CH689672A9 - Catalytic subunit of human telomerase. - Google Patents

Description

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

description

The present invention relates to novel nucleic acids and polypeptides that encode the catalytic subunit of telomerase. In particular, the present invention relates to the catalytic subunit of human telomerase. The invention provides methods and compositions related to medicine, molecular biology, chemistry, pharmacology, and medical, diagnostic, and prognostic methods.

The following discussion is intended to introduce the reader to the field to which the present invention relates. The citation of various references in this section should not be construed as an admission of discarding the invention.

It has long been known that the complete replication of the ends of eukaryotic chromosomes requires specialized cell components (Watson, Nature New. Biol. 239 (1972), 197 and Olovni-kov, J. Theor. Biol. 41 (1973), 181) . Replication of a linear strand of DNA by common DNA polymerases requires an RNA primer and can only proceed in the 5'-3 'direction. When the RNA bound to the outer 5'-end of chromosomal eukaryotic DNA strands is removed, a gap is introduced, which leads to a progressive shortening of the daughter strands with each round of replication. This shortening of telomeres, which represent the protein / DNA structures physically located at the ends of chromosomes, seems to be the reason for the phenomenon of cellular senescence or aging (see, for example, Goldstein, Science 249 (1990), 1129; Martin et al ., Lab. Invest. 23 (1979), 86; Goldstein et al., Proc. Nati. Acad. Sei. USA 64 (1969), 155 and Schneider and Mitsui, Proc. Nati. Acad. Sei. USA, 73 ( 1976), 3584) of normal human somatic cells in vitro and in vivo.

The length and integrity of telomeres is thus related to the entry of a cell into a senescent status (i.e. the loss of its ability to proliferate). Furthermore, the ability of a cell to maintain (or increase) the telomere length may allow the cell to escape senescence, i.e. to become immortal.

The structure of telomeres and telomeric DNA has been studied in numerous systems (see e.g. Harley and Villeponteau, Curr. Opin. Genet. Dev. 5 (1995), 249). In most organisms, telomeric DNA consists of a tandem array of very simple sequences; in humans and other verbrates, telomeric DNA consists of hundreds to thousands of tandem repeat units of the TTAGGG sequence. Methods for determining and modulating the telomer length in cells are described in PCT publications WO 93/23 572 and WO 96/41 016.

Telomer preservation is a function of a telomer-specific DNA polymerase known as telomerase. Telomerase is a ribonucleoprotein (RNP) that uses a portion of its RNA unit as a template for the synthesis of the DNA telomer repeat units (Morin, Eur. J. Cancer 33 (1997), 750; Yu et al., Nature 344 (1990) 126; Singer and Gottschling, Science 266

(1994) 404; Autexier and Greider, Genes Develop. 8: 563, 1994; Gilley et al., Genes Develop. 9

(1995), 2214; Mc Eachem and Blackbum, Nature 367 (1995), 403; Blackbum, Ann. Rev. Biochem. 61: 113 (1992); Greider, Ann. Rev. Biochem. 65 (1996), 337). The RNA components of human and other telomerases have been cloned and characterized (see PCT publication WO 96/01835 and Feng et al., Science 269 (1995), 1236). However, the characterization of the protein components of telomerase was difficult. This is partly due to the fact that it has proven difficult to purify Te-isomerase RNP, which is only present in extremely low concentrations in cells in which it is expressed. For example, it has been thought that human cells, which are known to express high levels of telomerase activity, have only about 100 enzyme molecules per cell.

Consistent with the relationship of telomeres and telomerase to a cell's ability to proliferate (i.e., the ability of a cell to divide indefinitely), telomerase activity has been demonstrated in immortal cell lines and in distinctly different types of tumor tissues, but it has not been found in normal somatic cell cultures or normal tissues in the vicinity of a tumor (see U.S. Pat. Nos. 5,629,154; 5,489, 508; 5,648,215 and 5,639,613; see also Morin, Cell 59 (1989) 521; Shay and Bacchetti, Eur. J. Cancer 33 (1997), 78 7; Kim et al., Science 266 (1994), 2011; Counter et al., EMBO J. 11 (1992), 1921; Counter et al., Proc. Nati. Acad USA 91 (1994), 2900 and Counter et al., J. Virol. 68 (1994), 3410) (ie was not present or was below the detection limit of the assay). A relationship between the level of telomerase activity in a tumor and the likely clinical course of the patient's disease has also been reported (e.g., U.S. Patent No. 5,639,613, supra, and Langford et al., Hum. Pathol. 28 (1997), 416). Telomerase activity has also been demonstrated in human gametes, proliferating stem cells or progeny thereof and activated lymphocytes. In somatic stem cells or their progeny and in activated lymphocytes, telomerase activity is typically either very low or only transiently expressed (see Chiù et al., Stem Cells 14 (1996), 239, Bodnar et., Exp. Cell Res. 228 (1996), 58 and Taylor et al., J. Invest. Dermatolo-gy 106 (1996), 759).

Human telomerase is an ideal target for the diagnosis and treatment of human diseases related to cellular proliferation and senescence, such as cancer. Procedure

2nd

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Other methods for diagnosing and treating cancer and other telomerase-related diseases in humans are described in U.S. Patent Nos. 5,489,508, 5,639,613 and 5,645,986. Methods of predicting tumor progression by observation of telomerase are described in U.S. Patent No. 5,639,613. Finding and characterizing the catalytic protein subunit of human telomerase would provide additional useful assertions for telomerase and for the diagnosis and therapy of diseases. In addition, cloning and determination of the primary sequence of the catalytic protein subunit would allow more effective therapies for human cancer and other diseases related to cell proliferation and senescence to be provided.

The present invention provides an isolated, substantially pure or recombinant protein preparation of a telomerase reverse transcriptase protein or a variant or a fragment thereof. In one embodiment, the protein has the following amino acid sequence:

Trp-RrX7-R1-RrRrX-Phe-Phe-Tyr-X-Thr-Glu-X8.9-R3-R3-Arg-R4-X2-Trp where X is any amino acid and a subscript is based on the number of successive residues, Ri is leucine or isoleucine, R2 glutamine or arginine, R3 phenylalanine or tyrosine and R4 lysine or histidine. In one embodiment, the protein has a sequence from human TRT. In other embodiments, the invention relates to peptides and polypeptides which essentially have sequence identity in common with a sub-sequence of such proteins.

In a related embodiment, the invention provides an isolated, substantially pure or recombinant nucleic acid encoding a telomerase reverse transcriptase protein. In one embodiment, the protein has the following amino acid sequence:

Trp-R, -X7-R, -R i -RrX-Phe-Phe-T yr-X-Thr-Glu-Xg.9-R3 -R3-Arg-R4-X2-Trp.

In one embodiment, the nucleic acid has a sequence of human TRT. In further embodiments, the invention relates to oligonucleotides and polynucleotides which have essential sequence identity in common with a subunit of such nucleic acids.

One aspect of the invention relates to human telomerase reverse transcriptase (hTRT). Thus, in one embodiment, the invention provides an isolated, substantially pure or recombinant protein preparation of a human telomerase reverse transcriptase (hTRT) protein or a variant or fragment thereof.

In one embodiment, the protein is characterized by an amino acid with at least about 75% or at least about 80% sequence identity to the hTRT protein from SEQ. ID. No. 2 (Fig. 17) or a variant or a fragment thereof. In a related embodiment, the hTRT protein has the sequence of SEQ. ID. # 2. In some embodiments, the protein has one or more telomerase activities, such as catalytic activity. In one embodiment, the hTRT protein fragment has at least 6 amino acid residues.

The invention also provides a composition comprising an hTRT protein and an RNA. The RNA can be a telomerase RNA, for example an RNA of human telomerase. In one embodiment, the hTRT protein and the human telomerase RNA form a ribonu-cleoprotein complex with telomerase activity.

One aspect of the invention relates to the provision of isolated human telomerase, for example an essentially pure human telomerase, which comprises human telomerase reverse transcriptase (hTRT) and human telomerase RNA (hTR). In one embodiment, the telomerase is at least about 95% pure. The telomerase can be isolated from a cell.

Another aspect of the invention relates to the provision of an isolated, synthetic, essentially pure or recombinant polynucleotide, which comprises a nucleic acid sequence encoding an hTRT protein. In one embodiment, the polynucleotide has a nucleotide sequence encoding an hTRT protein that contains one in SEQ. ID. No. 2 amino acid sequence indicated, or a sequence that includes one or more conservative substitutions in that amino acid sequence. A related aspect relates to the provision of a polynucleotide which, under strict conditions, contains a polynucleotide having the sequence of SEQ. ID. No. 1 hybridizes. In a further related embodiment, the nucleotide sequence of the polynucleotide has a smallest sum probability of less than about 0.5 compared to that in SEQ. ID. No. 1 specified nucleotide sequence using the BLAST algorithm using preset parameters.

Another aspect of the invention relates to the provision of a polynucleotide with a promoter sequence which is functionally linked to the sequence encoding the hTRT protein. The promoter can be a promoter that does not correspond to the naturally occurring hTRT promoter. A related aspect of the invention relates to the provision of an expression vector comprising the hTRT polynucleotide.

The invention also provides an isolated, synthetic, substantially pure or recombinant polynucleotide that is at least 10 nucleotides in length and comprises a sequential sequence of at least 10 nucleotides that results in a sequential sequence in ei3

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

nes naturally occurring hTRT gene or hTRT mRNA is identical or exactly complementary. In some embodiments, the polynucleotide is an RNA, a DNA, or it contains one or more non-naturally occurring synthetic nucleotides. In one embodiment, the polynucleotide is identical or exactly complementary to the sequential sequence of at least 10 consecutive nucleotides in a naturally occurring hTRT gene or hTRT mRNA. For example, the polynucleotide can be an "antisense" polynucleotide. In another embodiment, the antisense polynucleotide comprises at least about 20 nucleotides.

The invention also provides a method for producing recombinant telomerase by contacting a recombinant hTRT protein with a telomerase RNA component under conditions such that the recombinant protein and the telomerase RNA component associate to form a telomerase enzyme. that can catalyze the addition of nucleotides to a telomerase substrate. In one embodiment, the hTRT protein has the sequence of SEQ. ID. No. 2. The hTRT protein can be produced in an in vitro expression system and mixed with a telomerase RNA; in another embodiment, the telomerase RNA is co-expressed in the in vitro expression system. In one embodiment, the telomerase RNA is human telomerase RNA. In an alternative embodiment, the contacting takes place in a cell, for example a human cell. In one embodiment, the cell has no telomerase activity prior to contacting hTRT and the RNA of an hTRT polynucleotide (or introduction, e.g., by transfection). In one embodiment, the telomerase RNA is naturally expressed by the cell.

The invention also provides a cell, for example a human cell, mouse cell or yeast cell, which contains the recombinant polynucleotides according to the invention, for example a polynucleotide with a sequence which is functionally linked to a promoter and encodes an hTRT protein. In certain aspects, the cell is a vertebrate cell, e.g. a mammalian cell, e.g. of a human, and has an increased proliferation ability relative to the cell that is otherwise identical but does not include the recombinant polynucleotide, or has an increased level of telomerase activity relative to a cell that is otherwise identical but does not include recombinant polynucleotides . In some embodiments, the cell is immortal.

Related embodiments of the invention relate to the provision of organisms and cells comprising a polynucleotide that is a human telomerase reverse transcriptase polypeptide. These include a transgenic non-human organism, such as yeast, a plant or bacterium, or an animal, such as a mouse. The invention also provides transgenic animals and cells in which an hTRT gene has been knocked out or mutated so that the gene does not express a naturally occurring hTRT gene product. Thus, in alternative embodiments, the transgenic animal has a mutated telomerase gene, is an animal deficient in telomerase activity, is an animal whose TRT deficiency is the result of a mutated gene that compares a TRT with a reduced level of telomerase activity owns a wild-type TRT or an animal with a mutated TRT gene with one or more mutations, including "missense" mutations, "nonsense" mutations, insertions and deletions.

The invention also provides an isolated or recombinant antibody or fragment thereof that specifically binds to hTRT protein. In one embodiment, the antibody binds with an affinity of at least about 10®M "1. The antibody can be a monoclonal or polyclonal composition, for example a polyclonal antiserum. A related aspect of the invention relates to the provision of a cell which can secrete the antibody, for example a hybridoma.

The invention also provides a method of detecting whether a compound or treatment is a modulator of telomerase reverse transcriptase activity or hTRT expression by detecting or monitoring a change in activity or expression in a cell , an animal or a composition comprising an hTRT protein or corresponding polynucleotide after administration of the compound or treatment. In one embodiment, the method includes the following steps: providing a TRT composition, contacting the TRT with the test compound and measuring the activity of the TRT, a change in the TRT activity in the presence of the test compound being an indicator of whether the test compound is TRT Activity modulated. In certain embodiments, the composition is a cell, an organism, a transgenic organism or an in vitro system, for example an expression system, which contains a recombinant polynucleotide encoding an hTRT polypeptide. Thus, the human telomerase reverse transcriptase of this method can be a product of in vitro expression. In various embodiments, detection of telomerase activity or expression can be accomplished by detecting a change in the frequency of an hTRT gene product by monitoring the incorporation of a nucleotide label into a substrate for telomerase, the hybridization of a probe with an extended one Telomerase substrate, the amplification of an elongated telomerase substrate, the telomer length of a cell exposed to the test compound and the loss of the ability of the telomerase to bind to a chromosome or by measuring the accumulation or loss of a telomer structure.

One aspect of the invention relates to the provision of a method for the detection of a human telomerase reverse transcriptase (hTRT) gene product in a biological sample

4th

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Contacting the biological sample with a probe that specifically binds the gene product, the probe and the gene product forming a complex, and by detecting the complex, the presence of the complex correlating with the presence of the hTRT gene product in the biological sample. The gene product can be RNA, DNA or a polypeptide. Examples of probes that can be used for detection include, but are not limited to, nucleic acids and antibodies.

In one embodiment, the gene product is a nucleic acid that is detected by amplifying the gene and detecting the amplification product, the presence of the complex or the amplification product correlating with the presence of the hTRT gene product in the biological sample.

In one embodiment, the biological sample from a patient is, for example, a human patient. In a further embodiment, the biological sample contains at least one cell from an in vitro cell culture, for example a culture of human cells.

The invention further provides a method for detecting the presence of at least one immortal or telomerase-positive human cell in a biological sample comprising human cells, wherein the biological sample comprising human cells is obtained and the presence of a cell with a high level of an hTRT gene product is detected in the sample, the presence of a cell with a high level of the hTRT gene product correlating with the presence of immortal or telomerase positive cells in the biological sample. The invention also provides a method for diagnosing a telomerase-related condition in a patient by obtaining a cell or tissue sample from the patient, determining the amount of a human telomerase reverse transcriptase (hTRT) gene product in the cell or tissue and comparing the amount of the hTRT gene product in the cell or tissue with the amount in a healthy cell or tissue of the same type, with a different amount of the hTRT gene product in the sample from the patient compared to the healthy cell or is a diagnostic sign of a condition related to telomerase in healthy tissue. In one embodiment, the telomerase-related condition is cancer.

The invention further provides a method for diagnosing cancer in a patient by obtaining a sample from the patient and detecting a human telomerase reverse transcriptase (hTRT) gene product in the sample from the patient, wherein the detection of the hTRT- Gene product in the sample correlated with a diagnosis of cancer.

The invention also provides a method of diagnosing cancer in a patient by obtaining a patient sample, determining the amount of human telomerase reverse transcriptase (hTRT) gene product in the patient sample, and comparing the amount of the hTRT gene product to a normal value or Control value, where an amount of the hTRT gene product in the patient that is larger compared to the normal or control value is a diagnostic sign of cancer.

The invention also provides a method for diagnosing cancer in a patient by obtaining a patient sample containing at least one cell, determining the amount of the hTRT gene product in a cell in the sample and comparing the amount of the hTRT gene product in the Cell with a normal value for the cells, with an amount of the hTRT gene product which is above the normal value being a diagnostic sign of cancer. In one embodiment, it is assumed that the sample contains at least one malignant cell.

The invention also provides a method of providing a prognosis for a cancer patient wherein the amount of the hTRT gene product in a cancer cell obtained from the patient is determined and the amount of hTRT in the cancer cell with a prognostic value for hTRT per cancer cell consistent with is a cancer prognosis, with an amount of hTRT per cell in the sample that is at the prognostic value providing the particular prognosis.

The invention also provides a method for monitoring the effectiveness of an anti-cancer treatment to decrease the proliferation capacity of cancer cells in a patient by first measuring the amount of an hTRT gene product in at least one cancer cell by the patient, a second measurement in terms of the level of hTRT gene product is performed in at least one patient's cancer row, the anti-cancer treatment being administered to the patient before or at the same time and by comparing the first to the second measurement, with a low level of hTRT gene product correlated in the second measurement with the effectiveness of anti-cancer treatment to reduce the proliferation capacity of cancer cells in the patient.

The invention also provides kits for the detection of an hTRT gene or gene product. In one embodiment, the kit contains a container comprising a molecule selected from an hTRT nucleic acid or a sub-sequence thereof, an hTRT polypeptide or a sub-sequence thereof and an anti-hTRT antibody. The invention also provides methods of treating human diseases. One aspect of the invention relates to the provision of a method for increasing the proliferation capacity of a vertebrate cell, for example a mammalian cell, by introducing a recombinant polynucleotide into the cell, the polynucleotide being a sequence

5

CH 689 672 A9

comprising a human telomerase reverse transcriptase (hTRT) polypeptide. In one embodiment, the hTRT polypeptide has a sequence of SEQ. ID. No. 2 (Fig. 17). In one embodiment, the sequence is functionally linked to a promoter. In one embodiment, the hTRT has catalytic activity of telomerase. In one embodiment, the cell is human, for example a cell in a human patient.

In an alternative embodiment, the cell is cultivated in vitro. In a related embodiment, the cell is introduced into a human patient.

The invention further provides a method of treating a human disease by introducing a recombinant hTRT polynucleotide into at least one cell in a patient. In one embodiment, a vector is used for gene therapy. In a related embodiment, the method further consists in introducing into the cell a polynucleotide comprising a sequence encoding a human telomerase RNA, for example an hTRT polynucleotide operably linked to a promoter.

The invention also provides a method for increasing the proliferation ability of a verte-15-cell, the method comprising introducing an effective amount of a human telomerase reverse transcriptase (hTRT) polypeptide into the cell. In one embodiment, the hTRT polypeptide has catalytic activity from telomerase. The invention further provides cells and progeny of the cells with increased proliferation capacity.

The invention also provides pharmaceutical compositions containing a pharmaceutically acceptable carrier and a molecule selected from: an hTRT polypeptide, a polynucleotide encoding an hTRT polypeptide and an hTRT nucleic acid or a sub-sequence thereof.

The invention also provides a method of treating a condition associated with an increased level of telomerase activity within a cell, the method comprising introducing into the cell a therapeutically effective amount of an inhibitor of telomerase activity. wherein the inhibitor is an hTRT polypeptide or an hTRT polynucleotide. In one embodiment, the inhibitor is a polypeptide that comprises the sequence of SEQ. ID. No. 2 or 4 or a sub-sequence thereof. In further embodiments, the polypeptide inhibits TRT activity, for example the binding of endogenous TRT to telomerase RNA.

The invention also provides a vaccine comprising an hTRT polypeptide and an adjuvant.

30th

DESCRIPTION OF THE FIGURES FIG. 1

shows highly preserved residues in TRT motifs of humans, S. pombe (tez1), S. cerevisiae (EST2) and 35 Euplotes aediculatus (p123). Identical amino acids are marked with an asterisk (*), while similar amino acid residues are marked with a dot (•). Motif "0" in the figure is also called Motif T; Motif «3» is also called motif A.

Fig. 2

shows the location of telomerase-specific motifs and RT-specific sequence motifs of telomerase-40 proteins and other reverse transcriptases. The location of the telomerase-specific motif T and the conserved RT motifs 1, 2 and A-E are indicated by filled boxes. The HIV-1 RT marked with an open rectangle marks the portion of this protein shown in FIG. 3.

Fig. 3

45 shows the crystal structure of the p66 subunit of HIV-1 reverse transcriptase (“Brookhaven code” 1HNV). The view is shown from the back of the right hand, so that all motifs are recognizable.

Fig. 4

shows several sequence orientations of telomerase RTs (Sp_Trt1p, S. pombe TRT (here also denoted by 50 «tezlp»); bTRT, human TRT; Ea_p123, Euplotes p123; Sc_Est2p, S. cerevisiae Est2P) and members of other RT families (S_a1 , Cytochrome oxidase group II intron I-encoded protein from S. cerevisiae mitochondria, Dm_TART, reverse transcriptase from Drosophila melanogaster TART non-LTR retrotransposon element); HIV-1, reverse transcriptase from human HIV). TRT con and RT con represent “consensus” sequences for telomerase RTs and non-telomerase RTs. 55 amino acids are marked with an h, hydrophobic; p, polar; c, loaded. Triangles indicate residues that are conserved within the telomerase proteins, but are different in other RTs. The thick line under motif E denotes the "primer grip" area.

Fig. 5

shows the expression of hTRT-RNA in telomerase-negative mortal cell strains and telomerase-60 positive immortal cell lines, as described in Example 2.

Fig. 6

shows a possible phylogenetic family tree of telomerases and retro elements with RNA-dependent RNA polymerases as the root.

Fig. 7

65 shows a restriction map of the lambda clone G.o5.

6

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Fig. 8

shows a map of chromosome Sp with the location of the STS marker D5S678 (located near the hTRT gene).

Fig. 9

shows the construction of an hTRT promoter reporter plasmid.

Fig. 10

(two pages) shows the in vitro coexpression of hTRT and hTR for the production of catalytically active human telomerase.

Fig. 11

(two pages) shows the alignment of sequences of four TRT proteins and interesting motifs are marked. TRT con denotes a TRT “consensus” sequence. RT con denotes “con sensus” residues in other reverse transcriptases. “Consensus” remains with capital letters indicate absolute preservation in TRT proteins.

Fig. 12

shows a topoisomerase II cleavage site and NFicB binding site motifs in an hTRT intron, the sequence shown being SEQ. ID. No. 7 (Fig. 12) corresponds.

Fig. 13

(two pages) shows the sequence of the DNA encoding the Euplotes 123 kD telomerase protein subunit (Euplotes TRT protein).

Fig. 14

shows the amino acid sequence of the Euplotes 123 kD telomerase protein subunit (Euplotes TRT protein).

Fig. 15

(fourteen pages) shows the DNA and amino acid sequences of the catalytic subunit of S. pom-be-telomerase (S. pombe-TRT).

Fig. 16

(two pages) shows the hTRT cDNA sequence, the sequence shown SEQ. ID. No. 1 corresponds. Fig. 17

shows the hTRT protein encoded by the cDNA of Fig. 16. The protein sequence shown corresponds to SEQ. ID. No. 2.

Fig. 18

shows the sequence of clone 712562, the sequence shown SEQ. ID. No. 3 corresponds.

Fig. 19

Figure 25 shows a 259 residue protein encoded by clone 712562, the sequence shown being SEQ. ID. No. 10 corresponds.

Fig. 20

(seven pages) shows the sequence of an open reading frame nucleic acid encoding an a182-polypeptide variant, the sequence shown being SEQ. ID. No. 4 corresponds. This figure also shows the amino acid sequence of this Al82 polypeptide variant, the amino acid sequence shown SEQ. ID. No. 5 corresponds.

Fig. 21

(six pages) shows the sequence of a genomic hTRT clone, the sequence shown SEQ. ID. No. 6 corresponds. “Consensus” motifs and elements are given, including sequences that are characteristic of a topoisomerase II cleavage site, NFxB binding sites, an Alu sequence and other sequence elements.

Fig. 22

Figure 4 shows the effect of mutating the TRT gene in yeast as described in Example 1.

Fig. 23

shows the sequence of EST AA281296 SEQ. ID. No. 8 corresponds.

Fig. 24

shows the sequence of the 182 base pairs deleted in clone 712562, the sequence shown SEQ. ID. No. 9 corresponds.

Fig. 25

shows the results of an assay for telomerase activity from BJ cells transfected with an expression vector encoding an hTRT protein (pGRN133) or a control plasmid (pBBS212) as described in Example 13.

Fig. 26

Figure 12 is a schematic diagram of affinity purification of telomerase showing the binding steps and elution steps by displacement.

Fig. 27

is a photo of a Northern blot of telomerase preparations obtained during the purification protocol described in Example 1. Lane 1 contained 1.5 fMol telomerase RNA, lane 2 4.6 fMol, lane 3 14 fMol, lane 4 41 fMol, lane 5 nuclear extract (42 fMol telomerase), lane 6 Affi-gel heparin-purified telomerase (47 fmol telomerase), lane 7 affinity-purified telomerase (68 fMol) and lane 8 via glycerol gradient purified telomerase (35 fMol).

7

CH 689 672 A9

Fig. 28

shows telomerase activity during a cleaning protocol.

Fig. 29

is a photo of an SDS-PAGE gel showing the presence of an approximately 123 kD polypeptide and a 5 approximately 43 kD doublet from Euplotes aediculatus.

Fig. 30

is a graph showing the sedimentation coefficient of Euplotes aediculatus telomerase.

Fig. 31

is a photo of a polyacrylamide / urea gel with 36% formamide, which shows the substrate utilization of 10 Euplotes-Telomerase.

Fig. 32

shows the suspected alignments of telomerase RNA template and hairpin structure primers with telomerase RNA.

Fig. 33

15 is a photograph of lanes 25-30 of the gel shown in FIG. 31 with lower exposure.

Fig. 34

shows the DNA sequence of the gene encoding the 43 kD telomerase protein subunit of Euplotes. Fig. 35

(four pages) shows the DNA sequence and the amino acid sequences of all three open reading frames 20 of the 43 kD telomerase protein subunit from Euplotes.

Fig. 36

shows a sequence comparison between the 123 kD telomerase protein subunit from Euplotes (upper sequence) and the 80 kD polypeptide subunit from T. thermophila (lower sequence).

Fig. 37

25 shows a sequence comparison between the 123 kD telomerase protein subunit from E. aediculatus (upper sequence) and the 95 kD telomerase polypeptide from T. thermophila (lower sequence).

Fig. 38

shows the “best fit” alignment between a part of the “La domain” of the 43 kD telomerase protein subunit of E. aediculatus (upper sequence) and a part of the 95 kD polypeptide subunit of 30 T. thermophila (lower Sequence).

Fig. 39

shows the “best fit” alignment between a part, the “La domain” of the 43 kD telomerase protein subunit from E. aediculatus (upper sequence) and a part of the 80 kD polypeptide subunit from T. thermophila (lower sequence).

35 Fig. 40

shows the alignment and motifs of the polymerase domain of the 123 kD telomerase protein subunit of E. aediculatus and the polymerase domains of various reverse transcriptases, including a cytochrome oxidase group II intron 1-encoded protein from S. cerevisiae mitochondria (al Sc (group II)) count, dong (LINE) and yeast ESTp (L8543.12).

40 Fig. 41

shows the alignment of a domain of the 43 kD telamerase protein subunit with different La proteins.

Fig. 42

shows the nucleotide sequence encoding T. thermophila 80 kD protein subunit.

45 Fig. 43

shows the amino acid sequence of the T. thermophila 80 kD protein subunit.

Fig. 44

shows the nucleotide sequence encoding the T. thermophila 95 kD protein subunit.

Fig. 45

50 shows the amino acid sequence of the T. thermophila 95 kD protein subunit.

Fig. 46

shows the amino acid sequence of L8543.12 («Est2p»),

Fig. 47

shows the alignment of the amino acid sequence encoded by the Oxytricha PCR product with the Eu-55 plotes p123 sequence.

Fig. 48

shows the DNA sequence of Est2.

Fig. 49

shows the partial amino acid sequence of a cDNA clone encoding human telomerase 60 peptide motifs.

Fig. 50

shows the partial DNA sequence of a cDNA clone encoding human telomerase peptide motifs.

65

8th

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Fig. 51

shows the amino acid sequence of tez1, also known as S. pombe trt.

Fig. 52

(two pages) shows the DNA sequence of tez1. Intron areas and other non-coding areas are shown in lower case and exons (i.e. coding area) are shown in upper case.

Fig. 53

shows the alignment of EST2p, Euplotes and Tetrahymena sequences, as well as «consensus» sequences.

Fig. 54

shows the sequences of peptides useful for the production of anti-hTRT antibodies. Fig. 55

is a schematic summary of the tez1 + sequencing experiments.

Fig. 56

shows two degenerate primers that were used in the PCR to identify the S. pombe homologue to the E. aediculatus p123 sequences.

Fig. 57

shows the four major bands used in the PCR with the degenerate primers to identify the S. pombe homologue to the E. aediculatus p123 sequences.

Fig. 58

shows the alignment of the M2-PCR product with E. aediculatus p123, S. cerevisiae and Oxytricha telomerase protein sequences.

Fig. 59

Figure 3 is a diagram showing the 3 'RT-PCR strategy for the identification of the S. pombe homologue to the E. aediculatus p123.

Fig. 60

shows features of the libraries used to screen for S. pombe telomerase protein sequence and the results of the screening.

Fig. 61

shows the positive results obtained with the HindIII-cleaved positive genomic clones containing the S. pombe telomerase sequence.

Fig. 62

Fig. 5 is a schematic showing the 5'-RT-PCR strategy used to obtain the full length S. pombe TRT clone.

Fig. 63

shows the alignment of RT domains of catalytic subunits of telomerase for S. pombe (S.p.), S. cerevisiae (S.c.) and E. aediculatus (E.a.).

Fig. 64

shows the alignment of sequences from Euplotes («Ea_P123»), S. cerevisiae («Sc_Est2P») and S. pombe («Sp_Tez1p»). In part A, the shaded areas indicate residues that both sequences have in common. In Part B, the shaded areas indicate residues that all three sequences have in common.

Fig. 65

shows the disruption strategy used for the telomerase genes in S. pombe.

Fig. 66

shows the experimental results that confirm tez1's disruption.

Fig. 67

shows the progressive shortening of telomeres in S. pombe due to the disruption of tez1.

Fig. 68

(four pages) shows the DNA sequence and amino acid sequence of the ORF encoding a 63 kD teiomerase protein or a fragment thereof encoded by the EcoRI-Notl insertion of the Genbank clone # AA281296.

Fig. 69

shows an alignment of reverse transcriptase motifs from different sources.

Fig. 70

is a restriction map and a functional map of plasmid pGRN121.

Fig. 71

(two pages) shows the results of preliminary analyzes of nucleic acid sequencing of an hTRTcDNA sequence.

Fig. 72

(10 pages) shows the preliminary nucleic acid from hTRT and the derived ORF sequences in the three reading frames.

Fig. 73

is a restriction map and a functional map of plasmid pGRN121.

9

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Fig. 74

(8 pages) shows the improved nucleic acid sequence and derived ORF sequences from hTRT.

Fig. 75

shows a restriction map of the lambda clone 25-1.1.

Fig. 76

shows a map of the restriction sites and regions of DNA pGRN 144.4.

I. Introduction

Telomerase is a ribonucleoprotein complex (RNP) that comprises an RNA component and a catalytic protein component. The present invention relates to the cloning and characterization of the catalytic protein component of telomerase, hereinafter referred to as “TRT” (telomerase reverse transcriptase). This protein is referred to as TRT because it works as an RNA-dependent DNA polymerase, the RNA component of the telomerase (hereinafter referred to as "TR") being used to control the synthesis of the telomer DNA repeat sequences. In addition, TRT is evolutionarily related to other reverse transcriptases (see Example 12).

One aspect of the present invention relates to the cloning and characterization of the catalytic protein component of human telomerase, hereinafter referred to as "hTRT". Human TRT is of exceptional interest and value because, as noted below, telomerase activity in humans (and other mammalian cells) correlates with the property of cell proliferation, cell immortality and the development of a neoplastic phenotype. For example, in immortal human cells (e.g., malignant tumor cells and immortal cell lines), telomerase activity and, as shown in Example 2 below, the amount of gene products of human TRT are increased over mortal cells (e.g., most human somatic cells).

The present invention further provides methods and compositions of value for the diagnosis, prognosis, and treatment of human diseases and conditions, as will be described in detail below. In addition, methods and reagents are provided that are useful for immortalizing cells (in vivo and ex vivo), producing transgenic animals with desired features, as well as numerous other uses, which are largely described below. The invention also provides methods and agents useful for the production, cloning and re-cloning of TRT genes and proteins from ciliates, fungi, vertebrates, for example mammals, and other organisms.

As described in detail below, TRT was first characterized after purification of telomerase from the ciliate Euplotes aediculatus. The comprehensive purification of E. aediculatus telomerase using RNA affinity chromatography and other methods yielded the protein "p123". Surprisingly, it was found that p123 is not related to proteins previously thought to be protein subunits of the telomerase holoenzyme (i.e., the p80 and p95 proteins from Tetrahymena thermophila). Analysis of the sequences of p123 DNA and protein (Genbank accession number U95964; Figures 13 and 14) revealed the presence of reverse transcriptase (RT) motifs, consistent with the role of p123 as a catalytic subunit of telomerase (see e.g. Fig. 11). In addition, p123 is related to the S. cerevisiae (yeast) Estlp protein, which was known to play a role in the maintenance of telomeres in S. cerevisiae (Genbank accession number S5396). However, it was not recognized prior to the present invention that it encodes a protein of a telomerase catalytic subunit (see, for example, Lendvay et al., Genetics 144 (1996), 1399).

One aspect of the present invention relates to reagents and methods for identifying and cloning new TRTs using: nucleic acid probes and primers that are generated or derived from the disclosed TRT polynucleotides (e.g., for cloning TRT genes and cDNAs); Antibodies that specifically recognize motifs, motif sequences or other TRT epitopes (e.g. for the expression cloning of TRT genes or for the purification of TRT proteins); the screening of computer databases or other aids. For example, the PCR amplification (polymerase chain reaction) of DNA from S. pombe described in Example 1 with primers with degenerate sequences, which were designed on the basis of the Euplotes p123 RT motifs B 'and C, was selected. Of the four main products generated, one encoded a peptide sequence that is homologous to Euplotes p123 and S. cerevisiae Est2p. The complete sequence of the protein homologous to S. pombe TRT was obtained using this PCR product as a probe by screening cDNA and genomic banks of S. pombe and amplifying S. pombe RNA by reverse transcription and PCR (RT-PCR) . The complete sequence of the S. pombe gene (trt1 "; Genbank accession number AF015783; Fig. 15) showed that the homology between p123 and Est2p was particularly high in the reverse transcriptase motifs.

Amplification using degenerate primers derived from the Telomerase RT motifs was also used to obtain TRT gene sequences from Oxytricha trifallax and Tretrahy-mena thermophila as described in Example 1.

10th

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

The sequences Euplotes p123, S. pombe trt1 and S. cerevisiae Est2p according to the invention were searched for in computerized databases of human-expressed sequences “tags” (ESTs) using the program “BLAST” (Altschul et al., J. Mol. Biol 215 (1990), 403). The search in this database with the Est2p sequence did not result in a match, but a human EST (Genbank accession number AA281296; see SEQ. ID. No. 8) could be identified by searching with p123 and trtl sequences as described in Example 1 . This human EST is believed to encode a homologous protein. Complete sequencing of the cDNA clone containing the EST (hereinafter referred to as "clone 712562"; see SEQ. ID. No. 3) showed the presence of seven RT motifs. However, this clone could not encode a coherent human TRT, since motifs B ', C, D and E were contained in a different open reading frame (ORF) compared to the more NH2-terminal motifs. In addition, the distance between motifs A and B 'was significantly smaller than with the TRTs already characterized. (Clone 712562 was obtained from the I.M.A.G.E. Consortium; Lennon et al., Genomics 33 (1996), 151).

The cDNA clone pGRN121, which encodes a functional hTRT (SEQ. ID. No. 1), was isolated from a cDNA library which originated from the human cell line 293 (see Example 1). Comparison of clone 712562 with pGRN121 showed that clone 712562 has a deletion of 182 base pairs between motifs A and B '(SEQ. ID. No. 9). The additional 182 base pairs in pGRN121 mean that all TRT motifs are present in a single open reading frame and that the distance between the areas of motif A and motif B 'increases so that this is consistent with the distance at other known TRTs. SEQ encodes as described below in the examples (e.g. example 7). ID. No. 1 a catalytically active Teiomerase protein with the sequence of SEQ. ID. No. 2. The polypeptide from SEQ. ID. No. 2 has 1132 amino acids and an estimated molecular weight of about 127 kilodaltons (kD).

As discussed below and described in Example 9, the 182 base pair deletion of TRT cDNAs characteristic of clone 712562 is detected following reverse transcription of mRNA from telomerase positive cells (e.g. Testes and 293 cells). hTRT-RNAs that lack this sequence of 182 base pairs are commonly referred to as "al82 variants" and can represent one, two or more species. The hTRT variants, which lack the 182 base pair sequence found in the pGRN121 cDNA (SEQ. ID. No. 1), most likely do not encode a telomerase enzyme with complete catalytic activity, but they can play a role in telomerase regulation , as described below, and / or have partial telomerase activity, for example telomer binding or hTR binding activity, as discussed below.

One aspect of the present invention is thus to provide an isolated polynucleotide with a sequence of a naturally occurring human TRT gene or an mRNA, a polynucleotide with the sequence of SEQ. ID. No. 1 is included, but is not limited to this. A related aspect of the present invention relates to the provision of a polynucleotide encoding an hTRT protein, a fragment, a variant or a derivative. Another related aspect of the present invention relates to the provision of “sense” and “anisense” nucleic acids that bind to an hTRT gene or mRNA. The present invention further provides hTRT proteins that are either synthesized or purified from natural sources, as well as antibodies and other agents that specifically bind an hTRT protein or a fragment thereof. The present invention also provides many new methods, including methods using the above-mentioned compositions, for example by providing diagnostic and prognostic assays for human diseases, methods of drug development and therapeutic methods, of identification of telomerase-associated proteins and methods of screening for agents that can activate or inhibit telomerase activity. Numerous other aspects and embodiments of the present invention are provided below.

One aspect of the invention is the use of a polynucleotide that is approximately 10 kb or more in length and comprises a sequential sequence of at least 10 nucleotides that is identical or identical to a sequential sequence in a naturally occurring hTRT gene or hTRT mRNA is exactly complementary to examining or screening (for) an hTRT gene sequence or an hTRT mRNA or for producing a recombinant host cell. Another aspect of the invention is the use of an agent that increases the expression of hTRT in the manufacture of a medicament for the treatment of a condition treated by increasing the proliferation capacity of a vertebrate cell, optionally using the medicament to inhibit the effects of aging .

Another aspect of the invention relates to the use of an inhibitor of telomerase activity in the manufacture of a medicament for the treatment of a condition associated with an increased level of telomerase activity within a human cell. The proteins, variants and fragments according to the invention and the coding polynucleotides or fragments are also provided in a further aspect of this invention for use as a medicament.

The invention further relates to the use of a protein, a variant or a fragment thereof or a polynucleotide or fragment, as defined here, in the manufacture of a medicament.

11

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

ment, for example in the manufacture of a medicament for inhibiting an effect of aging or cancer.

In certain embodiments of the invention, the hTRT polynucleotides are not the 389 nucleotide polynucleotide of SEQ. ID. No. 8 and / or not clone 712562, the plasmid containing an insert with the sequence of SEQ. ID. No. 3 in Fig. 18.

The description below is organized by topic. Part II describes further amino acid motifs that are characteristic of TRT proteins. Parts III to VI describe, among other things, nucleic acids, proteins, antibodies and purified compositions of the invention, with a focus on compositions related to human TRT. Part VII describes, among other things, methods and compositions of the invention that are useful for the treatment of human diseases. Part VIII describes the production and identification of immortalized human cells. Part IX describes, among other things, the use of the nucleic acids, polynucleotides and other compositions according to the invention for the diagnosis of human diseases. Part X is a glossary of terms used in Parts I to IX. Part XI describes examples which relate to specific embodiments according to the invention. The description of the invention is structured according to topics and subtopics in order to make the subject more understandable, but is in no way a limitation.

II. TRT genes and proteins

The present invention provides isolated and / or recombinant genes and proteins having a sequence of a catalytic subunit of the telomerase protein (ie, telomerase reverse trans-scriptase), including, but not limited to, the naturally occurring forms of such genes and Proteins include in isolated or recombinant form. Typically, TRTs are large, basic proteins with amino acid motifs specific for reverse transcriptase (RT) and telomerase, as disclosed here. Since these motifs are conserved within different organisms, TRT genes can be obtained from numerous organisms using the methods according to the invention or using primers, nucleic acid probes and antibodies according to the invention, for example those which are specific for one or more of the motif sequences.

The seven RT motifs found in TRTs are similar to those found in other reverse transcriptases, but have special characteristics. For example, within the TRT-RT motifs, as shown in Figure 4, there are a number of amino acid substitutions (marked with an arrow) in amino acid residues that are highly conserved within the other RTs. For example, there are the two aspartic acid residues (DD) in motif C that coordinate the metal ions in the active center (see Kohlstaedt et al., Science 256 (1992), 1783; Jacobo-Molina et al., Proc. Nati. Acad. Sei USA 90 (1993), 6320; Patel et al., Biochemistry 34 (1995), 5351) in the context hxDD (F / Y) in the RTs of telomerase, while this in the other RTs in context (F / Y ) xDDh occur, where h is a hydrophobic amino acid and «x» is any amino acid; see Xiong et al., EMBO J. 9 (1990) 3353; Eickbush, in The Evolutionary Biology of Viruses (S. Morse ed. Raven-Press, NY, p. 121 (1994 ». Further systematic exchanges characteristic of the telomerase subgroups occur in the motif E, in which WxGxSx represents a« consensus »sequence or is conserved within the telomerase proteins while hLGxxh is characteristic of other RTs (Xiong et al., supra; Eickbush, supra). This motif E is referred to as the "primer grip" and has been described that mutations in this area influence "RNA priming", but not "DNA priming" (Powell et al., J. Biol. Chem. 272 (1997), 13262). Since telomerases are a DNA primer, ( it is not surprising that telomerase differs from other RTs in the area of the "primer handle". In addition, the distance between motifs A and B7 in the TRTs is larger as is typically the case is the case in the other RTs, where d This can represent an insertion within the "finger" area of the structure, which resembles a right hand (Fig. 3; see Kohlstaedt et al., op. cit .; Jacobo-Molina et al., Op. Cit .; and Patel et al., op. cit.).

Furthermore, the T motif, as mentioned above, is an additional characteristic of TRT proteins. For example, the T motif shown in Fig. 4 (W-L-X-Y-X-X-h-h-X-h-h-X-p-F-F-Y-X-T-E-X-p-X-X-X-p-X-X-X-Y-X-R-K-X-X-W, where X is any amino acid, includes the following, can be described using the following: h being hydrophobic and p polar, polar

T rp-R, -X7-R, -R, -R2-X-Phe-Phe-T yr-X-Thr-GIu -X j.9-Rj-R3-Arg-Rj-Xj-T rp where X is any amino acid and the subscript refers to a number of consecutive residues, Ri is leucine or isoleucine, R2 is glutamine or arginine, R3 is phenylalanine or tyrosine and R4 is lysine or histidine.

The T-motif can also be described using the following formula:

12th

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Trp-R, -X4-h-h-X-h-h-Rrp-Phe-Phe-Tyr-X-Thr-Glu-

Xp-X3-p-X3.3-Rj-Rj-Arg-Rj-Xj-Trp where X is any amino acid and a subscript refers to a number of consecutive residues, Ri is leucine or isoleucine, R2 is glutamine or arginine , R3 phenylalanine or tyrosine, R4 lysine or histidine, h a hydrophobic amino acid selected as Ala, Leu, Ile, Val, Pro, Phe, Trp, and Met and p a polar amino acid selected as Gly, Ser, Thr, Tyr, Cys, Asn and Gin is.

In a further embodiment, the present invention provides isolated, naturally occurring and recombinant TRT proteins that comprise one or more of the motifs shown in FIG. 11, for example,

Motif T

w-x12-ffy-x-te-x1 (vi rr-x, -w-x7-i

Motif r e-x2-v-x

Motif 1

x3-r-x2-pk-x3

Motif 2

x-r-x-r-x

Motif A

x, -f-x3-d-x4-yd-x2

Motif B '

y-x, -g-x2-qg-x3-s-x,

Motif C

x6-dd-x-l-x3

If the TRT protein shown contains more than one TRT motif, the order (NH2- + COOH) is as shown in FIG. 4.

In another embodiment, the present invention provides isolated, naturally occurring TRT proteins that include the following super motif:

(nh2) -x300 «owx, 2-ffy-x-te-xi0.irr-x3-w-x7-ix, .20-e-x2-vx-x5.20-x, -r-x2-pk- x4., 0-rxix-x60.i0-x «-f-xj-d-x4-yd-x; -xi0.130-y-x4-g-x2-qg-x, -s-xi-xj. 3y

x6-dd-x-l-x3-x10.20-x12-k

It will be apparent to those skilled in the art having the reagents disclosed herein, including the TRT sequences, that with these reagents and the methods and teachings provided herein (including specific procedures described below), those skilled in the art will have TRT genes and proteins obtained, isolated and can be prepared in recombinant form. For example, primers (e.g., degenerate amplification primers) are provided that hybridize to gene sequences that encode RT and T motifs characteristic of TRT. For example, one or more primers or degenerate primers that hybridize to sequences encoding the FFYXTE region of the T motif, other TRT motifs (as discussed below), or combinations of motifs or "consensus" sequences can be made based on the codon usage of the target organism, and to amplify the TRT gene sequence from genomic DNA or cDNA produced by the target organism. The use of degenerate primers is well known in the art. This also includes primer sets that hybridize to the set of nucleic acid sequences that may encode the amino acids of the target. The preference and use of certain codons within the target organism must be considered. Amplification (e.g. PCR) conditions are also used, which are suitable to allow base mismatches during the PCR attachment steps. Typically two primers are used, amplification systems with a single primer (or in this case a set with a single degenerate primer) are well known and can also be used to obtain TRT genes.

Table 1 shows examples of primers according to the invention which can be used for the amplification of new TRT nucleic acids, in particular those from vertebrates (e.g. mammals). “N” is an equimolar mixture of all four nucleotides and sequences in brackets represent equimolar mixtures of the specified nucleotides.

13

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Table 1

EXAMPLES OF DEGENERATED PRIMERS FOR AMPLIFICATING TRT

NUCLEIC ACIDS

Motif direction g'-sequence-3 '

a FFYVTE forward TT (CT) TT (CT) T A (CT) GTNACNGA b FFYVTE reverse TCNGTNAC (GA) TA (GA) AA (GA) AA

c RFIEKP Forward (CA) GNTT (CT) AT (ACT) CCNAA (AG) CC d RFIEKP Backward GG (TC) TTNGG (T G A) AT (G A) AANC

e AYPTI Forward GCNTA (CT) GA (CT) ACNAT f A YD TT Backward TANGT (GA) TC (GA) TANGC

g GIPQG Forward GGNAT (ACT) CCNCA (AG) GG h Ql? QQS Backward (GC) (AT) NCC (TC) TGNGG (TGA) ATNCC

i LVDDFL Forward (CT) TNGTNGA (CT) GA (CT) TT (CT) (CT) T j ÜDFLLVT Reverse GTNACNA (GA) NA (GA) (GA) AA (GA) TC (GA) TC

Allowed primer combinations (y = yes, n = no)

Backward

Forward h

d f

Hi

a -

n y

y y

y c -

n n

y y

y e -

n n

n y

y g-

n n

n n

y i -

n n

n n

n

In one embodiment, an amplified TRT nucleic acid is used as a hybridization probe for colonic hybridization with a gene bank (eg a cDNA bank) that has been produced from the target organism, a nucleic acid with which all or a substantial part of the TRT protein Part of the coding sequence is identified and isolated or cloned. Reagents and methods, such as those just described, are used in accordance with the methods described herein for obtaining TRT gene sequences from Oxytricha trifallax and Tetrahymena thermophila, such as

14

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

described in detail below. Of course, following the cloning of a previously uncharacterized TRT gene, the sequence can be determined by routine methods and the encoded polypeptide synthesized and examined for TRT activity, for example the catalytic activity of telomerase (as described here and / or by means of the Specialty of known telomerase assays).

It is also obvious to the person skilled in the art that TRT genes can be cloned using a large number of cloning methods according to the invention, since the TRT motif sequences and the nucleic acids according to the invention comprising such sequences can be used in a large number of such methods. For example, hybridization using a probe based on the sequence of a known TRT with DNA or other nucleic acid libraries from the target organism as described in Example 1 can be used. Degenerate PCR primers or their amplification products, for example those described above, can themselves be labeled and used as hybridization probes. In a further embodiment, expression cloning methods are used. For example, one or more antibodies that specifically bind peptides that span a TRT motif or other TRT epitope, such as specifically bind the FFYXTE motif (where X represents any of the 20 standard amino acids), can be used to isolate a ribosomal complex can be used which comprises a TRT protein and the mRNA encoding it. To produce the antibodies according to the invention, the peptide immunogens typically have a length of 6 to 30 amino acids, preferably 10 to 20 amino acids. The antibodies can also be used to screen a cDNA expression bank derived from the desired organism to identify a clone encoding a TRT sequence. In a further embodiment, in order to identify a clone encoding a TRT sequence, DNA databases can be searched by computer for DNAs which contain conserved sequences within known TRTs.

Another aspect of the present invention relates to the provision of compositions comprising an isolated or recombinant polypeptide having the sequence of a naturally occurring TRT protein. Typically, the naturally occurring TRT has a molecular weight between about 80,000 daltons (d) and about 150,000 d, preferably between about 95,000 d and about 130,000 d. Typically, the naturally occurring TRT has a positive total charge at pH 7 (the estimated p1 typically being over 9). In one embodiment, the polypeptide has a telomerase activity as defined here. In a related embodiment, the polypeptide has a sequence for a TRT-specific region (T motif) and a telomerase activity. The invention also provides fragments of such polypeptides. The present invention also provides isolated or recombinant polynucleotides with the sequence of a naturally occurring gene encoding a TRT protein. The invention also provides isolated TRT polynucleotides having a sequence of TRT from a non-vertebrate (e.g. yeast) and vertebrate, e.g. mammals (e.g. mice or humans). The isolated polynucleotides can be associated with other naturally occurring or vector nucleic acid sequences. Typically, the length of the nucleic acid isolated is less than about 300 kb, preferably less than about 50 kb, more preferably less than about 20 kb, and most preferably less than about 10 kb. Occasionally the length is less than about 5 kb or 2 kb. In some embodiments, the isolated TRT polynucleotide is even smaller, for example, a gene fragment, primer, or probe less than about 1 kb or 0.1 kb in length.

III. Nucleic acids

A) General

The present invention provides isolated and recombinant nucleic acids with a sequence of a polynucleotide encoding a telomerase protein (TRT) catalytic subunit, for example a recombinant TRT gene from Euplotes, Tetrahymena, S. pombe or humans. Examples of polynucleotides are shown in Fig. 13 (Euplotes), Fig. 15 (S. pombe) and Fig. 16 (human, Genbank accession number AF15950). The present invention provides “sense” and “anti-sense” polynucleotides with a TRT gene sequence, including probes, primers, TRT protein-encoding polynucleotides and the like.

B) Human TRT

The present invention provides nucleic acids with a sequence of a human telomerase catalytic subunit (i.e., hTRT).

One aspect of the invention relates to the provision of a polynucleotide with a sequence or sub-sequence of a human TRT gene or an RNA. In one embodiment, the polynucleotide according to the invention has a sequence of the SEQ. ID. No. 1 (Fig. 16) or a sub-sequence thereof. In a further embodiment, the polynucleotide has a sequence of the SEQ. ID. No. 3 (Fig. 18), SEQ. ID. No. 4 (Fig. 20) or sub-sequences thereof. The invention also provides polynucleotides

15

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

sequences essentially identical to the hTRT nucleic acid sequences disclosed here, for example SEQ. ID. No. 1 and the other sequences disclosed (e.g. SEQ. ID. Nos. 4, 6 (Fig. 21) and 7 (Fig. 12)). Thus, the invention provides naturally occurring alleles of the human TRT genes and variants of the polynucleotide sequences with one or more nucleotide deletions, insertions or substitutions based on an hTRT nucleic acid sequence disclosed herein. As described below, variants of the nucleic acids can be made using the recombination or synthetic methods described below, or by other means.

The invention also provides isolated and recombinant polynucleotides with a sequence of a flanking region of a human TRT gene. These polynucleotides include those derived from genomic sequences from untranslated regions of the hTRT mRNA. An example of a genomic sequence is SEQ. ID. No. 6 (Fig. 21). As described in Example 4, SEQ. ID. No. 6 was obtained by sequencing the clone> .G <i> 5, which was isolated from a human genome library. M3 <& 5 contains an insert with a length of 15 kilobase pairs (kbp), which include approximately 13,000 bases 5 'to the hTRT coding sequences. This clone contains hTRT promoter sequences and further regulatory sequences of the hTRT gene (e.g. enhancer).

The invention also provides isolated and recombinant polynucleotides with a sequence from an intron region of a human TRT gene. An example of an intron sequence is SEQ. ID. No. 7 (see Example 3 and Fig. 12). In some embodiments, hTRT introns are enclosed in "minigenes" for improved expression of hTRT proteins in eukaryotic cells.

A related aspect of the present invention relates to the provision of polynucleotides which encode hTRT proteins or protein fragments, including modified and modified hTRT polypeptides and variants of the hTRT polypeptides. In one embodiment, the encoded hTRT protein or fragment has one as in SEQ. ID. No. 2 (FIG. 17) shown amino acid sequence on or an amino acid sequence with conservative exchanges of the SEQ. ID. No. 2. In one embodiment, the encoded hTRT protein or fragment has exchanges that have activity of the protein (e.g., catalytic activity of telomerase). Of course, due to the degeneracy of the genetic code, the nucleic acids encoding the hTRT protein need not have the sequence of a naturally occurring hTRT gene, i.e. a variety of polynucleotides can be an hTRT polypeptide having an amino acid sequence of SEQ. ID. Code number 2. The present invention provides any possible variation of nucleotide sequences produced by the selection of combinations based on the selection of possible codons in accordance with the known triplet genetic code. All of these variations are hereby expressly disclosed. Thus, in some cases, nucleotide sequences encoding hTRT polypeptide which can hybridize with the nucleotide sequences of the naturally occurring sequence (under suitably selected conditions of stringency) are preferred, but in other cases it may be advantageous to produce nucleotide sequences encoding hTRT which one have essentially different condon usage.

In certain embodiments, the invention provides hTRT oligo- and polynucleotides that comprise a sub-sequence of an hTRT nucleic acid disclosed herein (e.g., SEQ. ID. Nos. 1, 4, 6, and 7). The nucleic acids according to the invention typically comprise at least about 10, preferably at least about 12 or about 15 consecutive bases of the illustrated hTRT polynucleotide. The nucleic acids according to the invention often also comprise a longer sequence, for example a sequence with a length of at least about 25, about 50, about 100, about 200 or at least about 500 bases, for example if expression of a polypeptide is desired. In some embodiments of the present invention, the hTRT polynucleotide differs from a polynucleotide having the sequence of EST AA281296 (SEQ. ID. No. 8).

Further embodiments of the present invention provide "Al82hTRT" polynucleotides with a sequence encoding naturally occurring or non-naturally occurring hTRT polynucleotides, for example SEQ. ID. No. 3 or SEQ. ID. No. 4, which does not contain the 182 base pair sequence found in pGRN121 (SEQ. ID. No. 9 (FIG. 24)), which is also missing in clone 712562). These polynucleotides are of interest in part because they encode polypeptides that contain combinations of TRT motifs that are different from those found in full-length hTRT polypeptide (SEQ. ID # 2), such as the polypeptide encoded by pGRN121 , differentiate. As discussed below, it is believed that these polypeptides can play a biological role in nature (e.g., in regulating the expression of telomerase in cells) and / or can be used as drugs (e.g., as dominant-negative products that function inhibit the wild-type protein) or that these polypeptides have further roles and uses, for example those described here.

For example, in contrast to clone pGRN121, clone 712562 encodes a protein with 259 amino acid residues and an estimated molecular weight of approximately 30 kD (hereinafter referred to as "712562 hTRT". The 712562 hTRT polypeptide (SEQ. ID. No. 10 (Fig. 19) ) contains the motifs T, 1, 2 and A, but not the motifs B ', C, D and E. Similarly, a variant of the hTRT polypeptide with therapeutic and other activities can be expressed by a nucleic acid which is the The pGRN121 cDNA is similar, but lacks the 182 base pairs that the clone 712562 does not have, for example with the sequence SEQ ID NO: 4. This nucleic acid (hereinafter referred to as "pro90hTRT"), which is produced using routine synthetic methods or recombinant methods as they

16

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

can be synthesized encodes a protein with 807 amino acid residues (estimated molecular weight about 90 kD) that has the same amino-terminal sequences as that of SEQ. ID. No. 1 encoded hTRT protein, but deviates at the carboxy-terminal region (the first 763 amino acid residues have in common both proteins, the last 44 amino acid residues of pro90hTRT differ from the full-length hTRT). The pro90hTRT polypeptide contains the motifs T, 1,2 and A, but not the motifs B, C, D and E and can therefore have some, but not all, telomerase activities.

C) Production of human TRT nucleic acids

There are numerous applications for the polynucleotides according to the invention. These include, but are not limited to, the expression of polypeptides encoding hTRT or fragments thereof, the use as “sense” or “antisense” probes or primers for the hybridization and / or amplification of naturally occurring hTRT genes or RNAs ( eg for diagnostic or prognostic applications) and as medicinal products (eg in «antisense», triplex or ribozyme compositions). After reading this disclosure, it is apparent that these uses have a tremendous impact on the diagnosis and treatment of human diseases related to aging, cancer and fertility, and also on the growth, reproduction and manufacture of products based on them Cells. As described in the sections below, the hTRT nucleic acids of the invention can be prepared using known techniques (e.g., by cloning, synthesis or amplification).

1) Cloning, amplification and recombinant production

In one embodiment, hTRT genes or cDNAs are cloned using a nucleic acid that specifically hybridizes to hTRT mRNA, cDNA or genomic DNA. A sample suitable for this purpose is a polynucleotide with the sequence set out in SEQ. ID. No. 1 is provided or a sub-sequence thereof. Typically, the genomic DNA or cDNA with the target hTRT is ligated into a vector (e.g. a plasmid, phage, virus, artificial yeast chromosome etc.) and it can be in a genomic or cDNA library (e.g. a human placenta cDNA library ) being found. After an hTRT nucleic acid is identified, it can be isolated according to standard procedures known to those skilled in the art. An example illustrating the screening of a human cDNA library for the hTRT gene is Example 1; a similar example relating to the screening of a human genomic library is Example 4. Cloning methods are well known and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Vols. 1-3, Cold Spring Harbor Laboratory, (1989); Berger and Kimmel, Methods In Enzymology, vol. 152: Guide To Molecular Cloning Techniques, San Diego: Academic Press, Inc. (1987); Ausubel et al., Current Protocols in Molecular Biology, Green Publishing and Wiley-Interscience, New York (1997); Cashion et al., U.S. Patent No. 5,017,478; and Carr. EP-B1 0 246 864.

The invention also provides genomic hTRT nucleic acids or cDNA-hTRT nucleic acids which can be obtained by amplification methods such as e.g. the polymerase chain reaction (PCR) was isolated. In one embodiment, the sequence encoding the hTRT protein is amplified from an RNA sample or cDNA sample (e.g., double-stranded placenta cDNA (Clontech, Palo Alto, CA) using the primers

Primer

5'-GTGAAGGCACTGTTCAGCG-3 '("TCPl.l") and

51-CGCGTGGGTGAGGTGAGGTG-3 ("TCPl.15").

In some embodiments, a third primer or a second pair of primers can be used, for example for nested PCR to increase specificity. An example of a second pair of primers is

51-CTGTGCTGGGCCTGGACGATA-31 ("billTCPö") and 51-AGCTTGTTCTCCATGTCGCCGTAG-3 '("TCPl.14").

It will be apparent to those skilled in the art that numerous other primers and combinations of primers useful for the amplification of hTRT nucleic acids are provided by the present invention.

In addition, the invention provides primers which amplify any specific region (for example coding regions, promoter regions and / or introns) or a sub17

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

sequence of genomic hTRTDNA, cDNA or RNA. For example, the hTRT intron at position 274/275 of SEQ. ID. No. 1 (see example 3) can be amplified (e.g. for the detection of genomic clones) using the primers TCP1.57 and TCP1.52 (primer pair 1) or the primers TCP1.49 and TCP1.50 (primer pair 2). (The designations for the primers refer to the primers listed in Table 2 below). The primer pairs can be used individually or in a «nested PCR» in which primer set 1 is used first. Another illustrative example relates to primers which specifically amplify and thus detect (for example, the size or completeness of a cDNA) the 5 'end of the hTRT mRNA or the exon which codes the 5' end of the hTRT gene -Clons to judge). The following primer pairs are useful for amplifying the 5 'end of hTRT: primers K320 and K321 (primer pair 3); Primers K320 and TCP1.61 (primer pair 4); Primers K320 and K322 (primer pair 5). The primer sets can be used in a “nested PCR” in the order of set 5, then set 4 or set 3, or set 4 and then set 3. Another illustrative example relates to primers selected for specific amplification or detection of the conserved hTRT-TRT motif region, which comprises approximately the middle third of the mRNA (eg for use as a hybridization probe to identify TRT clones from non-human organisms ). The following primer pairs are useful for amplifying the TRT motif range of hTRT nucleic acids: primers K304 and TCP1.8 (primer pair 6), or primers LT1 and TCP1.15 (primer pair 7). The primer sets can be used in an experiment with «nested PCR» in the order set 6, then set 7.

Suitable conditions for PCR amplification are known to the person skilled in the art. These include (without limitation) 1 unit of Taq polymerase (Perkin Elmer, Norwalk CT), each 100 pM dNTP (dATP, dCTP, dGTP, dTTP), 1 x PCR buffer (50 mM KCl, 10 mM Tris, pH- 8.3 at room temperature, 1.5 mM MgCl 2, 0.01% gelatin) and 0.5 nM primer, the amplification being about 30 cycles at 94 ° C for 45 seconds, 55 ° C for 45 seconds and 72 ° C for 90 seconds. It will be apparent to those skilled in the art that other thermostable DNA polymerases, reaction conditions and parameters related to the cycles also provide suitable amplification. Other suitable methods for in vitro amplification that can be used to obtain hTRT nucleic acids include, but are not limited to, the methods described below. After amplification, the hTRT nucleic acids, if desired, can be cloned into a variety of vectors using routine molecular biology techniques, detected, or otherwise used in accordance with the methods of the invention.

It is known to the person skilled in the art that the cloned or amplified hTRT nucleic acids obtained as described above can be prepared or propagated using further methods, for example by chemical synthesis or replication by transformation into bacterial systems such as E. coli (see for example Ausubel et al., loc. cit.) or eukaryotic expression systems, for example mammalian expression systems. Similarly, hTRT-RNA can be expressed in accordance with the in vitro methods of the invention or in bacterial systems such as E. coli, for example without using commercially available vectors containing promoters recognized by an RNA polymerase, such as for example, T7, T3 or SP6 or by transcription of DNA generated by PCR amplification using primers containing an RNA polymerase promoter.

The present invention further provides altered or modified hTRT nucleic acids. It is obvious to the person skilled in the art that the cloned or amplified hTRT nucleic acids obtained can be modified by generally known methods (for example site-directed mutagenesis, “linker scanning” mutagenesis), for example shortened, derivatized, changed), or simply de novo can be synthesized as described below. The altered or modified hTRT nucleic acids are useful for a variety of uses, including, but not limited to, facilitating cloning, manipulating an hTRT gene or gene product, or expressing a variant of an hTRT gene product. For example, in one embodiment, the hTRT gene sequence is altered to encode an hTRT polypeptide with altered properties or activities, as discussed in detail below, for example, by mutation in a conserved motif from hTRT. In another illustrative example, the mutations in the protein coding region of an hTRT nucleic acid can be used to change the glycosylation pattern, to change codon preferences, to produce splice variants, to remove protease-sensitive sites, to generate antigenic domains , to modify specific activity etc. In further embodiments, the sequence encoding hTRT and its derivatives is changed without changing the encoded amino acid sequences, for example to produce RNA transcripts with more desirable properties, such as, for example, increased translation efficiency or a longer or shorter half-life in comparison to transcripts that occur from the naturally occurring one Sequence were made. In a further embodiment, to increase the expression rate of the peptide in a special prokaryotic or eukaryotic expression host, modified codons are selected according to the frequency with which certain codons are used by the host. Useful in vitro and in vivo recombination methods which can be used to produce the hTRT polynucleotide variants according to the invention can be found in Sambrook et al. and Ausubel et al., supra.

18th

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

As mentioned above, the present invention provides nucleic acids with flanking (5 'or 3') and intron sequences of the hTRT gene. The nucleic acids include of interest because they contain promoters and other regulatory elements that are involved in hTRT regulation and are useful for the expression of hTRT and other recombinant proteins or RNA gene products. It is obvious that in addition to those in SEQ. ID. Nos. 6 (Fig. 17) and 7 provided nucleic acid sequences additional hTRT intron sequences and flanking sequences can be easily obtained using routine molecular biology methods. For example, an additional genomic hTRT sequence can be obtained by further sequencing of the lambda clone Go5, which is described above and in Example 4. In addition, other hTRT genomic clones and sequences can be screened by screening a human genomic library using an hTRT nucleic acid probe with a sequence or sub-sequence of SEQ. ID. # 1 can be obtained. Additional clones and sequences (e.g., still further upstream) can be obtained using labeled sequences or subclones derived from XGo5 to search for suitable gene banks. Other useful methods for further characterizing flanking hTRT sequences are the general methods described by Gobinda et al., PCR Meth. Applic. 2 (1993), 318; Triglia et al., 1988, Nucleic Acids Res. 16: 8186; Lagerstrom et al., PCR Methods Applic. 1: 111 (1991); and Parker et al., Nucleic Acids Res. 19 (1991), 3055.

Intron sequences can be identified by routine means, for example by comparing the genomic hTRT sequence with hTRT cDNA sequences (see for example Example 3), by S1 analysis (see Ausubel et al., Loc. Cit., Chapter 4), or numerous others means known in the art. Intron sequences can also be found in pre-mRNA (i.e. unspliced or incompletely spliced mRNA precursors). These can be amplified or cloned after reverse transcription of cellular RNA.

If desired, the cloned, amplified, or otherwise synthesized hTRT nucleic acid or other TRT nucleic acid can be determined or verified using well known methods for DNA sequencing (see, e.g., Ausubel et al., Supra). The useful sequencing methods use enzymes such as the Klenow fragment of DNA polymerase I, Sequenase (US Biochemical Corp., Cleveland OH), Taq DNA polymerase (Perkin Elmer, Norwalk CT), thermostable T7 polymerase (Amersham, Chicago IL), or combinations of recombinant polymerases and exonucleases with "proofreading" activity, for example the ELONGASE amplification system available from Gibco BRL (Gaithersburg MD). For sequencing or verifying the sequence of oligonucleotides (such as oligonucleotides produced by de novo chemical synthesis), the method of Maxam and Gilbert is preferred (Maxam and Gilbert, Meth. Enz. 65 (1980), 499; Ausubel et al., loc. cit., chapter 7).

The 5'-untranslated sequences of hTRT or other TRT mRNAs can be determined directly by cloning a "full length" hTRT or another cDNA using standard methods such as reverse transcription of mRNA and then by cloning and sequencing the cDNA obtained become. Preferred libraries obtained by oligo (dT) priming for screening or amplifying full-length cDNAs which have been selected for inclusion of larger cDNAs are preferred. Genebanks obtained by "random priming" are also suitable. These often contain a larger proportion of clones that contain 5 'regions of genes. Other generally known methods for obtaining 58 RNA sequences can be used, for example that of Frohman et al., Proc. Nat. Acad. Sci., USA 85 (1988), 8998, RACE protocol. If desired, the transcription start site of an hTRT or other TRTmRNA can be determined by routine methods using the nucleic acids provided herein (e.g., those with the sequence of SEQ. ID No. 1). One method is S1 nuclease analysis (Ausubel et al., Op. Cit.), In which a labeled DNA with a sequence of the 5 'region of SEQ. ID no. 1 is used.

2) Chemical synthesis of nucleic acids

The present invention also provides hTRT polynucleotides (RNA, DNA or modified) made by direct chemical synthesis. Chemical synthesis is generally preferred for the preparation of oligonucleotides or of oligonucleotides and polynucleotides containing non-standard nucleotides (e.g. probes, primers and "antisense" oligonucleotides). The direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, for example by the phosphotriester method of Narang et al., Meth. Enzymol. 68: 90 (1979); the Brown et al. phosphodiester method, Meth. Enzymol. 68: 109 (1979); the diethyl phosphoramidite method of Beaucage et al., Tetra. Lett. 22: 1859 (1981); and the method of U.S. Patent No. 4,458,066 using a solid support. Chemical synthesis typically produces a single-stranded oligonucleotide that can be converted to double-stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase and an oligonucleotide primer using the single strand as a template. The person skilled in the art is aware that the chemical synthesis of DNA is often based on sequences

19th

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

zen is limited to about 100 or 150 bases, but longer sequences can be obtained by ligation of the shorter sequences or by more sophisticated synthetic methods.

The hTRT polynucleotides and oligonucleotides (or hTRT or other polynucleotides or oligonucleotides) according to the invention can be achieved using non-standard bases (for example those which differ from adenine, cytidine, guanine, thymine and uridine) or non-standard backbone structures desired properties (eg increased nuclease resistance, closer binding, stability or a desired TM) can be produced. The processes described in the PCT publication WO 94/12 633 are among those for the production of nuclease-resistant oligonucleotides. A large number of useful modified oligonucleotides can be made, including oligonucleotides with a peptide nucleic acid (PNA) backbone (Nielsen et al., Science 254 (1991), 1497) or incorporation of 2'-0-methylribonucleotides, phosphorothioate -Nucleotides, methylphosphonate nucleotides, phosphotriester nucleotides, phosphorothioate nucleotides and phosphoramidates. Other useful oligonucleotides may contain alkyl and halogen substituted sugar moieties that contain one of the following groups at the 2 "position: OH, SH, SCH3, F, OCN, OCH3OCH3, 0CH30 (CH2) nCH3, 0 (CH2) nNH2 or 0 (CH2) nCH3, where n is from 1 to about 10; C1 to C10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl; CI; Br; CN; CF3; OCF3; O-, S-, or N-alkyl ; O-, S-, or N-alkenyl; SOCH3, SO2CH3; ONO2; NO2; N3, NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino, substituted silyl; an RNA-cleaving group, a cholesterol group, a folate group, a Reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents with similar properties, folate, cholesterol and other groups which facilitate the uptake of the oligonucleotide, for example Lipid analogs can be conjugated directly or via a linker at the 2-position of each nucleoside or at the 3'- or 5'-position of the 3'-terminal or 5'-terminal nucleoside. One or more such conjugates can be used. Oligonucleotides can also have sugar mimetics, for example cyclobutyls instead of the pentofuranosyl group. Further embodiments may include at least one modified base form or a "universal base" such as inosine or the inclusion of other non-standard bases such as queosine and wybutosine as well as acetyl, methyl, thio and similarly modified forms of adenine, cytidine, guanine, thymine and Uridine, which is not easily recognized by endonucleases. The invention further provides oligonucleotides with backbone analogs, including phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramate, alkylphosphotiester, sulfamate, 3'-thioacetyl, methylene (methylimino), 3'-N-carbamate, morpholine caramate, chiral Methylphosphonates, nucleotides with short-chain alkyl or cycloalkyl linkages between the sugars, short-chain links ("backbone links") between the sugars with different atoms or those that are heterocyclic or CH2-NH-OCH2, CH2-N (CH3) -OCH2, CH2 (- N (CH3) -CH2, CH2-N (CH3) -N (CH3) -CH2 and 0-N (CH3) -CH2-CH2 backbones (where phosphodiester is OPO-CH2), or mixtures of these compounds, are useful also oligonucleotides with a morpholino backbone structure (U.S. Patent No. 5,034,506).

Other useful literature includes: Oligonucleotides and Analogues, A Practical Approach, F. Eckstein (ed.) IRL Press at Oxford University Press (1991); Antisense Strategies, Annais of the New York Academy of Sciences, Vol. 600, Baserga and Denhardt (ed.) (NYAS 1992), Milligan et al., J. Med. Chem. 36 (14) (July 9, 1993), 1923-1937; Antisense Research and Applications (1993, CRC Press) published the entire publication, specifically Chapter 15 of Sanghvi, entitled "Heterocyclic base modifications in nucleic acids and their applications in antisense oligonucleotides." Antisense The-rapeutics, Sudhir Agrawal (Editor (Humana Press, Totowa, New Jersey, 1996).

D) The labeling of nucleic acids

It is often useful to label the nucleic acids according to the invention, for example if the hTRT or other oligonucleotides or polynucleotides are to be used as nucleic acid probes. The markings (see below) can be incorporated by any means well known to those skilled in the art. In one embodiment, an unamplified nucleic acid (e.g. mRNA, poly-A mRNA, cDNA) is labeled. Agents for the production of labeled nucleic acids are generally known to the person skilled in the art. These include, for example, nick translation, labeling using «random priming», end labeling (e.g. using a kinase) and chemical conjugation (e.g. photobiotinylation). In a further embodiment, the label is incorporated simultaneously during an amplification step in the preparation of the sample nucleic acids. Thus, for example, the polymerase chain reaction (PCR) or other nucleic acid amplification methods with labeled primers or labeled nucleotides provide a labeled amplification product. In a further embodiment, a label is incorporated into the transcribed nucleic acids by transcription amplification using a labeled nucleotide (e.g. fluorescein-labeled UTP and / or CTP). An amplification product can also or alternatively be marked after completion of the amplification.

20th

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

E) Examples of oligonucleotides

As noted above and discussed in detail below, oligonucleotides are used for a variety of uses, such as primers, probes, therapeutic or other "antisense" oligonucleotides, and triplex oligonucleotides. Numerous other possible uses result from the present description. Table 2 illustrates certain specific oligonucleotides that can be used in the practice of the invention. Of course, numerous other useful oligonucleotides according to the invention can be synthesized by the person skilled in the art using the teaching provided here.

In Table 2, “seq” means that the primer has been used or is useful for sequencing; “PCR” means that the primer has been used for PCR or is useful for it; “AS” means that the primer has been used or is useful in the antisense inhibition of telomerase activity; “CL” means that the primer has been used or useful for cloning regions of hTRT genes or RNA; “Mut” means that the primer was used to construct mutants for hTRT genes or gene products or is of use. "UC" means "Upper case" and "Ic" means "lower case", mismatches and insertions (relative to SEQ. ID No. 1) are underlined; Deletions are indicated by a «-». Of course, none of the information in Table 2 is intended to limit the use of a particular oligonucleotide to any single use or set of uses.

21

U1

A

o ro ro primer 5'-Seauen7-.V •

TCP 1.1 GTGAAGGC ACTGTTC AGCG

TCPl.2 GTGGATGATTICTTGTTGG

TCP 1.4 CTGGACACTCAGCCCTTGG

TCP 1.5 GGC AGGTGTGCTGG ACACT

TCP 1.6 TTTG ATG ATGCTGGCGATG

TCP 1.7 GGGQCTC GT CTT CTAC AGG

TCP 1.8 C AGCAGG AGG ATCTTGT AG

TCPl.9 TGACCCCAGGAGTGGCACG

TCPl. 10 TCA AGCTG ACT CG ACACCG

TCP1.I! CGGCGTGACAGGGCTGC

TCP 1.12 GCTGAAGGCTGAGTGTCC

TCP 1.13 TAGTCCATGTTCACAATCG

TCPl. 14 CTGTGCTGGGCCTGGACGATA

TCPl. 15 CGCGTGGGTGAGGTGAGGTG

TCP 1.16 1TTCCGTGTTGAGTGTTTC

TCP I I 7 GTCACCGTGTTGGGCAGG

TCP 1.19 GCTACCTGCCCAACACGG

TCPl. 20 GCGCGAAGAACGTGCTGG

TCPl.21 CA-CTGCTCCTTGTCGCCTG

TCPl.22 TTCCCAAGGACTTTGTTGC

TCPl.24 TGITCCTCAAGACGCACTG

TCP 1.25 TACT GCGT GCGTCGGT ATG

TCP 1.26 GG TCITGCGGCTG AAGTGT

TCPl .27 TGG rrCACCTGCTGGCACü

TCP 1.28 GTGGTTTCTGTGTGGTGTC

TCP1.29 GACACCACACAGAAACCAC

TCP 1.30 GTGCCAGCAGGTGAACCAG

TCP I 32n GCAGTGCGTCTTGAGGAGC

TCPl .33 TOGA ACCAT AGCGTCAGGG AG

TCPl.34 GGCCTCCCTGACGCTATGGTT

TCPl.35 GC (GT) CGGCGCTGCCACTCAGG

TCP 1.3 51 GCTCGGCGCTGCCACl CAGG

TCP 1.36 ACGCCGAG ACCA AGCAC TTC

co en

CJ O

ro o

Table 2 useful oligonucleotides

use

Comments Fehoaamnç? • see PCR AS CL MUT

Y

Y

seq

PCR

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

O T

en oo «o o> -j io>

(O

Ol o

U1 m

CTI

o en

■ fc. O

CJ

O

TCPl.38 CCAAAG AGGTGGCTTCTT CG

TCP 1.39 AAGGCCAGCACGTTCTTCGC

TCPl.40 CACGTTCGTGCGGCGCCTG

TCP 1.41 CCTTCACCACCAGCGTGCG

TCPI.42 GGCG ACGACGT GCTGGTT C

TCP1.43 GGCTCAGGGGCAGCGCCAC

TCP 1.44 CTGGCAGGTGTACGGCTTC

TCPl.45 (iCGTGGACCGAGTGACCGTGCirri'C

TCP 1.46 GACGTGGTGGCCGCGATGTGG

TCP 1.47 GAAGTCTGCCGTTGCCCAAGAG

TCP 1.48 GACACCACACAGAAACCACGGTCAC

TCP 1.49 CGCCCCCTCCTTCCGCCAGGT

TCPl.50 CGAAGCCGAAGGCCAGCACGTTCTT

TCP 1.51 GGTGC.CCCG AGTGCTGCAG AGG

TCPl.52 GTAGCIXJCGCACGCTGGTGGTGAAG

TCPl.53 TGGGCGACGACGTGCTGGTTCA

TCPl .54 TATGGTTCCAGGCCCGTrCGCATCC

TCPl.55 CCAGCTGCGCCTACCAGGTGTGC

TCPl.56 GGCCTCCCTGACGCTATGGTTCCAG

TCPl.57 GGTGCTGCCGCTGGCCACGTTCG

TCPl .58 TCCCAGGGCACGCACACCAGGCACT

TCP 1.59 GTACAGGGCACACCTTTGGTCACTC

TCPl.60 TCGACGACGTACACACTCATCAGCC

TCP 1.61 AGCGGCAGCACCTCGCGGT AGTGGC

TCPl.62 CCACCAGCrcCITCAGGCAGGACAC

TCP 1.63 CCAGGGCTTCCCACGTGCGCAGCAG

TCP 1.64 CGCACG AACGIGGCCAGCGGC AGCA

TCPl.65 TGACCGTGGTTTCTGTGTGGTGT

TCP1.66 CCCTCTTCAAGTGCTGTCTGATTCC

TCPl.67 ATCGCGGCCACCACGTCCCT

TCP 1.68 TGCTCC AG ACACTCGGCCGGTAG A A

TCP 1.69 ACGAAGCCGTACACCTGCC

TCP 1.72 CGACATCCCTGCGTTCTTGGCTTrC

TCP 1.73 CACTGCTGGCCTCATTCAGGG

TCPl.74 GCGACATGGAGAACAAGC

TCPl.75 GCAGCCATACTCAGGGACAC

TCP 1.76 CC ATCCTCTCC ACGCTGCTC

M ro en o

Table 2

(Continuation)

X X

X X

X X

X X

X X

X X

X X

X X

X X

X X

X X

X X

X X

y * ^

X x 2

x x en

X X g

X X _

O)

XX

TO

x x>

XX <0

X X

X X

X X

X X

X X

X X

X X

X X

X X

X X

X X

X X

X X

X X

X X

X X

X X

ro en ro o

Table 2

(Continuation)

TCP 1.77 GCGATGACCTCCGTGAGCCTG

TCP 1.78 CCCAGGACAüCiCTCACGG A

billTCPJ CCTCTTCAAGTGCTGTCTGATTCC

billTCP2 C AGCTCGACGACGT ACACACTCATC

bilITCP4 CTGACGTCCAGACTCCGCTTCAT

billTCP6 AGCTTGTTCTCCATGTCGCCGT AG

rpprimOl GACCTGAGCAGCTCGACGACGTACACACTCATC

1.11 GI CG 1CGAGCIGCICAGGTC

LÜ AGCACGCTGAACAGTGCCTr

Lt3 GACCTGAGCAGCTCGACGAC

Ll4 AAGGCACrGITCAGCGTGCT

Li 5 CGG ££ GAGTGTCTGGAGCAA

1.16 GGATGAAGCGGAGTCTGGA

HamH I L (7 ATGGATCCGTCGTCGAGCTGCTCAGGTCT

Sai I Ll8 ATCAGC TGAGCACGCTG AACAGTGCCTTC

K.303 GTCTCCGTGACATAAAAGAAAGAC

K304 GCCAAGTTCCTGCACTGGCT

K305 GCCTGTTCTTTTGAAACGTGGTCT

K.306 XXGCCTGTT CTTTTGAAACGTGGTCT

K311 GTCAAGATGCCTGAGATAGAAC

K3I2 TGCTT AGCTTGTGGGGGTGTCA

K3I3 TGCTT AGCTTGTGGGGGTGTCA

K.320 GCTGCGTCCTGCTGCGCACGT

K32I CAGCGGGGAGCGCGCGGCATC

K322 TGGGCCACCAGCGCGCGGAAA

stanti. 1 CGGCCGCAGCCCGTCAGGCrTGGGG

slanti.2 CCGACAQCTCCCGCAGCTGCACCC

slanti. 3 CGT AC AC ACTCATC AGCCAGT GCAGG AACTT GGC

slanli.4 CGCüCCCGCTCGTAGTTGAGCACGCTGAACAGTGCCTTC

slami 5 ÜCGGAGTCTGGACGTCAGCAGGGCGGGCCTGGCITCCCCi

UTR2 ATTTGACCCACAGGGACCCCCATCCAG

FW5 ATGACCGCCCTCCTCGTGAG

Naml GCCACCCCCGCGATGCC

Nam2 AGCCCTGGCCCCGGCCA

Nam3 TCCCACGTGCGCAGCAG

Nam4 AGCAGÜACGCAGCGCTG

PEU I CGCGGTAGTGGCTGCGCAGCAGGGAGCGCACGGC

BamHI stables

Pvu II position (not Sai I

X = biotin, = K305

<J \

O

CJ

Table 2

(Continuation)

PE02 LMIOl

LMI03

LM104 LMIOÎ

LMI06

LM107

LMI08

LMI09 LM_FFYT

TCF061: 1IUMOI:

HUM02:

HUM03:

HUM04:

SLW FIN

SLW FIC

SLW F2N SLW F2C

SLW F3N

CCAGGGCTTCCCACGTGCGCAGCAGGACÜCAGCGC CTAGTCTAGATCA / GCT AGCGTAATCTGGAACATCGT A TGGGTA / GTCCAGGATGGTCTTGAAGTC T ACCATGGGCT ACCCAT ACGACGTTCCAGATTACGCTCA

TATGAGCGTAATCTGGAACGTCGTATGGGTAGCCCATGG

GTGTACGTCGTCGAGCTCCTCAGGTCTGCCTTTT

ATGTCACGGAG

GTGT ACGTCGTCG AGCTCCTCAGGTCTTTCGCTT ATGTC ACGGAGACC

CCTCAGGTCTTTCnTGCTGTCACGG AG ACAACGTTT1 CAAAAGAACAG

GGTC'I I ICI I I IATGTCGCGGAGACAACGTTT CAAAAGAACAG

CTTTCTTTTATGTCACGGCGACAACGTTTCAAAAGAACA

ATGAGTGTGTACGTCGTCGAGCTCCTCAGGTCTACCACG

CAAAAGAACAGGCTCTTTTTC

GGCTGATGAGTGTGTACGTCGTCGA

ACGTGGTCTCCGTGACATAAAAGAA

AGGT CTTTCrTTT ATGTCACGGA

CACAGACCCCCGTCGCCTGGTC

CGGAGTCTGGACGTCAGCAGGGC

ceceealccelaaclaaaATGCCGCGCGCTCCCCGCTGC

Xba I si.fr/HA day / hTRT in pGRNI21 inaariwt HA was in «m» Nde I SWh at the 5 * endo of hTRT attachment to LM103 change = F559A

(phe> ala)

Change = F560A

(phe> ala)

Change = Y561A

(tyr> ala)

Change = T563A

(thr> ala)

Change = E564A deletion from FFYVTE (aa 559-554)

Complementary to TCP1 61 to DD-Motif, designed to attach to the mTRT to DD-Motif, designed to attach to the mTRT if necessary designed to attach to the mTRT if necessary designed to attach to the mTRT for a GST fusion construct (782- 1636)

ccEEaaltcellaeltacttaCAAAGAGGTGGcTICI TCGGC

UC ■ hTRT seq, Ic = BamH1 digit + 2 stop codons for a GST fusion construct (782-1636) LIC = hTRT seq, le = EcoR I digit + 3 stop codons SLW FIN / SLW FIC ampHfify a B93 nt piece of pGRN121 ( 782 -1636)

cgcgeatccetaaclaaaGCCACCTC I I I GGAGGGTGCG for a GST fusion construct (1625-2458)

UC = hTRT seq, Ic = BamH1 digit + 2 stop codons ccpeaaUcgttayttacttaAGACCTGAGCAGCTCGACGAC for a GST fusion construct (1625-2458)

UC = hTRT seq, Ic = EcoR I site + 3 stop codons SLW F2N / SLW F2C amplify an 872 nt piece of PGRN121 (1625 - 2458)

ceceeatccetaactaaaATGAGTGTGTACGTCGTCGAG for a GST fusion construct (2426-3274)

en en co o

Table 2

(Continued)

UC = hTRT seq, le = Ba / nHI position +2 stop codons for a GST fusion construct (2426-3274)

UC = hTRT seq, lc = EcoR I position +3 stop codons SLW F3N / SLW F3C amplify an 887 nt piece of pGRN1121 (2426-3274)

ceceeatccgtaactaaaATCCCGCAGGGCTCCATCCTC for a GST fuslon construct (3272-4177)

UC = hTRT seq, lc = BamHI digit + 2 stop codons ccggaaltcgttaBttacttaGTCCAGGATGGTCTTGAAGTC for a GST Fuskxis construct (3272-4177)

UC = hTRT seq, lc = EcoR I site + 3 stop codons SLW F4N / SLW F4C amplify a 994 nt piece of pGRN121 (3272-4177)

SLW F3C ccgeaattcettagttacttaGATCCCCTGGCACTGGACO

SLW F4N SLW F4C

40-60 GGCATCGCGGGGGTGGCCGGG

260-280 GGACACCTGGCGGAAGGAGGG

500-520 GCGTGCCAGCAGGTGAACCAG

770-790 CTCAGGGGCAGCGCCACGCCT

885-905 AGGTGGC1TC1TCGGCGGGTC

1000-1020 GGACAAGGCGTGTCCCAGGGA

1300-1320 GCTGGGGTG ACCGCAGCTCGC

1520-1540 GATGAACTTCTTGGTGTTCCT

2110-2130 GTGCGCC AGGCCCTGTGG ATA

2295-2315 GCCCATGGGCGGCCTTCTGGA

2450-2470 GAGGCCACTGCTGGCCTCATT

2670-2690 GGGTGAGGTGAGGTGTCACCA

3080-3110 GCTGCAGCACACATGCGTGAAACCTGTACGC

3140-3160 GACGCGCAGGAAAAATGTGGG

3690-3710 CCGAGCGCCAGCCTGTGGGGA

55-75 CAGCGGGGAGCGCGCGGCATC

151-171 CAGCACCTCGCGGTAGTGGCT

TP 1.1 TCAAGCCAAACCTGAATCTGAG

TP 1.2 CCCGAGTGAATCTTTCTACGC

TP 1.3 GTCTCTGGCAGTTTCCTCATCCC

TP 1.4 TTTAGGCATCCTCCCAAGCACA

Phosphorothioate Phosphorothloat Phosphorothioate Phosphorothioate Phosphorothioate Phosphorothioate Phosphorothioate Phosphorothioate Phosphorothioate Phosphorothioate Phosphorothioate Phosphorothioate Phosphorothioate Phosphorothioate Phosphorothioate Phosphorothioate Phosphorothioate

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

IV. TRT proteins and peptides

A) General

The invention provides a wide variety of hTRT proteins which are useful, inter alia, for inhibiting telomerase activity in a cell, for inducing an anti-hTRT immune response, as a therapeutic reagent, as a standard or as a control in a diagnostic assay , as a target in screening for activation or inhibition of an activity of hTRT or telomerase and numerous other possible uses are either described here or obvious to the person skilled in the art. The hTRT proteins according to the invention include functionally active proteins (which are useful, for example, for transferring telomerase activity into a telomerase-negative cell) and variants, inactive variants (which are useful, for example, for inhibiting telomerase activity in a cell). , hTRT polypeptides, proteins, and telomerase RNPs (eg, the protein-containing ribonucleo-protein complexes) that have one, different, or all functional activities of naturally occurring hTRT and telomerase, as discussed further below for illustration purposes.

In one embodiment, the hTRT protein according to the invention is a polypeptide with the sequence of SEQ. ID. No. 2 (Fig. 17) or a fragment thereof. In another embodiment, the hTRT polypeptide differs from SEQ. ID. No. 2 through internal deletion, insertion or conservative replacement of amino acid residues. In a related embodiment, the invention provides hTRT polypeptides that SEQ. ID. No. 2 essentially the same. The invention also provides hTRT polypeptides that are related to the amino acid sequence of SEQ. ID. No. 2 are modified, i.e. for example truncated, mutated, derivatized or fused to other sequences (for example to form a fusion protein). In addition, the present invention provides telomerase RNPs comprising an hTRT protein according to the invention which complexes with a template RNA (e.g. hTR). In further embodiments, one or more telomerase-associated proteins are associated with an hTRT protein and / or hTR.

The invention also provides other naturally occurring hTRT species or non-naturally occurring variants, for example proteins with the sequence of SEQ. ID. No. 5 (Fig. 20), SEQ. ID. No. 10 (Fig. 19) or a substantially identical sequence and fragments, variants or derivatives thereof.

However, the invention provides still further hTRT species and variants. An example of an hTRT variant may result from ribosomal reading frame shift of mRNA encoded by clone 712562 (SEQ. ID. No. 3 (FIG. 18)) or by the pro90 hTRT variant described in SEQ. ID. No. 4 (FIG. 20) and these result in the synthesis of hTRT polypeptides that contain all TRT motifs (see, for example, Tsuchihashi et al., Proc. Nati. Acad. Sci. USA 87 (1990 ), 2516; Craigengen et al., Cell 50 (1987), 1; Weiss, Cell 62 (1990), 117). Ribosomal reading frame shift can occur when specific mRNA sequences or secondary structures cause the ribosome to "freeze" and a nucleotide to jump forwards or backwards within the sequence. Thus, a ribosomal reading frame shift on the 712562 mRNA could result in the synthesis of a polypeptide with approximately 523 amino acid residues. Such a ribosomal reading frame shift on the pro90 sequence could result in a protein with about 1071 residues. It is apparent that the proteins resulting from a ribosomal reading frame shift can also be expressed by the synthetic methods or recombination methods provided by the invention.

Human TRT proteins, peptides, and functionally equivalent proteins can be obtained by purification, chemical synthesis, or recombinant production, as discussed in more detail below.

B) TRT protein activity

The TRT polypeptides according to the invention (including fragments, variants, products of alternative alleles and fusion proteins) can have one, more or all of the functional activities associated with native hTRT. Unless otherwise stated, it is assumed here that an hTRT or another TRT polypeptide has a stated activity if the activity is either from the hTRT protein without an associated RNA (eg HTR) or in a complex of hTRT with associated RNA (eg hTR) shows. The hTR-binding activity of hTRT is an example of an activity associated with the hTRT protein. Methods for producing complexes from nucleic acids (e.g. hTR) and the hTRT polypeptides according to the invention are described below.

Modification of the hTRT proteins (e.g., by chemical or recombinant means, including mutation or modification of a polynucleotide encoding the hTRT polynucleotide, or chemical synthesis of a polynucleotide with a sequence different from the native polynucleotide sequence) to achieve this a different set of activities compared to native hTRT may be useful in therapeutic applications or screening for specific modulators of hTRT or telomerase activity. It can also perform assays for a number

27

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

of hTRT activities to identify agents (e.g., activity modulating agents) that interact with hTRT or telomerase to alter telomerase activity.

The activities of native hTRT, as discussed below, include catalytic activity of telomerase (which can be either processive or non-processive); Telomerase processivity; Activity of conventional reverse transcriptase; nucleolytic activity; Primer or substrate (telomer, synthetic telomerase substrate or primer) binding activity, dNTP binding activity; RNA (i.e. hTR) binding activity; and protein binding activity (e.g. binding to telomerase-associated proteins, telomer-binding proteins or to a complex of protein and telomeric DNA). Of course, the present invention also provides hTRT compositions without special hTRT activity, but with useful activities related to hTRT or other TRT proteins (e.g. certain short immunogenic peptides, inhibitory peptides).

1) Catalytic activity of telomerase

As used herein, a polypeptide according to the invention has "catalytic activity of telomerase" if the polypeptide is capable of a DNA primer or substrate by adding a partial, one or more than one repeat with a sequence (eg TTAGGG) that is encoded by a matrix nucleic acid (eg hTR). This activity can be processive or non-processive. Processive activity occurs when a telomerase RNP adds multiple repeat units to a primer or telomerase before the DNA is released from the enzyme complex. Non-processive activity occurs when telomerase attaches a partial or a repeat unit to a primer and is then released. In vivo, however, a non-process response could add multiple repeat units through successive rounds of association, extension, and dissociation. However, this can also occur in vitro, but is typically not observed in standard assays due to the very large molar excess of primers compared to telomerase under standard assay conditions.

To characterize an hTRT polypeptide for non-process activity, a common telomerase reaction is carried out under conditions that favor a non-process reaction, e.g. at high temperatures, e.g. 35-40 ° C, typically 37 ° C, low dGTP concentrations (1 nM or less), high primer concentrations (5 pM or more), and high dATP / TTP concentrations (2 mM or more), the temperature and the dGTP typically have the greatest impact. To characterize an hTRT polypeptide in terms of process activity, a conventional telomerase reaction is carried out using conditions which favor a process reaction, for example 27 to 34 ° C., typically 30 ° C., high dGTP concentration (10 mM or more ), low primer concentration (1 nM or less) and / or low dATP and TTP concentrations (0.3-1 mM), whereby the temperature and the concentration typical for dGTP are most critical. Alternatively, a TRAP assay (for processive or moderate processive activity) or the dot blot and gel blot assay (for processive activity) can be used. The hTRT polypeptide according to the invention can have a non-processive activity but no processive activity (for example if a change in the hTRT polypeptide reduces or eliminates the ability to translocate), but it can also be only processive or both activities have.

a) Non-process activity

Non-processive catalytic activity of telomerase can extend the DNA primer from the position where the 3 'end attaches to the RNA template to the 5' end of the template; this extension typically ends with the addition of the first G residue (e.g. if the matrix is hTR). As shown in Table 3, the exact number of nucleotides added depends on the position of the 3'-terminal nucleotide of the primer within the TTAGG repeat sequence.

28

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

TABLE 3

NON-PROCESSIVE ACTIVITY

i)

TTAGGGttag (DNA)

3 'AUCCCAAUC

5 '(RNA)

ü)

TTAGggttag (DNA)

3 'AUCCCAAUC

5 '(RNA)

in DNA, UC = primer, le = attached nucleotides

Thus, 4 nucleotides are added to the -TTAGGG primer (i), while 6 nucleotides are added to the --TTAG primer (ii). The first repeat unit added in a process reaction by telomerase corresponds to this step; in a processive reaction, however, telomerase carries out a translocation step, whereby the 3 'end is released and is bound again at a position, at the 3' region of the template, which is sufficient to prime the addition of a further repeating unit (see Morin, Eur. J. Cancer 33: 750 (1997).

A completely non-processive reaction only results in the production of a band in a conventional assay using a single synthetic primer. Since this result can also be caused by other enzymes, for example the activity of a terminal transferase, it may be desirable in some applications to verify that the product is the result of a catalytic activity of telomerase. A band generated by telomerase (hTRT) can be distinguished by various additional features. The number of nucleotides added to the end of the primer should match the position of the primer terminus. Thus, a -TTAGGG primer should have 4 attached nucleotides and a -TTAG primer should have 6 nucleotides (see above). In practice, two or more primers permuted in the sequence, which have the same overall length but different 5 'and 3' end points, can be used. The following example is intended to illustrate this. The non-procedural extension of the primers TTAGGGTTAGGGTTAGGG and GTTAGGGTTAGGGTTAGG will produce products whose absolute length differs in one nucleotide (4 are added to TTAGGGTTAGGGTTAGGG to a total length of 22 nt and GTTAGGGTTAGGGTT are added to a total length of 5 nGTT. The nucleotide dependence of the reaction should match the position of the primer terminus. Thus, a product of the -TTAGGG primer should need dGTP, TTP and dATP but not dCTP and a product of the -AGGGTT primer should need dGTP and dATP but not TTP or dCTP. The activity should be sensitive to the pretreatment with RNAse or Micrococcus nuclease under conditions which lead to the degradation of hTR and thus to the elimination of the template (see Morin, Cell 59 (1989) 521).

b) Processive activity

In practice, process activity can easily be observed by the appearance of a 6 nucleotide ladder in a conventional assay, a TRAP assay, gel blot assay, or the dot blot assay. The common assay is described in Morin, Cell 59 (1989), 521, the entire contents of which are incorporated herein by reference; the TRAP assay is described in U.S. Patent No. 5,629,154; see also PCT publication WO 97/15 687, PCT publication WO 95/13 381; Krupp et al., Nucleic Acids Res. 25 (1997), 919 and Wright et al. Nuc. Acids Res. 23 (1995), 3794, to which reference is made here in full. The dot blot assay is described in detail in parallel U.S. patent application serial no. 08/833 377, filed on April 4, 1997, to which reference is hereby made in full. The references cited above are hereby incorporated by reference in their entirety and for all purposes. Other assays for processive catalytic activity of telomerase can also be used, for example the "Stretch-PCR" -As-say by Tatematsu et al., Oncogene 13 (1996), 2265. The gel blot assay, a combination of conventional ones and dot blot assays can also be used. In this variant, a conventional assay is carried out with a radioactively labeled nucleotide and with high dGTP concentrations (e.g. 0.1-2 mM). After performing the usual assay, the synthesized DNA is separated by denaturing PAGE and transferred to a membrane (e.g. nitrocellulose). Telomer DNA (the product of telomerase - an extended telomerase primer or substrate) can then be obtained by Ver29

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

drive like hybridization using labeled telomeric DNA probes (e.g. probes containing the CCCTAA sequence as used in the dot blot assay as above). An advantage of this method is that they are more sensitive than the usual assay and provide information about the size of the synthesized fragments and the processivity of the reaction.

c) Activity regulations

The telomerase activity of an hTRT polypeptide can be determined using an unpurified, partially purified or substantially purified hTRT polypeptide (e.g. in association with hTR) in vitro or after expression in vivo. For example, telomerase activity in a cell (e.g. a cell that expresses a recombinant hTRT polypeptide according to the invention) can be detected by determining the increase or decrease in the length of the telomeres. Typical assays for catalytic activity of telomerase can be performed using a complex of hTRT and hTR. However, alternative telomerase template RNAs can be substituted or one can carry out assays to determine another activity such as telomerase primer binding. Assays to determine the length of the telomeres are known in the art. This includes the hybridization of probes to telomer DNA (an amplification step can be included) and a TRF analysis, i.e. analysis of telomeric DNA restriction fragments (TRFs) after restriction endonuclease digestion (see PCT publications WO 93/235 572 and WO 96/41 016; Counter et al., EMBO J. 11 (1992), 1921; Allsopp et al., Proc. Nati. Acad. Sci. USA 89 (1992), 10114; Sanno, Am. J. Clin. Pathol. 106 (1996), 16 and Sanno, Neuroendocrinology 65 (1997), 229.

The catalytic activity of telomerase of an hTRT polypeptide can be detected by a number of methods, the assays described above and other assays relating to catalytic activity of telomerase can be used. In one method, the hTRT protein is expressed (e.g., as described below) in a telomerase negative human cell in which hTR is expressed (ie, either normally in the cell or by recombinant expression) and the presence or absence of telomerase activity is determined in the cell or in cell lysate. Examples of suitable telomerase negative cells are IMR 90 (ATCC, # CCL-186) or BJ cells (a human foreskin fibroblast line; see, for example, Feng et al., Science 269 (1995), 1236). Other examples include pigmented retinal epithelial cells (RPE), human umbilical cord artery epithelial cells (HUVEG; ATCC # CRL-1730), human aortic endothelial cells (HAEC; Clonetics Corp., # CC-2535) and human breast epithelial cells (HME; Hammond et al. , Proc. Nati. Acad. Sci. USA 81 (1984), 5435; Stampfer, T., Tissue Culture Methods 9 (1985), 107). In an alternative embodiment, the hTRT polypeptide is expressed in a telomerase-positive cell (eg by transfection with an hTRT expression vector) and an increase in the telomerase activity in the cell compared to a non-transfected control cell is then detected, if the polypeptide has a catalytic activity of telomerase. Usually, the catalytic activity of telomerase is significantly increased in a cell transfected with a suitable expression vector which expresses hTRT, that is to say it is at least about 2 times, at least about 5 times or even at least about 10 times to 100 times or even 1000 times higher in Comparison to untransfected (control) cells.

In a further alternative embodiment, the hTRT protein is expressed in a cell (eg a telomerase negative cell in which hTR is expressed) as a fusion protein (see below) with a label or an "epitope tag" for ease of purification. In one embodiment, the RNP is obtained from the cell using an antibody that specifically recognizes the "tag." Preferred "tags" are typically short or small and may have a cleavage site or other properties that allow the "tag" from the hTRT polypeptide. Examples of suitable tags include the Xpres epitope (Invitrogen, Inc. San Diego CA) and other portions that can be specifically bound by an antibody or nucleic acid. Other equivalent methods are described, for example, in Example 6. In an alternative embodiment, the tags include those from inserted sequences For example, of sequences that are in SEQ. ID. No. 1 upstream of the ATG codon, which translates the protein of SEQ. ID. No. 2 initiated, whereby the insertion of a (new) methionine initiation codon can be included in the upstream sequence.

Of course, when an hTRT variant is expressed in a cell (e.g., as a fusion protein) and the subsequent isolation (e.g., as a ribonucleoprotein complex), other cell proteins (ie, telomerase-associated proteins) can be associated with (directly or indirectly bound to) the isolated complex. . In such cases it will sometimes be desirable to determine telomerase activity in the complex containing hTRT, hTR and the associated proteins.

30th

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

2. Other activities of telomerase or TRT protein

The hTRT polypeptides according to the invention include variants which lack a catalytic activity of telomerase, but which have retained one or more other activities of telomerase. These other activities and methods according to the invention for measuring such activities include (but are not limited to) those discussed in the following sections:

a) Usual reverse transcriptase activity

The usual telomerase activity with regard to reverse transcriptase is described, for example, in Morin (1997), cited above, and Spence et al., Science 267 (1995) 988. Because hTRT contains conserved amino acid motifs necessary for the catalytic activity of reverse transcriptase, hTRT has the ability to transcribe exogenous (i.e., non-hTR) RNAs. In a conventional RT assay, the ability of the enzyme to transcribe an RNA template is measured by extending an attached DNA primer.

Reverse transcriptase activity can be determined by numerous means known in the art, for example by observing the increase in the size of a labeled nucleic acid primer (e.g. RNA or DNA) or the incorporation of a labeled dNTP. See, for example, Ausubel et al., Op. Cit.

Since hTRT is specifically associated with hTR, the DNA primer / RNA template can of course be modified for a common RT assay to achieve features related to hTR and a telomeric DNA primer. For example, the RNA can have the sequence (CCCTAA) n, where n is at least 1, at least 3, at least 10 or larger. In one embodiment, the (CCCTAA) n region is at or near the 5 'end of the RNA (similar to the 5' localization of template regions in telomerase RNAs). Similarly, the DNA primer may have a 3 'end that has portions of the TTAGGG telomeric sequence, e.g. XnTTAG, XnAGGG, Xn (TTAGGG) qTTAG, etc., where X is a non-telomeric sequence and n is 8- Is 20 or 60-30 and q is 1 to 4. In a further embodiment, the DNA primer has a 5 'end which is not complementary to the RNA template, so that the 5' end remains unbound when attached. Additional modifications of standard reverse transcription assays that can be used in the methods of the invention are known in the art.

b) Nucleolytic activity

Nucleolytic activity of telomerase is described, for example, in Morin, 1997, op. Cit. Collins and Grieder, Genes and development 7 (1993) 1364. Telomerase has nucleolytic activity (Joyce and Steitz, Trends Biochem. Sci. 12 (1987), 288), but the telomerase activity has special features. Telomerase removes nucleotides, usually only one, from the 3 ^ end when the 3 'end of the DNA is positioned at the 5' border of the DNA template. In humans and in Tetrahymena, this nucleotide is the first G of the telomer repeat unit (TTAGG in humans). Telomerase preferentially removes G residues, but has no nucleolytic activity towards other nucleotides. This activity can be monitored. Two different methods are described here for illustration. One method involves a common telomerase reaction with a primer that binds the entire template sequence (i.e., ends at the template boundary; -TAGGGATTAG in humans). Nucleolytic activity is detected by monitoring the exchange of the last dG residue for a radiolabelled dGTP provided in the assay. The exchange is monitored by the appearance of a band with the size of the initial primer in gel electrophoresis and autoradiography.

A preferred method uses a DNA primer that has a "blocked" 3 'end that cannot be extended by telomerase. The 3'-blocked primer can be used in standard telomerase assays, but is not extended until the 3 "nucleotide is removed by the nucleolytic activity of telomerase. The advantage of this method is that telomerase activity is reduced by various standard measures can be monitored, and the signal is strong and easier to quantify. The blocking of the 3 "end of the primer can be achieved in different ways. In one procedure, a 3'-deoxy-dNTP residue is added to the 3 'end of the primer using standard oligonucleotide synthesis procedures. This end has a 2'OH, but not the 3'OH required for telomerase. There are other ways to block the 3'-end, for example using a 3'-dideoxy end of a 3'-amine end, etc. An example of a primer for an assay for a nucleolytic activity of hTRT is 5'- TTAGGGTTAGGGTTA (G3 ') with the last residue corresponding to a 3'-deoxy-guanosine residue (Glen Research, Sterling, VA). Numerous other variants relating to a small primer, which are based on the teaching provided here, are known to the person skilled in the art.

31

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

c) Primer (Telomer) binding activity

Primer (telomer) binding activity of telomerase is described, for example, by Morin, 1997, op. Cit .; Collins et al., Celi 81 (1995), 667 and Ham'ngton et al., J. Biol. Chem. 270 (1995), 8893. Telomerase is believed to have two sites that bind a telomeric DNA primer. The RT motifs associated with primer binding show that hTRT and / or hTRT / hTR has DNA primer binding activity. There are several ways to determine primer binding activity. However, most methods have one step in common, namely the incubation of a labeled DNA primer with hTRT or hTRT / hTR or other TRT / TR combinations under suitable binding conditions. Most methods also use a means to separate unbound DNA from protein-bound DNA. These procedures include the following steps:

i) Gel shift assays (which are also referred to as electrophoretic / mobility shift assays) are assays in which unbound DNA primer is separated from protein-bound DNA primer by electrophoresis in a non-denaturing gel (Ausubel et al., Loc. Cit.).

ii) Matrix binding assays involve various variations in the basic procedure. This includes binding the hTRT or the hTRT / hTR complex to a matrix (e.g. nitrocellulose) either before or after incubation with the labeled primer. By binding the hTRT to a matrix, the unbound primer can be mechanically separated from the bound primer. Residual unbound DNA can be removed by washing the membrane before quantification. It is obvious to the person skilled in the art that there are various possibilities for coupling proteins to such matrices, solid supports and membranes, these include, for example, chemical, photochemical and UV crosslinking, antibodies / epitope and non-covalent, hydrophobic, (electrostatic, etc. ) Interactions.

Any DNA with an affinity for telomerase can be used as the DNA primer, for example a telomer DNA primer such as (TTAGGG) n, where n can be 1 to 10 and is typically 3 to 5. The 3 'and 5' ends can end at any position in the repeat sequence. The primers can also have 5 'or 3' extensions of non-telomeric DNA that can facilitate labeling or detection. The primers can also be derivatized, for example to facilitate detection or isolation.

d) dNTP binding activity Telomerase's dNTP binding activity is described, for example, in Morin, 1997, cited above, and Spence et al., cited above. described. Telomerase requires dNTPs for the synthesis of DNA. The hTRT protein has nucleotide binding activity and can be assayed for dNTP binding similarly to other nucleotide binding proteins (Kantrowitz et al., Trends Biochem. Sci. 5 (1980), 124). Typically, the binding of a labeled dNTP or a dNTP analog is monitored, as is known in the art for non-telomerase RT proteins.

e) RNA (i.e. hTR) binding activity

RNA (ie, hTR) binding activity of telomerase is described, for example, in Morin, 1997, loc. Cit., Harrington et al., Science 275 (1997), 973 and Collins et al., Cell 81 (1995), 677. The RNA binding activity of a TRT protein according to the invention can be detected in a manner similar to that described above with regard to the DNA primer binding assay, using a labeled RNA probe. Methods for separating bound and unbound RNA and for detecting RNA are well known in the art. These can be used for the activity assays according to the invention in a manner similar to that described for the DNA primer binding assay. The RNA can be full-length hTR, fragments of hTR, or other RNAs that can be shown to have an affinity for telomerase or hTRT. See U.S. Patent No. 5,583,016 and PCT Publication No. 96/40 868 (see also USSN 08/478,352, filed June 7, 1995).

3) Telomerase motifs as targets

As noted above, the present invention provides hTRT polypeptides that do not have the full set (as described above) of telomerase activities from naturally occurring telomerase, hTRT, or other TRTProteins. In view of the present description of RT and of telomerase-specific motifs of TRT, it is obvious that changes or mutations of conserved amino acid residues which can be found in the motif sequences discussed above lead to the formation of mutants with loss of activity which, for therapeutic purposes, are useful for screening and characterizing drugs and for other applications. For example, deletion of motifs B results in the RT domains of the endogenous TRT gene in s. pombe, as described in Example 1, to haploid cells in which a progressive telomer shortening to the point where hybridization with telomer repeat

32

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

units were almost no longer detectable, could be observed, which indicates a loss of catalytic activity of telomerase. Similarly, changes in the WxGxS site of motif E can affect DNA primer binding or telomerase function. In addition, changes in the amino acids in motifs A, B 'and C can influence the catalytic activity of telomerase. The mutation of the DDM motif of hTRT can significantly reduce or cancel telomerase activity (see Example 16).

C) The synthesis of hTRT and other TRT polypeptides

The invention provides a number of methods for making the hTRT and other TRT polypeptides disclosed herein. The chemical synthesis and recombinant expression of hTRT proteins, including fusion proteins, is described in detail in the following sections.

1) Chemical synthesis

The invention provides hTRT polypeptides that have been synthesized in whole or in part using general chemical methods well known in the art (see, for example, Caruthers et al., Nucleic Acids Res. Symp. Ser. (1980) 215-223 and Horn et al., Nucleic Acids Res. Symp. Ser. (1980), 225-232). For example, peptide synthesis can be performed using various solid phase methods (Roberge et al., Science 269 (1995) 202), including automatic synthesis (e.g. using the Perkin Elmer ABI 431A peptide synthesizer according to the manufacturer's instructions). If a full-length protein is desired, shorter polypeptides can be fused by condensing the amino terminus of one molecule with the car-boxyl terminus of the other molecule to form a peptide bond.

The newly synthesized peptides can be substantially purified, for example by preparative HPLC (see, for example, Creighton Proteins, Structures and Molecular Principles, WH Freeman and Co., New York NY (1983). The composition of the synthetic peptides (or any other peptides or polypeptides according to the invention ) can be confirmed by amino acid analysis or sequencing (eg the Edman degradation method; Creighton, op. cit.) It is important to note that the amino acid sequence of hTRT or any part thereof can be altered during synthesis and / or using chemical methods can be combined with sequences from other proteins, this can also be carried out otherwise and comprise any part of the amino acid sequences for any purpose for the production of a variant of the polypeptides according to the invention.

2) Recombinant expression of hTRT and other Trt proteins

The present invention provides methods, reagents, vectors and cells useful for expressing hTRT polypeptides and nucleic acids using in vitro (cell free), ex vivo or in vivo (cell or organism based) recombinant expression systems. In one embodiment, expression of the hTRT protein or a fragment thereof comprises inserting the coding sequence into an appropriate expression vector (i.e., a vector containing the elements necessary for the transcription and translation of the inserted coding sequence). Thus, one aspect of the invention relates to the provision of a polynucleotide which is essentially identical in sequence to a sequence of at least 25 nucleotides encoding an hTRT gene and preferably 50 to 100 nucleotides or more of the hTRT cDNAs or genes according to the invention for many possible uses, that is operably linked to a promoter to form a transcription unit that can express an hTRT polypeptide. Methods well known to those skilled in the art can be used to construct expression vectors containing an hTRT sequence provided by the present invention and suitable transcriptional or translational controls (see, for example, Sambrook et al., Supra, Ausubel et al., Supra, and the present specification ).

The hTRT polypeptides according to the invention include fusion proteins which contain hTRT polypeptides or fragments of the hTRT protein. The fusion proteins are typically produced recombinantly, but they can also be synthesized chemically. Fusion proteins may be useful for increasing expression of the hTRT polypeptide constructs or for producing hTRT polypeptides with other desired properties, for example comprise a label (e.g. an enzymatic reporter group), a binding group or an antibody epitope. An example of a fusion protein comprising hTRT and enhanced green fluorescent protein (EGFP) sequences is described in Example 15 below. It is obvious to the person skilled in the art that the uses and possible uses discussed in Example 15 and elsewhere here are not restricted to the specific fusion proteins, but rather are intended to illustrate the possible uses for the different fusion constructs.

The fusion protein systems according to the invention can also be used to facilitate the efficient production and isolation of hTRT proteins or peptides. For example, in

33

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

In some embodiments, the non-hTRT sequence portion of the fusion protein is a short peptide that can be specifically bound to an immobilized molecule so that the fusion protein can be separated from unbound components (such as unrelated proteins in a cell lysate). An example of this is a peptide sequence that was bound by a specific antibody. Another example is a peptide comprising traces of polyhistidine, for example (His) 6 or histidine tryptophan sequences, which can be bound by a resin containing nickel or copper ions (i.e. metal chelate affinity chromatography). Other examples include protein A domains or fragments that allow purification on immobilized immunoglobulin and the domain used in the "FLAGS" extension / affinity purification system (Immunex Corp., Seattle WA). In some embodiments, the fusion protein contains a cleavage site so that the hTRT sequence or other TRT polypeptide sequence can be easily separated from the non-hTRT peptide sequence or non-hTRT protein sequence. In this case, the cleavage can be carried out chemically (e.g. with cyanogen bromide, 2- (2-nitrophenylsulfonyl) -3-methyl-3'-bromoindolen, hydroxylamine or low pH) or enzymatically (e.g. with factor Xa, enterokinase). The selection of the fusion and cleavage system may depend in part on the proportion (i.e. the sequence) of the hTRT polypeptide to be expressed. Fusion proteins are generally described in Ausubel et al., Op. Cit., Chapter 16, Kroll et al., DNA Cell. Biol. 12 (1993), 441 and in the 1997 Invitrogen Catalog (Invitrogen Inc., San Diego CA). Further examples of fusion proteins according to the invention with epitope tags or tags and cleavage sites are provided in Example 6 below.

It is obvious to the person skilled in the art that the expression systems discussed in this section concentrate on the expression of hTRT polypeptides, but the same or similar cells, vectors and methods for expressing hTRT polypeptides according to the invention can be used also “sense” and “antisense” polynucleotides, the production of hTRT polypeptides not necessarily being desired. Typically, expression of a polypeptide requires a suitable initiation codon (e.g. methionine), an open reading frame, and translation regulatory signals (e.g. a ribosome binding site, a termination codon), which may be absent if translation of a nucleic acid sequence to produce a protein is not desired.

Expression of hTRT polypeptides and polynucleotides can be performed to take advantage of the benefits associated with the benefits provided by the present invention. An example of such an advantage is the expression of hTRT polypeptides, which are subsequently isolated from the cell in which they were expressed (e.g., for the production of large amounts of hTRT for use as a vaccine). A second example of such an advantage is the expression of hTRT in a cell to change the phenotype of the cell (as in applications in gene therapy). Non-mammalian cells can be used for expression of hTRT for purification, while eu-karyotic cells, particularly mammalian cells (e.g. human cells) can be used not only for isolation and purification of hTRT, but also for expression of hTRT when there is a change is desired in the phenotype in a cell (eg to achieve a change in the ability to proliferate, for example in applications in gene therapy). For example, but not limited to, hTRT polypeptides with one or more telomerase activities (e.g., the catalytic activity of telomerase) can be expressed in a host cell so as to increase a cell's ability to proliferate (e.g., to immortalize a cell) . In contrast, hTRT "antisense" polynucleotides or inhibitory polypeptides can typically be expressed to decrease a cell's ability to proliferate (e.g., in a telomerase positive malignant tumor cell). Numerous specific applications are described here, for example in the discussion below of the uses of the reagents and methods according to the invention for therapeutic applications.

Examples of useful expression systems according to the invention (cells, regulatory elements, vectors and expression) include a number of cell-free systems, such as, for example, a reticulocyte lysate and wheat germ systems, which use hTRT polynucleotides according to general methods well known in the art ( see for example Ausubel et al., loc. cit., chapter 10). In alternative embodiments, the invention provides reagents and methods for expressing hTRT in prokaryotic or eukaryotic cells. The present invention thus provides nucleic acids encoding hTRT polynucleotides, proteins, protein sub-sequences or fusion proteins, which are found in bacteria, fungi, plants, insects and animals, including humans, cell expression systems known in the art including isolated cells, cell lines, cell cultures, Tissues and whole organisms can be expressed. It will be apparent to those skilled in the art that the hTRT polynucleotides introduced into a host cell or cell-free expression system are usually functionally linked to a suitable expression control sequence for each host or cell-free system.

Useful bacterial expression systems include E. coli, Bacilli (such as Bacillus subtilis), other Enterobacteriaceae (such as Salmonella, Serratia and numerous Pseudomonas species) or other bacterial hosts (e.g. Streptococcus cremoris, Streptococcus lactis, Streptococcus thermo-philus, Leuconostocroost, Leuconostocroost mesenteroides », Lactobacillus acidophilus, Lactobacillus lactis, Bifidobacterium bifidum, Bifidobacterium breve and Bifidobacterium longum). To those in prokaryo-

34

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Useful hTRT expression constructs include recombinant bacteriophages, plasmid or cosmid DNA expression vectors, or the like, and typically include promoter sequences. Examples of promoters include inducible promoters, such as the lac promoter, the hybrid lacZ promoter of the “Bluescript7” phagemid (Stratagene, La Jolla CA) or pSportl (Gibco BRL); Phage lambda promoter systems; a tryptophan (trp) promoter system; ptrp-lac hybrids etc. The bacterial expression constructs optionally contain a ribosome binding site and regulatory signal sequences for the termination of the transcription. Illustrative examples of specific vectors useful for expression include pTrcHis2 (Invitrogen, San Diego CA), pThioHis A, B & C, and numerous other vectors known in the art or those that can be developed (see, for example Ausubel, loc. Cit.). Vectors useful for bacteria include those that facilitate the production of hTRT fusion proteins. Vectors useful for the expression of fusion proteins in high level bacterial cells include, but are not limited to, the multifunctional cloning and expression vectors for E. coli, for example the above-mentioned Bluescript7 (Stratagene), in which the hTRT protein, an hTRT fusion protein or an hTRT fragment coding sequence in the reading frame with sequences for the amino terminal Met and the subsequent 7 residues of the β-galactosidase can be ligated into the vector, so that a hybrid protein is produced (for example pIN vectors; van Heeke and Schuster, J. Biol. Chem. 264 (1989), 5503). Vectors such as pGEX vectors (e.g. pGEX-2TK; Pharmacia Biotech) can also be used to express foreign polypeptides such as an hTRT protein as fusion proteins with glutathione-S-transferase (GST). Such fusion proteins can be purified from lysed cells by adsorption on glutathione-agarose beads and subsequent elution in the presence of free glutathione. Proteins made in such systems often contain protease cleavage sites for enterokinase, thrombin or factor Xa so that the desired cloned polypeptide can be released from the GST unit, if desired, which is useful for purification or other uses. Other examples are fusion proteins which include hTRT and the E. coli maltose binding protein (MBP) or E. coli thioredoxin. Illustrative examples of hTRT expression constructs useful in bacterial cells are provided in Example 6 below.

The present invention further provides hTRT polypeptides which have been expressed in fungal systems such as Dictyostelium and preferably yeast such as Saccharomyces cerevisiae, Pichia pastoris, Torulopsi holmil, Saccharomyces fragilis, Saccharomyces lactis, Hansenula polymorpha and Candida pseudotropicalis. A number of suitable vectors are available for the expression of hTRT in yeast, including plasmid and YACs vectors (YACs = artificial yeast chromosomes). The vectors typically contain expression control sequences such as e.g. constitutive or inducible promoters (e.g. the a-factor, alcohol oxidase, PGH and 3-phosphoglycerate kinase or other glycolytic enzymes), an origin of replication, termination sequences etc., whichever is desired. Vectors suitable for use in Pichia include pPICZ, His6 / pPICZB, pPICZalpha, pPIC3.5K, pPIC9K, pA0815, pGAP2A, B & C, pGAP2alpha A, B and C (Invitrogen, San Diego, CA) and many others on the Vectors known in the art or to be developed. In one embodiment, the vector His6 / pPICZB (Invitrogen, San Diego, CA) is used to express a His6-hTRT fusion protein in the yeast Pichia pastoris. pYES2 (Invitrogen, San Diego, CA) is an example of a vector useful in Saccharomyces. Illustrative examples of hTRT expression constructs useful in yeast are provided in Example 6 below.

The hTRT polypeptides according to the invention can also be expressed in plant cell systems which have been transfected with plant or plant virus expression vectors (e.g. with cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (Ti or the plasmid pBR322). In cases where plant virus expression vectors are used, the expression of an hTRT coding sequence can be controlled by a number of promoters. For example, viral promoters such as the 35S and 19S promoters from CaMV (Brisson et al. Nature 310 (1984) 511-514) can be used alone or in combination with the omega leader sequence from TMV (Takamatsu et al., EMBO J. 6 (1987) 307-311). In an alternative embodiment, plant promoters such as the promoter of the RU-BISCO small subunit gene (Coruzzi et al., EMBO J. 3 (1984) 1671 to 1680 and Broglie et al., Science 224 (1984), 838- 843) or heat shock promoters (Winter and Sinibaldi, Results Probi. Cell Differ. 17 (1991), 85) or promoters of genes for storage proteins. These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection (for further study of these methods, reference is made to Hobbs or Murry (1992) in McGraw Hill Yearbook of Science and Technology, McGraw Hill New York NY, p. 191 -196 (1992), or Weissbach and Weissbach (1988), Methods for Plant Molecular Biology, Academic Press, New York NY, pp. 421-463).

Another expression system provided by the present invention for expressing an hTRT protein is an insect system. A preferred system uses a baculovirus polyhedrin promoter. In one of these systems, the nuclear polyhedrosis virus from Autogra-pha californica (AcNPV) is used as a vector for the expression of foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The sequence coding for the desired gene can be converted into a

35

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

non-essential region of the virus, for example the polyhedrin gene, are cloned and placed under the control of the polyhedrin promoter. The successful insertion of the sequence encoding, for example, the hTRT protein leads to the inactivation of the polyhedrogen wrestling and a recombinant virus is produced which lacks the coat protein. The recombinant viruses are then used to infect S. frugiperda cells or Trichoplusia larvae, in which the hTRT sequence is then expressed (see, for general procedures, Smith et al., J. Virol. 46 (1983), 584 and Engelhard et al., Proc. Nati. Acad. Sci. 91 (1994) 3224-7). Vectors useful for baculovirus expression include pBlueBacHis2 A, B &C;pBlueBac4.5; pMelBacB and numerous other vectors known in the art or vectors to be developed. Illustrative examples of hTRT expression constructs useful in insect cells are provided in Example 6 below.

The present invention also provides expression systems in mammals and mammalian cells. As noted above, hTRT polynucleotides can be expressed in mammalian cells (e.g. human cells) for the production of significant amounts of hTRT polypeptides (e.g. for purification) or to change the phenotype of a target cell (e.g. for gene therapy, cell immortilization, etc.) become. In the latter case, the expressed hTRT polynucleotide may or may not encode a polypeptide with a catalytic activity of telomerase. This means that a "sense" or "anti-sense" polynucleotide, an inhibitory or stimulating polypeptide, a polypeptide with zero, one or more telomerase activities can be expressed, other combinations and variants are known to the person skilled in the art after reading these Description obvious.

Tissue culture mammalian cells suitable for the expression of the nucleic acids according to the invention include any normal mortal, normal or abnormal immortal animal or human cell. These include: the CV1 monkey kidney line transformed with SV40 (COS-7, ATCC CRL 1651), the human embryonic kidney line (293) (Graham et al., J. Gen. Virol. 36 (1977), 59), the Babyham star kidney cells (BHK, ATCC CCL 10), CHO (ATCC CCL 61 and CRL 9618), mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23 (1980) 243-251), monkey kidney cells (CV1, ATCC CCL 70), kidney cells of the African Vervet Monkey (VERO-76, ATCC CRL 1587), human cervical carcinoma cells (He-La, ATCC CCL 2), dog kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138 , ATCC CCL 75), human liver cells (HEP G2, HB 8065), mouse breast tumor (MMT 060562, ATCC CCL 51) and TRI cells (Mather et al., Annais NY Acad. Sci. 383 (1982), 44- 46, MDCK (ATCC CCL 34 and CRL 6253), HEK 293 (ATCC CRL 1573), WI-38 (ATCC CCL 75) (ATCC: American Type Culture Collection Rockville, MD)). The use of mammalian tissue cell cultures to express polypeptides is generally discussed in Winnacker, FROM GENES TO CLONES (VCH Verlag, N.Y., N.Y., 1987).

Virus-based and non-viral expression systems are provided for mammalian host cells. Non-viral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for expression of a protein or an RNA, and human artificial chromosomes (see e.g. Harrington et al., Nat Genet 15 (1997), 345). Non-viral vectors useful for the expression of hTRT polynucleotides and polypeptides in mammalian cells (e.g. human cells) include pcDNA3.1 / His, pEBVHis A, B & C (Invitrogen, San Diego CA), MPSV vectors, others in the 1997 Invitrogen catalog (Invitrogen Inc., San Diego, CA), which is incorporated by reference in its entirety into the disclosure of the present specification, and numerous other vectors and systems known in the art for other proteins. Illustrative examples of hTRT expression constructs useful in mammalian cells are provided in Example 6 below.

Useful viral vectors include vectors based on retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, SV40-based vectors, papilloma viruses, HBP-Epstein Barr virus, vaccinia virus vectors and “Semliki Forest” virus (SFV) . SFV and vaccinia vectors are discussed generally in Ausubel et al., Op. Cit., Chapter 16. These vectors are often made up of two components, a modified viral genome and an envelope structure surrounding it (see generally Smith, Annu. Rev. Microbiol. 49 (1995), 807). However, viral vectors are sometimes introduced in the naked form or coated with proteins that are not viral proteins. However, the viral nucleic acid in a vector can be modified in a variety of ways, for example if this is desired for gene therapy. The goal of these changes is to prevent the virus from growing in target cells and to maintain its ability to grow in vector form in available pack or helper cells to provide space within the viral genome for insertion of exogenous DNA sequences and to insert new sequences that are desired Encode gene and allow suitable expression. Thus vector nucleic acids generally comprise two components: essential viral sequences acting in ice for replication and packaging in a helper line and the transcription unit for the exogenous gene. Other viral functions are expressed in trans in a specific packaging or helper cell line. Adenovirus vectors (e.g. for use in gene therapy in humans) are described, for example, in Rosenfeld et al., Cell 68 (1992), 143 and in PCT publications Wo 94/12 650; 94/12 649 and 94/12 629. In cases where an adenovirus is used as the expression vector, an hTRT coding sequence can be ligated into an ade novirus transcription / translation complex which consists of the late promoter and the three36

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

partial leader sequence. Insertion into a non-essential E1 or E3 region of the viral genome results in a viable virus that can express in infected host cells (Logan and Shenk, Proc. Nati. Acad. Sci. 81 (1984), 3655). Replication-deficient retroviral vectors that contain a therapeutic polynucleotide sequence as part of the retroviral genome are described, for example, in Miller et al., Mol. Cell. Biol. 10 (1990) 4239; Kolberg, J. NIH Res. 4 (1992), 43 and Cornette et al., Hum. Gene Ther. 2: 215 (1991).

Promoters of mammalian genes or mammalian viruses are often suitable in mammalian cell systems. Suitable promoters can be constitutive, cell type-specific, stage-specific and / or modulatable or regulatable (e.g. by hormones such as glucocorticoids). Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive major late promoter of adenovirus, the dexamethasone inducible MMTV, the SV40 promoter, the MRP pollll promoter, the constitutive MPSV promoter, the Tetracycline inducible CMV promoter (such as the human immediate early CMV promoter), the constitutive CMV promoter and promoter-enhancer combinations known in the art.

Further regulatory elements may also be required or desired for an efficient expression of an hTRT polynucleotide and / or the translation of an hTRT protein-coding sequence. For translation, these elements typically include an ATG initiation codon and an adjacent ribosome binding site or other sequences. If the initiation codon and upstream promoter sequences are inserted into an expression vector, no additional translation signals or other control signals are required for the hTRT protein coding sequences. However, in cases where only a coding sequence or a part thereof is inserted, exogenous transcription and / or translation control signals (e.g. the promoter, the ribosome binding site and the ATG initiation codon) often have to be provided. In addition, the initiation codon must typically be in the correct reading frame to ensure translation of the desired protein. Exogenous transcription elements and initiation codons can come from numerous sources, both natural and synthetic. In addition, the efficiency of expression can be increased by the insertion of enhancers suitable for the cell system used (Scharf et al., Results Probi. Cell Differ. 20 (1994), 125 and Bittner et al., Meth. Enzymol. 153 (1987) , 516). For example, the SV40 enhancer or the CMV enhancer can be used to increase expression in mammalian host cells.

Expression of hTRT gene products can also be accomplished by activating an hTRT promoter or enhancer in a cell, such as a human cell, e.g. a telomerase-negative cell line. Activation can be achieved through a number of different measures. These include the administration of an exogenous promoter activating agent or the inhibition of a cellular component that suppresses the expression of the hTRT gene. Conversely, of course, inhibition of promoter function, as described below, will decrease hTRT gene expression.

The invention provides the inducible and repressible expression of hTRT polypeptides using such systems as the Ecdyson-inducible expression system (Invitrogen) and the "Tet-On and Tet-off" tetracycline-regulated systems from Clontech. In the expression system inducible by ecdyson, an analogue of the steroid hormone ecdysone, muristerone A, is used to activate the expression of a recombinant protein via a heterodimeric nuclear receptor (No et al., Proc. Nati. Acad. Sei. USA 93 (1996), 3346) . In one embodiment, hTRT is cloned into the pIND vector (Clontech), which contains five modified Ecdyson reaction elements (E / GREs) upstream of a minimal heat shock promoter and the multiple cloning site. This construct is then transfected into cell lines stably expressing the ecdysone receptor. After the transfection, the cells are treated with Muristeron A to induce the intracellular expression of pIND. In another embodiment, the hTRT polypeptide is expressed using the "tet-on and tet-off" expression systems (Clontech) in addition to the regulated high-level gene expression systems described (Gossen et al., Proc. Nati. Acad. Sei. USA 89 (1992), 5547 and Gossen et al., Science 268 (1995), 1766).

The hTRT vectors according to the invention can be introduced into a cell, a tissue, organ, a patient or an animal by a number of methods. The nucleic acid re-expression vectors according to the invention (typically dsDNA) can be introduced into the selected host cell by generally known methods such as calcium chloride transformation (for bacterial systems), electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistic methods, virosomes , Immunoliposomes, polycation: nucleic acid conjugates, naked DNA, artificial virions, fusion to the herpes virus structural protein VP22 (Elliot and O'Hare, Cell 88, 223) can be transferred by agent-enhanced uptake of DNA and ex vivo transduction. Useful liposome-mediated DNA transfer methods are described in U.S. Patents 5,049,386, 4,946,787 and 4,897,355, PCT publications WO 91/17 424 and WO 91/16 024, and Wang and Huang, Biochem. Biophys. Res. Commun. 147: 980 (1987); Wang and Huang, 1989 Biochemistry 28: 9508; Litzinger and Huang, Biochem. Biophys. Acta 1113 (1992), 201; Gao and Huang, Biochem. Biophys. Res. Commun. 179 (1991), 280. Immunoliposomes as carriers for exogenous polynucleotides have also been described (Wang and Huang, Proc. Nati. Acad. Sci. USA 84 (1987), 7851 and Trubetskoy et al.,

37

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Biochem. Biophys. Acta 1131 (1992), 311). These should have improved cell type specificity compared to liposomes due to the inclusion of specific antibodies, which presumably bind to surface antigens on specific cell types, have improved cell type specificity. Behr et al., Proc. Nati. Acad. Be. USA 86: 6982 (1989) report the use of lipopolyamine as a reagent to mediate transfection itself, with no need to add any additional phospholipid to form liposomes. Suitable introductory methods can be selected by the person skilled in the art with regard to suitable practices and requirements with regard to regulation (e.g. for gene therapy or for the production of cell lines for the expression of recombinant proteins). Of course, the introductory methods listed above can also be used to transfer nucleic acids into cells for gene therapy, transfer into tissue culture cells, etc.

Stable expression is often desirable for the long-term production of recombinant proteins with high yield. For example, cell lines stably expressing hTRT can be produced using the expression vectors according to the invention which contain viral origins of replication or endogenous expression elements and a selectable marker gene. After the vector has been introduced, the cells can be allowed to grow in enriched media for 1 to 2 days before switching to selective media. The selectable marker is used to confer resistance to the selection. Its presence allows the growth of cells that successfully express the inserted sequences in selective media. Resistant and stably transfected cells can be grown using tissue culture techniques appropriate for the cell type. An amplification step can be included, for example, by administering methyl trexate to cells transfected with a DHFR gene according to methods well known in the art.

In addition, a host cell strain can be selected for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired manner. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, phosphorylation, lipidation, and acylation. Post-translational processing can also be important for correct insertion, folding and / or function. The different host cells have a cellular machinery and characteristic mechanisms for such post-translational activities that are specific to each cell. A specific cell can thus be selected to ensure the correct modification and processing of the introduced foreign protein.

The present invention also provides transgenic animals (i.e. mammals that are transgenic to a human or other TRT gene sequence) that express an hTRT or other TRT polynucleotide or polypeptide. In one embodiment, hTRT is secreted in the milk of a transgenic mammal such as a transgenic bovine, a goat or a rabbit. Methods for producing such animals can be found, for example, in Heyneker et al., PCT WO 91/08 216.

The hTRT proteins and complexes of the invention, including those produced by the expression systems described above, can be purified using a number of general methods known in the art in accordance with the specific methods provided by the present invention (such as described below) become. It is obvious to a person skilled in the art that after chemical synthesis, biological expression or purification, the hTRT protein can have a conformation that differs from the native conformation of naturally occurring telomerase. In some cases it may be helpful or even necessary to denature the polypeptide (which may include, for example, reducing disulfide or other bonds) and then refolding the polypeptide into the preferred conformation. Correct refolding may also require the presence of hTR (or hTR fragments). Methods for reducing and denaturing proteins and for inducing refolding are known to the person skilled in the art (see, for example, Debinski et al. J. Biol. Chem. 268 (1993), 14065; Kreitman and Pastan, Bioconjug. Chem. 4 (1993), 581 and Buchner et al. Anal Biochem. 205 (1992), 263 and McCaman et al., J. Biotech. 2 (1985), 177). (See also USSN 08/478 352, filed June 7, 1995, supra).

D. Complexes of human TRT and human telomeplase RNA, telomerase-associated proteins and other biomolecules, which are produced by coexpression and other means

HTRT polypeptides according to the invention can associate with other biomolecules in vivo and in vitro, including RNAs (eg hTR), proteins (eg telomerase-associated proteins), DNA (eg telomer DNA, [T2AGs] n) and nucleotides such as (deoxy) ribonucleotide -Triphosphate belong. These associations can be used to detect the presence or function of hTRT, to identify or purify hTRT or telomerase-associated molecules and to analyze the structure or function of hTRT or telomerase according to the methods according to the invention.

In one embodiment, the present invention provides hTRT that complexes (e.g., is associated with or bound to) a nucleic acid, usually an RNA. In one embodiment, the bound RNA can act as a template for telomerase-mediated DNA synthesis. Examples of RNAs that can form a complex with the hTRT polypeptide

38

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

include a naturally occurring host cell telomerase RNA, a human telomerase RNA (e.g. hTR; U.S. Patent No. 5,583,016), an hTR subsequence or domain, a synthetic RNA or other RNAs. The RNA-hTRT protein complex (an RNP) typically has one or more telomerase activities, for example, catalytic activities of telomerase. These hTRT-hTR-RNPs (or other hTRT-RNA complexes) can be prepared by a number of methods as described below by way of illustration, including in vitro reconstitution and co-expression of hTRT and hTR (and another RNA ) in vitro (ie in a cell-free system) in vivo or ex vivo.

Thus, in one embodiment, the present invention provides an hTRT-hTR complex (or other hTRT-RNA complex) that was formed in vitro by mixing separately purified components ("in vitro reconstitution"; see, for example, US Pat. No. 5,583 016 for a description of the reconstitution and USSN 08/478 352, filed June 7, 1995; see also Autexier et al., EMBO J. 15; 5928).

In an alternative embodiment, the invention provides telomerase RNPs made by coexpressing the hTRT polypeptide and an RNA (e.g. hTR) in vitro in a cell-free transcription / translation system (e.g. wheat germ lysate or rabbit reticulocyte lysate). As shown in Example 7, coexpression of a recombinant hTRT polypeptide and hTR in vitro results in the production of catalytic activity of telomerase (as measured by a TRAP assay).

The present invention also provides telomerase RNPs made by expressing the hTRT polypeptide in a cell, such as a mammalian cell, in which hTR is naturally expressed, or in the hTR (or other RNA associated with the hTRT -Protein can form a complex) is introduced, or is expressed by recombination. Thus, in one embodiment, hTRT is expressed in a telomerase negative human cell in which hTR is present (e.g., BJ or IMP90 cells), allowing both molecules to assemble into an RNP. In a further embodiment, hTRT is expressed in a human or non-human cell in which hTR is recombinantly expressed. Methods for expressing hTR in a cell can be found in US Patent 5,583,016. The clone pGRN33, which contains a cDNA encoding the RNA component of telomerase, was also deposited (ATCC 75926). The RNA component of genomic sequences coding for human telomerase were deposited as 15 kb SaulllA1 to Hindlll insertion of the clone 28-1 (ATCC 75925). For expression in eukaryotic cells, the hTRT sequence is typically functionally linked to a transcription initiation sequence (RNA polymerase binding site) and transcription termination sequences (see, for example, PCT publication WO 96/01 835; Feng et al., Science 269 (1995), 1236).

The present invention also provides recombinantly produced or substantially purified hTRT polypeptides which are co-expressed and / or associated with so-called “telomerase-associated proteins”. Thus, the present invention provides hTRT that is co-expressed or complexed with other proteins (e.g. telomerase-associated proteins). Telomerase-associated proteins are understood to mean those proteins which are purified with human telomerase and / or which can play a role in modulating the telomerase function or activity, for example in that they are involved in the association of telomerase with telomer DNA. Examples of, but not limited to, telomerase-associated proteins include the following proteins and / or their human homologs: nucleolin (see U.S. Patent Application Serial No. 08/833,377 and Srivastava et al., FEBS Letts. 250 (1989), 99); EF2H (the homolog to elongation factor 2; see U.S. Patent Application No. 08/833,377 and Nomura et al., DNA Res. (Japan) 1 (1994), 27, GENBANK accession number # D21163), TP1 / TLP1 (Harrington et al., Science 275 (1997), 973); Nakayama et al. (1997) Cell 88: 875; the human homologue to Tetrahymena p95 (Collins et al., Cell 81 (1995) 677); TPC2 (a telomere length regulating protein; ATCC accession number 97708 (see USSN 08/710 249 and 08/713 922, both filed on September 13, 1996)), TPC3 (also a telomer length regulating protein; ATCC accession number 97707 (see USSN 08/710 249 and 08/713 922, both filed on September 13, 1996)), DNA-binding protein B (dbpB; Horwitz et al., J. Biol. Chem. 269 (1994), 14130) and that Telomer repeat unit binding factor (TRF 1 &2; Chang et al., Science 270 (1995), 1663; Chong et al., Hum. Mol. Genet. 6 (1997), 69; EST1, 3 and 4 (Lendvay et al., Genetics 144 (1996), 1399; Nugent et al., Science 274 (1996), 249 and Lundblad et al., Cell 57 (1989), 633) and the end-capping factor (Cardenas et al ., Genes Dev. 7 (1993), 883).

Telomerase-associated proteins can be identified based on the co-purification with or binding to hTRT protein or the hTRT-hTR-RNP. In an alternative embodiment, they can be identified on the basis of binding to an hTRT fusion protein, for example a GST-hTRT fusion protein, etc., as can be done by affinity purification (see Ausubel et al., Chapter 20). A particularly useful method for determining protein / protein interactions and for identifying hTR-T-associated proteins is the “two hybrid screen” method by Chien et al. (Proc. Nati. Acad. Sci. USA 88 (1991), 9578; see also Ausubel et al., Op. Cit., Chapter 20). This screening allows protein / protein interactions to be identified in vivo by reconstitution of a transcription activator, the yeast Ga14 transcription protein (see Fields and Song, Nature 340 (1989), 245). This method is based on the properties of the yeast Ga14 protein that is separated from 39

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

domains that are responsible for DNA binding and transcriptional activation. Polynucleotides encoding “two hybrid” proteins, usually expression vectors, are constructed. A polynucleotide comprises the yeast Ga14 DNA binding domain fused to a polypeptide sequence of a protein to be tested for hTRT interaction (e.g. Nucleolin or EF2H). In an alternative embodiment, the yeast Ga14 DNA binding domain is fused to cDNAs from a human cell, thereby producing a library of human proteins fused to the Ga14DNA binding domain for screening for telomerase-associated proteins. The other polynucleotides include the Ga14 activation domain fused to an hTRT polypeptide sequence. The constructs are introduced into a yeast host cell. After expression, the intermolecular bond between hTRT and the test protein can reconstitute the Ga14 DNA-binding domain with the Ga14-activating domain. This leads to the transcriptional activation of a reporter gene (e.g. lacZ, His3) which is functionally linked to a Ga14 binding site. Reporter gene cell colonies containing an protein interacting with hTRT or a telomerase-associated protein can be identified by selection or examination. It is obvious to the person skilled in the art that there are numerous variations of the “2 hybrid screen” system, for example the LexA system (Bartel et al., In Cellular Interactions in Development: A Praticai Approach, Hartley (ed.), DA (Oxford Univ. Press (1993), pp. 153-179)).

Another useful method for identifying telomerase-associated proteins is a "three-hybrid" system (see, for example, Zhang et al., Anal. Biochem. 242 (1996), 68 and Licitra et al., Proc. Nati. Acad. Sci. USA 93 (1996), 12817). The telomerase RNA component can be used in this system with the TRT or the hTRT protein and a test protein. Another useful method for identifying interacting proteins (ie proteins that heterodimerize or form higher order heteromultimers) is in particular the E. coli / BCCP “interactive screening” system (see Germino et al., Proc. Nati. Acad Sei. USA 90 (1993), 933 and Guarente, Proc. Nati. Acad. Sei. (USA), 90 (1993), 1639).

The present invention also provides complexes of telomere binding proteins (which may or may not be telomerase-associated proteins) and hTRT (which may or may not be complexed with hTR, other RNAs, or one or more telomerase-associated proteins). Examples of telomer binding proteins include TRF1 and TRF2 (loc.) RnpA1, mpA2, RAP1 (Buchman et al., Mol. Cell. Biol. 8 (1988), 210, Buchman et al., Mol. Cell. Biol. 8 (1988), 5086), SIR3 and SIR4 (Aparicio et al., Cell 66 (1991), 1279), TELI (Greenwell et al., Cell 82 (1995), 823; Mor-row et al., Cell 82 (1995), 831); ATM (Savitsky et al., Science 268 (1995), 1749), “end-capping” factor (Cardenas et al., Genes Dev. 7 (1993), 883) and the corresponding human homologues. The above-mentioned complexes can generally be prepared as described above for complexes of hTRT and hTR or telomerase-associated proteins, for example by mixing or coexpression in vitro or in vivo.

V. Antibodies and other binding agents

A related aspect of the present invention relates to antibodies that are specifically immunoreactive with hTRT. These include polyclonal and monoclonal antibodies, antibody fragments, single chain antibodies, human and chimeric antibodies, including antibodies or antibody fragments fused to phage coat or cell surface proteins, and others known in the art and described herein. The antibodies according to the invention can specifically recognize and bind polypeptides that have an amino acid sequence that matches the amino acid sequence of SEQ. ID. No. 2 is essentially identical, or an immunogenic fragment thereof, or an epitope on the protein defined thereby. The antibodies according to the invention can have a specific binding affinity for hTRT of at least about 107, 108, 109 or 1010 M_1. They can be polycyclonic or monoclonal, recombinant or otherwise produced. The invention also provides anti-hTRT antibodies that recognize a conformational epitope of h TRT (e.g., an epitope on the surface of the hTRT protein or a telomerase RNP). Probable conformational epitopes, if desired, can be identified by computer-aided analysis of the hTRT protein sequence, by comparison to the conformation of related reverse transcriptases such as the p66 subunit of HIF-1 (see, e.g., Figure 3) or empirically. Anti-hTRT antibodies that recognize conformation epitopes are useful, among other things, in the detection and purification of human telomerase and in the diagnosis and treatment of human diseases.

For the production of anti-hTRT antibodies, hosts such as goats, sheep, cows, guinea pigs, rabbits, rats or mice can be injected with an hTRT protein or any portion, fragment or oligopeptide thereof that has retained immunogenic properties, be immunized. It is not necessary to maintain this biological activity for the selection of hTRT polypeptides for antibody induction, but the protein fragment or oligopeptide must be immunogenic and preferably antigenic. Immunogenicity can be demonstrated by injecting a polypeptide and an adjuvant into an animal (e.g. a rabbit) and by examining the appearance of antibodies directed against the injected polypeptide (see

40

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988), e.g., Chapter 5, and this document is incorporated by reference in its entirety and for all purposes with this description). Peptides used to induce specific antibodies typically have an amino acid sequence consisting of at least five amino acids, preferably at least 8 amino acids, and more preferably at least 10 amino acids. They usually take the entire amino acid sequence of the protein with SEQ. ID. No. 2 or a part thereof, or have an essentially identical sequence. Short stretches of the amino acid of the hTRT protein can be fused with those of other proteins, such as the keyhole limpet hemocyanin and an anti-hTRT antibody made against the chimeric molecule. Depending on the type of host used, various adjuvants can be used to increase the immune response.

The antigen is presented to the immune system in a manner determined by animal procedures. These and other parameters are generally well known to the immunologist. Typically, the injections are administered into the balls of the foot, intramuscularly, intradermally, into the perilymph nodes or intraperitoneally. The immunoglobulins produced by the host can be precipitated, isolated and purified according to routine procedures, which include affinity purification.

Illustrative examples of immunogenic hTRT peptides are provided in Example 8. In addition, Example 8 describes the preparation and use of polyclonal anti-hTRT antibodies.

A) Monoclonal antibodies

Monoclonal antibodies to hTRT proteins and peptides can be prepared in accordance with the methods of the invention using any method suitable for the production of antibody molecules by continuous cell lines in culture. These methods include, but are not limited to, the originally described hybridoma method by Koehler and Milstein (Nature 256 (1975), 495), the hybridoma method using human B cells (Kosbor et al., Immunol. Today 4 ( 1983), 72 and Cote et al., Proc. Nati. Acad. Sci. USA 80 (1983), 2026), and the EBV hybridoma method (Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss Inc New York NY, pp. 77-96 (1985)).

In one embodiment, suitable animals are selected and the appropriate immunization protocol to be used. The production of non-human monoclonal antibodies, for example from the mouse, from Lagomorpha and from the horse, is generally known and can be carried out, for example, by immunizing an animal with a preparation containing hTRT or fragments thereof. In one method, the spleen of the animals is cut out after a suitable period of time and individual spleen cells are typically fused with immortalized myeloma cells under suitable selection conditions. The clonai cells are then separated and the supernatants from each clone (e.g. hybridoma) tested for the production of an appropriate antibody specific for the desired region of the antigen. Methods for producing antibodies are well known in the art. See, for example, Goding et al., Monoclonal Antibodies: Principles and Practice (2nd Edition), Acad. Press, N.Y., and Harlow and Lane, op. Cit., Both publications are incorporated by reference in their entirety and for all purposes, as a part of this description. Other suitable methods include in vitro exposure of lymphocytes to the antigenic polypeptides or, alternatively, a selection of antibody banks in phage vectors or similar vectors (see the description below).

B) Human antibodies

Another aspect of the invention relates to the provision of human antibodies against an hTRT polypeptide. Human monoclonal antibodies to a known antigen can also be made using transgenic animals that have elements of a human immune system (see, for example, US Patent Nos. 5,569,825 and 5,545,806, both documents being incorporated by reference in their entirety and for all purposes as Are part of this description) or using human peripheral blood cells (Casali et al., Science 234 (1986), 476). Some human antibodies are selected by competitive binding experiments or so that they have the same epitope specificity as a specific mouse antibody.

In a further embodiment, human antibodies against an hTRT polypeptide can be produced by screening a DNA bank from human B cells according to the general protocol described by Huse et al., Science 246 (1989), 1275. This document is also to be considered part of the present description by reference. Antibodies binding to the hTRT polypeptide are selected. Sequences encoding these antibodies (or a binding fragment) are then cloned and amplified. The protocol described by Huse is often used with the «phage-display» technology.

41

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

C. Humanized or Chimeric Antibodies

The invention also provides anti-hTRT antibodies made as chimeric, human antibody-like or humanized antibodies so as not to lower their potential antigenicity to the target. The preparation of chimeras and humanized antibodies, or antibodies similar to human antibodies, has been described in the art (see, for example, U.S. Patent Nos. 5,585,089 and 5,530,101; Queen et al., Proc. Nati. Acad. Sci. USA 86 (1989), 10029 and Verhoeyan et al., Science 239 (1988), 1534, which documents are to be considered in their entirety and for all purposes as the subject of this description by reference. Humanized immunoglobulins have variable scaffold regions, which essentially come from a human immunoglobulin (with the name acceptor-immunoglobulin) and the complementarity of the determining regions, which essentially come from a non-human immunoglobulin (e.g. from the mouse) (with the designation donor immunoglobulin). The constant region (s), if present, originate essentially from a human immunoglobulin.

In some applications, for example when administered to human patients, the humanized (as well as the human) anti-hTRT antibodies according to the invention offer a number of advantages over antibodies of mice or other species: (1) the human immune system should be the basic structure or the constant Do not recognize the area of the humanized antibody as foreign and therefore the antibody response against such an injected antibody should be lower than against a completely foreign mouse antibody or a partially foreign chimeric antibody; (2) since the effector area of the humanized antibody is human, it is likely to interact better with other parts of the human immune system, and (3) injected humanized antibodies have a half-life that is essentially equivalent to that of naturally occurring human antibodies, which it is allows smaller and less frequent doses to be administered compared to antibodies from other species.

D) "Phage Display"

The present invention also provides anti-hTRT antibodies (or binding composition) made by phage display methods (see, for example, Dower et al. 1 WO 91/17 271 and McCafferty et al., WO 92/01 047 and Vaughan et al., Nature Biotechnology 14 (1996), 309, which documents are to be regarded in their entirety and for all purposes as a component of this description by reference. These processes produce phage banks in which members present different antibodies on their outer surfaces. Antibodies are usually presented as Fv or Fab fragments. Phages that present antibodies with a desired specificity are selected by affinity enrichment with an hTRT polypeptide.

In a variation of the "phage-display" process, humanized antibodies with the binding specificity of a selected mouse antibody can be produced. In this method, the variable region of either the heavy or the light chain of the selected mouse antibody is used as the starting material. For example, if the variable region of a light chain is selected as the starting material, a phage bank is created in which members present the variable region of the same light chain (i.e. the starting material from the mouse) and a variable region of a different heavy chain. The heavy chain variable regions are obtained from a bank with human rearranged heavy chain variable regions. A phage that shows strong specific binding to the hTRT polypeptide (e.g. at least 10® and preferably at least 109 M ~ 1) is selected. The variable region of the human heavy chain from this phage then serves as the starting material for the production of a further phage bank. In this bank, each phage presents the same variable region of the heavy chain, i.e. the area identified by the first presentation bench, and a different area of the light chain. The light chain variable regions are obtained from a bank of human rearranged light chain variable regions. Again, a phage that shows strong specific binding is selected. These phages present the variable regions of fully human anti-hTRT antibodies. These antibodies usually have the same or a similar epitope specificity as the mouse-derived starting material.

E) Hybrid antibodies

The invention also provides hybrid antibodies that share specificity with antibodies to an hTRT polypeptide, but can also specifically bind a second entity. In these hybrid antibodies, one pair normally comes from a heavy and light chain from an anti-hTRT antibody and the other pair from an antibody directed against another epitope or protein. This leads to the property of multifunctional valence, i.e. the ability to bind at least two different epitopes simultaneously, at least one epitope being the epitope to which the anti-complex antibody binds. Such hybrids can be created by merging the corresponding

42

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Hybridomas producing constituent antibodies are formed or by combination methods.

Immunoglobulins according to the invention can also be fused with functional areas of other genes (e.g. enzymes) for the production of fusion proteins (e.g. immunotoxins) with useful properties.

F) Anti-idiotypic antibodies

Anti-idiotypic antibodies that can be isolated using the methods described above are also useful. Anti-idiotypic antibodies can be prepared, for example, by immunizing an animal with the primary antibody (i.e. anti-hTRT antibody or hTRT-binding fragments thereof). For anti-hTRT antibodies, anti-idiotypic antibodies are selected whose binding to the primary antibody is inhibited by an hTRT polypeptide or fragments thereof. Since both the anti-idiotypic antibody and the hTRT polypeptide or fragments thereof bind the primary immunoglobulin, the anti-idiotypic immunoglobulin can represent the "internal picture" of an epitope and thus replace the hTRT polypeptide in assays, or it can do so be used to bind (ie inactivate) anti-hTRT antibodies, for example in a patient. Anti-idiotypic antibodies can also interact with telomerase-associated proteins. The administration of such antibodies could affect telomerase function by titrating out hTRT-associated proteins.

G) General

The antibodies according to the invention can belong to any isotype, for example IgM, IgD, IgG, IgA and IgE, with IgG, IgA and IgM being often preferred. Humanized antibodies can also include sequences from more than one class or isotype.

In a further embodiment according to the invention, fragments of the intact antibodies described above are provided. Typically, these fragments can compete with the intact antibody from which they are derived for specific binding to the hTRT polypeptide and bind with an affinity of 107, 108, 109 M ~ 1, or 1010 M ~ 1. Antibody fragments include separate heavy chains, light chains, Fab, Fab'F (ab ') 2, Fabc and Fv. Fragments can be made by enzymatic or chemical separation of intact immunoglobulins. For example, an F (ab ') 2 fragment from an IgG molecule by proteolytic cleavage with pepsin at a pH of 3.0-3.5 using standard methods, as described, for example, in Harlow and Lane, loc. Cit. be preserved. Fab fragments can be obtained from F (ab ') 2 fragments by limited reduction from a whole antibody by digestion with papain in the presence of reducing agents (see generally Paul, W. (ed.) Fundamental Immunology 2nd Raven Press, NY, 1989 , Chapter 7, this document being incorporated by reference in its entirety and for all purposes as part of this description). Fragments can also be made by recombinant DNA techniques. Nucleic acid segments encoding selected fragments are made by digesting full length coding sequences with restriction enzymes or by de novo synthesis. Fragments are often expressed in the form of a phage fusion coat protein.

Many of the immunoglobulins described above can undergo uncritical amino acid substitutions, additions or deletions in both the variable and constant regions without loss of binding specificity or effector functions or intolerable reduction in binding activity (i.e. below about 107 M ~ 1). Typically, immunoglobulins with such changes show a substantially identical sequence compared to a reference immunoglobulin from which they are derived. A mutant immunoglobulin can be selected that has the same specificity and increased affinity compared to a reference immunoglobulin from which it is derived. The «phage-display» technology offers useful methods for the selection of such immunoglobulins. See, for example, Dower et al., WO 91/17 271, McCafferty et al., WO 92/01 047 and Huse, WO 92/06 204.

The antibodies according to the invention can be used with or without modification. The antibodies are often labeled by either covalent or non-covalent addition of a detectable label. The antibodies according to the invention are particularly useful as labeled binding units in diagnostic applications.

The anti-hTRT antibodies according to the invention can be purified using generally known methods. The complete antibodies, their dimers, individual light and heavy chains, or other forms of immunoglobulin according to the present invention can be purified using the methods and reagents of the present invention in accordance with standard procedures common in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, etc. (see General Scopes, Protein Purification: Principiesand Practice, 3rd Edition (Springer-Verlag, NY; 1993), Preferred are essentially pure immunoglobulins with at least about 90 to 95% or even 98 to 99% or higher homogeneity.

43

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

VI. Purification of human telomerase

The present invention provides isolated human telomerase with unprecedented purity. In particular, the present invention provides: purified hTRT recombinant or non-recombinant in origin; purified hTRT-hTR complexes (i.e., RNPs), recombinant, non-recombinant, or mixed origin, optionally including one or more telomerase-associated proteins; purified naturally occurring human telomerase; etc. Furthermore, the invention provides methods and reagents for the above-described molecules and complexes including variants, fusion proteins, naturally occurring proteins etc. (collectively referred to as “hTRT and / or hTRT complexes”) in partially purified, to be able to win in a substantially purified or highly purified form. Before making this invention, attempts have been made to purify the telomerase-enzyme complex to homogeneity, but with limited success (see, for example, U.S. Patent Application Serial No. 08 / 833,377, filed April 4, filed 1997 and 08/510 736, filed August 4, 1995, and PCT Application No. 97/06 012, filed April 4, 1997. These documents are to be considered part of this description for useful cleaning procedures by reference). The methods provided in the applications listed above make it possible to purify telomerase up to about 60,000 times or higher compared to crude cell extracts. The present invention provides hTRT and hTRT complexes with even higher purity, in part thanks to the new immunoaffinity reagents according to the invention (eg anti-hTRT antibodies) and / or the reagents, cells and methods provided here for the recombinant expression of hTRT . The recombinant expression of hTRT and hTRT complexes facilitates purification because the desired molecules are produced in a significantly higher concentration compared to most naturally occurring expressing cells and / or because the recombinant hTRT molecules can be modified in this way (e.g. by Fusion with an epitope “tag” that they can be easily cleaned.

It is obvious that naturally occurring telomerase can be purified from a telomerase positive cell. Recombinant hTRT and hTRT complexes can include expressed and purified using any of the in vitro, in vivo, ex vivo, plant or animal expression systems described above, or other systems known in the art.

In one embodiment, the hTRT, telomerase and other compositions according to the invention are purified by means of an immunoaffinity step, either only this step or a combination with further purification steps being carried out. Typically, an immobilized or immobilizable anti-hTRT antibody, as provided by the present invention, is contacted with a sample, for example a cell lysate, which contains the desired hTRT or the hTRT-containing complex, under conditions in which a anti-hTRT antibody that binds hTRT antigen. After removal of unbound components of the sample by methods well known in the art, the hTRT composition can, if desired, be eluted from the antibody in substantially pure form. In one embodiment, immunoaffinity chromatography methods well known in the art are used (see, for example, Harlow and Lane, loc. Cit., Ausubel, loc. Cit., And Hermansanetal., 1992, Immobilized Affinity Ligand Techniques (Acade-micPress, SanDiego »in accordance with the methods of the invention. In one In another illustrative embodiment, immunoprecipitation of anti-hTRT-immunoglobulin-hTRT complexes is performed using immobilized protein A. Numerous variations and alternative immunoaffinity purification protocols suitable for use in accordance with the methods and reagents of the invention are well known to those skilled in the art.

In a further embodiment, recombinant hTRT proteins can be purified as a result of their high-level expression using routine protein purification processes, for example by ammonium sulfate precipitation, affinity columns (eg immunoaffinity), size-exclusion, anion and cations Exchange chromatography, gel electrophoresis, etc. (see generally R. Scopes, Protein Purification, Springer-Verlag, NY (1982) and Deutscher, Methods in Enzymology 182: Guide to Protein Purification, Academic Press, Inc. NY (1990)) instead of immunoaffinity testing. drive or in addition to it. Cation exchange methods can be particularly useful due to the basic pI value of the hTRT protein. For example, immobilized phosphate can be used as a functional cation exchange group (e.g. P-11 phosphocellulose, Whatman catalog # 4071 or cellulose phosphate, Sigma catalog #C 3145). Immobilized phosphate has two advantageous features in terms of hTRT purification - it is a cation exchange resin and it shows physically a similarity to the phosphate backbone of nucleic acids. Therefore, the "pseudo" AF-finite chromatography can be used because hTRT binds hTR and telomer DNA. Other nonspecific nucleic acid affinity chromatography methods are also useful for purification (see, for example, U.S. Pat. No. 08/833,377; Albers et al., Methods Enzymol. 21 (1971), 198; Arnt -Jovin et al., Eur. J. Biochem. 54 (1975), 411 and Pharmacia catalog # 27-5575-02). Further use of this probable binding function of hTRT would involve the use of specific nucleic acids (e.g. primer or hTR) in affinity chromatography for purification (Chodosh et al., Mol. Cell. Biol. 6 (1986), 4723; Wu et al., Science 238 (1987), 1247 and Kadonaga, Methods Enzymol. 208 (1991), 10); immobilized «Cibricon Blue» dye, the physical

44

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Kalisch resembles nucleotides, represents another useful resin for the purification of hTRT due to the hTRT binding of nucleotides (e.g. as substrates for DNA synthesis) (Pharmacia catalog # 17-0948-01 or Sigma catalog #C 1285) .

In one embodiment, hTRT proteins are isolated directly from an in vitro or in vivo expression system in which other telomerase components are not co-expressed. Isolated hTRT protein can of course also be easily removed from purified human telomerase or hTRT complexes, for example by breaking up the telomerase RNP (for example by exposure to a mild or other denaturing agent) and separating the RNP components (for example by routine measures such as chromatography or immunoaffinity chromatography). getting cleaned.

Telomerase purification can be monitored using an assay for telomerase activity (e.g., the TRAP assay, a conventional assay, or a primer binding assay) by determining the hTRT enrichment (e.g. by ELISA) by determining the hTR enrichment or by other methods known in the art.

The purified human telomerase provided by the present invention, the hTRT proteins and hTRT complexes, are highly purified in one embodiment (i.e. at least about 90% homogeneous, preferably at least about 95% homogeneous). Homogeneity can be determined by standard measures such as SDS polyacrylamide gel electrophoresis and other means known in the art (see, e.g., Ausubel et al., Supra). Although highly purified human telomerase, hTRT protein or hTRT complexes are occasionally desired, naturally purified (e.g. at least about 75% homogeneity) or partially purified (e.g. at least about 20% homogeneous) human telomerase, hTRT protein is also desirable or hTRT complexes also useful for many applications and these are also provided by the present invention. For example, partially purified telomerase is useful for screening test compounds for telomerase-modulating activity and for other uses (see, for example, U.S. Patent Application Serial No. 08/911312 (Attorney Register No. 015 389-002 500, filed August 14, 1997, cited above, and U.S. Patent No. 5,645,986, USSN 08/151,477, filed November 12, 1993, and USSN 08/288 501, filed August 10, 1994).

VII. The treatment of diseases related to telomerase

A) Introduction

The present invention provides hTRT polynucleotides, polypeptides and antibodies that are useful for the treatment of human diseases and conditions. The recombinant and synthetic hTRT gene products (protein and mRNA) according to the invention can be used to generate or increase telomerase activity in a cell, but also to inhibit telomerase activity in a cell in which this is not desired. Thus, inhibiting, inactivating, or otherwise altering a telomerase activity (e.g., a telomerase catalytic activity, accuracy, processivity, telomer binding, etc.) in a cell can be used to alter the cell's proliferation ability. For example, the reduction in telomerase activity in an immortalized cell, such as a malignant tumor cell, can mortalize the cell. Conversely, increasing telomerase activity in a mortal cell (which includes, for example, most human somatic cells) can increase the cell's ability to proliferate. For example, expression of an hTRT protein in skin fibroblasts, which increases the length of the telomer, leads to an increased proliferation capacity of the fibroblasts; such an expression can slow down or reverse the age-dependent slowdown in wound closure (see, for example, West, Arch. Derm. 130 (1994), 87).

Thus, one aspect of the present invention relates to the provision of reagents and methods that are useful in the treatment of diseases and conditions characterized by the presence, absence or amount of human telomerase activity in a cell and which are responsive to a Respond to treatment using the compositions and methods described herein. These diseases include, as described in detail and below, cancer, other diseases related to cell proliferation (especially diseases related to aging, immunological dysfunction, infertility (or fertility) etc.

B) Treatment of cancer

The present invention provides methods and compositions for reducing telomerase activity in tumor cells and for treating cancer. Cancer cells (e.g. malignant tumor cells) that express telomerase activity (telomerase positive cells) can be mortalized by reducing or inhibiting endogenous telomerase activity. In addition, any decrease in telomerase activity could change the aggressive nature of cancer to a more easily treatable disease status (by increasing the effectiveness of conventional treatment

45

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

methods) because telomerase levels correlate with disease characteristics such as the potential for metastasis (see, for example, U.S. Patent Nos. 5,639,613; 5,648,215; 5,489,508 and Pandita et al., Proc. Am. Ass. Cancer Res 37 (1996, 559).

The invention provides compositions and methods that are useful in treating a wide variety of different types of cancer, including solid tumors and leukemias. The types of cancer that can be treated include (but are not limited to): breast, prostate, and colon adenocarcinoma; all forms of lung cancer that originate from the bronchi; Bone marrow cancer, melanoma, hepatoma, neuroblastoma; the papilloma; the apudome, the choristome, the branchchioma; the malignant carcinoid syndrome; Karzi-noid heart disease; the carcinoma (e.g. Walke carcinoma, basal cell carcinoma, basosquamous carcinoma, Brown Pearce carcinoma, ductal carcinoma, Ehrlich tumor, insitu carcinoma, cancer 2 carcinoma, Merkel cell carcinoma, mucous carcinoma, non-small cell carcinoma , Oat cell carcinoma, papillary carcinoma, scirrhous carcinoma, bronchiolo-alveolar carcinoma, bronchial carcinoma, squamous cell carcinoma and transitional cell carcinoma); histiocytic dysfunction; Leukemia (e.g. in connection with B-cell leukemia, mixed-cell leukemia, zero-cell leukemia, T-cell leukemia, chronic T-cell leukemia, HTLV-II-associated leukemia, acute lymphocytic leukemia, chronic-lymphocytic leukemia , Mast cell leukemia and myeloid leukemia), malignant histiocytosis, Hodg-kin disease, small immunoproliferative; non-Hodgkin's lymphoma, solitary plasma cell tumor; Reticuloendo theliosis, melanoma, chondroblastoma; Chondroma, chondrosarcoma; Fibroma; Fibrosarcoma; Giant cell Tu-more; Histiocytoma, lipoma; Liposarcoma; Mesothelioma, myxoma; Myxosarcoma; Osteoma; Osteosarcoma; Ewing's sarcoma; Synoviom; Adenofibroma; Adenolymphoma; Carcinosarcoma, chordoma, craniopharyngioma, dysgerminoma, hamartoma; Mesenchymoma; Mesonephroma, myosarcoma, ameloblastoma, cementoma; Odontoma; Teratoma; Thymoma, chorioblastoma; Adenocarcinoma, adenoma; Cholangioma; Cholesteatoma; Cy-lindrome; Cystadenocarcinoma, cystadenoma; Granulosa cell tumor; Gynandroblastoma, hepatoma; Hidrade-nom; Islet cell tumor; Leydig cell tumor; Papilloma; Sertoli cell tumor, theca cell tumor, leiomyoma; Leio myosarcoma; Myoblastoma; Fibroid; Myosarcoma; Rhabdomyoma; Rhabdomyosarcoma; Ependymon; Ganglio-neuroma, glioma; Medulloblastoma, meningioma; Neurilemmoma; Neuroblastoma; Neuroepithelioma, neurofibroma, neuroma, paraganglioma, non-chromaffin paraganglioma, angiokeratome, angiolymphoid hyperplasia with eosinophilia; sclerosing angioma; Angiomatosis; Glomangioma; Hemangioendotheli-om; Hemangioma; Hemangiopericytoma, hemangiosarcoma; Lymphangioma, lymphangiomyoma, lymphangiosarcoma; Pinealom; Carcinosarcoma; Chondrosarcoma; Cystosarcoma phyllodes; Fibrosarcoma; Hemangiosarcoma; Leiomyosarcoma; -Leukosarcoma; Liposarcoma; Lymphangiosarcoma; Myosarcoma; Myxosarcoma, ovarian cancer, rhabdomyosarcoma; Sarcoma (e.g. Ewing's sarcoma, experimental, Kaposi's sarcoma and mast cell sarcoma); Neoplasms (e.g. bone neoplasms, breast neoplasms, neoplasms of the digestive system, colorectal neoplasms, liver neoplasms, pancreatic neoplasms, neoplasms of the appendages, testicular neoplasms, orbital neoplasms, neoplasms of the head and neck system, the neoplasms of the central, central, and neoplasms Hearing organ, pelvis, respiratory tract and urogenital tract); Neurofibromatosis and cervical squamous dysplasia). The invention provides compositions and methods useful for treating other conditions in which cells have become immortalized or hyperproliferative, for example by disregulating (e.g., abnormally high expression) hTRT, the telomerase enzyme or activity.

The present invention further provides compositions and methods for cancer prevention, including anti-hTRT, vaccines, gene therapy vectors that prevent telomerase activation, and gene therapy vectors that result in the specific death of telomerase positive cells . In a related aspect, the gene exchange therapy methods described below can be used to "treat" a genetic predisposition to cancer.

C) Treatment of other conditions

The present invention also provides compositions and methods useful for treating disease and disease conditions (in addition to cancer) characterized by under or over expression of telomerase or hTRT gene products. Examples include: diseases related to cell proliferation, diseases resulting from cell senescence (particularly diseases related to aging), immunological dysfunction and infertility, diseases related to an immune malfunction etc.

Certain diseases related to aging are characterized by changes associated with cell senescence due to reduced telomere length (compared to younger cells). These are the result of the absence (or a much lower level) of telomerase activity in the cell. Reduced telomere length and replication ability contribute to the diseases described below. Telomerase activity and telomer length can be increased, for example, by increasing the level of hTRT gene products (protein and mRNA) in the cell. Listed below are some of the disease states associated with cellular senescence where hTRT expression can have a therapeutic effect: Alzheimer's disease, Parkinson's disease, Huntington's disease and stroke related to age.

46

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

existing diseases of the integument, for example skin atrophy, elastolysis and wrinkling of the skin, sebaceous gland hyperplasia, formation of age spots, graying of the hair and hair loss, chronic skin ulcers, and deterioration in wound healing associated with aging; degenerative joint diseases, osteoporosis; aging-related weakening of the immune system (involving, for example, cells such as B and T lymphocytes, monocytes, neutrophils, eosinophils, basophils, NK cells and their corresponding precursors); diseases of the vascular system related to aging, including atherosclerosis, calcification, thrombosis and aneurysms; Diabetes, muscle atrophy; Respiratory diseases, diseases of the liver and intestinal tract, metabolic diseases, endocrine diseases (e.g. dysfunction of the pituitary gland and adrenal glands), reproductive diseases and macular degeneration associated with aging. These diseases and conditions can be treated by increasing the level of hTRT gene products in the cell, thereby increasing the length of the telomeres, restoring or increasing the cell's ability to replicate. Such methods can be carried out ex vivo on cultivated cells or in vivo on cells. In one embodiment, the cells are first treated to activate telomerase and extend the telomeres, and then treated to inactivate the hTRT gene and telomerase activity.

In a preferred embodiment, telomerase activity is generated by a vector according to the invention in an embryonic germ cell or stem cell (see USSN 08/591 246, filed on January 1, 1996; USSN 08/376 327, filed on January 20, 1995; Pederson et al .; USSN 08/665217, filed on June 14, 1996 and USSN 08/829 372, filed on March 31, 1997) before or during the differentiation.

The present invention also provides methods and compositions useful for treating infertility. Human germline cells (e.g. stem cells, their precursors or descendants) can proliferate indefinitely and are characterized by high telomerase activity. Abnormal or decreased concentrations of hTRT gene products, for example, can lead to inadequate or abnormal sperm production, leading to infertility or reproductive dysfunction. Thus, infertility based on "telomerase" can be treated using the methods and composition described here to increase telomerase concentrations. Since the inhibition of telomerase can have a negative effect on spermatogenesis, oogenesis and the viability of sperm and eggs, the telomerase-inhibiting compositions according to the invention can have contraceptive effects when used in germline cells to reduce the concentrations of hTRT gene products.

The invention also provides methods and compositions that can be used to reduce the proliferative potential of telomerase positive cells by reducing telomerase activity, for example in activated lymphocytes and hematopoietic stem cells. Thus the invention provides means for effecting immunosuppression. Conversely, the methods and reagents according to the invention are useful for increasing the telomerase activity and the proliferative potential in cells, such as stem cells, which express a low telomerase level or no telomerase before the therapeutic intervention.

D) Types of intervention

Based on the discussion above, it is clear that modulating a cell's telomerase level or activity can have a marked impact on the cell's proliferative potential and is therefore of great benefit in treating a disease. It is also clear that this modulation can be done by either reducing telomerase activity or increasing activity. The telomerase-modulating molecules according to the invention can act through a number of mechanisms; some of these mechanisms are described in this and the following subsections to help the user choose therapeutic agents. However, the users do not intend to limit the particular drugs, compositions, and methods described herein to any particular mechanism of action.

Telomerase activity can be reduced by various mechanisms or combinations of mechanisms. One mechanism is to decrease hTRT gene expression to decrease telomerase activity. This reduction can take place at the stage of transcription of the hTRT gene in mRNA, during processing (eg splicing), during nuclear transport or with regard to the stability of mRNA, with the translation of mRNA for the production of hTRTProtein or with regard to stability and function of hTRT protein. Another mechanism relates to disruption of one or more telomerase activities (eg the catalytic activity of reverse transcriptase or the hTR binding activity) by using inhibitory nucleic acids, polypeptides, or other agents (eg mimetics). small molecules, drugs and prodrugs that can be identified using the methods described herein or provided by the compositions described herein. Too far

47

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Other mechanisms include the sequestration of hTR and / or telomerase-associated proteins and the disruption of the assemblies of the telomerase-RNP from the constituents of the subunits. In a related mechanism, an hTRT promoter sequence is operably linked to a gene encoding a toxin and this construct is introduced into a cell. If or when transcriptional activators for hTRT are expressed or activated in the cell, the toxin is expressed, which leads to a specific killing of the cell.

A related method for reducing the proliferation capacity of a cell involves the introduction of an hTRT variant with low accuracy, i.e. a variant with a high error rate, for example over 1%, so that different telomer repeat units are synthesized. These different repeat units affect telomer-protein binding and lead to chromosomal rearrangements and anomalies and / or lead to cell death.

Similarly, telomerase activity can be increased by various mechanisms or a combination of mechanisms. These include increasing the amount of hTRT in a cell. Typically, this is accomplished by introducing a polynucleotide encoding an hTRT polypeptide into the cell (e.g., using a recombinantly produced polypeptide comprising an hTRT DNA sequence functionally linked to a promoter or a stable hTRT mRNA). In an alternative embodiment, a catalytically active hTRT polypeptide can itself be introduced into a cell or a tissue, for example by microinjection or other measures known in the art. In other mechanisms, the expression of the endogenous hTRT gene or the stability of hTRT gene products in the cell can be increased. Telomerase activity in a cell can also be increased by disrupting the interaction of endogenous telomerase inhibitors and the telomerase RNP or endogenous repressors of the hTRT transcription and the hTRT gene, and furthermore by increasing the expression or activity of activators of the hTRT -Transcription and other measures that are obvious to a person skilled in the art after reading this description.

E) Intervention means 1) TRT proteins and peptides

In one embodiment, the invention provides telomerase modulating polypeptides (i.e., proteins, polypeptides and peptides) that increase or decrease telomerase activity. These can be introduced directly into a target cell (e.g. by injection, liposome-mediated fusion, application of a hydrogel to the tumor surface (e.g. in the case of melanoma, fusion or attachment of the structural protein VP22 of the herpes virus and other methods described here and known in the art) In a second embodiment, telomerase-modulating proteins and peptides according to the invention are expressed in a cell by introducing a nucleic acid (for example a DNA expression vector or mRNA) which encodes the desired protein or peptide in the cell. The expression can either be constitutive or inducible, depending on the vector and choice of promoter (see discussion below) Messenger RNA preparations encoding hTRT are particularly useful when only transient expression (e.g. transient activation of Telomerase) is desirable Expression of nucleic acids into a cell is well known in the art (see also other places in this description, for example the sections on oligonucleotides and gene therapy methods).

One aspect of this invention relates to a telomerase-modulating polypeptide that increases telomerase activity in a cell. In one embodiment, the polypeptide is a catalytically active hTRT polypeptide that can direct the synthesis of human telomeric DNA (in conjunction with an RNA template, such as hTR). This activity can be measured as discussed above, for example using a telomerase activity assay such as a TRAP assay. In one embodiment, the polypeptide is a full-length hTRT protein with a sequence that corresponds to the sequence of 1132 residues of SEQ. ID. No. 2 corresponds or is essentially identical to it. In a further embodiment, the polypeptide is a variant of the hTRT protein from SEQ. ID. No. 2, for example a fusion polypeptide, derivatized polypeptide, truncated polypeptide, polypeptide with conservative substitutions etc. A fusion or derivatized protein may contain a "targeting" unit which increases or causes the polypeptide to penetrate the cell membrane the polypeptide is preferably supplied to a specific cell type (for example liver cells or tumor cells) or cell compartments (for example the nuclear compartment). Examples of such "targeting" units include lipid tails, amino acid sequences such as, for example, the "antennapedial peptide (see USSN 08 / 838,545, filed on April 9, 1997) or a nuclear localization signal (NLS; for example Xenopus nucloplasmin; Robbins et al. (1991) Cell 64: 615). Naturally occurring hTRT protein (e.g. with a sequence that corresponds to SEQ. ID. No. 2 or is essentially identical to it) acts in the cell nucleus. Therefore, it is likely that one or more sub-sequences of SEQ. ID. No. 2, for example residues 193 to 196 (PRRR) and residues 235-240 (PKRPRR) act as a nuclear localization signal. The small areas are probably NLSs. This is based on the observation that many NLSs include a four residue pattern, the

48

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

is composed of basic amino acids (K or R) or three basic amino acids (K or R) and H or P; a pattern starting with P followed by a basic segment within three residues containing 3 K or R residues from four residues (see for example Nakai et al., Genomics 14 (1992) 897). It is believed that the deletion of one of these sequences or both and / or additional localization sequences interferes with hTRT transport into the nucleus and / or increases hTRT conversion and thus to prevent telomerase from accessing its nuclear substrates of degradation of the proliferative potential is useful. In addition, an hTRT variant that lacks the NLS could assemble into an RNP that can no longer maintain the telomeric length because the resulting enzyme cannot get into the nucleus.

The hTRT polypeptides according to the invention are typically associated in the target cell with a telomerase RNA, such as hTR, if they are used to increase telomerase activity in a cell. In one embodiment, an introduced hTRT polypeptide associates with an endogenous hTR to form a catalytically active RNP (e.g., an RNP that includes the hTR and a full length polypeptide and a sequence of SEQ. ID No. 2). The RNP formed in this way can also associate with other, for example telomerase-associated, proteins. In further embodiments, the telomerase RNP (which contains the hTRT protein, hTR and optionally further constituents) is introduced as a complex into the target cell.

In a similar embodiment, an hTRT expression vector is introduced into a cell (or the progeny of a cell) into which a telomerase RNA (e.g. hTR) expression vector is introduced simultaneously, after, or before. In this embodiment, hTRT protein and telomerase RNA are co-expressed in the cell and they assemble to form a telomerase RNP. A preferred telomerase RNA is hTR together. An expression vector useful for the expression of hTR in a cell has been described above (see U.S. Patent 5,583,016). In a further embodiment, the hTRT polypeptide and the hTR-RNA (or an equivalent) are associated in vitro to form a complex which is then introduced into the target cells. Another aspect of the invention relates to the provision of hTRT polypeptides which are useful for reducing telomerase activity in a cell.

As described above, these "inhibitory" polypeptides can either be introduced directly into the cell or generated by expression of recombinant nucleic acids in the cell. Peptide "imitators" or polypeptides comprising non-standard amino acids (i.e. amino acids that do not correspond to the 20 amino acids encoded by the genetic code or their normal derivatives) are usually introduced directly.

In one embodiment, inhibition of telomerase activity results in the sequestration of an ingredient required for accurate telomere extension. Examples of such components are hTRT and hTR. Thus, administration of a polypeptide that binds hTR but has no catalytic activity of telomerase can decrease endogenous telomerase activity in the cell. In a related embodiment, the hTRT polypeptide can bind a cell component that is not hTR, for example a (or more) telomerase-associated protein, thereby disrupting telomerase activity in the cell.

In another embodiment, the hTRT polypeptides according to the invention disrupt the interaction of endogenously expressed hTRT protein and another cellular component required for the telomerase function, for example hTR, telomer DNA, telomerase-associated proteins, telomer-associated proteins, telomeres Cell cycle controlling proteins, DNA repair enzymes, histones or non-histone chromosomal proteins etc. (for example by competition).

By selecting molecules according to the invention (e.g. polypeptides) which affect the interaction of endogenously expressed hTRT protein and other cellular components, one can prefer molecules which, as described here, contain one or more of the conserved motifs of the hTRT protein. The evolutionary conservation of these areas shows that these motifs play an important role in the correct functioning of human telomerase. These motifs are thus generally useful areas for using the function of the hTRT protein to generate hTRT protein variants according to the invention. Thus, hTRT polypeptide variants with mutations in conserved motifs are particularly useful.

In a further embodiment, expression of the endogenous hTRT gene is repressed by introducing a large amount of hTRT polypeptide into the cell (e.g. typically at least about more than 2-fold compared to the endogenous level, preferably about at least 10 to about 100-fold). This acts via a test coupling loop and thus inhibits the transcription of the hTRT gene, the processing of the hTRT pre-m PNA, the translation of the hTRT mRNA or the assembly and transport of the telomerase RNP.

49

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

2) Oligonucleotides a) «Antisense» constructs

The invention provides methods and "antisense" oligonucleotides or polynucleotide reagents that can be used to decrease expression of hTRT gene products in vitro or in vivo. The administration of the “antisense” reagents according to the invention to a target cell leads to a reduced telomerase activity and is particularly useful for the treatment of diseases which are characterized by high telomerase activity (e.g. cancer). It is assumed that no limitation to a particular mechanism is intended here that “antisense” oligonucleotides bind to the “sense” hTRT mRNA and interfere with its translation. Alternatively, the “antisense” molecules can make the hTRT mRNA sensitive to cleavage with nuclease, interfere with transcription, processing or localization or otherwise interfere with RNA precursors (“pre-mRNA”), the transcription of mRNA of the hTRT Repress gene or act through some other mechanism. However, the specific mechanism by which the anti-sense molecules reduce hTRT expression is not critical.

The “antisense” polynucleotides according to the invention comprise an “antisense” sequence with at least 7 to 10 or more nucleotides which hybridize specifically with a sequence of mRNA which encodes human TRT or mRNA which is transcribed by the hTRT gene. The “antisense” polynucleotide according to the invention preferably has a length of about 10 to about 50 nucleotides or of about 14 to about 35 nucleotides. In other embodiments, the "antisen-se" polynucleotides are polynucleotides that are shorter than about 100 nucleotides or shorter than about 200 nucleotides. In general, the "antisense" polynucleotides should be long enough to form a stable double helix, but short enough (depending on the type of delivery) to be administered in vivo if desired. The minimum length of a polynucleotide required for a specific hybridization with a target sequence depends on various factors, for example the G / C content, the location of mismatched bases (if any), the extent of the uniqueness of the sequence compared to the population of target polynucleotides, the chemical nature of the polynucleotide (eg it can be a methylphosphonate backbone, a peptide nucleic acid, a phosphorothioate) etc. In general the "antisense" sequence is to ensure a specific hybridization to that Target hTRT mRNA sequence essentially complementary. In certain embodiments, the "antisense" sequence is exactly complementary to the target sequence. However, the “antisense” polynucleotides can also contain nucleotide substitutions, additions, deletions, transitions, transpositions or modifications, as long as the specific binding to the relevant target sequence, which corresponds to hTRT-RNA or its gene, is retained as a functional property of the polynucleotide .

In one embodiment, the "antisense" sequence is complementary to relatively accessible sequences of the hTRT mRNA (e.g. sequences that have relatively few secondary structures). This can be determined by analyzing the predicted RNA secondary structures, for example by means of the MFOLD program (Genetics Computer Group, Madison Wl) and by in vitro or in vivo tests known in the art. Examples of oligonucleotides that can be tested for an “antisense” suppression of the hTRT function are oligonucleotides that match the following positions of SEQ. ID. # 1 can hybridize (i.e. are essentially complementary): SEQ. ID. No. 1: 40-60; 260-280; 500-520; 770-790, 885-905; 1000-1020; 1300-1320; 1520-1540; 2110-2130; 2295-2315; 2450-2470, 2670-2690; 3080-3110; 3140-3160; and 3690-3710. Another useful method for identifying effective "antisense" compositions uses combinatorial arrays of oligonucleotides (see, for example, Milner et al., Nature Biotechnology 15 (1997), 537).

The invention also provides an "antisense" polynucleotide that has sequences in addition to the "an-tisense" sequence (e.g., in addition to the anti-hTRT "sense" sequence). In this case, the «antisense» sequence is contained in a polynucleotide with a longer sequence. In a further embodiment, the sequence of the polynucleotide essentially consists of the “antisense” sequence or it represents this.

The “antisense” nucleic acids (DNA, RNA, modified DNA or RNA analogs etc.) can be produced using any method suitable for producing nucleic acids, for example the methods described here. In one embodiment, “antisense” RNA molecules according to the invention can be produced, for example, by chemical synthesis de novo or by cloning. For example, an “antisense” RNA that hybridizes with hTRT mRNA can be produced by functionally linking an hTRT DNA sequence (for example SEQ. ID. No. 1 or a fragment thereof) to a promoter in reverse orientation a vector (eg a plasmid) is inserted (ligated). Provided that the promoter and preferably termination and polyadenylation signals are correctly positioned, the strand of the inserted sequence that corresponds to the non-coding strand is transcribed and this acts as an “antisense” oligonucleotide according to the invention.

The “antisense” oligonucleotides according to the invention can be used to inhibit telomerase activity

50

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

in cell-free extracts, cells and animals, including mammals and humans. The phosphorothioate «antisense» oligonucleotides:

A) GGCATCGCGGGGGTGGCCGGG

B) CAGCGGGGAGCGCGCGGCATC

C) CAGCACCTCGCGGTAGTGGCT

D) GGACACCTGGCGGAAGGAGGG

can be used, for example, to inhibit telomerase activity. At a concentration of 10 nM, each oligonucleotide inhibited; Mixtures of oligonucleotides A and B; A, B, C and D; and A, C and D the telomerase activity in 293 cells when treated once a day for 7 days. Inhibition was also observed with an “antisense” hTR molecule (5'-GCTCTAGAATGAAGGGTG-3 ') when this was done with oligonucleotides A, B and C; A, B and D; and A and C was combined. Control and oligonucleotides useful for such experiments include:

51) GCGACGACTGACATTGGCCGG

52) GGCTCGAAGTAGCACCGGTGC

53) GTGGGAACAGGCCGATGTCCC

In order to be able to determine the “antisense” oligonucleotides according to the invention that are optimal for a particular desired application, a scanning can be carried out using a set of “antisense” oligonucleotides according to the invention. An example of such a set is the set of 30-mer oligonucleotides that span the hTRT mRNA and one of the next is offset by 15 nucleotides (ie ON1 corresponds to positions 1-30 and is TCCCACGTGCGCAGCAGGACGCAGCGCTGC, ON2 corresponds to positions 16 -45 and is GCCGGGGCCAGGGCTTCCCACGTGCGCAGC, and ON3 corresponds to positions 31-60 and is GGCATCGCGGGGGTGGCCGGGGCCAGGGCT etc. until the end of the mRNA). Each member of this set can be tested for inhibitory activity as described here. Those oligonucleotides that show inhibitory activity under the desired conditions then identify a desired region. Further oligonucleotides according to the invention which correspond to the desired region (i.e. 8-mers, 10-mers, 15-mers etc.) can be tested to identify the oligonucleotide with the activity preferred for the application.

For general procedures related to antisense polynucleotides, see Antisense RNA and DNA (1988), D.A. Melton, (ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) and Dagle et al., Nucleic Acids Research 19 (1991), 1805. For a review article on antisense therapy, see, for example, Uhlmann et al ., Chem. Reviews 90 (1990) 543-584.

b) triplex oligo- and polynucleotides

The present invention provides oligonucleotides and polynucleotides (e.g., DNA, RNA, or PNA) that bind to double-stranded or duplex hTRT nucleic acids (e.g., in a folded region of the hTRT RNA or in the hTRT gene), thereby forming a nucleic acid that contains a triple helix or a “triplex” nucleic acid. The formation of a triple helix leads to the inhibition of hTRT expression, for example by preventing the transcription of the hTRT gene, as a result of which telomerase activity in a cell is reduced or eliminated. It is believed (but is not intended to be bound by any particular mechanism) that the triple helix pair will interfere with the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.

Triplex oligo- and polynucleotides according to the invention are constructed using the base pairing rules for the formation of triple helices (see for example Cheng et al., I. Biol. Chem.

1988, 263: 15110; Ferrin and Camerini Otero, Science 354 (1991), 1494; Ramdas et al., J.Biol. Chem.

1989, 264: 17395; Strobel et al., Science 254 (1991), 1639 and Rigas et al., Proc. Nati. Acad. Be. USA 83 (1986), 9591, each of which is to be considered part of this description by reference) and the hTRT mRNA and / or gene sequences. Typically, the triplex-forming oligonucleotides of the invention comprise a specific sequence from about 10 to at least about 25 nucleotides or a longer sequence that is "complementary" to a specific sequence in the hTRT-RNA or the gene (ie long enough to be a stable triple -Helix formation, but short enough, depending on the type of delivery, to be administered in vivo if desired). In this context, "complementary" means the ability to

51

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Formation of a stable triple helix. In one embodiment, oligonucleotides are designed to specifically target the regulatory regions of the hTRT gene (e.g., the hTRT-5 'flanking sequence, promoters and enhancers) or the transcription initiation site (e.g., between -10 and +10 from the transcription initiation site ) bind specifically. For a review of recent therapeutic advances using triplex DNA, see Gee et al., In Huber and Carr, 1994, Molecular and Immunologie Approaches, Futura Publishing Co., Mt. Kisco NY and Rininsland et al., Proc. Nati. Acad. Be. USA 94 (1997), 5854, both documents being incorporated by reference into this description.

c) Ribozymes

The present invention also provides ribozymes that are useful for inhibiting telomerase activity. The ribozymes according to the invention bind, cleave specifically and inactivate hTRT mRNA. Useful ribozymes can include 5 'and 3' terminal sequences complementary to the hTRT mRNA, and these can be constructed by those skilled in the art based on the hTRT mRNA sequence described herein (see PCT publication WO 93/23 572, loc. Cit.). The ribozymes according to the invention include ribozymes with the characteristics of the group I intron ribozymes (Cech, Biotechnology 13 (1995), 323) and “hammerhead” ribozymes (Edgington, Biotechnology 10 (1992), 256).

The ribozymes according to the invention include ribozymes with cleavage sites such as GUA, GUU and GUC. Other optimal cleavage sites for ribozyme-mediated inhibition of telomerase activity in accordance with the present invention include those described in PCT publications WO 94/02 595 and WO 93/23 569, both documents being incorporated by reference into this specification are to be seen. Short RNA oligonucleotides 15 and 20 ribonucleotides in length corresponding to a region of the target hTRT gene that contains a cleavage site can be evaluated for secondary structural features that lead to more desirable oligonucleotides. The suitability of the cleavage sites can also be assessed by testing accessibility to hybridization with complementary oligonucleotides using "ribonuclease protection" assays, or by testing ribozyme activity in vitro according to standard procedures known in the art.

As described by Hu et al., PCT publication WO 94/03 596, "antisense" and ribozyme functions can be combined in a single oligonucleotide. This document is also to be regarded as the subject of this description by reference. In addition, ribozymes can comprise one or more modified nucleotides or modified bonds between nucleotides, as described above in connection with the description of illustrative oligonucleotides according to the invention.

In one embodiment, the ribozymes according to the invention are generated in vitro and introduced into a cell or a patient. In another embodiment, gene therapy methods are used to express ribozymes in a target cell ex vivo or in vivo.

d) administration of oligonucleotides

Typically, the therapeutic methods according to the invention include the administration of an oligonucleotide which acts in such a way that it can inhibit or stimulate telomerase activity in vivo under physiological conditions and which is relatively stable under these conditions for a period of time which is sufficient for a therapeutic effect . As already stated above, modified nucleic acids can be useful in imparting such stability, but also for the targeted delivery of the oligonucleotide to the desired tissue, organ or cell.

Oligo- and polynucleotides can be supplied directly as drugs in a suitable pharmaceutical formulation, or indirectly by means of introducing a nucleic acid into a cell, including liposomes, immunliposomes, ballistic methods, direct uptake in cells, etc., as described here , belong. To treat a disease, the oligonucleotides according to the invention are administered to the patient in a therapeutically effective amount. A therapeutically effective amount is the amount sufficient to alleviate disease symptoms or to modulate telomerase activity in the target cell, such as using a TRAP assay or other suitable assay for the biological function of telomerase can be determined. Methods suitable for the delivery of oligonucleotides for therapeutic purposes are described in US Pat. No. 5,272,065, which is hereby incorporated by reference into this description. Further details regarding the administration of pharmaceutically active compounds are described below. In a further embodiment, oligonucleotides and polynucleotides can be supplied using gene therapy and the recombinant DNA expression plasmids according to the invention.

52

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

3) gene therapy

Gene therapy refers to the introduction of an otherwise exogenous polynucleotide that has a medically useful phenotypic effect on the (typically) mammalian cell (s) into which it is transferred. One aspect of the present invention relates to the provision of gene therapy methods and compositions for the treatment of telomerase associated conditions. In illustrative embodiments, gene therapy involves introducing into a cell a vector that expresses an hTRT gene product (e.g., an hTRT protein that is substantially similar to the hTRT polypeptide having a sequence of SEQ. ID No. 2, for example, by To increase telomerase activity, or an inhibitory hTRT polypeptide to reduce the activity), the expression of a nucleic acid with a sequence of an hTRT gene or an hTRT mRNA (for example an “antisense” RNA, for example to reduce telomerase activity) , the expression of a polypeptide or polynucleotide that otherwise influences the expression of hTRT gene products (eg a ribozyme which is directed against hTRT-MRNA to reduce telomerase activity) or the exchange, or the interruption of an endogenous hTRT sequence (e.g. «gene replacement» and «gene knockout»). Numerous other embodiments will be apparent to those skilled in the art after reading this description. In one embodiment, an vector encoding hTR is also introduced. In a further embodiment, vectors encoding telomerase-associated proteins are also introduced with or without a vector for hTR.

Vectors useful for hTRT gene therapy can be viral or non-viral. These include the vectors described above in connection with the hTRT expression systems according to the invention. It will be apparent to those skilled in the art that gene therapy vectors can include promoters and other regulatory or processing sequences, such as those described herein. The vectors usually comprise a promoter and, if appropriate, an enhancer (independent of an enhancer which is already present within the promoter sequences) which serve to control the transcription of an oligoribonucleotide, and further regulatory elements which are required for the maintenance of the vector as an episome or for that Provide high level chromosomal integration and transcription if desired. A plasmid useful for gene therapy can comprise further functional elements, for example selectable markers, identification regions and further sequences. Additional sequences can play a role in providing stability both outside and inside the cell in order to effect the targeted delivery of hTRT nucleotide sequences ("sense" or "antisense") to a desired organ, tissue or cell population, or to mediate entry into a cell, the nucleus of a cell and / or integration within nuclear DNA.

For example, aptamer-like DNA structures or other sites of the units that bind proteins can be used to mediate the binding of a vector to cell surface receptors or to serum proteins that bind to a receptor, thereby increasing the efficiency of DNA transfer into the Cell is increased. Other DNA sites and structures can bind directly or indirectly to receptors in the nuclear membrane or to other proteins that migrate to the nucleus, thereby facilitating the nuclear uptake of a vector. Other DNA sequences can directly or indirectly influence the efficiency of the integration.

Suitable vectors for gene therapy may or may not have an origin of replication. For example, it is useful for replication of the vector to incorporate an origin of replication into a vector prior to administration to a patient. However, the origin of replication can often be removed prior to administration if the vector is designed to integrate into the host's chromosomal DNA or to bind to the host's mRNA or DNA. In some situations (e.g. with tumor cells) it may not be necessary for the exogenous DNA to integrate stably into the transduced cell, since transient expression can be sufficient to kill cells.

As already mentioned, the present invention also provides methods and reagents for gene replacement therapy (i.e. the replacement of an endogenous hTRT gene with a recombinant gene by homologous recombinaton). Vectors specially designed for integration by homologous recombination can be used for this. The factors important for the optimization of the homologous recombination include the extent of the sequence identity and the length of the homology to chromosomal sequences. The specific sequence mediating homologous recombination is also important, since integration into transcriptionally active DNA takes place much more easily. Methods and substances for constructing constructs for targeted homologous recombination are described, for example, in Mansour et al., Nature 336 (1988), 348; Bradley et al., Bio / Technology 10 (1992), 534 and beyond in U.S. Patent No. 5,627,059; 5,487,992, 5,631,153 and 5,464,764. In one embodiment, gene replacement therapy comprises the modification or replacement of all or part of a regulatory sequence which controls the expression of the hTRT gene to be regulated. For example, the hTRT promoter sequences (for example, those found in SEQ. ID No. 6 (FIG. 21)) can be interrupted (in order to reduce the hTRT expression or to eliminate a control point for the transcription) or against an exogenous one Promoter be replaced, for example to increase hTRT expression.

The invention also provides methods and reagents for the hTRT gene knockout (i.e., the

53

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Deletion or disruption of an endogenous hTRT gene by homologous recombination using a recombinantly produced vector). In this "gene knockout", the sequences considered as targets can be regulatory sequences (e.g. the hTRT promoter) or sequences coding for RNA or protein. The change in expression of endogenous genes by homologous recombination is described in detail in US Pat. No. 5,272,071 (and the US patents cited above), WO 91/09 955, where 93/09 222, WO 96/29 411 , WO 95/31 560 and WO 91/1250. See also Moynahan et al., Hum. Mol. Genet. 5 (1996) 875.

The present invention further provides methods of specifically killing telomerase-positive cells or preventing the transformation of telomerase-negative cells to a telomerase-positive status, using the hTRT gene promoter to regulate the expression of a protein toxic to the cell. As shown in Example 14, an hTRT promoter sequence can be operably linked to a reporter gene so that activation of the promoter leads to expression of the protein encoded by the reporter gene. If, instead of a reporter protein, the encoded protein is toxic to the cell, activation of the promoter leads to cell morbidity or cell death. In one embodiment according to the invention, a vector which comprises a gene encoding a toxic protein which is functionally linked to an hTRT promoter is introduced into cells, such as human cells, for example cells in a human patient, which leads to cell death in those cells, in which hTRT promoter activating factors are expressed, such as cancer cells. In a related embodiment, the encoded protein itself is not toxic to a cell, but encodes an activity that makes the cell sensitive to an otherwise non-toxic agent. Tumors can be treated, for example, by introducing an hTRT promoter herpes thymidine kinase (TK) gene fusion construct into tumor cells and administering gangcyclovir or the equivalent (see, for example, Moolton and Wells, J. Nati. Cane. Inst. 82 (1990 ), 297). Numerous other suitable toxic or potentially toxic proteins and systems are known in the art (using promoter sequences that differ from hTRT), which can be modified and administered by a person skilled in the art after reading this description in accordance with the present invention.

Vectors for gene therapy can be introduced into cells or tissues in vivo, in vitro or ex vivo. For ex vivo therapy, vectors can be introduced into stem cells taken from the patient and clonai propagated for autologous back transplantation in the same patient (see, for example, U.S. Patents 5,399,493 and 5,437,994, reference being incorporated by reference in these documents the description can be viewed). The cells that may be targeted for hTRT gene therapy with the goal of increasing telomerase activity in a target cell include, but are not limited to, embryonic or germ cells, particularly primate or human cells, as noted above, hematopoietic Stem cells (AIDS and after chemotherapy), vascular endothelial cells (diseases of the cardiovascular and cerebral vessels), skin fibroblasts and subcutaneous keratinocytes (wound healing and burns), chondrocytes (arthritis), brain astrocytes and microglial cells (Alzheimer's disease), osteoblasts (osteopellenosis) Eye diseases) and pancreatic islet cells (type I diabetes) and the cells listed in list 1 below.

In an embodiment according to the invention, an inducible promoter functionally linked to a TRT-coding sequence (or a variant thereof) is used to modulate the proliferation capacity of cells in vivo or in vitro. In a particular embodiment, for example, insulin-producing pancreatic cells, which are transfected with an hTRT expression vector under the control of an inducible promoter, are introduced into a patient. The proliferation ability of the cells can then be controlled by administering the promoter activating agent (e.g. tetracycline) to the patient, whereby the cells can multiply more than would otherwise have been possible. Cell proliferation can then be stopped, continued, or initiated again, as desired by the attending physician.

4) vaccine and antibodies

Immunogenic peptides or polypeptides with an hTRT sequence can be used to elicit an anti-hTRT immune response in a patient (i.e., they can act as a vaccine). Examples of immunogenic hTRT peptides and polypeptides are described in Examples 6 and 8 below. An immune response can also be triggered by the delivery of plasmid vectors which encode the desired polypeptide (i.e. the administration of "naked DNA"). The desired nucleic acids can be delivered by injection, liposomes or other administration measures. In one embodiment, types of immunization are selected which trigger a response of cytotoxic lymphocytes to telomerase-expressing cells that is restricted to MHC class I in the test subject or the patient. After immunization, an increased immune response against cells that express high concentrations of telomerase (e.g. malignant cells) is elicited in the person or animal.

Anti-hTRT antibodies, for example from the mouse, from humans, or humanized monoclonal

54

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Antibodies can also be administered to achieve an immune response to cells that express telomerase (e.g., passive immunization).

F) Medicines

Related aspects of the present invention relate to the provision of drugs that contain hTRT oligo- and polynucleotides, polypeptides and antibodies, agonists, antagonists or inhibitors, alone or in combination with at least one other agent such as one stabilizing compound, diluent, carrier, or other active ingredient or agent.

The pharmaceuticals according to the invention can be administered in a sterile, biocompatible pharmaceutical carrier, which includes, for example, but not limited to, saline, buffered saline, dextrose and water. Each of these molecules can be administered to the patient alone or in combination with other agents, drugs or hormones, in drugs where it is mixed with one or more suitable excipients, excipients and / or pharmaceutically acceptable carriers. In an embodiment according to the invention, the pharmaceutically acceptable carrier is pharmaceutically inert.

The drugs can be administered orally or parenterally. Methods for parenteral administration include topical, intra-arterial (e.g., directly to the tumor), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration. In addition to the active ingredients, these drugs can include suitable pharmaceutically acceptable carriers that include excipients and other compounds that facilitate the processing of the active ingredients into formulations that can be used pharmaceutically. Further details regarding formulation and administration procedures can be found in the latest edition of "Remington's Pharmaceutical Sciences" (Maack Publishing Co., Easton PA).

Drugs for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in doses suitable for oral administration. Such carriers allow the formulation of the drugs as tablets, pills, dragees, capsules, liquids, gels , Syrups, slurries, suspensions, etc., which are suitable for ingestion by the patient. See PCT publication WO 93/23 572.

Drugs for oral use can be obtained by combining active ingredients with a solid excipient, optionally by grinding a mixture obtained, and further processing the mixture of granules after adding suitable additional compound to obtain tablets or dragee cores if so desired. Suitable excipients are carbohydrate or protein fillers, including, but not limited to, sugars, including lactose, sucrose, mannitol or sorbitol; Starch from corn, wheat, rice, potatoes or other plants; Cellulose such as methyl cellulose, hydroxypropyl methyl cellulose or sodium carboxymethyl cellulose and gums, which include gum arabic and tragacanth resin, and proteins, such as gelatin and collagen. If desired, disintegrating or solubilizing agents can be added, for example cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof, for example sodium alginate.

Drage cores are provided with suitable coatings, for example concentrated sugar solutions, they can also contain gum arabic, talc, polyvinylpyrrolidone, “Carbopol” gel, polyethylene glycol and / or titanium dioxide, lacquer solutions and suitable organic solvents or solution mixtures. Dyes or pigments can be added to the coatings for the tablets or coated tablets to identify the product or to indicate the amount of active ingredient (i.e. the dosage).

Oral drugs include plug-in capsules made from gelatin and soft sealed capsules made from gelatin and a coating of glycerin or sorbitol. Push-fit capsules can contain the active ingredients mixed with a filler or binder, including lactose or starches, lubricants such as tallow or magnesium stearate, and optionally stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, for example in fatty acids, liquid paraffin or liquid polyethylene glycol with or without stabilizers. Medicines for parenteral administration include aqueous solutions with active compounds. For the injection, the medicament according to the invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers, such as, for example, Hank's solution, Ringer's solution or in physiologically buffered saline. Aqueous suspensions for injection may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. In addition, suspensions of the active compounds can be prepared as suitable oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty acids such as sesame oil or synthetic fatty acid esters such as ethyl oleate or triglycerides or liposomes. If appropriate, the suspensions may also contain suitable stabilizers or agents which increase the solubility of the compounds so as to allow the production of highly concentrated solutions.

55

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

For topical or nasal administration, penetrants are used in the formulation which are suitable for the specific barriers to be penetrated. Such penetrants are well known in the art.

The pharmaceuticals according to the invention can be prepared by methods which are similar to those known in the art (for example by customary mixing, solution, granulation, dragee production, pulverization, emulsification, encapsulation, inclusion or lyophilization processes). .

The drugs can be provided as a salt and can be prepared with many acids, including, but not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, etc. Salts tend to be more soluble in aqueous or other protonic solvents to be as the corresponding form of the free base. In other cases, the preferred preparation may be a lyophilized powder in 1mM-50mM histidine, 0.1% to 2% sucrose, and 2% to 7% mannitol at a pH in the range 4.5 to 5.5 which is combined with a buffer before use.

After the preparation of the medicaments, which comprise a compound according to the invention formulated in a compatible carrier, they can be placed in a suitable container and labeled according to the treatment of a specified disease state. When administering human telomerase proteins and nucleic acids, such a label would include an indication of the amount and frequency of administration and the method of administration.

Medicaments suitable for the use according to the invention include compositions in which the active constituents are contained in an effective amount in order to achieve the desired purpose. “Therapeutically effective amount” or “pharmacologically effective amount” are commonly used terms and refer to the amount of an agent that can effectively produce the desired pharmacological result. Thus, a therapeutically effective amount is an amount sufficient to improve the symptoms of the disease to be treated.

A useful assay to determine an effective amount for a given application (e.g., a therapeutically effective amount) is to measure the effect on telomerase activity in a target cell. The amount actually administered depends on the person to be treated and is preferably an amount optimized so that the desired effect is achieved without significant side effects. The determination of a therapeutically effective dose is within the skill of the person skilled in the art.

For any compound, the therapeutically effective dose can initially be determined either in cell culture assays or using a suitable animal model. The animal model is also used to determine an appropriate concentration range and route of administration. Information thus obtained can then be used to determine useful doses and routes of administration in humans.

A therapeutically effective amount refers to the amount of protein, polypeptide, peptide, antibody, oligo- or polynucleotide, agonist or antagonist that ameliorates the symptoms or condition. The therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or on experimental animals (e.g. ED50, the dose which is therapeutically effective in 50% of the population; and LD50, the dose which is lethal in 50% of the population). The therapeutic index indicates the dose ratio between therapeutic and toxic effects and it can be expressed as the ratio ED50 / LD50. Pharmaceutical compositions that have a large therapeutic index are preferred. The data obtained from cell culture assays and animal studies are used to determine the dose range for human use. The dosage of such compounds is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending on the dosage form used, the sensitivity of the patient and the route of administration.

The exact dosage is selected individually by the respective doctor for the patient to be treated. The dosage and administration are adjusted so that they provide a sufficient level of the active unit or maintain the desired effect.

Additional factors to consider include the severity of the condition (e.g., the size and location of the tumor; the age, weight, and gender of the patient; diet, timing, and frequency of administration, the combination (s) of Drugs, sensitivity to reactions and tolerance towards or response to therapy). Long-acting drugs can be administered every 3 to 4 days, every week, or once every 2 weeks depending on the half-life and the clearance rate of the particular formulation. Instructions regarding specific dosages and methods of administration can be found in the literature (see US Pat. Nos. 4,657,760, 5,206,344 and 5,225,212, which documents may be incorporated by reference into this specification). Those skilled in the art will use different formulations for nucleotides than for proteins or their inhibitors. In the same way, the administration of polynucleotides or polypeptides is specific for certain cells, conditions, locations, etc.

56

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Vili. The increase in the ability to proliferate and the production of immortalized cells,

Cell lines and animals

As discussed above, most vertebrate cells show senescence after a limited number of divisions in the culture (e.g. 50 to 100 divisions). However, certain cell variants can divide indefinitely in culture (e.g. HeLa cells, 293 cells) and are therefore useful for research and industrial use. Such immortal cell lines are usually obtained from spontaneously developing tumors or after transformation by exposure to radiation or a tumor-inducing virus or a tumor-inducing chemical. Unfortunately, there is only a limited selection of cell lines available, especially human cell lines that show differentiated cell function. In addition, the immortal cell lines currently available are characterized by chromosomal abnormalities (e.g. aneuploidy, gene rearrangements or mutations). Furthermore, many of the long-established cell lines are relatively undifferentiated (e.g. they do not produce such highly specialized products that are unique to certain tissues or organs). There is therefore a need for new methods for producing immortal cells, in particular human cells. Use of immortalized cells refers to the production of natural proteins and recombinant proteins (e.g. therapeutic polypeptides) or antibodies for which a stable genetically normal cell line is preferred. Specialized cell types may also be preferred for the production of some recombinant proteins (e.g. pancreatic cells for the production of human insulin). A further possible use for immortalized cells is the introduction into a patient for gene therapy or for the exchange of diseased or damaged cells or tissues. For example, autologous immune cells which, for example, contain or express a recombinant hTRT gene or polypeptide according to the invention can be used for cell exchange in a patient after aggressive cancer therapy, for example after irradiation of the entire body. Another possible use for immortalized cells is the ex vivo production of "artificial" tissues or organs (e.g. skin) for therapeutic use. Another use of such cells is screening for active substances or confirmation of the action of active substances, for example telomerase-inhibiting active substances, or in the production of vaccines. Further possible uses of the cells according to the invention are obvious to the person skilled in the art.

The immortalized cells and cell lines according to the invention, as well as those with only increased replication ability, are generated in the cell by increasing telomerase activity. Any method described here for increasing telomerase activity can be used. Thus, in one embodiment, cells are immortalized by increasing the amount of hTRT polypeptide in the cell. In one embodiment, the hTRT level is increased by introducing an hTRT expression vector into the cell (with stable transfection sometimes preferred). As already discussed above, the hTRT coding sequence is normally functionally linked to a promoter that can be inducible or constitutively active in the cell.

In one embodiment, a polynucleotide comprises a sequence that is a polypeptide of SEQ. ID. No. 2, wherein the sequence is functionally linked to a promoter (e.g. a constitutively expressed promoter, e.g. a sequence of SEQ. ID No. 6 (Fig. 6)) and the polynucleotide is introduced into the cell. In one embodiment, the polynucleotide comprises a sequence of SEQ. ID. No. 1. Preferably the polynucleotide contains polyadenylation and termination signals. In other embodiments, additional elements such as enhancers or those discussed above are included. In an alternative embodiment, the polynucleotide does not contain a promoter sequence, in which case such a sequence is provided by the endogenous genome of the target cell after integration (e.g. by recombination, e.g. homologous recombination) of the introduced polynucleotide. The polynucleotide can be introduced into the target cell by any method, including the methods described herein (such as lipofection, electroporation, virosomes, liposomes, immunliposomes, polycation / nucleic acid conjugates, naked DNA).

Using the method according to the invention, any vertebrate cell can be caused to show an increased ability to proliferate or even to be immortalized and to remain in culture indefinitely. In one embodiment, the cells are mammalian, with human cells being preferred for many applications. Human cells that can be immortalized include the cells listed in Listing 1.

Of course, the “diagnostic” assays according to the invention described below can be used to identify and characterize the immortalized cells according to the invention.

57

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Listing 1

Human cells in which hTRT expression can be increased Horn-forming epithelial cells

Keratinocytes of the epidermis (differentiating epidermal cells)

Basal cells of the epidermis (stem cells)

Keratinocytes of the fingernails and toenails

Basal cells of the nail bed (stem cells)

Cells of the hair shaft medullary, cortical, cuticular cells; Cells of the hairline sheath, cuticular cells, cells of the Hux-ley layer, external cells of the Henle layer; Hair germ cells (stem cells)

Cells of the moist multilayered border epithelium

Surface epithelial cells of the multilayered squamous epithelium of the tongue, oral cavity, esophagus, anal canal, distal urethra, vagina, basal cells of these epithelium (stem cells)

Cells of the outer corneal epithelium

Cells of the urinary epithelium (which lines the bladder and ureters)

Epithelial cells specializing in exocrine secretion Salivary gland cells Mucus-secreting cells (secretion rich in polysaccharides)

serous gland cells (secretion rich in glycoprotein enzymes)

Cells of the von Ebner glands in the tongue (secretions wash around the taste buds)

Breast cells secrete milk

Lacrimal gland cells secrete tears

Cells of the ceruminal gland of the ear, secrete cerumen

Cells of the eccrine sweat glands, secrete glycoproteins (dark cells)

Cells of the eccrine sweat glands, secrete small molecules (bright cells)

Apocrine sweat gland cells (scent secretion, sex hormone sensitive)

Cells of the minor glands in the eyelid (specialized sweat gland)

Cells of the sebum glands secrete lipid-rich sebum

Bowman gland cells in the nose (secretions wash around the olfactory epithelium)

Brunner gland cells in the duodenum secrete alkaline solution consisting of mucus and enzymes

Cells of the seminal vesicle, secrete components of the seminal fluid including fructose (as a fuel for sperm floating in it)

Prostate cells, secrete other constituents of the seminal fluid Bulbourethral gland cells, secrete mucus Bartholin's gland cells, secrete vaginal secretion, Littré gland cells, secrete mucus

Cells of the uterine mucosa, secrete primarily carbohydrates isolated goblet cells of the respiratory and digestive tract,

secrete mucus secreting cells of the gastric mucosa main cells of the gastric glands, secrete pepsinogen parietal cells of the gastric glands, secrete HCl

Acinus cells of the pancreas, secrete digestive enzymes and bicarbonate paneth cells of the small intestine, secrete lysozyme

Type II alveolar cells of the lung secrete surfactant clara cells of the lung cells that specialize in the secretion of hormones

Anterior lobe cells, secrete growth hormone, follicle-stimulating hormone, luteinizing hormone, prolactin, adrenocorticotropic hormone and thyreotropic hormone cells of the pituitary central lobe, secrete melanocyte-stimulating hormone cells of the pituitary gland, secrete vasophysinophenin

Intestinal cells secrete serotonin, endorphin, somatostatin, gastrin, secretin, cholecystokinin, insulin and glucagon

Cells of the thyroid gland, secrete thyroid hormone, calcitonin

Parathyroid cells secrete parathyroid hormone, oxyphile cells

Adrenal gland cells secrete epinephrine, norepinephrine and steroid hormones

58

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Mineralocorticoide Glucocorticoide

Cells of the gonads, secrete testosterone (Leydig cells of the testicles) estrogen (inner theca cells of the egg follicle) progesterone (luteal cells of the broken egg follicle)

Cells of the juxtaglomerular apparatus of the kidney juxtaglomerular cells (secrete renin)

Macula densa cells peripolar cells

Mesangial cells

Absorbing epithelial cells in the intestine, exocrine glands and urogenital tract

Brush hem cells of the intestine (with small villi)

multilayered duct cells of exocrine glands gallbladder epithelial cells

Brush border cells of the proximal tubule of the kidney. Distal tubule cells of the kidney. Non-ciliated cells of the ductulus efferens. Epididymal main cells. Epididymal basal cells

Cells specialized in metabolism and storage

Hepatocytes (liver cells)

Fat cells white fat cells brown fat cells lipocytes of the liver

Epithelial cells, which primarily have a protective function and which line the lungs, the intestine, the exocrine glands and the urogenital tract

Type I pneumocytes (lining the air space of the lungs)

Pancreatic duct cells (centroazine cells)

non-multilayered duct cells of the sweat gland, the salivary gland, the mammary gland parietal cells of the renal glomerulus podocytes of the renal glomerulus

Henle Loop Thin Segment Cells (in the Kidney)

Manifold cells (in the kidney)

Ductal cells of the seminal vesicle, prostate

Epithelial cells that line closed internal body cavities line vascular endothelial cells of the blood vessels and lymphatic vessels fenestrated cells continuous cells spleen cells

Synovial cells (line the joint cavities, primarily secrete hyaluronic acid)

Serosa cells (line the peritoneal, pleural and pericardial cavity)

Squamous cells that line the perilymphatic space of the ear. Cells that line the endolymphatic space of the ear. Squamous cell highly prismatic epithelial cells of the endolymphatic sac with small villi without small villi "dark" cells

Reissner membrane cells (similar to choroid plexus cells)

vascular stria-basal cells vascular stria-von-ebner-crescent-cells

Claudius cells

Böttcher cells

Choroid plexus cells (secrete brain and spinal fluid)

Squamous cells of the soft brain and spinal cord skin

Ciliary epithelial cells of the eye cells containing pigment non-pigmented cells

Cells of the inner corneal epithelium

59

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Cilia-bearing cells with propulsive function of the respiratory tract of the fallopian tubes and the endometrium (in women)

of the Haller network and the duct efferens (in men)

of the central nervous system (ependymal cells that line the brain cavity)

Cells that specialize in the secretion of extracellular matrix

Epithelial cells:

Ameloblasts (secrete the tooth enamel)

Ebner crescent cells of the equilibrium organ of the ear (secrete proteoglycan)

Interdental cells of the Corti organ (secrete membrane tectoria, which covers the hair cells of the Corti organ)

Non-epithelial cells (connective tissue)

Fibroblasts (various fibroblasts of the loose connective tissue, the cornea, the tendons, the reticular connective tissue of the bone marrow, etc.)

Pericytes of the blood capillaries nucleus pulposus cells of the intervertebral disc

Cementoblasts / Cementocytes (secrete bone-like cementum of the tooth root)

Odontoblasts / Odontocytes (secrete the dentine)

Chondrocytes

Hyaline cartilage, fibrous cartilage, elastic cartilage osteoblasts / osteocytes

Osteoprogenitor cells (stem cells of the osteoblasts)

Hyalocytes of the vitreous body of the eye Star cells deg perilymphatic space of the ear

Contractive cells

Skeletal muscle cells red cells (slow)

white cells (fast)

intermediate cells

Muscle spindle core cells

Core chain cells of the muscle spindle

Satellite cells (stem cells)

Cardiac muscle cells normal cells

Node cells

Purkinje fiber cells

Smooth muscle cells

Myoepithelial cells of the iris of the exocrine glands

Blood and immune system cells Red blood cells Megakaryocytes Macrophages Monocytes

Connective tissue macrophages (various types)

Langerhans cells (in the epidermis)

Osteoclasts (in the bone)

dendritic cells (in the lymphatic tissue)

Microglial cells (in the central nervous system)

Neutrophils

Eosinophils

Basophils

Mast cells

T lymphocytes

T helper cells

T suppressor cells cytotoxic T cells

B lymphocytes

60

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Igm IgG IgA

igE

Killer cells

Stem cells for the blood and immune system (various types)

Sensory cells

Photoreceptor cells rod cells cone cells blue-sensitive cells green-sensitive cells red-sensitive cells cells of the hearing organ inner hair cell of the Corti organ outer hair cell of the Corti organ

Cells for the perception of acceleration and gravity Type I hair cells of the balance organ of the ear Type II hair cells of the balance organ of the ear Taste cells Type II taste bud cells Cells for the perception of smell olfactory neurons

Basal cells of the olfactory epithelium (stem cells for olfactory neurons)

Cells for determining the pH of the blood Carotid body cells Type I cells Type II cells

Cells for the perception of touches Merkel cells of the epidermis primary sensitive neurons, perception Cells for the perception of temperature primary sensitive neurons, perception for cold-sensitive cells Heat-sensitive cells Cells for the perception of pain primary sensitive neurons, perception Structures and forces in the musculoskeletal system proprioceptive primary sensitive neurons

Autonomous neurons, cholinergic neurons, adrenergic neurons, peptide-neurons

Cells that support the sensory organs and peripheral neurons

Supporting cells of the Corti organ inner pillar cells outer pillar cells inner phalangeal cells outer phalangeal cells border cells Hensen cells

Support cells of the balance organ

Support cells of the taste buds (type I taste bud cells)

Supporting cells of the olfactory epithelium

Schwann cells

Satellite cells (enclosing the peripheral nerve cells) intestinal glial cells

Neurons and glial cells of the central nervous system specialize in touch, specialize in temperature, specialize in pain

61

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Neurons glial cells astrocytes oligodendrocytes

Cells of the lens

Epithelial cells of the anterior lens Lenticular fiber cells (crystalline-containing cells)

Pigment cells

Melanocytes pigmented retinal epithelial cells

Germ cells

Oogonium, oocyte spermatocyte

Spermatogonium (stem cells for spermatocytes)

Nurse cells

Egg follicle cells

Sertoli cells (in the testicles)

Thymic epithelial cells

Stem cells embryonic stem cells embryonic germ cells adult stem cells fetal stem cells

IX. Diagnostic assays

A. Introduction

1) TRT assays

The present invention provides a large number of assays for TRT, preferably hTRT, and telomerase. These assays provide, among other things, the basis for sensitive, inexpensive, easy-to-use and widely applicable assays for the diagnosis and prognosis of a number of human diseases, including cancer as an illustrative example. As noted above, the amount of hTRT gene products (protein and mRNA) in immortal human cells is usually increased compared to most normal mortal cells (i.e., telomerase negative cells and most adult telomerase positive normal somatic cells). Thus, one aspect of the insertion is to provide assays for detection to determine the presence, absence or amount of an hTRT gene product in a sample so as to make the cells as immortal (e.g. a malignant tumor cell), mortal (as for example, most somatic cells in adults) to be able to characterize as telomerase positive or telomerase negative, the sample being derived from or containing human or other mammalian cells or eukaryotic cells.

Any condition characterized by the presence or absence of an hTRT gene product (i.e., protein or RNA) can be diagnosed using the methods and substances described herein. These include, as described in more detail below, cancer, other diseases associated with accelerated cell proliferation, immunological dysfunction, fertility, infertility, etc. Since the extent to which telomerase activity in cancer cells increases, with the characteristics of the tumor, e.g. Potential for metastasis formation, correlated, can be assessed and predicted by monitoring the hTRT, mRNA or protein levels, the likely future development of a tumor.

In one aspect, the diagnostic and prognostic methods according to the invention include determining whether a human TRT gene product is present in a biological sample (e.g. from a patient). A second aspect relates to the determination of the amount of an hTRT gene product in a biological sample (e.g. from a patient) and the comparison with the amount in a Kon62

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

troll test (e.g. normal cells or tissue). A third aspect relates to the determination of the cellular or intracellular localization of an hTRT gene product in a cell or tissue sample. A fourth aspect relates to the study of host cells (e.g. a patient) to identify nucleic acids with sequences that are characteristic of an inheritable tendency for abnormal hTRT gene expression (abnormal amount, regulation or abnormal product), which is useful genetic screening or genetic counseling. A fifth aspect relates to the use of the assays according to the invention for detecting the presence of anti-hTRT antibodies (e.g. in patient serum). The procedures described in more detail below are useful assays that can be performed using the sequences and relationships described herein. Numerous variations or other possible uses of these assays are of course obvious to the person skilled in the art.

Although the assays described above are described in connection with diagnostic and prognostic methods, they can always be used when an hTRT gene, gene product or a variant is to be detected, quantified or characterized. Thus, for example, the «diagnostic» methods described below for examining hTRT or telomerase during the production and purification of hTRT or human telomerase, for the characterization of cell lines derived from human cells (eg for the identification of immortal lines), for the characterization of Cells, animals, plants, fungi, bacteria, or other organisms that contain a human TRT gene or gene product (or fragments thereof) are useful.

The term "diagnostic" used here has its normal meaning, namely the identification of the presence or nature of the disease (e.g. cancer), a condition (e.g. sterile, activated), a status (e.g. fertile) and the term "prognostic" also has its usual meaning, namely the prognosis of the likely development and / or the outcome of a disease or condition. These two terms are sometimes used differently in a clinical setting. Any of the "diagnosis" assays or assay formats described below are equally suitable for making a prediction, since it is now clear that higher levels of telomerase activity are associated with a poorer prognosis for cancer patients, and because the present invention has detection methods provides that are specific for hTRT expressed at concentrations that closely correlate with telomerase activity in a cell.

2) Diagnosis and prognosis of cancer

The determination of an hTRT gene, mRNA or protein level which is above the normal or standard range indicates the presence of telomerase-positive cells or immortal cells, which include, for example, certain tumor cells. Since certain embryonic and fetal cells as well as certain "adult" stem cells express telomerase, the present invention also provides methods for determining other conditions such as e.g. Pregnancy, by detecting or isolating telomerase positive fetal cells from the mother's blood. These values can be used to make a diagnosis, or can be helpful in making a diagnosis, even if the cells could not have been classified as cancerous or otherwise detected or classified using traditional methods. Thus, the methods according to the invention allow the detection or verification of cancerous or other states associated with telomerase with increased reliability and possibly at an earlier stage. The assays according to the invention allow a distinction to be made between classes and stages of human tumors or other diseases associated with cell proliferation by providing quantitative assays for the hTRT gene and gene products and thus facilitating the selection of suitable treatment procedures and precise diagnoses. Because the level of telomerase activity can be used to distinguish between benign and malignant tumors (e.g., U.S. Patent No. 5,489,508 and Hiyama et al., Proc. Am Ass. Cancer Res. 38 (1997), 637 ) to predict the impending invasion (e.g., U.S. Patent No. 5,639,613 and Yashima et al., Proc. Am. Ass. Cancer Res. 38 (1997), 326) and to correlate with the potential for metastasis (e.g. US Pat. No. 5,648,215 and Pandita et al., Proc. Am Ass. Cancer Res. 37 (1996), 559), these assays are also used for the prophylaxis, detection and treatment of a large number of human cancers.

A prognostic value for an hTRT gene product (mRNA or protein) or the activity for a specific tumor type, tumor class or tumor stage, such as, is used for the prognosis of cancer diseases (or other diseases or conditions which are characterized by an increased telomerase concentration) described below. The level of hTRT protein or mRNA or telomerase activity in a patient can be determined, for example, using the assays described here and compared to the prognostic level.

Depending on the assay used, the frequency of an hTRT gene product in a sample will be considered increased in some cases whenever detectable by the assay

63

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

is. Due to the small amount of hTRT mRNA and protein even in telomerase-positive cells and the rarity or absence of these gene products in normal or telomerase-negative cells, sensitive assays are required in order to be able to prove whether the hTRT gene products are in normal cells at all are found. If less sensitive assays are selected, hTRT gene products will not be detectable in healthy tissue, but in telomerase-positive cancer or other telomerase-positive cells. Typically, the amount of an hTRT gene product in a sample with an increased concentration is at least about 5 times, more often at least about 10 times, more often at least 50 times and very often at least about 100 to 1000 times compared to the level of telomerase-negative control cells or Cells from healthy adult tissues, with the percentage of telomerase positive normal cells being very low.

The diagnostic and prognostic methods according to the invention can be used with any cell or tissue type of any origin and they can be used for the detection of an immortal or neoplastic cell, a tumor tissue or a cancer of any origin. The types of cancer that can be detected include, but are not limited to, all of those listed above for discussion of the therapeutic use of hTRT.

The assays according to the invention are also useful for monitoring the effectiveness of a therapeutic intervention in patients who are being treated for cancer. Treatments for cancer that can be monitored include all currently recommended forms of therapy (including chemotherapy, radiation therapy, and surgical measures), as well as future treatments, such as the therapies described here, that relate to the inhibition or activation of telomerase (see for example, PCT Publication Nos. 96/01 835 and 96/40 868 and U.S. Patent No. 5,583,016; and U.S. Patent Applications Serial No. 08/472 802 and 08/482 115, both of which issued on July 7, June 1995; 08/521 634, filed August 1, 1995; 08/714 482, filed September 16, 1996 and 08/770 564 and 08/770 565, both filed on December 20, 1996, wherein all of these documents may be considered part of this description based on the reference in their entirety).

In another aspect, the assays described below are useful for the detection of certain variations in the hTRT gene sequence (mutations and inheritable hTRT alleles) which indicate a predisposition to cancer or other conditions associated with abnormal regulation of telomerase activity are (infertility, premature aging).

3) Diagnosing conditions that are not cancer

In addition to the diagnosis of cancer, the assays according to the invention have numerous other possible uses. The present invention provides reagents and methods for diagnosing conditions or diseases which are characterized by under- or overexpression of telomerase or hTRT gene products in cells. In adults, a low level of telomerase activity is normally found in a limited type of normal human somatic cells, such as stem cells, activated lymphocytes and germ cells. Other somatic cells lack telomerase activity. Thus, the detection of hTRT or telomerase * activity in cells where they are normally absent or inactive, or the detection of an abnormal level (i.e. higher or lower compared to normal) in cells in which hTRT is normally low Level (such as stem cells, activated lymphocytes and germ cells) is a diagnostic sign of a telomerase-related disease or condition. This detection can also be used to identify or isolate specific cell types. Examples of such diseases and conditions include: diseases relating to cell proliferation, immunological functional disorders, infertility, diseases relating to immune cell function, pregnancy, fetal abnormality, premature aging etc. In addition, the assays according to the invention for monitoring the effectiveness of a therapeutic intervention ( including, but not limited to, agents that modulate telomerase activity) in a patient or in a cell or animal based assay. One aspect of the invention relates to the provision of assays useful for diagnosing infertility. Human germ cells (e.g. stem cells, progenitors or progeny) can proliferate indefinitely and are characterized by a high telomerase activity. Abnormal or decreased levels of hTRT gene products or abnormal products can lead to inadequate or abnormal sperm production, leading to infertility or reproductive dysfunction. The invention thus provides assays (methods and reagents) for the diagnosis and treatment of “telomerase-based” functional disorders with regard to production. In a similar application, the assays can be used to monitor the effectiveness of contraceptives (e.g. male or female contraceptives) that target or indirectly affect sperm production (and that reduce hTRT levels or telomerase activity) should).

64

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Another aspect of the invention relates to the provision of assays for analyzing the telomerase and hTRT levels and their function in stem cells, fetal cells, embryonic cells, activated lymphocytes and hematopoietic stem cells. For example, assays for the detection of hTRT gene products can be used to generally monitor immune function (e.g., to monitor the prevalence of activated lymphocytes or the accumulation of progenitor stem cells) to identify, select, or isolate activated lymphocytes or stem cells based on increased levels of hTRT, and to monitor the effectiveness of therapeutic interventions targeting these tissues (e.g., immunosuppressive agents or therapeutic efforts to increase a stem cell population).

The invention also provides assays that are useful for the identification of anti-telomerase and anti-TRT immunoglobulins (found in a patient's serum). The substances and assays described here can be used to identify patients in whom such autoimmune antibodies are found, which allows diagnosis and treatment of the condition associated with the immunoglobulins.

4) Monitoring cells in culture

The assays described here are also useful for monitoring the expression of hTRT gene products and the characterization of hTRT genes in cells ex vivo or in vitro. Since elevated hTRT levels are characteristic of immortalized cells, the assays according to the invention can be used, for example, to screen for immortalized cells or to identify them, or to identify an active ingredient that can mortalize immortalized cells again by inhibiting hTRT expression or function. For example, this assay is useful for the identification of cells that have been immortalized by increased expression of hTRT in the cell, e.g. expression of a recombinant hTRT, or increased expression of an endogenously encoded hTRT (e.g. by promoter activation).

Similarly, these assays can also be used to monitor hTRT expression in transgenic animals or cells (e.g., in yeast or human cells containing an hTRT gene). In particular, the effects of certain treatments (for example the administration of a known or suspected telomerase antagonist) with regard to the hTRT level in humans and non-human cells which express the hTRT according to the invention can be used to identify useful active substances and drug candidates (for example modulating telomerase activity Active ingredients) can be used.

B) Normal, diagnostic and prognostic values

Assays for the presence or amount of an hTRT gene product can be performed in a variety of ways and the results interpreted in a number of ways, depending on the assay format, the nature of the sample to be assayed, and the information desired. For example, the amount of hTRT gene products in most human somatic tissues in the steady state is so small that they cannot be detected by certain assays. In addition, there is generally no telomerase activity in these cells, so the verification of the activity is fairly easy. Conversely, there is a sufficient amount of hTRT protein and / or hTRT mRNA or telomerase in other telomerase-positive tissues, for example malignant tumors, so that these can be detected using the same assays. Even in those somatic cell types in which a low level of telomerase activity can normally be detected (e.g. stem cells and certain activated cells of the hematopoietic system), the level of hTRT mRNA and telomerase activity is very low (e.g. estimated at about 1% or less) compared to the level in immortal cells; immortal and mortal cells can thus be easily distinguished using the methods according to the invention. If a "less sensitive" assay is used, the mere detection of the hTRT gene product in a biological sample can be used for diagnosis itself, without the need for additional analysis. Although the assays described below can be endowed with an excellent sensitivity, they can also, if desired, be endowed with a lower sensitivity (for example by suitable selection of buffers, washing conditions, number of rounds of amplification, reagents and / or selection of signal amplifiers). . In principle, each assay can be designed in such a way that it only detects hTRT gene products in the biological samples in which they are present in a certain concentration, for example in a higher concentration than in healthy or another control tissue. In this case, any detectable level of hTRT mRNA or hTRT protein is considered to be elevated in cells from post-natal human somatic tissue (in contrast to hematopoietic cells and other stem cells).

In some cases, however, it may be desirable to establish normal levels or baseline (or ranges) for hTRT gene product expression levels, especially when using very sensitive assays that have a very low level of hTRT gene products found in normal

65

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

somatic cells can be present. Normal levels of expression or of normal expression products can be determined for any particular population, subpopulation or groups of organisms according to standard procedures well known to those skilled in the art. In general, basic levels (normal levels) of hTRT protein or hTRT mRNA are determined by quantifying the amount of hTRT protein and / or mRNA in biological samples (e.g. liquids, cells or tissues) that are obtained from normal (healthy) individuals ( e.g. a human). For certain samples and purposes, it may be desirable to quantify the amount of an hTRT gene product per cell or per tumor cell. In order to determine the cell amount of a sample, the level of a constitutively expressed gene product or of another gene product which is expressed in cells of the type from which the sample was taken can be determined with a known level. Alternatively, normal values of an hTRT protein or hTRT mRNA can be determined by determining the amount of hTRT protein / RNA in cells or tissues known to be healthy from the same patient from from which the diseased (or possibly diseased) cells were taken, or from a healthy individual.

Alternatively, in some cases, base levels can be defined as the level that is present in non-immortal human somatic cells in culture. It is possible that normal values (base values) differ somewhat between different cell types (e.g. the hTRT mRNA level in the testes is higher than in the kidney) or also according to the age, gender or physical condition of a patient. Thus, for example, if an assay is used to determine changes in hTRT levels associated with cancer, the cells used to determine the normal range of hTRT gene product expression may be from people of the same or different ages, depending according to the type of examination. The use of standard statistical methods used in molecular genetics allows the determination of base expression levels and of significant deviations from such base levels.

When carrying out the diagnostic and prognostic methods according to the invention as described above, it can sometimes be expedient to refer to “diagnostic” and “prognostic” values. The term "diagnostic value" as used herein refers to a value determined for the hTRT gene product detected in a sample and which, when compared to a normal range (or "base range") for the hTRT gene product, indicates the presence of a disease displays. This disease can be characterized by high telomerase activity (e.g. cancer), the lack of telomerase activity (e.g. infertility) or by an intermediate value.

The term "prognostic value" refers to an amount of the hTRT gene product that has been detected in a given cell type (e.g. a malignant tumor cell) and which is consistent with a specific diagnosis or prognosis for the disease (e.g. cancer). The amount of hTRT gene product detected in a sample (including zero amount) is compared to the prognostic value for the cell so that the relative comparison of the values indicates the presence of a disease or the outcome of the disease (e.g. cancer). In one embodiment, data are collected, for example, to carry out a tumor prognosis in order to obtain a statistically significant correlation to hTRT levels with different tumor classes or stages. A predetermined range of hTRT levels is established for the same cell sample or tissue sample derived from individuals with known clinical results. A sufficient number of measurements are carried out in order to determine a statistically significant value (or a range of values) with which a comparison is then carried out. The predetermined range of hTRT levels or hTRT activity for a given cell sample or tissue sample can then be used to determine a value or range for the level of hTRT gene products that has a favorable (or unfavorable) prognosis (eg, a "low level »In the case of cancer) would correlate. A region corresponding to a “high level” that correlates with one (or more) unfavorable prognosis (s) in the case of cancer can be determined in a similar way. The level of an hTRT gene product of a biological sample (e.g., a patient sample) can then be determined, compared to the low and high ranges, and used to predict a clinical course.

Although the discussion above relates to cancer as an illustration, diagnostic and prognostic values can also be determined for other diseases (eg diseases related to cell proliferation) and conditions, and for diseases or conditions that are not cancer, a « correlate a high »mirror with the desired curve and a« low »mirror with an unfavorable curve. For example, some diseases may be characterized by a deficiency (e.g. low level) of telomerase activity in stem cells, activated lymphocytes or germline cells. In such cases, "high" levels of hTRT gene products based on cells of the same age and / or of the same type (e.g. from other patients or other tissues in a particular patient) can correlate with a favorable course.

Of course, the assay methods do not necessarily require the determination of absolute values of hTnT, unless it is desired because relative values are sufficient for many applications of the methods according to the invention. In cases where quantification is desirable, the

66

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

present invention reagents ready so that virtually any known method for quantifying gene products can be used.

The assays according to the invention can also be used to assess the effectiveness of a specific therapeutic treatment procedure in animal studies, in clinical tests or in monitoring the treatment of individual patients. In these cases, it may be desirable to establish the baseline level for the patient prior to initiating therapy and to repeat the assays once or more throughout the course of treatment, usually at regular intervals to check if the hTRT level is up a desired endpoint (e.g., decreased TRT expression if it is an assay for cancer) as a result of the treatment.

It is obvious to the person skilled in the art that, in addition to the quantity or frequency of hTRT gene products, different or abnormal expression patterns (for example abnormal amounts of RNA splice variants) or abnormal expression products or expression product variants, for example mutated transcripts, are shortened or «non-sense »-Polypeptides) can be identified by comparison with normal expression levels and normal expression products. In these cases, the determination of "normal" or "basic" includes the identification of healthy organisms and / or tissues (ie organisms and / or tissues without dysregulation of hTRT expression or neoplastic growth) and the determination of the expression level of hTRT gene product variants ( splice variants) or the sequencing or detection of the hTRT gene, mRNA or reverse transcribed cDNA in order to obtain or determine typical (normal) sequence variants. The use of statistics and standard methods used in molecular genetics allows significant deviations from such base levels to be determined.

C) Detection and quantification of TRT gene products

As emphasized here, hTRT gene products are usually found in most normal somatic cells with an extremely low level. For example, the mRNA encoding hTRT protein is extremely rare or is absent in all telomerase-negative cell types examined to date. In immortal cells, for example 293 cells, hTRT mRNA can only be present in a number of approximately 100 copies per cell, whereas normal somatic cells have only one or no copy per cell. It is thus apparent that it will sometimes be advantageous to include signal or target amplification methods in the as-say format when highly sensitive assays for hTRT gene products are desired. See, for example, Plenat et al., Ann. Pathol. 17 (1997), 17 (fluoresceinyl tyramide signal amplification); Zehbe et al., J. Pathol. 150 (1997), 1553 ("catalyzed reporter de-position"); further references listed here (e.g. for bDNA signal amplification, for PCR and other target amplification formats), as well as other methods known in the art.

As already noted above, it is often not necessary to quantify the hTRT mRNA or the hTRT protein in the assays described here, since the detection of an hTRT gene product (under assay conditions in which the product is in a control, for example Telomerase negative cells) is undetectable, in itself sufficient for a diagnosis. In another example, quantification may be superfluous, namely if the product levels found in a test sample (e.g. tumor) and control sample (healthy cells) are compared directly.

If desired, however, the amounts of hTRT gene product measured in the assays described here can be described in various ways, depending on the measurement method and the expediency. Thus, normal, diagnostic, prognostic, high or low amounts of hTRT protein / mRNA can be expressed as standard weight units per amount of a biological sample (e.g. picogram per gram of tissue, picogram per 1012 cells) as a number of molecules per amount of a biological Sample (eg transcripts / cell, mole / cell), as units of activity per cell or per amount of another unit or by similar methods. The amount of an hTRT gene product can also be expressed in relation to the amount of another molecule. Examples include the number of hTRT transcripts in a sample / the number of 28S rRNA transcripts in a sample; Nanogram hTRT protein / nanogram total protein, etc.

When hTRT gene products are determined in two (or more) different samples, it is sometimes useful to have a common basis of comparison for the two samples. For example, when comparing a sample with normal tissue and a sample with cancer tissue, equal amounts of tissue (in terms of weight, volume, number of cells, etc.) can be compared. In an alternative embodiment, equivalents of a marker molecule (e.g. 28S RNA, hTR, telomerase activity, telomer length, actin) can be used. For example, the amount of hTRT protein in a healthy tissue sample containing 10 picograms of 28S rRNA can be compared to a diseased tissue sample containing the same amount of 28S rRNA.

It will be apparent to those skilled in the art that virtually any of the assays described herein can be designed as a quantitative assay. Typically, a known amount or source of an hTRT gene product (e.g., made using the methods and compositions of the invention) can be used to calibrate the assay.

In certain embodiments, assay formats are selected that reflect the presence,

67

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

or the frequency of an hTRT allele or gene product in each cell in a sample (or in a representative sample). Examples of such assay formats include those with which a signal can be detected using histology (e.g. immunohistochemistry with signal amplifiers or target-amplifying amplification steps), fluorescence-activated cell analysis or cell sorting (FACS). These assay formats are particularly advantageous when dealing with a highly heterogeneous cell population (e.g. a cell population that contains many different cell types in which only one cell type or only a few elevated hTRT levels occur, or a population of similar cells that Express telomerase with different levels).

D) Sampling

The hTRT gene or gene product (i.e. mRNA or polypeptide) is preferably detected and / or quantified in a biological sample. Such samples include, but are not limited to, cells (including whole cells, cell fractions, cell extracts and cultured cells or cell lines), tissues (including blood), blood cells (e.g. white blood cells), tissue samples, e.g. fine needle biopsy samples (e.g. from the prostate, breast , Thyroid gland, etc.), body fluids (e.g. urine, sputum, amniotic fluid, blood, peritoneal fluid, pleural fluid, semen) or cells derived therefrom (e.g. bladder cells from urine, lymphocytes from blood), media (from cultured cells or cell lines) and washing liquids (e.g. from the bladder and lungs). The biological samples can also include tissue sections, for example frozen sections, which were taken for histological purposes. For the diagnosis and prognosis of cancer, a sample is taken from a cancerous, procancerous or presumably cancerous tissue or tumor. It may occasionally be desirable to freeze a biological sample for later analysis (e.g. when monitoring the effectiveness of drug treatments).

In some cases, the cells or tissues can be fractionated before analysis. For example, in a tissue biopsy from a patient, a cell sorter (e.g., a fluorescence activated cell sorter) can be used to sort cells by features such as expression of a surface antigen (e.g., a tumor specific antigen) according to well known methods.

Although the sample is usually taken from a human patient or cell line, the assays can also be used to detect homologous hTRT genes or corresponding gene products in animal samples. Alternatively, hTRT genes and gene products can be examined in transgenic animals or organisms which express a human TRT protein or corresponding nucleic acid sequence.

If necessary, the sample can be pretreated by dilution in a suitable buffer solution or concentrated if desired. A number of standard aqueous buffer solutions at physiological pH can be used, whereby a number of buffers can be used, for example phosphate, Tris buffer etc.

The term "biological sample" obtained from a patient can be referred to as either "biological sample" or "patient sample". When analyzing a “patient sample”, cells or tissues do not necessarily have to be removed from the patient. For example, appropriately labeled hTRT-binding agents (e.g. antibodies or nucleic acids) can be injected into the patient and visualized (after binding to the target) using standard imaging techniques (e.g. CAT, NMR etc.).

E) Nucleic acid assays

In one embodiment, the invention provides methods for detecting and / or quantifying the expression of hTRT mRNAs (including splice variants or sequence variants and alternative alleles). In an alternative embodiment, the invention provides methods for the detection and analysis of normal or abnormal hTRT genes (or a fragment thereof). The formats of such qualitative or quantitative assays include, but are not limited to, amplification-based assays with or without signal amplification, hybridization-based assays, and amplification / hybridization-based assays. It will be apparent to those skilled in the art that the distinction between hybridization and amplification is made for convenience only: As illustrated in the examples below, many assay formats contain elements of hybridization as well as amplification, so in some cases the categorization is somewhat arbitrary.

1) Production of nucleic acids

In some embodiments, nucleic acid assays are performed with a nucleic acid sample isolated from the cell or cell line or tissue or organism to be tested. The nucleic acid (e.g. genomic DNA, RNA or cDNA) can be "isolated" from a sample according to a number of methods known to those skilled in the art. Related in this context

68

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

the term "isolates" from any separation of the species or target to be detected from any other substance in the mixture, but does not necessarily indicate a substantial degree of purity of the target. It will be apparent to those skilled in the art that when changes in the number of copies of the hTRT gene are detected, the target to be detected is genomic DNA. Conversely, the target to be detected in a nucleic acid-based assay is RNA, if expression levels of a gene or of genes are to be detected. In a preferred embodiment, the nucleic acid sample is total mRNA (i.e. poly (A) + RNA) in a biological sample. Methods for isolating nucleic acids are well known to those skilled in the art and are described, for example, in Tijssen, P. (ed.) In Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, Elsevier, N.Y. (1993), Chapter 3, which document is to be considered part of this description by reference. In one embodiment, total nucleic acid is isolated from a sample using an acidic guanidine / phenol / chloroform extraction method and poly (A) + mRNA by oligo-dT column chromatography or by using magnetic (dT) n beads (see e.g. Sambrook et al., and Ausubel et al., loc. cit.).

In alternative embodiments, isolation of nucleic acids (e.g. total RNA or poly-A + RNA) from the biological sample is not required prior to performing amplification, hybridization, or other assays. These embodiments have certain advantages in the event that hTRT-RNA is to be measured because they reduce the likelihood that hTRT-mRNA will be lost during isolation and handling. For example, many amplification methods such as PCR and RT-PCR (reverse transcriptase-PCR) can be carried out using permeabilized cells (histological samples and FACS analyzes) lysed whole cells or high cell fractions, such as certain cell extracts. Preferably steps are taken to preserve the integrity of the target nucleic acid (e.g. mRNA) if necessary (e.g. by adding RNAase inhibitors). Amplification and hybridization assays can also be carried out in situ, for example in thin tissue sections of a biopsy sample or a single-layer cell layer (e.g. blood cells or separate cells of a tissue culture). The amplification can also be carried out in intact whole cells or fixed cells. For example, PCR, RT-PCR, or LCR amplification methods, as is known in the art, can be performed in situ, for example using a polymerase or ligase, a primer or primer and (deoxy) ribonucleoside triphosphates (when using a polymerase) and reverse transcriptase and a primer (when transcribing RNA and detecting the cDNA) on fixed, permeabilized or micro-injected cells so as to amplify the target hTRT RNA or DNA. Cells containing hTRT-RNA (e.g. telomerase positive cells) or a desired hTRT-DNA sequence can then be detected. This method is often useful when fluorescence-labeled dNTPs, primers or other components are used in connection with microscopy, FACS analysis or equivalent methods.

2) Assays based on amplification

In one embodiment, the assays according to the invention for the detection of an hTRT gene or gene product are assays based on amplification. In an assay based on amplification, an entire hTRT gene or transcript or a part thereof (e.g. mRNA or cDNA, hereinafter referred to as “target”) is amplified and the amplified product is then directly or indirectly detected. If there is no gene or gene product that can act as a template, no amplification product is produced (e.g. with the expected size) or the amplification is unspecific and usually no single amplification product is produced. In contrast, the target sequence is amplified when the gene or gene product in question is present, thus indicating the presence and / or the amount of the corresponding gene or mRNA. Assays based on amplification are well known to those skilled in the art.

The present invention provides a wide variety of primers and probes for the detection of hTRT genes and gene products. Such primers and samples are sufficiently complementary to the hTRT gene or gene product to be able to hybridize to the target nucleic acid. Primers are usually at least 6 bases in length, preferably from about 10 to about 100 bases, more preferably from about 12 to about 50 bases, and most preferably between about 14 and about 25 bases. After reading this description, the person skilled in the art will be able, using routine methods, to select primers with which the entire hTRT gene or gene product or a portion thereof can be amplified or with which a distinction can be made between gene product variants, hTRT alleles, etc. can be. Table 2 lists examples of primers that are useful for PCR amplification of the hTRT or specific hTRT gene products or regions. It is known in the art that single oligomers (see, for example, U.S. Patent No. 5,545,522), nested sets of oligomers, or even a pool of degenerate oligomers, can be used for amplification, for example, as described below Amplification of the Tetrahymena TRT cDNA is demonstrated.

The invention provides a variety of methods for the amplification and detection of an hTRT

69

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Gene or gene product. These include the polymerase chain reaction (including all variants, for example reverse transcriptase PCR; the “Sunrise Amplification System” (Oncor, Inc., Gaithersburg MD) and numerous other methods known in the art. In an illustrative embodiment, the PCR -Amplification carried out in a solution of 50 nl, which contains the nucleic acid sample (eg a cDNA obtained by reverse transcription of hTRT-RNA), each 100 nM dNTP (dATP, dCTP, dGTP and dTTP; Pharmacia LKB Biotechnology, NJ) (die) hTRT-specific PCR primer, 1 unit Taq polymerase (Perkin Elmer, Norwalk CT), 1 x PCR buffer (50 mM KCl, 10 mM Tris, pH 8.3 at room temperature, 1, 5mM M9Cb and 0.01% gelatin), amplification being performed with about 30 cycles, 45 seconds each at 94 ° C, 45 seconds at 45 ° C and 90 seconds at 72 ° C. However, it can of course be used for certain reactions numerous variants for optimizing the PCR amplification ations are introduced.

Other suitable target amplification methods include ligase chain reaction (LCR) (see, for example, Wu and Wallace, Genomics 4 (1989), 560; Landegren et al., Science 241 (1988), 1077, Barany, Proc. Nati. Acad. Sci. USA 88 (1991), 189 and Barringer et al., Gene 89 (1990), 117), strand displacement amplification (SDA) (see, for example, Walter et al., Proc. Nati. Acad. Sci. USA 89 (1992) 392 -396); transcription amplification (see, for example, Kwoh et al., Proc. Nati. Acad. Sei. USA 86 (1989), 1173), "self-sustained-sequence" replication (3SR) (see, for example, Fahy et al., PCR Methods Appi. 1 (1992) 2 and Guatelli et al., Proc. Nat. Acad. Sci. USA 87 (1990) 1874); nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario; see e.g. Compton, Nature 350 (1991) 91); the transcription-based amplification system (TAS); and the “self-sustained-sequence” replication system (SSR). Each of the above publications is to be regarded as part of this description by reference. A convenient PCR variant is the PCR-ELISA (e.g. Boehringer Mannheim Cat. No. 1 636 111), whereby digoxigenin-dUTP is incorporated into the PCR product. The PCR reaction mixture is denatured and hybridized with a biotin-labeled oligonucleotide designed to bind to an internal sequence of the PCR product. The hybridization products are immobilized on streptavid in coated supports and detected using anti-digoxigenin antibodies. Examples of in vitro amplification methods that give the person skilled in the art sufficient guidance can be found in the following publications: PCR Technology: Principles and Applications for DNA Amplification, H. Erlich (ed.) Freeman Press, New York, NY (1992); PCR Protocols: A Guide to Methods and Applications, Ed. Innis, Gelfland, Snisky, and White, Academic Press, San Diego, CA (1990); Mattila et al., Nucleic Acids Res. 19 (1991), 4967; Eckert and Kunkel, PCR Methods and Applications 1 (1991), 17; PCR, ed. McPherson, Quirkes and Taylor, IRL Press, Oxford; U.S. Patent Nos. 4,683,195, 4,683,202 and 4,965,188; Barringer et al., 1990, Gene 89: 117; Kwoh et al., Proc. Nati. Acad. Be. USA 86: 1173; Guatelli et al., Proc. Nati. Acad. Be. USA 87 (1990) 1874; Lomell et al., J. Clin. Chem. 35 (1989), 1826. Each of these documents is to be considered part of this description by reference.

Amplified products can be analyzed directly (e.g. based on the size determined by gel electrophoresis), by hybridization to a target nucleic acid immobilized on a solid support, e.g. a bead, membrane, disc or chip; by sequencing; immunologically (e.g. by PCR-ELISA), by detection of a fluorescent phosphorescent or radioactive signal or by a number of other generally known measures. In an illustrative example of a detection method, PCR primers are used, for example, which were enlarged with the aid of hairpin loops, which are linked to fluorescein and a benzoic acid derivative, which serves as a quenching compound, so that only then Fluorescence is emitted when the primers unfold to bind their targets and replication takes place.

Since hTRT mRNA is usually expressed as an extremely rare transcript that is only present in very low concentrations even in telomerase positive cells, it is often desirable to optimize or amplify the signal obtained by an amplification step. One way to do this is to increase the number of amplification cycles. For example, although 20 to 25 cycles are sufficient for the amplification of most mRNAs by means of the polymerase chain reaction under standard reaction conditions, the detection of hTRT-mRNA can require 30 to 35 amplification cycles in many samples, depending on the detection format. By carefully selecting the amplification condition, including the number of amplification cycles, an assay can be designed that only leads to an amplification product if a threshold amount of the target is present in the test sample (ie, so that only samples with a high level of hTRT mRNA are " positive »show result). In addition, methods are known for amplifying the signal generated by amplifying the target sequence. Methods for improving the ability to detect an amplified target include signal amplification systems, for example: signal amplification with branched DNA "branched DNA signal amplification" (see, for example, US Pat. No. 5,124,246 and Urdea, Bio / Tech. 12 (1994) , 926); the «tyramide» signal amplification system (TSA, Du-Pont); Amplification with a catalytic signal ("catalytic signal amplification" (CSA) (Dako); Q beta replicase systems (Tyagi et al., Proc. Nat. Acad. Sci. USA 93 (1996), 5395) etc.

It will be apparent to those skilled in the art that regardless of the amplification method used, a variety of quantitative methods known in the art for quantification are used

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

can be used if desired. For example, if desired, two or more polynucleotides can be amplified together in a single sample. This method can be used as a convenient method for quantifying the amount of hTRT mRNA in a sample since both reverse transcription and amplification reactions are carried out in the same reaction mixture for a target and control polynucleotide. Common amplification with the control polynucleotide (which is normally present in a known concentration or number of copies) can be used to normalize the number of cells in the sample compared to the amount of hTRT in the sample. Suitable control polynucleotides for co-amplification reactions include DNA expressed from housekeeping genes, constitutively expressed genes and RNAs or DNAs synthesized in vitro that are added to the reaction mixture. Endogenous control polynucleotides include those that are already present in the sample, while exogenous control polynucleotides are added to a sample, creating a "spiked" reaction. Examples of control RNAs include β-actin RNA, GAPDH RNA, snRNAs, hTR and endogenously expressed 28S rRNA (see Khan et al., Neuro. Lett. 147 (1992), 114). Exogenous control polynucleotides include a synthetic AW106 cRNA that can be synthesized by pAW 106 as a “sense” strand using T7 polymerase In order for the co-amplification method to be used for quantification, both the control nucleotides and the target polynucleotides must be used Usually amplified in a linear region. Detailed protocols for quantitative PCR can be found in PCR Protocols, A Guide to Methods and Applications, Innis et al., Academic Press, Inc. NY (1990) and Ausubel et al., loc 15) and Diaco, R. (1995) Practical Considérations for the Design of Quantitative PCR Assays, in PCR Stratégies, pp. 84-108, Innis et al. Ed. Academic Press, New York.

Depending on the sequence of the endogenous or exogenous standard, different primer sets can be used for the coamplification reaction. In one method, so-called competitive amplification, quantitative PCR involves co-amplification with a known amount of a control sequence, using the same primers used for the target nucleic acid (a pair with two primers). In an alternative embodiment, known as non-competitive amplification, the control sequence and the target sequence (e.g. hTRT cDNA) are amplified using different primers (i.e. 2 pairs of 2 primers). In a further alternative embodiment, the so-called semi-competitive amplification, three primers are used, one being hTRT-specific, one control-specific and one being able to attach to both target sequences and control sequences. Semi-competitive amplification is described in U.S. Patent No. 5,629,154, which is hereby incorporated by reference into this specification.

3) Assays based on hybridization a) General

A large number of methods for the specific measurement of DNA and RNA using nucleic acid hybridization methods are known to the person skilled in the art (see Sambrook et al., Cited above). Assays based on hybridization relate to assays in which a nucleic acid probe is hybridized to a target nucleic acid. The nucleic acid hybridization probes according to the invention are usually completely or essentially identical to a coherent sequence of the hTRT gene or the corresponding RNA sequence. Preferably, the nucleic acid probes are at least about 10 bases long, more preferably at least about 20 bases, and most preferably about 200 bases or longer. Methods for selecting nucleic acid probe sequences for use in nucleic acid hybridization are described in Sambrook et al., Supra. discussed. In some formats, either the target or the probe or both are immobilized. The immobilized nucleic acid can be a DNA, RNA or another oligonucleotide or polynucleotide. These can include natural or non-naturally occurring nucleotides, nucleotide analogs or backbones. Such assays can be used in various formats, for example: Southern, Northern "dot" and "slot" blots, high-density polynucleotide or oligonucleotide arrays (e.g. GeneChips \ Affymetrix), measuring sticks, measuring pins ("pins"), chips or Globules. All of these methods are well known in the art and are the basis of many commercially available diagnostic kits. Hybridization techniques are generally described in Harnes et al., Eds. Nucleic Acid Hybridization, A Practical Approach IRL Press (1985); Gali and Pardue, Proc. Nati. Acad. Be. USA, 63 (1969), 378-383 and John et al., Nature, 223 (1969) 582-587.

A variety of formats for nucleic acid hybridization are known to those skilled in the art. For example, direct hybridization is a common format in which a target nucleic acid is hybridized with a labeled complementary probe. Labeled nucleic acids are usually used for hybridization, the label providing the detectable signal. One method of assessing the presence, absence, or amount of hTRT mRNA is to perform Northern transfer of RNA from a sample and hybridize with a labeled hTRT-specific nucleic acid probe, as illustrated in Example 2. As already mentioned

71

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Note that, if at all, hTRT mRNA is only present in very small amounts in most cells. Thus, when using Northern hybridization, it is often desirable to use an amplification step or, alternatively, large amounts of starting RNA. A convenient method for assessing the presence, absence, or amount of DNA encoding hTRT proteins in a sample involves Southern transfer of DNA from a sample and hybridization with a labeled hTRT-specific nucleic acid probe.

Other common hybridization formats include "sandwich" assays and competition or displacement assays. The "sandwich" assays are commercially available, convenient hybridization assays for the detection and isolation of nucleic acid sequences. In such assays, a "capture" nucleic acid covalently immobilized on a solid support and a labeled "signal" nucleic acid in solution are used. The biological or clinical sample provides the target nucleic acid. The "capture" nucleic acid and the "signal" nucleic acid probes hybridize to the target nucleic acid, forming a "sandwich" hybridization complex. Efficacy requires that the signal nucleic acid can hybridize with the "capture" nucleic acid.

b) Assays based on a chip and on a disk

The present invention also provides probe-based hybridization assays for hTRT gene products using arrays of immobilized oligonucleotides or oligonucleotides to which an hTRT nucleic acid can hybridize (ie, to some, but not usually all, or not even all of them) most of the immobilized oligo- or polynucleotides). High-density oligonucleotide arrays or polynucleotide arrays are a means for the efficient detection of the presence and characteristics (eg the sequence) of a target nucleic acid (eg an hTRT gene, mRNA or cDNA). Techniques are known with which can produce arrays which contain thousands of oligonucleotides complementary to defined sequences at defined positions on a surface, using photolithographic methods for the synthesis in situ (see, for example, US Pat. Nos. 5,578,832, 5,556,752 and 5,510 270; F odor et al., Science 251 (1991), 767; Pease et al., Proc. Nati. Acad. Sci. USA 91 (1994), 5022 and Lockhart et al., Nature Biotech 14 (1996); 1675 ) or other methods for the rapid synthesis and attachment of defined oligonucleotides (Blanchard et al., Biosensors & Bioelectronics 11 (1996), 687). Using these methods, oligonucleotides (e.g. 20-mers) with a known sequence are synthesized directly on a surface, for example on a disk with derivatized glass. Typically, the array produced is redundant with some on-chip oligonucleotide probes specific for the TRT polynucleotide to be detected.

Combinations of oligonucleotide probes can be designed so that alternatively spliced mRNAs can be detected or to determine which of different hTRT alleles is expressed in a particular sample.

In an illustrative embodiment, reverse transcription of total RNA from a test cell prepares cDNA (e.g., using PCR). The amplified product is normally labeled, for example by incorporating a fluorescence-labeled dNTP. The labeled cDNAs are then hybridized to a chip that includes oligonucleotide probes that are complementary to various sub-sequences of the hTRT gene. The hybridization positions are determined (for example according to the general methods of Shalon et al., Genome Research 6 (1996), 639 or Schena et al., Genome Res. 6 (1996) 639) and the sequence (or other information) from the hybridization pattern ) derived by methods well known in the art.

In one embodiment, two cDNA samples are hybridized to the same chip, each labeled with a different fluorescent group. The ratio of hybridization of each labeled sample to sites complementary to the hTRT gene is then examined. If both samples contain the same amount of hTRT mRNA, the ratio of the two fluorescence signals is 1: 1. The signal of the fluorescence does not have to be set so that any difference in the molar sensitivity of the fluorescence signals is taken into account.

In contrast, if the first sample is from healthy (or control) tissue and the second sample from cancerous tissue, the fluorescence signal used in the second sample will prevail.

c) In situ hybridization

An alternative method for detecting the expression of a gene encoding an hTRT protein is in situ hybridization. In situ hybridization assays are well known and generally described in Angerer et al., Methods Enzymol. 152: 649-660 (1987) and Ausubel et al., Op. Cit. In an in situ hybridization assay, cells or tissue samples are fixed to a solid support that is typically in a permeabilized state, typically on a glass sheet. The cells are then hybridized with a hybridization solution at a moderate temperature to allow the attachment of labeled nucleic acid probes (e.g. 35S-labeled riboprobes, fluorescence72

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

labeled probes) that are complementary or substantially complementary to hTRT. Free probe is removed by washing and / or cleaving with nuclease and the bound probe is visualized directly on the disc by autoradiography or an appropriate imaging technique as is known in the art.

4) Specific detection of variants

As noted above and illustrated in the examples (e.g., Example 9), amplification primers or probes can be selected to provide amplification products that span specific deletions, truncations, and insertions, thereby detecting specific variants or abnormalities in the hTRT -mRNA is facilitated. An example of a gene product of an hTRT variant that can be detected is an hTRT-RNA, such as a product described above and in Example 9 (SEQ. ID. No. 4). The biological function of the 182 variant (s) is not known, if one is available at all. The shortened hTRT protein presumably encoded by the variant could, however, be involved in regulating telomerase activity, for example by assembling a nonfunctional telomerase RNP which titrates telomerase components. Alternatively, negative regulation of telomerase activity could be achieved by controlling the hTRT pre-mRNA (nascent mRNA) in a manner that leads to elimination of the mRNA and a reduction in the hTRT mRNA level. For these and other reasons, it is useful to be able to discover 182 variants. In addition, it may occasionally be desirable in samples in which two species of hTRT-RNA are present (eg, a 182-hTRT-RNA and an hTRT-RNA encoding the full-length hTRT protein), their relative and / or absolute frequency to compare.

The invention provides a variety of methods for the detection of 182 variants. For example, amplification using primer pairs spanning the 182 base pair deletion results in products of different sizes which correspond to the deleted and undeleted hTRT-RNAs (if both are present) and these can be distinguished on the basis of their size (for example by gel electrophoresis) . Examples of primer pairs useful for amplifying the region spanning the 182 bp deletion include TCP.14 and TCP1.15 (primer set 1) or TCP1.25 and bTCP6 (primer set 2) (see Table 2) . These primer pairs can be used separately or in a PCR experiment with «nested» primers, whereby primer set 1 is used first. It is also obvious to the person skilled in the art that hybridization methods (e.g. Northern hybridization) or RNAse protection assays for the detection of hTRT-RNA variants or in order to be able to differentiate between such variants can be used using the hTRT nucleic acid probe according to the invention.

Another suitable method involves PCR amplification (or an equivalent method) with three primers. A primer is specific to each hTRT-RNA species (e.g. as illustrated in Table 4) and a primer is complementary to both species (e.g. TCP1.25 (2270-2288)) - analogous to the semi-competitive quantitative PCR method, which is described in more detail above. An example of a primer that is specific to SEQ. ID No. 1 is a primer that attaches within the sequence of 182 nucleotides (i.e. nucleotides 2345 to 2526 of SEQ. ID. No. 1), e.g. TCP1.73 (2465-2445). A primer that goes to SEQ. ID. No. 4 (an Al82 variant) is, for example, a primer attached to nucleotides 2358-2339 of SEQ. ID. No. 4 (i.e. the site corresponding to the insertion of 182 nucleotides in SEQ. ID. No. 1). The absolute abundance of the Al82-hTRT mRNA species or its relative abundance compared to the species encoding the full-length hTRT protein can be analyzed for correlation with cell status (eg, ability to proliferate indefinitely) . Numerous other primers can be selected based on the present description.

73

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

TABLE 4

PRIMER EXAMPLES

182 species (e.g. SEQ. ID. No. 4) specific primer: 5'-GGCACTGGACGTAGGACGTG-3 hTRT (SEQ. ID. No. 1) specific primer (TCPl.73):

5'-CACTGCTGGCCTCATTCAGGG-3 common (forward) primer (TCPl.25):

5'-TACTGCGTGCGTCGGTATG-3

The further hTRT genes of variants or the corresponding gene products which can be detected include those which are characterized by codons which lead to an early stop, deletions, substitutions or insertions. Deletions can be detected based on the reduced size of the gene, the mRNA transcript or the cDNA. In a similar manner, insertions can be detected based on the increased size of the gene, the mRNA transcript or the cDNA. Insertions and deletions can also cause a shift in the reading frame, which leads to the creation of codons for an early stop or longer open reading frames. Substitutions can be detected by hybridization with a probe. These changes are demonstrated by examining changes in the size of the hTRT polypeptide variant (e.g. by Western analysis) or by hybridization or specific amplification, depending on what appears appropriate. Alternatively, mutations can be determined by sequencing the gene or gene product according to standard procedures. In addition, and as noted above, amplification assays and hybridization probes can be selected to target specific abnormalities. For example, nucleic acid probes or amplification primers can be selected that hybridize specifically to the region that contains or specifically amplifies the deletion, substitution or insertion. Where the hTRT gene contains such a mutation (1) the probe either does not hybridize, or the amplification reaction does not provide specific amplification or will result in a change in the size of the amplification product or the hybridization signal; or (2) the probe or amplification reaction comprises the entire deletion or both ends of the deletion (deletion connection point) or (3) similarly probes and amplification primers can be selected which specifically target point mutations or insertions.

5) Detection of HTRT mutant alleles

Mutations in the hTRT gene could be responsible for the development of a disease or could contribute to a disease state. Changes in the genomic DNA of hTRT could affect the level of gene transcription, lead to changes in amino acid residues in the hTRT protein, lead to the production of truncated hTRT polypeptides, which change the processing routes of the pre-mRNA (which the hTRT-mRNA- Mirror can change) and also have further consequences.

Changes in genomic DNA in non-hTRT loci can also affect expression of hTRT or telomerase by altering the enzymes or cellular processes responsible for regulating expression of hTRT, hTR and telomerase-associated proteins, as well as processing and RNP assembly and transportation. Changes affecting hTRT expression, processing or RNP assembly could also be important for the course of cancer, for old-age diseases, for diseases related to DNA damage, etc.

The detection of mutations in hTRT mRNA or the corresponding gene and gene control elements can be carried out in a variety of ways in accordance with the methods described here. These include the following illustrative examples: a method called primer screening can be used; PCR primers are designed whose 3 'ends attach to nucleotides in a sample DNA (or RNA) that may be mutated. If the DNA (or RNA) is amplified by the primers, the 3 'ends will pair with the nucleotides in the gene, if the DNA is not amplified then either or both ends will not pair with the nucleotides in the gene, indicating the presence of one Mutation indicates. Similarly, primers can be designed to target point mutations using the ligase chain reaction (LCR, supra

74

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

described) can be demonstrated. Another technique that can be used in the present method is based on restriction fragment length polymorphism, RFLP (Pourzand, C., Cerutti, P. Mutat. Res. 288 (1993), 113-121). A Southern blot of human genomic DNA, which was cleaved with various restriction enzymes, is hybridized with an hTRT-specific probe. Differences in the number or size of fragments between the sample and a control indicate a change in the experimental sample, usually an insertion or deletion. Single strand conformation polymorphism, SSCP, (Orrita, M. et. Al., PNAS USA 86 (1989), 2766-70) is another technique that can be used for the present method. SSCP is based on the different migration of denatured single-stranded wild-type or mutant DNAs (which were normally generated by PCR). Single-stranded DNA adopts a three-dimensional conformation that is sequence-specific. Sequence differences that only affect a single base can also lead to a mobility shift in a non-denaturing gel. Because of its simplicity, SSCP is one of the most commonly used methods for mutation screening. Another technique that can be used in the present method is denaturing gradient gel electrophoresis, DGGE (Myers et al., Methods in Enzymology 155 (1987), 501-527). Using DGGE, mutations can be identified based on the melting behavior of double-stranded DNA. Special equipment for denaturing electrophoresis is used to analyze the melting profile of experimental DNA and control DNA: A DNA containing a mutation has a different mobility in these gel systems compared to the control. The examples discussed illustrate common methods, and there are numerous other methods known to those skilled in the art.

F. Analysis of the karyotype

The present invention further provides methods and reagents for karyotype analysis or other chromosome analysis using hTRT sequence probes and / or the detection or localization of hTRT gene sequences in chromosomes of a patient, human cell line or non-human cell . In one embodiment, the amplification (ie, a change in the number of copies), deletion (ie, partial deletion), insertion, substitution, or changes in the chromosomal localization (eg translocation) of an hTRT gene with the presence of a pathological condition or a predisposition for it Development of a pathological condition (e.g. cancer) must be correlated.

It was found in the present invention that in normal human cells the hTRT gene was mapped near the telomer of chromosome 5p (see Example 5 below). The closest neighboring STS marker is D5S678. Its location can be used to identify markers that are closely linked to the hTRT gene. These markers can be used to identify YACs, STSs, cosmids, BACs, lambda phages or P1 phages, or other clones that contain hTRT genomic sequences or controls. The marker or location of the gene can be used to examine human tissue samples for changes in the normal location of the hTRT gene, its organization, or sequence associated with the occurrence of a cancer type or disease. This information can then be used to diagnose or predict the disease or cancer involved. In addition, the nature of any change in the hTRT gene can provide information about the causes by which cells become immortal. For example, a translocation event could indicate that activation of hTRT expression occurs in some cases by replacing the hTRT promoter with another promoter that controls hTRT transcription in an unfavorable manner. Methods and reagents of this type according to the invention can be used to develop strategies with which hTRT activation processes can be combated. The location is also useful for determining the nature of hTRT gene repression in normal somatic cells, for example when examining whether the location is within a portion of non-expressing heterochromatin. Assays based on nuclease hypersensitivity to distinguish between heterochromatin and dichromatin are described, for example, in Wu et al., Cell 16 (1979), 797; Groudine and Weintraub, Cell 30 (1982), 131 and Gross and Garrard, Ann. Rev. Biochem. 57: 159 (1988).

In one embodiment, changes in the hTRT gene are identified by analysis of the karyotype, and numerous methods known in the art can be used. A convenient method is in situ hybridization (ISH). When in situ hybridization methods are used for karyotype analysis, a detectable or detectably labeled sample is usually hybridized with a chromosomal sample in situ to locate an hTRT gene sequence. Generally, ISH comprises one or more of the following steps: (1) fixation of the tissue, cell or other biological structure to be analyzed; (2) Prehybridization treatment of the biological structure to increase the accessibility of the target DNA (e.g. denaturation with heat or alkali) and to reduce non-specific binding (e.g. by blocking the hybridization ability of repetitive sequences (e.g. using human genomic DNA); (3) Hybridization of one or more nucleic acid probes (e.g. common nucleic acids, PNAs or other

75

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

their nucleic acid analogs) with the nucleic acid in the biological structure or the tissue; (4) post-hybridization wash steps to remove nucleic acid fragments that were not bound during hybridization; and (5) detection of the hybridized nucleic acid fragments. The reagents and conditions to be used in each of these steps depend on the application. Of course, these steps can be modified in a variety of ways, as is known to those skilled in the art.

In one embodiment of the ISH, the hTRT probe is labeled with a fluorescence marker (in situ fluorescence hybridization; “FISH”). It is usually desirable to use a “two-color” fluorescence in situ hybridization that uses two probes labeled with a different fluorescent dye. A test probe that hybridizes with the desired hTRT sequence is labeled with one dye, and a control probe that hybridizes with another region is labeled with a second dye. A nucleic acid which hybridizes with a stable region of the desired chromosome, for example the centromer region, can be used as the control probe. In this way, differences in the efficiency of hybridization, which can vary from sample to sample, can be taken into account.

The ISH methods for the detection of chromosomal anomalies (e.g. FISH) can be carried out on nanograms of the nucleic acids in question. Normal tissues or tumor sections embedded in paraffin can be used, but also fresh or frozen material, tissues or sections. Since FISH can be used with this limited material, preparations of uncultivated primary tumors that can only be produced in traces (“touch” preparations) can also be used (see, for example, Kallioniemi et al., Cytogenet. Cell Genet. 60 (1992 ) 190). For example, small biopsy tissue samples from tumors can be used for "touch" preparations (see e.g. Kallioniemi et al., Op. Cit.). Small numbers of cells obtained by suction biopsy or cells in body fluids (e.g. blood, urine, sputum etc.) can also be analyzed. Suitable prenatal diagnoses include amniotic fluid, maternal blood, etc. Appropriate hybridization protocols that can be used for the methods and reagents described here are described in Pinkel et al., Proc. Nati. Acad. Be. USA 85: 9138 (1988); EP-A1 0 430 402; Choo (Ed. Methods in Molecular Biology Vol. 33: In Situ Hybridization Protocols, Humana Press-Totowa, New Jersey (1994) and Kallioniemi et al., Op. Cit.

Other methods useful for karyotype analysis include, for example, methods such as quantitative Southern blotting, quantitative PCR or comparative genomic hybridization (Kallioniemi et al., Science 258 (1992), 818), the hTRT probes and primers according to the invention for identification amplification, deletion, insertion, substitution of hTRT sequences in chromosomes in a biological sample or other rearrangements of hTRT sequences can be used.

G. TRT polypeptide assays

1. General

The present invention provides methods and reagents for the detection and quantification of hTRT polypeptides. These methods include analytical biochemical methods, for example electrophoresis, mass spectroscopy, "gel shift", capillary electrophoresis, chromatographic methods, for example size exclusion, high pressure liquid chromatography (HPLC), (TLC), hyperdiffusion chromatography, etc., or various immunological methods, for example liquid or gel precipitin -Reaction, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescence assays, Western blotting, mass spectrometry and other methods, which are described below and for the person skilled in the art after reading this description are obvious.

2) Electrophoretic assays

In one embodiment, the hTRT polypeptides are detected by electrophoretic protein separation. One aspect relates to a two-dimensional electrophoresis system.

Means for the detection of proteins using electrophoresis methods are well known to the person skilled in the art (see generally R. Scopes (1982) Protein Purification, Springer-Verlag, NY; Deutscher, Methods in Enzymology Vol. 182 (1990): Guide to Protein Purification, Academic Press, Inc., NY).

In a similar embodiment, a mobility shift assay is used (see e.g. Ausubel et al., Supra). For example, labeled hTR associates with hTRT and migrates with changed mobility in electrophoresis in a non-denatured polyacrylamide gel etc. Thus, for example, if a labeled hTR probe or a labeled telomerase primer is mixed with a sample containing hTRT or co-expressed with hTRT the presence of hTRT protein (or a polynucleotide encoding hTRT) in the sample (eg in a cell-free expression system) results in a detectable change in hTRT mobility.

76

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

3) Immunoassays A) General

The present invention also provides methods for the detection of hTRT polypeptides using one or more antibody reagents according to the invention (i.e. immunoassays). As used herein, an immunoassay refers to an assay that uses an antibody (including fragments, chimeric, and other binding agents, as defined herein in detail) that specifically binds to an hTRT polypeptide or epitope. Antibodies according to the invention can be produced by a large number of methods known to the person skilled in the art, for example those described above.

A number of well-established immunological binding assay formats are known which are suitable for practicing the invention (see, for example, U.S. Patents 4,366,241, 4,376,110, 4,517,288 and 4,837,168 and Methods in Cell Biology Volume 37: Antibodies in Cell Biology, Asai, ed. Academic Press, Inc. New York (1993); Basic and Clinical Immunology, 7th edition, Stites & Terr Ed. (1991) Harlow and Lane, op Chapter 14) and Ausubel et al., Loc. Cit. (e.g. Chapter 11), each of which may be considered part of this description by reference in its entirety and for all purposes. Typically, a "capture" agent is used in immunological binding assays (or immunoassays) for specific binding to the analyte and often also its immobilization. In one embodiment, the "capture" agent is a unit that specifically binds to an hTRT polypeptide or a sub-sequence, for example an antihTRT antibody. In an alternative embodiment, the "capture" agent can bind to hTRT-associated protein or RNA under conditions in which the hTRT-associated molecule remains bound to the hTRT (so that if the hTRT-associated molecule is immobilized, the hTRT -Protein is similarly immobilized). In assays in which an hTRT-associated molecule is captured, the associated hTRT protein will normally be detected, for example by means of an anti-hTRT antibody or the like. Immunoassays for the detection of protein complexes are known in the art (see e.g. Harlow and Lane, op. Cit., P. 583).

Normally, the hTRT gene product to be examined is detected directly or indirectly by means of a detectable label. The specific label or detectable group used in the assay is normally not a critical aspect of the invention as long as it does not substantially interfere with the specific binding of the antibody used in the assay or the antibody. The label can be covalently linked to the “capture” agent (for example an anti-TRT antibody) or it can be linked to a third unit, for example another antibody that specifically binds to the hTRT polypeptide, for example (to another Epitope as that recognized by the "capture" agent, the "capture" agent (e.g. an anti (first antibody) immunoglobulin); an anti-TRT antibody; an antibody linked to an anti-TRT Antibody binds or an antibody / telomerase complex (eg via binding to an associated molecule, for example a telomeraseas-associated protein). Other proteins which can bind to the antibody used in the assay, for example protein A or protein G, may also be labeled In some embodiments, it may be appropriate to use more than one labeled molecule (ie, those that can be distinguished from one another) otein or the RNA that is associated with the hTRT protein can be used if this is caused by the "capture" agent (e.g. an anti-hTRT antibody) bound (e.g. immobilized) target is a complex (ie a complex of hTRT and a TRT-associated protein, hTR or another TRT-associated molecule. If the complex is a protein / nucleic acid- Complex (for example TRT-hTR), the reporter molecule can be a polynucleotide or another molecule (for example an enzyme) which recognizes the RNA component of the complex.

Some immunoassay formats do not require the use of labeled components. For example, agglutination assays can be used to detect the presence of the target antibody. In this case, antigen-coated particles are agglutinated by the samples containing target antibodies. In this assay format, the components need not be labeled and the presence of the target antibody can be verified by simple visual inspection.

b) Non-competitive assay formats

The present invention provides methods and reagents for competitive and non-competitive immunoassays for the detection of hTRT polypeptides. Non-competitive immunoassays are assays in which the amount of analyte captured (in this case hTRT) is measured directly. An example of such an assay is a monoclonal antibody-based "twosite" immunoassay that uses monoclonal antibodies that are reactive with two non-interfering epitopes on the hTRT protein. See, for example, Maddox et al, i. Exp. Med. 158 (1983), 1211 for background information. In a preferred "sandwich" assay, the "capture" agent (e.g. an anti-TRT antibody) is bound directly to a solid substrate where it is immobilized. These immobilized antibodies then capture any hTRT present in the test sample.

77

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Protein. The hTRT immobilized in this way can then be labeled, i.e. by binding to a second anti-hTRT antibody bearing a label. In an alternative embodiment, the second anti-hTRT antibody may not have a label, but may be bound by a labeled third antibody that is specific for antibodies of the species from which the second antibody is derived. In a further alternative embodiment, the second antibody can be modified with a detectable unit, for example biotin, which can specifically bind a third labeled molecule, for example enzyme-labeled streptavidin.

c) Competitive assay formats

In competitive assays, the amount of hTRT protein present in a sample is determined indirectly by measuring the amount of (exogenous) hTRT added, which is caused by the hTRT protein present in the sample from a "capture" agent (eg an anti-TRT -Antibody) was displaced, or was competed away. In one of these competitive assays, a known amount of a labeled hTRT protein is added to the sample and then the sample is contacted with a "capture" agent (e.g. an antibody that specifically binds hTRT protein). The amount of exogenous (labeled) hTRT protein bound to the antibody is inversely proportional to the concentration of hTRT protein present in the sample. In one embodiment, the antibody is immobilized on a solid substrate. The amount of hTRT protein bound to the antibody can either be determined by measuring the amount of hTRT protein present in a TRT-antibody complex or, alternatively, by determining the amount of remaining uncomplexed TRT -Protein. The amount of hTRT protein can be detected by providing a labeled hTRT molecule.

A hapten inhibition assay is another example of a competitive assay. In this assay, hTRT protein is immobilized on a solid substrate. A known amount of anti-TRT antibody is added to the sample and then the sample is contacted with the immobilized hTRT protein. In this case, the amount of anti-TRT antibody bound to the immobilized hTRT protein is inversely proportional to the amount of hTRT protein present in the sample. The amount of immobilized antibody can be determined either by detection of the immobilized antibody fraction or the antibody fraction that remains in solution. The detection can be direct, in the case when the antibody is labeled, or indirect, in the event that the label is bound to a molecule which specifically binds to the antibody described above.

d) Other assay formats

The invention also provides reagents and methods for detecting and quantifying the presence of hTRT in the sample using an immunoblot (Western blot) format. In this format, hTRT polypeptides in a sample of other components of the sample are transferred by gel electrophoresis (e.g. based on molecular weight), the separated proteins onto a suitable solid support (e.g. a nitrocellulose filter, a nylon filter, a derivatized nylon filter etc.) and the carrier is incubated with anti-TRT antibodies according to the invention. The anti-TRT antibodies specifically bind to hTRT or other TRT on the solid support. These antibodies can either be directly labeled or alternatively they can subsequently be detected using labeled antibodies (e.g. labeled sheep anti-mouse antibodies) or using other labeled reagents that specifically bind to the anti-TRT antibody.

Other assay formats include liposome immunoassays (LIA) that use liposomes that have been designed to bind specific molecules (e.g. antibodies) and release encapsulated reagents or markers. The chemicals released can then be detected according to standard methods (see Monroe et al., Amer. Clin. Prod. Rev. 4 (1986), 34).

As mentioned above, assay formats that use FACS (and equivalent instruments or methods) have advantages when measuring hTRT gene products in a heterogeneous sample (e.g., a biopsy sample containing both normal and malignant cells).

e) substrates, solid supports, membranes and filters

As noted above, depending on the assay, various components, including the antigen, target antibody, or anti-hTRT antibody, may be bound to a solid surface or support (i.e., a substrate, membrane, or filter paper). Many methods for immobilizing biomolecules on a variety of solid surfaces are known in the art. The solid surface can be, for example, a membrane (e.g. nitrocellulose), a microtiter plate (e.g. PVC, polypropylene or polystyrene), a test tube (glass or plastic), a test strip (e.g. glass, PVC, polypropylene, polystyrene, latex etc.) , a microcentrifuge tube or a glass or plastic bead. The desired constituent can be bound covalently or linked non-covalently through non-specific binding.

78

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

A large number of different organic and inorganic polymers, which can be both natural and synthetic, can be used as the material for the solid surface. Such polymers include polyethylene, polypropylene, poly (4-methylbutene), polystyrene, polymethacrylate, poly (ethylene terephthalate), rayon, nylon, poly (vinyl butyrate), polyvinylidene difluoride (PVDF), silicones, poly-formaldehyde, cellulose, cellulose acetate, Nitrocellulose etc. Other materials that can be used include paper, glasses, ceramics, metals, metalloids, materials with semiconductor properties, cements etc. In addition, gel-forming substances can be used, for example proteins (eg gelatins), lipopolysaccharides, silicates, agarose and polyacrylamides. Polymers which form several aqueous phases are also suitable, for example dextrans, polyalkylene glycols or tensides, for example phospholipids, long-chain (12 to 24 carbon atoms) alkylammonium salts etc. In the case of porous solid surfaces, different pore sizes can be used, depending on the type of system become.

A variety of different substances can be used to obtain different properties to produce the surface, in particular as laminates. For example, protein coatings such as gelatin can be used to avoid non-specific binding, to simplify covalent conjugation, to enhance signal detection, etc.

If a covalent bond between a compound and the surface is desired, the surface is usually polyfunctional or can be made polyfunctional. The functional groups that are present on the surface and can be used for a link include carboxylic acids, aldehydes, amino groups, cyano groups, ethylene groups, hydroxyl groups, mercapto groups, etc. The way in which such linkages can be carried out is large The number of compounds on different surfaces is generally known and is described in detail in the literature, see for example Immobilized Enzymes, Ichiro Chibata, Halsted Press, New York, 1978 and Cuatrecasas, J. Biol. Chem. 245 (1970) 3059.

In addition to covalent binding, various methods for non-covalently binding an assay component can be used. A non-covalent bond is typically the non-specific absorption of a compound on the surface.

Of course, reducing non-specific binding in immunoassays is desirable. Particularly when the assay includes an antigen or an antibody immobilized on a solid substrate, minimizing the amount of non-specific binding to the substrate is desirable. Means to reduce such non-specific binding are well known to those skilled in the art. This typically involves coating the substrate with a protein composition. In particular, protein compositions such as bovine serum albumin (BSA), non-fat milk powder and gelatin are frequently used, with milk powder occasionally preferred. In an alternative embodiment, the surface can be designed such that it binds one component non-specifically, but does not essentially bind another. For example, a surface bearing a lectin such as Concanavalin A binds a carbohydrate-containing compound, but not an unglycosylated labeled protein. U.S. Patent Nos. 4,447,576 and 4,254,082 provide an overview of various solid surfaces for use in the non-covalent linking of assay components.

H. Assays for Anti-TRT Antibodies

The present invention also provides reagents and assays for the detection of hTRT-specific immunoglobulins. In one embodiment, immobilized hTRT (e.g., recombinant hTRT bound to a microtiter plate) is incubated with serum from a patient under conditions where anti-hTRT antibodies, when present, bind the immobilized hTRT. After washing to remove unspecifically bound immunoglobulin, bound serum antibodies, if present, can be detected by adding detectably labeled anti-human Ig) antibodies (alternative embodiments or variations are well known to the person skilled in the art; see, for example, Harlow, loc. Cit., Chapter 14). These assays are useful for the detection of anti-hTRT antibodies in any source including animal or human serum or a vehicle such as saline. In one embodiment, the assays are used to detect or monitor an immune response to hTRT proteins in a patient, particularly an autoimmune response (e.g. anti-telomerase). Anti-hTRT antibodies can be present in the serum, tissues or fluids of a patient suffering from an autoimmune disease or other condition.

I) Assay combinations

The diagnostic and prognostic assays described here can be carried out in various combinations and also in conjunction with other diagnostic or prognostic tests. For example, if the present methods are used to detect the presence of cancer cells in a patient sample, the presence of hTRT can be used to determine the stage of the disease, whether a particular tumor is likely to

79

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

penetrates adjacent tissue or metastasizes to a distant location and whether the cancer is likely to recur. The tests, which can provide additional information, include microscopic analysis of biopsy samples, the detection of antigens (e.g. cell surface markers) that are associated with possible tumor development (e.g. using histocytochemistry, FACS etc.), imaging methods (e.g. after Administration of a labeled anti-tumor antibody to a patient), telomerase activity assays, telomerase length assays, hTR assays, etc. Such combined tests can provide useful information regarding disease progression.

The combination of assays can also provide useful information. For example, as noted above, assays for hTRT mRNA can be combined with assays for hTR (RNA) or telomerase activity (i.e., TRAP) to provide information about telomerase assembly and function.

J) kits

The present invention also provides kits that are useful in screening, monitoring, diagnosis and prognosis in patients with a telomerase-related condition or for determining the level of expression of hTRT in cells or cell lines. The kits contain one or more reagents for determining the presence or absence of an hTRT gene product (RNA or protein) or for quantifying the expression of the hTRT gene. Preferred reagents include nucleic acid primers and probes that bind to the hTRT gene, RNA, cDNA or portions thereof, but also proteins, peptides, antibodies and control primers, probes, oligonucleotides, proteins, peptides and -Antibody. Other substances can be included, including enzymes (e.g. reverse transcriptases, DNA polymerases, ligases), buffers and reagents (labels, dNTPs).

The kits may alternatively or in combination with any of the other ingredients described herein contain an antibody that specifically binds to hTRT polypeptides or sub-sequences thereof. It can be a monoclonal or polyclonal antibody. The antibody can be conjugated to a further unit, for example a label and / or it can be immobilized on a solid support (substrate). The kit (s) may also contain a second antibody for the detection of hTRT polypeptide / antibody complexes or for the detection of hybridized nucleic acid probes, as well as one or more hTRT peptides or proteins for control or others Reagents.

The antibody or hybridization probe can be free or immobilized on a solid support, such as a test tube, microtiter plate, test stick, etc. The kit can also include instructions for using the antibody or hybridization probe in an assay to detect a predisposition to Describe TRT. The kit can contain suitable reagents for the detection of labels or for labeling positive and negative controls, washing solutions, dilution buffers etc.

In one embodiment, the kit contains a pair of primers for the amplification of hTRT mRNA. Such a kit can also contain a probe for amplified hTRT-DNA and / or a polymerase, buffer, dNTPs etc. In a further embodiment, the kit comprises a probe, optionally a labeled probe. In another embodiment, the kit comprises an antibody.

X. Identification of modulators of telomerase activity

A. General

The invention provides compounds and treatments that modulate the activity or expression of a telomerase or a telomerase component (e.g., hTRT protein). The invention also provides assays and screening methods (including high throughput screening methods) for identifying compounds and treatments that modulate telomerase activity or expression. These modulators of telomerase activity and expression (hereinafter referred to as "modulators") include telomerase agonists (which increase telomerase activity and / or expression) and telomerase antagonists (which telomerase activity and / or expression reduce).

The modulators according to the invention have a large number of possible uses. For example, it is believed that telomerase modulators are effective therapeutic agents for the treatment of human diseases. Screening for agonistic activity and transcription or translation activators allows compositions to be provided that can increase telomerase activity in a cell (including a telomer-dependent replication ability or "partial" telomerase activity). Such agonistic compositions also allow methods to be provided to immortalize otherwise normal untransformed cells, including cells that can express useful proteins. Such agonists also allow methods to regulate cellular senescence to be provided. Conversely allowed

80

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

screening for antagonistic activity, providing compositions that reduce telomer-dependent replication ability and thereby mortalize cells that are otherwise immortal, for example cancer cells. Screening for antagonistic activity allows compositions to be provided that reduce telomerase activity and thereby prevent unlimited cell division from cells that show unregulated cell growth, such as cancer cells. Examples of diseases and conditions that can be treated by means of the modulators are listed here, for example in sections VII and IX above. In general, the modulators according to the invention can be used whenever an increase or a decrease in a telomerase activity in a cell or an organism is desired. Thus, a modulator that increases hTRT expression levels, in addition to use in disease treatment, can be used to produce a cultured human cell line that has properties generally described in Section VIII above and has numerous other uses that are useful for are obvious to the expert.

A compound or treatment modulates the «expression» of telomerase or a telomerase component, if the administration of the compound or treatment changes the transcription rate or the transcription level of the gene encoding a telomerase component (eg gene encoding hTRT-mRNA) affects the stability or the post-transcriptional processing of RNA encoding a telomerase component (e.g. transport, splicing, polyadenylation or another modification), affects translation, stability, post-translational processing or the modification of a coded protein (e.g. hTRT) or changes the level in some other way on functional (eg catalytically active) telomerase RNP. A compound or treatment affects telomerase "activity" when the administration of the compound or treatment alters a telomerase, for example those described in Section IV (B) above (e.g., includes processive or non-processive catalytic activity of) Telomerase; telomerase processivity, conventional reverse transcriptase activity; nucleolytic activity; primer or substrate-binding activity; dNTP-binding activity; RNA-binding activity; influencing the telomerase-RNP assembly and protein-binding activity). There is not necessarily a clear distinction between changes in "activity" and changes in "expression". These terms are used to facilitate discussion, but not to limit it. The modulators according to the invention should influence telomerase activity or expression (e.g. without generally changing the expression of housekeeping proteins such as actin) and should not, for example, reduce the expression of a telomerase component by non-specific poisoning of a target cell.

B. Assays to identify telomerase modulators

The invention provides methods and reagents for screening compositions or compounds that can affect the expression of a telomerase or a telomerase component, modify the replication ability of telomerase with respect to DNA, or otherwise affect the ability of the telomerase enzyme and the TRT protein telomerase. To be able to synthesize DNA (“full activity”). The invention also provides screening methods for modulators of any "partial activity" of hTRT or all of these activities. Thus, the present invention provides assays that can be used to screen for agents that increase telomerase activity, for example, by causing hTRT protein or telomerase to be expressed in a cell in which it does not normally express or by increasing levels of telomerase activity in telomerase positive cells.

Telomerase or proteins of a telomerase subunit, their catalytic or immunogenic fragments, or oligopeptides thereof, can be used to screen for therapeutic compounds in a variety of methods for screening drugs. The fragment used in such a test can be freely in solution, fixed to a solid support, carried by a cell surface, or located intracellularly. The formation of binding complexes between telomerase or the subunit protein and the agent to be tested can be measured.

In various embodiments, the invention includes methods of screening for antagonists that bind to the active site of the enzyme; inhibit the association of its RNA moiety, telomerase-associated proteins, nucleotides or telomeric DNA with telomerase or hTRT protein; promote the dissociation of the enzyme complex; interfere with the transcription of the telomerase RNA unit (e.g. hTR) or inhibit one of the «partial activities» described here. The invention also relates to screening for compositions that associate nucleic acid and / or telomerase-associated compositions with hTRT, e.g. inhibit the association of hTR with hTRT, or the association of hTRT with the human homologue of p80 or other associated protein, or the association of hTRT with a telomer or nucleotide; screening for compositions that promote dissociation or association (i.e., assembly) of the enzyme complex, for example an antibody directed against hTR or hTRT; screening for agents that affect the processivity of the enzyme; and screening for nucleic acids and other compounds that bind telomerase, such as one to hTR complex81

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

mental nucleic acid. The invention also contemplates screening for compositions that increase or decrease the transcription of the hTRT gene and / or the translation of the hTRT gene product. The invention also contemplates a screening method for telomerase modulators in animals, in one embodiment by the reconstitution of a telomerase activity or an anti-telomerase activity in an animal, for example in a transgenic animal. The invention also provides in vivo assay systems that include knockout models in which one or different endogenous telomerase, telomerase RNA unit, and / or telomeraseas-associated proteins have been deleted or inhibited. The endogenous telomerase activity can be retained completely or partially. In one embodiment, an exogenous telomerase activity is fully or partially reconstituted.

In one embodiment of the invention, a series of assays for partial telomerase activity are provided which allow the identification of a variety of different classes of modulators of telomerase activity. The “partial activity” assays according to the invention allow the identification of modulator classes of telomerase activity which otherwise could not have been detected in a “full activity” telomerase assay. In a partial activity assay, the non-processive activity of TRT and telomerase is involved. The processive nature of telomerase is described by Morin, Cell 59 (1989), 521-529; see also Prowse “Identification of a nonpro-cessive telomerase-activity from mouse cells” Proc. Nati. Acad. Sci. USA90 (1993), 1493-1497. The “reverse transcriptase-like” activity of telomerase is used in a further partial activity assay according to the invention. These assays examine the reverse transcriptase activity of the hTRT protein; see Lingner "Reverse transcriptase motifs in the catalytic subunit of telomerase", Science 276 (1997), 561-567. In a further partial activity assay according to the invention, the “nucleolytic activity” of hTRT and telomerase, for which the enzyme involves the removal of at least one nucleotide from the 3 ′ strand, typically guanosine, is used. This nucleolytic activity was observed in Tetrahymena telomerase by Collins "Tetrahymena telomerase catalyzes nu-cleolytic cleavage and nonprocessive elongation" Genes Dev. 7 (1993), 1364-1376. In a further partial activity assay according to the invention, an analysis of the ability of hTRT and telomerase is designated as part of their activity with regard to processive DNA polymerization. Another partial activity assay according to the invention involves analysis of the ability of hTRT or telomerase to bind their RNA moiety, i.e. hTR human cells, which is used as a template for telomer synthesis. Further partial activity assays according to the invention include the analysis of the ability of hTRT and telomerase chromosomes to bind in vivo or to bind oligonucleotide primers in vitro or in reconstituted systems or to bind proteins which are associated with chromosomal structures (see for an example of a such protein Harrington, J. Biol. Chem. 270 (1995), 8893-8901). The chromosomal structures that bind hTRT include, for example, DNA of the telomer repeat unit, telomer proteins, histones, nuclear matrix protein, control proteins for cell division or the cell cycle, etc.

In one embodiment, an assay to identify modulators includes contacting one or more cells (ie, "test cells") with a test compound and determining whether the test compound affects the expression or activity of a telomerase (or a telomerase component) in the cell . Typically, this determination involves comparing the activity or expression in the test cell with a similar cell or cells (i.e. control cells) that have not been contacted with the test compound. In an alternative embodiment, cell extracts can be used instead of intact cells. In a related embodiment, the test compound is administered to a multicellular organism (e.g. a plant or an animal). The telomerase or the telomerase component can be completely endogenous to the cell or the multicellular organism (ie encoded by naturally occurring endogenous genes), or it can also be a recombinant cell or a transgenic organism which contains one or more recombinantly expressed telomerase genes. Components comprises (for example hTRT, hTR, telomerase-associated proteins) or the cell or the multicellular organism can have both endogenous and recombinant components. Thus, in one embodiment, telomerase activity modulators are administered to mortal cells. In another embodiment, telomerase activity modulators are administered on immortal lines. For example, antagonists of telomerase-mediated DNA replication can be identified by administering the presumptive inhibitory composition to a cell known to have significant amounts of telomerase activity, such as cancer cells.

In another embodiment, a modulator is identified by monitoring a change in a telomerase activity of a ribonucleoprotein complex (RNP) that includes a TRT (eg hTRT) and a template RNA (eg hTR), the RNP being reconstituted in vitro (as described, for example, in Example 7 below).

In another embodiment, the modulator is identified by monitoring a change in the expression of a TRT gene product (e.g., RNA or protein) in a cell, animal, in vitro expression system, or other expression system.

In a further embodiment, the modulator is identified on the basis of the change in the expression of a reporter gene, for example one such as is described in Example 15, where

82

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

the expression of which is wholly or partly regulated by a naturally occurring TRT regulatory element, for example a promoter or enhancer. In a related embodiment, the ability of a test compound to bind a telomerase component (e.g. hTRT), RNA or a gene regulatory sequence (e.g. the TRT gene promoter) is examined.

In a further embodiment, the modulator is identified by observing changes in the hTRT pre-mRNA processing, this relating, for example, to alternatively spliced products, alternative polyadenylation events, RNA cleavage, etc. In a related embodiment, the activity of the modulator can be examined by monitoring the production of hTRT polypeptides, some of which may have activity for dominant-negative telomerase regulation.

Assay formats for identifying compounds that affect protein expression and activity are well known in the biotechnology and pharmaceutical industries, and additional assays and variations of the assays provided above for illustration will be apparent to those skilled in the art.

Changes in telomerase activity or expression can be measured by any suitable method. Changes in the expression level of a telomerase component (e.g. the hTRT protein) or a precursor (e.g. hTRT mRNA) can be examined using methods well known to those skilled in the art. Some of these methods are described here, for example in Section IX, and this includes monitoring the levels of TRT gene products (for example protein and RNAs) by hybridization (for example using the TRT probes and primers according to the invention), immunoassays (for example using those according to the invention) anti-TRT antibody), RNAse protection assays, amplification assays, or any other means useful for detection described herein or known in the art. Quantifying the amounts of nucleic acid in a sample (e.g. determining RNA levels, e.g. hTR or hTRT mRNA) is also useful for determining transcriptional regulators in ice or trans.

Similarly, changes in telomerase activity can be measured using the methods described here (e.g. in Section IV (B), supra) or other assays for telomerase function. The quantification of telomerase activity can, if desired, be carried out by any method, including those described herein. Telomerase antagonists that cause or accelerate the loss of a telomer structure can be identified by monitoring or measuring their effect on telomerase activity in vivo, ex vivo or in vitro due to their effects on telomer length (by staining or the use of “tagged” hybridization probes) or simply by examining the cell division of telomerase-positive cancer cells (critical shortening of telomeres leads to a phenomenon called “Kriese” or to M2 senescence (Shay, Biochem. Biophys. Acta 1072 ( 1991) 1-7), whereby this is avoided by activating telomerase in the cancer cells, but in the absence of telomerase leads to their senescence or to their death due to a chromosomal deletion and a chromosomal rearrangement). Reconstitution of human telomerase activity in vivo provides a method of screening for telomerase modulators in cells or animals of any origin. Such agonists can be identified in an activity assay according to the invention, which includes the determination of measurable changes in the telomer length, as described above. Further examples of assays for measuring telomerase activity in cells include the accumulation or loss of telomer structures, the TRAP assay, or an assay for a quantitative polymerase chain reaction.

In one embodiment, the assays according to the invention also comprise a method in which the test compound provides a statistically significant reduction in the activity of hTRT compared to the relative amounts of an incorporated label in a parallel reaction in which the agent is absent, which can be used to detect, that the agent is a telomerase inhibitor.

The methods according to the invention can be adapted on the basis of protocols which are described in the scientific literature and the patent literature and are known in the art. For example, if a telomerase according to the invention or a TRT protein is used to identify compositions which act as modulators of telomerase activities, a large number of potentially useful molecules can be screened in a single test. The modulators can have an inhibitory (antagonist) or potentiating (agonist) effect on telomerase activity. For example, if a field of 1000 inhibitors is to be screened, in principle all 1000 inhibitors can be placed in a microtiter plate well and tested simultaneously. If such an inhibitor is discovered, the pool of 1000 can then be divided into 10 pools of 100 and the process repeated until an individual inhibitor is identified.

When screening active substances, a large number of compounds are investigated with regard to their ability to act as telomerase modulators, this process being greatly accelerated by methods of high throughput screening. The assays for full or partial telomerase activity described here can be adapted for use in a high throughput method. It will be apparent to those skilled in the art that numerous methods are available for this purpose.

83

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Another method for screening active substances which can be used for the screening of compounds with suitable binding affinities for telomerase or a protein of a telomerase subunit with high throughput is described in detail in "Determination of Amino Acid Sequence Antigenicity" by Geysen (Geysen , PCT publication WO 84/034 564, published September 13, 1984, which document may be incorporated by reference into this description). In summary, it can be said that a large number of different small peptide test compounds are synthesized on a solid substrate, for example plastic sticks or another surface. The peptide test compounds are reacted with fragments of telomerase or protein subunits of telomerase and washed. Bound telomerase or a telomerase protein subunit is then detected by well known methods. Substantially purified telomerase or a telomerase protein subunit can also be applied directly to plates for use in the aforementioned methods for screening active substances as a coating. In an alternative embodiment, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.

This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies that can bind telomerase or (a) subunit protein) specifically with a test compound for binding to telomerase or the subunit protein compete. In this way, the antibodies can be used to detect the presence of a peptide that shares one or more antigenic determinants with the telomerase or a protein subunit.

Other methods for identifying modulators of telomerase activity are described in US Pat. Nos. 5,645,986 and USSN 08/151,477, filed November 12, 1993, and USSN 08 288 501, filed August 10, 1994, which documents Reference can be viewed as part of this description. The present invention also provides improvements to previously known methods, in part by providing reagents such as hTRT polynucleotides, probes and primers, highly purified hTR, hTRT and telomerase, and anti-telomerase and anti-TRT antibodies all of which can be used in the assays, for example as controls, standard, binding or hybridization agents, or otherwise.

The recombinantly produced telomerase and TRT (e.g. hTRT) according to the invention is of course useful in assays for the identification of modulators. The screening assays can also use telomerase or hTRT derived from full or partial reconstitution of telomerase activity or from an increase in existing activity. The assays or screening methods provided by the invention can be used to test the ability of telomerase to synthesize telomeric DNA or to test for one or all of the "partial activities" of hTRT and TRTs, as generally described above. The assay can involve the ex vivo modification of cells manipulated to express telomerase with or without its RNA moiety or associated proteins, and can be re-implanted into an animal that can be used for an in vivo test. The invention thus provides in vivo as-say and transgenic animals useful therefor. Knockout cells in which one or more units of the endogenous telomerase-enzyme complex have been deleted or inhibited and cells in which exogenous or endogenous telomerase activity has been reconstituted can be used in these in vivo assay systems or is activated.

Telomerases and TRT proteins that have been modified site-specifically (e.g. by site-directed mutation) to modify or delete one or all functions of the telomerase enzyme or the TRT protein can also be used to discover therapeutic agents in the screening methods according to the invention. For example, TRT can be manipulated to lose its ability to bind substrate DNA, to bind its RNA moiety (such as hTR), to catalyze the addition of telomeric DNA, to bind deoxynucleotide substrates, to have nucleolytic activity, telomer -binding associated proteins or chromosomal structures, etc. The “mutant proteins” or “muteins” obtained can be used to identify compounds which specifically modulate one, more or all functions or activities of the TRT protein or telomerase.

C. Examples of Telomerase Modulators 1) General

The test compounds referred to above can be a large variety of compounds, both naturally occurring and synthetic, organic and inorganic compounds, as well as polymers (e.g. oligopeptides, polypeptides, oligonucleotides and polynucleotides) as well as small molecules, antibodies (as defined in detail here), sugars, fatty acids, nucleotides and nucleotide analogs, analogs of naturally occurring structures (eg peptide “imitators”, nucleic acid analogs etc.) and numerous other compounds.

The invention provides all types of modulators without being limited to any particular wir84

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

mechanism. For purposes of illustration, the following are examples of modulators that include compounds or treatments:

(i) they bind to the hTRT polypeptide (e.g. the active center of the enzyme) or to another telomerase component and affect telomerase activity;

(ii) inhibit or promote association or inhibit or promote dissociation of a telomerase component (e.g. hTRT or the hTRT-hTR-RNP) with a telomerase-associated protein (which include, for example, those in section IV (D) above) ;

(iii) inhibit or promote association or inhibit or promote dissociation of telomerase polypeptides (e.g. hTRT) with a telomerase RNA (e.g. hTR);

(iv) inhibit or promote association or inhibit or promote dissociation of telomerase polypeptides (e.g. hTRT) with chromosomes (e.g. telomeres) or chromosomal DNA (e.g. telomeric DNA);

(v) they increase or decrease the expression of a gene product of a telomerase component (eg products of the hTRT gene), including changes in the rate or level of transcription of the TRT gene or translation, the transport or the stability of a gene product etc. by binding to the gene or gene product (for example by interaction with a factor (for example a transcription regulating protein) which influences the transcription of the hTRT gene or another telomerase component).

2) Peptide modulators

Possible modulators of telomerase activity also include peptides (e.g. inhibitory (antagonist) and activating (agonist) peptide modulators). For example, oligopeptides can be screened with randomly generated sequences so that peptide modulators (agonists or inhibitors) of telomerase activity can be found. Such peptides can be used directly as drugs, or to find the orientation or location of a functional group that can inhibit telomerase activity, which in turn leads to the design and testing of an inhibitory small molecule, or where the peptide is the backbone for chemical modification will increase its pharmacological uses. The peptide can be structural “imitators” and molecular modulation programs can be used to design “imitators” based on the characteristic secondary and / or tertiary structure of a telomerase enzyme and hTRT protein. Such structural “imitators” can also be used in vivo as modulators of telomerase activity (agonists and antagonists). Structural “imitators” can also be used as immunogens to generate anti-telomerase or anti-TRT protein antibodies.

3) Inhibitory natural compounds as modulators of telomerase activity

In addition, a large number of potentially useful activity modifying compounds can be screened in extracts from natural products as a starting material. Such extracts can come from a large number of sources, namely the species fungi, actinomyces, algae, insects, protozoa, plants and bacteria. The extracts that show inhibitory activity can then be analyzed to isolate the active molecule. See for example Turner, i. Eth-nopharmacol. 51 (1-3) (1996), 39-43 and Suh, Antcancer Res. 15 (1995) 233-239.

4) Inhibitory oligonucleotides

A particularly useful set of inhibitors provided by the present invention include oligonucleotides that can either bind to an mRNA encoding hTRT protein or to the hTRT gene, in both cases preventing the production of functional hTRT protein or is inhibited. Other oligonucleotides according to the invention interact with the RNA unit of telomerase, for example hTR, or can prevent the binding of telomerase or hTRT to its respective DNA target, or from one telomerase component to another or to a substrate. Such oligonucleotides can also inhibit the telomerase enzyme hTRT protein or protein and RNA and, as already described above, a partial activity (such as their process activity, reverse transcriptase activity, nucleolytic activity etc.). The association can be via a sequence-specific hybridization to another nucleic acid or through a general binding, such as an aptamer, or both.

Telomerase activity can be inhibited by targeting the hTRT mRNA with “anti-sense” oligonucleotides which can bind the hTRT mRNA.

Another useful class of inhibitors include oligonucleotides that inactivate or cleave hTRT mRNA or hTR. The oligonucleotide is chemically modified or has enzymatic activity, which causes such cleavage. Such oligonucleotides include ribozymes or EDTA bound to an oligonucleotide. It can also be a covalent one

85

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Act binding, for example psoralen or cross-linking reagents that are bound to an oligonucleotide. As noted above, a pool of many different such oligonucleotides can be screened for those with the desired activity.

Another class of useful inhibitors include oligonucleotides that bind polypeptides. Double or single stranded DNA molecules or double or single stranded RNA molecules that bind to specific polypeptide targets are called "aptamers". The specific oligonucleotide / poly-peptide association can be mediated by electrostatic interactions. For example, aptamers bind specifically to anion-binding outer sides on thrombin which physiologically binds to the polyanionic heparin (Bock, Nature 355 (1992), 564-566). Since hTRT protein binds to both hTR and its DNA substrate, and since the present invention provides hTRT and other TRT proteins in large quantities in purified form, the person skilled in the art can easily screen for TRT-binding aptamers using the methods according to the invention .

Oligonucleotides (e.g. RNA oligonucleotides) that bind telomerase, hTRT, HTR or portions thereof can be generated using the SELEX method (Tuerk, Methods Mol. Biol. 67 (1997), 2190). In this method, a very large pool (106-109) of nucleic acids with random sequences is bound to the target under conditions (e.g. hTRT) which result in a very strong distinction between molecules with high affinity and low affinity with regard to the binding of the target. The bound molecules are separated from unbound ones and the bound molecules are amplified by means of a specific nucleic acid sequence which is contained in their ends and by means of suitable amplification reagents. This process is repeated several times until a relatively small number of molecules remain that have a high binding affinity for the target. These molecules can then be tested for their ability to modulate telomerase activity as described here.

Antagonists of telomerase-mediated DNA replication can also be based on inhibition of hTR (Norton, Nature Biotechnology 14 (1996) 615-619) via complementary sequence recognition or cleavage, as is the case with ribozymes.

The inhibitory oligonucleotides of the invention can be transferred into the cell using a variety of methods well known in the art. For example, oligonucleotides can be transported into the plasma spontaneously without specific modification. In an alternative embodiment, they can be delivered by means of liposomes that fuse with the cellular membrane or are taken up by endocytosis, i.e. by using the ligand attached to the liposome or by direct attachment to the oligonucleotide, which then bind to surface membrane protein receptors of the cell, resulting in endocytosis. In an alternative embodiment, the cells can be permeabilized to increase the transport of the oligonucleotides into the cell without damaging the host cells. A DNA binding protein, for example HBGF-1, which is known to transport an oligonucleotide into a cell, can also be used.

5) Inhibitory ribozymes

Ribozymes act by binding to a target RNA via the portion of a ri-enzyme that binds the target RNA, which is kept in close proximity to an enzymatic portion of the RNA that cleaves the target RNA. Thus, the ribozyme normally recognizes and cleaves a target RNA via pairing with complementary bases and, once bound in the right place, acts as an enzyme, cleaving and inactivating the target RNA. Cleaving a target RNA in such a manner destroys its ability to direct the synthesis of a coded protein, provided that the cleavage occurs in the coding sequence. After a ribozyme has bound and cleaved its RNA target, it is typically released from the RNA so that it can repeatedly bind and cleave new targets.

6) The identification of telomerase-associated proteins for use as modulators

In one embodiment of the invention, telomerase is used to identify telomerase-associated proteins, i.e. Telomerase-associated proteins that modulate or otherwise complement telomerase activity. As noted above, these proteins or fragments thereof can modulate a function by causing the telomerase-enzyme complex to dissociate or prevent association, prevent assembly of the telomerase complex, hTRT from binding to their one prevent complementary unit-forming nucleic acid or prevent it from binding to its DNA template, hTRT from binding nucleotides, or prevent, increase or increase one or more or all of the partial activities of the telomerase enzyme or the hTRT protein as already described above inhibit.

The person skilled in the art can use the methods according to the invention for the detection of which fractions (e.g. domains) of these telomerase-associated proteins come into contact with telomerase. In an embodiment according to the invention, these telomerase-associated proteins or fragments thereof are used as modulators of telomerase activity.

86

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

7) Telomerase-associated proteins as dominant-negative mutants

In an embodiment according to the invention, telomerase-associated proteins are used as modulators of telomerase activity. Telomerase-associated proteins include chromosomal structures, for example histones, proteins of the nuclear matrix, control proteins of cell division / cell cycle, etc. Other telomerase-associated proteins which can be used as modulators for the purpose according to the invention! include p80 and p95, the human protein and their human homologs, for example TP1 and TRF-1 (Chong, Science 270 (1995), 1663-1667). In addition, fragments of these telomerase-associated proteins can be identified by the person skilled in the art in accordance with the methods according to the invention and used as modulators of telomerase activity.

8) Dominant-negative mutants

Eight highly conserved motifs were identified within the TRTs of various non-human species, as already described above (see also Lingner, Science 276 (1997), 561-567). Figure 4 shows a schematic of the human TRT (hTRT) coding cDNA sequence (from pGRN121) and RT motifs compared to S. pombe-Trtlp, Euplotes p123 and S. cerevisiae Est2p. The present invention provides recombinant and synthetic nucleic acids in which the codons for the conserved amino acid residues in each of the eight motifs have been exchanged for each of the other codons. A large number of the coded sequences obtained express a non-functional hTRT. See, for example, Example 16. Thus, for example, the present invention provides a large number of “mutated” telomerase enzymes and TRT proteins which have partial activity of telomerase but not all of the activity. For example, such a polymerase can bind telomeric structures, but not telomerase-associated RNA (i.e. hTR). Such a telomerase mutant, when expressed at a high enough concentration, can deplete a necessary telomerase component (e.g. hTR) and thereby act as an inhibitor of wild-type telomerase activity. A mutant telomerase acting in this way represents an antagonist or a so-called "dominant-negative" mutant.

9) Antibodies

The antibodies of the invention can generally be used to identify, purify or inhibit any or all of the activities of a telomerase enzyme and hTRT protein. Antibodies can act as antagonists of telomerase activity in various ways, for example, by preventing the telomerase complex or the nucleotide from binding to their DNA substrates, by preventing the telomerase components from being active Complex by maintaining a functional (telomerase complex) quaternary structure or by binding to one of the active centers of the enzyme or to other sites that have allosteric effects on activity (the different partial activities of telomerase are described in detail elsewhere in this description) .

D) Designing modulators

The telomerase modulators according to the invention can be designed or manufactured using methods that are well known in pharmacy, including combinatorial methods and “rational drug design” methods.

1) Methodology of combinatorial chemistry

The production and simultaneous screening of large banks of synthetic molecules can be carried out using well known combinatorial chemistry techniques, see for example van Breemen, Anal. Chem. 69 (1997), 2159-2164 and Lam, Anticancer Drug Des. 12: 145-167 (1997).

As noted above, combinatorial chemistry methodology can be used to generate a huge number of oligonucleotides that can be screened very quickly for specific oligonucleotides that have suitable binding affinities and specificities against any target, such as the inventive TRT -Proteins can be used (see for a general background information Gold, J. of Biol. Chem. 270 (1995), 13581-13584).

2) "Rational Drug Design"

"Rational drug design" includes an integrated set of methods, including structural analysis of target molecules, synthetic chemistry and advanced computer processes. When used to design modulators, such as antagonists / inhibitors of protein targets, such as the telomerase enzyme and hTRT protein, the goal of "rational drug design" is to understand the three-dimensional shape and chemistry of a molecule. «Rational drug de87

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

sign »is supported by data obtained by X-ray structure analysis or NMR and which can now be determined for the hTRT protein and telomerase enzyme according to the methods provided by the invention and using the reagents according to the invention. Calculations with regard to electrostatic properties, hydrophobicity and solvent access are also helpful. See, for example, Coldren, Proc. Nati. Acad. Be. USA 94: 6635-6640 (1997).

D) kits

The invention also provides kits that can be used to determine whether a test compound is a modulator of TRT activity. The kit typically includes one or more of the following: an essentially purified TRT polypeptide or polynucleotide (including probes and primers), a plasmid that can express a TRT (e.g. hTRT) when in a cell or a cell-free expression system is introduced; a plasmid that can express a TR (e.g. hTR) when introduced into a cell or cell-free expression system; Cells or cell lines; a composition for detecting a change in TRT activity and a guide showing how a change in TRT activity can be detected and measured, indicating that a change in telomerase activity in the presence of the test compound is an indicator of this is whether the test compound modulates telomerase activity and a container. The kit may also include agents such as a TRAP assay or a quantitative polymerase chain reaction based assay so as to measure a change in TRT activity. The kit may also include instructions on how to detect and measure a change in TRT activity, indicating that a change in telomerase activity in the presence of the test compound is an indicator of whether the test compound is the telomerase Activity modulated.

XI. Transgenic organisms (telomerase “knockout” cells and animal models)

The invention also provides transgenic non-human multicellular organisms (e.g. plants and animals) or unicellular organisms (e.g. yeast) which contain an exogenous TRT gene sequence which is a coding sequence or a regulatory sequence (e.g. a promoter) can act. In one embodiment, the organism expresses an exogenous TRT polypeptide with a sequence from a human TRT protein. In a related embodiment, the organism also expresses a telomerase RNA component (e.g. hTR).

The invention also provides unicellular and multicellular organisms (or cells thereof) in which at least one gene encoding a telomerase subunit (e.g. TRT or TR) or a telomerase-associated protein is mutated or deleted (ie in a coding or regulatory region ), so that no native telomerase is expressed or that it is expressed with a reduced level or is expressed in comparison with wild-type cells or organisms with different activities. Such cells and organisms are often referred to as “gene knock-out” cells or organisms.

The invention further provides cells and organisms in which an endogenous telomerase gene (e.g. mouse TRT) is mutated or deleted and an exogenous telomerase gene (e.g. for human TRT) is introduced and expressed. For example, cells and organisms of this type are useful as model systems to: identify modulators of hTRT activity or expression; to be able to determine the effects of mutations in genes for telomerase components and, for example, to be able to determine the timing with regard to the state of development and the tissue localization of telomerase activity (in order to be able to assess when a telomerase modulator should be administered and possible side effects to be able to determine).

Examples of multicellular organisms include plants, insects and animals such as mice, rats, rabbits, monkeys, apes, pigs and other mammals. An example of a unicellular organism is yeast.

Methods for changing or disrupting a specific gene (e.g. endogenous TRT genes) are well known to those skilled in the art, see for example Baudin et al., Nucl. Acids Res. 21 (1993), 3329; Wach et al. , Yeast 10 (1994) 1793; Rothstein, Methods Enzymol. 194: 281; Anderson, Methods Cell Biol. 48 (1995), 31; Pettitt et al., Development 122 (1996), 4149-4157; Ramirez-Solis et al., Methods Enzymol. 225 (1993), 855 and Thomas et al., Cell 51 (1987), 503, each of which is to be considered by reference in its entirety and for all purposes as a part of the description.

The “knockout” cells and animals according to the invention include cells and animals in which one or more units of the endogenous telomerase-enzyme complex have been deleted or inhibited. Reconstitution of telomerase activity prevents the cell or animal from inevitable cell death due to its inability to preserve telomeres. Methods for changing the expression of endogenous genes are well known to those skilled in the art. Typically, such methods involve the modification or replacement of all regulatory sequences that express expression.

88

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

control certain genes to be regulated, or a portion thereof. The regulatory sequences, for example the native promoter, can be changed. The common method for targeted mutation of genes involves inserting a genomic DNA fragment containing the desired gene into a vector, followed by cloning two genomic arms around a selectable neomycin resistance cassette in a vector containing thymidine kinase. A suitable cell is then transfected with this «knock-out» construct, i.e. an embryonic stem cell (ES) of the mouse, the cells then being subjected to a positive selection (using G418 and, for example, for neomycin resistance) and a negative selection (using, for example, FIAU to exclude cells which lack thymidine kinase). This allows the selection of cells that have made a homologous recombination with the “knockout” vector. This procedure leads to the inactivation of the desired gene. See, for example, U.S. Patents 5,464,764; 5,631,153; 5,487,992 and 5,627,059.

Knocking out expression of an endogenous gene can also be achieved by means of homologous recombination, which is used to introduce a heterologous nucleic acid into the regulatory sequences (e.g. a promoter) of the desired gene. In order to prevent the expression of functional enzyme or product, simple mutations are suitable which either change the reading frame or interrupt the promoter. To upregulate expression, a native promoter can be exchanged for a heterologous promoter that induces higher levels of transcription. Gene trap insertion can also be used to interrupt a host gene. Mouse embryonic stem cells (ES) can be used to produce transgenic “knockout” animals, as described, for example, in Holzschu Transgenic Res. 6 (1997), 97-106.

The change in expression of endogenous genes by homologous recombination can also be achieved using nucleic acid sequences that contain the structures in question. Upstream sequences are used to target constructs for heterologous recombination. Using the information regarding the TRT structural gene, for example SEQ. ID. No. 1, the person skilled in the art can produce constructs for a homologous recombination, which only requires routine experiments. Homologous recombination for changing the expression of endogenous genes is described in US Pat. No. 5,272,071 and WO 91/09 955, WO 93/09 222, WO 96/29 411, WO 95/31 560 and WO 91/12 650. Homologs Recombination in Myco bacteria is described in Azad Proc. Nati. Acad. Be. USA 93 (1996), 4787; Baulard, J. Bacteriol. 178 (1996), 3091 and Pelicic, Mol. Microbiol. 20 (1996), 991. Homologous recombination in animals is described by Moynahan, Hum. Mol. Genet. 5 (1996), 875 and in plants from Offringa, EMBO J. 9 (1990) 3077.

XI. glossary

The following terms are defined below to provide additional guidance to those skilled in the art when practicing the invention: adjuvant, allele (& allele sequence), amino acids (including hydrophobic, polar, charged), conservative substitution, control elements (and regulatory sequences), de ri vatisie rt, detectable labeling, elevated level, epitope, favorable and unfavorable prognosis, fusion protein, gene product, hTR, immortal, immunogen and immunogenic, isolated, modulator, motif, nucleic acid (& polynucleotide), oligonucleotides (& oligomers), functionally linked, Polypeptides, probe (including nucleic acid probes and antibody probes), recombinant, selection system, sequence, specific binding, stringent hybridization conditions (& stringency), essential identity (& essential similarity), essentially pure (& essentially purified), telomerase negative and Telomerase positive cells, catalytic activity of telomerase , Telomerase-related and test compound.

The term "adjuvant" as used here refers to any substance that increases the immune response to an antigen with which it is mixed. Adjuvants useful in the present invention include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, and surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum are also useful adjuvants.

The term "allele" or "allele sequence" used here refers to an alternative form of a nucleic acid sequence (i.e. a nucleic acid encoding hTRT protein). Alleles are the result of mutations (i.e. changes in the nucleic acid sequence) and this generally leads to the production of modified and / or differently regulated mRNAs or polypeptides, the structure and / or function of which may or may not be changed. Common mutation-based changes that lead to the formation of alleles are generally attributed to natural deletions, additions, or substitutions of nucleotides that may or may not affect the encoded amino acids. Each type of change can occur alone, in combination with the others, or once or more within a given gene, chromosome, or other cellular nucleic acid. Any given gene can have none, one, or many allelic forms. The term “allele” used here refers to either a gene or an mRNA transcribed by the gene or both.

89

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

The “amino acids” described here are sometimes referred to by the standard one-letter code: Àlanin (A), serine (S), threonine (T), aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q), Arginine (R), Lysine (K), Isoleucine (I), Leucine (L), Methionine (M), Valine (V), Phenylalanine (F), Tyrosine (Y), Tryptophan (W), Proline (P), Glycine (G), histidine (H), cysteine (C). This also includes synthetic and non-naturally occurring amino acid analogs (and / or peptide linkages).

The term "hydrophobic amino acids" used here refers to A, L, I, V, P, F, W and M. The term "polar amino acids" used here refers to G, S, T, Y, C, N and Q. The term "charged amino acids" used here refers to D, E, H, K and R.

The term “conservative substitution” used here refers to a change in the description of a protein in a change in the amino acid composition of the protein that does not significantly change the activity of the protein. Thus, the term "conservatively modified variations" of a particular amino acid sequence refers to amino acid substitutions in which amino acids are exchanged that are not critical for protein activity, or to the substitution of amino acids with other amino acids with similar properties (acidic, basic, positive or negative charged, polar or non-polar, etc.), so that the substitutions even of critical amino acids do not significantly change the activity. Conservative substitution tables that list functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D) glutamic acid (E) 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K), 5) isoleucine (I), leucine (L) methionine (M); Valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W) (see also Creighton, Proteins, (1984), W.H. Freeman and Company). It will be apparent to those skilled in the art that the above exchanges are not the only possible conservative substitutions. For example, replacing a charged amino acid with another charged amino acid could be considered a conservative substitution, regardless of whether it is positively or negatively charged. In addition, individual substitutions, deletions, or additions that change, add, or delete a single amino acid or a small percentage of amino acids in a coded sequence are also “conservatively modified variations”. A "conservative substitution" can be introduced into a recombinant protein by using one or more codons that differ from the codons used by the native or wild-type gene. In this case, conservative substitution also includes substitution of one codon for an amino acid with another codon for the same amino acid.

The terms “control elements” or “regulatory sequences” used here include enhancers, promoters, transcription terminators, origins of replication, chromosome-integrated sequences, 5'- and 3'- untranslated regions with which proteins or other biomolecules interact in order to carry out transcription and translation. In the case of eukaryotic cells, the control sequences contain a promoter and preferably an enhancer, which is derived, for example, from immunoglobulin genes, SV40, cytomegalovirus and a polyadenylation sequence. They can also contain donor and acceptor sequences for the splicing. Depending on the vector system used and the host used, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used.

The term “derivatized (s)” polynucleotide, oligonucleotide or nucleic acid used here refers to an oligo- or polynucleotide that comprises a derivatized substituent. In some embodiments, the substituent is essentially non-interfering with hybridization with complementary polynucleotides. Derivatized oligonucleotides or polynucleotides which have been modified with added chemical substituents (eg by modification of an already synthesized oligo or polynucleotide or by incorporation of a modified base or a backbone analog during synthesis) can be introduced into a metabolically active eukaryotic cell hybridize with an hTRT-DNA, -RNA or an hTRT-protein, whereby they produce a change or chemical modification to a DNA, RNA or a protein there. In an alternative embodiment, the derivatized oligo- or polynucleotides may interact and change with hTRT polypeptides, Te-isomerase-associated proteins, or other factors that interact with hTRT-DNA or hTRT gene products, or the expression or function of Modify or modulate hTRT DNA or RNA or proteins. Examples of attached chemical substituents include: Europium (III) texaphyrin, cross-linking agents, psoralen, metal chelates (e.g. iron / EDTA chelate for iron-catalyzed cleavage), topoisomerases, endonucleases, exonucleases, ligases, phosphodiesterases, photodynamic porphyrins , chemotherapeutic agents (eg adriamycin, doxirubicin), intercalating agents, base modifying agents, immunoglobulin chains and oligonucleotides. Iron / EDTA chelates are chemical substituents that are often used when local cleavage of a polynucleotide sequence is desired (Hertzberg et al., J. Am. Chem. Soc. 104 (1982), 313; Hertzberg and Dervan, Biochemistry 23 (1984), 3934; Taylor et al., Tetrahedron 40 (1984), 457 and Dervan, Science 232 (1986), 464). Examples of chemical methods of attachment include: direct connection e.g. via an attached reactive amino group (Corey and Schultz, Science 238 (1988), 1401, which document is to be considered part of this description by reference) and other chemical processes for direct compounds, all of which

90

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

However, connection methods for streptavidin / biotin and digoxigenin / anti-digoxigenin antibodies can also be used. Methods for coupling chemical substituents are provided in U.S. Patents 5,135,720, 5,093,245, and 5,055,556, which are incorporated by reference into this specification. Other chemical coupling methods are at the discretion of those skilled in the art.

The term "detectable label" as used herein has the usual meaning in the art and relates to an atom (eg a radionuclide), molecule (eg fluorescein) or a complex which can be used or can be used to detect the presence of a molecule (e.g. due to a physical or chemical property) or to indicate, or to enable the binding of another molecule to which it is covalently bound or closely associated. The term “label” also refers to covalently bound or closely associated molecules (e.g. a biomolecule, for example an enzyme) that acts on a substrate to produce a detectable atom, molecule or complex. Detectable markings suitable for the use according to the invention include all compositions which can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. The labels useful for the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g. DynabeadsV), fluorescent dyes (e.g. fluorescein, Texas red, rhodamine, green fluorescent protein) green fluorescent protein («enhanced green fluorescent protein»), lissamin, phycoery-thrin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, «FluorX» (Amersham), «SyBR Green I & II» ( Molecular Probes), etc.), radioactive labels (e.g. 3H, 125 |, 35S, 14C or 32P), enzymes (e.g. hydrolases, especially phosphatases such as alkaline phosphatase, esterases and glycosidases or oxidoreductases, especially peroxidases such as horseradish peroxidase and other enzymes commonly used in an ELISA), substrates, cofactors, inhibitors, chemiluminescent groups, chromogenic agents and colorimetric labels such as colloidal gold or colored glass or plastic (e.g. Polystyrene, polypropylene, latex etc.) beads. Patents whose teaching relates to the use of such markers include U.S. Patent 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. Means for detecting such markings are well known to those skilled in the art. Thus, for example, radioactive markings and chemiluminescent markings can be detected using a photographic film or scintillation counters and fluorescent markers can be detected with a photodetector (e.g. in the case of fluorescence-activated cell sorting) to detect emitted light. Enzymatic labels are typically detected in that the enzyme is provided with a substrate and that the reaction product produced is detected by the action of the enzyme on the substrate. Colorimetric markings are detected simply by making the colored marking visible. Thus, a label is any composition that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. The label can be directly or indirectly coupled to the desired assay component according to methods well known in the art. Non-radioactive labels are often applied by indirect means. In general, a ligand molecule (e.g. biotin) is covalently attached to the molecule. The ligand then binds an anti-ligand molecule (e.g. streptavidin) that is either inherently detectable or is covalently bound to a signal generating system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. A number of ligands and anti-ligands can be used. In cases where a ligand has a natural anti ligand, for example biotin, thyroxine or cortisone, it can be used in conjunction with the labeled, naturally occurring anti ligands. Alternatively, any hapten or antigenic compound can be used in combination with an antibody. The molecules can also be conjugated directly to signal-generating compounds, for example by conjugation with an enzyme or fluorophore. Means for the detection of markings are well known to the person skilled in the art. Thus, for example, if the marking is a radioactive marking, the detection means include a scintillation counter, photographic film, such as in autoradiography, or imaging using stored phosphorus (“storage phosphor imaging”). A fluorescent label can be detected by excitation of the fluorochrome with light of a suitable wavelength and the detection of the fluorescence obtained. The fluorescence can be detected visually, using a photographic film, using an electronic detector, such as charge-coupled devices (CCDs) or secondary electron multipliers, etc. Similarly, enzymatic labels can be detected by providing the appropriate substrates for the enzyme, and the reaction product obtained is detected. Simple colorimetric markings can also be detected by observing the color associated with the marking. When pairs of fluorophores are used in an assay, it is often preferred that they have different emission patterns (wavelengths) so that they can be easily distinguished.

The term "elevated level" refers to an amount of hTRT gene product (or other specified substance or activity) in a cell that is elevated, or higher compared to the level in a reference standard, to the level in normal telomerase negative cells in a

91

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

a person or in other people who do not suffer from the corresponding condition, for example for a diagnosis and for a prognosis, for the level in tumor cells of a number of stages, or classes of for example tumors.

The term "epitope" used here has its usual meaning, i.e. it refers to a location on an antigen that is recognized by an antibody. Epitopes are typical sections of amino acids that represent a small fraction of the total protein. Epitopes can affect the conformation (i.e. discontinuously). This means that they can be formed by amino acids which are encoded by non-contiguous parts of the primer sequence and which come to lie side by side due to the protein folding.

The terms "favorable prognosis" and "unfavorable prognosis" are known in the art. In the context of cancer, “favorable prognosis” means that a tumor is likely to regress, or there is a longer chance of survival for patients with a favorable prognosis compared to those with an unfavorable prognosis, while the term “unfavorable prognosis” means that the tumor is likely is more aggressive, which leads to poor disease progression for the patient or faster progression of the disease.

The term "fusion protein" as used herein refers to a composite protein, i.e. a single contiguous amino acid sequence constructed from two (or more) different heterologous polypeptides that are normally not fused together in a single amino acid sequence. Thus, a fusion protein can include a single amino acid sequence that contains two completely different amino acid sequences, or two similar or identical polypeptide sequences, provided that these sequences are not normally found together in a single amino acid sequence. Fusion proteins can generally be made using either nucleic acid recombination techniques, i.e. as the result of transcription and translation of a recombinant gene fusion product, the fusion comprising a portion encoding a polypeptide of the invention and a portion encoding a heterologous protein, or by chemical synthesis methods well known in the art. The non-hTRT region (s) of the fusion protein can be fused to the amino terminus of the hTRT polypeptide, the carboxy terminus, or both.

The term “gene product” as used herein refers to an RNA molecule transcribed from a gene or a protein that is encoded by the gene or translated by the RNA.

The term “hTR” (human telomerase RNA) used here refers to the RNA component of human telomerase and all naturally occurring alleles and variants or recombined variants. hTR is described in detail in U.S. Patent No. 5,583,016, which may be incorporated by reference in its entirety and for all purposes, as a part of this specification.

The term "immortal" as used herein, when referring to a cell, has its normal meaning in the field of telomerase and refers to cells that have an apparently unlimited replicative potential. The term "immortal" can also refer to cells with increased proliferation ability compared to their unmodified counterparts. Examples of immortal human cells are malignant tumor cells, germline cells and certain transformed human cell lines that are cultured in vitro (e.g. cells that became immortal after transformation by viral oncogenes). In contrast, most normal human somatic cells are mortal, i.e. they have limited replicative potential and become senescent after a similar number of cell divisions.

The terms "immunogen" and "immunogen" used here have the usual meaning in this field, i.e. an immunogen is a molecule, such as a protein or other antigen, that triggers an adaptive immune response after injection into a person or animal.

The term “isolated” as used here, when it refers to a molecule or a composition, for example an RNP (for example at least one protein and at least one RNA), means that the molecule or the composition of at least one further compound (for example a protein , other RNAs or other contaminants with which it is associated in vivo or in its naturally occurring state has been separated off, so an RNP is considered "isolated" if, for example, it was isolated from a cell membrane, for example from a cell extract however, the isolated composition can also be substantially pure.

The term “modulator” as used herein refers to any synthetic or natural compound or composition that can alter in any way both the “full” or any “partial activity” of a telomerase reverse transcriptase (TRT). A modulator can be an agonist or an antagonist. A modulator can be, but is not limited to, any organic or inorganic compound. These include, for example, small molecules, peptides, proteins, sugars, nucleic acids, fatty acids etc. The term “motif” used here refers to a sequence of successive amino acids (or to a nucleic acid sequence that codes a sequence of successive amino acids) that have a characteristic or defines a structure in a protein that is common to all proteins of a defined class or a defined type, or is conserved within these proteins. The motif or the “consensus” sequence can contain both conserved and non-conserved residues, the conserved residues in the motif sequence92

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

However, the sequence indicates that the conserved residue or class of residues (ie, hydrophobic, polar, non-polar, or another class) is typically at the indicated position in each protein (or gene or mRNA) of the class of proteins that are characterized by the Motive are defined, is present. Motifs can differ according to the protein class. Thus, for example, the reverse transcriptase enzymes form a class of proteins, which can be defined by one or more motifs, and telomerase enzymes also belong to this class. However, the telomerase enzymes can be defined as the class of enzymes with motifs that are characteristic of this class. It is obvious to a person skilled in the art that the identification of a residue as a conserved residue in a motif does not mean that every member of the class defined by the motif has this indicated residue (or class of residues) in the indicated position and that one or more members the class may have a different remainder at the conserved position.

The terms "nucleic acid" and "polynucleotide" used here are used interchangeably. The use of the term "polynucleotide" is not intended to exclude oligonucleotides (i.e. short polynucleotides) and this term may also refer to synthetic and / or non-naturally occurring nucleic acids (i.e. include nucleic acid analogs or modified backbone residues or bonds).

The terms "oligonucleotides" or "oligomers" as used herein refer to a nucleic acid sequence of about 7 nucleotides or more and up to about 700 nucleotides that can be used as a probe or "amplimer". Oligonucleotides are preferably between about 10 and about 50 nucleotides in length, more preferably from about 14 to about 35 nucleotides, and most preferably from about 15 to about 25 nucleotides, and this term can also refer to synthetic and / or non-naturally occurring nucleic acids refer (ie include nucleic acid analogs or modified backbone residues or bonds).

The term "functionally linked" as used herein refers to a functional relationship between two or more nucleic acid segments (eg DNA): For example, a promoter or enhancer is functionally linked to a coding sequence if it transcribes the sequence in a suitable host cell or another Expression system stimulated. Sequences that are functionally linked are generally sequential and in the case of a signal sequence both sequential and in the reading frame. However, enhancers do not have to be in close proximity to the coding sequences, the transcription of which they are intended to increase.

The term "polypeptide" and the term "protein" used here are used interchangeably and refer to a polymer composed of amino acid residues, including synthetic, naturally occurring and non-naturally occurring analogs thereof. Peptides are examples of polypeptides.

The term “probe” used here refers to a molecule that specifically binds another molecule. An example of a probe is a "nucleic acid probe" that specifically binds (i.e., attaches or hybridizes) to an essentially complementary nucleic acid. Another example of a probe is an "antibody probe" that specifically binds to a corresponding antigen or epitope.

The term "recombinant" as used herein refers to a polynucleotide that has been synthesized in vitro or otherwise manipulated (eg, a "recombinant polynucleotide"), to methods that relate to the use of recombinant polynucleotides to produce gene products in cells or others biological systems or a polypeptide ("recombinant protein") encoded by a recombinant polynucleotide.

The term “selection system” used here refers in connection with stably transformed cell lines to a method for identifying and / or selecting cells which contain a desired recombinant nucleic acid. A wide variety of selection systems for the identification of transformed cells are known and these are suitable for use in the present invention. For example, cells transformed by plasmids or other vectors can be selected by the resistance to antibiotics conferred by genes contained on the plasmids, for example the well-known genes amp, gpt, neo and hyg or other genes, such as the herpes simplex virus -Thymidine kinase genes (Wigler et al., Cell 11 (1977), 223-32 and the adenine phosphoribosyltransferase gene (Lowy et al., Cell 22 (1980), 817), which are used in tk ~ and aprt "cells Resistance to an antimetabolite, an antibiotic or herbicide can also be used as the basis for a selection, for example dhfr, which confers resistance to metotrexate and is also useful for gene amplification (Wigler et al. Proc. Nati. Acad ., 77 (1980), 3567); npt, which confers resistance to the aminoglycosides neomycin and G-418 (Col-bere-Garapin et al., J. Mol. Biol. 150 (1981), 1) and as or pat , the resistance to chlorosulfuron or Grant phosphinotricin acetyltransferase (Murry, in McGraw Hill Yearbook of Science and Technology, McGraw Hill, New York NY, pages 191-196 (1992)). Other selectable genes are described, for example trpB, a gene which confers resistance to hygromycin and allows cells to use indole instead of tryptophan, or hisD, which allows cells to use histinol instead of histidine (Hartman and Mulligan, Proc. Nati. Acad Se., 85 (1988), 8047). Recently, the use of visible markers has become very popular, with markers such as anthocyanins, ß-

93

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Glucuronidase and its substrate, GUS and luciferase and its substrate, luciferin, are not only used to identify transformants, but also to quantify the amount of transient or stable protein expression that can be attributed to a specific vector system (Rhodes et al., Meth. Mol. Biol 55 (1995), 121).

The term "sequence" of a gene, a nucleic acid, a protein or a peptide, as used herein, refers (unless specifically stated otherwise) to the arrangement of nucleotides in either one or both strands of a double-stranded DNA molecule, for example the sequence of both coding strand as well as the complementary strand, or in a single-stranded nucleic acid molecule, or on the arrangement of amino acids in a peptide or protein.

The term "specifically binding" as used herein refers to the ability of a molecule, typically an antibody or polynucleotide, to contact and associate with another specific molecule even in the presence of many other different molecules. For example, a single-stranded polynucleotide can specifically bind to a single-stranded polynucleotide that is complementary in sequence, and an antibody specifically binds to (or "is specifically immunoreactive therewith" with) its corresponding antigen.

The term “stringent hybridization conditions” or “stringency” used here refers to conditions in the range from about 5 ° C. to about 20 ° C. or 25 ° C. below the melting temperature (Tm) of the target sequence and a probe with an exact or almost exact complementarity to the goal. The term "melting temperature" as used herein refers to the temperature at which a population of double-stranded nucleic acid molecules half dissociates into single strands. Methods for calculating the Tm of nucleic acids are well known in the art (see, e.g., Berger and Kimmel (1987) Methods in Enzymology, Vol. 152: Guide to Molecular Cloning Techniques, San Diego: Academic Press, Inc. and Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Volumes 1-3, Cold Spring Harbor Laboratory (hereinafter referred to as "Sambrook"), both of which may be considered part of this description by reference). As indicated in standard references, a simple determination of the Tm value can be calculated using the equation: Tm = 81.5 + 0.41 (% G + C) when a nucleic acid is in aqueous solution at 1 M NaCI ( see, for example, Anderson and Young, Quantitative Filter Hybridization in: Nucleic Acid Hybridization (1985)). Other references contain refined calculations, whereby both structural features and sequence features are taken into account when calculating the Tm. The melting temperature of a hybrid (and thus the conditions for stringent hybridization) are influenced by various factors, e.g. the length and type (DNA, RNA, base composition) of the probe and the type of target (DNA, RNA, base composition, in solution or immobilized etc.) and the concentration of salts and other components (e.g. the presence or absence of formamide, dextran sulfate, polyethylene glycol). The effects of these factors are well known and are discussed in standard references related to this subject; see e.g. Sambrook, op. Cit., And Ausubel et al., Op. Cit. Typically, stringent hybridization conditions correspond to salt concentrations below about 1.0 M sodium ions, typically about 0.01 to 1.0 M sodium ions at pH 7.0 to 8.3 and temperatures of at least about 30 ° C for short probes (e.g. 10 to 50 nucleotides) and at least about 60 ° C for long probes (e.g. over 50 nucleotides). As already noted, stringent conditions can also be achieved by adding destabilizing agents such as formamide, in which case lower temperatures can be used.

The term "essential sequence identity" used here in connection with nucleic acids refers to the extent of sequence similarity between two polynucleotides. Significant sequence identity can be determined by hybridization under stringent conditions, by direct comparison or other measures. For example, two polynucleotides can be identified as having essential sequence identity if they can specifically hybridize with one another under stringent hybridization conditions. Other degrees of sequence identity (eg, less than "essential" can be characterized by hybridization under different stringency conditions. Alternatively, essential sequence identity can be described as a percentage of identity between two nucleotide sequences (or polypeptide sequences). Two sequences are then considered to be essentially identical, if they are at least about 60% identical, preferably at least about 70%, at least about 80%, at least about 90% or at least about 95 or 98 to 100% The percent sequence identity (nucleotide or amino acid) is typically determined by determining the optimal alignment between two sequences or by comparing the two sequences. For example, an exogenous transcript used for sense suppression may have a certain percentage of identity or similarity compared to a reference sequence (eg the corresponding endogenous sequence), to be discribed. The optimal alignment of sequences can be done using the algorithm of Smith and Waterman, Adv. Appi. Math. 2 (1981), 482, which relates to local homology ("local homology algorithm") can be carried out by the algorithm, which relates to homology alignment ("homology alignment algorithm") by Needleman and Wunsch , J. Mol. Biol. 48 (1970), 443, by the “search for similarity” method by Pearson and Lipman, Proc. Nati. Acad. Be. USA 85 (1988), 2444, by designing the

94

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

algorithms using computers (GAP, BESTFIT, FASTA and TFASTA in the software package from Wisconsin Genetics, Genetics Computer Group, 575 Science Dr., Madison, Wl) or by viewing. The best targeting (which gives the highest percentage of identity) generated by the various methods is selected. Typically, these algorithms compare two sequences using a "comparison window" (usually at least 18 nucleotides in length) in order to be able to identify and compare local areas of sequence similarity, allowing small additions or deletions (i.e. gaps). Additions and deletions are typically 20% or less of the length of the sequence relative to the reference sequence that contains no additions or deletions. It is sometimes desirable to describe sequence identity between two sequences in terms of a particular length or range (e.g., two sequences can be described as having at least 95% identity over a length of at least 500 base pairs). Usually the length is at least about 50, 100, 200, 300, 400 or 500 base pairs, amino acids or other residues. The percentage of sequence identity is calculated by comparing two optimally aligned sequences over the comparison range, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G or U) occurs in both sequences, so the number determine paired positions, and by determining the number (or percentage) of paired positions compared to the total number of bases in the reference sequence or the comparison region. An additional algorithm that is suitable for determining sequence similarity is the BLAST algorithm, which is described in Altschul, J. Mol. Biol. 215 (1990), 403-410 and Shpaer, Genomics 38 (1996), 179- 191. Software for performing BLAST analysis is available publicly from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm first includes the identification of sequence pairs with which many hits are achieved ("high scoring sequence pairs, HSPs)") by the identification of short words with the length W in the query sequence that match or meet a positively evaluated point T, if they are aligned against a word of the same length in a database sequence. T refers to the neighborhood word score threshold (Altschul et al., Op. Cit.). These initial hits of the neighborhood word act as starting points for initiating searches which are intended to find longer HSPs containing them. The word hits are lengthened in both directions along each sequence as far as the hit can be increased in terms of cumulative alignment. The prolongation of the word hits in each direction comes to a standstill if: the hit with regard to the cumulative alignment decreases by the amount X of the maximum achievable value; the cumulative hit goes to 0 or below due to the accumulation of one or more alignments of remainder hits; or the end of each sequence has been reached. The parameters W, T and X of the BLAST algorithm determine the sensitivity and the speed of the alignment. The BLAST program uses a word length (W) of 11 by default, the "BLOSUM62 scoring matrix" (see Heni-koff, Proc. Nati. Acad. Sei. USA 89 (1992), 10915-10919), alignments (B) of 50, expectation (E) of 10, M = 5, N = -4, and a comparison of both strands. The term BLAST refers to the BLAST algorithm, which performs a statistical analysis of the similarity between two sequences; see for example Karlin, Proc. Nati. Acad. Be. USA 90: 5873-5787 (1993). A similarity measurement provided by the BLAST algorithm is the smallest sum probability (P (N)), which indicates the probability that a pairing between two nucleotides or amino acid sequences would happen accidentally. For example, a nucleic acid can be considered similar to a TRT nucleic acid if the smallest sum probability when comparing the test nucleic acid to a TRT nucleic acid is less than about 0.5, 0.2, 0 , 1, 0.01 or 0.001. Alternatively, another indication that two nucleic acid sequences are similar is that the polynucleotide encoding the first nucleic acid cross-reacts immunologically with the polypeptide encoded by the second nucleic acid.

The terms "essential identity" or "essential similarity" used in connection with a polypeptide refer to the degree of similarity between two polypeptides in which a polypeptide has a sequence of at least 70%, 80%, 85% or up to 100% Sequence identity includes, based on a reference sequence or most preferably 90% identity over a comparison window of about 10 to 20 amino acid residues. Amino acid sequence similarity or sequence identity is determined by optimizing the pairing of the residues, if necessary by introducing gaps, if necessary. See Needleham et al., J. Mol. Biol 48 (1970), 443-453; Sankoff et al. (1983) Time Warps, String Edits, and Macromolecules: The Theory and Practice of Sequences Com-parison, Chapter One, Addison-Wesley, Reading, MA; and software packages from IntelliGenetics, Mountain View, CA and the University of Wisconsin Genetics Computer Group, Madison, Wl. It is obvious to a person skilled in the art that the terms “essential identity”, “essential similarity” and “essential sequence identity” can be used interchangeably with respect to polypeptides or polynucleotides.

The term "substantially pure" or "substantially purified" as used herein, when referring to a composition containing a specified reagent, such as an antibody (e.g., an anti-hTRT antibody), means that the specified reagent is at least about 75 %, at least about 90%, at least about 95%, at least about 99% or more of the composition

95

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

(without buffer, for example). Thus, for example, a preferred immunoglobulin preparation according to the invention which specifically binds an hTRT polypeptide is essentially purified.

As used here, a "telomerase negative" cell is a cell in which telomerase is not expressed, i.e. no catalytic activity of telomerase can be detected using a conventional assay or a TRAP assay for catalytic activity of telomerase. As used here, a "telomerase positive" cell is a cell in which telomerase is expressed (i.e., telomerase activity can be detected).

As used herein, a "telomerase-related" disease or condition is a disease or condition in a subject that correlates with an abnormally high level of telomerase activity in the subject's cells, which is any telomerase activity for most normal somatic cells, or that correlates with a low level of telomerase activity that results in impairment of normal cell function. Examples of conditions related to telomerase include cancer (high telomerase activity in malignant cells) and infertility (low telomerase activity in germline cells).

The term "test compound" as used herein refers to any synthetic or natural compound or composition. This expression includes all organic and inorganic compounds including, for example, small molecules, peptides, proteins, sugars, nucleic acids, fatty acids etc.

XII. Examples

The following examples are intended to illustrate the present invention and are not intended to be limiting.

The following abbreviations are used in the following sections: eq (equivalents); M (molar); pM (micromolar); N (normal), mole (mole), mmole (millimole) jiMol (micromole); nmoles (nanomoles); g (grams), mg (milligrams); ng (micrograms), ng (nanograms); I or L (liter), ml (milliliter), nl (microliter); cm (centimeters); mm (millimeters); um (micrometer), nm (nanometer); ° C (degrees Celsius); RPN (ribonucleoprotein); remN (2'-0-methylribonucleotides); dNTP (deoxyribonucleotide); dh ^ O (distilled water); DDT (dithiothreitol); PMSF (phenylmethylsulfonyl fluoride); TE (10mM Tris-HCl; 1mM EDTA, pH about 7.2); KGIu (potassium glutamate); SSC (salt and sodium citrate buffer); SDS (sodium dodecyl sulfate); PAGE (polyacrylamide gel electrophoresis); Novex (Novex, San Diego, CA); BioRad (Bio-Rad Laboratories, Hercules, CA), Pharmacia (Pharmacia Biotech, Piscataway, NJ), Boehringer-Mannheim (Boeh-ringer-Mannheim Corp., Concord, CA); Amersham (Amersham, Inc., Chicago, IL); Stratagene (Strategist-Cloning Systems, La Jolla, CA); NEB (New England Biolabs, Beverly, MA); Pierce (Pierce Chemical Co., Rockford IL); Beckman (Beckman Instruments, Fullerton, CA), Lab Industries (Lab Industries, Inc., Berkeley, CA), Eppendorf (Eppendorf Scientific, Madison, Wl); and Molecular Dynamics (Molecular Dynamics, Sunnyvale, CA).

example 1

Isolation of telomerase proteins and clones

The following example describes in detail the isolation of telomerase proteins and clones from different organisms, including the hTRT and S. pombe telomerasec DNA clones

A. Background i) Introduction

The section provides an overview of purification and cloning, which is described in more detail in subsequent sections of this example. Although telomerase RNA subunits have been identified in ciliates, yeast and mammals, protein subunits of the enzyme as such were not identified prior to the present invention. Purification of telomerase from the protozoan Euplotes aediculatus yielded two proteins, designated p123 and p43 (see below; Lingner, Proc. Nati. Acad. Sci. USA 93 (1996) 10712). Euplotes aediculatus is a hypotricher ciliate with a macronucleus that contains about 8 x 107 telomeres and about 3 x 105 telomerase molecules. After purification, the active telomerase complex showed a molecular weight of approximately 230 kD, which corresponds to an RNA subunit of 66 kD and two proteins of approximately 123 kD and 43 kD (see Lingner (1996) above). Experiments with photo cross-linking indicated that the larger p123 protein was involved in the specific binding of the telomeric DNA substrate (Lingner (1996), op. Cit.).

The p123 and p43 proteins were sequenced and the cDNA clones encoding these proteins were isolated. It was found that these Euplotes sequences are not related to the telomerase-associated proteins of Tetrahymena p80 and p95. Sequence analysis of Euplotes-p123 revealed the presence of reverse transcriptase (RT) motifs. Furthermore, by sequence analysis

96

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

of the Euplotes p123 found a homologous yeast protein which is referred to as Est2 protein (Lingner, Science 276 (1997), 561). Yeast Est2 has recently been shown to be essential for the maintenance of telomeres in vivo (Lendvay, Genetics 144 (1996), 1399), but has not been identified as a catalytic teiomerase protein. Site-directed mutagenesis showed that the RT motifs of yeast Est2 are essential for the synthesis of telomer DNA in vivo and in vitro (Lingner (1997) cited above).

ii) Identification and characterization of S. pombe telomerase

PCR amplification of S. pombe DNA was performed with degenerate sequence primers designed according to the Euplotes p123 RT motifs as described below. One of the four major PCR products generated was a 120 base pair band encoding a peptide sequence homologous to p123 and Est2. This PCR product was used as a probe in colony hybridization and thus two overlapping clones from a S. pombe genomic library and three from a S. pombe cDNA library could be identified. Sequence analysis revealed that none of the three S. pombe cDNACIones were full length, so reverse transcriptase (RT) -PCR was used to obtain sequences encoding the N-terminus of the protein.

The complete sequencing of these clones showed a putative S. pombe telomerase RT gene, trtl. The full nucleotide sequence of trtl has been deposited with GenBank under accession number AF015783. Analysis of the sequence revealed that trtl encodes a basic protein with an estimated molecular weight of 116 kD. The homology to p123 and Est2 in the seven reverse transcriptase motifs was found to be exceptionally high. These motifs are underlined and designated as motifs 1, 2, A, B, C, D and E (see Fig. 1). An additional telomerase-specific motif was found, which was referred to as the T motif. 15 introns in the size range from 36 to 71 base pairs interrupted the coding sequence.

To test S. pombe-trtl as a catalytic subunit, two deletion constructs were created. In one, only motifs B to D in the RT domains were removed. The second removed 99% of the open reading frame.

Haploid cells that had grown from S. pombe spores from both mutants showed progressive telomeric shortening to the point where hybridization to telomer repeat units was practically undetectable. A trt1 + / trt1 "diploid was sporulated and the resulting tetrads were parsed and germinated on a yeast extract medium supplemented with amino acid (YES plate, Alfa, (1993) Experiments with Fission Yeast, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) The colonies obtained from each spore were grown for 3 days at 32 ° C. and streaked on fresh YES plates every three days, one colony from each round was placed in 6 ml of a YES at 32 ° C. "Liquid culture added and grown to stationary phase. Genomic DNA was prepared. After cleavage with Apal, the DNA was electrophoresed on a 2.3% agarose gel to confirm that approximately equal amounts were given in each lane, stained with ethidium bromide , then transferred to a nylon membrane and hybridized with a telomeric DNA probe.

Senescence was indicated by the delayed onset of inability to grow on agar (typically the fourth streak after germination), by colonies with margins that were increasingly frayed (the colony morphology is shown in Figure 22B) and by a high number of elongated cell fractions that increased more and more (as shown in Figure 22C). Cells were plated on minimal medium (Alfa, (1993), op. Cit.), With glutamic acid replaced by ammonium chloride, at 32 ° C. for 2 days prior to photography.

The separation of individual enlarged cells under the dissection microscope showed that the majority of the cells were not subject to any further division. The same telomerase negative (trt1 ~) cell population always contained cells of normal size that continued to divide but often produced non-dividing offspring. The telomerase-negative surviving cells could use a recombination mode of telomer preservation, as documented for budding yeast strains with which various genes for telomer replication have been deleted (Lendvay (1996) op. Cit. And Lundblad Cell 73 (1993), 347.

iii) The identification and characterization of human telomerase

An EST (expressed sequence "tag") derived from human telomerase reverse transcriptase (hTRT) cDNA was identified by a BLAST search of the dbEST (expressed sequence "tag") gene bank database and designated as Genbank AA28196, the Euplotes-123 kD peptide and the corresponding nucleic acid sequences as well as the Schizosaccharomyces protein and corresponding cDNA (tezl) sequences were used. The AA281296 EST is 389 nucleotides in length and the positions of its residues in the hTRTcDNA clone (Fig. 16) lie between residues 1679 to 2076. A clone containing the EST sequence which is called clone # 712562 (Fig. 18) was obtained from the IMAGE Consortium (Human Genome Center, DOE, Lawrence Livermore National Laboratory, Livermore, CA). This clone was made from a cDNA

97

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Bank of germinating B cells obtained from a flow sorting of almond cells. Complete sequencing of this hTRT cDNA clone revealed the presence of all eight telomerase RT (TRT) motifs, as shown in Figure 1. This hTRT clone did not encode a contiguous region of TRT since the RT motifs B ', C, D and E were contained in a different open reading frame compared to the more N-terminally located RT motifs. In addition, the distance between RT motifs A and B was much smaller than that of the three previously known (non-human) TRTs.

To isolate a full length cDNA clone, a cDNA library derived from human cell line 293 (described above) that expresses high levels of telomerase activity was screened. A lambda cDNA library from cell line 293 was divided into 25 pools, each containing approximately 200,000 plaques. Each pool was screened by PCR with the primer pairs 5'-CGGAAGAGTGTCTGGAGCAA-3 'and 5'-GGATGAAGCGGAGTCTGGA-3'. Six sub-pools of a positive primary pool were further screened by PCR using the same pair of primers. When screening the primary pool and the secondary sub-pool, hTRT was amplified with a total of 31 cycles, 45 seconds at 94 ° C, 45 seconds at 60 ° C and 90 seconds at 72 ° C. As a control, RNA of the house-keeping enzyme GAPDH was amplified using the prime rpaa re 5'-CTCAGACACCATGGGGAAGGTGA-3 'and 5 -ATGATCI l GAGGCTGT 1GTCATA-3' with a total of 16 cycles, each 45 seconds at 94 ° C, 45 Seconds at 55 ° C and 90 seconds at 72 ° C.

An hTRT positive sub-pool from the secondary screening was then screened by plaque hybridization with a probe from the 5 'region of clone # 712562. One phage could be identified as positive (with the designation Lambda phage 25-1.1, ATCC 209024, deposited on May 12, 1997). It contained an insert of about 4 kilobases, which was cut out and subcloned into the EcoRI site of the “Bluescript II SK +” vector (Stratagene, San Diego, CA) as an EcoRI fragment. This plasmid containing the cDNA clone was named pGRN121. The cDNA insertion is approximately 4 kilobase pairs in length. The complete nucleotide sequence of the human hTRT cDNA (pGRN121) was deposited with Genbank (accession number AF015950) and with ATCC accession number ATCC 209016 on May 6, 1997.

B. Cultivation of Euplotes aediculatus

The cultures of E. aediculatus used in this example were developed by Dr. David Prescott, MCDB, University of Colorado. Dr. Prescott originally isolated this culture from water in a pond, but this organism is also available from the ATCC (ATCC # 30859). Cultures were grown as in Swanton et al., (Swanton et al., Chromosoma 77 (1980), 203) in 15 liter glass containers containing chlorogonium as a food source. The organisms were harvested from the cultures after reaching a density of about 104 cells / ml.

C. Preparation of Nuclear Extracts

In this example, nuclear extracts from E. aediculatus were prepared using the method of Lingner et al., (Lingner et al., Genes Develop. 8 (1994) 1984) with the minor modifications described below. In summary, cells grown as described in Part B were concentrated with 15 µm Nytex filters and cooled on ice. The cell pellet was resuspended to a final volume of 110 ml TMS / PMSF / spermidine phosphate buffer. The TMS / PMSF / spermidine phosphate storage buffer was prepared by adding 0.075 g of spermidine phosphate (USB) and 0.75 ml of PMSF (from a 100 mM stock solution prepared in ethanol) to 150 ml of TMS. TMS contained 10 mM Tris acetate, 10 mM MgCh, 85.5752 g sucrose / liter and 0.33297 g CaCfe / liter, pH 7.5.

After resuspending in TMS / PMSF / spermidine phosphate buffer, 8.8 ml of 10% NP-40 and 94.1 g of sucrose were added. The mixture was placed in a siliconized glass beaker with a stainless steel stir bar attached to a motor above it. The mixture was stirred until the cells were completely lysed (about 20 minutes). The mixture was then centrifuged for 10 minutes at 7500 rpm (8950 x g) at 4 ° C using a Beckman JS-13 swing-out rotor. The supernatant was removed and the pellet from nuclei was resuspended in TMS / PMSF / spermidine phosphate buffer, then centrifuged again at 7500 rpm (8950 x g) at 4 ° C. for 5 minutes using a Beckman JI-D-13 swing-out rotor.

The supernatant was removed and the pellet was resuspended from nuclei in a buffer containing 50 mM Tris acetate, 10 mM MgCl2, 10% glycerol, 0.1% NP-40, 0.4 M KGIu, 0.5 mM PMSF, pH -Value 7.5, using a buffer volume of 0.5 ml per 10 g of cells harvested. The resuspended nuclei were then homogenized in a “dounced” glass homogenizer with about 50 up and down movements and then centrifuged for 25 minutes at 14,000 rpm at 4 ° C. in an Eppendorf centrifuge. The supernatant containing the nuclear extract was collected, frozen in liquid nitrogen and stored at -80 ° C until used.

98

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

D. Purification of Telomerase

In this example, as described in Part C, prepared nuclear extracts for the purification of

E. aediculatus telomerase used. In this purification protocol, telomerase was first enriched by chromatography on an Affi-Gel heparin column and then extensively purified by affinity purification with an "antisense" oligonucleotide. Since the template region of telomerase RNA in the telomerase RNP particle is amenable to hybridization, an "antisense" oligonucleotide (ie the "affinity oligonucleotide") was synthesized as an "affinity bait" for the telomerase, this oligonucleotide being complementary to this template region . A biotin residue was added to the 5 'end of the oligonucleotide so that it could be immobilized on an avidin column.

After binding the telomerase to the oligonucleotide and washing thoroughly, the telomerase was eluted using a displacement oligonucleotide. The affinity oligonucleotide contained DNA bases that were not complementary to the telomerase RNA 5 'to the telomerase-specific sequence. Because the displacement oligonucleotide was complementary to the affinity oligonucleotide along its entire length, it was able to form a thermodynamically more stable duplex than the telomerase bound to the affinity oligonucleotide. Thus, the addition of the displacement oligonucleotide resulted in the elution of the telomerase from the column.

In this example, nuclear extract prepared from 45 liter cultures was frozen until a total of 34 ml of nuclear extract could be obtained. This corresponded to 630 liters of culture (approximately 4 x 109 cells). The nuclear extract was diluted to 410 ml with a buffer, with final concentrations of 20 mM Tris acetate, 1 mM MgCb, 0.1 mM EDTA, 33 mM KGIu, 10% (v / v) glycerin, 1 mM Dithiothreitol (DTT) and 0.5 mM phenylmethylsulfonyl fluoride (PMSF) were obtained at a pH of 7.5.

The diluted nuclear extract was applied to an Affi-Gel heparin gel column (Bio-Rad) with a final volume of 230 ml and a diameter of 5 cm, equilibrated with the same buffer and with a 2 liter gradient from 33 to 450 mM KGIu eluted. The column was operated at a flow rate of one column volume / hour at 4 = C. Fractions of 50 ml each were collected and examined for telomerase activity as described in Part D.

Telomerase eluted from the column at approximately 170 mM KGIu. Fractions containing telomerase (approximately 440 ml) were pooled and adjusted to 20 mM Tris acetate, 10 mM M9Cb, 1 mM EDTA, 300 mM KGIu, 10% glycerin, 1 mM DTT and 1% Nonidet P-40. This buffer was called «WB».

For this preparation, 1.5 mmol of the two competitive DNA oligonucleotides (5'-TAGACCTGTTAGTGTACATTTGAATTGAAGC-3 ') and

(5'-TAGACCTGTTAGGTTGGAl I l GTGGCATCA-3 '), 50 ng yeast RNA (Sigma) and 0.3 nMol biotin-labeled telomerase-specific oligonucleotide

(5'-Biotin-TAGACCTGTTTA- (rmeG) 2- (rmeU) 4- (rmeG) 4-rmeG-3 ')) per ml of the pool. The 2-O-methylribonucleotides of the telomerase-specific oligonucleotides were complementary to the template region of the telomerase RNA; the deoxyribonucleotides were not complementary. The inclusion of competitive, non-specific DNA oligonucleotides increased the effectiveness of the purification because the effects of nucleic acid binding proteins and other components in the mixture that would either bind to the affinity nucleotide or would remove the telomerase from the mixture were minimized.

This material was then added to an "Ultralink" immobilized "Neutravidin plus" (Pierce) column material at a volume of 60 nl suspension per ml pool. The column material was pre-blocked twice for 15 minutes each time using a preparation of WB containing 0.01% Nonidet P-40, 0.5 mg BSA, 0.5 mg / ml lysozyme, 0.05 mg / ml glycogen and 0 , 1 mg / ml yeast RNA contained. The blocking was carried out by means of a rotating disc so as to block the column material thoroughly. After the first and before the second blocking step, the column material was centrifuged at 200 x g for 2 minutes to pellet the matrix.

The mixture of pool and column was incubated on a rotating disk (about 10 rpm; lab industries) for 8 minutes at 30 ° C. and then again for 2 hours at 4 ° C., in order to allow binding. The pool and column mixture was then centrifuged at 200 x g for 2 minutes and the supernatant containing unbound material was removed. The pool and column mixture was then washed. This washing process comprised the following steps: rinsing the mixture of pool and column at 4aC with WB, 15 minutes washing the mixture at 4 ° C with WB, rinsing with WB, 5 minutes washing at 30 ° C with 0.6 M KGIu and no nonidet P-40 containing WB, 5 minutes washing at 25 ° C with WB and finally rinsing again with WB. The volume remaining after the last washing step was kept low so as to achieve a ratio of buffer to column material of approximately 1: 1.

Telomerase was incubated from the column material by adding 1 nmol of displacement oligonucleotide (5'-CA4C4A4C2TA2CAG2TCTA-3 '), per ml of column material and 30 minutes incubation at 25cC. The material was centrifuged at 14,000 rpm in a microcentrifuge (Eppendorf) for 2 minutes and the eluate was collected. The elution procedure was repeated two more times, each time using fresh displacement oligonucleotide. As noted above, the crowding out

99

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

oligonucleotide with the affinity oligonucleotide a thermodynamically more stable complex compared to telomerase, since it was complementary to the affinity oligonucleotide. Thus, by adding the displacement oligonucleotide to an affinity-bound telomerase, effective elution of telomerase was achieved under native conditions. At this stage the polymerase appeared to be about 50% pure, as determined by analysis on a protein gel. Affinity purification of telomerase and elution with a displacement oligonucleotide is shown in Figure 26 (lanes A and B, respectively). In this figure, the 2'-0-methyl sugars of the affinity oligonucleotide are indicated by the thick line. The black and shaded oval shapes are intended to graphically represent the protein subunits according to the invention in this figure.

The protein concentrations of the extract and the material obtained after Affi-Gel heparin column chromatography were determined by the method of Bradford (Bradford, Anal. Biochem. 72 (1976), 248), using BSA for the standards. Only a fraction of the telomerase preparation was further purified on a glycerol gradient.

The sedimentation coefficient of telomerase was determined by glycerol gradient centrifugation as described in Part I.

Table 5 shows a telomerase related to a purification for telomerase that was purified according to the procedures of this example. The telomerase was 12-fold enriched in nuclear extracts compared to the total cell extracts, with a yield of 80% being achieved; 85% of the telomerase was solubilized after extraction from nuclei.

Table 5

Purification of telomerase

fraction

protein

(mg)

Telomerase (pMol RNP)

Telomerase / Protein / pmol RNP / mg

yield

(%)

Nuclear extract cleaning factor

2020

1720

0.9

100

1

Heparin

125

1040

8.3

60

10th

affinity

0.3 "

680

2270

40

2670

Glycerin gradient

N / A*

N / A*

N / A*

25th

N / A*

* NA = Not available

** This value was calculated from the measured amount of telomerase (680 pmol), with an assumed purity of 50% (based on a protein gel).

E. Telomerase activity

At each step in the purification of telomerase, the preparation was analyzed using three separate assays, one of which was based on the determination of activity as described in this example. In general, telomerase assays were carried out in a volume of 40 nl, the 0.003 to 0.3 nl nuclear extract, 50 mM Tris-Cl (pH 7.5), 50 mM KGIu, 10 mM MgCb, 1 mM DTT, Contained 125 mM dTTP, 125 ^ M dGTP and about 0.2 pmole 5'-32P-labeled oligonucleotide substrate (ie about 400,000 cpm). Oligonucleotide primers were heat denatured before addition to the reaction mixture. The reactions were assembled on ice and incubated at 25 ° C for 30 minutes. The reactions were stopped by adding 200 μl of 10 mM Tris-Cl (pH 7.5), 15 mM EDTA, 0.6% SDS and 0.05 mg / ml Proteinase K and incubated at 45 ° C. for at least 30 minutes . After ethanol precipitation, the products were analyzed on 8% denaturing PAGE gels as is known in the art (see e.g. Sambrook et al., 1989).

F. Quantification of Telomerase Activity

This example describes the quantification of telomerase activity during the purification procedure. The quantification was achieved by examining the extension of oligonucleotide primes in the presence of dGTP and [u-32P] dTTP.

Briefly summarized: 1 ^ M 5 '- (G4T4) 2-3'-oligonucleotide was in a 20 nl reaction mixture in the presence of 2 ni [a-32P] dTTP (10 mCi / ml, 400 Ci / mmol; 1 Ci = 37 GBq) and 125 nM dGTP as described in Lingner et al. (Genes Develop. 8 (1994), 1984) and applied to an 80% PAGE sequencing gel as is known in the art ( see for example Sambrook et al., 1989).

The results of this study are shown in Fig. 28. Lane 1 had no telomerase (i.e. negative control), lanes 2, 5, 8 and 11 contained 0.14 fmol telomerase; lanes 3, 6, 9 and 12 contained 0.42 fmol telomerase and lanes 4, 7, 10 and 13 contained 1.3 fmol telomerase. The activity was determined using a “phosphor leaner” (Molecular Dynamics) according to the instructions

100

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

solutions of the manufacturer quantified. The measurement showed that under these conditions 1 fmol of affinity-purified telomerase incorporated 21 fmol of dTTP in 30 minutes.

As shown in this figure, the specific activity of telomerase did not change significantly during the purification procedure. Affinity-purified telomerase was completely active. However, inhibitory activity could be demonstrated at high concentrations and the activity of the crude extracts was not linear. Therefore, in the assay shown in Fig. 28, the high extract was diluted 700-7000 times. The inhibitory activity was removed by the purification and no inhibitory effect was found in the preparations of purified telomerase even at high enzyme concentrations.

G. Gel electrophoresis and Northern blots

As indicated in Part E, the preparation of each telomerase purification step was analyzed using three separate assays. This example describes gel electrophoresis procedures and blotting procedures that were used to quantify telomerase RNA contained in fractions and to analyze the integrity of the telomerase ribonucleoprotein particle.

i) Denaturing gels and Northem blots

In this example, a synthetic T7-transcribed telomerase RNA with known concentration served as the standard. During this study, the RNA component was used as a measure of telomerase.

A construct for the transcription of E. aediculatus telomerase RNA by the T7 phage RNA polymerase was produced by means of the polymerase chain reaction (PCR). The telomerase RNA gene was amplified with primers that attached to both ends of the gene. The primers that attached to the 5 'end also encoded a hammerhead ribozyme sequence to produce the natural 5' end after cleaving the transcribed RNA, a T7 promoter sequence, and an EcoRI site for subcloning .

The sequence of this 5 'primer was 5'

GCGGGAATTCTAATACGACTCACTATAGGGAAGAAACTCTGATGAGGCCGA AAGGCCGAAACTCCACGAAAGTGGAGTAAGTTTCTCGATAATTGATCTGTA

G-3 '. The 3 'primer contained an Earl site for termination of transcription at the natural 3 end and a BamHI site for cloning. The sequence of this 3 "primer was 5'-CGGGGATCC-TCTTCAAAAGATGAGAGGACAGCAAAC-3 '. The PCR amplification product was digested with EcoRI and BamHI and subcloned into the corresponding sites of pUC19 (NEB) to give" pEaT7 ". The correct insertion was confirmed by sequencing. T7 transcription was carried out with an Earl linearized plasmid as described by Zaug et al., Biochemistry 33 (1994) 14935. The RNA was gel-purified and the concentration determined (for A260 of 1 = 40 ^ g The RNA was used as a standard for determining the telomerase RNA present in various preparations of telomerase as a standard.

The hybridization signal was proportional to the amount of telomerase RNA and the deduced RNA concentrations were in principle consistent with the concentrations obtained by native gel electrophoresis, but were slightly higher. Comparison of the amount of total telomerase RNA in total cell RNA with serial dilutions of known T7 RNA transcript concentrations indicated that each E. aediculatus cell contained approximately 300,000 telomerase molecules.

Visualization of the telomerase was achieved by Northern blot hybridization to its RNA component, the method described by Lingner et al. described methods were used (Lingner et al., Genes Develop. 8 (1994), 1984). Briefly summarized: RNA (0.5 ng / lane or less) was separated by 8% PAGE and electroblotted onto a «Hybond-N» membrane (Amersham), as is known in the art (see eg Sambrook et al 1989) The blot was hybridized overnight in 10 ml of 4 x SSC, 10 x Denhardt's solution, 0.1% SDS and 50 n / ml denatured herring sperm DNA. After 3 hours of pre-hybridization, 2 x 106 cpm probe / ml hybridization solution were added. The randomly labeled probe was a PCR product that covered the entire telomerase RNA gene. The blot was washed with different buffer exchanges for 2 minutes 2 x SSC, 0.1% SDS and then for 1 hour in 0.1 x SSC and 0.1% SDS at 45 ° C.

ii) Native gels and Northem blots

In this experiment, the preparation of purified polymerase was separated on native (ie non-denaturing) gels made from 3.5% polyacrylamide and 0.33% agarose, as is known in the art and described by Lamond and Sproat (Lamond and Sproat (1994), loc. Cit.) The telomerase comigrated with the dye xylene cyanole.

101

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

The results with native gels indicated that telomerase was retained as an RNP during the purification process. Figure 27 is a photo of a Northem blot showing the mobility of telomerase in various fractions on a non-denaturing gel and the mobility of in vitro transcribed telomerase. Lane 1 in this figure contained 1.5 fmol telomerase RNA, lane 2 4.6 fmol telomerase RNA, lane 3 14 fmol telomerase RNA, lane 4 41 fmol telomerase RNA, lane 5 nuclear extract (42 fmol telomerase) , Lane 6 Affi-gel heparin-purified telomerase (47 fMol telomerase), lane 7 affinity-purified telomerase (68 fMol) and lane 8 via a glycerol gradient purified telomerase (35 fMol).

As shown in Figure 27, in nuclear extracts, the telomerase was assembled into an RNP particle that migrated more slowly than unassembled telomerase RNA. Less than 1% free RNA was detected by this method. However, a slow migrating telomerase-RNP complex was occasionally detected in extracts. After purification on the Affi-Gel-Heparin column, the telomerase RNP particle showed no change in mobility (FIG. 27, lane 6). However, after affinity purification, the mobility of the RNA particle was slightly increased (Fig. 27, lane 7), which may indicate that a protein subunit or fragment was lost. The affinity-purified telomerase did not change its size on glycerol radii, but about 2% free telomerase RNA was detectable (FIG. 27, lane 8), which suggests that there was little disassembly of the RNP particle.

G. Telomerase Protein Composition

This example describes the analysis of the purified telomerase protein composition.

In this example, the glycerol gradient fractions obtained in Part D were separated on a 4 to 20% polyacrylamide gel (Novex). After electrophoresis, the gel was stained with Coomas Sie Brilliant Blue. Figure 29 shows a photo of this gel. Lanes 1 and 2 contained molecular weight markers (Pharmacia) as indicated on the left side of the gel shown in FIG. 29. Lanes 3 through 5 contained pooled gradient glycerin fractions as indicated above the gel (i.e. lane 3 contained fractions 9-14, lane 4 fractions 15-22 and lane 5 fractions 23-32). Lane 4 contained the pool with 1 pmole of telomerase RNA. In lanes 6 through 9, BSA standards were run at the concentrations indicated above the gel in Figure 29 (ie lane 6 contained 0.5 pmol BSA, lane 7 1.5 pmol BSA, lane 8 4.5 pmol BSA and lane 9 15 pmol BSA).

As shown in Figure 29, 120 and 43 kD molecular weight polypeptides were purified along with the telomerase. The 43 kD polypeptide appeared as a doublet. It was found that the approximately 43 kD polypeptide in lane 3 migrated differently from the doublet in lane 4, and it could be an unrelated protein. The 120 kD protein and the 43 kD Du blette both show a level of about 1 pmole after staining with "Coomassie Brilliant Blue" compared to BSA standards. Since this fraction contained 1 pmole of telomerase RNA, which was completely assembled into an RNP particle (see FIG. 27, lane 8), there appear to be two polypeptide subunits that are stoichiometric to the telomerase RNA. However, the two proteins may also be enzyme subunits separated by 43 kD.

Affinity-purified telomerase, which was not subjected to fractionation on a glycerol gradient, contained additional polypeptides with an estimated molecular weight of 35 or 37 kD. It has been estimated that this latter fraction is at least 50% pure. However, the 35 kD and 37 kD polypeptides present here in the affinity-purified material were not reproducibly separated by centrifugation with glycerin gradients. These polypeptides can be contaminants because they were not visible in all activity-containing preparations.

H. Sedimentation coefficient

The sedimentation coefficient for telomerase was determined by glycerol gradient centrifugation. In this example, nuclear extract and affinity-purified telomerase were fractionated on 15-40% glycerol gradients containing 20 mM Tris acetate, 1 mM MgCh, 0.1 mM EDTA, 300 mM KGIu and 1 mM DTT, pH 7. 5, contained. Glycerin gradients were placed in 5 ml tubes (13 x 51 mm) and centrifuged for 14 hours at 55,000 rpm at 4 ° C. using a SW55Ti rotor (Beckman).

Marker proteins were run in a parallel gradient; there was a sedimentation coefficient of 7.6 S for alcohol dehydrogenase (ADH), 113 S for catalase, 17.3 S for apoferritin and 19.3 S for thyroglobulin. The telomerase peak was identified by electrophoresis of gradient fractions on a native gel and then blot hybridization on their RNA component.

Fig. 30 is a graph showing the sedimentation coefficient for telomerase. As shown in this figure, affinity-purified telomerase co-sedimented with catalase at 11.5 S, while telomerase from nuclear extracts sedimented somewhat faster with the peak around 12.5 S. Thus, purified telomerase appears to lose a proteolytic fragment or a loosely associated subunit to have what is consistent with the mobility of the enzyme in native gels.

102

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

The estimated molecular weight for telomerase is 229 kD in total, assuming that it consists of a 120 kD protein subunit, a 43 kD protein subunit, and a 66 kD RNA subunit. This is in close agreement with the molecular weight of 232 kD of catalase. However, the sedimentation coefficient is not only a function of the molecular weight, but also of the specific partial volume and the coefficient of friction of the molecule, neither parameter for the telomerase RNP being known.

I. substrate recycling

In this example, the requirements of telomerase for the substrate were examined. A simple model for the replication of DNA ends assumes that after semi-conservative DNA replication, telomerase extends double-stranded, blunt-ended DNA molecules. A variation of this model assumes that a single-stranded 3 "end is generated by a helicase or nuclease after replication. This 3 'end is then used by the telomerase for binding and extension.

To determine whether telomerase can elongate blunt-ended molecules, model hairpin structures with telomer repeat units located at their 3 'ends were synthesized. These primer substrates were gel cleaned, 5'-end labeled with polynucleotide kinase, heated at 80 ° C and 0.4 nM for 5 minutes and then slowly cooled to room temperature in a heat block to allow renaturation and helix formation of the hairpin structures. The substrate mobility on a non-denaturing gel showed that there was very efficient hairpin formation compared to dimerization.

In this example, assays were carried out with unlabeled 125 nM dGTP, 125 nM dTTP and 0.02 nM 5'-end-labeled primer (5'-32P-labeled oligonucleotide substrate) in 10 pl reaction mixtures containing 20 mM Tris acetate with 10 mM MgCfe, 50 mM KGIu and 1 mM DTT at pH 7.5. These mixtures were incubated at 25 ° C for 30 minutes. The reactions were stopped by adding formamide application buffer (i.e. TBE, formamide, bromothymol blue and cyanol, Sambrook, 1989, op. Cit.).

Primers were incubated without telomerase («->»), with 5.9 fMol of affinity-purified telomerase («+»), or with 17.6 fMol of affinity-purified telomerase («+++ •>). The affinity-purified telomerase used in this assay was dialyzed with a membrane with a molecular cutoff of 100 kD to remove the displacement oligonucleotide. The reaction products were separated on an 8% PAGE (urea gel, which contained 36% formamide to denature the hairpin structure). The sequences of the primers used in this study and their track assignments are shown in Table 6.

103

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Table 6 Primer sequences

track

Primer sequence (5 'after 3')

1-3

C4 (A4C4) 3CACA (G4T4) 3G4

4-6

C2 (A4C4) 3CACA (G4T4) 3G4

7-9

(A4C4) 3CACA (G4T4) 3G4

10-12

A: C4 (A4C4) 2CACA (G4T4) 3G4

13-15

C4 (A4C4), CACA (G4T4),

16-18

(A4C4) 3CACA (G4T4) 3

19-21

A3C4 (A4C4): CACA (G4T4) 3

22-24

C4 (A4C4) 2CACA (G4T4) 3

25-27

C2 (A4C4) 2CACA (G4T4) j

28-30

(A4C4) 2CACA (G4T4) 3

The results obtained with the gels are shown in Fig. 31. Lanes 1-15 contained substrates with telomer repeat units that ended with four G residues. Lanes 16-30 contained substrates with telomer repeat units that ended with four T residues. The putative orientation on the telomerase RNA template is shown in FIG. 32. It is believed that the primer sets are at two different positions in the template shown in Figure 32 (i.e. 32A and 32B, respectively). This could have affected their retention and / or renewal rates.

Fig. 33 shows a lower exposure of lanes 25-30 of Fig. 31. The lower exposure of Fig. 33 was therefore made to allow visualization of the nucleotides that were added and the delay positions in extended product. The percentage of elongated substrate was quantified for every third line of a set on a "phosphor lean" as shown in Figure 31 below.

The effectiveness of this hairpin structure as a substrate was compared with double-stranded telomer-like structures with overhangs of different lengths. A model substrate that ended with four G residues (see lanes 1 to 15 of Figure 31) was not elongated if it contained a smooth end (see lanes 1-3). However, a slight elongation with an overhang length of two bases was observed. However, the extension became efficient when the overhang was about 4 bases long. Telomerase worked similarly with a double-stranded substrate that ended with four T residues, requiring a 6 base overhang for highly efficient elongation. The weak bands among the primers in lanes 10-15 of FIG. 31, which are independent of telomerase, represent shorter oligonucleotides in the primer preparations.

The weaker exposure of lanes 25-30 in Fig. 33 shows a ladder of elongated products, the darkest bands correlating with the presumed 5 'boundary of the matrix (as described by Lingner et al., Genes Develop. 8 (1994) , 1984). The frequent occurrence of products that correspond to other positions in the template suggested that "pausing" and / or "dissociating" with the purified polymerase occurs at sites that do not correspond to the translocation site.

As shown in Figure 31, double-stranded, blunt-ended oligonucleotides were not substrates for telomerase. A competition experiment was carried out to find out whether these molecules could bind to telomerase. In this experiment, 2 nM 5 'end-labeled substrate was extended with the sequence (G-tT ^ or a hairpin substrate with an overhang of six bases with 0.125 mM telomerase (Fig. 31, lanes 25-27). Although the same unlabeled oligonucleotide -

104

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

substrate competitively competed for elongation with the labeled substrate, no decrease in activity was observed when the double-stranded, smooth-ended hairpin oligonucleotides were used as competitors, even in the presence of a 100-fold excess of hairpin structures.

These results indicated that double-stranded, blunt-ended oligonucleotides cannot bind at the concentrations of telomerase tested in this example, i.e. a single-stranded 3 'end is required for binding. This 3 'end is probably required for base pairing with the telomerase RNA template.

J. Cloning & sequencing of the 123 kD polypeptide

This example describes the cloning of the 123 kD polypeptide from telomerase (i.e. the 123 kD protein subunit). In this study, an internal fragment of the Telomera gene was amplified using PCR. Oligonucleotide primers were used that were designed to match the peptide sequences obtained from the purified polypeptide obtained in Part D above. The polypeptide sequence was determined using the "nanoES tandem" mass spectroscopy method known in the art and described by Calvio et al., RNA 1 (1995), 724-733. The oligonucleotide primers used in this example had the following sequences, with degenerate positions shown in parentheses:

5 1 - rCT (G / A) AA (G / A) TA (G / A) TG (T / G / A) GT (G / A / T / C) A (T / G / A) (G / A) TT (G / A) TTCA t - 3 'and

5'- GCGGATCCATGAA (T / C) CC (A / T) GA (G / A) AA (T / C) CC (A / T) AA (T / C) GT-3 '

A 50 nl reaction contained 0.2 mM dNTPs, 0.15 ng E. aediculatus chromosomal DNA, 0.5 n 'Taq (Boehringer Mannheim), each 0.8 ng primer and 1 x reaction buffer (Boehringer Mannheim). The reaction was incubated in a "thermal cycler" (Perkin-Elmer), incubating for 5 minutes at 95 ° C, followed by 30 cycles of 1 minute each at 94 ° C, 1 minute at 52 ° C and 2 minutes at 72 ° C. The reaction was completed by incubation at 72 ° C for 10 minutes.

A genomic DNA library was prepared from the chromosomal DNA of E. aediculatus by cloning DNA with blunt ends into the site of the “pCR-Script” plasmid vector (Stratagene) called Smal. This bank was screened by colony hybridization with the radiolabelled gel-purified PCR product. The plasmid DNA from positive clones was prepared and sequenced according to the dideoxy method (Sanger et al., Proc. Nati. Acad. Sei., 74 (1977), 5463) or manually using an automatic sequencer (ABI). The DNA sequence of the gene encoding this polypeptide is shown in Figure 13. The start codon derived from the DNA sequence is in this sequence at the nucleotide at position 101 and the open reading frame ends at position 3193. The genetic code of Euplotes differs from other organisms in that the “UGA” codon is a cysteine Rest coded. The amino acid sequence of the polypeptide derived from the DNA sequence is shown in FIG. 14. It was assumed that no unusual amino acids were incorporated during translation and that there were no post-translational modifications.

K. Cloning and sequencing of the 43 kD polypeptide

This example describes the cloning of the 43 kD polypeptide from telomerase (i.e. the 43 kD protein subunit). In this study, an internal fragment of the telomerase gene was PCR amplified using oligonucleotide primers designed to match the peptide sequences obtained from the purified polypeptide obtained in Part D above. The polypeptide sequence was determined using the "nanoES tandem" mass spectroscopy methods known in the art and described by Calvio et al., RNA 1 (1995), 724-733. The oligonucleotide primers used in this example had the following sequences:

5'-NNNGTNAC (C / T / A) GG (Cn "/ A) AT (C / T / A) AA (C / T) AA-3 'and 5' - (T / G / A) GC (T / G / A) GT (C / T) TC (T / C) TG (G / A) TC (G / A) TT (G / A) T A-3 ".

In this sequence, "N" means the presence of one of four nucleotides (i.e. A, T, G or C).

A 50 nl reaction contained 0.2 mM dNTPs, 0.2 ng E. aediculatus chromosomal DNA, 0.5 nl Taq (Boehringer Mannheim), each 0.8 pg primer and 1 x reaction buffer (Boehringer Mannheim). The reaction was incubated in a "thermal cycler" (Perkin-Elmer), incubating for 5 minutes at 95 ° C, followed by 30 cycles of 1 minute each at 94 ° C, 1 minute at 52 ° C and 1 minute at 72 ° C. The reaction was completed by incubation at 72 ° C for 10 minutes.

A genomic DNA library was created from the chromosomal DNA of E. aediculatus by cloning

105

CH 689 672 A9

Preparation of DNA with smooth ends into the Smal site of the «pCR-Script» plasmid vector (Stratagene). This bank was screened by colony hybridization with the radioactively labeled gel-purified PCR products. The plasmid DNA from positive clones was prepared and sequenced according to the dideoxy method (Sanger et al., Proc. Nati. Acad. Sei., 74 (1977), 5463) or ma-5 manually using an automatic sequencer (ABI) . The DNA sequence of the gene encoding this polypeptide is shown in Figure 34. Three possible reading frames are shown for this sequence, as shown in FIG. 35. For the sake of clarity, the amino acid sequence is shown under the nucleotide sequence for all three reading frames. These reading frames are labeled "a", "b" and "c". A possible start codon is co-doped at nucleotide position 84 in the reading frame “c”. The coding area could end at position 1501 in reading frame "b". Premature stop codons, indicated with asterisks in this figure, occur in all three reading frames between nucleotide positions 337-350.

The “La domain” is identified by bold letters. Further downstream, the protein sequence appears to be encoded by different reading frames, since none of these reading frames is interrupted by 15 stop codons. In addition, peptide sequences from the purified protein are encoded in all three reading frames. Thus this gene appears to contain intron sequences or, alternatively, the RNA is edited. Other options include ribosomal reading frame shifting or sequencing errors. However, the homology to the sequence of the La protein remains of great interest. It should be noted once again that the "UGA" codon in Euplotes co-co-20 a cysteine residue.

L. Comparisons of amino acids and nucleic acids

In this example, comparisons are made between published sequences and the sequences of the 25 123 kD and 43 kD telomerase polypeptide subunits.

i) Compare to the 123 kD telomerase subunit of E. aediculatus

The amino acid sequence of the 123 kD polypeptide from Euplotes aediculatus was compared to the sequence 30 of the 80 kD telomerase protein subunit from Tetrahymena thermophila (GenBank accession number # U25641) to investigate their similarity. The nucleotide sequence encoding this protein obtained from GenBank is shown in Figure 42. The amino acid sequence of this protein as obtained from GenBank is shown in Figure 43. The sequence comparison between the 123 kD protein from E. aediculatus and the 80 kD protein from T. thermophila is shown in FIG. 36. In this figure, the sequence of E. aediculatus represents the upper sequence and the sequence of T. thermophila the lower sequence. In this, as well as in FIGS. 37-39, identities are indicated by vertical bars, while individual dots between the sequences indicate amino acids with somewhat similarity and colons between the sequences indicate amino acids with greater similarity. The proven identity was about 19%, while the percentage of similarity was about 45%. 40 These values are similar to those observed with any random protein sequence.

The amino acid sequence of the 123 kD polypeptide from Euplotes aediculatus was also compared to the sequence of the 95 kD telomerase protein subunit from Tetrahymena thermophila (GenBank accession number # U25642) to investigate the similarities. The nucleotide sequence encoding this protein obtained from GenBank is shown in FIG. 44. The amino acid sequence of the protein obtained from Gen-Bank er-45 protein is shown in Fig. 45, and the sequence comparison in Fig. 37. In this figure, the sequence of E. aediculatus is the upper sequence and the sequence of T. thermophila that lower sequence. Identities are indicated by vertical bars. The identity observed was about 20%, while the percentage of similarity was about 43%. These values are similar to the values observed with any random protein sequence.

50 It is striking that the amino acid sequence of the 123 kD protein from E. aediculatus contains the five motifs which are characteristic of reverse transcriptases. The 123 kD polypeptide was also compared to the polymerase domains of various reverse transcriptases. Figure 40 shows the alignment of the 123 kD polypeptide with the putative yeast homolog (L8543.12 or ESTp). The amino acid sequence of L8543.12 (or ESTp) obtained from GenBank is shown in Figure 46. 55 Four motifs (A, B, C and D) were part of this comparison. In Fig. 40 highly preserved residues are identified by white letters on a black background. Remains of the sequences of E. aediculatus that are conserved in the other sequence are marked in bold. The «h» indicates the presence of a hydrophobic amino acid. The digits between the amino acid residues of the motifs indicate the amount of gaps within the sequences. For example, the "100" between motifs A and B affects a gap of 100 amino acids in the sequence between motifs.

By searching the gene bank, a yeast protein (GenBank accession number # u20618) and the gene "L8543.12" (Est2) could be identified, which shows some homology to the 123 kD telomerase subunit of E. aediculatus. Based on the observation that both proteins contain reverse transcriptase-65 motifs in their C-terminal regions; that both proteins have in common that they are similar106

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

show sensitivity in areas outside the reverse transcriptase motif; that the proteins are similarly basic (pl = 10.1 for E. aediculatus and pl = 10.0 for yeast); and both proteins are large (123 kD for E. aediculatus and 103 kD for yeast) it can be concluded that these sequences encompass the catalytic core of their corresponding telomerases. Based on this observation regarding the homology between two phylogenetically removed organisms such as E. aediculatus and yeast, it is assumed that human telomerase contains a protein that has the same characteristics (ie has reverse transcriptase motifs, is basic and large (> 100 kD)) .

ii) Compare with the 43 kD telomerase subunit of E. aediculatus

The amino acid sequence of the "La domain" of the 43 kD polypeptide from Euplotes aediculatus was compared to the sequence of the 95 kD telomerase protein subunit from Tetrahymena thermophila (described above) to examine their similarity. The sequence comparison is shown in Fig. 38. In this figure, the E. aediculatus sequence is the top sequence and the T. thermophila sequence is the bottom sequence. Identities are marked with vertical bars. The observed identity was about 23%, while the percentage of similarity was about 46%. These values are similar to those that you would observe with any random protein sequences.

The amino acid sequence of the "La domain" of the 43 kD polypeptide from Euplotes aediculatus was compared to the sequence of the 80 kD telomerase protein subunit from Tretrahymena thermophila (described above) to examine their similarity. The sequence comparison is shown in Fig. 39. In this figure, the sequence of E. aediculatus is the upper sequence and the sequence of T. thermophila is the lower sequence. Identities are marked with vertical bars. The observed identity was about 26%, while the percentage of similarity was about 49%. These values are similar to those that would be observed with any random red sequences.

The amino acid sequence of a domain of the 43 kD polypeptide from E. aediculatus was also compared to La proteins from numerous other organisms. These comparisons are shown in Fig. 41. In this figure, highly preserved residues are identified by white letters on a black background. Remains of the sequences of E. aediculatus that are conserved in the other sequence are marked in bold.

M. Identification of telomerase protein subunits in another organism

In this example, the sequences identified in the previous examples were used to identify the telomerase protein subunits of Oxytricha trifallax. This is a ciliate that is only distantly related to E. aediculatus. In this example, primers were selected based on the conserved region of the 123 kD polypeptide from E. aediculatus, which included the motifs of the reverse transcriptase domain. Suitable primers were synthesized and used in a PCR reaction with total DNA from Oxytricha. Oxytricha DNA was prepared according to methods known in the art. The PCR products were then cloned and sequenced using methods known in the art.

The oligonucleotide sequences used for the primers were:

5 '- (T / C) A (A / G) AC (T / A / C) AA (G / A) GG (T / A / C) AT (T / C) CC (C / T / A) (C / T) A (G / A) GG-3 '5' - (G / A / T) GT (G / A / T) ATNA (G / A) NA (G / A) (G / A) TA (G / A) TC (G / A) TC-3 '

Positions that were degenerate are shown in parentheses with the alternative bases shown in parentheses. “N” corresponds to any of the four nucleotides.

The PCR reaction (50 pl) contained 0.2 mM dNTPs, 0.3 ng chromosomal DNA from Oxytricha trifallax, 1 pl Taq polymerase (Boehringer Mannheim), each 2 nM primer, 1 x reaction buffer (Boehringer Mannheim). The reaction mixture was incubated in a "thermocycler" (Perkin-Elmer) under the following conditions: 1x5 minutes at 95 ° C, 30 cycles each 1 minute at 94 ° C, 1 minute at 53 ° C and 1 minute at 72 ° C, then 1x10 minutes at 72 ° C. The PCR product was gel purified and sequenced using the dideoxy method (see, e.g., Sanger et al., Proc. Nati. Acad. Sci. 74 (1977), 5463-5467) or other methods known in the art.

The amino acid sequence was derived from the PCR product and compared with the sequence of E. aediculatus. Fig. 47 shows the alignment of these sequences, the sequence of 0. Trifallax being shown in the top row and the E. aediculatus sequence being shown in the bottom row. As can be seen from this figure, there is a high degree of homology between the 0th trifallax polypeptide sequence identified in this example and the polypeptide sequence of E. aediculatus. Thus, it is clear that the sequences identified in the present invention are useful for the identification of homologous telomerase protein subunits in other eukaryotic organisms. In fact, due to the development according to the invention, homologous telomerase sequences could be identified in numerous different species.

107

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

N. Identification of Tetrahymena Telomerase Sequences

In this example, a Tetrahymena clone was created that has homology with the Euplotes sequences and EST2p.

In this experiment, a PCR was used with degenerate oligonucleotide primers, which were directed against conserved motifs, in order to be able to identify regions of homology between tetrahymena, Euplotes and EST2p sequences. The PCR method used in this example represents a new method that was designed for the specific amplification of rare DNA sequences from complex mixtures. This method avoids the problem of amplifying DNA products with the same PCR primer at both ends (i.e. products, a single primer) that is commonly encountered in PCR cloning procedures. These products of a single primer create an undesirable background and this can often complicate the amplification and detection of the desired product from two primers. This method used in these experiments is preferably selected for products from two primers. The special thing is that one primer is biotinylated and the other is not. After several rounds of PCR amplification, the products are purified using magnetic streptavidin beads and products from two primers are specifically eluted by means of heat denaturation. This method is also used in other experiments that were not described in these examples. Indeed, this method is in use in applications where it is desirable to amplify rare DNA sequences, including the initial steps in cloning methods, e.g. 5 'and 3, RACE and any method that uses degenerate primers in PCR.

A first PCR run was performed using Tetrahymena macronuclear DNA as a template, which was isolated according to methods known in the art. A 24-mer forward primer was used with the sequence

5'-Biotin-GCCTATTT (TC) TT (TC) TA (TC) (GATC) (GATC) (GATC) AC (GATC) GA-3 ', which is referred to as "K231" and the FFY> 0 "E Area corresponds and the 23-mer reverse primer with the sequence 5'-CCAGATAT (GATC) A (TGA) (GATC) A (AG) (AG) AA (AG) TC (AG) TC-3 ', the is referred to as “K220” and corresponds to the DDFL (FIL) I. This PCR reaction contained 2.5 pl of DNA (50 ng), each 4 pl of primer (20 pM), 3 pl of 10 × PCR buffer, 3 pl 10 x dNTPs, 2 pl Mg, 0.3 pl Taq and 11.2 pl dh ^ O. 8 cycles were carried out with the mixture, 45 seconds each at 94 ° C, 45 seconds at 37 ° C and 1 minute at 72 ° C.

The mixture of the PCR reaction was bound to 200 pl magnetic streptavidin beads, washed with 200 pi TE, resuspended in 20 pl dH2Ü and then heat-denatured by boiling at 100 ° C for 2 minutes. The beads were pulled down and the eluate was removed. Thereafter, 2.5 pl of this eluate was amplified again under the above conditions, except that 0.3 pl a-32P dATP was contained and 33 PCR cycles were carried out. The reaction mixture was separated on a denaturing 5% polyacrylamide gel and the appropriate area was cut out of the gel. These products were re-amplified with another 34 cycles under the conditions listed above, except that the annealing temperature was 42 ° C.

A second PCR run was performed using Tetrahymena macronuclear DNA isolated using methods known in the art as a template, the 23-mer forward primer having the sequence 5 '

AC AAT G (C A) G (G AT C) (TC A) T (GAT C) (T C A) T (G AT C) C C (G AT C) AA (AG) AA-3 '

with the designation “228” was used, which corresponds to PKK after the region R (LI) (LI) and a reverse primer with the sequence 5′-

ACGAATC (GT) (GATC) GG (TAG) AT (GATC) (GC) (TA) (AG) TC (AG) TA (AG) CA 3 '

with the designation «K224», which corresponds to the CYDSIPR range. This PCR reaction contained 2.5 pl DNA (50 ng), each 4 pl primer (20 pM), 3 pl 10 x PCR buffer, 3 pl 10 x dNTPs, 2 pl Mg, 0.3 pl a-32P- dATP, 0.3 pl Taq and 10.9 pl dH2Ü. The reaction mixture was separated on a denaturing 5% polyacrylamide gel and the appropriate area was cut out of the gel. These products were amplified again with 34 additional cycles under the conditions given above, except that the annealing temperature was 42 ° C.

10 pl of the reaction product from Run 1 was bound to magnetic streptavidin-coated beads in 200 pi TE. The beads were washed with 200 pi TE and then resuspended in 20 pl dH2Ü, heat denatured and the eluate was removed. The reaction product from Run 2 was then added to the beads and diluted with 30 pl 0.5 x SSC. The mixture was heated to 94 ° C and allowed to cool to 50 ° C. The eluate was removed and the beads were washed three times in 0.5 x SSC at 55 ° C. The beads were then resuspended in 20 pl dH2Ü, heat denatured, and the eluate labeled "Round 1 eluate" was removed and stored.

108

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

To isolate the Tetrahymena band, the round 1 eluate with the forward primer K228 and the reverse primer K227 with the sequence 5'-

CAATTCTC (AG) TA (AG) CA (GATC) (CG) (TA) (CT) TT (AGT) AT (GA) TC-3 '(SEQ ID

which corresponds to the DIKSCYD range, amplified again. The PCR reactions were carried out as described above. The reaction products were separated on a 5% polyacrylamide gel, the band corresponding to approximately 295 nucleotides was cut out of the gel and sequenced.

The clone labeled 168-3 was sequenced. The following DNA sequence (including the primer sequences) was found:

GATTACTCCCGAAGAAAGGATCTTTCCGTCCAATCATGACTTTCTTAAGAAA

GGAC AAGC AAAAAAAT ATT AAGTT AAAT CT AAATT AAATT CT AAT GGAT AG

CC AACTTGT GTTT AGGAATTT AAAAG AC AT GCTGGGAT AAAAGAT AGGAT A

CTCAGTCTTTGATAATAAACAAATTTCAGAAAAATTTGCCTAATTCATAGAG

AAATGGAAAAATAAAGGAAGACCTCAGCTATATTATGTCACTCTAGACATA

AAGACTTGCTAC

An additional sequence of this gene was obtained by PCR, using a new primer designed to match the sequence of 168-3 («K2-97» with the sequence 5'-GAGTG ACAT AAT AT ACGT GA- 3 '; mated and primer K231 (FFYXTE). The sequence of the fragment obtained from this reaction, together with 1683, is as follows (without the primer sequences):

AAACACAAGGAAGGAAGTCAAATATTCTATTACCGTAAACCAATATGGAA ATT AGT G AGT AAATT AACT ATT GT C AAAGT AAG AATTT AGTTTT CT G AAAAG AAT AAAT AAAT G AAAAAT AATTTTT AT C AAAAAATTT AGCTT G AAG AGG AG AATTT GG AAAAAGTT G AAG AAAAATT G AT ACC AG AAG ATT C ATTTT AG AAA TACCCTCAAGGAAAGCTAAGGATTATACCTAAAAAAGGATCTTTCCGTCCA ATC AT G ACTTT CTT AAG AAAGG AC AAGC AAAAAAAT ATT AAGTT AAATCTA AATT AAATT CT AAT GGATAGC C AACTT GT GTTT AGG AATTTAAAAG AC ATG CT GGG ATAAAAG AT AGG ATACTC AGT CTTT G AT AATAAAC AAATTT C AGAA AAATTTGATACAGTAAAAT

The amino acid sequence corresponding to this DNA fragment is:

KHKEGSQIFYYRKPIWKLVSKLTIVKVRIQFSEKNKQMKNNFYQKIQLEEENLE KVEEKLIPEDSFQKYPQGKLRIIPKKGSFRPIMTFLRKDKQKNIKLNLNQILMDS QLVFRNLKKQYQIEQ

This amino acid sequence was aligned against other telomerase genes (EST2p and Euplotes). The orientation is shown in Fig. 53. A "consensus" sequence is also shown in this figure.

109

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

P. Identification of Schizosaccharomyces pombe Telomerase Sequences

In this example, the S. pombe tez1 sequence was identified as a homologue of E. aediculatus p123 and S. cerevisiae Est2p.

55 shows a summary of these experiments. In this figure, the upper region (part A) shows the relationship between the two overlapping genomic clones and the 5825 bp region that was sequenced. The region labeled “tez1” corresponds to the protein coding region, the flanking sequences also being shown. The box below the 5825 bp range is an approximately 2 kb HindIII fragment used to prepare the tez1 disruption construct as described below.

The lower half of Figure 55 (Part B) is an "broken down" scheme of the same DNA region. The sequence referred to as the "original PCR" is the original degenerate PCR fragment generated with the degenerate oligonucleotide primer pair based on the sequence of motif 4 (B ') and motif 5 (C) from Euplotes, such as described in the previous examples.

i) PCR with degenerate primers

PCR using the genetic primers was used to find the protein homologous to p123 of E. aediculatus in S. pombe. Figure 56 shows the sequences of the degenerate primers used in this reaction (referred to as "poly 4" and "poly 1"). The PCR runs were carried out under the same buffer conditions as described in the examples above (see, for example, Part K above) with a 5-minute ramp time of 94 ° C, after which cycles were carried out for 30 seconds at 94 ° C, 45 seconds Connect at 50 ° C and seconds at 72 ° C, then 7 minutes at 72 ° C, and then the reaction mixture was kept at 4 ° C. The PCR runs were carried out under different conditions (i.e. different concentrations of S. pombe DNA and different MgCb concentrations). The PCR products were separated on agarose gels and stained with ethidium bromide as described above. Different PCR runs resulted in the production of 3 bands (labeled "T", "M1" and "B"). These bands were amplified again under the same conditions as described above and separated on gels. After this renewed amplification, four bands were observed ("T", "M1", "M2" and "B") as shown in Fig. 57. These four bands were then re-amplified under the same conditions as described above. The third band from the top of the lane in Figure 57 was identified as a band that contained the correct sequence for a teiomerase protein. It was found that the PCR product labeled M2 fits relatively well with other telomerase proteins, as shown in FIG. 58. In addition to the orientation shown, this figure also shows the actual sequence of tez1. In this figure, asterisks denote residues that have all four sequences in common (Oxytricha "OT"; E. aediculatus "Ea_p123", S. cerevisiae "Sc_p103" and M2), and the circles (i.e. dots) denote similar amino acid residues.

ii) 3'-RT-PCR

In order to obtain additional sequence information, 3 'and 5' RT-PCR were carried out on the telomerase candidate shown in FIG. 58. 59 shows a diagram of the 3'-RT-PCR strategy used. First, the oligonucleotide primer «Qt» (5'-CCA GTG AGC AGA GTG ACG AGG ACT CGA GCT CAA GCT IIIIII TTT III TT-3 '; was prepared from mRNA cDNA, then this cDNA as a template for OCR with «Qo» ( 5'-CCA GTG AGC AGA GTG ACG-3 '; used and a primer designed on the basis of the original degenerate PCR reaction (ie «M2-T» with the sequence 5'-G TGT CAT TTC TAT ATG GAA GAT TTG ATT GAT G-3 '). The second PCR reaction (ie «nested» PCR) was carried out using «Qi» (5'-GAG GAC TCG AGC TCA AGC-3'); and another PCR primer that was designed with a sequence that originated from the original degenerate PCR reaction or “M2-T2” with the sequence 5'-AC CTA TCG TTT ACG AAA AAG AAA GGA TCA GTG-3 ';. The buffers used in this PCR corresponded to those as described above, the amplification carried out starting with a 5-minute ramp up at 94 ° C., after which 30 cycles of 30 seconds each at 94 ° C., 30 seconds at 55 ° C and 3 minutes at 72 ° C, then incubated at 72 ° C for 7 minutes. The reaction products were stored at 4 ° C until used.

iii) Screening aenomic and cDNA libraries

After receiving this additional sequence information, various genomic and cDNA libraries were screened in order to be able to identify all banks which contained this candidate for the telomerase gene. The procedure used as well as the gene banks and results are shown in FIG. 60. In this figure, part A lists the gene banks tested in this experiment; Part B shows the areas used; parts C and D show those obtained with these banks

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

results of dot blot hybridization. Positive libraries were then screened to obtain the tez1 genomic gene and a cDNA version of the tez1 gene by colony hybridization. In this experiment, approximately 3 x 104 colonies were screened from the Hindlll genomic library and six positive clones were identified (approximately 0.01%). Then DNA from two independent clones (A5 and B2) was prepared. Figure 61 shows the results obtained with Hindill-cleaved positive genomic clones A5 and B2.

In addition, cDNA REP banks were used. About 3 x 105 colonies were screened and 5 positive clones (0.002%) were identified. DNA was prepared from three independent clones (2-3, 4-1 and 5-20). In later experiments it was found that 2-3 and 5-20 contained identical insertions.

iv) 5'-RT-PCR

Since the cDNA version of the gene product was not complete up to this point, a 5 'RT-PCR was carried out to obtain a full-length clone. The strategy is shown schematically in Fig. 62. In this experiment, cDNA was prepared using the DNA oligonucleotide primer «M2-B» (5'-CAC TG A TCC TTT CTT TTT CGT AAA CGA TAG GT-3 '); and "M2-B2" (5'-C ATC AAT CAA ATC TTC CAT ATA GAA ATG ACA3 '); which were designed by tezi based on known and previously identified areas. An oligonucleotide linker "PCR Adapt Sfil" with a 5 'phosphorylated end ("P") (p-GGG CCG TGT TGG CCT AGT TCT CTG CTC3') was then ligated to the 3 'end of this cDNA and this construct was used as the template for a “nested” PCR. In the first round of PCR, PCR Adapt SFI and M2-B were used as primers. In the second round, PCR Adapt Sfili (5-GAG GAG GAG AAG AGC AGA GAA CTA GGC CAA CAC GCC CCS); and M2-B2 (5'-ATC AAT CAA ATC TTC CAT ATA GAA ATG ACA-3 '); used as primer. "Nested" PCR was used to increase the specificity of the reaction.

v) Sequence alignments

After the sequence was determined by tezi, it was compared to previously described sequences. Figure 63 shows the alignment of reverse transcriptase (RT) domains from telomerase catalytic subunits of S. pombe ("S.p. Tezlp"), S. cerevisiae "S.c. Est2p »), and E. aediculatus p123 (E.a. p123»). In this figure, "h" denotes hydrophobic residues, "p" small polar residues and "c" charged residues. The amino acid residues indicated above the alignment show the “Consensus” RT motif by Y. Xiong and T.H. Eickbush (Y. Xiong and T.H. Eickbush, EMBO J. 9 (1990) 3353-3362). The asterisks indicate the residues that are conserved in all three proteins. "Motif 0" was identified here as a motif that is specific for this telomerase subunit and is generally not found in reverse transcriptases. It is therefore valuable in order to be able to identify other amino acid sequences as good candidates for catalytic telomerase subunits.

64 shows the alignment of the entire sequences of Euplotes ("Ea_P123"), S. cerevisiae ("Sc_Est2P") and S. pombe ("Sp_Tez1p"). In Part A, shaded areas indicate residues that both sequences have in common. In Part B, shaded areas denote residues that all three sequences have in common.

vi) Tezi's genetic disruption

In this example, the effects of tezi's disruption were examined. Since telomerase is involved in the maintenance of telomeres, it was assumed that if tezi is actually a component of telomerase, tezi's disruption should lead to a gradual telomer shortening.

In these experiments homologous recombination was used for the specific disruption of the tez1 gene in S. pombe. This procedure is shown schematically in FIG. 65. As indicated in Figure 65, wild-type tez1 was exchanged for a fragment containing the ura4 or LEU2 marker.

Disruption of the tez1 gene was confirmed by PCR (Fig. 66) and a Southern blot was performed to check the telomer length. Figure 67 shows the Southern blot results of this experiment. Since an Apal restriction enzyme site is immediately adjacent to a telomer sequence in S. pombe, the cleavage of genomic DNA preparations from S. pombe allows the telomer length to be analyzed. Thus, S. pombe DNA was cut with Apal, the cleavage products were separated on an agarose gel and these were then hybridized with a probe specific for a telomer sequence in order to find out whether the telomeres of the disrupted S. pombe cells were shortened. The results are shown in Fig. 67. From these results it could be concluded that disruption of the tez1 gene caused a shortening of the telomeres.

111

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Q. Cloning and characterization of human teiomerase protein and cDNA

In this example, information regarding the nucleic acid sequence and amino acid sequence for human telomerase was obtained. Partially homologous sequences were first identified using a BLAST search using the Euplotes 123 kD peptide and nucleic acid sequences as well as the sequences of the Schizosaccharomyces protein and the corresponding cDNA (tezl) sequence. The human sequences (also referred to as “hTCP1.1”) were determined from a partial cDNA clone (clone 712562). Sequences from this clone were aligned against the sequences determined as described in previous examples.

1 shows the sequence alignment of Euplotes ("p123") Schizosaccharomyces ("tezi"), Est2p (ie the protein of S. cerevisiae encoded by the Est2 nucleic acid sequence, which is also referred to here as "L8543.12") and the like human homologues identified in this comparison search. Figure 51 shows the amino acid sequence of tezi and Figure 52 shows the DNA sequence of tezi. In Fig. 52, the introns and other non-coding areas are shown in small letters and the exons (i.e. coding areas) in capital letters.

As shown in Fig. 75, there are areas that are highly conserved among these proteins. For example, this figure shows that there are areas of identity "Motif 0", "Motif 1", "Motif 2" and "Motif 3". The identical amino acids are marked with an asterisk (*) and the similar amino acid residues with a circle (•). This shows that there are areas within the telomerase motifs that are conserved within a large number of eukaryotes, which range from yeast to ciliate to human. It is believed that other organisms also contain such conserved sequence regions. Fig. 49 shows the partial amino acid sequence of the clone encoding human telomerase motifs and Fig. 50 the corresponding DNA sequence of the Genbank clone # AA281296.

To complete the information on the sequence of Genbank clone # AA281296, Sanger dideoxy sequencing and other methods known in the art were used. Some of the primers used in sequencing are shown in Table 7. These primers were designed to hybridize to the clone (GenBank accession number # AA281296); this was based on sequence complementarity to either the sequence of the plasmid backbone or the sequence of insertion of the human cDNA into the clone.

112

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Table 7 Primers

Primer

sequence

TCP1.I

GTGAAGGCACTGTTCAGCG

TCP 1.2

GTGGATGATTTCTTGTTGG

TCPl.3

ATGCTCCTGCGTTTGGTGG

TCP 1.4

CTGGACACTCAGCCCTTGG

TCPl.5

GGCAGGTGTGCTGGACACT

TCPl.6

TTTGATGATGCTGGCGATG

TCPl.7

GGGGCTCGTCTTCTACAGG

TCPl.8

CAGCAGGAGGATCTTGTAG

TCPl.9

TGACCCCAGGAGTGGCACG

TCPl.10

TCAAGCTGACTCGACACCG

TCPl.11

CGGCGTGACAGGGCTGC

TCPl.12

GCTGAAGGCTGAGTGTCC

TCPl.13

TAGTCCATGTTCACAATCG

Based on these experiments, it was found that the EcoRI Not insertion of the Genbank clone # AA281296 contains only a partial open reading frame for the human teiomerase protein, but it could encode an active fragment of this protein. The open reading frame in the clone encodes a protein of approximately 63 kD. The sequence of the longest open reading frame found is shown in FIG. 68. The open reading frame (ORF) begins at the ATG codon with the “met” marked in the figure. The poly A tail at the 3 'end of the sequence is also shown. 69 shows a preliminary alignment of reverse transcriptase proteins from telomerase from the human sequence (human telomerase “core protein 1”, “HS TCP1”), E. aediculatus p123 (“Ep p123”), S. pombe tezi (“ Sp Tezi »), S. cerevisiae EST2 (« Sc Est2 ») and the« Consensus »sequence. Various motifs are given in this figure.

In order to obtain a complete clone, the hybridization of a cDNA library and 5'-RACE was carried out, so as to obtain clones which encode regions of regions which have not yet been cloned. In these experiments, RACE was used to generate material for a sequence analysis (rapid amplification of cDNA ends, “Rapid Amplification of cDNA Ends”; see, for example, MA Froh-man, “RACE: Rapid Amplification of cDNA Ends”, in Innis et al . (Ed.) PCR-Protocols: A Guide to Methods and Applications (1990) p. 2838, and Frohman et al., Proc. Nati. Acad. Sci. 85 (1988) 8998-9002). Four such clones were generated and used to provide additional 5 'sequence information (pFWRP5, 6, 19 and 20).

In addition, human cDNA libraries (inserted in lambda) were hybridized with the EcoRI-NotI fragment of the clone (# AA281296). A lambda clone called "lambda 25-1.1" (ATCC accession number # 209024) was found which contained complementary sequences. Figure 75 shows a restriction map of this lambda clone. The insertion of human cDNA was subcloned from this clone as an EcoRI restriction fragment into the EcoRI site of the commercially available "phagemid" pBluescriptIISK + (Stratagene), creating the plasmid "pGRN121" which was deposited with the ATCC (ATCC- Accession number # 209016). Preliminary results showed that the plasmid pGRN121 contains the complete sequence of the open reading frame (ORF), which encodes the human Teiomerase protein.

The cDNA insertion of plasmid pGRN121 was performed using art113

CH 689 672 A9

known method sequenced. 70 shows a map of the plasmid pGRN121 with regard to restriction sites and function, the determination of which is based on this preliminary work. The results of this preliminary sequence analysis are shown in Fig. 71. Based on this analysis, and as shown in FIG. 70, a putative starting site for the coding region was found at a position approximately 50 5 nucleotides from the EcoRI site (located at position 707) and the location of the telomerase specific motifs «FFYVTE», «PKP», «AYD», «QG» and «DD». In addition, a putative stop point was found at nucleotide # 3571 (see Fig. 72). Fig. 72 shows the DNA sequences and corresponding amino acid sequences for the open reading frames in the sequence ("a", "b" and "c"). However, due to the tentative nature of this early sequencing work, 10 it emerged that the reading frames for the various motifs were not properly aligned.

An additional analysis performed with pGRN121 revealed that the plasmid contained significant regions of the 5 'end of the coding sequences which are not present on the Genbank clone with the accession number # AA281296. It was further found that pGRN121 contains a variant of the coding sequence which contains an insertion of approximately 182 nucleotides. It was found that 15 this insertion is missing in the Genbank clone with the accession number # AA281296. Such variants, like the E. aediculatus sequences, can be tested in functional assays, for example telomerase assays, in order to be able to detect the presence of a functional telomerase in a sample.

Further sequence analysis determined the cDNA sequence of pGRN121, where-20 was found in a contiguous open reading frame encoding a protein with a molecular weight of approximately 127,000 daltons and 1132 amino acids as shown in Fig. 74. An improved map of pGRN121 based on this analysis is shown in FIG. 73. The results of the additional sequence analysis of the hTRT cDNA are shown in FIG. 16.

25 Example 2

The correlation between hTRT frequency and cell immortality

The relative abundance of hTRT mRNA was assessed in six strains of telomerase-negative mortal cells and six cell lines of telomerase-positive immortal cells (Fig. 5). The steady state of minor levels of hTRT mRNA was significantly increased in immortal cells that were previously shown to have active telomerase. Lower levels of hTRT mRNA were found in some strains of telomerase negative cells.

RT-PCR was performed for hTRT, hTR, TP1 (telomerase-associated protein, which is related to Tetrahymena p80 35 (Harrington et al., Science 275 (1997) 973 and Nakayama et al., Cell 88 (1997), 875) ) and GAPDH (to normalize for equal amounts of RNA template) with RNA derived from the following cells: (1) human fetal lung fibroblast GFL, (2) human fetal skin fibroblast GFS, (3) adult prostate stromal -Fibroblasts 31 YO, (4) human fetal Kniesyn-oval fibroblasts HSF, (5) neonatal foreskin fibroblasts Bi, (6) human fetal lung fibroblasts 40 IMR90 and the immortalized cell lines: (7) melanoma LOX IMVI, (8) leukemia U251, (9) lung cancer NCI H23, (10) colon adenocarcinoma SW620, (11) breast tumor MCF7, (12) 293 adenovirus E1-transformed human embryonic kidney cell line.

hTRT nucleic acid was amplified by cDNA using the oligonucleotide primers LT5 and LT6 (Table 2) with a total of 31 cycles (94 ° C. 45 s, 60 ° C. 45 s, 72 ° C. 90 s). GAPDH was amplified using the primer 45 K136 (CTCAGACACCATGGGGAAGGTGA) and K137 (ATGATCTTGAGGCTGTTGTCATA) with a total of 16 cycles (94 ° C 45 s, 55 ° C 45 s, 72 ° C 90 s). hTR was amplified with the primers F3B (TCTAACCCTAACTGAGAAGGGCGTAG) and R3c (GTTTGCTCTAGAATGAACGGTGGAAG) with a total of 22 cycles (94 ° C 45 s, 55 ° C 45 s, 72 ° C 90 s). TP1 mRNA was amplified with the primers TP1.1 and TP1.2 in 28 cycles (the cycles were the same as for hTRT). The reaction 50 products were separated on an 8% polyacrylamide gel, stained with “SYBR Green” (Molecular Probes) and visualized by scanning on a “Storm 860” (Molecular Dynamics). The results shown in Figure 5 show that hTRT mRNA levels correlate directly with the levels of telomerase activity in the cells tested.

55 Example 3

Characterization of an hTRT intron sequence

A suspected intron was first identified by PCR amplification of human genomic DNA as described in this example, and the result was then confirmed by sequencing the genomic clone XGo5 (see Example 4). PCR amplification was carried out using the forward primer TCP1.57, which in combination with the individual reverse primers TCP1.46, TCP1.48, TCP1.50, TCP1.52, TCP1.54, TCP1.56 and TCP1.58 was (see Table 2). The products of genomic DNA amplified with TCP1.57 / TCP1.46, TCP1.48, 65 TCP1.50, TCP1.52, TCP1.54 or TCP1.56 were about 100 base pairs longer than the products of

114

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

pGRN121 amplifications. The amplification with TCP1.57 / TCP1.58 was the same when using genomic DNA and pGRN121-DNA. This indicated that the genomic DNA contained an insert between the sites for TCP1.58 and TCP1.50. The PCR products of TCP1.57 / TCP1.50 and TCP1.57 / TCP1.52 were directly sequenced with the primers TCP1.39, TCP1.57 and TCP1.49 without subcloning.

As shown below, the intron sequence of 104 bases (SEQ. ID NO: 7) (Fig. 12) in the hTRT mRNA (shown in bold) is on the compound that is linked to bases 274 and 275 of SEQ. ID. No. 1 corresponds to:

CCCCCCGCCGCCCCCTCCTTCCGCCAG / GTGGGCCTCCCCGGGGTCGGCG

TCCGGCTGGGGTTGAGGGCGGCCGGGGGGAACCAGCGACATGCGGAGAG

CAGCGCAGGCGACTCAGGGCGCTTCCCCCGCAG / GTGTCCTGCCTGAAGG AGCTGGTGGCCCG A.GTGCTGCAG

«/» Indicates the spitting points, the sequence shows good correspondences with the «Consen-sus» -5 'and 3' splicing point sequences, which are typical for human introns.

This intron contains motifs which are characteristic of a cleavage site for topoisomerase II and an NFKB binding site (see FIG. 21). These motives are partly of interest because the expression of topoisomerase II is highly regulated in most tumors. Topoisomerase II relaxes DNA by cutting and retrieving the DNA, which increases the expression of certain genes. It has been shown that inhibitors of topoisomerase II act as anti-tumor agents. In the case of NFKB, this transcription factor could play a role in the regulation of telomerase during terminal differentiation. NFKB could play a role in the early repression of telomerase during development and is therefore another target for therapeutic intervention in regulating telomerase activity in cells.

Example 4

Cloning of the lambda phage G <t> 5 and characterization of genomic hTRT sequences a) Lambda Gtf> 5

A human genomic DNA library was screened by PCR and hybridization, and a genomic clone containing sequences encoding hTRT-RNA was found. The bank is a genomic bank of human fibroblasts that was made using DNA from W138 lung fibroblast cells (Stratagene, catalog # 946204). In this bank, partial Sau3AI fragments are inserted into the Xhol site of the lambda vector FIXRII (Stratagene) with an insertion size of 9-22 kb.

This genomic bank was divided into pools of 150,000 phages each and each pool was screened by “nested” PCR (outer primer pair TCP1.52 &TCP1.57; inner primer pair TCP1.49 & TCPl.50, see Table 1). These primer pairs span a suspected intron (see Example 3 above) in the genomic DNA of hTRT and ensure that the PCR product was from a genomic source and not from contamination by the hTRTcDNA clone. Positive pools were further divided until a pool of 2000 phages was obtained. This pool was plated out at low density and by hybridization with a DNA fragment that the base pairs 1552-2108 of SEQ. ID. No. 1 includes (restriction sites Sphl or EcoRV) screened.

Two positive clones were isolated and screened again using nested PCR as described above. Both clones were positive on the edge of the PCR. One of the clones (XGo5) was cleaved with Noti, with an insertion size of about 20 kb. The subsequent mapping (see below) showed an insertion size of 15 kb and showed that the phage Go> 5 contains approximately 13 kb of DNA upstream from the starting point of the cDNA sequence.

The phage G <d5 was mapped by restriction enzyme cleavage and DNA sequencing. The map obtained is shown in FIG. 7. The phage DNA was digested with Ncol and the fragments cloned into pBBS167. The subclones obtained were screened by PCR to identify those containing sequences corresponding to the 5 'region of the hTRT cDNA. A subclone (pGRN140) containing a 9 kb Ncol fragment (with the hTRT gene sequence and 4 to 5 kb of the lambda vector sequence) was partially sequenced to determine the orientation of the insert. pGRN 140 was cut with Sali to remove lambda vector sequences to give pGRN144. pGRN144 was then sequenced. Preliminary results of sequencing are shown in Figure 76 and the results of further sequencing are shown in SEQ. ID. No. 6 (Fig. 21). The

115

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

The 5 'end of the hTRT mRNA corresponds to base 2441 of SEQ. ID. No. 6 (Fig. 21). As shown in Figure 7, two Alu sequence elements are 1700 base pairs upstream of the 5 'end of the hTRT cDNA and these are likely to provide the upstream limit on the promoter region of hTRT. The sequence also has an intron located at base 4173 (SEQ ID NO: 6) (Fig. 21), 3 'to the intron described in Example 3 above.

b) Additional genomic clones

In addition to the genomic clone described above, two P1 bacteriophage clones and a human BAC clone are provided as illustrative embodiments of the invention. The P1 insertions usually have a length of 75 to 100 kb and the BAC insertions usually exceed 100 kb.

The P1 clones (DMPC-HFF # 1-477 (F6) -GS # 15371 and DMPC-HEF # 1-1103 (H6) -GS # 15372) were obtained by PCR screening of a human P1 library by human Foreskin fibroblast cells were derived (Shepherd et al., PNAS USA 91 (1994) 2629) using primers TCP1.12 and UTR2 which amplify the 3 'end of hTRT. These clones were both negative with primers that amplify the 5 'end of hTRT (i.e., no amplification took place).

The human BAC clone (326 E 20) was hybridized to a human genomic BAC library using an 1143 bp Sphl / Xmnl fragment from SEQ. ID. No. 1 (bases 1552-2695) obtained, which includes the RT motif area. This clone is believed to contain the 5 '. It is believed that the genomic hTRT clones in this example encompass the entire hTRT gene.

Example 5

Chromosomal location of the hTRT gene

The hTRT gene was located on chromosome 5p by radiation hybrid mapping (Boehnke et al., Am. J. Hum. Genet. 49 (1991) 1174 and Walter et al., Nature Genet. 7 (1994) 22 ) using the “medium resolution Stanford G3 panel” of 83 RH clones of the entire human genome (generated at the Stanford Human Genome Center). A human lymphoblastoid cell (donor; rM) was exposed to 10,000 rad X-rays and then fused to non-irradiated hamster recipient cells (A3). 83 independent hybrid clones from somatic cells were isolated, each clone representing a fusion event between an irradiated donor cell and a hamster recipient cell. The DNA from panel G3 was used to order markers in the desired area and to establish the distance between these markers.

The primers used for RH mapping were TCP1.12 and UTR2, using 45 cycles with Boehringer Mannheim Taq buffers and Perkin-Elmer Taq polymerase, 45 seconds 94 ° C, 45 seconds 55 ° C and 45, respectively Seconds 72 ° C. The 83 pools were amplified independently and 14 (17%) were positive for hTRT (by occurrence of a 346 bp band). The amplification results were entered into the “Stanford RH server”, which then provided the card position 5p and the next marker, STS D5S678.

By querying the "Genethon genom mapping" website, a YAC was identified during the mapping, which contains the STS marker D5S678: CEPH YAC 780_C_3, size: 390 660 kb. This YAC also contained markers for chromosome 17. This result indicated that the hTRT gene is on chromosome 5, near the telomer end. In a number of tumors there are increased number of copies for 5p. The cat cry syndrome has also been mapped to deletions in this area.

Example 6

Design and construction of vectors for the expression of hTRT proteins and polynucleotides Expression of hTRT in bacteria

The following example describes in detail the design of hTRT-expressing bacterial expression vectors with which large amounts of biologically active full-length hTRT (SEQ. ID. No. 2) can be produced. The production of biologically active hTRT protein in this manner is useful for telomerase reconstitution assays, for the study of modulators of telomerase activity, for the analysis of the activity of newly isolated species of hTRT, for the identification and isolation of compounds that associate specifically with hTRT, for the analysis of the activity of an hTRT protein variant that has been site-specifically mutated as described above, and as an immunogen, to name just a few examples.

116

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

The bacterial expression vector pThioHis A / hTRT

In order to be able to produce large amounts of full-length hTRT (SEQ. ID. No. 2), the bacterial expression vector pThioHis A (Invitrogen, San Diego, CA) was selected as the expression system. The hTRT-encoding insert contains nucleotides 707 to 4776 of the hTRT insertion in the plasmid pGRN121 (SEQ. ID. No. 1). This nucleotide sequence contains the complete coding sequence of the hTRT protein (SEQ. ID. No. 2).

This expression vector according to the invention can be designed for inducible expressions in bacteria. Expression can be induced to encode high levels of a fusion protein in E. coli, which is composed of a cleavable, “HIS-tagged” thioredoxin unit and the full-length hTRT protein. The expression system was used essentially according to the manufacturer's instructions. The amino acid sequence of the vector-encoded fusion protein obtained according to the invention is shown below: (- * -) denotes a cleavage site for enterokinase;

msdkiihltddsfd7dvlkadgailvdfwahwcgpckmiapildeiadeyqgkltvax lridhnpgtapkygirgiptlllfkngevaatxvgalskgqlkefldanlagsgsgdd ddk - * -vpmheleifefaaastqrcvllrtwealapatpampraprcravrsllrshy revlplatfvrrlgpqgwrlvqrgdpaafralvaqclvcvpwdarpppaapsfrqvsc lkelvarvlqrlcergaknvlafgfalldgarggppeapttsvrsylpntvtdalrgs gawglllrrvgddvlvhllarcalfvlvapscayqvcgpplyqlgaatqarppphasg prrrlgcerawnksvreagvplglpapgarrrggsasrslplpkrprrgaapepertp vgqgswahpgrtrc-psdrgfcwsparpaeeatslegalsgtrkshpsvgrqhhagp? ? Stsrpprpwdtfcppvyaetkhflyssgdkeqlrpsfllsslrpsltgarrlvetifl gsrpwmpgtprrlprlpqrywqmrplflellgnhaqcpygvllkthcplraavtpaag vcarexpqgsvaapeeedtdprrlvqllrqhsspwqvygfvraclrrlvppglwgsrh nerrflr-n'tkkf i slgkha: <lslqeltw: o: svrdcawlrrs gvgcvpaaz: - rlrez i lakflkwlmsvywellrsffyvtettfqknrlffyrksvwsklqsigirqhlxrvql relseaevrqhrearpalltsrlrfipkpdglrpivnmdywgartfrrexraerlts rvkalfsvlnyerarrpgllgasvlglddihrawrtfvlrvraqdpppelyfvkvdvt gaydtipqdrltevi as 11kpqntycvrryawqkaahghvrkafkshvstltdlqpy mrqfvaklqetsplrdawieqssslneassglfdvflrfmchhavrirgxsyvqcqg ipqgsilstllcslcygdmenklfagirrdglllrlvddfllvtphlthaxtflrtlv rgvpeygcwnlrktwnf pvedealggtafvqmpahglf pwcgllldtrtlevqsdy ssyartsirasltfnrgfkagrnmrrklfgvlrlkchslfldlqvnslqtvctniyki lllqayrfhacvlqlpfhqqvwknptfflrvisdtaslcysilkaknagmslgakgaa gplpseavqwlchqafllkltrhrvtyvpllgslrtaqtqlsrklpgttltaleaaan palpsdfktild

117

CH 689 672 A9

pGEX-2TK with hTRT nucleotides 3272-4177 from pGRN121

This construct according to the invention is used for the production of fusion protein, for example in order to be able to obtain polyclonal and monoclonal antibodies against the hTRT protein.

5 fragments of hTRT can also be used for other purposes, e.g. for modulating telomerase activity, e.g. as a dominant-negative mutant or to prevent association of telomerase with other proteins or nucleic acids.

For the production of large amounts of an hTRT protein fragment, the E. coli expression vector pGEX-2TK (Pharmacia Biotech, Piscataway N.J.) was selected and used essentially according to the instructions of the manufacturer. An expression system according to the invention was thus produced. The construct obtained contains an insert derived from nucleotides 3272 to 4177 of the hTRT insertion in the plasmid pGRN121. The vector controls the expression of high levels of a fusion protein in E. coli, consisting of the glutathione-S-transferase sequence (underlined below), the cleavage sequence for thrombin (double underlined), the recognition sequence for cardiac muscle protein ki-15 nose (italics), residues introduced by cloning (in brackets, GSVTK) and the hTRT protein fragment (in bold) (SEQ. ID. No. 38) as shown below:

MSPILGYWKIKGLVOPTRLLLEYLES: <YEEHLYERDgGnKWRNK: <? ET. (; Rf: fpkn.pw 20 ISGDVKLTOSMAIIRYIADKKNMLGGCPKE ^ AET.qMLEGAVLDIRYGV.qSTAy.qyn LSLYDMLD? 3rd

KLVCr: <KRI5AIPOIDKYLKSSKYIAWPLOGWQATFGGGDK? P: <SDLVPRGfigga. <7y f

GSVTK] IPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKT 25 FLRTLVRGVPEYGCWNLRKTWNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTL EVQSDYSSYARTSIRASVTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVC TNIYXILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILXAKNAGMSL GAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTA

30 LEAAANPALPSDFKTILD

When this fusion protein was expressed, insoluble aggregates were formed. It was treated generally as described above in the section “Purifying proteins from inclusion bodies”. In this case, induced cells were suspended in PBS (20 mM sodium phosphate, pH 7.4 and 150 mM NACI) and broken up by ultrasound treatment. NP-40 was added up to 0.1% and the mixture incubated for 30 minutes at 4 ° C with gentle mixing. The insoluble material was obtained by centrifugation at 25,000 g at 4 ° C for 30 minutes. The insoluble material was washed once in 4 M urea in PBS, collected by centrifugation and then washed again in PBS. The pellet recovered was estimated to contain more than 75% fusion protein. This material was quickly dried in a vacuum, then suspended in an adjuvant for injection into mice and rabbits to generate antibodies. The recombinant protein is separated from the glutathione S-transferase unit by site-specific proteolysis using thrombin in accordance with the manufacturer's instructions.

45

pGEX-2TK with nucleotides 2426 to 3274 of pGRN121 with an «HIS-8 tag»

Another E. coli expression vector pGEX-2TK (Pharmacia Biotech, Piscataway N.J.) construct was produced to produce large quantities of a fragment of hTRT. This construct contains 50 an insert that consists of nucleotides 2426 to 3274 of hTRT insertion in plasmid pGRN121 (SEQ. ID. No. 1) and a sequence encoding eight consecutive histidine residues (HIS-8 tag). To insert this “HIS-8 tag”, the vector pGEX-2TK was linearized with the hTRT nucleotides 2426 to 3274 of pGRN121 with BamHI. This opens the plasmid at the junction between GST-thrombin-myocardial proteinase and the sequence encoding hTRT. A double-stranded oligonucleotide with BamHI-compatible ends, as seen in the sequence shown below, was ligated to the linearized plasmid 55, which resulted in the insertion of eight histidine residues upstream of the hTRT sequence in the reading frame.

In E. coli, the vector controls the expression of high levels of a fusion protein which is composed of the glutathione-S-transferase sequence (underlined); the cleavage sequence for Throm-60 bin (double underlined); the recognition sequence for cardiac muscle protein kinase (italic); a set of three and a set of five residues introduced by cloning (GSV and GSVTK) eight on successive histidines (also underlined) and the hTRT protein fragment (in bold) (SEQ. ID. No. 39):

65

118

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

mspilgywkikglvoptrllleyleekyeehlyerdf.gdkwrnkkfelgi.f.fp nt.pyyidodvkt.tosmaiiryiadkhnmlggcpkeraeismlegavldirygvs rtayskdfetlkvdflskt.pemlkmff.dri.chktylngdhvthpdfmlydal dvvlymdpmct.dafpklvcfkkrieaîpoidkylksskytawplogwqatfgg gdhppksplvprgsflflawgsvlhhhhhhhhrgsvtklmsvyvvellrsffyv tettfqknrlffyrpsvwsklqsigirqhlkrvqlrelseaevrqhrearp alltsrlrfipkpdglrpivnmdywgartfrrekraerltsrvkalfsvl nyerarrpgllgasvlglddihrawrtfvlrvraqdpppelyfvkvdvtg

AYDTIPQDRLTEVIASnKPQNTYCVRRYAWQKAAHGHVRKAFKSHVSTL

tdlqpymrqfvahlqetsplrdawteqssslneassglfdvflrfmchha vrirgksyvqcqgi

This vector can be used to produce fusion protein to generate polyclonal and monoclonal antibodies to an hTRT protein. This fusion protein can also be used to affinity purify antibodies directed against hTRT peptides contained within the fusion protein. The recombinant protein can be separated from the glutathione-S-transferase unit by site-specific proteolysis using thrombin in accordance with the manufacturer's instructions.

pGEX-2TK with the hTRT nucleotides 2426 to 3274 of pGRN121 without «HIS-8-TAG»

Another E. coli expression vector pGEX-2TK (Pharmacia Biotech, Piscataway N.J.) construct was produced to produce large amounts of an hTRT fragment. This vector construct can be used to produce fusion protein for the generation of polyclonal and monoclonal antibodies against the hTRT protein. This fusion protein can also be used to affinity purify antibodies directed against hTRT peptides contained in the fusion protein.

This construct contains an insert derived from nucleotides 2426 to 3274 of the hTRT insertion in plasmid pGRN121 (SEQ. ID. No. 1), but without the "His-8-tag". In E. coli, the vector controls the expression of high levels of a fusion protein which is composed of glutathi-on-S-transferase (underlined), a cleavage sequence for thrombin (double underlined), a recognition sequence for the heart muscle protein kinase (italic), residues introduced by cloning (in brackets, GSVTK) and the hTRT protein fragment (in bold) (SEQ. ID. No. 40):

MSPILGYWKIKGLVOPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYY IDGDVKLTOSMAIIRYIADKHNMT.GGCPKERAEISMLEGAVLDIRYGVSRTAYSKDFF. TLKVDFLSKLPEMLKMFEDRLCHKTYLNGDKVTHPDFMLYDALDVVLYMPPMCLPAFP KLVCFKKRIEAIPOrDKYLKSSKYIAWPLOGWOATFGGGDHPPKSnT.VPRGSgRASVr GSVTK] MSVYWELLRSFFYVTETTFQKNRLFFYRPSVWSKLQSIGIRQHLKRVQLRE L S EAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMD YWGARTFRREKRAERLTSRK ALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPEYFVKVDVTGAYD TIPQDRLTEVIASIIKPQNTYCVRRYAWQKAAHGVRKAFKSHVSTLTDLQPYMRQFV AHLQETSPLRDAWIEQSSSLNEASGLFDVFLRFMCHHAVRIRGKSYVQCQGI

The recombinant protein can be separated from the glutathione S-transferase unit by site-specific proteolysis using thrombin in accordance with the manufacturer's instructions.

pGEX-2TK with the hTRT nucleotides 1625 to 2458 of gPRN121

Another E. coli expression vector pGEX-2TK (Pharmacia Biotech, Piscataway N.J.) construct was produced to produce large amounts of an hTRT protein fragment. This vector construct can be used to produce fusion protein for generating polyclonal and monoclonal antibodies against an hTRT protein. In addition, this fusion protein can be used to affinity purify antibodies directed against hTRT peptides contained in the fusion protein.

This construct contains an insert from nucleotides 1625 to 2458 of the hTRT insertion

119

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

in plasmid pGRN121 (SEQ. ID. No. 1). In E. coli, the vector controls the expression of high levels of a fusion protein which is composed of glutathione-S-transferase (underlined), a cleavage sequence for thrombin (double underlined), a recognition sequence for cardiac muscle protein kinase (italics) Cloning introduced residues (in brackets, GSVTK) and the hTRT protein fragment (in bold) (SEQ. ID. No. 41):

MSPII-GYWKIKGLVOPTRLI .LEYLEEKYEEHLYERDEGDKWRNKKFF.T .GT .F.FP

NLPYYTDGDVKLTQSMAITRYIADKHNMLGGCPKERAEISMLEGAVT.DTRYGV

SRIAYSKDFETLKVDFLSKLPF.MLKMFEDRLCHKTYLNGDHVTHPDFMI.YDA

LDWLYMDPMCLDAFPKLVCFKKRIEAIPOIDKYLKSSKYIAWPLOGWOATFG

GGDHPPKSDLVPRGSfl & 4SVrGSVTKlATSLEGALSGTRHSHPSVGROHHAGP

PSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGÀRR

LVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLL

KTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPW

QVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELT

WKMSVRDCAWLRJRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLR

S

The recombinant protein is separated from the glutathione-S-transferase unit by site-directed proteolysis using thrombin in accordance with the manufacturer's instructions.

pGEX-2TK with hTRT nucleotides 782-1636 from pGRN121

Another E. coli expression vector pGEX-2TK (Pharmacia Biotech, Piscataway N.J.) construct was produced to produce large amounts of an hTRT protein fragment. This vector construct can be used to produce fusion protein for generating polyclonal and monoclonal antibodies against an hTRT protein. In addition, this fusion protein can be used to affinity purify antibodies directed against hTRT peptides contained in the fusion protein.

This construct contains an insert derived from nucleotides 782 to 1636 of hTRT insertion in plasmid pGRN121 (SEQ. ID. No. 1). In E. coli, the vector controls the expression of high levels of a fusion protein which is composed of glutathione-S-transferase (underlined), a spa sequence for thrombin (double underlined), a recognition sequence for cardiac muscle protein kinase (italics) Cloning introduced residues (in brackets, GSVTK) and an hTRT protein fragment (in bold) (SEQ. ID. No. 42):

MSPILGYWXIKGT.VOPTRLLLEYLEEXyEEHLYERPF.GDKWRNXKFELGLEFPNLPYY IDGDVKLTOSMATIRYIADXHNMLGGCPKERAEISN'.LEGAVLDIRYGVSRTAYSXDFE TLXLKDTVFDMLRLCLKKDFLFLMLDLCLKKDFLFLMDG

GSVTK] MPRAPRCRAVRSLLSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQ CLVCVPWDARPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPP EATTSVRSYL PNTVTD ALRGS GAWGL L LRRVGDD VLVHLLARCAL FVL VAP CAYQ VC G PPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASR SLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCWSPARPAEEATSL

The recombinant protein is separated from the glutathione-S-transferase unit by site-specific proteolysis using thrombin in accordance with the manufacturer's instructions.

pT7FLhTRT with hTRT cDNA lacking the 5 'non-coding sequence

As described above, in one embodiment, the invention provides an hTRT that is site-modified to facilitate cloning in bacterial, mammalian, yeast, and insect expression vectors and does not contain a 5'-untranslated hTRT sequence. In some circumstances, minimizing the amount of non-protein coding sequence allows for improved protein production (yield) and leads to increased mRNA stability. In this invention

120

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

According to the embodiment, the 5'-non-coding region of hTRT was removed in the bacterial expression vector before cloning.

This was accomplished by creating an additional restriction site immediately upstream (5 ') from the start codon (ATG) of the hTRT coding sequence (Fig. 16). The creation of a restriction site immediately 5 'to the coding region of the protein allows the efficient production of a large number of vectors which encode fusion proteins, for example fusion proteins which contain labels and peptide "TAGs" for immunodetection and purification.

In this case, the oligonucleotide 5'-CCGGCCACCCCCCATATGCCGCGCGCTCCC-3 'as described above was used to modify the hTRT cDNA nucleotides 779 to 781 of the hTRT cDNA (Fig. 16) from GCG to CAT. These 3 nucleotides are the last nucleotides before the ATG start codon, so this change does not lead to modification of the protein sequence. The sequence change leads to the creation of a single Ndel restriction site in the hTRT cDNA. Single stranded hTRT DNA was used as a source of DNA for site-directed mutagenesis. The plasmid obtained was sequenced to confirm successful mutagenesis.

This modification allowed the construction of the plasmid according to the invention described below with the name pT7FLhTRT. The site-specifically modified hTRT sequence (addition of the Ndel restriction site) was cleaved with Ndel and Noti, an hTRT fragment being generated. This fragment was then cloned into a pSL3418 plasmid which had previously been cut with Ndel and Smal (also a restriction enzyme whose cleavage results in blunt ends). pSL 3418 is a modified paED4 plasmid into which a FLAG sequence (Immunex Corp. Seattle WA) and an enterokinase sequence have been inserted immediately upstream of the aforementioned Ndel site. This plasmid, called pT7FLhTR, allows full-length expression of hTRT (with a "flag tag" at its 5 'end) in an E. coli strain that expresses T7 RNA polymerase.

Vector in which an hTRT coding sequence is controlled by the MPSV promoter

The invention also provides a mammalian expression vector in which hTRT is oriented so that the hTRT coding sequence is controlled by the MPSV promoter (described below). An EcoRI restriction fragment from pGRN137 containing the hTRT ORF was cloned into the EcoRI site of pBBS212 (see below), thereby removing the 5'-untranslated region (5'-UTR) from hTRT (pGRN137 was excised) a Sall-Sse8387 I fragment of pGRN130 (described below) that contains the Kozak mutation of hTRT, constructed in the Sall-SSE 83871 sites of pGRN136, to produce a mammalian expression plasmid called hTRT (a «Kozak- Consensus sequence contains) from the MPSV promoter (pGRN136 was constructed by cutting out a Hindlll-Sall fragment from pGRN126 containing the hTRT-ORF and cloning into the Hindlll-Sall sites of pBBS242. This produces a mammalian expression plasmid expressing hTRT from the MPSV promoter). This gave a mammalian expression plasmid called pGRN145, which expresses hTRT with a “Kozak Consensus” sequence using the MPSV promoter. See also the mammalian expression vector, pGRN152-MPSV promoter, which is described below.

Plasmids with hTRT cDNA lacking the 3 'non-coding sequence

As discussed above, the invention provides expression vectors containing TRT-encoding nucleic acids in which some or all of the non-coding sequences have been deleted. In some circumstances, minimizing the amount of non-protein coding sequence allows for improved protein production (yield) and increases mRNA stability. In this embodiment according to the invention, the 3 "untranslated region of hTRT was deleted before cloning into the bacterial expression plasmid.

Plasmid pGRN121, which contains full length hTRT cDNA as discussed above, was first deleted for all APAI sites. This was followed by the deletion of the MscI-Hincll-hTRT restriction fragment, which contains the 3-'UTR. The Ncol-Xbal fragment containing the stop codon of hTRT was then inserted into the Ncol-Xbal site of pGRN121 to give a plasmid called pGRN124, which is equivalent to pGRN121, except that it was 3 "- UTR is missing.

Bacterial expression vectors with antibiotic selection markers

The invention also provides bacterial expression vectors containing selection markers for conferring a selectable phenotype in transformed cells and sequences encoding the maintenance of the vector as an episome and replication so that integration into the host genome is not required. For example, the marker can encode biotic resistance, for example resistance to chloramphenicol (see Harrod, Nucleic Acids Res. 25 (1997), 1720-1726), Kanamycin, G418, bleomycin and hygromycin, which allows the selection of the cells that contain the desired DNA -Se-

121

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

sequences were transformed; see, for example, Blondelet-Rouault, Gene 190 (1997), 315-317 and Mahan, Proc. Nati. Acad. Be. USA 92: 669-673 (1995).

In one embodiment according to the invention, the full-length hTRT (SEQ. ID. No. 1) was cloned into a modified “BlueScript” plasmid vector (Stratagene, San Diego, CA), which bears the designation pBBS235, and in which a resistance gene for the Antibiotic chloramphenicol had been inserted. The NotI fragment of pGRN124 (discussed above) containing the hTRT ORF was cloned into the NotI site of pBBS235 so that the TRT ORF is in the opposite direction to the lac promoter of the vector. This creates a plasmid which is suitable for the mutagenesis of plasmid insertions, for example the TRT nucleic acids according to the invention. This plasmid construct with the name pGPN125 is used in the methods according to the invention which include mutagenesis of telomerase enzyme and sequences coding for TRT protein. It can also be used for the in vitro transcription of hTRT using the T7 promoter and the in vitro transcription of "antisense" hTRT using the T3 promoter.

In a further embodiment according to the invention, the hTRT-ORF-containing NotI restriction fragments of pGRN124 were subcloned into the Notl site of pBBS235 (described below), so that the TRT-ORF has the same orientation as the lac promoter of the vector . This creates a plasmid called pGRN126 that can be used to express full-length hTRT in E. coli. The expressed product contains 29 amino acids encoded by the vector pBBS235, followed by 18 amino acids encoded by the 5'-UTR of hTRT, followed by the full-length hTRT protein.

In a further embodiment according to the invention, an in vitro mutagenesis was carried out for converting the ATG initiation codon from hTRT in a “Kozak consensus” sequence and for generating EcoRI and BglII restriction interfaces to facilitate cloning into the bacterial expression vectors carried out by pGRN125.

In this mutagenesis procedure, the oligonucleotide

5'-T GCGCACGTGGGAAGCCCT GGCagatctgAatt CcaCcATGCCGCGCGCTCCCCGCTG-3 '

(SEQ. ID No. 43) is used. This new expression vector was named pGRN127.

In a further embodiment according to the invention, the second Asp of the TRT “DD motif” (described above) was converted to an alanine in order to generate a non-functional telomerase enzyme and thus a mutant TRT protein for use in experiments with dominant / negative mutants. hTRT (SEQ. ID No. 1) was mutagenized in vitro with the oligonucleotide

5 '- CGGGACGGGCTGCTCCTGCGTTTGGTGGAcGcgTTCTTGTTGGTGACACCTCA C CTCACC-3 "

(SEQ. ID # 44) to convert the asparagine at residue 869 (Asp869) (from SEQ. ID # 2) to an alanine (Ala) codon. This also created a Mlul restriction site. This expression plasmid was given the designation pGRN130 and also contains the “Kozak Consensus” sequence as described for pGRN127.

The invention also provides a vector designed to express an "antisense" sequence fragment from hTRT. The plasmid pGRN126 was with the restriction enzymes Mscl and Sma! completely cleaved and religated, with over 95% of the hTRT ORF being deleted in mammalian cells. A Smal-MscI fragment was reinserted during this process to generate CAT activity again. This unpurified plasmid was then cut again with Sali and EcoRI and the fragment containing the initiation codon of hTRT was inserted into the SalI-EcoRI sites of pBBS212 (described above). This created an "antisense" expression plasmid that expresses the "antisense" sequence that spans the 5'-UTR and 73 base pair residues of the hTRT-ORF. This plasmid was named pGRN135.

Expression of hTRT telomerase in yeast

In the following example, the construction of the hTRT-expressing yeast expression vectors according to the invention for the production of large amounts of biologically active hTRT (SEQ. ID. No. 2) is shown in full length. The use of biologically active hTRT in many embodiments according to the invention has been described above.

Pichia pastoris full length pPICZ B and hTRT expression vector

The Pichia pastoris expression vector PPICZ B (Invitrogen, San Diego, CA) was selected for the production of large amounts of biologically active hTRT. The insertion for the hTRT coding Se122

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

The sequence was derived from nucleotides 659 to 4801 of the hTRT insertion in plasmid pGRN121 (SEQ. ID. No. 1). This nucleotide sequence contains the full-length hTRT coding sequence (SEQ. ID. No. 2). This expression vector was designed for the inducible expression of large amounts of unmodified hTRT protein (SEQ. ID. No. 2) with full length in P. pastoris. Expression is controlled by a yeast promoter, but the expressed sequence uses the hTRT initiation and termination codons. No exogenous codons were introduced by cloning. The resulting PPICZ B / hTRT vector (Invitrogen, San Diego, CA) was used to transform the yeast.

Pichia pastoris expression vector hTRT-His6 / pPICZ B

A second Pichia pastoris expression vector according to the invention, which was derived from pPICZ B (Invitrogen, San Diego, CA), also contains the full-length hTRT coding sequence (SEQ. ID. No. 2), which is derived from nucleotides 659 to 4801 of the hTRT insertion in plasmid pGRN121 (SEQ. ID. No. 1). This hTRT-His6 / pPICZ B expression vector encodes the full-length hTRT protein (SEQ. ID. No. 2) and fuses with the Myc epitope and His6 reporter sequences at its C-terminus. The hTRT stop codon was removed and replaced by vector sequences encoding the Myc epitope and the His6 reporter tag, as well as a stop codon. This vector was designed to control the inducible expression of the subsequent high level fusion protein in yeast, this fusion protein comprising the hTRT sequence (underlined), vector sequences (in brackets L and NS-AVD), the Myc epitope (double underlined) and the His6 - «day» (italics) (SEQ. ID. No. 45) exists:

mpraprcravrsllrshyrevlplatfvrrlgpogwrlvorodpaafraiva oci.vcvpwdarpppaapsfrovsclkelvarvi.orlcergaknvlafgfat.r.

dgarggppeafttsvrsylpntvtdalrgsgawglllrrvgddvlvhllarc alfvlvapscayovcgpplyolgaatoarppphasgprrrlgcerawhhsvr eagvplglpapgarrrggsasrslplpkrprrgaapepertpvgogswahpgr trgpsdrgfcwsparpaefatslegalsgtrhshpsvgrohhagppstsrppr pwdtpcppvyaetkhflyssgdkfolrpsfllsslrpsltgarrlvetiflgsrp wmpgtprrlprlporywomrplflellgnhaocpygvllkthcplraavtpa agvcarekpogsvaapeeedtdprrlvqllrohsspwovygfvraclrrlvp pglwgsrhnerrflrntkkfislgkhaklsloeltwkmsvrdcawlrrspgv gcvpaaehrlrffii.akflhwlmsvywellrsffyvtettfoknrlffyrks vwsklosrgirohlkrvolrelseaevrohrearpalltsrlrftpkpdglrprv nmdywgartfrrekraerltsrvkalfsvlnyf.rarrpgllgasvlglddih rawrtfvlrvraodpppelyfvkvdvtgaydtipodrlteviashkpontycv rryavvokaahghvrkafkshvsti.tdlopymrofvahloftsplrdawie ossslneassglfdvflrfmchhavrirgksyvocogipogsilstllcslcygd menklfagirrdgrjj-rivddfllvtphlthaktflrtlvrgvpfygcvvnt.r ktvvnfpvedeai.ggtafvqmpahglfpwcgllr.dtrtlevosdyssyart.si rasltfnrgfkagrnmrrklfgvlrlkchslfldlovnslotvctn1ykillt.o

AGPLPSEAVOWTCHOAFLLKr.TRHRVTYVPr.T GST.RTA.OTOLSRKLPGTTI.TA LEAAAXPAl.PSnFKTU nrLlEOKLISEF.Dr.rNSAVTÏÏHHHHHH

Expression of hTRT in insect cells

In the following example, the construction of hTRT-telomerase-expressing expression vectors for insect cells for the production of large amounts of biologically active hTRT with full length (SEQ. ID. No. 2 and SEQ. ID. No. 4) is described in detail.

123

CH 689 672 A9

Full-length baculovirus expression vector pVL1393 and hTRT

The desired telomerase coding sequence was cloned into the baculovirus expression vector pVL1393 (Invitrogen, San Diego, CA). This construct was then cotransfected in Spodoptera fungupei-da (sf-9) cells with linearized DNA from the Autograph California nuclear-polyhedrosis virus (Baculogold-AcMNPV). The recombinant baculoviruses obtained were then plaque-cleaned and expanded in accordance with published protocols.

This expression vector allows the expression of high levels of full-length hTRT protein in insect cells. Expression is controlled by a baculovirus polyhedrin gene promoter. No exogenous codons were introduced by cloning.

Full-length baculovirus expression vector pBlueBacHis2 B and hTRT

The baculovirus expression vector pBlueBacHis2 B (Invitrogen, San Diego, CA) was selected as a source of control elements to produce large amounts of biologically active full-length hTRT (SEQ. ID. No. 2). The hTRT-coding insert consisted of nucleotides 707 to 4776 from the hTRT insertion in plasmid pGRN121 (SEQ. ID. No. 1), which contains the full-length hTRT coding sequence (SEQ. ID. No. 2).

A full length hTRT with His6 and anti-Xpress tags (Invitrogen) was also constructed. This vector contains an insert consisting of nucleotides 707 to 4776 of the hTRT insertion of plasmid pGRN121 (SEQ. ID. No. 1). This vector controls the expression of high levels of full-length hTRT protein fused to a cleavable 6-histidine and anti-Xpress tags in insect cells. The amino acid sequence of the fusion protein is shown below; (- * -) indicates a cleavage site for enterokinase:

MPRGSHHHHHHGMASMTGGQQMGRDLYDDDDL - * - DPSSRSAAGTMEFAAAS

TQRCVLLRTWEALAPATPAMPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQ

GWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQ

RLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGL

LLRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHAS

GPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAP

EPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGTRHSHPS

VGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRP

SLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPY

GVLLKTHCPLRAAVTPÂAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSP

WQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTW

KMSVRDCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFY

VTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPAL

LTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERA

RRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQD

RLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQF

VAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVR1RGKSYVQCQ

GIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFL

RTLVRGVPEYGCWNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLD

TRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQ

VNSLQTVCTNIYK1LLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCY

SILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSL

RTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD

Baculovirus expression vector pBlueBac4.5 and full-length hTRT protein

A second baculovirus expression vector, pBlueBac4.5 (Invitrogen, San Diego, CA), was constructed to produce large amounts of full-length biologically active hTRT (SEQ. ID. No. 2). The hTRT coding insert consisted of nucleotides 707 to 4776 from the hTRT from the plasmid

124

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

pGRN121 (SEQ. ID. No. 1) This nucleotide sequence contains the full-length hTRT coding sequence (SEQ. ID. No. 2).

Baculovirus expression vector pMelBacB and full-length hTRT protein

A third baculovirus expression vector pMelBacB (Invitrogen, San Diego, CA) was constructed to produce large amounts of biologically active full-length hTRT (SEQ. ID. No. 2). The hTRT-encoding insert also consists of nucleotides 707 to 4776 from the hTRT insert from plasmid pGRN121 (SEQ. ID. No. 1). This nucleotide sequence contains the full-length hTRT coding sequence (SEQ. ID. No. 2).

pMelBacB controls the expression of full-length hTRT (SEQ. ID. No. 2) in insect cells into the extracellular medium via the secretory metabolic pathway using the melittin signal sequence. High levels of hTRT with full length (SEQ. ID. No. 2) are thus secreted. The melittin signal sequence is cleaved after excretion, but is part of the protein pool that remains intracellular. For this reason, it is given in parentheses in the following sequence. The sequence of the fusion protein (SEQ. ID No. 38) encoded by the vector is shown below:

(Mkflvnvalvfmvvyisyiya) - * - dpssrsaagtmefaaastqrcvllrt \ vea lapatpampraprcravrsllrshyrevlplatfvrrlgpqgwrlvqrgdpa afralvaqclvcvpwdarpppaapsfrqvsclkelvarvlqrlcergaknvl afgfalldgarggppeafttsvrsylpntvtdalrgsgawglllfüivgddvl vhllarcalfvlvapscayqvcgpplyqlgaatqarppphasgprrrlgcera wnhsvreagvplglpapgarrrggsasrslplpkrprrgaapepertpvgqgs wahpgrtrgpsdrgfcwsparpaeeatslegalsgtrhshpsvgrqhhagpp stsrpprpwdtpcppvyaetkhflyssgdkeqlrpsfllsslrpsltgarrlvet iflgsrpwmpgtprrlprlpqrywqmrplflellgnhaqcpygvllkthcplr aavtpaagvcarekpqgsvaapeeedtdprrlvqllrqhsspwqvygfvrac lrrlvppglwgsrhnerrflrntkkfislgkhaklslqeltwkmsvrdcawl rrspgvgcvpaaehrlreeilakflhwlmsvywellrsffyvtettfqknrl ffyrksvwsklqsigirqhlkrvqlrelseaevrqhrearpalltsrlrpipkpd glrpivnmdyvvgartfrrekraerltsrvkalfsvlnyerarrpgllgasvl glddihrawrtfvlrvraqdpppelyfvkvdvtgaydtipqdrlteviasiikp qntycvrryawqkaahghvrkafkshvstltdlqpymrqfvahlqetsplr davvieqsssineassglfdvflrfmchhavrjrgksyvqcqgipqgsilstllc slcygdmenklfagirrdglllrlvddfllvtphlthaktflrtlvrgvpeyg cwnlrktwnfpvedealggtafvqm pahglfpwcgllldtrtlevqsdys syartsirasltfnrgfkagrnmrrklfgvlrlkchslfldlqvnslqtvctni ykilllqayrfhacvlqlpfhqqvwknptfflrvisdtaslcysilkaknagps lgaklltakllqqlqqlqlqql

Expression of hTRT in mammalian cells

The recombinant hTRT according to the invention can be produced in large quantities as a biologically active full-length hTRT (SEQ. ID. No. 2) in a large number of mammalian cell lines. This biologically active, mammalian hTRT is useful in many embodiments of the invention as discussed above.

MPSV hTRT expression plasmids

The invention also provides an expression system for use in mammalian cells that provides the highest possible expression of a recombinant protein, such as telomerase, without the need to modify the coding sequence (for example, by optimizing codon usage). In one embodiment, the invention provides an MPSV mammalian ex

125

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

ready systems that can express the TRTs of plasmid pBBS212 according to the invention, described as pMPSV-TM in Lin J.H., Gene 47 (1994), 287-292. The MPSV plasmids can be expressed as either stable or transient clones.

In this expression system, exogenous control elements for transcription are incorporated into the vector, while the hTRT coding sequence (SEQ. ID. No. 1) itself remains unchanged. The “myeloproliferative sarcoma virus (MPSV) LTR (MPSVLTR) promoter, which is enhanced by the cytomegalovirus (CMV) enhancer, is incorporated for the initiation of transcription. This promoter shows consistently higher expression levels in cell lines (see Lin, J.H (1994) op. Cit.). A “Kozak consensus” sequence can be incorporated for the initiation of translation (see Kozak, Mamm. Genome 7 (1996), 563-574). Any extra 5 'and 3' hTRT sequences that are not translated can be removed to ensure that these sequences do not interfere with expression, as previously discussed. The MPSV plasmid containing the complete hTRT coding sequence, which contains all the extra sequences, is called pGRN133. A control hTRT "antisense" plasmid was also constructed. This vector is identical to pGRN133, except that the TRT insertion is the antisense sequence of hTRT SEQ. ID. No. 1 (this «antisense» control vector is called pGRN134). The MPSV plasmid which contains the complete hTRT coding sequence, but from which all extra sequences have been removed, and which contains the “Kozak Consensus” sequence, is designated pGRN155.

Two selection markers, PAC (puromycin-N-acetyl-transferase = puromycin resistance) and HygB (hy-gromycin B = hygromycin resistance) are available for the selection of the plasmids after transfection (see the discussion above, which relates to selectable markers ). Double selection using markers on both sides of the vector polylinker should increase the stability of the hTRT coding sequence. A sequence coding for DHFR (dihydrofolate reductase) is inserted in order to allow the amplification of the expression cassette after the generation of stable clones. Further measures for gene amplification can also be used to increase the yields of recombinant proteins.

The invention also provides MPSV mammalian expression plasmids that contain hTRT fusion proteins. In one embodiment, the hTRT sequence, which has retained its 5 'untranslated region, is linked to an epitope "flag", for example in the IBI FLAG (Intranational Biotechnologies Inc. (IBI) Kodak, New Haeven, CT) and inserted into the MPSV expression plasmid (called pGRN147). This special construct contains a "Kozak" initiation site for translation. The expressed fusion protein can be purified using the M-1 anti-FLAG octa-peptide monoclonal antibody (IBI, Kodak, supra).

In a further embodiment, hTRT is changed in a site-specific manner. An amino acid residue codon is mutagenized, whereby aspartic acid at position 869 (SEQ. ID. No. 1) has been converted into an alanine. This Asp869- »Ala-hTRT mutant, which had retained its 5'-untranslated region and into which a« Kozak »sequence had been inserted, was inserted into an MPSV expression plasmid and was designated pGRN146. The Asp869-> Ala-hTRT mutant was further modified to contain the FLAG sequence as described above and the cloned insert was inserted into an MPSV expression plasmid. This expression plasmid was designated pGRN154. In particular, for pGRN154, an Eam1105l restriction fragment of pGRN146, which contained the “Kozak” sequence, which in turn contained the “front end” (5 ′ segment) of hTRT, is cloned into the Eam11051 sites of pGRN147 (see above), so as to construct an MPSV expression plasmid which can express hTRT together with a "Kozak" sequence and the D869- "A mutation described above and the" IBI flag ".

A further embodiment according to the invention provides a further expression plasmid which is derived from pGRN146. The mammalian expression plasmid, designated pGRN152, was generated by cutting out the EcoRI fragment from plasmid pGRN146 (containing the hTRT ORF) and cloned into the EcoRI site of pBBS212 to remove the 5'-UTR of hTRT. The hTRT is oriented so that its expression is controlled by the MPSV promoter. This creates a mammalian expression plasmid that expresses hTRT together with a “Kozak Consensus” sequence and the D869-> A mutation using the MPSV promoter.

Expression of hTRT in 293 cells using the episomal vector pEBVHis

The episomal vector, pEBVHis (Invitrogen, San Diego, CA), was modified to express an hTRT (SEQ. ID. No. 2) fusion protein that hTRT with an epitope "tag" of an N-terminal extension that " Xpress »-Epitap (Invitrogen, San Diego, CA), expressed (labeled pGRN122). The NotI-hTRT fragment of pGRN121, which contains the open reading frame (ORF) of hTRT, was cloned into the NotI site of pEBVHisA so that the hTRT-ORF is in the same orientation as the Rous Sarcoma Virus (RSV) Promoter of the vector. In this orientation, the His6 flag was relatively closer to the N-terminus of the hTRT.

A control vector was also constructed which contained the "antisense" sequence of hTRT and the epitope "tag" (the control plasmid was designated pGRN123). The vector was transfected into 293 cells and the translated hTRT was identified and analyzed for the “Xpress” epi-

126

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

top specific antibody isolated. pEBVHis is a hygromycin resistant episomal EBV vector that expresses the desired protein fused to an N-terminal peptide. The cells carrying the vector are selected and expanded and then nuclear and cytoplasmic extracts are prepared. These both control extracts are immunoprecipitated with anti-Xpress antibody and the immunoprecipitated beads are tested for telomerase activity using a typical assay.

Expression of recombinant hTRT in mortal normal diploid human cells

In an embodiment according to the invention, recombinant hTRT and necessary telomerase enzyme complex components can be expressed in normal dipolar mortal cells in order to achieve their direct immortalization or to facilitate their immortalization. This would allow one to obtain diploid immortal cells with an otherwise normal phenotype and karyotype. As discussed above, this use of telomerase is of enormous commercial importance.

"Sense" -hTRT (Fig. 16) and "antisense" -hTRT (SEQ. ID No. 45) were cloned in a CMV vector. These vectors were purified and transfected into two clones from normal mortal diploid human cells. The human clones were the freshly passed diploid human cell strains BJ and IMR90.

Analysis of telomerase activity using a TRAP assay (using the “TRAPeze” kit (Oncor, Inc., Gaithersburg, MD)) showed that when transfected with “sense” -hTRT, both in the cell strain BJ and IMR90 telomerase- Activity was generated, but not when transfected with «antisense» hTRT.

Expression of recombinant hTRT in immortalized human IMR90 cells

Using the same hTRT "sense" construct cloned into the CMV vectors used in the studies described above with diploid human BJ and IMR90 cell strains, the immortalized SW13 ALT "pathway" cell line (an IMR90 cell immortalized with SV40 antigen) transiently transfected. Using a TRAP assay (TRAPeze, Oncor, Inc., Gaithersburg, MD) it was possible to show that telomerase activity was generated in transfected cells with the “sense” construct.

Vectors for the regulated expression of hTRT in mammalian cells: inducible and repressible expression of hTRT

The invention provides vectors which can be manipulated so as to be able to induce or repress the expression of the TRTs according to the invention, for example hTRT. For example, hTRT (SEQ. ID. No. 1) was incorporated into the Invitrogen's ecdysone-inducible expression system (San Diego, CA) and the "Tet-On and Tet-Off" tetracycline-regulated systems from Clontech Laboratories, Inc. ( Palo Alto, CA) cloned. Such inducible expression systems are provided for those methods according to the invention in which it is important to be able to control the level or the rate of transcription of transfected TRT. For example, the invention provides cell lines that are immortalized by the expression of hTRT. Such cells can be made "mortal" by inhibiting hTRT expression by the vector using its transcriptional control elements, for example the "tet-off" system. The invention also provides methods in which TRT is only expressed transiently so as to avoid the constitutive expression of hTRT, which could lead to an undesired "immortalization" of the transfected cells, as already discussed above.

The ecdysone-inducible mammalian expression system was designed to allow for the regulated expression of the desired gene in mammalian cells. The system is characterized by its tightly regulated mechanism, which allows practically no basal transcription in mammalian cells and is more than 200 times inducible. The expression system is based on the Drosophila heterodimeric ecdysone receptor. In the Ecdyson-inducible expression system, an analog of the steroid hormone Ecdyson, Muristeron A is used to activate the expression of hTRT via a heterodimeric nuclear receptor. Expression levels were reported to be more than 200-fold above base levels with no effect on the physiology of the mammalian cell (No, "Ecdysone-Inducible Gene Expression in Mammalian Cells and Transgenic Mice", Proc. Nati. Acad. Sei. USA 93: 3346-3351 (1996). After binding of Ecdyson or Muristeron, an analog of Ecdyson, the receptor activates an ecdysone-responsive promoter, which leads to the controlled expression of the desired gene. In the ecdysone-inducible mammalian expression system, both monomers of the heterodimeric receptor are constitutively expressed by the same vector pV-gRXR. The ecdysone-responsive promoter that ultimately controls expression of the desired gene is located on a second vector pIND that controls the transcription of the desired gene.

hTRT is cloned into the vector pIND (Clontech Laboratories, Inc., Palo Alto, CA), which modifies 5

127

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

contains ecdysone responsive elements (E / GREs) upstream of a minimal heat shock promoter and the multiple cloning site. This construct is then used to transfect cell lines that have previously been modified so that they stably express the ecdysone receptor. After transfection, the cells are treated with Muristeron A to induce intracellular expression of pIND.

The “Tet-on and Tet-off” expression systems (Clontech, Palo Alto, CA) allow access to the regulated gene expression systems with high-level expression, which Gossen says “Tight control of gene expression in mammalian cells by tetracycline responsive promoters », Proc. Nati. Acad. Be. USA 89 (1992), 5547-5551, with regard to the “tet-off” transcriptional repression system, see also Gossen, “Transcriptional activation by tetracycline in mammalian cells”, Science 268 (1995), 1766-1769 with regard to the “ Tet-On »inducible transcription system. In "Tet-Off" transformed cell lines, gene expression is switched on when tetracycline (Tc) or doxycycline ("Dox", a Tc derivative) are removed from the culture medium. In contrast, in "Tet-On" cell lines the expression is switched on by the addition of Tc or "Dox" to the medium. Both systems allow the tightly regulated expression of cloned genes in response to different concentrations of Tc or «Dox».

In this system, "pTRE" is used as a "response" plasmid that can be used to express a desired gene. pTRE contains a multiple cloning site (MCS) immediately downstream of the Tet responsive PhCMV * -1 promoter. Desired cDNAs or genes that have been inserted into one of the sites in the MCS respond to the regulatory proteins tTA and rtTA in the “tet-off” and “tet-on” systems. PhCMV * -1 contains the Tet Responsive Element (TRE), which consists of seven copies of the tet operator sequence at 42 bp (teto). The TRE element is immediately upstream of the minimal CMV promoter (PminCMV), which lacks the enhancer that is part of the full CMV promoter in the pTet plasmids. As a consequence, PhCMV * -l is switched off in the absence of binding of regulatory proteins to the tetO sequences. The cloned insert must have an initiation codon. In some cases, the addition of a Kozak Consensus ribosome binding site can improve expression levels. However, many cDNAs were efficiently expressed in Tet systems without the addition of a "Kozak" sequence. “PT-RE-Gen X” plasmids are co-transfected with pTK-Hyg, which allows the selection of stable transfectants.

Establishing a "tet-off" or "tet-on" expression system generally requires two consecutive stable transfections to create a "double-stable" cell line that contains integrated copies of genes that contain the appropriate regulatory protein and TRT the control of a responsive element (TRE) included. In the first transfection, a suitable regulatory protein is introduced into the cell line of choice by transfecting a "regulatory plasmid", for example a "pTet-Off" or "pTet-On" vector, which expresses the suitable regulatory proteins. The hTRT cloned into the pTRE “response plasmid” is then introduced in the second transfection in order to generate the double-stable “tet-off” or “tet-on” cell line. Both systems result in very precise on / off control of gene expression, regulated dose-dependent induction and high absolute levels of gene expression.

Expression of recombinant hTRT using DHFR and adenovirus sequences

The pGRN155 plasmid construct was designed for the transient expression of hTRT cDNA in mammalian cells. A "Kozak consensus" sequence is inserted at the 5 'end of the hTRT sequence. The hTRT insertion contains no 3 'or 5' untranslated sequences (UTR). The hTRT cDNA is inserted into the EcoRI site of p91023 (B) (Wong, Science 228 (1985), 810-815). The hTRT insertion is in the same orientation as the DHFR open reading frame (ORF). This makes the expression vector particularly useful for transient expression.

pGRN15 contains the SV40 origin of replication and enhancer immediately upstream of an adenovirus promoter, a gene for tetracycline resistance, an E. coli origin of replication and a VAI and VAII gene region of adenovirus. This expression cassette contains in the following order: the late major adenovirus promoter; the trilateral leader of adenovirus; a hybridin-tron consisting of a 5 'splice from the first exon of the three-sided leader and a 3' splice from the mouse immunoglobulin gene; the hTRT cDNA; the mouse DHFR coding sequence and the SV40 polyadenylation signal.

It has now been described that the tripartite leader of adenovirus and the VA RNAs increase the efficiency with which polycistronic mRNAs are translated. The DHFR sequence has also been reported to enhance the stability of hybrid mRNA. The DHFR sequence can also provide a marker for selection and amplification of vector sequences (see Logan, Proc. Nati. Acad. Sci. USA 81 (1984) 3655; Kaufman, Proc. Nati. Acad. Sci. USA 82 (1985), 689 and Kaufman (1988) personal communication, Focus (Life Technologies, Inc.), Vol. 10 No. 3).

The following list of expression plasmids is provided for purposes of illustration.

128

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

pGRN121

The EcoRI fragment of lambda clone 25-1.1.6, which contains all of the cDNA encoding the hTRT protein, is inserted into the EcoRI site of pBluescriptIISK + so that the 5 'end of the cDNA is near the T7 promoter is in the vector. The selectable marker used in this vector is ampicillin.

pGRN122

The NotI fragment of pGRN121, which contains the sequence coding for the hTRT-ORF (open reading frame), is inserted into the NotI site of pEBVHisA, so that the coding sequence is functionally linked to the RSV promoter. This plasmid expresses a fusion protein which is composed of a “His6 flag” fused to the N-terminus of the hTRT protein. The marker that can be used with this vector is ampicillin or hygromycin.

pGRN123

The NotI fragment of pGRN121, which contains the sequence encoding the hTRT-ORF (open reading frame), is inserted into the NotI site of pEBVHisA so that the coding sequence is oriented in the opposite direction compared to the RSV promoter that «antisense» hTRT is expressed.

pGRN124

All APAI sites in pGRN121 were deleted and then the Mscl-Hincll fragment containing the 3'-UTR (“untranslated region”) was deleted. The Nco-Xbal fragment containing the stop codon of the hTRT coding sequence was then inserted into the Nco-Xbal sites of pGRN121, creating a plasmid that is equivalent to pGRN121 except that it lacks the 3'-UTR. This plasmid can preferably be used for increased expression levels in some cells.

pGRN125

The Not I fragment of pGRN124 containing the hTRT coding sequence is inserted into the NotI site of pBBS235 so that the open reading frame is in the opposite orientation compared to the lac promoter. The detectable marker used with this vector is chloram phenicol.

pGRN126

The NotI fragment of pGRN124 containing the hTRT coding sequence is inserted into the NotI site of pBBS235 so that the hTRT coding sequence is inserted in the same orientation as the lac promoter.

pGRN127

The oligonucleotide

5 1 - TGCGCACGTGGGAAGCCCTGGCagatctgAattCCaCcA

TGCCGCGCGCTCCCCGC TG-3 »

was used for the in vitro mutagenesis of pGRN125 to convert the initiation codon ATG of the hTRT coding sequence into a “Kozak consensus” sequence and to generate EcoRI and BglII sites for cloning. COD2866 was also used to convert AmpS to AmpR (am-picillin resistance) and CODI941 was used to convert CatR (chloramphenicol resistance, to CatS (chloramphenicol sensitivity).

pGRN128

The oligonucleotide 5 '-

TGCGCACGTGGGAAGCCCTGGCagatctgAattCCaCcATGCCGCGCGCTCCCCGC TG-3 1

129

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

was used for in vitro mutagenesis in order to convert the initiation codon ATG of hTRT into a “Kozak consensus” sequence and to generate EcoRI and BglII sites for cloning. It also became the oligonucleotide

5 '-

CTGCCCTCAGACTTCAAGACCATCCTGGACTACAAGGACGACGATGACAAA TGAATTCAGATCTGCGGCCGCCACCGCGGTGGAGCTCCAGC-3 '

used to insert the “IBI Flag” (International Biotechnologies Inc. (IBI) Kodak, New Häven, CT) at C-Ter-minus and to create EcoRI and Bglll sites for cloning. COD2866 was also used to convert AmpS to AmpR and CODI941 to convert CatR to CatS.

pGRN129

The oligonucleotide 5 '-

CGGGACGGGCTGCTCCTGCGTTTGGTGGAcGcgTTCTTGTTGGTGACACCTCA CCTCACC-3 '

was used for in vitro mutagenesis to convert Asp869 to an Ala codon (i.e. the second Asp of the DD motif was converted to an alanine for dominant / negative experiments). This also created a Mlul site. The oligonucleotide too

5 '-

CTGCCCTCAGACTTCAAGACCATCCTGGACTACAAGGACGACGATGACAAA

TGAATTCAGATCTGCGGCCGCCACCGCGGTGGAGCTCCAGC-3 '

was used to insert the “IBI Flag” (IBI, New Häven, CT) at the C-terminus and to create EcoRI and Bglll sites for cloning. COD2866 was also used to convert AmpS to AmpR and CODI941 to convert CatR to CatS.

pGRN130

The oligonucleotide

CGGGACGGGCTGCTCCTGCGTTTGGTGGAcGcgTTCTTGTTGGTGACACCTCA CCTCACC-3 '

was used in an in vitro mutagenesis to convert the Asp869 codon to an Ala codon (i.e. the second Asp of the DD motif was converted to an alanine to produce a dominant / negative protein variant). This also created a Mlul site. The oligonucleotide too

5 1 -

TGCGCACGTGGGAAGCCCTGGCagatctgAattCCaCcATGCCGCGCGCTCCCCGC TG-3 '

was used in an in vitro mutagenesis to convert the initiation codon ATG of the hTRT coding sequence into a “Kozak consensus” sequence and to generate EcoRI and BglII sites for cloning. COD2866 was also used to convert AmpS to AmpR (ampicillin resistance) and CODI 941 to convert CatR (chloramphenicol resistance) to CatS (chloramphenicol sensitivity).

pGRN131

The EcoRI fragment from pGRN128, which contains the hTRT ORF with a “Kozak” sequence and “IBI Flag” (IBI, New Haven, CT) mutations, is inserted into the EcoRI site of pBBS212 so that the hTRT ORF is expressed by the MPSV promoter.

pGRN132

The EcoRI fragment from pGRN128, which contains the hTRT ORF with a “Kozak” sequence and “IBI Flag”

130

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

(IBI, New Häven, CT) contains mutations inserted into the EcoRI site of pBBS212 so that the “anti-sense” sequence of the hTRT-ORF is expressed by the MPSV promoter.

pGRN133

The EcoRI fragment from pGRN121, which contains the hTRT coding sequence, inserts into the EcoRI site of pBBS212 so that the hTRT protein is expressed under the control of the MPSV promoter.

pGRN134

The EcoRI fragment from pGRN121, which contains the hTRT coding sequence, is inserted into the EcoRI site of pBBS212 so that the “antisense” sequence of the hTRT coding sequence is expressed under the control of the MPSV promoter. The selectable markers used with this vector are chlorine / HygB / PAC.

pGRN135

pGRN126 was completely cut with Mscl and Smal and re-ligated to delete more than 95% of the inserted hTRT coding sequence. A Smal-MscI fragment was reinserted during this process to re-generate Cat activity for selection. This unpurified plasmid was then cut again with Sali and EcoRI and the fragment containing the initiation codon of the hTRT coding sequence inserted into the Sall-EcoRI sites of pBBS212. An "antisense" expression plasmid was then produced which expresses the "antisense" sequence of the 5'-UTR and of 73 bases of the coding sequence. The selectable markers that can be used with this vector are chlorine / HygB / PAC.

pGRN136

The HindIII-SalI fragment of pGRN126, which contains the hTRT coding sequence, inserts into the HindIII-SalI sites of pBBS242.

pGRN137

The Sall-Sse8387l fragment of pGRN130, which contains the "Kozak" sequence, is inserted into the Sall-Sse8387l sites of pGRN136.

pGRN138

The EcoRI fragment of pGRN124 containing hTRT without the 3'-UTR inserts into the EcoRI site of pEGFP-C2 so that the orientation of hTRT is the same as for the EGFP domain.

pGRN139 The oligonucleotide 5 1 -

CTGCCCTCAGACTTCAAGACCATCCTGGACTACAAGGACGACGATGACAAA

TGAATTCAGATCTGCGGCCGCCACCGCGGTGGAGCTCCAGC-3 '

was used to insert the “IBI Flag” (IBI, New Häven, CT) at the C-terminus of hTRT in pGRN125 using in vitro mutagenesis and to generate EcoRI and Bglll sites for cloning. COD2866 was also used to convert AmpS to AmpR (ampicillin resistance) and CODI941 to convert CatR (chloramphenicol resistance) to Cats (chloramphenicol sensitivity).

pGRN140

The Ncol fragment, which contains the upstream sequence of genomic hTRT and the first intron of hTRT from lambdaG55, inserts into the Ncol site of pBBS167. The fragment is oriented so that hTRT is in the same direction as the lac promoter.

131

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

pGRN141

The Ncol fragment, which contains the upstream sequences of the genomic hTRT and the first intron of hTRT from lambdaG55, is inserted into the Ncol site of pBBS167. This fragment is oriented so that hTRT is in the opposite direction compared to the lac promoter.

pGRN142

The NotI fragment from lambdaG45, which contains the complete genomic -15 kbp insert, including the hTRT gene promoter region, was inserted into the NotI site of plasmid pBBS185. The fragment is oriented so that the hTRT ORF is in an opposite orientation to the lac promoter.

pGRN143

The NotI fragment from lambdaG45, which contains the complete genomic -15 kbp insert, including the hTRT gene promoter region, was inserted into the NotI site of plasmid pBBS185. The fragment is oriented so that the hTRT ORF is in the same orientation as the lac promoter.

pGRN144

SALL deletion of pGRN140 to remove lambda sequences.

pGRN145

This vector was designed to express hTRT sequences in mammalian cells. The EcoRI fragment from pGRNl37 containing the hTRT coding sequence inserts into the EcoRI site of pBBS212 to remove the portion of the sequence that corresponds to the 5'-UTR of hTRT mRNA. The hTRT coding sequence is oriented so that it is expressed under the control of the MPSV promoter. The selectable markers used with this vector are chlorine / HygB / PAC.

pGRN146

This vector was constructed for the expression of hTRT sequences in mammalian cells. The Sse8387l-Notl fragment of pGRN130, which contains the D869A mutation of hTRT, inserts into the Sse8387l-Notl sites of pGRN137. The selectable markers used with this vector are ampicillin / HygB / PAC.

pGRN147

The Sse8387l-Notl fragment of pGRN139, which contains the “IBI Flag” (IBI, New Häven, CT), inserts into the Sse8387l-Notl sites of pGRN137.

pGRN148

The Bglll-Eco47lll fragment of pGRN144, which contains the promoter region of hTRT, is inserted into Bglll-Nrul sites of pSEAP2, resulting in an hTRT promoter / reporter construct.

pGRN149

This vector is an intermediate vector for the construction of an expression vector for an hTRT fusion protein. The mutagenic oligonucleotide

3 'was used to add a Csp45l site to the C-terminus of hTRT by in vitro mutagenesis of pGRN125. The “Stop” codon from hTRT was deleted and exchanged for a Csp451 site.

The selectable marker used with this vector is ampicillin.

pGRN150

The Gblll-Fspl fragment of pGRN144, which contains the promoter region of hTRT, is inserted into the Bglll Nrul sites of pSEAP2, resulting in an hTRT promoter / reporter construct.

132

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

pGRN151

This vector was designed to express hTRT sequences in mammalian cells. The EcoRI fragment from pGRN147 containing the hTRT coding sequence inserts into the EcoRI site of pBBS212 so as to remove the portion of the sequence which corresponds to the 5'-UTR of the hTRT mRNA. The hTRT coding sequence is oriented so that it is expressed under the control of the MPSV promoter. The markers used with this vector are chlorine / HygB / PAC.

pGRN152

The EcoRI fragment from pGRN146, which contains the hTRT coding sequence, inserts into the EcoRI site of pBBS212 to remove the portion of the sequence that corresponds to the 5'-UTR of hTRT. The hTRT coding sequence is oriented so that it is expressed under the control of the MPSV promoter.

pGRN153

The styl fragment of pGRN130 containing the D869-> A mutation of hTRT (a sequence coding for an hTRT variant) was inserted into the styl sites of pGRN158, whereby a plasmid was obtained which contained the hTRT coding sequence with a « Kozak Consensus »sequence at its 5 'end, an" IBI FLAG "sequence at its 3' end, which contains the region coding for the C-terminus and the D869-> A mutation.

pGRN154

The EcoRI fragment of pGRN153 containing the hTRT gene was inserted into the EcoRI site of plasmid pBS212 so that the hTRT ORF is in the same orientation as the MPSV promoter. This creates an MPSV-controlled expression plasmid that expresses the hTRT protein with a “Kozak consensus” sequence at its amino terminal end, an “IBI FLAG” at its C-terminus and the D869- “A mutation.

pGRN155

This vector was designed to express hTRT sequences in mammalian cells. The insert contained full-length hTRT cDNA without 5 'and 3' UTR and "Kozak" sequences. The EcoRI fragment from pGRN145, which contains the hTRT cDNA with the "Kozak Consensus" sequence but no 3'- or 5'-UTR, was inserted into the EcoRI site of p91023 (B) so that the hTRT is in the same orientation as the DHFR-ORF. This creates an expression vector for the transient expression of hTRT. The selectable marker used with this vector is tetracycline.

pGRN156

This vector was designed to express hTRT sequences in mammalian cells. The EcoRI fragment from pGRN146, which contains the D869A mutation of the hTRT cDNA with the "Kozak Consensus" sequence without 3'- or 5'-UTR, is inserted into the EcoRI site of p91023 (B), so that hTRT is in the same orientation as the DHFR ORF. This creates an expression vector for the transient expression of hTRT. The insert contained full length hTRT cDNA without 5 'and 3' UTR, D869A and "Kozak" sequences. The selectable marker used with this vector is tetracycline.

PGRN157

This vector was constructed for the expression of hTRT sequences in mammalian cells. The EcoRI fragment from pGRN147, which contains the hTRT cDNA with the “Kodak flag” at the C-terminus, and the “Kozak Consensus” sequence without 3'- or 5'-UTR, inserted into the EcoRI site of p91023 (B) so that hTRT is in the same orientation as the DHFR ORF. This creates an expression vector for the transient expression of hTRT. The insert contained full length hTRT cDNA without 5'- and 3'-UTR, a "FLAG" sequence (Immunex Corp., Seattle, WA) and "Kozak" sequences. The selectable marker used with this vector is tetracycline.

pGRN158

This vector was constructed for the expression and mutagenesis of TRT sequences in E. coli. The EcoRI fragment from pGRN151, which contains the hTRT-ORF, inserts into the EcoRI site of pBBS183 so that the hTRT-ORF is in the opposite orientation compared to the lac promoter

133

CH 689 672 A9

is present. The insert contained full length hTRT cDNA without 5'- and 3'-UTR, a "FLAG" sequence (Immunex Corp., Seattle, WA) and "Kozak" sequences. The coding sequence is controlled by a T7 promoter. The selectable marker used with this vector is ampicillin.

5

PGRN159

This vector was constructed for the expression and mutagenesis of TRT sequences in E. coli. The Hel-Kpnl fragment from pGRN138, which contains the fusion of EGFP to hTRT, inserts into the Xbal-10 Kpnl sites of pBlueScriptllKS +. This results in a T7 expression vector for the fusion protein (the coding sequence is controlled by a T7 promoter). This insert contains full length hTRT cDNA without the 3'-UTR as a fusion protein with EGFP. The selective marker used with this vector is ampicillin.

15 pGRN160

This vector was constructed for the expression of “antisense” hTR sequences in mammalian cells. The coding sequence is controlled by an MPSV promoter. The Xhol-Nsil fragment of pGRN90 containing the full length hTR inserted into the Sall SSE / 8387I sites of pBBS295. This er-20 gives a transient stable vector which expresses hTR in the «antisense» orientation. A GPT marker was built into the vector. The selectable markers used with this vector are chlorine / gpt / PAC.

pGRN161

25th

This vector was constructed for the expression of "sense" -HT sequences in mammalian cells. An Xhol-Nsil fragment from pGRN89 containing full-length hTR inserts into the Sall SSE8387I sites of pBBS295. This results in a transient / stable vector that expresses hTR in the “sense” orientation. The coding sequence is controlled by an MPSV promoter. A GPT-30 marker was built into the vector. The markers used with this vector are chlorine / gpt / PAC.

pGRN162

An Xhol-Nsil fragment from pGRN87 containing full length hTR inserts into the Sali 35 SSE8387I sites of pBBS295. This results in a transient / stable vector which expresses shortened hTR (from position +108 to +435) in the «sense» orientation.

pGRN163

40 This vector was constructed for the expression and mutagenesis of TRT sequences in E. coli. The coding sequence is controlled by a T7 promoter. RA45

(5'-gccacccccgcgctgcctcgagctccccgctgc-3 ')

45 is used in an in vitro mutagenesis to convert the initiation Met into hTRT to Leu and to introduce an Xhol site in the next two codons after the Leu. COD 1941 was also used for the transfer from CatR to CatS and the introduction of a BSPHI position and COD 2866 for the transfer from AmpS to AmpR, whereby an Fspl point was introduced. The selectable marker used with this vector is ampicillin.

50 pGRN164

This vector was constructed for the expression of hTR sequences in E. coli. The primers hTR + 1 ggggaagctttaatacgactcactatagggttgcggagggtgggcctg-3 '

55

and hTR +44 5

ccccggatcctgcgcatgtgtgagccgagtcctggg-3 '

were used for amplification of a fragment of pGRN33 by means of PCR which contains hTR with a complete length of 60 and the T7 promoter at its 5 'end (as in hTR + 1). A BamHI HindIII cleavage of the PCR product was inserted into the BamHI HindIII sites of pUC119. The coding sequence is controlled by a T7 promoter. The selectable marker used with this vector is ampicillin.

65

134

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

pGRN165

This vector was used for the expression and mutagenesis of hTRT sequences in E. coli. The coding sequence is controlled by a T7 promoter. An EcoRI fragment from pGRN145, which contains the hTRT-orf with a “Kozak” sequence at the front end, is inserted into the EcoRI site of pBluescriptIISK +, so that hTRT is in the same orientation as the T7 promoter. The selectable marker used with this vector is ampicillin.

pGRN166

This vector was constructed for the expression and mutagenesis of TRT sequences in mammalian cells. The coding sequence is controlled by a T7 promoter. The EcoRI fragment from pGRN151, which contains the hTRT ORF with a “Kozak” sequence at the front end and an “IBI flag” at the rear end, is inserted into the EcoRI site of pBluescriptllSK + so that hTRT is in the same orientation as the T7 promoter. The insert contained full-length hTRT cDNA without 5 'and 3' UTR, a "FLAG" sequence (Immunex Corp. Seattle, WA) and "Kozak" sequences. The selectable marker used with this vector is ampicillin.

pGRN167

An Avrll-Stul fragment from pGRN144 containing the 5 'end of the hTRT ORF inserts into the Xbal-Stul sites of pBES161.

pGRN168

The EcoRI fragment from pGRN145 containing the optimized hTRT expression cassette was inserted into the EcoRI site of pIND so that the hTRT coding sequence is in the same orientation as the miniCMV promoter.

pGRN169

The EcoRI fragment of pGRN145 containing the optimized hTRT expression cassette was inserted into the EcoRI site of pIND in such a way that the hTRT is in the opposite orientation with respect to the miniCMV promoter.

pGRN170

The EcoRI fragment from pGRN145 containing the optimized hTRT expression cassette was inserted into the EcoRI site of pIDN (spl) in such a way that the hTRT is in the opposite orientation, based on the miniCMV promoter.

pGRN171

The Eco47ll-Narl fragment from pGRN163 inserts into the Eco47111-Narl sites of pGRN167. The M1L mutation is thus introduced into a fragment of the genomic hTRT DNA.

pGRN172

The BamHI-Stul fragment from pGRN171, which contains the Met to Leu mutation in hTRT, inserts into the Bglll-Nrul sites of pSEAP2- «Basic».

pGRN173

The EcoRV-Eco47lll fragment from pGRN144, which contains the 5 'end of the hTRT promoter region, was inserted into the Srfl-Eco47lll sites of pGRN172. This resulted in a promoter / reporter plasmid which contains the promoter region of hTRT of approximately 2.3 kb upstream of the start of the hTRT-ORF until immediately after the first intron in the coding region with the Met1- »Leu mutation.

pGTN174

The EcoRI fragment from pGRN145 containing the “optimized” hTRT expression cassette was inserted into the EcoRI site of pIDN (spl) in such a way that the hTRT has the same orientation as the miniCMV promoter.

135

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Example 7

Reconstitution of telomerase activity a) Co-expression of hTRT and hTR in vitro

In this example, the co-expression of hTRT and hTR is described using a cell-free in vitro expression system. The results show that the hTRT polypeptide encoded by pGRN121 encodes a catalytically active teiomerase protein and that the in vitro reconstitution (IVR) of the telomerase RNP can be achieved using recombinantly expressed hTRT and hTR.

Telomerase activity was increased by adding linearized plasmids with hTRT (pGRN121; 1 ng DNA, cleaved with Xbal) and hTR (pHTR + 1; 1 ng, cleaved with Fspl) to a coupled transcription / translation reticulocyte lysate (Promega TNT®) reconstituted. pHTR + 1 is a plasmid which, after linearization with Fspl and subsequent transcription by T7 RNA polymerase, generates a transcript with 445 nucleotides, which begins with nucleotide +1 and extends to nucleotide 446 of hTR (Autexier et al. , EMBO J. 15 (1996), 5928). For a 50 nl reaction, the following ingredients were added: 2 ngl TNT® buffer, 1 n 'TNT® T7 RNA polymerase, 1 nl 1 mM amino acid mixture, 40 units RNasin® RNase inhibitor, each 1 ng linearized matrices -DNA and 25 nl TNT® reticulocyte lysate. The ingredients were added in the ratio recommended by the manufacturer and incubated at 30 ° C for 90 minutes. The transcription was controlled by the T7 promoter and could also be carried out with similar results before the addition of reticulocyte lysate. After incubation, 5 and 10 n 'of the programmed transcription / translation response for Te-isomerase activity were examined using the TRAP assay described above (Autexier et al., Op. Cit.) Using 20 PCR cycles for signal amplification.

The results of the reconstitution are shown in Fig. 10. There are three lanes for each transcription-translation reaction. The first two lanes are duplicate assays and the third lane is a duplicate sample heat-denatured (953C, 5 minutes) to exclude PCR-generated artifacts before the TRAP phase.

As shown in Figure 10, the reticulocyte lysate has no detectable telomerase activity (lane 6). Similarly, no detectable activity is observed when either hTR alone (lane 1) or the full length hTRT gene (lane 4) is added to the lysate. When both components are added (lane 2), telomerase activity is generated, as shown by the characteristic pattern of repeats. If the carboxy-terminal region of the hTRT gene is removed by cleavage of the vector with Ncol («shortened hTRT»), telomerase activity is abolished (lane 3). Lane 5 shows that translation of the truncated hTRT alone does not produce telomerase activity. Lane "R8" shows a positive control for a telomerase product ladder generated by "TRAP" from TSR8 (a synthetic telomerase product with a nucleotide sequence of the standard for quantification)

C5'-ATTCCGTCGAGCAGAGTTAG [GGTTAG] r3 ')).

B) Mixing hTRT and hTR in vitro

The in vitro reconstitution of telomerase activity was also achieved by mixing. hTRT was transcribed and translated as described above, but without the addition of the hTR plasmid. Reconstitution of the TelomeraseRNP was then accomplished by mixing the hTRT translation mixture with hTR (previously generated by transcription with T7 RNA polymerase from phTR + 1-Fsp) in the ratio of 2 Al hTRT translation mixture to 2 n 'hTR (1 ng) and subsequent 90-minute incubation at 30 ° C. This process of hTRT-hTR reconstitution is referred to as “linked reconstitution” or “linked IVR”. Telomerase activity is present in this mixture (i.e. can be detected). An improved signal was observed after partial purification of the activity by DEAE chromatography. In this case, a “Millipore Ultrafree-MC DEAE central filter” device was used according to standard procedures (i.e. according to the manufacturer's instructions). The buffers used were: hypoO.1, hypo0.2 and hypol.0, with hypo 20 mM Hepes-KOH, pH 7.9, 2 mM MgCh, 1 mM EGTA, 10% glycerol, 0.1% NP- 40, 1mM DTT, 1mM sodium metabisulfite, 1mM benzamidine and 0.2mM phenylmethylsulfonyl fluoride (PMSF) and where 0.1, 0.2 and 1.0 refer to 0.1, 0.2 or 1.0 M KCl. The filters were preconditioned with hpyol.O, washed with hypoO.1, then the reconstituted telomerase was charged, the column was washed with hypoo.1 and then hypo0.2, and the reconstituted telomerase was compared with half the volume using hypo 1.0 to the charging volume eluted. This preparation could be stored frozen at -70 ° C and it remains active.

Telomerase activity was examined in a two-step procedure. The first step was a

136

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Usual telomerase assay as described by Morin, Cell 59 (1989), 521, except that no radioactive labeling was used. In the second step, an aliquot was examined using the “TRAP” method for 20 to 30 cycles (as described above). The usual telomerase assay was carried out in such a way that 1 to 10 μl of reconstituted telomerase were investigated in 25 mM Tris-HCl, pH 8.3, 50 mM potassium acetate, 1 mM EGTA, 1 mM MgCfe, 2 mM DATP, 2 mM TTP, 10 nM dGTP and 1 nM primer (usually M2, AATCCGTCGAGCAGAGTT) in a final volume of 40 to 50 nl at 30 ° C and for 60 to 180 minutes. The reaction was stopped by heating at 95 ° C. for 5 minutes and the second step, the “TRAP” reaction (50 μl), was carried out with 1-10 ml of the mixture from the first step.

In additional experiments, the synthesis of hTRT and hTR was monitored during an in vitro reconstitution using the incorporation of 35S methionine and Northern blotting. Proteins of approximately the predicted size were synthesized for hTRT (127 kD), hTRT-Nco (85 kD) and pro90hTRT (90 kD) in approximately equimolar amounts (relative to one another). Northern analysis showed that hTR synthesis was the correct size (445 nucleotides) and was predominantly intact.

Variations on the reconstitution protocol described above will be apparent to those skilled in the art. For example, the following parameters can be varied: reconstitution time and temperature, the presence or the concentration of constituents such as monovalent salts (e.g. NaCl, KCl, potassium acetate, potassium glutamate etc.), divalent salts (MgCfe, MnCb, MgSO-t, etc.), denaturing agents (Urea, formamide etc.), detergents (NP-40, Tween, CHAPS, etc.). Alternative improved purification methods (e.g. immunoprecipitation, affinity chromatography or standard chromatography) can also be used. These and other parameters can be systematically varied so as to optimize the conditions for certain assays or other reconstitution protocols.

C) Reconstitution using hTRT variants and fusion proteins

Reconstitution of catalytic telomerase activity occurred when EGFP-hTRT co-expressed a fusion of the en-hanced green fluorescent protein to hTRT (see Example 15) or epitope-tagged hTRT (IBI FLAG, see Example 6) with hTR , both of which reconstituted telomerase activity to approximately wild-type levels.

In contrast, telomerase activity was not reconstituted when the hTRT variant pro90hTRT (which lacks the RT motifs B ', C, D and E) was used. This shows that pro90hTRT has no catalytic telomerase activity, which could have other activities (e.g. the ability to bind RNA (i.e. hTR) and it could also act as a dominant-negative regulator of telomerase in vivo as described above.

D) The investigation of in vitro reconstituted telomerase activity using the gel blot and the usual telomerase assay

The following example shows that in vitro reconstituted telomerase (IVR) can be tested in addition to amplification based assays (i.e. TRAP) using standard telomerase assays. Telomerase was reconstituted in vitro (IVR) as described in part (B) above (the "linked reconstitution" procedure), followed by DEAE purification as described above. The IVR telomerase was examined by means of the gel blot assay under the following reaction conditions: 1 to 10 nl IVR telomerase in 25 mM Tris-HCl, pH 8.3, 50 mM potassium acetate, 1 mM EG-TA, 1 mM MgCI2, 0.8 mM dATP, 0.8 mM TTP, 1.0 mM dGTP and 1 nM primer (M2, AATCCGTCGAGCAGAGTT; or H3.03, TTAGGGTTAGGGTTAGGG) for 180 minutes at 30 ° C and a final volume of 40 nl- The synthesized telomeric DNA was isolated using standard procedures, separated on a gel with 8% polyacrylamide and 8 M urea, transferred to a nylon membrane and using the 32P- (CCCTAA) used in the dot blot assay. n-Ribonucleotide probe searched. Using this probe, a ladder of six nucleotides in the lane could be identified that represented 10 n 'IVR telomerase, which was equivalent to the ladder observed for 5 nl native nuclear telomerase purified with "mono Q" and heparin chromatography. The results show that IVR telomerase, equivalent to native telomerase, has a processive catalytic telomerase activity.

IVR telomerase (“linked reconstitution”) was also examined using the usual telomerase assay based on the incorporation of 32P-dGTP. The telomerase thus reconstituted, which was then purified via DEAE purification as described above, was examined under both process and non-process reaction conditions. The assay conditions were: 5-10 n 'reconstituted telomerase ("linked reconstitution") in 25mM Tris-HCl, pH 8.3, 50mM potassium acetate, 1mM EGTA, 1mM MgCfe, 2mM dATP, 2mM TTP with 10 nM 32P-dGTP (72 Ci / mmol) (for the assay with regard to process conditions) or 1 nM 32P-dGTP (720 Ci / mmol) (for non-process conditions) and 1 nM primer (ie H3.03 TTAGGGTTAGGGTTAGGG) at 30 ° C (for the process reaction) or 37 ° C for the non-process reaction for 180 minutes in a final volume of 40 nl. The synthesized telomeric DNA was isolated by standard methods and on a

137

CH 689 672 A9

10th

30th

50

55

Sequencing gel separated with 8% polyacrylamide and 8 M urea. The process response showed a weaker ladder of six nucleotides, which corresponds to a process telomerase reaction, and a repeat unit was added to the non-process reaction, a pattern equivalent to the control reaction with a preparation of native telomerase. Common assays using telomerase "linked reconstitution" are useful in the search for telomerase modulators as described here, as well as in other applications such as the elucidation of structural and functional properties of telomerase.

E) In vitro reconstituted telomerase recognizes 3 'ends of primers

This experiment shows that "linked reconstitution" recognizes telomerase 3 "ends of primers, equivalent to native (purified) telomerase. Telomerase forms a base-paired duplex between the 3 'end of a primer and the template region of hTR and appends the next proposed nucleotide (Morin, 1989, loc. cit.) To verify that (recombinant) IVR-15 telomerase ("linked reconstitution") has the same property, the reactions of primers with —GGG or —TAG- 3 'ends (AATCCGTCGAGCAGAGGG or AATCCGTCGAGCAGATAG) with a primer compared to a —GTTâ' terminus (M2; AATCCGTCGAGCAGAGTT; the standard "TRAP" primer) using in vitro reconstituted and native telomerase used by the The two-step assay (common / TRAP) described above in Figure 20. The product ladders of the primers —GGG and —TAG shifted +4 and +2, respectively, compared to the standard prim he (--GTT 3 'end); the same effect was observed with native telomerase. This experiment shows that in vitro reconstituted ("linked reconstitution") and native telomerase recognize primer ends in the same way.

These results (along with the results above, which show that IVR telomerase 25 has both processive and non-processive catalytic activity) indicate that IVR telomerase has a similar structure and properties compared to native or purified telomerase.

Example 8

Production of anti-hTRT antibodies A) The production of anti-hTRT antibodies or hTRT peptides

35 To produce anti-hTRT antibodies, the following peptides of hTRT were synthesized with the addition of C (cysteine) as the amino-terminal residue (see Fig. 54):

S-l: FFY VTE TTF QKN RLF FYR KSV WSK

40 S-2: RQH LKR VQL RDV SEA EVR QHR EA

S-3: ART F RR EKR AER LTS RVK ALF SVL NYE

A-3: PAL LTS RLR FIP KPD GLR PIV NMD YVV

45

The cysteine unit was used to immobilize (i.e. covalently link) the peptides to BSA and KLH (keyhole limpet hemocyanin) carrier proteins. The KLH peptides were used as the antigen. The BSA peptide conjugates served as material for ELISAs to test the specificity of immune antisera.

The KLH peptide conjugates were injected into New Zealand White rabbits. The introductory injections were given proximal to the armpit and groin lymph nodes. Subsequent injections were given intramuscularly. The antigen was emulsified with complete Freund's adjuvant for introductory injections, and incomplete Freund's adjuvant was used for subsequent injections. The rabbits followed a 3 week booster cycle, with 50 ml of blood delivering 20 to 25 ml of serum drawn 10 days after each booster. Antisera against each of the four peptides recognized the hTRT unit of recombinant hTRT fusion protein (GST-HIS8-hTRT fragment 2426 to 3274) on Western blots (see example

6) -

60 Using a partially purified telomerase fraction of human 293 cells (approximately 1000-fold purification compared to the crude nuclear extract), which is described in the parallel US patent application with serial no. 08 / 833.37 (see also PCT Application No. 97/06012) and affinity-purified with anti-S-2 antibodies, a 130 kD protein duplicate could be detected on a We-Stern blot. A sensitive chemilumi-

65 nescent detection methods applied (“SuperSignal” chemiluminescent substrates, Pierce), the signal

138

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

however, the blot was weak, suggesting that hTRT is present in low or very low frequency in these immortal cells. The occurrence of a duplicate is consistent with a post-translational modification of hTRT, i.e. Phosphorylation or glycosylation.

For affinity purification, the S-2 peptide was immobilized on “SulfoLink” (Pierce, Rockford, IL) via its N-terminal cysteine residue in accordance with the manufacturer's protocol. First blood serum from a rabbit immunized with the KLH-S-2 peptide was loaded via an S-2 "SulfoLink" and antibodies specifically bound to the S-2 peptide were eluted.

B) Production of anti-hTRT antibodies against hTRT fusion proteins

GST-hTRT fusion proteins were expressed in E. coli as the GST-hTRT fragment # 4 (nucleotides 3272-4177) and the GST-His8-hTRT fragment # 3 (nucleotides 2426 to 3274) as described in Example 6. The fusion proteins were purified as an insoluble protein and the purity of the antigen was examined via SDS-polyacrylamide gels and checked for approximately 75% purity with respect to the recombinant protein GST-hTRT fragment # 4 and over 75% purity for the recombinant protein GST-His8-hTRT fragment # 3 estimated. (Routine procedures can be used to obtain these and other fusion proteins with a purity greater than 90%). These recombinant proteins were used to immunize both New Zealand White rabbits and female Balb / c mice. The antigen was emulsified with complete Freund's adjuvant for introductory injections and incomplete Freund's adjuvant was used for subsequent injections. A 3 week booster cycle was performed for the rabbits and mice with blood drawn 10 days after each booster.

The first and second blood draws from both mice and rabbits were tested for the presence of anti-hTRT antibodies after removal of anti-GST antibodies using an immobilized matrix containing GST. The antisera were tested for anti-hTRT antibodies by Western blots using an immobilized recombinant GST-hTRT fusion protein and by immunoprecipitation using the partially purified native telomerase enzyme. No signal was observed at these early blood draws; It is assumed that the titer of anti-hTRT antibodies increases with subsequent blood samples.

Example 9

Detection of an HTRT-MRNA that corresponds to the RNA of the Al82 variant

Poly-A + RNA from human testes and the 293 cell line was analyzed for hTRT mRNA using RT-PCR and «nested» primers. The first set of primers was TCP1.1 and TCP1.15, the second set of primers TCP1.14 and billTCP6. The amplification with both sets of primers resulted in two products which differed by 182 bp. The larger and smaller products of testicular RNA were sequenced and it turned out that they corresponded exactly to pGRN121 (FIG. 16) and clone 712562 (FIG. 18). The variant hTRT-RNA product was observed in mRNA from SW39i, OVCAR4, 293 and testes.

Additional experiments were carried out to show that the A182 cDNA was not an artifact of reverse transcription. Briefly summarized, hTRT-RNA (i.e. without deletion) was generated by in vitro transcription of pGRN121 for use as a template for RT-PCR. Separately, cDNA synthesis reactions were carried out using the “Superscript®” reverse transcriptase (Bethesda Research Laboratories, Bethesda MD) at 42 ° C or 50 ° C with random primers or a specific primer. After 15 PCR cycles, the longer product was detectable, but not the smaller product (i.e. the one corresponding to the deletion, even after 30 or more cycles. This indicates that the RTPCR product is not an artifact.

Example 10

Sequencing of testicular hTRT mRNA

The sequence of the form of hTRT-RNA found in testes was determined by direct manual sequencing of DNA fragments, which were carried out by means of PCR of testicular cDNA (“marathon” testicular cDNA, Clontech, San Diego, CA) using a "ThermoSequenase radiolabeled terminator cycle" sequencing kit (Amersham Life Science). The PCR reactions were carried out using “ne-sted” PCR, as shown in Table 7, unless stated otherwise. In all cases, a negative control reaction was carried out with primers, but without cDNA. The lack of a product in the control reaction indicated that the products derived from the reaction with cDNA were not present due to contamination with hTRT from pGRN121 or other cell sources (e.g. 293 cells). The DNA fragments were excised from agarose gels to purify the DNA prior to sequencing.

139

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

The sequence of the testicular mRNA, which corresponds to bases 27 to 3553 of the insertion sequence of pGRN121 (SEQ. ID. No. 1) and the entire hTRT-ORF (bases S6 to 3451) was obtained. In this area there were no differences between the testicle and pGRN121 sequences.

140

Oî o en o en o

Primer set 1

Primer set 2

l) A

nn

K320 / K322

A

K320 /TC1M.-I3

TCP 1.'10 / TCP 1.3-1

11

TCIM .'12 / TCPl 3211

'l'CPI .3 5 / TCPl .21

C.

TCP 1.65 / I CI '1.66

l'CPI.67 /TCI'1.68

1) 2

K30-I / l) i! ITCP6

LT 1 / TCP 1.6

1) 3

TCPl.12 / TCPl.7

TCIM.M /TCIM.15

i: i:

nn

TCP 1.7-1 / TCIM.7

i:

I CI '13 / ICI' 1 -1

TCP 1.2 / TCIM.9

i:

TCIM .26 / IJTU2

TCIM.M) /TCIM.-1

co co ro f \ 3

en o en o

Table 8

Final size

2 OS 556 <192 HIB 516

60-1

201

687

177

Primer for SEQ

K320.K322

TCI't .52, '1'CiM .3'.). K322, TCP1.-1I) .TC1 'I <11 .'l'CIM.BO.TCP 1.3'I.TCPl. <19 TCPl .3 5 .TCP 1.28, TCIM .38, TCIM .2 I .TCIM.-16.TCPl .33.MCIM .48

I CI '1.67.TCP 1.32. I CI '1.69.1 CI' 1.68.1 Cl '1.2-1.1Cl' [.'1A.K303

i.'n.i.Ti .rcp i, 6.trrci'-i; icp i. 13, tcp i .77. ree 1.1

TCP 1.6, TCP 1.l-I.TCP 1.73.1 Cl '1.7H.TCP 1.25.TCP 1.15.1 Cl' l .76

TCP 1.7'I.TCIM .7.TCP 1.75, TCIM. 15.TCIM .3

TCPl .2.TCT1.8.TCIM .9.TCIM .26

TCPI.'l.TCPI.M) TCI'I.I I

CH 689 672 A9

Example 11

Detection of hTRT mRNA by RNase protection reaction

5 RNase protection assays can be used to detect, monitor or diagnose for the presence of an hTRT mRNA or the mRNA of a variant. An illustrative probe for such an assay is an in vitro synthesized RNA comprising sequences complementary to hTRT mRNA sequences and additional non-complementary sequences. The latter sequences are therefore advantageous in order to be able to distinguish the full-length probe from fragment 10 of the probe, which results from a positive result in the assay. In a positive assay, the complementary sequences of the probe are protected from cleavage with RNase, since they are hybridized with the hTRTmRNA. The non-complementary sequences are cleaved from the probe in the presence of RNase and complementary target nucleic acid.

Two probes for the RNase protection assays are described for illustration, each of which can be used in the assay. The probes differ in their sequences complementary to hTRT, but contain identical non-complementary sequences which in this embodiment derive from the late leader sequence of the SV40 mRNA. In the 5'-3 'direction, the first probe comprises 33 nucleotides of a non-complementary sequence and 194 nucleotides of a sequence that is complementary to hTRT nucleotides 2513 to 2707 for a full-length probe 20 of 227 nucleotides. In the 5'-3 'direction, the second probe comprises 33 nucleotides of a non-complementary sequence and 198 nucleotides of a sequence that is complementary to the hTRT nucleotides 2837 to 3035 for a full-length probe of 231 nucleotides. To carry out the assay, each of the two probes can be used with RNA, i.e. PolyA + RNA are hybridized from a test sample and then T1 ribonuclease and RNase A are added. After cleavage 25, the probe RNA is purified and analyzed by gel electrophoresis. Detection of a 194 nucleotide fragment of the nucleotide probe with a length of 227 nucleotides or a 198 nucleotide fragment of the probe with a length of 231 nucleotides is a sign of the presence of hTRT-mRNA in the sample.

The illustrative probes for the RNase protection assay described in this example can be generated by in vitro transcription using T7 RNA polymerase. Radioactive or otherwise labeled ribonucleotides can be included for the synthesis of labeled probes. The matrices for the in vitro transcription reaction for the production of the RNA probes are PCR products. These illustrative probes can be synthesized with T7-35 polymerase after PCR amplification of pGRN121 DNA using primers that span the corresponding complementary region of the hTRT gene or the hTRT mRNA. In addition, the downstream primer contains T7 RNA polymerase promoter sequences and the non-complementary sequences.

The PCR product from the following primer pair T701 and reverse 01) is used to generate the first probe for the RNase protection assay:

40 T701 5'-GGGAGATCT TAATACGACTCACTATAG ATTCA GGCCATGGTG

CTGCGCCGGC TGTCA GGCTCCC ACGACGTAGT CCATGTTCAC-3 '

and reverse 01

45 5'-GGGTCTAGAT CCGGAAGAGTGT CTGGAGCAAG-3 '

The PCR product from the following pair of primers (T702 and reverse 02) is used to generate the second probe for the RNase protection assay:

50

T702 5'-GGGAGATCT TAATACGACTCACTATAG ATTCA GGCCATGGTG CTGCGCCGGC TGTCA GGGCG GCCTTCTGGA CCACGGCATA CC-3 "

55 and reverse 02

5'-G GTCTAGA CGATATCC ACAGGGCCTG GCGC-3 '

60

65

142

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Example 12

Construction of a phylogenetic family tree by comparison of hTRT with other reverse transcriptases

A phylogenetic family tree (Fig. 6) was constructed by comparing the seven Xiong and Eickbush (EMBO J. 9 (1990), 3353) RT domains. After sequence alignment of motifs 1, 2 and AE of 4 TRTs, 67 RTs and 3 RNA polymerases, the family tree was created using the «NJ (« Neighbor joining ») method (Saitou and Nei, Mol. Biol. Evol. 4 (1987 ), 4406). Elements from the same class that are located on the same branch of the family tree are shown simply as boxes. The length of each box corresponds to the most divergent element within the box.

The TRTs appear to be more closely related to the RTs that are associated with msDNA, group II introns and non-LTR (long terminal repeat unit) retrotransposons than with the LTR retrotransposon and viral RTs. The relationship of the telomerase RTs to the non-LTR branch of retro elements is very interesting, given that the latter elements have exchanged telomerase for telomer retention in Drosophila. The most striking observation, however, is that the TRTs form a discrete subset that is almost as closely related to the RNA-dependent RNA polymerases of +-stranded RNA viruses, such as poliovirus, as to one of the previously known RTs . Considering that the four telomerase genes come from evolutionarily distant organisms - protozoa, fungi and mammals - this separate grouping cannot be explained by the lack of phylogenetic diversity in the data set. Instead, this early fork suggests that the telomerase RTs are a very old group that may have arisen with the first eukaryote.

GenBank protein identification numbers or access numbers used for phylogenetic analysis are:

msDNAs (94535, 134069, 134074, 134075, 134078), Group II introns (483039, 101880, 1332208, 1334433, 1334435, 133345, 1353081), mitochondrial plasmid / RTL (903835, 134084), non-LTR re-trotransposons (140023 , 84806, 103221, 103353, 134083, 435415, 103015, 1335673, 85020, 141475, 106903, 130402, U0551, 903695, 940390, 2055276, L08889), LTR-Retrotransposons (74599, 85105, 130582, 99712, 83512 479443, 224319, 130398, 130583, 1335652, 173088, 226407, 101042, 1078824), hepadnaviruses (1 18876, 1706510, 118894), Caulimoviruses (331554, 130600, 130593, 93553), retroviruses (130601, 13054655, 7454 130671, 130607, 130629, 130589, 130631, 1346746, 130651, 130635, 1780973, 130646). Alignment was determined using “ClustalW 1.5” (JD Thompson, DG Higgins, TJGibson, Nucleic Acids Res. 22, (1994), 4673) and “PHYLIP.3.5” (J. Felsenstein, Cla-disfics 5 (1989), 164 ) analyzed.

Example 13

Transfection of cultured human fibroblasts (BJ) with a control plasmid and a plasmid encoding hTRT

This example shows that expression of a recombinant hTRT protein in a mammalian cell leads to the generation of an active telomerase.

Sub-confluent bi-fibroblasts were trypsinized and resuspended in fresh medium (DMEM / 199, containing 10% fetal calf serum) at a concentration of 4x10® cells / ml. The cells were transfected by electroporation with BioRad's Gene Pulser ™ electroporator. If necessary, cells can also be transfected using the «Superfect ™» reagent (Quiagen) according to the manufacturer's instructions. For the electroporation, 500 ^ l of cell suspension were placed in an electroporation cuvette (BioRad, 0.4 cm electrode spacing). Plasmid DNA (2 ng) was added to the cuvettes and the suspension was mixed gently and incubated on ice for 5 minutes. The control plasmid (pBBS212) contained no insert behind the MPSV promoter and the experimental plasmid (pGRN133) expressed hTRT from the MPSV promoter. The cells were electroporation at 300 V and 960 nFD. After the pulse was delivered, the cuvettes were kept on ice for approximately 5 minutes before being plated on 100 mm tissue culture plates in medium. After 6 hours the medium was exchanged for new medium. 72 hours after the transfection, the cells were trypsinized, washed once with PBS, pelleted and stored in the frozen state at -80 = 0. Cell extracts were prepared at a concentration of 25,000 cells / nl by a modified lysis method using detergents (see Bodnar et al., Exp. Cell Res. 228 (1996), 58 and Kim et al., Science 266 (1994) 2011 and the Description in patents and publications relating to the above TRAP assay) and telomerase activity in the cell extracts was determined by means of a modified PCR-based TRAP assay (Kim et al., 1994, Bodnar et al., 1996). In short, 5 x 104 cell equivalents were used for the telomerase primer extension part of the reaction. While the extract is typically added directly from the telomerase extension reaction to the PCR amplification, one can also use phenol / chloroform and chloro143

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Extract form before PCR amplification. One fifth of the material was used in the PCR amplification portion of the TRAP reaction (approximately 10,000 cell equivalents). Half of the TRAP reaction was loaded on a gel for analysis so that each trace in Figure 25 represents reaction products of 5000 cell equivalents. Extracts from cells transfected with pGRN133 were positive for telomerase activity, while extracts from untransfected cells (not shown) or cells transfected with the control plasmid showed no telomerase activity. Similar experiments with RPE cells gave the same result.

Reconstitution in BJ cells was also performed using other hTRT constructs (i.e. pGRN145, pGRN155 and pGRN138). Reconstitution using these constructs appeared to result in increased telomerase activity compared to cells transfected with pGRN133.

The highest level of telomerase activity was achieved with pGRN155. As already discussed above, pGRN155 is a vector which contains the late main promoter of adenovirus as a control element for the expression of hTRT and has been shown to be able to reconstitute telomerase activity upon transfection in BJ cells. Remarkably, reconstitution using the hTRT-GFP fusion protein pGRN138 (located in the nucleus, see Example 15 below) gave either in vitro (see Example 7) or with in vivo reconstitution systems (transfection in bi-cells) Telomerase activity. When transfected into BJ cells as described above, for example, the telomerase activity was comparable to that resulting from reconstitution with pGRN133 or pGRN145.

Similar results were obtained after transfection of normal human pigment-containing retinal epithelial cells (RPE) with the hTRT expression vectors according to the invention. RES cell senescence is believed to lead to or contribute to macular degeneration associated with age. RPE cells treated according to the method according to the invention with the hTRT expression vectors according to the invention should have a delayed senescence compared to untreated cells and should therefore be useful in transplantation therapies for the treatment or for the prevention of age-related macular degeneration.

Example 14

Promoter-reporter construct

This example describes the construction of plasmids in which reporter genes are functionally linked to upstream hTRT sequences containing promoter elements. These vectors have numerous possible uses, including the identification of regulatory factors of transcription acting in ice and trans in vivo and the screening for agents that can modulate hTRT expression (e.g. activation or inhibition) (e.g. screening of active substances). Although a number of reporter genes can be used (for example filament luciferase, β-glucuronidase, β-galactosidase, chloramphinicol acetyltransferase and GFP), the secreted human alkaline phosphatase (SEÄP; CloneTech) was used for preliminary experiments. The SEAP reporter gene encodes a truncated form of the placenta enzyme that lacks the domain for membrane anchoring, thereby enabling efficient protein secretion from transfected cells. It could be shown that the levels of SEAP activity detected in the culture medium are directly proportional to changes in intracellular concentrations of SEAP mRNA and protein (Berger et al., Gene 66 (1988), 1 and Cullen et al., Meth. Enzymol 216 (1992), 362).

Four constructs (pGRN148, pGRN150, "pSEAP2 basic" (no promoter sequence = negative control) and "pSEAP2 control" (which contains the early SV40 promoter and enhancer) were transfected in triplicate into mortal and immortal cells.

Plasmid pGRN148 was constructed as illustrated in Figure 9. Briefly, a Bglll-Eco47lll fragment from pGRN144 was digested and cloned into the Bglll-Nrul site of pSEAp2Basic (Clontech, San Diego, CA). A second reporter promoter, plasmid pGRN150, contains the sequences from the hTRT intron described in Example 3, so that regulatory sequences which may be present in the intron can be used. The initiation codon Met has mutated to Leu, so that the second ATG following the promoter region is the initiation ATG of the SEAP-ORF.

Constructs pGRN148 and pGRN150 containing the hTRT promoter were transfected into mortal (BJ cells) and immortal (293) cells. All transfections were carried out in parallel with two control plasmids. One plasmid as a negative control (pSEAP basic) and one plasmid as a positive control («pSEAP control», which contains the early SV40 promoter and the SV40 enhancer).

In immortal cells, constructs pGRN148 and pGRN150 seem to control SEAP expression as efficiently as positive pSEAP2 control (which contains the early SV40 promoter and enhancer). In contrast, only the «pSEAP2 control» showed detectable activity in mortal cells. These results show that, as expected, hTRT promoter sequences are active in tumor cells, but not in mortal cells.

Similar results were obtained with another normal cell line (RPE), (pigment-containing, retinal epi

144

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

received cell line). In RPE cells transfected with pGRN150 (which contains 2.2 kb of upstream genomic sequence), the hTRT promoter region was inactive while the plasmid «pSEAP2 control» was active.

As noted above, plasmids in which reporter genes are functionally linked to the upstream hTRT sequence containing promoter elements are extremely useful for the identification and screening of telomerase activity modulating agents, using both transient and stable methods Transfection can be used. In one procedure, for example, stable transformants of pGRN148 in telomerase-negative and telomerase-positive cells are produced by cotransfection with a eukaryotic selectable marker (such as neo) according to Ausubel et al., 1997, cited above. The cell lines obtained are then used for the screening of suspected telomerase-modulating agents, for example by comparing the hTRT promoter-controlled expression in the presence or absence of a test compound.

The promoter-reporter vectors (and others) according to the invention are also used to identify regulatory elements acting in trans and in ice for transcription and translation. Examples of regulatory elements of transcription acting in ice include promoters and enhancers of the telomerase gene. The identification and isolation of regulatory agents acting in ice and in trans leads to the provision of further methods and reagents for the identification of agents which modulate the transcription and translation of telomerase.

Example 15

Subcellular localization of hTRT

A fusion protein with hTRT and enhanced green fluorescent protein (EGFP; Cormack et al., Gene 173 (1996), 33) areas was constructed as described below. The EGFP unit provides a detectable “tag” or signal, which makes it easy to determine the presence or location of the fusion protein. Since EGFP fusion proteins are located in the correct cellular compartments, this construct can be used to determine the subcellular location of the hTRT protein.

A. Construction of PGRN138

A vector for expressing an hTRT-EGFP fusion protein in mammalian cells was constructed by integrating the EcoRI insert of pGRN124 (see Example 6) into the EcoRI site of pEGFP-C2 (Clontech, San Diego, CA). The amino acid sequence of the fusion protein is shown below. EGFP residues are in bold, residues encoded by the 5'-untranslated region of hTRT mRNA are underlined and the hTRT protein sequence is given in normal script.

145

CH 689 672 A9

MVSKGEELFTGWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPT

LVTTLTYGVQCFSRYPDHMRQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL

VNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLA

DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITI.GMDELYKS

GRTQISSSSFEFAAASTORCVLLRTWEALAPATPAMPRAPRCRAVRSLLRSHYREVLPI.A

TFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVL

QRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVG

DDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNH

SVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGP

SDRGFCWSPARPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVY

AETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQR

YWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDP

RRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSL

QELTWKMSVRDCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYWELLRSFFYVTE

TTFQKNRLFFYRPSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIP

KPDGLRPIVNMDYWGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDD

IHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYA

WQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAWIEQSSSLNEASSGL

FDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLR

LVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCWNLRKTWNFPVEDEALGGTAFVQMPAH

GLFPWCGLLLDTRTLEVQSDYSSYARTSIRASVTFNRGFKAGRNMRRKLFGVLRLKCHSL

FLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYS

ILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSR

KLPGTTLTALEAAANPALPSDFKTILD

Additional EGFP fusion constructs were made using a partial (e.g., truncated) hTRT coding sequence and, as described below, used to identify activities of certain regions of the hTRT polypeptide.

B. Nuclear localization and uses for DGRN138

The transfection of NIH 293 and BJ cells with pGRN138 allowed the nuclear localization of recombinantly expressed hTRT to be confirmed. Cells were transfected with pGRN138 (EGFPhTRT) and a control construct (only EGFP expressed). The nuclear localization of EGFP-hTRT is evident in both cell types (determined by fluorescence microscopy). As noted above, the pGRN138 hTRT-GFP fusion protein aids in the reconstitution of telomerase activity in both an in vitro transcription / translation system and in vivo when transfected into BJ cells.

hTRT-EGFP fusion proteins (or similar detectable fusion proteins) are used for a variety of applications. For example, the fusion construct described in this example or an EGFP construct and a truncated form of hTRT is used to assess the ability of hTRT and variants to enter a cell nucleus and / or to attach to the chromosome ends. In addition, cells transfected stably or transiently with pGRN138 are used for screening for suspected telomerase-modulating active substances or compounds. Agents that interfere with nuclear or telomer localization are identified as telomerase inhibitors. Tumor cell lines which stably express EGFP-hTRT are used for this purpose. Potential modulators of telomerase are administered to these transfected cells and the location of the EGFP-hTRT can be assessed. In addition, FACS or other fluorescence-based methods are used to select cells expressing hTRT, which provides homogeneous populations for the screening of active substances, especially when transient cell transfection is used.

In other applications, areas of hTRT to identify areas (eg residues 193-196 (PRRR) and residues 235-240 (PKRRRR)) that are required for nuclear localization can be mutagenized, these areas being targets for anti-telomerase agents (Modulators of telomerase activity). Other applications include:

Use of the fusion protein as a fluorescent marker for efficient cell transfection, both for

146

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Experiments regarding transient transfection as well as for the establishment of EGFP-hTRT-express-the stable cell lines;

Expression of an hTRT-EGFP fusion protein with mutated signals for nuclear localization (deficient for nuclear localization) in immortal cells so that the hTRT mutant EGFP captures all hTR of the immortal cells, retains them in the cytoplasm and prevents telomer maintenance;

The GFP can also be used as a "tag" for immunoprecipitation.

Example 16

The effect of a mutation on the catalytic activity of telomerase

This example describes experiments regarding the modification of hTRT amino acids, as well as the effect of amino acid exchanges in hTRT on the catalytic activity of telomerase. The exchange of amino acids and then the functional analysis is a standard procedure for evaluating the meaning and function of a polypeptide sequence. This example shows that changes in the reverse transcriptase (RT) and telomerase T motifs affect the catalytic activity of telomerase and provides additional confirmation of the catalytic nature of the hTRT polypeptide.

The usual nomenclature is used to describe the mutants: the target residue in the native molecule (hTRT) is indicated by the one-letter code and the position, and the corresponding residue in the mutant protein is indicated by the one-letter code. Thus, for example, the expression “K626A” denotes a mutant in which the lysine at position 626 (i.e. in motif 1) is replaced by an alanine by hTRT.

A) Mutagenesis of the FFYxTE motif from hTRT

In preliminary experiments, a mutated hTRT protein coding vector, "F560A", was produced in which the amino acid 560 from SEQ. ID. No. 2 was changed from phenylalanine (F) to alanine (A) by site-directed mutagenesis of pGRN121 using standard procedures. This mutation interrupts the TRT FFYxTE motif. The mutant F560A polynucleotide obtained was shown to control the synthesis of a full-length hTRT protein, as determined using a cell-free reticulocyte lysate transcription / translation system in the presence of 35S methionine.

When the mutant polypeptide was translated together with hTRT as described in Example 7, no telomerase activity was detected by means of the TRAP assay using 20 PCR cycles, while the control cotranslation of hTRT-hTR activity reconstituted. When using 30 PCR cycles in the TRAP assay, telomerase activity was detectable with the mutated hTRT, but was significantly lower compared to the control (wild-type) hTRT.

B) Additional site-directed mutagenesis of hTRT amino acid residues

Conserved amino acids in six RT motifs were changed to alanine to assess their contribution to catalytic activity using standard site-directed mutagenesis techniques (see, for example, Ausubel, op. Cit.). The mutants were examined using IVR telomerase, using the two-step assay (common / TRAP) described in detail in Example 7.

Mutants K626A (motif 1), R631A (motif 2), D712A (motif A), Y717A (motif A) and D868A (motif C) showed greatly reduced or undetectable telomerase activity, while the mutants Q833A (motif B) and G932A (Motif E) had moderate activity levels. Two mutations outside of the RT motifs (R688A and D897A) showed activity that was equivalent to the activity of wild-type hTRT. These results were consistent with analogous mutations in reverse transcriptase (Joyce et al., Ann. Rev. Biochem. 63 (1994), 777), and were similar to the results obtained with Est2P (see Lingner, Science 276 (1997), 561) . These experiments identified residues in the RT motifs that are critical for enzymatic activity. These experiments also show the hTRT, which is the catalytic protein of human telomerase. The mutations produce hTRT polypeptide variants which can be used as dominant-negative regulators of telomerase activity.

The telomerase-specific motif T (see above) was identified by amino acid alignment of the known TRTs. To determine the catalytic role of this motif in hTRT, a six amino acid deletion in this motif (A560-565; FFYxTE) was constructed using standard site-directed mutagenesis techniques (Ausubel, op. Cit.). The deletion was examined using IVR telomerase by means of two-step assays (common / TRAP) described in detail in Example 7. Mutant A560-565 showed no detectable telomerase activity after 25 PCR cycles, while wild-type hTRT-IVR telomerase generated a strong signal. Everyone

147

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

Amino acid residue in motif T was examined independently in a similar manner. The mutants FS60A, Y562A, TS64 and E565A maintained moderate levels of telomerase activity, while the control mutant F487A had minimal effect on activity. Remarkably, the mutant F561A showed a greatly reduced or undetectable telomerase activity, while in its "revertant" F561A561P activity was fully restored. With the F561A561F, the original phenylalanine is again at the mutated position. This is a control that shows that there were no further amino acid changes in the plasmid that could have explained the reduced activity observed. Thus, the T motif is the first non-RT motif that has been shown to be required for telomerase activity.

Motif T is useful for the identification of TRTs from other organisms and as a potential dominant / negative regulator of telomerase activity. Unlike most other RTs, telomerase stably associates with a small portion of a single RNA (ie hTR) and copies it processively, so the motif T could be involved in mediating hTR binding, processivity of the reaction, or other functions that are unique for the Telomerase-RT.

Example 17

Screen for modulators of telomerase activity using recombinantly expressed telomerase components

This example describes the use of in vitro reconstituted telomerase to screen and identify modulators of telomerase activity. The described assay can easily be adapted for high throughput processes (e.g. using multiple well plates and / or robotic technology systems). The numerous possible variations with regard to the assay steps are obvious to the person skilled in the art.

Recombinant clones for telomerase components (e.g. hTRT and hTRR) are produced in an in vitro reaction as follows and in Example 7 described above using the TNT® T7-coupled reticulocyte lysate system (Promega; described in US Pat. No. 5 324 637 and was used according to the manufacturer's instructions) transcribed and translated (only hTRT):

Reagent

Amount per reaction (ml)

TNT rabbit reticulocyte lysate

25th

TNT reaction buffer

2nd

TNT T7 RNA polymerase

1

AA mixture (complete)

1

"Prime" RNase inhibitor

1

Nuclease-free water

16

Xbal-cut pGRN121 (hTRT, 0.5 ng)

2nd

FSPL-cut pGRN164 (hTR, 0.5ng)

2nd

The reaction is incubated at 30 ° C for 2 hours. The product is then cleaned on an “ultrafree-MC” DEAE filter (Millipore).

The recombinant telomerase product (IVRP) is tested in the presence or absence of multiple concentrations of test compounds that have been solubilized in DMSO (e.g. 10 nM to 100 nM). The test compounds are preincubated for 30 minutes at room temperature in a total volume of 25 n ', in the presence of 2.5 nl IVRP, 2.5% DMSO and 1 x TRAP buffer (20 mM Tris-HCl, pH 8.3 , 1.5 mM MgCfe, 63 mM KCl, 0.05% Tween 20, 1.0 mM EGTA and 0.1 mg / ml bovine serum albumin). After the preincubation, 25 nl TRAP assay reaction mixture are added to each sample. The TRAP assay reaction mixture consists of 1 x TRAP buffer, 50 nl dNTP, 2.0 nQ / ml primer ACX, 4 ng / ml primer U2, 0.8 attomol / ml TSU2, 2 units 50 nl Taq polymerase ( Perkin Elmer) and 2 ng / ml [32P] 5'-end-labeled primer TS (3000 Ci / mmol). The tubes with the reaction mixture are then placed in the PCR thermal cycler (MJ Research) and the PCR is carried out as follows: 60 minutes at 30 ° C, 20 cycles of 30 seconds each at 94 ° C, 30 seconds at 60 ° C and 30 seconds at 72 ° C, then 1 minute at 72 ° C and cooling to 10 ° C. As mentioned above, the TRAP assay is described in U.S. Patent No. 5,629,154. The primers and substrates used have the sequences:

148

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

CH 689 672 A9

TS primer (5'-aATCCGTCGAGCaGAGTT-3 ');

ACX primer (5th.GCGCGG [CTTACq3CTAACC-3 ');

U2 primer (S'-ATCGCTTCTCGGCCTTTTo);

TSU2 (5, -AATCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGAT-3 ')

After completion of the PCR step, 4 n '10 x bromophenol blue containing application buffer are added to each reaction tube and the products (20 pl) on a 12.5% non-denaturing PAGE gel in 0.5 x TBE at 400 V let run. After the gel run has ended, the gel is dried and the TRAP products are made visible using the “phosphor imager” or by autoradiography. The telomerase activity in the presence of the test compound is measured by comparing the incorporation of the label into the reaction product with a parallel reaction without the agent.

The following clones described in the examples were deposited with the American Type Culture Collection, Rockville, MD20852, USA.

Lambda-Phage X 25-1.1 ATCC accession number 209024

pGRN121 ATCC accession number 209016

Lambda phage xG <u5 ATCC accession number 98505

These deposits under the Budapest Treaty were made on May 12, 1997 for ATCC 209024, on May 6, 1997 for ATCC 209016 and on August 14, 1997 for ATCC 98505.

The present invention provides new methods and substances for the diagnosis and treatment of diseases related to telomerase. Specific examples have been provided, but the above description is illustrative only and is not intended to be limiting. After reading the description, many possibilities of variation with regard to the invention are obvious to the person skilled in the art. The scope of the invention is thus not determined by the foregoing description, but is intended to be determined by the claims below, along with the full scope of its equivalents.

All publications and patent documents cited in this application are to be regarded as part of the description by reference and for all purposes, to the same extent as if each individual publication or patent document had been individually designated.

Claims (53)

Claims 1. Human telomerase reverse transcriptase (hTRT) polypeptide or a variant thereof or a fragment thereof, wherein the polypeptide or variant thereof or the fragment thereof has at least one of the functional activities associated with native hTRT and the activity or activities from the Group are selected, which consists of: the activity that extends a DNA primer or substrate by at least one TTAGGG repeat, i.e. the catalytic activity of telomerase, the activity of conventional reverse transcriptase, the activity that exogenous RNA transcribes, - the nucleolytic activity, - the primer- or substrate-binding activity, - protein binding activity, - the activity that binds human telomerase (hTR) RNA. 2. hTRT polypeptide or variant thereof or fragment thereof according to claim 1, with at least 75% sequence identity to the hTRT polypeptide SEQ. Id. No. 2. 3. hTRT polypeptide according to claim 1, with the sequence of SEQ. Id. No. 2 and with complete Län-9e- 4. hTRT polypeptide according to any one of claims 1 to 3, wherein the polypeptide or the variant or the fragment thereof have catalytic activity of telomerase. 5. A composition comprising an hTRT polypeptide according to any one of claims 1 to 4 and an RNA. 6. The composition of claim 5, wherein the RNA is a human telomerase RNA. 7. The composition of claim 6, wherein the hTRT polypeptide and human telomerase RNA form a ribonucleoprotein complex with telomerase activity. 149 CH 689 672 A9 8. A nucleic acid sequence encoding an hTRT polypeptide or a fragment thereof according to claim 1. 9. The nucleic acid sequence according to claim 8, which encodes an hTRT polypeptide, wherein the hTRT polypeptide is one as in SEQ. Id. No. 2 has given amino acid sequence, or a sequence that includes one or more conservative substitutions in the amino acid sequence. 5 10. A nucleic acid sequence encoding an hTRT polypeptide according to claim 3. 11. The nucleic acid sequence according to claim 10, comprising the sequence encoding the hTRT polypeptide of the insertion of pGRN121 (ATCC accession number 209016). 12. Nucleic acid sequence according to claim 10, with the sequence shown in Fig. 16 (SEQ. Id. No. 1). 13. A nucleic acid sequence according to claim 8, which is the sequence of SEQ. Id. No. 1. 10th 14. Nucleic acid sequence according to claim 8, which hybridizes under stringent conditions with a nucleic acid sequence which is exactly complementary to the sequence of SEQ. Id No. 1 is. 15. A polynucleotide comprising a nucleic acid sequence according to one of claims 8 to 14. 16. Nucleic acid sequence that is exactly or almost exactly complementary to the nucleic acid sequence according to one of claims 8 to 14. 15 17. A polynucleotide comprising a nucleic acid sequence according to claim 16. 18. A polynucleotide according to claim 15 or 17, comprising a promoter sequence functionally linked to the nucleic acid sequence encoding the hTRT polypeptide. 19. A substantially pure polynucleotide according to claim 15 or 17, which has a length of at least 10 nucleotides and a sequential sequence of at least 12 nucleotides. 20 that is identical or exactly complementary to a successive sequence in a naturally occurring hTRT gene or an hTRTmRNA. 20. Polynucleotide according to claim 19, with at least 15 consecutive nucleotides. 21. Polynucleotide according to claim 19, with at least 25 consecutive nucleotides. 22. Nucleic acid sequence or polynucleotide according to one of claims 8 to 15, which / to the 25 contiguous sequence in a naturally occurring hTRT gene or an hTRT mRNA is identical. 23. The nucleic acid sequence or polynucleotide according to claim 16 or 17, which is exactly complementary to the connected sequence in a naturally occurring hTRT gene or an hTRT mRNA. 30th 24. Expression vector comprising a nucleic acid sequence or a polynucleotide according to any one of claims 8 to 23. 25. Ex vivo cell or cell line transfected with a nucleic acid sequence or a polynucleotide according to any one of claims 8 to 23. 26. The cell of claim 25 which is a human cell. 35 27. The cell of claim 25 or 26, which has an increased proliferation capacity compared to a cell which is otherwise identical but does not contain the nucleic acid sequence or the polynucleotide. 28. Antibodies or fragments thereof that specifically bind to an hTRT polypeptide according to any one of claims 1 to 4. 40 29. Ex vivo cell which can secrete the antibody of claim 28. 30. A method of testing whether a compound is a modulator of telomerase reverse transcriptase activity or hTRT expression, comprising contacting an ex vivo cell, a cell extract or a composition containing an hTRT polypeptide according to claim 1 or a polynucleotide according to claim 15 with the connection and the detection of a change in the 45 Telomerase reverse transcriptase activity or hTRT expression in the ex vivo cell, the cell extract or the composition. 31. A method of examining whether a test compound is a modulator of telomerase reverse transcriptase activity, the method comprising the steps of: a) providing a polypeptide according to any one of claims 1 to 4; 50 b) contacting the polypeptide with the test compound; and c) measuring the activity of the hTRT polypeptide, wherein a change in the telomerase reverse transcriptase activity measured in the presence of the test compound compared to the activity in the absence of the test compound provides evidence that the test compound detects the telo -merase reverse transcriptase activity modulated. 55 32. A method for producing recombinant telomerase according to any one of claims 1 to 4, which comprises culturing a cell according to claim 26 or 27 and isolating the polypeptide from the culture. 33. The method of claim 32, further comprising contacting the recombinant hTRT polypeptide with a telomerase RNA component under conditions such that the recombinant Associate 60 nante polypeptide and the telomerase RNA component to form a telomerase enzyme that can catalyze the attachment of nucleotides to a telomerase substrate. 34. A method for the detection of an hTRT gene product in a biological sample, comprising: a) contacting the biological sample with a probe which hybridizes specifically to the hTRT gene product, the probe and the gene product forming a complex, and detection of the complex 65 xes; or 150 5 10th 15 20th 25th 30th 35 40 45 50 55 60 65 CH 689 672 A9 b) specific amplification of the gene product in the biological sample, and detection of the amplification product, the presence of the complex or the amplification product being correlated with the presence of the hTRT gene product in the biological sample, and wherein the gene product is a polynucleotide according to claim 15 or 17 is. 35. A method for detecting an hTRT polypeptide according to claim 1 in a biological sample, comprising: Contacting the biological sample with a probe which specifically binds the hTRT polypeptide, the probe and the hTRT polypeptide forming a complex, and detection of the complex, the presence of the complex being correlated with the presence of the hTRT polypeptide in the biological sample. 36. The method according to claim 35, characterized in that the probe is a monoclonal antibody. 37. A method for detecting the presence of at least one telomerase-positive human cell in a biological sample containing human cells according to claim 23, the method comprising the following steps: a) determining the amount of hTRT gene product in the sample, b) comparison of the measured amount with a control which correlates with a sample containing no telomerase positive cells, the presence of a higher level of the hTRT gene product in the sample compared to the control being correlated with the presence of telomerase positive cells in the biological sample. 38. The method of claim 37, wherein the telomerase positive cells are cancer cells. 39. A method of diagnosing a telomerase-related condition on a patient's cell sample or tissue sample, comprising: a) determining the amount of hTRT gene product in the sample, b) Comparison of the amount of hTRT gene product in the sample with the amount in a healthy cell of the same type, wherein an amount of hTRT gene product in the sample which differs from the amount in the healthy cell is a diagnostic indicator of a telomerase in Related state, wherein the gene product is a polynucleotide according to claim 15. 40. Kit for the detection of an hTRT gene or gene product, the kit containing one or more reagents selected from the group consisting of the polynucleotides according to claim 15 or 17, the polypeptides according to one of claims 1 to 7 and the Antibody or fragment thereof according to claim 28. 41. A method for increasing the proliferation capacity of a vertebrate cell ex vivo, characterized in that the expression of hTRT according to claim 1 is increased in the cell. 42. A method for increasing the proliferation capacity of a vertebrate cell ex vivo by introducing a recombinant polynucleotide into the cell, the polynucleotide comprising a sequence encoding an hTRT polypeptide according to claim 1 and wherein the sequence is functionally linked to a promoter. 43. The method of claim 42, wherein the hTRT polypeptide has the sequence of the hTRT polypeptide encoded by pGRN121 (ATCC accession number 209016). 44. Medicament, comprising a molecule selected from the group consisting of the hTRT polypeptides according to one of claims 1 to 4, the polynucleotide according to claim 15, which encodes an hTRT polypeptide, and the antibody or fragment thereof according to claim 28. 45. A pharmaceutical composition comprising an inhibitor of telomerase activity, optionally in combination with a pharmaceutically acceptable carrier, the inhibitor being an hTRT polypeptide according to claim 1, an hTRT antibody according to claim 28 or an hTRT polynucleotide according to claim 15 or 17 . 46. The use of an inhibitor of telomerase activity in the manufacture of a medicament for the treatment of a condition associated with an increased level of telomerase activity within a human cell, said inhibitor: a) is a polypeptide according to claim 1 having an amino acid sequence which is identical to a successive sequence of at least six amino acids in hTRT; b) is an hTRT binding antibody; c) is a polynucleotide with a nucleotide sequence which is identical or complementary to a successive sequence of at least 12 nucleotides in an hTRT coding sequence; or d) a compound identified by screening comprising contacting a test compound with a polypeptide having an amino acid sequence that is identical to a sequential sequence of at least six amino acids in hTRT. 47. The use of the protein according to any one of claims 1 to 4, the polynucleotide according to claim 15 or 17, or the antibody or fragment according to claim 28 for the manufacture of a medicament for inhibiting cancer or aging. 48. Use of a polynucleotide that is at least 6 bases long and sufficiently complementary to SEQ. No. 1, in order to hybridize with the latter, is a primer in the amplification of the polynucleotides according to claim 15 or 17. 49. Use according to claim 48, wherein the polynucleotide is at least 15 bases long. 151 CH 689 672 A9 50. Use according to claim 48, wherein the polynucleotide is at least 25 bases long. 51. Use of a polynucleotide that is at least 10 bases long and attached to SEQ. Id. No. 1 or its complement hybridizes as a probe for the detection of the polynucleotide according to claim 15 or 17. 5 52. Use according to claim 51, wherein the polynucleotide is at least 20 bases long. 53. Use according to claim 51, wherein the polynucleotide is labeled and the label provides the detectable signal. 10th 15 20th 25th 30th 35 40 45 50 1 ! 60 152