figure 3 represents a functional analysis of 5' and/or 3' terminal deletions of hTR.
  • Figure 4 represents a mutational analysis of hTR residues 170-199.
  • Figure 5 is a linear representation of full length hTR which includes the template region (white box) and positions of several restriction sites present in the gene encoding hTR.
  • Figure 6 is the nucleotide sequence (SEQ ID NO:l) of the gene encoding the human RNA component of telomerase with the template boxed. The cleavage sites for several restriction endonucleases are marked.
  • Figure 7 is the hTR sequence used for several hTR reconstitution experiments with the template and cleavage sites for restriction endonucleases marked.
  • This invention provides isolated DNA encoding portions of the RNA component of human telomerase (hTR) that are essential to produce a biologically active human telomerase enzyme.
  • hTR human telomerase
  • the term "hTR” is used interchangeably for the RNA component or the gene encoding the RNA component.
  • This invention also provides truncated human telomerase RNA which, in combination with telomerase protein, produces biologically active human telomerase (i.e., one which catalyzes the addition of deoxyribonucleotides to the telomeres of chromosomes, thereby elongating the telomeres of these chromosomes) .
  • the essential oligonucleotides described herein are substantially shorter (comprise fewer nucleotides) than the endogenous human RNA component.
  • the term "essential" oligonucleotides refers to oligonucleotides which, when coupled with the human telomerase protein, form biologically active telomerase and without which biologically active telomerase is not produced. Both RNA that is essential to functional telomerase and DNA encoding RNA that is essential are referred to as "essential oligonucleotides" (essential DNA, essential RNA) .
  • Essential DNA of this invention includes isolated DNA sequences of hTR selected from the group consisting of: a) nucleotides 44-204 of hTR; b) nucleotides 1-203, 1-273, or 1-418 of hTR; and c) DNA encompassing nucleotides 44-204 and sequential deoxyribonucleotides but shorter than 1-445 of hTR. It further includes nucleotides 170-199 of hTR which are essential for telomerase activity although additional nucleotides are required to provide a biologically active RNA component.
  • This invention encompasses isolated DNAs whose sequences are provided ( Figures 5 and 6) and other DNAs which encode the same RNA sequences.
  • This invention further provides DNA which hybridizes to the essential DNA described above, especially under stringent conditions such as those described in Ausubel, et al . (1995) Current Protocols in Molecular Biology - A Laboratory Manual , Chapter 6, John Wiley & ⁇ Sons, NY, and DNA sequences which, but for the degeneracy of the genetic code, would hybridize to the essential DNA described above.
  • RNA component of human telomerase as shown in Figure 6 is not required for telomerase activity. In fact, only certain portions of the RNA component are essential to produce an active telomerase (e.g., by combining with human telomerase protein) . These encompass the template of the RNA component and a minimum number (160) of additional ribonucleotides upstream and downstream along the molecule (See encoding DNA molecule in Figure 6, nucleotides 44-204) .
  • This invention also provides, for the first time, functional vertebrate telomerase, produced with the complete nucleotide sequence of the RNA component or with the essential oligonucleotides of the RNA component (sequences ranging from 160 to 445 nucleotides in length) which have been delineated by the Applicants.
  • telomeres constructed with an RNA component comprising fewer ribonucleotides than the endogenous RNA component of the same species
  • telomerases modified by deletion of nonessential ribonucleotides and permit the production of telomerase variants which retain telomerase binding activity. These variants are useful in the treatment of conditions such as cell senescence (ageing) and in diseases as anti-tumor drugs.
  • telomere activity The following generally describes the reconstitution of recombinant human telomerase and the discovery of the essential oligonucleotides for telomerase activity. More specific methodology can be found in the examples.
  • telomerase activity was followed by a modification of the TRAP assay (Kim et al . , supra) (see Exemplification) . After nuclease digestion, which abolished endogenous telomerase activity, activity was restored by incubating MNase-treated telomerase with EDTA and an in vi tro transcribed hTR transcript (hTRl-557) , followed by the addition of Mg 2+ ( Figure 1) .
  • the hTRl-557 was transcribed from plasmid pGEM33 digested with Ec ⁇ RV .
  • the hTRl-557 contains the entire hTR (445 nucleotides (nt) ) plus downstream sequences (112 nt) .
  • Vector sequences 5' and 3' to hTR genomic sequence were also transcribed so that the total length of the transcript is 630 nt .
  • no RNA was added, no telomerase activity was restored.
  • two different concentrations of extract (6 ⁇ l and 12 ⁇ l) were used and to test for linearity in the reconstitution, two different concentrations of RNA were added (0.4 ⁇ g and 0.8 ⁇ g) .
  • the amount of reconstituted activity increased with the increased level of both the extract and hTR indicating that reconstitution was dependent on the added RNA ( Figure 1) .
  • RNAs tested were E. coli 5S, E. coli 16S and 23S RNAs, and Tetrahymena telomerase RNA ( Figure 1) . No activity was seen when these RNAs were added instead of hTR in the reconstitution assay. Also, no T 2 G 4 repeats were generated by adding Tetrahymena telomerase RNA to MNase-treated human extract, using the C-strand primer C 4 A 2 to detect the presence of amplified elongation products (see below and Exemplification) .
  • telomere activity was isolated which synthesized the expected mutant telomere repeats (Blasco et al . , 1995 Science 269 : 1267-1270 ; Feng, et al . (1995) Science 269 : 1236 -1241 .
  • hTR mutants were used in the in vi tro reconstitution experiments.
  • the sequence encoding the template region of hTR was changed from CTAACCCTA to CAAACCCAA (encoding hTR-C 3 A 3 ) and in pGEM36 to CCAACCCCA (encoding hTR-C crampA 2 ) , which should specify TTGGGG and TTTGGG repeats, respectively.
  • the RNA transcribed from these plasmids, hTR-C 3 A 3 and hTR-C 4 A 2 like hTRl-557, contain sequences downstream of hTR and vector sequences (see Exemplification) .
  • telomeres To assay the products of the mutant telomerases, a two step amplification protocol was used as described (Feng, et al . (1995) Science 269 : 1236 -1241) .
  • dATP was omitted from the initial telomerase reaction.
  • wild type (naturally-occurring) telomerase will not generate elongation products, however if the mutant RNAs (hTR-C 3 A 3 and hTR-C 4 A 2 ) are functional, they should generate telomerase products.
  • dATP was added and the C-strand primer corresponding to the appropriate mutant was used for PCR amplification (see Exemplification) .
  • telomere RNA The 450 nucleotide human telomerase RNA is much larger than the Tetrahymena (160 nt) and other ciliate telomerase RNAs (147-209 nt) , however it is significantly smaller than the yeast telomerase RNA (1300 nt) (Greider and Blackburn . (1989) Nature 337:331-337; Lingner et al . (1994) Genes & Dev. 8:1984-1998; Singer and Gottschling (1994) Science 266: 404 -409 ; Feng, et al . , supra ; McEachern and Blackburn (1995) Nature 376: 403 -409 ; McCormick-Graham and Romero (1996) Mol . Cell . Biol . 15:1871-1879; Zaug et al . (1996)
  • telomerase activity was reconstituted with RNAs deleted at the 5' and/or 3' ends ( Figures 3 and 5) .
  • a plasmid encoding only hTR was constructed that will generate an RNA without downstream genomic sequences or vector sequences (upstream or downstream) (phTR+1; see Exemplification) .
  • phTR+1 a plasmid encoding only hTR was constructed that will generate an RNA without downstream genomic sequences or vector sequences (upstream or downstream) (phTR+1; see Exemplification) .
  • phTR+1 genomic sequences or vector sequences
  • hTRl-445 Full length wild-type hTR is denoted as hTRl-445.
  • the numbers refer to the position of residues within the full length hTR.
  • Each RNA, with the enzyme used to cut the plasmid (in brackets) is denoted as hTRl-445 (Fspl), hTRl-418 (ApaLI) , hTRl-273 (BspEl) , hTRl-203 (S al ) , hTRl-182 (PvuII) , hTRl-169 (Bbvl) and hTRl-159 (Xbal) .
  • RNAs were then tested which were truncated at both the 5' and 3' ends that contained residue 44 through to either residue 184 or 204. Both of these RNAs were active in reconstitution, although they had reduced activity compared to the addition of full length hTR.
  • An hTR truncation starting at position 44 and ending at residue 170 (hTR44-170) was not active in reconstitution, indicating that a region of hTR, approximately 33 residues in length, between 170 and 203 is important for hTR function.
  • the ability of RNAs containing only residues 44-184 and 44-204 to reconstitute activity suggest that the 44 residues preceding the template are not essential for activity.
  • a 30 nucleotide region of hTR spanning residues 170-199 is essential for activity
  • substitutions were made spanning residues 170 to 179 (hTR170*) , 180 to 189 (hTR180*) and 190 to 199 (hTR190*) .
  • telomerase activity reconstituted with 2.5 pmol of these RNAs were compared to that reconstituted with three 3' deletions (hTRl-159, hTRl-169 and hTRl-182) or hTRl-445 ( Figures 4 and 5) .
  • Reconstitution with either hTR170*, hTR180*, hTR190* restored little activity, comparable to the activity restored by hTRl-169 and hTRl-182 (less than 8% of activity restored by hTRl-445) .
  • this region contains the site of the 17 nucleotide insertion that disrupted the ability of hTR to function in vi tro and in vivo .
  • these mutants define an essential functional region of the hTR.
  • this invention delineates the essential oligonucleotides necessary to reconstitute a functional human RNA component.
  • the findings described herein are summarized as follows. Human telomerase activity was restored to MNase-treated partially purified human telomerase by the addition of EDTA and in vi tro transcribed human telomerase RNA, as previously described for Tetrahymena telomerase (Autexier and Greider (1994) Genes & Dev. 8:563-575) .
  • telomere levels varied, but were always lower (less than 10%) , as was the case for levels of reconstituted Tetrahymena telomerase, suggesting that the added hTR may not be completely functional compared to endogenous telomerase RNA.
  • the transcribed RNA may lack some modifications or assume incorrect conformations which prevent it from forming a functional RNP (Autexier and Greider (1994) Genes & Dev. 8:563-575) .
  • the extra sequences downstream of hTR and the transcribed vector sequences did not inhibit the ability of hTRl-557 to reconstitute telomerase activity, compared to full length hTRl-445.
  • telomere RNA mouse telomerase RNA
  • mTR mouse telomerase RNA
  • the telomerase enzyme is about 250 kDa and consists of two proteins, of 80 and 95 kDa (Collins et al . (1995) Cell 81:677-686) .
  • the predicted sizes of the human (750 kDa) and mouse telomerase (>1000 kDa) enzymes differ from each other, and are larger than the Tetrahymena telomerase enzyme (Greider et al .
  • the mouse telomerase enzyme may consist of more or larger protein components than the human enzyme and consequently, at least in vi tro, mTR may be unable to form a functional complex with the human telomerase proteins.
  • telomere sequence changed the sequence of the elongation products generated by reconstituted telomerase, as seen with Tetrahymena , in vivo and in vi tro, (Yu et al . (1990) Nature 344:126-132; Autexier and Greider (1994) Genes & Dev. 8:563-575) and human and mouse (Blasco et al . , 1995, supra; Feng, et al. (1995) Science 269 : 1236-1241, in vivo, confirming the requirement and specificity of hTR in the in vi tro reconstitution of human telomerase activity.
  • the specificity and fidelity of reconstituted activity suggests that the reconstitution assay will be a useful biochemical tool for dissecting native human telomerase function, as it has been for Tetrahymena (Autexier and Greider, (1995) Genes & Dev. 15:2227-2239) .
  • telomere activity in vi tro is not absolutely required for telomerase activity in vi tro, including residues 5' to the template.
  • hTR approximately 159-203 nt length. This minimal function region is similar in size to the full length telomerase R ⁇ As from ciliates, which range in size from 147-208 nt (Greider, e ⁇ al . (1996) ; McCormick-Graham and Romero, supra) .
  • telomere reconstituted with the deleted hTRs which are still functional, compared to activity reconstituted with full length hTR, suggests that the deleted regions may contain sequences or potential secondary structures important for binding of telomerase protein components, for assembly and for overall structure and function of the telomerase complex. It is also possible that the remainder of the R ⁇ A plays some role in vivo, perhaps by binding proteins important in the regulation of telomerase.
  • This deletional analysis of hTR, and the size of the telomerase R ⁇ As in S. cerevisiae and K. lactis (1300 nt) suggest that the entire telomerase R ⁇ A in these organisms may not be needed for function.
  • the U2 snR ⁇ A is 1175 nt long compared to in most other organisms where it is about 190 nt long (Ares, (1986) Cell 47:49-59) .
  • Internal deletions which reduce the length of the yeast U2 snRNA to that of other U2 snRNAs are still active in splicing and yeast with the deleted U2 snRNA have normal growth rates (Igel and Ares (1988) Nature 334:450- 453; Shuster and Guthrie (1988) Cell 55:41-48) .
  • the yeast Ul snR ⁇ A is also larger (568 nt) than in metazoans where it is 165 nt.
  • Yeast cells carrying a deletion of 316 internal residues allows wild-type growth (Siliciano et al . (1991) Nucleic Acids Res . 19 : 6367-6372 ) .
  • Deletional analysis of hTR (in vivo) and yeast telomerase R ⁇ A will be required to further elucidate the role of the extra residues in these longer telomerase R ⁇ As. It is not clear, for hTR, mTR, or the yeast telomerase R ⁇ As, if there is a minimal core conserved secondary structure for all of these R ⁇ As which is essential, like with R ⁇ ase P (Waugh et al . (1989) Science 244 : 1569 -1571) .
  • telomere RNAs In Tetrahymena and other ciliate telomerase RNAs, there is a conserved region upstream of the template, which plays a role in determining the 5' boundary of the template, and the sequence synthesized by telomerase in vitro (Romero and Blackburn (1991) Cell 67 :343-353; Autexier and Greider, 1995, supra) . This sequence is absent in hTR, mTR and yeast telomerase RNAs (Singer and Gottschling (1994) Science 266: 404 -409 ; Blasco et al . , 1995, supra ; Feng, et al . , supra; McEachern and Blackburn (1995) Nature 376: 403 -409 .
  • telomere RNAs contain templates which are located approximately 50 nt from the 5' end, except for yeast where the template is more centrally located (Singer and Gottschling, supra ; McEachern and Blackburn, supra) .
  • Tetrahymena telomerase RNA which is only 159 nt in length, deletions of as little as 19 residues from the 5' end abolish activity indicating that residues 5' to the template are essential.
  • residues 5' to the template are not essential in vi tro. However, the 5' end may play some role, in vivo, perhaps by maintaining a correct RNA structure for proteins to interact with other sequences or structures of hTR.
  • this invention provides nucleic acid hybridization probes or primers which hybridize to a sample nucleotide sequence, its complement or to a fragment of either of these.
  • Methods of detecting telomerase with an essential oligonucleotide (DNA or RNA) in a cell, tissue, or fluid sample include the steps of: preparing the sample so that the essential oligonucleotide will hybridize to telomerase in the sample; combining or contacting the sample with the DNA or RNA under conditions under which hybridization of complementary nucleic acids occurs; and detecting hybridization wherein if hybridization occurs, telomerase is present in the sample. Further assays can be carried out to confirm whether telomerase is active. An additional step can also be taken to measure the amount of hybridization to determine the amount of telomerase in the sample.
  • These essential oligonucleotide probes can be detectably labeled (for example with radioactive or fluorescent materials, or with biotin or avidin) by methods known to those of skill in the art.
  • primers which are all or a portion of essential oligonucleotides can be used to initiate DNA synthesis for amplification or diagnostic procedures. If a primer is a portion of an essential oligonucleotide, it must be of sufficient length to hybridize to DNA and remain hybridized under the conditions used, although the nucleotides may not be identical in sequence. In general, a primer will be at least 12 nucleotides and can be up to 100 nucleotides in length. Preferably primers will be 18 to 30 in length. The primers may be labeled before hybridization so that detection of labeled hybridized material correlates with the presence and/or amount telomerase in a sample taken from an individual.
  • RNA component for early detection of diseases, such as cancers or parasites, where only a few cells may be present in the sample. They also relate to procedures wherein recombinant telomerase is synthesized (with a whole or truncated RNA component) and an active telomerase enzyme produced.
  • telomeres of chromosomes are not lengthened.
  • Another aspect of this invention relates to the use of the isolated DNA sequences in antisense therapy to block telomerase activity.
  • Antisense therapy refers to administration of or in si tu generation of oligonucleotides or their derivatives which specifically hybridize with the endogenous telomerase RNA component and/or which hybridize with genomic DNA encoding the RNA component so as to inhibit expression of that enzyme, e.g., by inhibiting transcription and/or translation.
  • This invention also relates to antisense constructs that can be delivered, for example, in an expression vector that, when transcribed in the cell, produces RNA which is complementary to at least the essential portions of the telomerase RNA component.
  • Expression vectors such as plasmids, are capable of directing the expression of genes to which they are operatively linked.
  • the antisense construct is an oligonucleotide which is generated ex vivo and which, when introduced into the cell, causes inhibition of expression by hybridizing with the telomerase RNA component or by hybridizing with genomic sequences encoding the RNA component, thus preventing telomerase from serving as a template for telomeric DNA synthesis.
  • oligonucleotides are preferably modified oligonucleotides which are resistant to endogenous nucleases and therefore stable in vivo.
  • General approaches to constructing oligomers useful in antisense therapy have been described, for example, in Inouye, U.S. Patent No. 5,272,065, incorporated herein by reference, and reviewed by Stein, et al . (1988) Cancer Res . 48 : 2659-2668.
  • telomeres that bind to essential telomerase protein
  • a DNA construct encoding an RNA essential oligonucleotide can be linked to a strong promoter to express excess sense strands at a high level which competitively inhibit the specific binding of an essential protein.
  • Other sense sequences may be constructed that will bind to the DNA essential oligonucleotides to form a triple helix and prevent transcription.
  • Knowledge of the essential portions of the RNA component increases the likelihood of success for these endeavors because these constructs contain nucleic acid sequences that will bind.
  • Oligonucleotides that bind to the RNA component of telomerase may also be combined with ribozyme sequences to produce molecules that not only bind but specifically cleave the RNA component, thus inactivating telomerase.
  • RNA and protein components are involved in telomeric primer recognition and binding by telomerase (Collins and Greider (1993) Genes & Dev. 7:1364-1376) .
  • the ability to reconstitute human telomerase activity from partial RNA component sequences and the telomerase protein not only facilitates the structural and functional dissection of this ribonucleoprotein, it allows the production of fundamental synthesized enzymes with multiple applications.
  • Truncated or recombinant human telomerase in all of the disclosed forms can be used to design drugs or produce pharmaceutical compositions for treating disorders in which telomerase activity would be beneficial.
  • Functional telomerase molecules can be delivered to cells to stimulate telomerase activity in cells normally lacking detectable telomerase or in cells which are abnormal because telomerase activity is present. Telomerase can be used to extend replicative cell life span and deter cell senescence and possible subsequent immortalization of cells.
  • modified oligomers and telomerase enzymes of the invention are useful in therapeutic, diagnostic and research contexts.
  • Recombinant telomerase can be especially useful in therapy where it is important to slow the loss of telomere sequences (i.e., preventing senescence of cells) .
  • compositions containing the telomerases of this invention can be used to treat conditions such as those described above. Additionally, the telomerase molecules can be used in screening other agents, for example, in binding assays, to identify compounds which inhibit or stimulate the activity of telomerase in vi tro or in vivo.
  • Pharmaceutical compositions containing the telomerases or essential oligonucleotides of this invention may also contain pharmaceutically acceptable carriers, diluents, fillers, salts, buffers, stabilizers and/or other materials well known in the art.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients (s) .
  • the characteristics of the carrier or other material will depend on the route of administration which can be carried out in a variety of conventional ways.
  • the amount of the active ingredient (s) in the pharmaceutical composition of this invention will depend upon the nature and severity of the condition being treated, and may depend on the nature of any prior treatments which the individual has undergone. In any event, such methods require the administration of a therapeutically effective amount of the active ingredient (s) which is at least the minimum amount necessary to effect a beneficial change in the condition being treated. It will be appreciated that the actual preferred amounts of active compound in a specific case will vary according to the specific compound being utilized, the particular compositions formulated, the mode of application, the particular situs of application, and the individual being treated. Dosages for a given recipient will be determined on the basis of individual characteristics, such as body size, weight, age and the type and severity of the condition being treated.
  • formulations described herein may be used for veterinary as well as human applications and that the term “individual” or “host” should not be construed in a limiting manner.
  • telomere activity has been demonstrated in human ovarian carcinoma cells, but not in normal cervical endothelial cells.
  • Telomere shortening before crisis may be lethal, but those cells that can reactivate telomerase maintain telomere length and survive crisis. This model suggests that if telomerase is required for the growth of immortalized cells, telomerase inhibitors may be excellent anti-cancer drugs.
  • This invention provides a method by which cancers may be diagnosed prior to or during clinical manifestation of symptoms by means of detecting telomerase activity in somatic cells that normally do not express telomerase.
  • Telomerase RNA expression in a sample of somatic cells or tissue can be detected using the DNA or RNA probes described herein; this is indicative of expression of telomerase which, in turn, is an indication of immortal cancer cells since most somatic cells do not normally produce telomerase.
  • Detection of hybridization in tissues that normally lack telomerase is an indication of a predisposition to cellular immortalization or cancer, or to the presence of cancer or immortal cells.
  • such methods of detecting the presence of immortal cells or a predisposition to immortalization in a eukaryotic cell, tissue or fluid sample can include: obtaining a cell, tissue or fluid sample; and using the essential oligonucleotides to determine the presence of telomerase in the sample (for example, by hybridization with a labeled probe) , wherein if the sample demonstrates the presence of telomerase, immortal cells or the predisposition to immortalization is present.
  • the same method may be used to detect a predisposition to cancer or the presence of cancer cells or tissue.
  • the recombinant telomerase can be used to produce polyclonal or monoclonal antibodies to the telomerase protein.
  • These antibodies allow detection of telomerase in vivo or in vi tro at minute levels and can serve to indicate the presence of abnormal telomerase activity due to tumor cell growth or other conditions such as parasitism by foreign eukaryotic organisms (i.e., yeasts, protozoa) , and the like. Because antibodies can accurately detect small amounts of antigen, early diagnosis of these disorders is possible.
  • the present invention also provides a means for developing drugs and pharmaceutical compounds that destroy or otherwise inactivate or interfere with the activity of telomerase.
  • the truncated or recombinant telomerase, or the essential oligonucleotides of this invention can be used to screen for potential new drugs and pharmaceutical compounds effective as anti-cancer and anti-microbial agents, as described below.
  • additional telomerase activity may have an anti-aging effect and result in restoration of cells by stabilizing telomere length, compounds which stimulate or trigger telomerase activity can be identified.
  • a method for screening agents which inhibit, prevent, or stimulate telomerase activity can comprise the steps of: contacting the potential agent with truncated or recombinant telomerase under conditions wherein telomerase is active; and determining whether the activity of telomerase is decreased or increased; whereby if the telomerase activity is decreased, the agent is identified as a telomerase inhibitor and, if the telomerase activity is increased, the agent is identified as a telomerase stimulator.
  • the telomerase protein can also be combined with the RNA component of telomerase to produce a functional recombinant telomerase molecule which can be delivered to cells by conventional methods.
  • DNA encoding a telomerase molecule can be introduced into target cells by recombinant DNA methods and transformation technology.
  • the incorporation of extra copies of functional telomerase molecules may extend the replicative life span of the host cell by stabilizing telomere length.
  • this invention includes methods for targeted gene therapy in individuals. Another application of this invention is the detection of eukaryotic disease-causing organisms in somatic cells and tissues of vertebrates and treatment of the resulting disease.
  • telomeres There are many fungi, protozoa, and even algae that invade the cells and tissues of vertebrates and are the cause of various diseases.
  • diseases include, but are not limited to, aspergillosis, histoplasmosis, candidiasis, paracoccidioidomycosis, malaria, trichinosis, filariasis, trypanosomiasis (sleeping sickness) , schistosomiasis, toxoplasmosis, and leishmaniasis.
  • telomerase probably require telomerase and express this enzyme as they multiply inside host cells which do not normally produce telomerase.
  • the above- described methods to detect telomerase can be used to develop early detection and diagnosis procedures for these eukaryotic microbial parasites.
  • An example of such a method to detect a disease caused by a eukaryotic microbial organism in a sample of cells from an individual may comprise the steps of: obtaining a sample of cells from the individual ; and determining if microbial telomerase is present in the sample; wherein if the sample demonstrates telomerase of a eukaryotic microbe, a disease caused by a eukaryotic microbial organism is present. If telomerase is normally present in the cells of the individual, e.g., germline cells, the microbial telomerase can be distinguished by determining if hybridization occurs with a probe specific for non-human telomerase.
  • telomeres may inhibit telomerase and thus prevent the multiplication of species of this parasite in an individual without affecting the host's somatic cells and tissues.
  • antisense nucleic acids complementary to essential oligonucleotides
  • the present invention provides a process for producing a recombinant product comprising: producing an expression vector which includes DNA which encodes a telomerase molecule; transfecting or infecting a host cell with the vector; and culturing the transfected or infected cell line to produce the encoded telomerase molecule (recombinant telomerase) .
  • the standard techniques of molecular biology can be used to prepare DNA sequences coding for the RNA and protein components of telomerase, and for construction of vectors with appropriate promoters for enzyme expression in a host cell. Suitable host cell/vector systems, transfection or infection methods and culture methods are well known in the art. These systems may also be used to produce antibodies to telomerase.
  • telomerase activity was eluted with buffer A containing 0.3 M NaCl.
  • the active fractions were pooled and concentrated with 50% ammonium sulfate and applied to a Toyopearl HW-65F column equilibrated in buffer A containing 0.1 M NaCl and eluted with the same buffer. Active fractions (1.7 mg/ml total protein) were pooled and used in reconstitution.
  • the C-strand primers, C 3 TA 2 primer, C 3 A 3 primer, or C 4 A 2 primer were used to detect the corresponding G-rich telomerase elongation products T 2 AG 3 , T 3 A 3 and T 2 G 4 .
  • These primers are modified versions of the Cx primer (Kim et al. , supra) and contain three repeats of the appropriate telomeric sequence plus some additional sequences at the 5' end (Trap-ezeTM kit, Oncor, Inc., 209 Perry Parkway, Gaithersberg, MD 20877, www.oncorinc.com/home) .
  • telomerase a human telomerase fraction of human telomerase fractions (in 1 mM EGTA) were treated for 10-15 min at 30 * C with 1.9-2.1 mM CaCl 2 and 1.0-1.15 Unit of micrococcal nuclease (MNase) (Pharmacia) per ⁇ l of extract. The MNase was inactivated by the addition of 1.5 mM EGTA. The extract was then incubated with in vi tro transcribed RNA and 5 mM EDTA for 5 min at 37"C. 8mM MgCl 2 was added prior to assaying for telomerase activity.
  • Mock-treated telomerase consists of telomerase treated as described above, with the addition of EGTA prior to MNase.
  • telomerase assays were performed with telomerase pretreated as indicated.
  • MNase-treated telomerase was reconstituted: without addition of RNA (lanes 1 and 2); with 0.4 ⁇ g hTRl-557
  • a 480 bp fragment containing the T7 promoter and positions +1 to 445 of the gene encoding hTR was generated by PCR from the cloned hTR gene (Feng, et al . , supra) , digested with Hindlll and BamHI, and cloned into pUC119 digested with the same enzymes.
  • the template used in PCR was a 794 bp EcoRI-FspI fragment from pGRN33, which contains a 2.5 kb genomic fragment including the hTR coding region (Feng, et al . , supra) .
  • the sequences of primers hTR+1 and hTR+445 used in PCR were
  • hTR+1 contains the T7 promoter and a Hindlll site at the 5' end.
  • hTR+445 contains a BamHI site and an engineered Fspl site at +445 at the 5' end.
  • PCR conditions were the following: 1 X Taq extender buffer (Stratagene) , 0.5 uM primers, 1 ng template, 200 uM dNTPs (Pharmacia) , 5U Taq extender (Stratagene) , 5U Taq polymerase (Perkin-Elmer) , 5 ⁇ g T4 gene 32 product (Boehringer Mannheim) , 30 cycles at 94 * C for 40 sec, 58 * C for 20 sec and 72 * C for 60 sec.
  • the resulting clone, phTR+1 contained hTR downstream of the T7 promoter as confirmed by sequencing both strands of the inserted DNA by the dideoxy-mediated chain termination method as per the manufacturer's instructions (U.S.
  • phTR170, phTR180 and phTR190 were constructed by replacing a 114 bp Xbal-BspEl fragment in phTR+l by the same fragments (generated by PCR) containing 10 base pair mutations spanning positions 170-179, 180-189 or 190-199 of hTR respectively.
  • phTR+l digested with Hindlll and BamHI was used as a template in PCR.
  • the 177 bp PCR fragment was digested with Xbal and BspEl and the resulting 114 bp fragment cloned into the phTR+l Xbal and BspEl restriction sites.
  • PCR conditions were as described for phTR+l except 10 ng of the template fragment was used and the cycling conditions were the following: 5 cycles at 94 * C 40 sec, 54 * C 20 sec, 72 * C 60 sec, followed by 25 cycles 94"C 40 sec, 60 * C 20 sec, 72 * C 60 sec.
  • Primers hTR170 and TRC31 were used in PCR for constructing phTR170. The sequences of hTR170 and TRC31 are
  • phTRl ⁇ O and phTR190 were constructed in a manner similar to phTR170, using primers hTR180 and hTR190, respectively, instead of hTR170.
  • the sequences of hTR180 and hTR190 are 5' -GGGGTCTAGAGCAAACAAAAAATGTGTCGACGACCCCCGTTCGCCTCCCGG-3' and 5' -GGGGTCTAGAGCAAACAAAAAATGTCAGCTGCTGGGGGCAAGCGGTCCCGGGGACC TGCG-3', respectively.
  • the resulting clones, phTR170, phTRl ⁇ O and phTR190 contained the expected substitutions within the inserted Xbal/B ⁇ pEI fragment, as confirmed by sequencing the inserted DNA.
  • telomerase was re- programmed to synthesize mutant telomere repeats and the activity of the reconstituted telomerase with telmerase RNA mutations was assayed in the absence or presence of ATP as indicated: no RNA, lane 1; hTRl-557, lanes 2 and 3; hTRl-557 with a 17 base insertion at position 176 (hTR+17) , lanes 4 and 5; hTRl-557 with a modified template (C 3 A 3 ) encoding T 3 G 3 repeats (hTR-C 3 A 3 ) , lanes 6 and 7; hTRl-557 with a modified template (C 4 A 2 ) encoding T 2 G 4 repeats (hTR-C 4 A 2 ) , lanes ⁇ and 9.
  • different C-strand oligonucleotides were used in the PCR assay to detect the appropriate telomerase elongation products, and 3 pmoles of RNA were added to each reaction
  • telomerase was reconstituted with hTR of various sizes as indicated: no RNA, lane 1; hTRl-159 (159 nt) , lane 2; hTRl-169 (169 nt) , lane 3; hTRl-l ⁇ 2 (182 nt) , lane 4; hTRl-203 (203 nt) , lane 5; hTRl-273 (273 nt) , lane 6; hTRl-445 (445 nt) , lane 7; hTR 44-184 (140 nt) , lane 6; hTR 44-204 (160 nt) , lane 9; hTRl-445 (445 nt) , lane 10.
  • Each reaction in lanes 2-7 included 2.5 pmoles of RNA, and 3 pmoles of RNA were added to reactions shown in lanes
  • Lanes 1-7 were exposed for 2 days and lanes 8-10 for 5 days.
  • RNAs used in reconstitution were in vi tro transcribed with SP6 or T7 RNA polymerase (Stratagene) using pGEM33 (encoding wild-type hTR plus downstream sequences-total length 557 nt-hTRl-557) digested with £coRV, pGEM34 (encoding hTRl-557 with a C 3 A 3 -containing template) , pGEM36 (encoding hTRl-557 with a C 4 A 2 -containing template) or pGEM38 (encoding hTRl-557 with a 17 bp insertion at residue 176) digested with Ec ⁇ RV ( Figures 1 and 2) .
  • RNAs used in reconstitution contained 5' (34 nt) and 3' (41nt) flanking RNA from the pGEM vector which does not encode the telomerase RNA.
  • the RNAs made from phTR+l, phTR170, phTRl ⁇ O and phTR190 contained only hTR sequences.
  • the hTR44-170, hTR44-164 and hTR44-204 hTRs were made using DNA fragments generated by PCR. For all three, the 5' primer was T7hs4 ⁇
  • RNAs 5' -GGAGGGGCGAACGGGCCAGCA-3' . Standard in vi tro transcription reaction conditions recommended by the RNA polymerase manufacturer were used. The RNAs were either gel purified or the transcription reactions treated with 3U RNase-free DNase (Pharmacia) per ⁇ g of DNA for 10 min. The RNA concentrations were determined by specific activity determination of RNA synthesized with radionucleotides. The integrity and size of the RNAs were determined by Northern analysis or staining with ethidium bromide.
  • RNA Size of the RNAs are the following, with the actual number of residues of hTR and the enzyme used in parentheses: hTRl-557, 630 nt (EcoRV 557) for all hTRs made from pGEM based vectors (+17 nt for hTR+17) .
  • RNAs made from pUC119 based plasmids were the following: hTRl-159 (Xbal 159) , hTRl-169 (B vl 169), hTRl-l ⁇ 2 (PvuII 182) , hTRl-203 (Smal 203), hTRl-273 (BspEl 273) and hTRl-445 (Fspl 445) .
  • the Fspl site at position 445 was created by site-directed mutagenesis.
  • the TGCAGT spanning nucleotides 443 to 448 was altered to TGCGCA which is cut by Fspl.
  • Tetrahymena telomerase RNA used as a control was in vi tro transcribed as previously described (Autexier and Greider (1994) Genes & Dev. 8:563-575) .
  • the 5S and 16S, 23S E. coli rRNAs were from Boehringer Mannheim and Sigma, respectively.
  • Mouse RNase P RNA and mouse telomerase RNA were a gift of Maria Blasco.
  • telomerase was reconstituted with hTR of various sizes and sequence as indicated: mock-treated telomerase, lane 1; no RNA, lane 2; hTRl-159 (159 nt) , lane 3; hTRl-169 (169 nt) , lane 4; hTRl-162 (162 nt) , lane 5; hTR170* (445 nt), lane 6; hTRl ⁇ O* (445 nt) , lane 7; hTR190* (445nt) , lane 8; hTRl-445 (445 nt) , lane 9.
  • the reactions shown in lanes 3-9 had 2.5 pmoles of RNA added to them.
  • the gel was exposed to X-ray film for 2 days, except for lane 1, which was exposed for 18 hours.
  • Figure 5 is a linear representation of full-length hTR.
  • the schematic includes the template region (white box) and positions of several restriction sites present in the gene encoding hTR.
  • the Fspl site was engineered into the gene.
  • the 5' and 3' deletions and substitutions in hTR are indicated (stippled boxes) , along with the relative activities these RNAs restore when added back to MNase-treated extract.
  • the size of the transcribed RNAs are also indicated.
  • activity of hTR+17 which has a 17 nucleotide insertion at position 176 in hTRl-557 is included.
  • the transcribed RNA in this case includes sequences downstream of hTR, plus vector sequences 5' and 3' to hTR.
  • the hTR sequence shown in Figure 7 was used to generate some of the reagents for the hTR reconstitution assays.
  • the actual hTR sequence, discovered at Cold Spring Harbor Laboratory is shown in Figure 6.

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Abstract

L'invention porte sur des séquences d'ADN codant pour des oligonucléotides d'un ARN tronqué correspondant à la partie ARN de la télomérase de vertébré qui est essentielle pour son fonctionnement, ainsi que l'utilisation de ces oligonucléotides. L'invention porte également sur la télomérase de vertébrés produite en combinant l'oligonucléotide d'ARN ou la partie ARN isolée avec la protéine de cette télomérase de vertébré.
PCT/US1996/009517 1995-06-07 1996-06-06 Oligonucleotides essentiels de la telomerase de vertebre WO1996040868A1 (fr)

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WO1998028442A1 (fr) * 1996-12-20 1998-07-02 Geron Corporation Procedes de detection et d'inhibition de la telomerase dans l'arn
WO1998045450A1 (fr) * 1997-04-04 1998-10-15 Geron Corporation Telomerase purifiee
WO1998059040A2 (fr) * 1997-06-20 1998-12-30 Bayer Aktiengesellschaft Sous-unite de telomerase catalytique humaine et son utilisation therapeutique et diagnostique
WO1999035243A2 (fr) * 1998-01-12 1999-07-15 Cold Spring Harbor Laboratory Allongement de la duree de vie d'une cellule, et procedes et reactifs associes
DE19804372A1 (de) * 1998-02-04 1999-08-05 Michael W Dr Dr Dahm Verfahren zur quantitativen Bestimmung von Tumorzellen in einer Körperflüssigkeit und dazu geeignete Testkits
US5968506A (en) * 1995-08-04 1999-10-19 Geron Corporation Purified telomerase
US6261836B1 (en) 1996-10-01 2001-07-17 Geron Corporation Telomerase
US6475789B1 (en) 1996-10-01 2002-11-05 University Technology Corporation Human telomerase catalytic subunit: diagnostic and therapeutic methods
US6517834B1 (en) 1995-08-04 2003-02-11 Geron Corporation Purified telomerase
US6545133B1 (en) 1995-08-04 2003-04-08 Geron Corporation Methods for purifying telomerase
US6610839B1 (en) 1997-08-14 2003-08-26 Geron Corporation Promoter for telomerase reverse transcriptase
US6808880B2 (en) 1996-10-01 2004-10-26 Geron Corporation Method for detecting polynucleotides encoding telomerase
US6927285B2 (en) 1996-10-01 2005-08-09 Geron Corporation Genes for human telomerase reverse transcriptase and telomerase variants
US7378244B2 (en) 1997-10-01 2008-05-27 Geron Corporation Telomerase promoters sequences for screening telomerase modulators
US7413864B2 (en) 1997-04-18 2008-08-19 Geron Corporation Treating cancer using a telomerase vaccine
US7585622B1 (en) 1996-10-01 2009-09-08 Geron Corporation Increasing the proliferative capacity of cells using telomerase reverse transcriptase
US7622549B2 (en) 1997-04-18 2009-11-24 Geron Corporation Human telomerase reverse transcriptase polypeptides

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US7285639B2 (en) 1996-10-01 2007-10-23 Geron Corporation Antibody to telomerase reverse transcriptase
US6617110B1 (en) 1996-10-01 2003-09-09 Geron Corporation Cells immortalized with telomerase reverse transcriptase for use in drug screening
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WO1998028442A1 (fr) * 1996-12-20 1998-07-02 Geron Corporation Procedes de detection et d'inhibition de la telomerase dans l'arn
US7585966B1 (en) * 1996-12-20 2009-09-08 Geron Corporation Inhibitory polynucleotides directed against the RNA component of telomerase
WO1998045450A1 (fr) * 1997-04-04 1998-10-15 Geron Corporation Telomerase purifiee
US7622549B2 (en) 1997-04-18 2009-11-24 Geron Corporation Human telomerase reverse transcriptase polypeptides
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US7750121B2 (en) 1997-04-18 2010-07-06 Geron Corporation Antibody to telomerase reverse transcriptive
WO1998059040A3 (fr) * 1997-06-20 1999-07-22 Bayer Ag Sous-unite de telomerase catalytique humaine et son utilisation therapeutique et diagnostique
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