Antisense compositions for detecting and inhibiting telomerase reverse transcriptase

Abstract

The present invention provides TRT antisense oligonucleotides, methods of detecting TRT, methods of diagnosing telomerase-related conditions, methods of diagnosing and providing a prognosis for cancer, and methods of treating telomerase-related conditions, including cancer.

Description

FIELD OF THE INVENTION

The present invention provides TRT antisense oligonucleotides, methods of detecting TRT, methods of diagnosing telomerase-related conditions, methods of diagnosing and providing a prognosis for cancer, and methods of treating telomerase-related conditions, including cancer, with TRT antisense oligonucleotides.

BACKGROUND OF THE INVENTION

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

It has long been recognized that complete replication of the ends of eukaryotic chromosomes requires specialized cell components (Watson, 1972, Nature New Biol., 239:197; Olovnikov, 1973, J. Theor. Biol., 41:181). Replication of a linear DNA strand by conventional DNA polymerases requires an RNA primer, and can proceed only 5′ to 3′. When the RNA bound at the extreme 5′ ends of eukaryotic chromosomal DNA strands is removed, a gap is introduced, leading to a progressive shortening of daughter strands with each round of replication. This shortening of telomeres, the protein-DNA structures physically located on the ends of chromosomes, is thought to account for the phenomenon of cellular senescence or aging of normal human somatic cells in vitro and in vivo. The length and integrity of telomeres is thus related to entry of a cell into a senescent stage (i.e., loss of proliferative capacity), or the ability of a cell to escape senescence, i.e., to become immortal. The maintenance of telomeres is a function of a telomere-specific DNA polymerase known as telomerase. Telomerase is a ribonucleoprotein (RNP) that uses a portion of its RNA moiety as a template for telomeric DNA synthesis (Morin, 1997, Eur. J. Cancer 33:750).

Consistent with the relationship of telomeres and telomerase to the proliferative capacity of a cell (i.e., the ability of the cell to divide indefinitely), telomerase activity is detected in immortal cell lines and an extraordinarily diverse set of tumor tissues, but is not detected (i.e., was absent or below the assay threshold) in normal somatic cell cultures or normal tissues adjacent to a tumor (see, U.S. Pat. Nos. 5,629,154; 5,489,508; 5,648,215; and 5,639,613; see also, Morin, 1989, Cell 59: 521; Shay and Bacchetti 1997, Eur. J. Cancer 33:787; Kim et al., 1994, Science 266:2011; Counter et al., 1992, EMBO J. 11:1921; Counter et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91, 2900; Counter et al., 1994, J. Virol. 68:3410). Moreover, a correlation between the level of telomerase activity in a tumor and the likely clinical outcome of the patient has been reported (e.g., U.S. Pat. No. 5,639,613, supra; Langford et al., 1997, Hum. Pathol. 28:416). Human telomerase is thus an ideal target for diagnosing and treating human diseases relating to cellular proliferation and senescence, such as cancer.

SUMMARY OF THE INVENTION

The present invention provides TRT antisense polynucleotides, which are useful for detecting, diagnosing, and treating telomerase-related conditions.

In one aspect, the present invention provides an isolated, synthetic, substantially pure, or recombinant polynucleotide having a sequence that is at least about ten nucleotides in length to at least about 100 nucleotides in length. This polynucleotide comprises a sequence that is substantially complementary or substantially identical to a contiguous sequence of an hTRT nucleic acid that has the nucleotide sequence of FIG. 1 .

In one aspect, the present invention provides an isolated, synthetic, substantially pure, or recombinant polynucleotide having a sequence that is at least about ten nucleotides in length to at least about 100 nucleotides in length. This polynucleotide comprises a sequence exactly complementary or identical to a contiguous sequence of a nucleic acid encoding the hTRT protein of FIG. 2 .

In one embodiment, the hTRT polynucleotide comprises a sequence that is exactly complementary or identical to a contiguous sequence of an hTRT nucleic acid having the nucleotide sequence of FIG. 1 .

In one embodiment, the polynucleotide is a DNA or an RNA. In one embodiment, the polynucleotide comprises one or more non-naturally occurring, synthetic nucleotides.

In one embodiment, the polynucleotide is identical to said contiguous sequence of a nucleic acid encoding the hTRT protein of FIG. 1 . In one embodiment, the polynucleotide is exactly complementary to said contiguous sequence of a nucleic acid encoding the hTRT protein of FIG. 1 .

In one embodiment, the polynucleotide is an antisense polynucleotide. In one embodiment, the polynucleotide is at least about 20 nucleotides in length to at least about 50 nucleotides in length.

In one embodiment, the polynucleotide inhibits telomerase activity by at least about 50% in transformed cells ex vivo, as compared to control cells that are not treated with the polynucleotide. In one embodiment, the polynucleotide inhibits telomerase expression by at least about 50% in vitro, as compared to control expression reactions that lack the polynucleotide. In one embodiment, the polynucleotide is selected from the group consisting of PS-ODN number 3, 4, 7, 8, 16, 21, 25, 26, 27, 28, 29, 33, 40, 41, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 62, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 80, 81, 82, 83, 84, 85, 86, 87, 88, 93, 94, 96, 100, 112, 114, 130, 143, 144, 151, 152, 201, 202, 203, 208, 209, 210, 211, 212, 213, 230, 237, and 241.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents the nucleotide sequence of a cDNA (SEQ ID NO:1) encoding a naturally occurring human telomerase reverse transcriptase (hTRT) protein.

FIG. 2 presents the amino acid sequence (SEQ ID NO:2) of a naturally occurring, 1132-residue human telomerase reverse transcriptase (hTRT) protein.

FIG. 3 shows inhibition of hTRT expression in vitro by hTRT sequence-specific antisense phosphorothioate oligonucleotides (PS-ODN). Each bar in the graph represents the in vitro inhibitory activity of a specific oligonucleotide, numbered starting with P,S-ODN #1. The PS-ODN are a series of 30-mers that span the hTRT mRNA and are offset one from the next by fifteen nucleotides. For example, ODN #1 corresponds to positions 16-35 of hTRT and is TCCCACGTGCGCAGCAGGACGCAGCGCTGC. ODN #2 corresponds to positions 31-60 and is GGCATCGCGGGGGTGGCCGGGGCCAGGGCT (SEQ ID NO:4), and so one to the end of the RNA (see the cDNA sequence of FIG. 1 , which represents an hTRT RNA sequence). The data are presented as a normalized percentage of the control with no added PS-ODN.

DETAILED DESCRIPTION

I. Introduction

Telomerase is a ribonucleoprotein complex (RNP) comprising an RNA component and a catalytic protein component. The catalytic protein component of human telomerase, hereinafter referred to as telomerase reverse transcriptase (“hTRT”), has been cloned, and protein, cDNA, and genomic sequences determined. See, e.g., Nakamura et al., 1997, Science 277:955, and copending U.S. patent applications Ser. Nos. 08/912,951 and 08/974,549. The sequence of a full-length native hTRT has been deposited in GenBank (Accession No. AF015950), and plasmid and phage vectors having hTRT coding sequences have been deposited with the American Type Culture Collection, Rockville, Md. (accession numbers 209024, 209016, and 98505). The catalytic subunit protein of human telomerase has also been referred to as “hEST2” (Meyerson et al., 1997, Cell 90:785), “hTCS1” (Kilian et al., 1997, Hum. Mol. Genet. 6:2011), “TP2” (Harrivigton et al., 1997, Genes Dev. 11:3109), and “hTERT” (e.g., Greider, 1998, Curr. Biol. 8:R178-R181). The RNA component of human telomerase (hTR) has also been characterized (see U.S. Pat. No. 5,583,016).

Human TRT is of extraordinary interest and value because, inter alia, telomerase activity in human cells and other mammalian cells correlates with cell proliferative capacity, cell immortality, and the development of a neoplastic phenotype. hTRT antisense polynucleotides, including the exemplary polynucleotides described herein, hybridize to and/or amplify naturally occurring hTRT genes or RNA. Such oligonucleotides are thus useful for diagnostic or prognostic applications to telomerase related conditions, including cancer. The hTRT antisense polynucleotides of the invention are also useful as therapeutic agents, e.g., antisense oligonucleotides, ribozymes, or triplex compositions, for inhibition of telomerase expression and activity (e.g., telomerase catalytic activity, infra).

The invention thus provides antisense oligonucleotide reagents, which can be used to detect expression of hTRT or reduce expression and activity of hTRT gene products in vitro, ex vivo, or in vivo. Administration of the antisense reagents of the invention to a target cell results in reduced telomerase activity, and is particularly useful for treatment of diseases characterized by high telomerase activity (e.g., cancers). Detection and inhibition of hTRT expression can be performed in a cell or cell extracts from a human, a mammal, a vertebrate, or other eukaryote.

The antisense polynucleotides of the invention are characterized by their ability to specifically hybridize to naturally occurring and synthetic hTRT nucleic acids, e.g., the hTRT gene, including any upstream, flanking, noncoding, and transcriptional control elements, hTRT pre-mRNA, mRNA, cDNA and the like. The hTRT antisense polynucleotides of the invention are typically at least 7-10 nucleotides in length to typically more 20 nucleotides up to about 100 nucleotides in length, preferably approximately 30 nucleotides in length. Such antisense oligonucleotides are used to detect the presence of hTRT nucleic acid in a biological sample, for diagnosis and/or prognosis of telomerase related conditions, e.g., cancers of any of a wide variety of types, including solid tumors and leukemias, diseases of cell proliferation, disease resulting from cell senescence (particularly diseases of aging), immunological disorders, infertility, disease of immune dysfunction, etc.

The antisense polynucleotides of the invention also can be used to inhibit telomerase expression in vitro, to inhibit telomerase expression and activity in cells ex vivo, and can be used in vivo as therapeutic agents for the treatment of telomerase-related conditions listed above, including cancers of a wide variety of types (see, e.g., exemplary cancers listed in U.S. patent application Ser. No. 08/974,549; and U.S. patent application Ser. No. 08/974,584). In one embodiment of the invention, the antisense polynucleotides are 30 nucleotides in length, and have the ability to inhibit telomerase expression at least by 50% in vitro (see, e.g., the antisense oligonucleotides of FIG. 3 ). In another embodiment of the invention, the antisense polynucleotides are 30 nucleotides in length, and have the ability to inhibit telomerase expression and activity at least 50% in transformed cells ex vivo (see, e.g., exemplary antisense hTRT oligonucleotides listed in Table 1).

II. Definitions

As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

As used herein, the terms “nucleic acid” and “polynucleotide” are used interchangeably. Use of the term “polynucleotide” includes oligonucleotides (i.e., short polynucleotides). This term also refers to deoxyribonucleotides, ribonucleotides, and naturally occurring variants, and can also refer to synthetic and/or non-naturally occurring nucleic acids (i.e., comprising nucleic acid analogues or modified backbone residues or linkages), such as, for example and without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like, as described herein.

As used herein “oligonucleotides” or “oligomers” refer to a nucleic acid sequence of approximately 7 nucleotides or greater in length, and up to as many as approximately 100 nucleotides in length, which can be used as a primer, probe or amplimer. Oligonucleotides are often between about 10 and about 50 nucleotides in length, more often between about 14 and about 35 nucleotides, very often between about 15 and about 30 nucleotides, and the terms oligonucleotides or oligomers can also refer to synthetic and/or non-naturally occurring nucleic acids (i.e., comprising nucleic acid analogies or modified backbone residues or linkages).

A polynucleotide “specifically hybridizes” or “specifically binds” to a target polynucleotide if the polynucleotide hybridizes to the target under stringent conditions. As used herein, “stringent hybridization conditions” or “stringency” refers to conditions in a range from about 5° C. to about 20° C. or 25° C. below the melting temperature (T m ) of the target sequence and a probe with exactly or nearly exactly complementarity to the target. As used herein, the melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half-dissociated into single strands. Methods for calculating the T m 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.; Sambrook et al. (1989) Molecular Cloning,: A Laboratory Manual, 2nd Ed., Vols. 1-3, Cold Spring Harbor Laboratory hereinafter, “Sambrook”); and Current Protocols in Molecular Biology (Ausubel et al., eds. through and including the 1997 supplement), incorporated herein by reference). As indicated by standard references, a simple estimate of the T m value may be calculated by the equation: T m =81.5+0.41(% G+C), when a nucleic acid is in aqueous solution at 1 M NaCl (see e.g., Anderson and Young, Quantitative Filter Hybridization in Nucleic Acid Hybridization (1985)). Other references include more sophisticated computations, which take structural as well as sequence characteristics into account for the calculation of T m . The melting temperature of a hybrid (and thus the conditions for stringent hybridization) is affected by various factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, and the like), 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 in the art, e.g., Sambrook, supra and Ausubel et al. supra. Typically, stringent hybridization conditions are salt concentrations less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion at pH 7.0 to 8.3, and temperatures at least about 30° C. for short nucleic acids (e.g., 7 to 50 nucleotides) and at least about 60° C. for long nucleic acids (e.g., greater than 50 nucleotides). As noted, stringent conditions may also be achieved with the addition of destabilizing agents such as formamide, in which case lower temperatures may be employed.

An “identical” polynucleotide refers to a polynucleotide that has the same sequence as the reference nucleotide subsequence to which the polynucleotide is being compared. An “exactly complementary” polynucleotide refers to a polynucleotide whose complement has the same sequence as the reference nucleotide subsequence to which the polynucleotide is being compared.

A “substantially complementary” polynucleotide and a “substantially identical” polynucleotide have the ability to specifically hybridize to a reference gene, DNA, cDNA, or mRNA, e.g., the hTRT nucleotide sequence of FIG. 1 and its exact complement.

An “antisense” polynucleotide is a polynucleotide that is substantially complementary to a target polynucleotide and has the ability to specifically hybridize to the target polynucleotide.

A “telomerase-related condition” refers to a diseases and disease conditions in a patient and/or a cell, characterized by under- or over-expression of telomerase or hTRT gene products. In addition to cancer, which is characterized by over-expression of telomerase, such conditions include diseases of cell proliferation, e.g., hyperplasias, disease resulting from cell senescence (particularly diseases of aging), immunological disorders, infertility, etc.

As used herein, “isolated,” when referring to a molecule or composition, such as, for example, an oligonucleotide, means that the molecule or composition is separated from at least one other compound, such as other oligonucleotides or other contaminants with which it is associated in vivo or in its naturally occurring state or synthetic state. An isolated composition can also be substantially pure.

A “synthetic” oligonucleotide refers to a polynucleotide synthesized using in vitro chemical methods, e.g., by using a machine that synthesizes polynucleotides using the phosphodiester method, the diethylphosphoramidite method, the phosphotriester methods, the solid support method, and other methods known to those skilled in the art.

As used herein, “recombinant” refers to a polynucleotide synthesized or otherwise manipulated in vitro (e.g., “recombinant polynucleotide”), to methods of using recombinant polynucleotides to produce gene products in cells or other biological systems, or to a polypeptide (“recombinant protein”) encoded by a recombinant polynucleotide.

As used herein, the term “substantially pure,” or “substantially purified,” when referring to a composition comprising a specified reagent, such as an oligonucleotide, means that the specified reagent is at least about 75%, or at least about 90%, or at least about 95%, or at least about 99% or more of the composition (not including, e.g., solvent or buffer). Thus, for example, an antisense oligonucleotide preparation that specifically binds an hTRT gene or mRNA is substantially purified.

“TRT” activity refers to one or more of the activities found in naturally-occurring full-length TRT proteins. These activities include “telomerase catalytic activity” (the ability to extend a DNA primer that functions as a telomerase substrate by adding a partial, one, or more than one repeat of a sequence, e.g., TTAGGG, encoded by a template nucleic acid, e.g., hTR), “telomerase conventional reverse: transcriptase activity” (see Morin, 1997, supra, and Spence et al., 1995, Science 267:988); “nucleolytic activity” (see Morin, 1997, supra; Collins and Grieder, 1993, Genes and Development 7:1364; Joyce and Steitz, 1987, Trends Biochem. Sci. 12:288); “primer (telomere) binding activity” (see, Morin, 1997, supra; Collins et al., 1995, Cell 81:677; Harrington et al., 1995, J. Biol. Chem. 270:8893); “dNTP binding activity” (Morin, 1997, supra; Spence et al., supra); and “RNA (e.g., hTR) binding activity” (see Morin, 1997, supra; Harrington et al., 1997, Science 275:973; Collins et al., 1995, Cell 81:677,).

“TRT” refers to telomerase reverse transcriptase protein, and “hTRT” refers to human telomerase reverse transcriptase protein.

The term “hTRT” is intended to refer to alleles, conservatively modified variants, polymorphic variants, and interspecies homologues of hTRT encoded by nucleic acids that specifically hybridize to the hTRT nucleic acid sequence provided in FIG. 1 .

“Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids that encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art (see, e.g., Creighton (1984) Proteins, W. H. Freeman and Company.

III. How to Make Antisense Polynucleotides

As described herein, the present invention provides antisense polynucleotides, which have the ability to specifically hybridize to hTRT. Without intending to be limited to any particular mechanism, it is believed that antisense oligonucleotides bind to, and interfere with the translation of, the sense hTRT mRNA. Alternatively, the antisense molecule may render the hTRT mRNA susceptible to nuclease digestion, interfere with transcription, interfere with processing, localization or otherwise with RNA precursors (“pre-mRNA”), repress transcription of mRNA from the hTRT gene, or act through some other mechanism. However, the particular mechanism by which the antisense molecule reduces hTRT expression is not critical.

Generally, to assure specific hybridization, the antisense sequence is substantially complementary to the target hTRT mRNA sequence. In certain embodiments, the antisense sequence is exactly complementary to the target sequence. The antisense polynucleotides may also include, however, nucleotide substitutions, additions, deletions, transitions, transpositions, or modifications, or other nucleic acid sequences or non-nucleic acid moieties so long as specific binding to the relevant target sequence corresponding 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., relatively devoid of secondary structure). These sequences can be determined by analyzing predicted RNA secondary structures using, for example, the MFOLD program (Genetics Computer Group, Madison Wis.) and testing in vitro or in vivo as is known in the art. FIG. 3 and TAble 1 show examples of oligonucleotides that are useful in cells for antisense suppression of hTRT function and are capable of hybridizing to hTRT (i.e., are substantially complementary to hTRT). Another useful method for identifying effective antisense compositions uses combinatorial arrays of oligonucleotides (see, e.g., Milner et al., 1997, Nature Biotechnology 15:537).

A. Triplex-forming Antisense Polynucleotides

As one embodiment of the antisense molecules described herein, the present invention provides polynucleotides 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), forming a triple helix-containing, or “triplex” nucleic acid. Triple helix formation results in inhibition of hTRT expression by, for example, preventing transcription of the hTRT gene, thus reducing or eliminating telomerase activity in a cell. Without intending to be bound by any particular mechanism, it is believed that triple helix pairing compromises the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules to occur.

Triplex oligo- and polynucleotides of the invention are constructed using the base-pairing rules of triple helix formation (see, e.g., Cheng et al., 1988, J. Biol. Chem. 263: 15110; Ferrin and Camerini-Otero, 1991, Science 354:1494; Ramdas et al., 1989, j. Biol. Chem. 264:17395; Strobel et al., 1991, Science 254:1639; and Rigas et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83: 9591; each of which is incorporated herein by reference) and the hTRT MnRNA and/or gene sequence. Typically, the triplex-forming oligonucleotides of the invention comprise a specific sequence of from about 10 to at least about 25 nucleotides or longer “complementary” to a specific sequence in the hTRT RNA or gene (i.e., large enough to form a stable triple helix, but small enough, depending on the mode of delivery, to administer in vivo, if desired). In this context, “complementary” means able to form a stable triple helix. In one embodiment, oligonucleotides are designed to bind specifically to the regulatory regions of the hTRT gene (e.g., the hTRT 5′-flanking sequence, promoters, and enhancers) or to the transcription initiation site, (e.g., between −10 and +10 from the transcription initiation site). For a review of recent therapeutic advances using triplex DNA, see Gee et al., in Huber and Carr, 1994, Molecular and Immunologic Approaches, Futura Publishing Co, Mt Kisco N.Y. and Rininsland et al., 1997, Proc. Natl. Acad. Sci. USA 94:5854, which are both incorporated herein by reference.

B. Ribozymes

In another embodiment, the present invention provides ribozymes useful for inhibition of hTRT telomerase activity. The ribozymes of the invention bind and enzymatically cleave and inactivate hTRT mRNA. Useful ribozymes can comprise 5′- and 3′-terminal sequences complementary to the hTRT mRNA and can be engineered by one of skill on the basis of the hTRT mRNA sequence disclosed herein (see PCT publication WO 93/23572, supra). Ribozymes of the invention include those having characteristics of group I intron ribozymes (Cech, 1995, Biotechnology 13:323) and others of hammerhead ribozymes (Edgington, 1992, Biotechnology 10:256).

Ribozymes of the invention include those having cleavage sites such as GUA, GUU and GUC. Other optimum cleavage sites for ribozyme-mediated inhibition of telomerase activity in accordance with the present invention include those described in PCT publications WO 94/02595 and WO 93/23569, both incorporated herein by reference. Short RNA oligonucleotides between 15 and 20 ribonucleotides in length corresponding to the region of the target hTRT gene containing the cleavage site can be evaluated for secondary structural features that may render the oligonucleotide more desirable. The suitability of cleavage sites may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assay, or by testing for in vitro ribozyme activity in accordance with standard procedures known in the art.

As described by Hu et al., PCT publication WO 94/03596, incorporated herein by reference, antisense and ribozyme functions can be combined in a single oligonucleotide. Moreover, ribozymes can comprise one or more modified nucleotides or modified linkages between nucleotides, as described above in conjunction with the description of illustrative antisense oligonucleotides of the invention.

C. Synthesis of Antisense Polynucleotides

The antisense nucleic acids (DNA, RNA, modified, analogues, and the like) can be made using any suitable method for producing a nucleic acid, such as the chemical synthesis and recombinant methods disclosed herein and known to one of skill in the art. In one embodiment, for example, antisense RNA molecules of the invention may be prepared by de novo chemical synthesis or by cloning. For example, an antisense RNA that hybridizes to hTRT mRNA can be made by inserting (ligating) an hTRT DNA sequence in reverse orientation operably linked to a promoter in a vector (e.g., plasmid). Provided that the promoter and, preferably termination and polyadenylation signals, are properly positioned, the strand of the inserted sequence corresponding to the noncoding strand will be transcribed and act as an antisense oligonucleotide of the invention.

The present invention also provides hTRT antisense polynucleotides (RNA, DNA. or modified) that can be produced by direct chemical synthesis. Chemical synthesis is generally preferred for the production of oligonucleotides or for oligonucleotides and polynucleotides containing nonstandard nucleotides (e.g., probes, primers and antisense oligonucleotides). Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiester method of Brown et al., Meth. Enzymol. 68:109 (1979); the diethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859 (1981); and the solid support method of U.S. Pat. No. 4,458,066.

Chemical synthesis typically produces a single stranded oligonucleotide, which may be converted into 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. One of skill will recognize that while chemical synthesis of DNA is often limited to sequences of about 100 or 150 bases, longer sequences may be obtained by the ligation of shorter sequences or by more elaborate synthetic methods.

It will be appreciated that the hTRT polynucleotides and oligonucleotides of the invention can be made using nonstandard bases (e.g., other than adenine, cytidine, guanine, thymine, and uridine) or nonstandard backbone structures to provides desirable properties (e.g., increased nuclease-resistance, tighter-binding, stability or a desired T M ). Techniques for rendering oligonucleotides nuclease-resistant include those described in PCT publication WO 94/12633. A wide variety of useful modified oligonucleotides may be produced, including oligonucleotides having a peptide-nucleic acid (PNA) backbone (Nielsen et al., 1991, Science 254:1497) or incorporating 2′-O-methyl ribonucleotides, phosphorothioate nucleotides, methyl phosphonate nucleotides, phosphotriester nucleotides, phosphorothioate nucleotides, phosphoramidates. Still other useful oligonucleotides may contain alkyl and halogen-substituted sugar moieties comprising one of the following at the 2′ position: OH, SH, SCH 3 , F, OCN, OCH 3 OCH 3 , OCH 3 O(CH 2 ) n CH 3 , O(CH 2 ) n NH 2 or O(CH 2 ) n CH 3 , where n is from 1 to about 10; C 1 to C 10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF 3 ; OCF 3 ; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH 3 ; SO 2 CH 3 ; ONO 2 ; NO 2 ; N 3 ; NH 2 ; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a cholesteryl 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 having similar properties. Folate, cholesterol or other groups that facilitate oligonucleotide uptake, such as lipid analogs, may be conjugated directly or via a linker at the 2′ position of any nucleoside or at the 3′ or 5′ position of the 3′-terminal or 5′-terminal nucleoside, respectively. One or more such conjugates may be used. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group. Other embodiments may include at least one modified base form or “universal base” such as inosine, or inclusion of other nonstandard bases such as queosine and wybutosine as well as acetyl-, methyl-, thio- and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.

The invention further provides oligonucleotides having backbone analogues such is phosphodiester, phosphorothioate, phosphorothioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3′-thioacetal, methylene(methylimino), 3′-N-carbamate, morpholino carbamate, chiral-methyl phosphonates, nucleotides with short chain alkyl or cycloalkyl intersugar linkages, short chain heteroatomic or heterocyclic intersugar (“backbone”) linkages, or CH 2 —NH—O—CH 2 , CH 2 —N(CH 3 )—OCH 2 , CH 2 —O—N(CH 3 )—CH 2 , CH 2 —N(CH 3 )—N(CH 3 )—CH 2 and O—N(CH 3 )—CH 2 —CH 2 backbones (where phosphodiester is O—P—O—CH 2 ), or mixtures of the same. Also useful are oligonucleotides having morpholino backbone structures (U.S. Pat. No. 5,034,506).

Useful references include Oligonucleotides and Analogues, A Practical Approach, edited by F. Eckstein, IRL Press at Oxford University Press (1991); Antisense Strategies, Annals of the New York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan et al., Jul. 9, 1993, J. Med. Chem. 36(14):1923-1937; Antisense Research and Applications (1993, CRC Press), in its entirety and specifically Chapter 15, by Sanghvi, entitled “Heterocyclic base modifications in nucleic acids and their applications in antisense oligonucleotides;” and Antisense Therapeutics, ed. Sudhir Agrawal (Humana Press, Totowa, N.J., 1996).

D. Labeled Antisense Oligonucleotides

It is often useful to label the antisense polynucleotides of the invention, for example, when the hTRT polynucleotides are to be used for detection of hTRT expression, and for diagnosis and prognosis of telomerase related conditions. The labels may be incorporated by any of a number of means well known to those of skill in the art. Suitable labels are any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include 32 P, 35 S, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin-streptavadin, digoxigenin, haptens and proteins for which antisera or monoclonal antibodies are available, or nucleic acid molecules with a sequence complementary to a target. The label often generates a measurable signal, such as radioactivity, that can be used to quantitate the amount of bound detectable moiety.

The label can be incorporated in or attached to a polynucleotide either covalently, or through ionic, van der Waals or hydrogen bonds, e.g., incorporation of radioactive nucleotides, or biotinylated nucleotides that are recognized by streptavadin. The detectable moiety may be directly or indirectly detectable. Indirect detection can involve the binding of a second directly or indirectly detectable moiety to the detectable moiety. For example, the detectable moiety can be the ligand of a binding partner, such as biotin, which is a binding partner for streptavadin, or a nucleotide sequence, which is the binding partner for a complementary sequence, to which it can specifically hybridize. The binding partner may itself be directly detectable, for example, an antibody may be itself labeled with a fluorescent molecule. The binding partner also may be indirectly detectable, for example, a nucleic acid having a complementary nucleotide sequence can be a part of a branched DNA molecule that is in turn detectable through hybridization with other labeled nucleic acid molecules.

IV. Exemplary Antisense Polynucleotides

A series of 30-mer antisense oligonucleotides, which span the entire hTRT sequence, are exemplary embodiments of the present invention (see FIG. 3 ). These oligonucleotides were systematically assayed for the ability to inhibit hTRT expression in vitro. The results of the experiment are presented in FIG. 3 (see also Example I). Any suitable series of hTRT antisense oligonucleotides can be tested in a similar fashion. For example, a series of 20-mer antisense oligonucleotides, offset one from the next by 10 nucleotides can be synthesized and tested in the same manner. A series of 25-mer, 35-mer, or 15-mer oligonucleotides can be examined in the same manner.

Selected oligonucleotides from the series of FIG. 3 were then tested ex vivo for their ability to inhibit hTRT expression in tumor cells (see Example II). The hTRT antisense oligonucleotides active for inhibiting telomerase activity ex vivo in tumor cells were than assayed for their long term cell culture effects on hTRT expression, telomerase activity, telomere dynamics, and cell proliferation (see Example II). The oligonucleotides of Table I represent exemplary oligonucleotides that inhibited telomerase activity ex vivo.

TABLE I
hTRT AS 30-mers:
PS-	Position	5′-AS sequence-3′
ODN#	* (3′-5′)	SEQ ID NO;
3	31-60	SEQ ID NO: 4
		GGCATCGCGGGGGTGGCCGGGGCCAGGGCT
4	46-75	SEQ ID NO: 5
		CAGCGGGGAGCGCGCGGCATCGCGGGGGTG
7	91-120	SEQ ID NO: 6
		AGCACCTCGCGGTAGTGGCTGCGCAGCAGG
8	106-135	SEQ ID NO: 7
		AACGTGGCCAGCGGCAGCACCTCGCGGTAG
16	226-255	SEQ ID NO: 8
		GCGGGGGGCGGCCGTGCGTCCCAGGGCACG
21	301-330	SEQ ID NO: 9
		CCGCGCTCGCACAGCCTCTGCAGCACTCGG
25	361-390	SEQ ID NO: 10
		GGGGGGCCCCCGCGGGCCCCGTCCAGCAGC
26	376-405	SEQ ID NO: 11
		GTGGTGAAGGCCTCGGGGGGGCCCCCGCGG
27	391-420	SEQ ID NO: 12
		TAGCTGCGCACGCTGGTGGTGAAGGCCTCG
28	406-435	SEQ ID NO: 13
		ACCGTGTTGGGCAGGTAGCTGCGCACGCTG
29	421-450	SEQ ID NO: 14
		CGCAGTGCGTCGGTCACCGTGTTGGGCAGG
33	481-510	SEQ ID NO: 15
		AGGTGAACCAGCACGTCGTCGCCCACGCGG
40	586-615	SEQ ID NO: 16
		GGGGGCCGGGCCTGAGTGGCAGCGCCGAGC
41	601-630	SEQ ID NO: 17
		CCACTAGCGTGTGGCGGGGGCCGGGCCTGA
43	631-660	SEQ ID NO: 18
		GCCCGTTCGCATCCCAGACGCCTTCGGGGT
44	646-675	SEQ ID NO: 19
		ACGCTATGGTTCCAGGCCCGTTCGCATCCC
45	661-690	SEQ ID NO: 20
		ACCCCGGCCTCCCTGACGCTATGGTTCCAG
46	676-705	SEQ ID NO: 21
		GGCAGGCCCAGGGGGACCCCGGCCTCCCTG
47	691-720	SEQ ID NO: 22
		CTCGCACCCGGGGCTGGCAGGCCCAGGGGG
48	706-735	SEQ ID NO: 23
		CTGCCCCCGCGCCTCCTCGCACCCGGGGCT
49	721-750	SEQ ID NO: 24
		AGACTTCGGCTGGCACTAGCCCCCGCGCCTC
50	736-765	SEQ ID NO: 25
		CTCTTGGGCAACGGCAGACTTCGGCTGGCA
51	751-780	SEQ ID NO: 26
		GCGCCACGCCTGGGCCTCTTGGGCAACGGC
52	766-795	SEQ ID NO: 27
		TCCGGCTCAGGGGCAGCGCCACGCCTGGGC
53	781-810	SEQ ID NO: 28
		CCAACGGGCGTCCGCTCCGGCTCAGGGGCA
54	796-825	SEQ ID NO: 29
		GCCCAGGACCCCTGCCCAACGGGCGTCCGC
62	916-945	SEQ ID NO: 30
		GGGTGGGAGTGGCGCGTGCCAGAGAGCGCA
68	1006-1035	SEQ ID NO: 31
		TCGGCGTACACCGGGGACAAGGCGTGTCC
69	1021-1050	SEQ ID NO: 32
		AGGAAGTGCTTGGTCTCGGCGTACACCGGG
70	1036-1065	SEQ ID NO: 33
		TCGCCTGAGGAGTAGAGGAAGTGCTTGGTC
71	1051-1080	SEQ ID NO: 34
		CGCAGCTGCTCCTTGTCGCCTGAGGAGTAG
72	1066-1095	SEQ ID NO: 35
		AGTAGGAAGGAGGGCCGCAGCTGCTCCTTG
73	1081-1110	SEQ ID NO: 36
		GGCCTCAGAGAGCTGAGTAGGAAGGAGGGC
74	1096-1125	SEQ ID NO: 37
		GCGCCAGTCAGGCTGGGCCTCAGAGAGCTG
75	1111-1140	SEQ ID NO: 38
		TCCACGAGCCTCCGAGCGCCAGTCAGGCTG
76	1126-1155	SEQ ID NO: 39
		CCCAGAAAGATGGTCTCCACGAGCCTCCGA
77	1141-1170	SEQ ID NO: 40
		ATCCAGGGCCTGGAACCCAGAAAGATGGTC
80	1186-1215	SEQ ID NO: 41
		CAGTAGCGCTGGGGCAGGCGGGGCAACCTG
81	1201-1230	SEQ ID NO: 42
		AGGGGCCGCATTTGCCAGTAGCGCTGGGGC
82	1216-1245	SEQ ID NO: 43
		AGCAGCTCCAGAAACAGGGGCCGCATTTGC
83	1231-1260	SEQ ID NO: 44
		TGCGCGTGGTTCCCAAGCAGCTCCAGAAAC
84	1246-1275	SEQ ID NO: 45
		ACCCGTAGGGGCACTGCGCGTGGTTCCCA
85	1261-1290	SEQ ID NO: 46
		TGCGTCTTGAGGAGCACCCCGTAGGGGCAC
86	1276-1305	SEQ ID NO: 47
		GCTCGCAGCGGGCAGTGCGTCTTGAGGAGC
87	1291-1320	SEQ ID NO: 48
		GCTGGGGTGACCGCAGCTCGCAGCGGGCAG
88	1306-1335	SEQ ID NO: 49
		GCACAGACACCGGCTGCTGGGGTGACCGCA
93	1381-1410	SEQ ID NO: 50
		AGCAGCTGCACCAGGCGACGGGGGTCTGTG
94	1396-1425	SEQ ID NO: 51
		CTGCTGTGCTGGCGGAGCAGCTGCACCAGG
96	1426-1455	SEQ ID NO: 52
		GCCCGCACGAAGCCGTACACCTGCCAGGGG
100	1486-1515	SEQ ID NO: 53
		AAGCGGCGTTCGTTGTGCCTGGAGCCCCAG
112	1666-1695	SEQ ID NO: 54
		CAGTGCAGGAACTTGGCCAGGATCTCCTCA
114	1696-1725	SEQ ID NO: 55
		AGCAGCTCGACGACGTACACACTCATCAGC
130	1936-1965	SEQ ID NO: 56
		TCCATGTTCACAATCGGCCGCAGCCCGTCA
143	2131-2160	SEQ ID NO: 57
		GGGTCCTGGGCCCGCACACGCAGCACGAAG
144	2146-2175	SEQ ID NO: 58
		TACAGCTCAGGCGGCGGGTCCTGGGCCCGC
151	2251-2280	SEQ ID NO: 59
		CGCACGCAGTACGTGTCTGGGGTTTGATG
152	2266-2295	SEQ ID NO: 60
		ACCACGGCATACCGACGCACGCAGTACGTG
201	3001-3030	SEQ ID NO: 61
		TTCACCTGCAAATCCAGAAACAGGCTGTGA
202	3016-3045	SEQ ID NO: 62
		ACCGTCTGGAGGCTGTTCACCTGCAAATCC
203	3031-3060	SEQ ID NO: 63
		TAGATGTTGGTGCACACCGTCTGGAGGCTG
208	3106-3135	SEQ ID NO: 64
		TTCCAAACTTGCTGATGAAATGGGAGCTGC
209	3121-3150	SEQ ID NO: 65
		AAAAATGTGGGGTTCTTCCAAACTTGCTGA
210	3136-3165	SEQ ID NO: 66
		GAGATGACGCGCAGAAAAATGTGGGGTTC
211	3151-3180	SEQ ID NO: 67
		AGGGAGGCCGTGTCAGAGATGACGCGCAGG
212	3166-3195	SEQ ID NO: 68
		AGGATGGAGTAGCAGAGGGAGGCCGTGTCA
213	3181-3210	SEQ ID NO: 69
		GCGTTCTTGGCTTTCAGGATGGAGTAGCAG
230	3436-3465	SEQ ID NO: 70
		GCGGGTGGCCATCAGTCCAGGATGGTCTTG
237	3541-3570	SEQ ID NO: 71
		CAGACTCCCAGCGGTGCGGGCCTGGGTGTG
241	3601-3630	SEQ ID NO: 72
		AGCCGGACACTCAGCCTTCAGCCGGACATG

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

EXAMPLES

The following examples are provided by way of illustration only and not by way of limitation. Those of skill will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.

Example I

Inhibition of hTRT Expression in vitro

In this example, inhibition of hTRT expression was examined using an in vitro cell-free expression system. A series of 30-mer antisense phosphorothioate oligonucleotides (PS-ODNs), which span the entire hTRT sequence, was systematically assayed for the ability to block hTRT expression in vitro (see FIG. 3 ). Co-expression of luciferase was used to normalize the samples and demonstrate the specificity of inhibition.

For inhibition of hTRT expression in vitro, an hTRT transcription/expression plasmid was prepared according to standard methodology for in vitro transcription and translation of hTRT RNA. Coupled transcription-translation reactions were performed with a reticulocyte lysate system (Promega TNT™) according to standard conditions (as performed in Example 7, U.S. patent application Ser. No. 08/974,549). Each coupled transcription/translation reaction included hTRT RNA transcribed from the expression plasmid, and a test antisense polynucleotide at a range of standard test concentrations, as well as the luciferase transcription/translation internal control (see, e.g., Sambrook et al., supra, Ausubel et al., supra). The translation reaction can also be performed with hTRT RNA that is synthesized in vitro in a separate reaction and then added to the translation reaction. 35 S-Met was included in the reaction to label the translation products. The negative control was performed without added PS-ODN.

The labeled translation products were separated by gel electrophoresis and quantitated after exposing the gel to a phosphorimager screen. The amount of hTRT protein expressed in the presence of hTRT specific PS-ODNs was normalized to the co-expressed luciferase control. The data are presented in FIG. 3 as a percentage of the control, which is without added PS-ODN.

Example II

Inhibition of hTRT Expression Ex Vivo

A. Reagents

Cells: ACHN cells, NCI, catalogue #503755; 293 cells, ATCC; BJ (see, e.g., Kim et al., Science 266: 2011-2015 (1994)); additional cells from the ATCC or NCI.

Media and solutions: RPMI 1640 medium, BioWhitaker; DMEM/M199 medium, BioWhitaker; EMEM, BioWhitaker; Fetal Bovine Serum, Summit (stored frozen at −20° C., stored thawed at 4° C.); Trypsin-EDTA, GIBCO (catalogue #25300-054) (stored frozen at −20° C., stored thawed 4° C.; Isoton II (stored at RT); DMSO (stored at RT); oligonucleotides (see Table 1 and FIG. 3 , stored in solution at −20° C.); PBS (Ca ++ /Mg ++ free); TE; 10 mM Tris-HCL, pH 8.0; 1 mM EDTA.

To prepare oligonucleotide stocks: Dissolve oligonucleotide nucleotides (PS-ODNs) in the appropriate amount of TE to make a concentrated stock solution (1-20 mM).

B. Treatment of Cells Ex Vivo with Antisense hTRT Oligonucleotides

1. For plating cells prior to oligonucleotide treatment, stock cultures of cells in log-phase growth (in T75 flask) were used. ACNH, 293, and BJ cells were used in this assay. The media was removed by aspiration, and the cells were rinsed with 2-5 ml of PBS. 1 ml of trypsin-EDTA was added to the cells, swirled to distribute, and incubated for 2 minutes. The trypsin was inactivated with 9 ml of media. The cells were gently triturated with media. 200 μL of the cells were then counted with a Coulter counter and diluted to the appropriate volume and number of cells per well.

2. For 6-well dishes, 1.1×10 5 cells total per well, 2 ml/well were added. The cells were allowed to settle 4-6 h prior to any treatment with oligonucleotides. The amount of cells can be scaled up or down proportionally for 12-well, 100 mm, or 150 mm dishes. For example, for 12-well dishes, use 4.6×10 4 cells in 2 ml media; for 100 mm dishes use 6×10 5 cells in 10 ml media; for 150 mm dishes use 1.7×10 6 cells in 35 ml media.

3. Oligonucleotides were diluted in media and fed to the cells at a range of standard test concentrations. Serial, sterile dilutions of the ODNs (see, e.g., Table 1) were prepared in sterile, filtered media for feeding the cells. The cells were treated in single, duplicate, or triplicate wells. Control wells were treated with TE diluted in media.

4. The cells were fed daily with freshly diluted PS-ODN-media by aspirating the media and then feeding with 2 ml of freshly diluted oligonucleotide in media.

5. When cells were near 70-80% confluent (3-4 days), the number of cells was determined per well. The media was removed by aspiration, and the cells were rinsed twice with 2 ml PBS. 0.5 ml trypsin-EDTA was added to the cells, swirled, and incubated for 2 minutes. The cells were triturated gently with 2 ml media per well. 200 μL of cells were counted in a Coulter counter. If necessary, the cells are replated at 1.1×10 5 cells per well, 2 ml media per well, and fed with PS-ODN as described above.

6. Samples of the cells were also harvested for analysis of telomerase activity by TRAP activity. The cells can also be analyzed by isolating RNA and performing RT-PCR, by TRF measurement, or by telomere length measurement (see, e.g., Example section, U.S. patent application Ser. No. 08/974,549 for assay protocols).

7. The cell population doublings (PDLs) were calculated for each timepoint according to the following formula. PDLs (P): Pn=Pn-1+[((ln(Total # cells))−(ln(# cells plated))/ln(2)].

8. Graph PDL vs. time (in days) for the full dose range of each PS-ODN as compared to control untreated cells.

9. Steps 2-8 were repeated for the desired duration (usually 2-4 weeks) or until cell growth was inhibited significantly.

10. Table 1 shows exemplary oligonucleotides that were tested using this assay, and which inhibited telomerase expression and activity by approximately 50% or more.

72 4015 base pairs nucleic acid single linear cDNA CDS 56..3454 /product= “human telomerase reverse transcriptase (hTRT)” 1GCAGCGCTGC GTCCTGCTGC GCACGTGGGA AGCCCTGGCC CCGGCCACCC CCGCG ATG 58 Met 1CCG CGC GCT CCC CGC TGC CGA GCC GTG CGC TCC CTG CTG CGC AGC CAC 106Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ser Leu Leu Arg Ser His 5 10 15TAC CGC GAG GTG CTG CCG CTG GCC ACG TTC GTG CGG CGC CTG GGG CCC 154Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe Val Arg Arg Leu Gly Pro 20 25 30CAG GGC TGG CGG CTG GTG CAG CGC GGG GAC CCG GCG GCT TTC CGC GCG 202Gln Gly Trp Arg Leu Val Gln Arg Gly Asp Pro Ala Ala Phe Arg Ala 35 40 45CTG GTG GCC CAG TGC CTG GTG TGC GTG CCC TGG GAC GCA CGG CCG CCC 250Leu Val Ala Gln Cys Leu Val Cys Val Pro Trp Asp Ala Arg Pro Pro 50 55 60 65CCC GCC GCC CCC TCC TTC CGC CAG GTG TCC TGC CTG AAG GAG CTG GTG 298Pro Ala Ala Pro Ser Phe Arg Gln Val Ser Cys Leu Lys Glu Leu Val 70 75 80GCC CGA GTG CTG CAG AGG CTG TGC GAG CGC GGC GCG AAG AAC GTG CTG 346Ala Arg Val Leu Gln Arg Leu Cys Glu Arg Gly Ala Lys Asn Val Leu 85 90 95GCC TTC GGC TTC GCG CTG CTG GAC GGG GCC CGC GGG GGC CCC CCC GAG 394Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro Glu 100 105 110GCC TTC ACC ACC AGC GTG CGC AGC TAC CTG CCC AAC ACG GTG ACC GAC 442Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr Asp 115 120 125GCA CTG CGG GGG AGC GGG GCG TGG GGG CTG CTG CTG CGC CGC GTG GGC 490Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val Gly130 135 140 145GAC GAC GTG CTG GTT CAC CTG CTG GCA CGC TGC GCG CTC TTT GTG CTG 538Asp Asp Val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val Leu 150 155 160GTG GCT CCC AGC TGC GCC TAC CAG GTG TGC GGG CCG CCG CTG TAC CAG 586Val Ala Pro Ser Cys Ala Tyr Gln Val Cys Gly Pro Pro Leu Tyr Gln 165 170 175CTC GGC GCT GCC ACT CAG GCC CGG CCC CCG CCA CAC GCT AGT GGA CCC 634Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro Pro His Ala Ser Gly Pro 180 185 190CGA AGG CGT CTG GGA TGC GAA CGG GCC TGG AAC CAT AGC GTC AGG GAG 682Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp Asn His Ser Val Arg Glu 195 200 205GCC GGG GTC CCC CTG GGC CTG CCA GCC CCG GGT GCG AGG AGG CGC GGG 730Ala Gly Val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg Gly210 215 220 225GGC AGT GCC AGC CGA AGT CTG CCG TTG CCC AAG AGG CCC AGG CGT GGC 778Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg Gly 230 235 240GCT GCC CCT GAG CCG GAG CGG ACG CCC GTT GGG CAG GGG TCC TGG GCC 826Ala Ala Pro Glu Pro Glu Arg Thr Pro Val Gly Gln Gly Ser Trp Ala 245 250 255CAC CCG GGC AGG ACG CGT GGA CCG AGT GAC CGT GGT TTC TGT GTG GTG 874His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val Val 260 265 270TCA CCT GCC AGA CCC GCC GAA GAA GCC ACC TCT TTG GAG GGT GCG CTC 922Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala Leu 275 280 285TCT GGC ACG CGC CAC TCC CAC CCA TCC GTG GGC CGC CAG CAC CAC GCG 970Ser Gly Thr Arg His Ser His Pro Ser Val Gly Arg Gln His His Ala290 295 300 305GGC CCC CCA TCC ACA TCG CGG CCA CCA CGT CCC TGG GAC ACG CCT TGT 1018Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg Pro Trp Asp Thr Pro Cys 310 315 320CCC CCG GTG TAC GCC GAG ACC AAG CAC TTC CTC TAC TCC TCA GGC GAC 1066Pro Pro Val Tyr Ala Glu Thr Lys His Phe Leu Tyr Ser Ser Gly Asp 325 330 335AAG GAG CAG CTG CGG CCC TCC TTC CTA CTC AGC TCT CTG AGG CCC AGC 1114Lys Glu Gln Leu Arg Pro Ser Phe Leu Leu Ser Ser Leu Arg Pro Ser 340 345 350CTG ACT GGC GCT CGG AGG CTC GTG GAG ACC ATC TTT CTG GGT TCC AGG 1162Leu Thr Gly Ala Arg Arg Leu Val Glu Thr Ile Phe Leu Gly Ser Arg 355 360 365CCC TGG ATG CCA GGG ACT CCC CGC AGG TTG CCC CGC CTG CCC CAG CGC 1210Pro Trp Met Pro Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gln Arg370 375 380 385TAC TGG CAA ATG CGG CCC CTG TTT CTG GAG CTG CTT GGG AAC CAC GCG 1258Tyr Trp Gln Met Arg Pro Leu Phe Leu Glu Leu Leu Gly Asn His Ala 390 395 400CAG TGC CCC TAC GGG GTG CTC CTC AAG ACG CAC TGC CCG CTG CGA GCT 1306Gln Cys Pro Tyr Gly Val Leu Leu Lys Thr His Cys Pro Leu Arg Ala 405 410 415GCG GTC ACC CCA GCA GCC GGT GTC TGT GCC CGG GAG AAG CCC CAG GGC 1354Ala Val Thr Pro Ala Ala Gly Val Cys Ala Arg Glu Lys Pro Gln Gly 420 425 430TCT GTG GCG GCC CCC GAG GAG GAG GAC ACA GAC CCC CGT CGC CTG GTG 1402Ser Val Ala Ala Pro Glu Glu Glu Asp Thr Asp Pro Arg Arg Leu Val 435 440 445CAG CTG CTC CGC CAG CAC AGC AGC CCC TGG CAG GTG TAC GGC TTC GTG 1450Gln Leu Leu Arg Gln His Ser Ser Pro Trp Gln Val Tyr Gly Phe Val450 455 460 465CGG GCC TGC CTG CGC CGG CTG GTG CCC CCA GGC CTC TGG GGC TCC AGG 1498Arg Ala Cys Leu Arg Arg Leu Val Pro Pro Gly Leu Trp Gly Ser Arg 470 475 480CAC AAC GAA CGC CGC TTC CTC AGG AAC ACC AAG AAG TTC ATC TCC CTG 1546His Asn Glu Arg Arg Phe Leu Arg Asn Thr Lys Lys Phe Ile Ser Leu 485 490 495GGG AAG CAT GCC AAG CTC TCG CTG CAG GAG CTG ACG TGG AAG ATG AGC 1594Gly Lys His Ala Lys Leu Ser Leu Gln Glu Leu Thr Trp Lys Met Ser 500 505 510GTG CGG GAC TGC GCT TGG CTG CGC AGG AGC CCA GGG GTT GGC TGT GTT 1642Val Arg Asp Cys Ala Trp Leu Arg Arg Ser Pro Gly Val Gly Cys Val 515 520 525CCG GCC GCA GAG CAC CGT CTG CGT GAG GAG ATC CTG GCC AAG TTC CTG 1690Pro Ala Ala Glu His Arg Leu Arg Glu Glu Ile Leu Ala Lys Phe Leu530 535 540 545CAC TGG CTG ATG AGT GTG TAC GTC GTC GAG CTG CTC AGG TCT TTC TTT 1738His Trp Leu Met Ser Val Tyr Val Val Glu Leu Leu Arg Ser Phe Phe 550 555 560TAT GTC ACG GAG ACC ACG TTT CAA AAG AAC AGG CTC TTT TTC TAC CGG 1786Tyr Val Thr Glu Thr Thr Phe Gln Lys Asn Arg Leu Phe Phe Tyr Arg 565 570 575AAG AGT GTC TGG AGC AAG TTG CAA AGC ATT GGA ATC AGA CAG CAC TTG 1834Lys Ser Val Trp Ser Lys Leu Gln Ser Ile Gly Ile Arg Gln His Leu 580 585 590AAG AGG GTG CAG CTG CGG GAG CTG TCG GAA GCA GAG GTC AGG CAG CAT 1882Lys Arg Val Gln Leu Arg Glu Leu Ser Glu Ala Glu Val Arg Gln His 595 600 605CGG GAA GCC AGG CCC GCC CTG CTG ACG TCC AGA CTC CGC TTC ATC CCC 1930Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro610 615 620 625AAG CCT GAC GGG CTG CGG CCG ATT GTG AAC ATG GAC TAC GTC GTG GGA 1978Lys Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val Val Gly 630 635 640GCC AGA ACG TTC CGC AGA GAA AAG AGG GCC GAG CGT CTC ACC TCG AGG 2026Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser Arg 645 650 655GTG AAG GCA CTG TTC AGC GTG CTC AAC TAC GAG CGG GCG CGG CGC CCC 2074Val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg Pro 660 665 670GGC CTC CTG GGC GCC TCT GTG CTG GGC CTG GAC GAT ATC CAC AGG GCC 2122Gly Leu Leu Gly Ala Ser Val Leu Gly Leu Asp Asp Ile His Arg Ala 675 680 685TGG CGC ACC TTC GTG CTG CGT GTG CGG GCC CAG GAC CCG CCG CCT GAG 2170Trp Arg Thr Phe Val Leu Arg Val Arg Ala Gln Asp Pro Pro Pro Glu690 695 700 705CTG TAC TTT GTC AAG GTG GAT GTG ACG GGC GCG TAC GAC ACC ATC CCC 2218Leu Tyr Phe Val Lys Val Asp Val Thr Gly Ala Tyr Asp Thr Ile Pro 710 715 720CAG GAC AGG CTC ACG GAG GTC ATC GCC AGC ATC ATC AAA CCC CAG AAC 2266Gln Asp Arg Leu Thr Glu Val Ile Ala Ser Ile Ile Lys Pro Gln Asn 725 730 735ACG TAC TGC GTG CGT CGG TAT GCC GTG GTC CAG AAG GCC GCC CAT GGG 2314Thr Tyr Cys Val Arg Arg Tyr Ala Val Val Gln Lys Ala Ala His Gly 740 745 750CAC GTC CGC AAG GCC TTC AAG AGC CAC GTC TCT ACC TTG ACA GAC CTC 2362His Val Arg Lys Ala Phe Lys Ser His Val Ser Thr Leu Thr Asp Leu 755 760 765CAG CCG TAC ATG CGA CAG TTC GTG GCT CAC CTG CAG GAG ACC AGC CCG 2410Gln Pro Tyr Met Arg Gln Phe Val Ala His Leu Gln Glu Thr Ser Pro770 775 780 785CTG AGG GAT GCC GTC GTC ATC GAG CAG AGC TCC TCC CTG AAT GAG GCC 2458Leu Arg Asp Ala Val Val Ile Glu Gln Ser Ser Ser Leu Asn Glu Ala 790 795 800AGC AGT GGC CTC TTC GAC GTC TTC CTA CGC TTC ATG TGC CAC CAC GCC 2506Ser Ser Gly Leu Phe Asp Val Phe Leu Arg Phe Met Cys His His Ala 805 810 815GTG CGC ATC AGG GGC AAG TCC TAC GTC CAG TGC CAG GGG ATC CCG CAG 2554Val Arg Ile Arg Gly Lys Ser Tyr Val Gln Cys Gln Gly Ile Pro Gln 820 825 830GGC TCC ATC CTC TCC ACG CTG CTC TGC AGC CTG TGC TAC GGC GAC ATG 2602Gly Ser Ile Leu Ser Thr Leu Leu Cys Ser Leu Cys Tyr Gly Asp Met 835 840 845GAG AAC AAG CTG TTT GCG GGG ATT CGG CGG GAC GGG CTG CTC CTG CGT 2650Glu Asn Lys Leu Phe Ala Gly Ile Arg Arg Asp Gly Leu Leu Leu Arg850 855 860 865TTG GTG GAT GAT TTC TTG TTG GTG ACA CCT CAC CTC ACC CAC GCG AAA 2698Leu Val Asp Asp Phe Leu Leu Val Thr Pro His Leu Thr His Ala Lys 870 875 880ACC TTC CTC AGG ACC CTG GTC CGA GGT GTC CCT GAG TAT GGC TGC GTG 2746Thr Phe Leu Arg Thr Leu Val Arg Gly Val Pro Glu Tyr Gly Cys Val 885 890 895GTG AAC TTG CGG AAG ACA GTG GTG AAC TTC CCT GTA GAA GAC GAG GCC 2794Val Asn Leu Arg Lys Thr Val Val Asn Phe Pro Val Glu Asp Glu Ala 900 905 910CTG GGT GGC ACG GCT TTT GTT CAG ATG CCG GCC CAC GGC CTA TTC CCC 2842Leu Gly Gly Thr Ala Phe Val Gln Met Pro Ala His Gly Leu Phe Pro 915 920 925TGG TGC GGC CTG CTG CTG GAT ACC CGG ACC CTG GAG GTG CAG AGC GAC 2890Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu Glu Val Gln Ser Asp930 935 940 945TAC TCC AGC TAT GCC CGG ACC TCC ATC AGA GCC AGT CTC ACC TTC AAC 2938Tyr Ser Ser Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu Thr Phe Asn 950 955 960CGC GGC TTC AAG GCT GGG AGG AAC ATG CGT CGC AAA CTC TTT GGG GTC 2986Arg Gly Phe Lys Ala Gly Arg Asn Met Arg Arg Lys Leu Phe Gly Val 965 970 975TTG CGG CTG AAG TGT CAC AGC CTG TTT CTG GAT TTG CAG GTG AAC AGC 3034Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gln Val Asn Ser 980 985 990CTC CAG ACG GTG TGC ACC AAC ATC TAC AAG ATC CTC CTG CTG CAG GCG 3082Leu Gln Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu Gln Ala 995 1000 1005TAC AGG TTT CAC GCA TGT GTG CTG CAG CTC CCA TTT CAT CAG CAA GTT 3130Tyr Arg Phe His Ala Cys Val Leu Gln Leu Pro Phe His Gln Gln Val1010 1015 1020 1025TGG AAG AAC CCC ACA TTT TTC CTG CGC GTC ATC TCT GAC ACG GCC TCC 3178Trp Lys Asn Pro Thr Phe Phe Leu Arg Val Ile Ser Asp Thr Ala Ser 1030 1035 1040CTC TGC TAC TCC ATC CTG AAA GCC AAG AAC GCA GGG ATG TCG CTG GGG 3226Leu Cys Tyr Ser Ile Leu Lys Ala Lys Asn Ala Gly Met Ser Leu Gly 1045 1050 1055GCC AAG GGC GCC GCC GGC CCT CTG CCC TCC GAG GCC GTG CAG TGG CTG 3274Ala Lys Gly Ala Ala Gly Pro Leu Pro Ser Glu Ala Val Gln Trp Leu 1060 1065 1070TGC CAC CAA GCA TTC CTG CTC AAG CTG ACT CGA CAC CGT GTC ACC TAC 3322Cys His Gln Ala Phe Leu Leu Lys Leu Thr Arg His Arg Val Thr Tyr 1075 1080 1085GTG CCA CTC CTG GGG TCA CTC AGG ACA GCC CAG ACG CAG CTG AGT CGG 3370Val Pro Leu Leu Gly Ser Leu Arg Thr Ala Gln Thr Gln Leu Ser Arg1090 1095 1100 1105AAG CTC CCG GGG ACG ACG CTG ACT GCC CTG GAG GCC GCA GCC AAC CCG 3418Lys Leu Pro Gly Thr Thr Leu Thr Ala Leu Glu Ala Ala Ala Asn Pro 1110 1115 1120GCA CTG CCC TCA GAC TTC AAG ACC ATC CTG GAC TGATGGCCAC CCGCCCACAG 3471Ala Leu Pro Ser Asp Phe Lys Thr Ile Leu Asp 1125 1130CCAGGCCGAG AGCAGACACC AGCAGCCCTG TCACGCCGGG CTCTACGTCC CAGGGAGGGA 3531GGGGCGGCCC ACACCCAGGC CCGCACCGCT GGGAGTCTGA GGCCTGAGTG AGTGTTTGGC 3591CGAGGCCTGC ATGTCCGGCT GAAGGCTGAG TGTCCGGCTG AGGCCTGAGC GAGTGTCCAG 3651CCAAGGGCTG AGTGTCCAGC ACACCTGCCG TCTTCACTTC CCCACAGGCT GGCGCTCGGC 3711TCCACCCCAG GGCCAGCTTT TCCTCACCAG GAGCCCGGCT TCCACTCCCC ACATAGGAAT 3771AGTCCATCCC CAGATTCGCC ATTGTTCACC CCTCGCCCTG CCCTCCTTTG CCTTCCACCC 3831CCACCATCCA GGTGGAGACC CTGAGAAGGA CCCTGGGAGC TCTGGGAATT TGGAGTGACC 3891AAAGGTGTGC CCTGTACACA GGCGAGGACC CTGCACCTGG ATGGGGGTCC CTGTGGGTCA 3951AATTGGGGGG AGGTGCTGTG GGAGTAAAAT ACTGAATATA TGAGTTTTTC AGTTTTGAAA 4011AAAA 4015 1132 amino acids amino acid linear protein 2Met Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ser Leu Leu Arg Ser 1 5 10 15His Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe Val Arg Arg Leu Gly 20 25 30Pro Gln Gly Trp Arg Leu Val Gln Arg Gly Asp Pro Ala Ala Phe Arg 35 40 45Ala Leu Val Ala Gln Cys Leu Val Cys Val Pro Trp Asp Ala Arg Pro 50 55 60Pro Pro Ala Ala Pro Ser Phe Arg Gln Val Ser Cys Leu Lys Glu Leu 65 70 75 80Val Ala Arg Val Leu Gln Arg Leu Cys Glu Arg Gly Ala Lys Asn Val 85 90 95Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro 100 105 110Glu Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr 115 120 125Asp Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val 130 135 140Gly Asp Asp Val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val145 150 155 160Leu Val Ala Pro Ser Cys Ala Tyr Gln Val Cys Gly Pro Pro Leu Tyr 165 170 175Gln Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro Pro His Ala Ser Gly 180 185 190Pro Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp Asn His Ser Val Arg 195 200 205Glu Ala Gly Val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg 210 215 220Gly Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg225 230 235 240Gly Ala Ala Pro Glu Pro Glu Arg Thr Pro Val Gly Gln Gly Ser Trp 245 250 255Ala His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val 260 265 270Val Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala 275 280 285Leu Ser Gly Thr Arg His Ser His Pro Ser Val Gly Arg Gln His His 290 295 300Ala Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg Pro Trp Asp Thr Pro305 310 315 320Cys Pro Pro Val Tyr Ala Glu Thr Lys His Phe Leu Tyr Ser Ser Gly 325 330 335Asp Lys Glu Gln Leu Arg Pro Ser Phe Leu Leu Ser Ser Leu Arg Pro 340 345 350Ser Leu Thr Gly Ala Arg Arg Leu Val Glu Thr Ile Phe Leu Gly Ser 355 360 365Arg Pro Trp Met Pro Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gln 370 375 380Arg Tyr Trp Gln Met Arg Pro Leu Phe Leu Glu Leu Leu Gly Asn His385 390 395 400Ala Gln Cys Pro Tyr Gly Val Leu Leu Lys Thr His Cys Pro Leu Arg 405 410 415Ala Ala Val Thr Pro Ala Ala Gly Val Cys Ala Arg Glu Lys Pro Gln 420 425 430Gly Ser Val Ala Ala Pro Glu Glu Glu Asp Thr Asp Pro Arg Arg Leu 435 440 445Val Gln Leu Leu Arg Gln His Ser Ser Pro Trp Gln Val Tyr Gly Phe 450 455 460Val Arg Ala Cys Leu Arg Arg Leu Val Pro Pro Gly Leu Trp Gly Ser465 470 475 480Arg His Asn Glu Arg Arg Phe Leu Arg Asn Thr Lys Lys Phe Ile Ser 485 490 495Leu Gly Lys His Ala Lys Leu Ser Leu Gln Glu Leu Thr Trp Lys Met 500 505 510Ser Val Arg Asp Cys Ala Trp Leu Arg Arg Ser Pro Gly Val Gly Cys 515 520 525Val Pro Ala Ala Glu His Arg Leu Arg Glu Glu Ile Leu Ala Lys Phe 530 535 540Leu His Trp Leu Met Ser Val Tyr Val Val Glu Leu Leu Arg Ser Phe545 550 555 560Phe Tyr Val Thr Glu Thr Thr Phe Gln Lys Asn Arg Leu Phe Phe Tyr 565 570 575Arg Lys Ser Val Trp Ser Lys Leu Gln Ser Ile Gly Ile Arg Gln His 580 585 590Leu Lys Arg Val Gln Leu Arg Glu Leu Ser Glu Ala Glu Val Arg Gln 595 600 605His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile 610 615 620Pro Lys Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val Val625 630 635 640Gly Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser 645 650 655Arg Val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg 660 665 670Pro Gly Leu Leu Gly Ala Ser Val Leu Gly Leu Asp Asp Ile His Arg 675 680 685Ala Trp Arg Thr Phe Val Leu Arg Val Arg Ala Gln Asp Pro Pro Pro 690 695 700Glu Leu Tyr Phe Val Lys Val Asp Val Thr Gly Ala Tyr Asp Thr Ile705 710 715 720Pro Gln Asp Arg Leu Thr Glu Val Ile Ala Ser Ile Ile Lys Pro Gln 725 730 735Asn Thr Tyr Cys Val Arg Arg Tyr Ala Val Val Gln Lys Ala Ala His 740 745 750Gly His Val Arg Lys Ala Phe Lys Ser His Val Ser Thr Leu Thr Asp 755 760 765Leu Gln Pro Tyr Met Arg Gln Phe Val Ala His Leu Gln Glu Thr Ser 770 775 780Pro Leu Arg Asp Ala Val Val Ile Glu Gln Ser Ser Ser Leu Asn Glu785 790 795 800Ala Ser Ser Gly Leu Phe Asp Val Phe Leu Arg Phe Met Cys His His 805 810 815Ala Val Arg Ile Arg Gly Lys Ser Tyr Val Gln Cys Gln Gly Ile Pro 820 825 830Gln Gly Ser Ile Leu Ser Thr Leu Leu Cys Ser Leu Cys Tyr Gly Asp 835 840 845Met Glu Asn Lys Leu Phe Ala Gly Ile Arg Arg Asp Gly Leu Leu Leu 850 855 860Arg Leu Val Asp Asp Phe Leu Leu Val Thr Pro His Leu Thr His Ala865 870 875 880Lys Thr Phe Leu Arg Thr Leu Val Arg Gly Val Pro Glu Tyr Gly Cys 885 890 895Val Val Asn Leu Arg Lys Thr Val Val Asn Phe Pro Val Glu Asp Glu 900 905 910Ala Leu Gly Gly Thr Ala Phe Val Gln Met Pro Ala His Gly Leu Phe 915 920 925Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu Glu Val Gln Ser 930 935 940Asp Tyr Ser Ser Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu Thr Phe945 950 955 960Asn Arg Gly Phe Lys Ala Gly Arg Asn Met Arg Arg Lys Leu Phe Gly 965 970 975Val Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gln Val Asn 980 985 990Ser Leu Gln Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu Gln 995 1000 1005Ala Tyr Arg Phe His Ala Cys Val Leu Gln Leu Pro Phe His Gln Gln 1010 1015 1020Val Trp Lys Asn Pro Thr Phe Phe Leu Arg Val Ile Ser Asp Thr Ala1025 1030 1035 1040Ser Leu Cys Tyr Ser Ile Leu Lys Ala Lys Asn Ala Gly Met Ser Leu 1045 1050 1055Gly Ala Lys Gly Ala Ala Gly Pro Leu Pro Ser Glu Ala Val Gln Trp 1060 1065 1070Leu Cys His Gln Ala Phe Leu Leu Lys Leu Thr Arg His Arg Val Thr 1075 1080 1085Tyr Val Pro Leu Leu Gly Ser Leu Arg Thr Ala Gln Thr Gln Leu Ser 1090 1095 1100Arg Lys Leu Pro Gly Thr Thr Leu Thr Ala Leu Glu Ala Ala Ala Asn1105 1110 1115 1120Pro Ala Leu Pro Ser Asp Phe Lys Thr Ile Leu Asp 1125 1130 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 3TCCCACGTGC GCAGCAGGAC GCAGCGCTGC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 4GGCATCGCGG GGGTGGCCGG GGCCAGGGCT 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 5CAGCGGGGAG CGCGCGGCAT CGCGGGGGTG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 6AGCACCTCGC GGTAGTGGCT GCGCAGCAGG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 7AACGTGGCCA GCGGCAGCAC CTCGCGGTAG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 8GCGGGGGGCG GCCGTGCGTC CCAGGGCACG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 9CCGCGCTCGC ACAGCCTCTG CAGCACTCGG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 10GGGGGGCCCC CGCGGGCCCC GTCCAGCAGC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 11GTGGTGAAGG CCTCGGGGGG GCCCCCGCGG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 12TAGCTGCGCA CGCTGGTGGT GAAGGCCTCG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 13ACCGTGTTGG GCAGGTAGCT GCGCACGCTG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 14CGCAGTGCGT CGGTCACCGT GTTGGGCAGG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 15AGGTGAACCA GCACGTCGTC GCCCACGCGG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 16GGGGGCCGGG CCTGAGTGGC AGCGCCGAGC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 17CCACTAGCGT GTGGCGGGGG CCGGGCCTGA 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 18GCCCGTTCGC ATCCCAGACG CCTTCGGGGT 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 19ACGCTATGGT TCCAGGCCCG TTCGCATCCC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 20ACCCCGGCCT CCCTGACGCT ATGGTTCCAG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 21GGCAGGCCCA GGGGGACCCC GGCCTCCCTG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 22CTCGCACCCG GGGCTGGCAG GCCCAGGGGG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 23CTGCCCCCGC GCCTCCTCGC ACCCGGGGCT 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 24AGACTTCGGC TGGCACTGCC CCCGCGCCTC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 25CTCTTGGGCA ACGGCAGACT TCGGCTGGCA 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 26GCGCCACGCC TGGGCCTCTT GGGCAACGGC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 27TCCGGCTCAG GGGCAGCGCC ACGCCTGGGC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 28CCAACGGGCG TCCGCTCCGG CTCAGGGGCA 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 29GCCCAGGACC CCTGCCCAAC GGGCGTCCGC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 30GGGTGGGAGT GGCGCGTGCC AGAGAGCGCA 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 31TCGGCGTACA CCGGGGGACA AGGCGTGTCC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 32AGGAAGTGCT TGGTCTCGGC GTACACCGGG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 33TCGCCTGAGG AGTAGAGGAA GTGCTTGGTC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 34CGCAGCTGCT CCTTGTCGCC TGAGGAGTAG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 35AGTAGGAAGG AGGGCCGCAG CTGCTCCTTG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 36GGCCTCAGAG AGCTGAGTAG GAAGGAGGGC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 37GCGCCAGTCA GGCTGGGCCT CAGAGAGCTG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 38TCCACGAGCC TCCGAGCGCC AGTCAGGCTG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 39CCCAGAAAGA TGGTCTCCAC GAGCCTCCGA 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 40ATCCAGGGCC TGGAACCCAG AAAGATGGTC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 41CAGTAGCGCT GGGGCAGGCG GGGCAACCTG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 42AGGGGCCGCA TTTGCCAGTA GCGCTGGGGC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 43AGCAGCTCCA GAAACAGGGG CCGCATTTGC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 44TGCGCGTGGT TCCCAAGCAG CTCCAGAAAC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 45ACCCCGTAGG GGCACTGCGC GTGGTTCCCA 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 46TGCGTCTTGA GGAGCACCCC GTAGGGGCAC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 47GCTCGCAGCG GGCAGTGCGT CTTGAGGAGC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 48GCTGGGGTGA CCGCAGCTCG CAGCGGGCAG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 49GCACAGACAC CGGCTGCTGG GGTGACCGCA 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 50AGCAGCTGCA CCAGGCGACG GGGGTCTGTG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 51CTGCTGTGCT GGCGGAGCAG CTGCACCAGG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 52GCCCGCACGA AGCCGTACAC CTGCCAGGGG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 53AAGCGGCGTT CGTTGTGCCT GGAGCCCCAG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 54CAGTGCAGGA ACTTGGCCAG GATCTCCTCA 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 55AGCAGCTCGA CGACGTACAC ACTCATCAGC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 56TCCATGTTCA CAATCGGCCG CAGCCCGTCA 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 57GGGTCCTGGG CCCGCACACG CAGCACGAAG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 58TACAGCTCAG GCGGCGGGTC CTGGGCCCGC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 59CGCACGCAGT ACGTGTTCTG GGGTTTGATG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 60ACCACGGCAT ACCGACGCAC GCAGTACGTG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 61TTCACCTGCA AATCCAGAAA CAGGCTGTGA 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 62ACCGTCTGGA GGCTGTTCAC CTGCAAATCC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 63TAGATGTTGG TGCACACCGT CTGGAGGCTG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 64TTCCAAACTT GCTGATGAAA TGGGAGCTGC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 65AAAAATGTGG GGTTCTTCCA AACTTGCTGA 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 66GAGATGACGC GCAGGAAAAA TGTGGGGTTC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 67AGGGAGGCCG TGTCAGAGAT GACGCGCAGG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 68AGGATGGAGT AGCAGAGGGA GGCCGTGTCA 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 69GCGTTCTTGG CTTTCAGGAT GGAGTAGCAG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 70GCGGGTGGCC ATCAGTCCAG GATGGTCTTG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 71CAGACTCCCA GCGGTGCGGG CCTGGGTGTG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 72AGCCGGACAC TCAGCCTTCA GCCGGACATG 30

Claims

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

2. The oligonucleotide of claim 1 that hybridizes to the target DNA at 5° C. below Tm.

3. The oligonucleotide of claim 1 that is DNA.

4. The oligonucleotide of claim 1 that is RNA.

5. The oligonucleotide of claim 1 that comprises one or more synthetic nucleotides.

6. The oligonucleotide of claim 5 that comprises a phosphorothioate oligonucleotide.

7. The oligonucleotide of claim 1 that is from 20 to 100 nucleotides in length.

8. The oligonucleotide of claim 7 that is 30 nucleotides in length.

9. The oligonucleotide of claim 1 that is from 10 to 50 nucleotides in length.

10. The oligonucleotide of claim 1 that comprises a sequence of about 7 to about 100 nucleotides that is exactly complementary to SEQ. ID NO:1.

11. The oligonucleotide of claim 10 that is from 20 to 100 nucleotides in length.

12. The oligonucleotide of claim 11, wherein the oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS:4-72.

13. The oligonucleotide of claim 12, that is 30 nucleotides in length.

14. The oligonucleotide of claim 1, wherein said oligonucleotide reduces telomerase activity in a cell by at least 50%.