WO1998059040A2 - Sous-unite de telomerase catalytique humaine et son utilisation therapeutique et diagnostique - Google Patents

Sous-unite de telomerase catalytique humaine et son utilisation therapeutique et diagnostique Download PDF

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WO1998059040A2
WO1998059040A2 PCT/EP1998/003468 EP9803468W WO9859040A2 WO 1998059040 A2 WO1998059040 A2 WO 1998059040A2 EP 9803468 W EP9803468 W EP 9803468W WO 9859040 A2 WO9859040 A2 WO 9859040A2
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telomerase
sequence
leu
arg
htc
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PCT/EP1998/003468
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German (de)
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WO1998059040A3 (fr
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Gustav Hagen
Hans-Ulrich Siegmund
Walter Weichel
Maresa Wick
Dmitry Zubov
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Bayer Aktiengesellschaft
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Priority claimed from DE1998116496 external-priority patent/DE19816496A1/de
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Priority to AU82149/98A priority Critical patent/AU745420B2/en
Priority to CA002294646A priority patent/CA2294646A1/fr
Priority to EP98932142A priority patent/EP0990037A2/fr
Priority to JP50368599A priority patent/JP2002508662A/ja
Publication of WO1998059040A2 publication Critical patent/WO1998059040A2/fr
Publication of WO1998059040A3 publication Critical patent/WO1998059040A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

  • telomeres The genetic material of eukaryotic cells is distributed on linear chromosomes.
  • the ends of the genes are derived from the Greek words telos (end) and meros (part, segment) as telomeres.
  • Most telomeres consist of repetitions of short sequences, which are mainly composed of thymine and guanine (Zakian, 1995).
  • the telomeric sequences of related organisms are often similar and even conserved between more distant species. It is remarkable that in all vertebrates examined so far, the telomeres are built up from the sequence TTAGGG (Meyne et al., 1989).
  • telomeres perform various important functions. They prevent the fusion of chromosomes (McClintock, 1941) and thus the development of dicentric inheritance. Such chromosomes with two centromeres can lead to the development of cancer by loss of heterozygosity or doubling or loss of genes.
  • telomeres serve to distinguish intact hereditary systems from damaged ones. For example, yeast cells stopped dividing when they contained a chromosome without telomer (Sandeil and Zakian, 1993).
  • telomeres Another important task is performed by telomeres in the replication of eukaryotic DNA
  • RNA primers are required to initiate DNA replication. After cleavage of the RNA primer, extension of the Okazaki fragments and subsequent ligation, the newly synthesized DNA strand lacks the 5 'end, because there the RNA
  • telomeres also play an important role in regulating cellular aging (Olovnikov, 1973). Human somatic cells show a limited culture
  • telomeres have central functions in the aging of cells and the stabilization of genetic material and prevention of cancer.
  • telomeres synthesize the telomeres
  • telomere As described above, organisms with linear chromosomes can only partially replicate their genome without a special protective mechanism. Most eukaryotes use a special enzyme, telomerase, to regenerate the telomer sequences. Telomerase is constitutively expressed in the unicellular organisms examined so far. In contrast, telomerase activity was only measured in germ cells and tumor cells in humans, whereas neighboring somatic tissue contained no telomerase (Kim et al, 1994).
  • telomere like the telomeres, was first identified in the ciliate Tetrahymena thermophila. Telomerase activity was demonstrated by extending the single-stranded oligonucleotide d (TTGGGG) 4 in the presence of dTTP and dGTP (Greider and Blackbum, 1985). The 7etr 2y “2e” ⁇ -telomer sequence TTGGGG was repeatedly attached to the primer. Even if as a starting material
  • telomeres Oligonucleotide with the irregular telomer sequence from Saccharomyces cerevisiae, T (G) ⁇ _3, the telomerase extended the primer with the telomer sequence von Tetrahymena (Greider and Blackburn, 1985) From these results it was concluded that the telomerase itself carries with it the template for the sequence of the telomeres
  • RNA component in telomerase After the existence of an RNA component in telomerase could first be demonstrated (Greider and Blackburn, 1987), the gene for the RNA subunit of telomerase was cloned a short time later (Greider and Blackburn, 1989).
  • This RNA contains a region with the Complement to the telomeric sequence of Tetrahymena (hereinafter referred to as "complement region")
  • complement region Complement region
  • telomerase belongs to the class of RNA-dependent DNA polymerases
  • telomeres The first protein subunits of 7etr? Y / we «telomerase, p80 and p95, were identified in 1995 (Collins et al, 1995) The observation that p95 anchors the enzyme to the DNA and p80 binds the RNA component led to following model The complement region of the telomerase RNA attaches to the single-stranded 3 'overhang. The 3' overhang is extended by incorporating the corresponding nucleotides in the 5 '-3' direction. The de «ovo synthesis of telomeres likely includes an elongation and a translocation step.
  • telomere Once a telomeric sequence has been synthesized, the telomerase will likely move along the DNA until it is back in position to add a complete telomeric sequence. This model does not have to be general, since telomerases of different species exist large differences in the number of nucleotides that the enzyme adds before it dissociates from the telomer (Prowse et al, 1993)
  • telomerase subunits from other organisms were also recently identified. Two protein subunits, pl23 and p43, were found in the ciliate Euplotes aediculatus, which have no homology to the Tetrahymena T ⁇ omerasQ-
  • the telomerase subunit pl23 has one at its N-terminus basic domain and a domain for a reverse transcriptase (RT) at the C-terminus, which indicates a catalytic function of this protein (Lingner et al, 1997). Furthermore, a significant homology of pl23 to the protein Est2 found by Lundblad from Saccharomyces cerevisiae was found described (Lingner et al, 1997)
  • telomeres While no essential function for telomerase activity has been demonstrated for p80 and p95, a key function was clearly demonstrated for the potential catalytic subunits of telomerase pl23 / est2p. A mutation of the RT activity center of est2p led to a significant shortening of the telomeres in yeast cells (Lingner et al, 1997)
  • RNA components of telomerases have been cloned from various organisms, including Saccharomyces cerevisiae, mice and humans (Singer and Gottschling, 1994, Blasco et al, 1996, Feng et al, 1995). All previously known telomerase RNAs contain a region that is complementary to the telomeric sequence of the respective organism
  • the primary sequence of human telomerase RNA (hTR) is not similar to the RNA components of the ciliates or Saccharomyces cerevisiae. In contrast, conserved areas exist between human and murine telomerase.
  • hTR human telomerase RNA
  • telomerase-associated protein hTPI
  • telomeres are composed of 2627 amino acids and shows three domains in N-Teminus, which are maximally 46% homologous to p80.
  • 16 repeats of the amino acids could be found in the C-terminal region Tryptophan and asparagine are shown, which presumably mediate a protein-protein interaction Activation of telomerase in human tumors
  • telomere et al An activity of telomerase was originally only detectable in germline cells, but not in normal somatic cells (Hastie et al, 1990, Kim et al, 1994). After the development of a more sensitive detection method (Kim et al,
  • telomere activity was not yet clear in 1994, low telomerase activity was also detected in hematopoietic cells (Broccoli et al, 1995, Counter et al, 1995, Hiyama et al, 1995) However, these cells nevertheless showed a reduction in telomeres (Vaziri et al, 1994, Counter et al , 1995) It is not yet clear whether the amount of enzyme in these cells is not sufficient to compensate for telomer loss, or whether the measured telomerase activity stems from a subpopulation, e.g. incompletely differentiated CD34 + 38 + precursor cells (Hiyama et al, 1995) For clarification, evidence of telomerase activity in a single cell would be necessary
  • telomere activity was detected in a large number of the tumor tissues tested to date (1734/2031, 85%, Shay, 1997), while no activity was found in normal somatic tissue (1/196, ⁇ 1%, Shay, 1997)
  • telomeres continued to shrink and telomerase could only be discovered in the subpopulation that survived the growth crisis (Counter et al, 1992). These cells were also in these immortalized cells the telomeres are stable (Counter et al, 1992)
  • Similar findings from studies on mice support the assumption that reactivation of telomerase is a late event in tumorigenesis
  • telomerase hypothesis was developed that links the loss of telomer sequences and cell aging with the activity of telomerase and the development of cancer.
  • shrinking of telomeres can be seen as a mechanism for tumor suppression Cells that do not contain telomerase stop their cell division at a certain length of the telomeres. If such a cell mutates, a tumor can only develop from it if the cell can extend its telomeres. Otherwise the cell became telomer sequences continue to lose until their chromosomes become unstable and they eventually perish. Reactivation of telomerase is believed to be the main mechanism for tumor cells to stabilize their telomeres.
  • telomere inhibition should allow therapy of tumors.
  • Conventional cancer therapies with cytostatics or short-wave radiation damage not only the tumor cells, but all the cells that divide in the body.
  • telomerase inhibitors would attack the tumor cells more specifically and thus cause fewer undesirable side effects.
  • Telomerase activity has been demonstrated in all tumor tissues tested so far, so that these therapeutic agents could be used against all types of cancer.
  • the effect of telomerase inhibitors would occur when the telomeres of the cells have shortened to such an extent that the genome becomes unstable. Since tumor cells usually have shorter telomeres than normal somatic cells, cancer cells would first be
  • telomeres like the germ cells, would only be damaged much later. Telomerase inhibitors are therefore a pioneering way of treating cancer.
  • telomerase inhibitors will only be possible if the protein structures of the enzyme with their functions have been identified and the knowledge about various telomer-binding proteins has been deepened.
  • the invention relates to the catalytically active human telomerase subunit (phTC), if appropriate in purified form, active parts of the protein, modulators, in particular agonists of the protein, substances imitating the function of the protein and combinations of these components. continues to affect
  • genomic sequences of hTC homologous genes the genomic sequences of hTC homologous genes, the cDNA sequences of hTC homologous genes, the sequences of the mRNAs which are transcribed by hTC homologous genes,
  • the phTC protein labeled with a detection reagent, the detection reagent preferably being an enzyme, a radioactively labeled element or a fluorescent chemical.
  • this is a polyclonal antibody.
  • this is a monoclonal antibody.
  • Such antibodies can be produced, for example, by injecting a substantially immunocompetent host with an amount of a phTC polypeptide or a fragment thereof which is effective for antibody production and by subsequently obtaining this antibody.
  • an immortalized cell line which produces monoclonal antibodies can be obtained in a manner known per se.
  • the antibodies can optionally be labeled with a detection reagent.
  • fragments can also be used which have the desired specific binding properties.
  • Preferred examples of such a detection reagent are enzymes, radioactively labeled elements, fluorescent chemicals or biotin. Oligonucleotides in purified form with a sequence which is identical or exactly complementary to a 10 to 500 nucleotide long, contiguous sequence of the above-described genomic DNA, cDNA or mRNA.
  • Such an oligonucleotide can in particular be an oligodeoxyribonucleotide or an oligoribonucleotide or a peptide nucleotide acid (PNA)
  • PNA peptide nucleotide acid
  • oligonucleotides which inhibit, repress or block the activity of the telomerase when they bind to the hTC mRNA.
  • a recombinant DNA molecule which contains a DNA sequence or a degenerate variation of this sequence which encodes phTC or a fragment of phTC, the latter sequence preferably containing the DNA sequence from FIG. 1, or which contains such a DNA sequence which corresponds to the preceding one DNA sequence listed hybridized under standard hybridization conditions.
  • the DNA described is preferably linked to an expression control sequence.
  • Particularly preferred expression control sequences are e.g. the early or late
  • Promoter of SV40 or adenovirus the lac system, the trp system, the TAC system, the TRC system, the main operator and promoter regions of the phage ⁇ , the control regions of the fd coat protein, the promoter of the 3-phosphoglycerate kinase, the promoter the acid phosphatase and the promoter of the yeast ⁇ -mating factor.
  • a unicellular host which has been transformed with a recombinant DNA molecule described above, which contains the DNA sequence or a degenerate variant of this sequence which codes for the phTC protein or a part of this protein.
  • the said DNA sequence is also in this recombinant DNA molecule linked to an expression control sequence
  • Preferred examples of the unicellular host are E. coh, Psendomonas, Bacilhis, Streptomyces, yeasts, CHO, Rl 1, B-W, L-M, COS 1, COS 7, BSC1, BSC40 and BMT10 cells, plant cells, insect cells and sucker cells in cell culture
  • a method for inhibiting telomerase activity in human cells preferably neoplastic cells, in which an exogenous polynucleotide consisting of a transcription unit is transferred into the cells.
  • This transcription unit contains a polynucleotide sequence of at least 29 consecutive nucleotides that are substantially identical or substantially complementary to the hTC RNA Is sequence and is linked to a heterologous transcription regulatory sequence which said transcription of the linked polynucleotide in said
  • heterologous transcription regulatory sequence preferably contains a promoter which is constitutively active in human cells
  • the heterologous transcription-regulatory sequence can contain a promoter that can be induced or repressed in human cells by adding a regulatory substance.
  • a promoter that can be induced or repressed in human cells by adding a regulatory substance.
  • inducible and repressible tetracycline-dependent promoters Heatshock promoters, metal ion-dependent promoters
  • the above-mentioned exogenous polynucleotide can be, for example, a viral genome with a transcription unit from the human hTC DNA component
  • the said transcription unit particularly preferably produces antisense RNA, which is substantially complementary to the human hTC RNA component
  • exogenous polynucleotide can particularly preferably contain the sequence from FIG. 1
  • Polynucleotide consists of a transcription unit which contains a polynucleotide sequence of at least 9 consecutive nucleotides, which is substantially identical or substantially complementary to the hTC RNA sequence and which is linked to a heterologous transcription regulatory sequence which controls the transcription of the linked polynucleotide in said cells
  • a method for the detection of telomerase-associated conditions in a patient comprising the following steps
  • C detection of diagnostic values that are higher or lower than standard comparison values indicate a telomerase-associated state, which in turn indicates a pathogenic state
  • This method is preferably used for the detection of a neoplastic disease of a patient.
  • the method then comprises the following steps A detection of the phTC protein in cellular samples to make a diagnostic
  • a method of determining the presence of the phTC protein in a cell or cellular sample that is complementary to the amplification of an hTC polynucleotide or hybridization of an hTC polynucleotide, primer, or hTC
  • a test kit for the detection of phTC in cellular samples and body fluids, whereby labeled, immunochemically reactive components can be, for example, polyclonal antibodies against phTC, monoclonal antibodies against phTC,
  • Telomerase its functional equivalents or its catalytically active fragments is also conceivable the use of a substance that requires the production and / or activity of phTC, a substance that can mimic the activity of phTC, a substance that inhibits the production and / or Can inhibit the activity of phTC or a mixture of these substances. Furthermore, a specific binding partner can be used
  • the method is preferably used to prevent or treat aging or cancer
  • telomerase assays are given in the context of example 15
  • Modulators of phTC are interesting for the treatment of diseases with
  • telomerase The prevention or treatment of aging processes or cancer can be mentioned here in particular
  • An antisense nucleic acid against the hTC mRNA which contains a nucleotide sequence which hybridizes with said mRNA, the antisense nucleic acid being a
  • the antisense nucleic acid preferably binds to the start codon of the respective mRNAs
  • a recombinant DNA molecule with a DNA sequence from which an antisense ribonucleic acid is produced against the hTC mRNA during transcription contains a nucleic acid sequence which can hybridize with the said hTC mRNA
  • Such a DNA molecule can be used to produce a cell line with reduced expression of phTC by transfecting a phTC-producing cell line with this recombinant DNA molecule
  • This is preferably a Tetrahymena-type ribozyme or a hammerhead-type ribozyme
  • This recombinant DNA molecule can be used to transfect a phTC-producing cell line
  • a compilation consisting of a pair of human hTC polynucleotide PCR primers, the primers preferably consisting of sequences which correspond to the sequence of the human hTC mRNA or are complementary to this sequence
  • telomerase / telomer regulation Animal models with which the telomerase / telomer regulation can be investigated in vivo. For example, tumor formation and aging can be examined directly with knockout or transgenic animals
  • Fig. 1 cDNA sequence of the human catalytic telomerase subunit (hTC) (SEQ ID No. 1).
  • Fig. 2 Derived amino acid sequence from the hTC DNA sequence shown in Fig. 1 (SEQ ID No. 2).
  • the DNA sequence shown in FIG. 1 can be completely translated into an amino acid sequence from position 64 to position 3461.
  • the amino acid residues are shown according to their one-letter code.
  • the picture shows an 0.8% agarose gel stained with ethidium bromide.
  • Two different DNA size standards are plotted in lanes 1 and 8, the DNA fragment lengths 3, 2, 0.5 and 0.4 kb being highlighted.
  • the AA281296 DNA in pT7T3D was with the restriction enzymes Eco RI / Not I (lane 3), Pst I
  • Lane 6 (Lane 6) and Xho 1 (lane 7) digested. Undigested AA281296 DNA was applied to lane 2 in pT7T3D. Lanes 4 and 5 were 1/10 of a PCR approach (1 minute 94 ° C, 2 minutes 60 ° C, 3 minutes 72 ° C) with the hTC cDNA in pT7T3D and the primers 1 (5 'GAGTGTGTACGTC-GTCGAGCTGCTCAGGTC 3 ') and 4 (5' CACCCTCGAGGTGAGACGCTCGGCC 3 ' ) [lane 4] or with the
  • Fig. 4 Detail from a protein sequence comparison of the catalytic telomerase subunits of Euplotes pl23 ( ⁇ l23) and humans (phTC).
  • Rectangles symbolize sequence sections coding for protein, while the light gray areas 5 'and 3' symbolize untranslated cDNA regions or represent intron sequences.
  • the dark gray blocks in the rectangle for the filling length cDNA either represent the telomerase-specific motif (T) or the seven Reverse transcriptase motifs (number 1-7)
  • the DNA fragments that are necessary to display the complete hTC cDNA are also shown as rectangles and marked according to their origin. All rectangles are arranged in their position relative to each other.
  • the origin of the DNA fragment, for which the rectangle AA261296 stands, is in Describe Example 2
  • Fig. 13 Detail sections from a protein sequence comparison of the catalytic telomerase subunits of Euplotes and humans (hTC).
  • the figure shows excerpts from a protein sequence comparison between the catalytic telomerase subunits of Euplotes and humans (hTC).
  • the motifs for reverse transcriptase are highlighted in the outlined boxes. The numbers below the borders relate to the respective amino acid position in FIG. 2.
  • the amino acid residues are shown in accordance with their one-letter code. Identical amino acids are printed in bold.
  • RT consensus consensus sequence for the reverse transcriptase
  • telomerase-specific motif is described in Example 9.
  • Fig. 14 Generated DNA sequence from Example 11 (3 'variant) (SEQ ID No. 7). The region that is not homologous to the DNA sequence shown in FIG. 1 is highlighted in bold.
  • Fig. 15 hTC expression in cancer cells and in normal human tissue.
  • Fig. A According to the manufacturer (Clontech), about 2 ⁇ g poly A + RNA from various human cell lines were immobilized on the Northern blot. Specifically, the RNA came from melanoma (G361), lung carcinoma (A549), colon adenocarcinoma (SW480), Burkitt's lymphoma Raji, leukemia cell line (MOLT-4), chronic leukemia cell line (K - 562), from a cervical tumor (HeLa) and from the leukemia cell line HL60. The labeled 4.4 kb, 6 kb and 9.5 kb transcripts are specific for hTC (see Example 10).
  • Fig. B According to the manufacturer (Clontech), approximately 2 ⁇ g poly A + RNA from various human tissues were immobilized on the Northern blot. In detail, the RNA from the heart, brain, placenta,
  • RNA size standard is shown.
  • Fig. 16 Western blot analysis of the rabbit sera against peptides from the human telomerase amino acid sequence (Example 12). In each case 20 ⁇ l of the bacterial lysates from Example 13 were analyzed with the aid of the antisera from Example 12 in a Western blot (Ausubel et al, 1987). In lanes 1, 2, 6 and 7, lysates from bacteria containing the pMALEST construct were applied. Lanes 3 from bacteria, which contain the pMALAl construct, were applied in lanes 3, 4, 8 and 9. In lanes 1, 3, 6 and 8, lysates are not made with IPTG
  • Fig. A with lanes 1 to 4 was incubated with preimmune sera against peptide B (compare example 12).
  • the PNDF membrane in Fig. B with lanes 6 to 9 was incubated with preimmune sera against peptide C (see example 12).
  • the PVDF membrane in Fig. B with lanes 1 to 4 was incubated with immune sera against peptide B (see example 12).
  • Fig. 17 Autoradiogram of 35 S-labeled, in vitro translated protein. The complete in vitro translated hTC was plotted in lane 1 (see example
  • Lane 3 shows one supplied by the manufacturer (see example 15) Positive control for in vitro translation
  • a protein size standard is marked on the right side
  • TRAP assay approach with partially purified human telomerase from HeLa cells was applied as a positive control.
  • lanes 5 and 6 a TRAP assay approach with in vitro translated was applied undiluted phTC
  • lanes 7 and 8 a TRAP assay approach with in vitro translated phTC was applied in a 1 4 dilution.
  • lanes 9 and 10 a TRAP assay approach with in vitro translated phTC in a 1 16 dilution was applied In lanes 1 1 and 12, a TRAP assay approach with in vitro translated luciferase was applied as a negative control
  • NCBI National Center for Biotechnology Information
  • Reading frame +1 is referred to below as EST +] .
  • the homology between pl23 and the EST + i is most noticeable in two sequence regions, which are separated by 30 amino acids.
  • the second homology block extends from amino acid 513 to 530 in the pl23 protein and has a 44% identity to the corresponding sequence section in the identified EST + i, taking amino acid residues into account similar properties there is a match of 61%
  • P Probabi ty
  • P indicates the probability with which a specific segment pair was also found in a BLAST search with a random sequence and ranges numerically between 0 (result highly significant) and 1 (result irrelevant)
  • hTPl Telomerase-associated protein 1
  • the corresponding gene is therefore abbreviated to hTC (human telomerase, catalytic) and the derived protein to phTC.
  • the EST identified by comparison with pl23 was entered in the EST database on April 2, 1997 and is not published in any journal.
  • the cDNA library in which this EST clone is present was prepared as follows, according to the National Center for Biotechnology Information:
  • Restriction enzyme interfaces Not I and Eco RI cloned into the vector pT7T3D-Pac.
  • the 389 bp fed into the EST database was sequenced using the - 28ml3 rev2 primer from Amersham (DNA sequence see FIG. 1 position 1685 to 2073).
  • the derived protein sequence from EST + i is composed of 129 amino acids, including 27 basic, 11 acidic, 51 hydrophobic and 28 polar amino acid residues.
  • the EST (AA 281296) identified in Example 1 was purchased commercially from Research Genetics, Inc. (Huntsville) in the form of a plasmid transformed in E. coli and analyzed experimentally: As shown in the ethidium bromide-stained agarose gel of FIG. 3, an approximately 2.2 kb fragment from the vector pT7T3D is released after restriction digestion of the plasmid DNA produced by EST AA 281296 using a polymerase chain (PCR) reaction carried out in parallel with specific internal reactions The AA AA121296 was checked for primers. The length of the expected PCR products is 325 and 380 bp and corresponds to the length of the fragments found experimentally (cf. lanes 4 and 5 in FIG. 3). It was thus possible to show that the length obtained by Research Genetics, Int (Huntsville) sent E coli clone contains the identified EST as a plasmid
  • the DNA sequence of clone AA281296 is thus composed of the sequence information from FIG. 1 (positions 1685 to 2351 and positions 2534 to 4042)
  • pl23 from Euplotes aediculatus and est2p from Saccharomyces cerevisiae are homologous to each other around the degree of relationship
  • the region of pl23 (amino acids 437-554) described above was also compared with est2p using the Lipman-Pearson protein comparison using identical parameters. It was found that p123 and est2p are 21% identical in this selected region or 22% identical amino acids or biochemically similar amino acid residues (see FIG. 5). Accordingly, the homology between EST +] and the pl23 of Euplotes is significantly higher than between the pl23 and est2p.
  • est2p was compared to EST + i under the conditions mentioned in Example 3 (see FIG. 6). It turned out that EST + i has 20% identity to est2p, a comparable one
  • hydrophobic amino acids such as leucine and isoleucine and the amino acids lysine and arginine in certain positions (Fig. 7 and 13)
  • Subunit of human telomerase represents
  • sequence information from the RACE rounds was used to amplify the individual fragments as a coherent cDNA clone by PCR
  • the PCR product was diluted 1,50. Five ⁇ l of this dilution were mixed in a second “nested” PCR together with 10 pmol dNTP mix in 1 ⁇ Kien Taq PCR reaction buffer and 1 ⁇ Advantage Kien Taq polymerase mix as well 10 pmol of the primer GSP2 and 10 pmol of the “nested” marathon adapter primer AP2 (5'-ACTCACTATAGGGCTCGAGCGGC-3 ', Clontech) were used.
  • the PCR conditions corresponded to the parameters selected in the first PCR. The only exception were in the last PCR step only 16 cycles selected instead of 22 cycles A 1,153 bp DNA fragment was obtained as the product of this nested RACE PCR. This was cloned into the TA cloning vector pCR2 1 from InVitrogen and completely double-stranded sequenced (FIG. 8 and SEQ ID No. 3).
  • Nucleotides 974 to 1153 represent the nucleotide region 1629 to 1808 of the hTC cDNA shown in FIG. 1.
  • the nucleotide region extending from bp 1-973, which has no homology to the hTC cDNA sequence shown in FIG. 1, is intron sequences of the hTC gene (data not shown)
  • a 3'-splice consensus sequence can be found at the exon-intron junction.
  • the presence of intron sequences could be due to incompletely spliced mRNA as the starting substance for cDNA synthesis. Genomic DNA contamination in the cDNA could explain the discovery of intron sequences
  • a second RACE was carried out with the gene-specific primer GSP5 from the 5 'region of RACE product 1 (5'-GGCAGTGACCAGGAGGCAACGAGAGG-3') and the API primer.
  • Marathon was used as the cDNA source -Ready cDNA from human testis (Clontech, catalog number 7414-1) was used.
  • the same PCR conditions were used as in the first
  • PCR selected in RACE round 1 Also in RACE round 2, a 2 "nested” PCR with diluted PCR product was connected to the 1 PCR as a cDNA source.
  • the gene-specific primer GSP6 from FIG. 5 ' was used as the "nested” PCR primer.
  • Region of RACE product 1 (5'-GGCACACTCGGCAGGAAACGCACATGG-3 ') and the AP2 primer used. The conditions corresponded to the parameters of the nested PCR from the RACE round
  • the 412 bp long PCR product of the nested PCR from RACE round 2 was cloned into the TA cloning vector pCRII-Topo from Invitrogen and completely sequenced (FIG. 9 and SEQ ID No 4).
  • the sequence section from bp 267 to bp 412 is completely homologous to the 5 'area of the product from RACE 1
  • This RACE product 2 is probably completely an intron region of the hTC gene (data not shown).
  • a third round of RACE led to the identification of further 5'-located hTC cDNA regions.
  • a gene-specific primer GSP9 (5'-CCTCCTCTGTTCACTGCTCTGGCC-3 ') from the 5' range of RACE product 2 was selected and together with the API primer and marathon-ready cDNA from human Testis (from Clontech) used in a new RACE.
  • RACE conditions were the same as for the 1st PCR in RACE 1 and 2.
  • a 1012 bp fragment (Fig. 10 and SEQ ID No. 5) was amplified and into the TA-cloning vector pCRII-
  • Consensus sequence can be found at the exon-intron junction.
  • a PCR was carried out on RACE 2 and the clone AA281296. Marathon-Ready cDNA from human testis (Clontech; catalog number 7414-1) was used as the cDNA source. The PCR was carried out as described under RACE 1 (compare Example 6), but with the primers C5F (5'-CGAGTGGACACGGTGATCTCTGCC-3 ') from the 5' region of RACE 2 and the primer C3B (5'-GCACACCTTTGGTCACTCCAAATTCC-
  • a 2486 bp DNA fragment hereinafter referred to as the C5F fragment, was obtained as the product of this PCR. This was used in the TA cloning vector pCRII-TOPO
  • Clontech, cat. No. HL5016b from the human leukemia cell line K562. About 3x10 p soda of this random and oligo dT primed library were plated as in Ausubel et al, (1987) and used for screening. A 719 bp long (position 1685 to 2404, corresponding to FIG. 1) radioactively labeled hTC-DNA fragment was used as the sample.
  • ⁇ clone 12 was verified as positive after rescreening with the same hTC probe. After plaque purification and ⁇ DNA preparation (Ausubel et al, 1987), the 4 kb insert was cloned into the vector pBluescript and sequenced (FIG. 11 and SEQ ID No. 6). A comparison of the ⁇ clone 12 sequence with the sequences of the RACE clones and the DNA sequence from clone AA281296 showed that this clone identified in the homology screening codes for a 5 ' part of the hTC cDNA and a putative ATG start codon in position 63 corresponding to FIG. 1.
  • Reading frame over about 400 bp is identical to various ESTs that are not related to the hTC cDNA.
  • the ⁇ clone 12 is thus a chimeric clone, which is composed essentially of the 5 'end of the hTC cDNA and another cDNA clone of unknown function.
  • FIG. 1 A summary schematic representation with the relative orientation of the RACE products and the homology screening is shown in FIG. The complete sequence of the hTC cDNA (FIG. 1) was obtained from the ⁇ clone 12 (positions 21 to 1655 corresponding to FIG. 11), the PCR product C5F (positions 1636 to 3908 corresponding to FIG. 1) and the EST AA281296 (Position 3909 to 4042 according to FIG. 1) composed.
  • RT motifs motifs for reverse transcriptases
  • FIG. 13 A consensus sequence of reverse transcriptases (Poch et al., 1989, Xiong and Eickbush, 1990) identified a total of seven motifs for reverse transcriptases (RT motifs) (FIG. 13). Within these motifs, some amino acids are highly conserved not only between the RT consensus sequence and the phTC, but also in comparison to the telomerase protein from Euplotes. For example, in RT motif 5 two aspartic acids
  • telomerase motif positions 553 and 565 in FIG. 2 is a structure specific for this protein family, since it does not occur in any protein known to date.
  • Another feature only identified in the catalytic telomerase proteins is the distance between the RT motifs 3 and 4, which at 107 amino acids is significantly larger than in other RTs.
  • telomerase RNA subunit does not correlate with the telomerase activity, but is observed ubiquitously (Feng et al, 1995). This raises the question of whether the expression of the catalytic telomerase subunit is associated with the telomease activity.
  • Northern blot experiments (Ausubel et al, 1987) were carried out to analyze the level of hTC expression.
  • the commercially available Northern blots were either with a series of RNA preparations from normal human tissue (Clontech; catalog number 7760-1) or with RNA samples from human cancer cells (Clontech;
  • RNA transcripts were approximately the size 9.5 kb and 4.4 kb and an RNA subtranscript of approximately 6 kb were detected, which cross-hybridize with the sample (FIG. 15, FIG. A).
  • the hTC mRNA was most strongly expressed in the leukemia cell lines K-562 and HL-60 (FIG. 15, FIG. A).
  • the hTC transcript could not be detected in the normal tissues tested (heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas)
  • PCR reaction took place in 3 steps. First the DNA was denatured for 3 min at 94 ° C. 34 cycles followed, in which the DNA was denatured successively for 45 sec at 94 ° C, then for 1 min at 68 ° C the primer was attached and then the DNA chain was extended for 3 min at 72 ° C. In the last PCR step, a final chain extension was carried out for 10 min at 72 ° C. The resulting PCR products were added to the TA cloning vector pCR2 1 cloned by InVitrogen
  • the cDNA synthesis kit from Boehringer Mannheim made from 2 ⁇ g DNasel-treated poly A-RNA of the human pramyeloid cell line, was first used
  • HL60 a cDNA synthesis was carried out according to the manufacturer's instructions. In a final volume of 50 ⁇ l, 1 ⁇ l of this HL60 cDNA was then mixed with 10 pmol each of the primers GSPlvor and HTRT3A and 10 pmol of dNTP mix and after addition of 1.25 ⁇ l of DMSO A PCR reaction was carried out in 1 x Kien Taq PCR reaction buffer and 1 x Advantage Kien Taq Polymerase Mix (Clontech). The PCR reaction was carried out in 3 steps.
  • Variant 1 of the human hTC cDNA is distinguished by a 182 bp deletion of nucleotides 2345 to 2526. This deletion results in a shift in the ORF and a shortened hTC protein is read, which lacks the RT motifs 4 to 7
  • Variant 2 of the human hTC cDNA has a 36 bp deletion of nucleotides 2184 to 2219.
  • the RT motif 3 is lost as a result of this deletion. However, the reading frame is retained and a protein is produced which selectively lacks the RT motif 3
  • Variant 3 of the human hTC cDNA represents a combination of variants 1 and 2. It has both a deletion of bp 2184 to 2219 and bp 2345 to 2526
  • Variant 4 of the human hTC cDNA is characterized by the loss of the nucleotide region from bp 3219 to 3842. This missing sequence is replaced by a sequence which is not homologous to hTC. From bp 3843, the sequence is again completely identical to the hTC shown in FIG Sequence The sequence of variant 4 is shown in FIG. 14. According to the selected 5 'primer, it begins with bp 1783 of the hTC cDNA shown in FIG 14 and SEQ ID No 7) at DNA level 98.7% with an EST (Accession No. AA299878) from a human uterine tumor
  • FIG. 1 In order to obtain antisera with specificity for the catalytic subunit of human telomerase, the existing nucleotide sequence (FIG. 1) was translated into an amino acid sequence (FIG. 2) with the aid of a program for secondary structure prediction (PROTEAN, from the software package DNAStar, DNASTAR Ine, Madison, WI, USA), two peptides were selected that are likely to elicit an immune response The following peptides are shown in the one-letter code for amino acids:
  • underlined cysteines do not come from the telomerase sequence, but have been added as linkers for the coupling
  • the peptides were isolated via the thiol-reactive coupling reagent m-maleimido-benzoyl-N-
  • MMS Hydroxysuccinimide ester
  • KLH keyhole limpet hemocyanin
  • pMalAl contains the nucleotide sequence of FIG. 1 from position 1789 to position 3908. This DNA fragment was PCR-primed with the primers C5A (5'- ACCGGAAGAGTGTCTGGAGCAAGTTG-3 ') and C3B (5'-
  • TA cloning vector pCRII-TOPO from Invitrogen.
  • the PCR conditions were carried out as described in Example 7.
  • the insert was cut out with the restriction enzyme Eco RI, the cut-outs filled in with the Klenow fragment (Ausubel et al, 1987) and in the bacterial expression vector pMAL-C2 (Fa.
  • the protein expression using these constructs was carried out in the bacterial strain E. coli DH5 ⁇ .
  • the expression conditions were as described in the operating instructions from New England Biolabs (catalog number 800).
  • the bacterial lysates produced were tested in a Western blot experiment (Ausubel et al, 1987).
  • Example 13 The bacterial lysates from Example 13 were analyzed with the aid of the antisera from Example 12 in a Western blot (Ausubel et al, 1987).
  • fusion proteins in the size of approximately 74 kDa and 106 kDa are expected for the constructs pMalEST and pMalAl, respectively.
  • Protein component can be reconstituted together with the RNA component in vitro
  • HTR2BAM (5'-CGCGGATCCCGGCGAGGGGTGACGGATGC-3) from a 293 cell cDNA library containing a PCR of 293 cell cDNA in one of the nucleotides HTRotid 100 AMP 3 ⁇ l cDNA from 293 cells were mixed with 10 pmol dNTP mix and in 1 x PCR reaction buffer with 0.5 ⁇ l
  • Taq polymerase (Gibco) carried out a PCR reaction. 10 pmol each of the primers HTR9BAM and HTR2BAM were added. The PCR was carried out in 3 steps. A ten-minute denaturation at 94 ° C was followed by 35 PCR cycles, in which the DNA was first denatured for one minute at 94 ° C and then the primers were attached for 2 min at 62 ° C and the DNA chain was extended This was followed by a chain extension at 72 ° C for 4 min. The resulting PCR products were cloned into the Bam HI site of the vector pUC19 after restriction digestion with Bam HI, so that the RNA component is under the control of the T7 promoter. This construct is referred to below as HTR504
  • the 341 1 bp long cDNA fragment (position 60 to position 3470, FIG. 1) was cloned into the vector PCRII TOPO (Invitrogen). Detailed information on the cloning is provided described in Examples 8 and 7, and in Fig. 12, respectively.
  • the T7 promoter lies 5 'in front of the hTC cDNA.
  • the catalytic telomerase protein component was synthesized after addition of the hTC FL construct in a commercially available transcription / translation system according to the manufacturer's instructions (Promega; catalog number L4610).
  • the successful in vitro translation of the expected 127 kDa product was checked using 35 S-labeled cysteine in an SDS-PAGE (Ausubel et al, 1987) (FIG. 17).
  • telomerase RNA component was carried out with a transcription system according to the manufacturer (Ambion; catalog number 1344) or according to the method described by Pokrovskaya and Gurevich (1994).
  • Telomere length predicts replicative capacity of human fibroblasts. Proc. Natl. Acad. Be. 89, 10114-10118.
  • RNA component of human telomerase Science 269, 1236-1241.
  • telomere terminal transferase of Tetrahymena IS a ⁇ bonucleoprotein enzyme with two kinds of p ⁇ mer specificity Cell 51, 887-898
  • TCGTGGAGAC CATCTTTCTG GGTTCCAGGC CCTGGATGCC AGGGACTCCC CGCAGGTTGC 1200
  • TGCAAGCCAC CGTGCCCGGC ATACCTTGAT CTTTTAAAAT GAAGTCTGAA ACATTGCTAC 540
  • TTTTATGTCA CGGAGACCAC GTTTCAAAAG AACAGGCTCT TTTTCTACCG GAAGAGTGTC 1140
  • MOLEK type cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • GCCGCTGTAC CAGCTCGGCG CTGCCACTCA GGCCCGGCCC CCGCCACACG CTAGTGGACC 660
  • CCGAAGGCGT CTGGGATGCG AACGGGCCTG GAACCATAGC GTCAGGGAGG CCGGGGTCCC 720 CCTGGGCCTG CCAGCCCCGG GTGCGAGGAG GCGCGGGGGC AGTGCCAGCC GAAGTCTGCC 780
  • CTCTGTGGCG GCCCCCGAGG AGGAGGACAC AGACCCCCGT CGCCTGGTGC AGCTGCTCCG 1440
  • GGTGTGCACC AACATCTACA AGATCCTCCT GCTGCAGGCG TACAGGTTTC ACGCATGCGT 1320

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Abstract

L'invention concerne la séquence nucléotide et la séquence protéine qui en dérive, codant pour la sous-unité de télomérase catalytique humaine. L'invention concerne en outre des procédés impliquant une utilisation pharmaceutique, thérapeutique ou diagnostique de ce gène/protéine, principalement pour le traitement du cancer et du vieillissement.
PCT/EP1998/003468 1997-06-20 1998-06-09 Sous-unite de telomerase catalytique humaine et son utilisation therapeutique et diagnostique WO1998059040A2 (fr)

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AU82149/98A AU745420B2 (en) 1997-06-20 1998-06-09 Human catalytic telomerase sub-unit and its diagnostic and therapeutic use
CA002294646A CA2294646A1 (fr) 1997-06-20 1998-06-09 Sous-unite de telomerase catalytique humaine et son utilisation therapeutique et diagnostique
EP98932142A EP0990037A2 (fr) 1997-06-20 1998-06-09 Sous-unite de telomerase catalytique humaine et son utilisation therapeutique et diagnostique
JP50368599A JP2002508662A (ja) 1997-06-20 1998-06-09 ヒトテロメラーゼの触媒サブユニット並びにその診断的及び治療的使用

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WO1999040221A3 (fr) * 1998-02-04 1999-10-14 Michael W Dahm Procede pour la determination quantitative de cellules tumorales dans un fluide corporel et trousses d'essai appropriees a cet effet
WO2000063429A2 (fr) * 1999-04-15 2000-10-26 Bayer Aktiengesellschaft Test rapide automatisable pour la detection de cancers a base d'arnm de telomerase(htc), ainsi qu'amorces et sondes specifiques
US6261836B1 (en) 1996-10-01 2001-07-17 Geron Corporation Telomerase
US6440735B1 (en) 1998-03-31 2002-08-27 Geron Corporation Dendritic cell vaccine containing telomerase reverse transcriptase for the treament of cancer
US6582904B2 (en) 1995-11-16 2003-06-24 Michael W. Dahm Method of quantifying tumour cells in a body fluid and a suitable test kit
US6610839B1 (en) 1997-08-14 2003-08-26 Geron Corporation Promoter for telomerase reverse transcriptase
US6617110B1 (en) 1996-10-01 2003-09-09 Geron Corporation Cells immortalized with telomerase reverse transcriptase for use in drug screening
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
EP1571215A2 (fr) * 1997-07-01 2005-09-07 Cambia Biosystems LLC Gènes et protéines de télomérases de vertèbres et leurs utilisations
US7195911B2 (en) 1996-10-01 2007-03-27 Geron Corporation Mammalian cells that have increased proliferative capacity
US7262288B1 (en) 1997-04-18 2007-08-28 Geron Corporation Nucleic acids encoding human telomerase reverse transcriptase and related homologs
US7378244B2 (en) 1997-10-01 2008-05-27 Geron Corporation Telomerase promoters sequences for screening telomerase modulators
US7402307B2 (en) 1998-03-31 2008-07-22 Geron Corporation Method for identifying and killing cancer cells
US7413864B2 (en) 1997-04-18 2008-08-19 Geron Corporation Treating cancer using a telomerase vaccine
US8017387B2 (en) 2006-10-12 2011-09-13 Istituto Di Ricerche Di Biologia Molecolare P. Angeletti Spa Telomerase reverse transcriptase fusion protein, nucleotides encoding it, and uses thereof
US8709995B2 (en) 1997-04-18 2014-04-29 Geron Corporation Method for eliciting an immune response to human telomerase reverse transcriptase

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US6582904B2 (en) 1995-11-16 2003-06-24 Michael W. Dahm Method of quantifying tumour cells in a body fluid and a suitable test kit
US6261836B1 (en) 1996-10-01 2001-07-17 Geron Corporation Telomerase
US7005262B2 (en) 1996-10-01 2006-02-28 Geron Corporation Methods for detecting nucleic acids encoding human telomerase reverse transcriptase
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
US6808880B2 (en) 1996-10-01 2004-10-26 Geron Corporation Method for detecting polynucleotides encoding telomerase
US7195911B2 (en) 1996-10-01 2007-03-27 Geron Corporation Mammalian cells that have increased proliferative capacity
US7056513B2 (en) 1996-10-01 2006-06-06 Geron Corporation Telomerase
EP1333094A3 (fr) * 1996-10-01 2005-02-09 Geron Corporation Sous-unité catalytique de la télomérase humaine
US6927285B2 (en) 1996-10-01 2005-08-09 Geron Corporation Genes for human telomerase reverse transcriptase and telomerase variants
US8236774B2 (en) 1997-04-18 2012-08-07 Geron Corporation Human telomerase catalytic subunit
US7413864B2 (en) 1997-04-18 2008-08-19 Geron Corporation Treating cancer using a telomerase vaccine
US8709995B2 (en) 1997-04-18 2014-04-29 Geron Corporation Method for eliciting an immune response to human telomerase reverse transcriptase
US7262288B1 (en) 1997-04-18 2007-08-28 Geron Corporation Nucleic acids encoding human telomerase reverse transcriptase and related homologs
EP1571215A3 (fr) * 1997-07-01 2007-10-31 Cambia Biosystems LLC Gènes et protéines de télomérases de vertèbres et leurs utilisations
EP1571215A2 (fr) * 1997-07-01 2005-09-07 Cambia Biosystems LLC Gènes et protéines de télomérases de vertèbres et leurs utilisations
US6610839B1 (en) 1997-08-14 2003-08-26 Geron Corporation Promoter for telomerase reverse transcriptase
US7199234B2 (en) 1997-08-14 2007-04-03 Geron Corporation Regulatory segments of the human gene for telomerase reverse transcriptase
US7378244B2 (en) 1997-10-01 2008-05-27 Geron Corporation Telomerase promoters sequences for screening telomerase modulators
WO1999027113A1 (fr) * 1997-11-26 1999-06-03 Geron Corporation Transcriptase inverse de telomerase de souris
US6821726B1 (en) 1998-02-04 2004-11-23 Michael W. Dahm Method for quantitatively analyzing tumor cells in a body fluid and test kits suited therefor
WO1999040221A3 (fr) * 1998-02-04 1999-10-14 Michael W Dahm Procede pour la determination quantitative de cellules tumorales dans un fluide corporel et trousses d'essai appropriees a cet effet
US6440735B1 (en) 1998-03-31 2002-08-27 Geron Corporation Dendritic cell vaccine containing telomerase reverse transcriptase for the treament of cancer
US7402307B2 (en) 1998-03-31 2008-07-22 Geron Corporation Method for identifying and killing cancer cells
US7824849B2 (en) 1998-03-31 2010-11-02 Geron Corporation Cellular telomerase vaccine and its use for treating cancer
US6808883B1 (en) 1999-04-15 2004-10-26 Bayer Ag Automatable rapid test for detection of cancer, based on telomerase (hTC) mRNA with specific primers and probes
WO2000063429A3 (fr) * 1999-04-15 2002-01-03 Bayer Ag Test rapide automatisable pour la detection de cancers a base d'arnm de telomerase(htc), ainsi qu'amorces et sondes specifiques
WO2000063429A2 (fr) * 1999-04-15 2000-10-26 Bayer Aktiengesellschaft Test rapide automatisable pour la detection de cancers a base d'arnm de telomerase(htc), ainsi qu'amorces et sondes specifiques
US8017387B2 (en) 2006-10-12 2011-09-13 Istituto Di Ricerche Di Biologia Molecolare P. Angeletti Spa Telomerase reverse transcriptase fusion protein, nucleotides encoding it, and uses thereof

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