WO2010043243A1 - Novel diagnostic and therapeutic agents - Google Patents

Abstract

The invention relates to a diagnostic agent comprising a polynucleotide and to a therapeutic agent comprising a polyribonucleotide capable of inhibiting and/or reducing the activity of a telomerase for use in medicine. The invention further provides a method of diagnosing a proliferative disorder. Also provided are therapeutic agents and methods of using the same.

Description

NOVEL DIAGNOSTIC AND THERAPEUTIC AGENTS

The present invention relates to a diagnostic and/or therapeutic agent comprising a polynucleotide capable of inhibiting and/or reducing the activity of a telomerase for use in medicine.

All vertebrates, including mammals, have telomeres consisting of non-coding TTAGGG repeats that are bound by the multi-protein complex "shelterin", protecting chromosome ends from DNA repair mechanisms and degradation. Mammalian telomeric chromatin is enriched for the constitutive heterochromatin marks H3K9me3, H4K20me3 and HP1. Similar to pericentric heterochromatin, telomeric heterochromatin is thought to be fundamental for the maintenance of chromosomal integrity.

Telomeres shorten with age, which is thought to underlie the aging process. During DNA replication, the end of chromosomes cannot be completely replicated (a situation known as the "end replication problem") resulting in a loss of telomeric sequence after every cell division.

Telomerases are intracellular enzymes comprising a reverse-transcriptase activity which permits synthesis of DNA sequence and facilitates the addition of telomeric repeats de novo to chromosome ends, thereby maintaining or extending the telomere length at the chromosome ends. Accordingly, telomerase is capable of compensating for the loss of telomeric sequences; however, telomerase activity is usually restricted to early developmental stages (i.e. embryonic stages and for a short period after birth) and to stem cells; in adult, differentiated cells, telomerase activity is dramatically reduced and cannot compensate telomere loss upon cell division. The erosion of chromosomal DNA results in a reduced protection of chromosome ends, resulting in the exposition of open

DNA ends, leading to a DNA damage response and subsequent activation of p53 and

RB. The final result is senescence and apoptosis (see, for a review, Deng et a/., 2008, Nature Reviews Cancer, 8:450-458).

In general, telomerase activity is needed in a cell with high proliferative potential, to ensure a sufficient length of telomeres (as short telomeres lose their protective function, resulting in a DNA damage response). Importantly, 80-90% of human tumours (which have a high rate of cell proliferation) show telomerase activity which ensures sufficient telomere length. Against this background, the inventors have surprisingly discovered that the telomeric repeats are transcribed by DNA-dependent RNA polymerase II, which in turn interacts with the TRF1 shelterin protein. Telomeric RNAs (TeIRNAs) contain UUAGGG repeats, are polyadenylated and transcribed from the telomeric C-rich strand and block the activity of telomerase indicating that TeIRNAs regulate telomerase activity at chromosome ends.

Accordingly, in a first aspect, the present invention provides a diagnostic agent comprising a polynucleotide capable of hybridising to the sequence UUAGGG, or a fragment or variant thereof, which fragment or variant is capable of hybridising to the sequence UUAGGG.

Preferably, the polynucleotide of the diagnostic agent is single-stranded and, in a preferred embodiment, comprises or consists of the sequence CCCTAA or, more preferably, the sequence (CCCTAA)₄ (that is, the sequence 5'- CCCTAACCCTAACCCTAACCCTAA-S').

Conveniently, the diagnostic agent comprises a detectable moiety, preferably phosphorous-32 (³²P), which may conveniently be conjugated to the 5'-end of the polynucleotide of the diagnostic agent. Alternatively, the diagnostic agent comprises 16- biotin-CTP as the detectable moiety (which allows non-radioactive detection of the diagnostic agent), preferably conjugated to the 5'-end of the polynucleotide of the diagnostic agent. Other detectable moieties suitable for the detection of probes or polynucleotides are known in the art. Methods for labelling polynucleotides with such detectable moieties, and methods for detecting those detectable moieties, are well- known to those in the art of molecular biology and biochemistry.

As discussed below, the polynucleotide of the diagnostic agent of the invention may comprise modifications (for example, it may be a peptide nucleic acid (PNA) or a locked nucleic acid (LNA)).

The term "polynucleotide" refers to a heteropolymer of nucleotides or the sequence of these nucleotides. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), to locked nucleic acid (LNA), or to any DNA-like or RNA-like material. In the sequences herein A is adenine, C is cytosine, T is thymine, G is guanine and N is A, C, G or T (U). It is contemplated that where the polynucleotide is RNA, the T (thymine) in the sequences provided herein is substituted with U (uracil). Generally, polynucleotides provided by this invention may be assembled from fragments of the genome and short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides, to provide a synthetic polynucleotide sequence.

In one embodiment, the polynucleotide of the diagnostic agent of the invention comprises or consists of non-natural or modified nucleotide bases or nucleotide linkages, as are known in the art. For example, the polynucleotide of the diagnostic agent of the invention may comprise or consist of: phosphorothioate (PO) linkage and/or boranophosphate (BO) linkage and/or locked nucleic acids (LNA) and/or ribo-difluorotolyl nucleotides and/or uncharged nucleic acid mimics; alternatively, or in addition, the polynucleotide may comprise a modification such as: 2'-modified RNA (such as 2'-O- methoxyethyl RNA (2'-MOE), 2'-fluoro RNA, or 2'-O-methyl RNA) and/or 2'-deoxy-2'- fluoro-beta-D-arabinonucleic acid (FANA) and/or 4-thio modified RNA.

This invention also includes the reverse or direct complement of any of the polynucleotide sequences of the invention; cloning or expression vectors containing the polynucleotide sequences; and host cells or organisms transformed with those expression vectors. Polynucleotide sequences (or their reverse or direct complements) according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology, such as use as hybridization probes, use as primers for PCR, use in an array, use in computer-readable media, use in sequencing full-length genes, use for chromosome and gene mapping, use in the recombinant production of protein, and use in the generation of anti-sense DNA or RNA, their chemical analogs and the like.

The polynucleotide of the invention may be obtained from any appropriate source, such as cDNA, genomic DNA, chromosomal DNA, micro-dissected chromosome bands, cosmid or YAC inserts, and RNA, including mRNA without any amplification steps. For example, Sambrook et al. (1989) describes three protocols for the isolation of high molecular weight DNA from mammalian cells (p. 9.14-9.23).

Alternatively, the polynucleotide of the invention may be synthesised using chemical- synthesis approaches (such as, for example, chemical synthesis using oligonucleotide synthesising machines) as are known in the art In a further aspect, the invention provides a method for detecting the presence of a proliferative disorder in a test individual comprising the steps of:

(i) providing a test sample comprising or consisting of one or more cell from an individual to be tested;

(ii) determining the amount and/or concentration in the test sample of a polyribonucleotide comprising or consisting of the sequence UUAGGG, or a fragment or variant thereof, which fragment or variant is capable of hybridising to the sequence UUAGGG; (iii) detecting the presence of a proliferative disorder in the test individual if the amount and/or concentration of the test polyribonucleotide in the test sample in step (ii) is lower than the amount and/or concentration of that test polyribonucleotide in a control sample.

As discussed above, cells with proliferative disorders typically exhibit telomerase activity, which ensures sufficient telomere length associated with rapid and sustained cellular proliferation. Many tumour cells exhibit telomerase activity which is required for continued tumour growth. A low amount or concentration of TeIRNA (i.e. which is a polyribonucleotide comprising or consisting of the sequence UUAGGG) in a cell will therefore result in telomerase activity (or elevated telomerase activity) which is indicative of rapid and sustained cellular proliferation and the onset and continuance of a proliferative disorder in an individual.

Typically, the presence of a proliferative disorder in the test individual will be detected if the amount and/or concentration of the test polyribonucleotide in the test sample in step (ii) is 60% or less (for example, 50% or less, or 40% or less, or 30% or less, or 20% or less, or 10% or less) of the amount and/or concentration of that test polyribonucleotide in a control sample comprising one or more cell from a healthy individual.

Preferably, the test polyribonucleotide in the test sample comprises or consists of the TeIRNA sequence. As discussed above, TeIRNA comprise UUAGGG sequence repeats (for example, arranged as tandem repeats) and may comprise other sub-telomeric sequences and polyadenylated tail sequence. Typically, human TeIRNA contains between 1 and 3000 UUAGGG sequence repeats and mouse TeIRNA contains between 1 and 10000 UUAGGG sequence repeats. In one embodiment, the control sample comprising an amount and/or concentration of the test polyribonucleotide is a control sample comprising or consisting of one or more cell from a healthy individual.

By "healthy individual" we include an individual not affected by the proliferative disorder for which the test individual is being examined. Accordingly, the one or more cell from the healthy individual would ^' comprise an amount of the test polyribonucleotide associated with a "normal" (i.e. healthy) cell, and exhibiting a normal level or rate of cellular proliferation. Methods for measuring the rate and extent of cellular proliferation are well known in the art of biochemistry and cellular biology.

in another embodiment, the control sample comprises or consists of an amount of the test polyribonucleotide known to be associated with a "normal" (Ae. healthy) cell of that type, and which would be associated with a normal level or rate of cellular proliferation. An amount of the test polyribonucleotide may be isolated or purified from a standard primary cell-line or be chemically-synthesised by methods known in the art.

It will be appreciated that, when conducting the method for detecting the presence of a proliferative disorder in a test individual, an appropriate control sample will need to be selected. For example, if the one or more cell from the test individual is an adult differentiated cell from a breast biopsy, an appropriate control sample would be an adult differentiated cell from a breast biopsy taken from a healthy individual and/or an amount of the test polynucleotide known to be associated with an adult differentiated cell from a breast biopsy taken from a healthy individual.

Preferably, the method comprises the step of obtaining RNA from the one or more cell from the test individual, after step (i) and before step (ii). Exemplary methods for obtaining RNA are described in the accompanying Examples and are known in the art.

Typically, the invention provides a method wherein the amount and/or concentration of the test polyribonucleotide comprising or consisting of the sequence UUAGGG, or a fragment or variant thereof, which fragment or variant is capable of hybridising to the sequence UUAGGG₁ is detected using a diagnostic agent according to the invention and/or a probe capable of hybridising to the test polyribonucleotide.

The terms "probe" or "oligonucleotide fragment" or a "polynucleotide fragment", "portion," or "segment" or "primer" are used interchangeably and refer to a sequence of nucleotide residues which are at least about 5 nucleotides, more preferably at least about 7 nucleotides, more preferably at least about 9 nucleotides, more preferably at least about 11 nucleotides and most preferably at least about 17 nucleotides. The fragment is preferably less than about 1600 nucleotides, more preferably less than about 500 nucleotides, more preferably less than about 200 nucleotides, more preferably less than about 100 nucleotides and most preferably less than about 50 nucleotides. Preferably the probe is from about 6 nucleotides to about 200 nucleotides, preferably from about 15 to about 50 nucleotides, more preferably from about 17 to 30 nucleotides and most preferably from about 20 to 25 nucleotides. Preferably the fragments can be used in polymerase chain reaction (PCR), various hybridization procedures or microarray procedures to identify or amplify identical or related parts of mRNA or DNA molecules. A fragment or segment may uniquely identify each polynucleotide sequence of the present invention.

Probes may, for example, be used to determine whether specific mRNA molecules are present in a cell or tissue or to isolate similar nucleic acid sequences from chromosomal DNA as described by Walsh et al. (Walsh, P. S. et al., 1992, PCR Methods Appl 1 :241- 250). For example, they may be labelled by end-labelling, nick translation, Klenow fill-in reaction, PCR, or other methods well known in the art. Probes of the present invention, their preparation and/or labelling are elaborated in Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; or Ausubel, F. M. et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York N. Y., both of which are incorporated herein by reference in their entirety.

By "capable of hybridising to the test polyribonucleotide" we include the ability of the diagnostic agent according to the invention and/or a probe to hybridise specifically to the test polynucleotide, e.g. under highly stringent hybridisation conditions or very highly stringent hybridisation conditions (such as those described in Sambrook et al., Molecular Cloning Laboratory Manual, Cold Spring Harbour Laboratory Press, New York). "Highly stringent hybridisation conditions" refer to hybridisation in 6x SSC at about 45⁰C, followed by one or more washes in 0.2x SSC, 0.1% SDS at 65⁰C. ""Very highly stringent hybridisation conditions " refer to hybridisation in 0.5M sodium phosphate, 7% SDS at 65'C, followed by one or more washes at 0.2x SSC, 1% SDS at 65⁰C. Exemplary hybridisation conditions are described in the accompanying examples.

Preferably, the probe is a polynucleotide comprising or consisting of the sequence (CCCTAA)₄ (that is, the ' sequence 5'-CCCTAACCCTAACCCTAACCCTAA-S''). Conveniently the probe is labelled (preferably at the 5'-end of the probe) with a detectable moiety such as phosphorous-32 (³²P) and/or 16-biotin-CTP (which allows non-radioactive detection of the diagnostic agent). Other detectable moieties suitable for the detection of probes or polynucleotides are known in the art. Methods for labelling polynucleotides with such detectable moieties, and methods for detecting those detectable moieties, are well-known to those in the art of molecular biology and biochemistry.

Alternatively, the amount and/or concentration of the test polyribonucleotide comprising or consisting of the sequence UUAGGG₁ or a fragment or variant thereof, which fragment or variant is capable of hybridising to the sequence UUAGGG, is detected using quantitative RT-PCR (real-time polymerase chain reaction), a technique known in the art of molecular biology.

Preferably, the invention provides a method wherein the test individual is a mammal. It is preferred that the mammal is a human, or a non-human mammal such as a horse, pig, sheep, goat, dog, cat, rabbit, rat, mouse, or any other domesticated mammal, preferably of agricultural or commercial significance.

In a further aspect, the present invention provides a therapeutic agent comprising or consisting of a polyribonucleotide capable of inhibiting and/or reducing the activity of a telomerase.

It will be appreciated that the polynucleotide of the diagnostic agent of the invention and the polyribonucleotide of the therapeutic agent of the invention are distinct.

The term "polyribonucleotide" refers to a heteropolymer of nucleotides or the sequence of these nucleotides in an RNA molecule. These phrases also refer to RNA of cellular or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), to locked nucleic acid (LNA), or to any RNA-like material. Generally, polyribonucleotides provided by this invention may be assembled from fragments of the genome and short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides, to provide a synthetic polyribonucleotide sequence.

In an embodiment, the polyribonucleotide of the therapeutic agent of the invention comprises or consist of non-natural or modified nucleotide bases or nucleotide linkages, as are known in the art. For example, the polyribonucleotide of the therapeutic agent of the invention may comprise or consist of: phosphorothioate (PO) linkage and/or boranophosphate (BO) linkage and/or locked nucleic acids (LNA) and/or ribo-difluorotolyl nucleotides and/or uncharged nucleic acid mimics; alternatively, or in addition, the polyribonucleotide may comprise a modification such as: 2'-modified RNA (such as 2'-O- methoxyethyl RNA (2'-MOE), 2'-fluoro RNA, or 2'-O-methyl RNA) and/or 2'-deoxy-2'- fluoro-beta-D-arabinonucleic acid (FANA) and/or 4-thio modified RNA.

This invention also includes the reverse or direct complement of any of the polyribonucleotide sequences of the invention; cloning or expression vectors containing the polyribonucleotide sequences; and host cells or organisms transformed with those expression vectors. Polyribonucleotide sequences (or their reverse or direct complements) according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology, such as use as hybridization probes, use as primers for PCR, use in an array, use in computer-readable media, use in sequencing full-length genes, use for chromosome and gene mapping, use in the recombinant production of protein, and use in the generation of anti-sense DNA or RNA, their chemical analogs and the like.

The polyribonucleotide of the therapeutic agent of the invention may be obtained from any appropriate source, such as cellular RNA, including mRNA without any amplification steps, or may be synthesised using chemical-synthesis approaches (such as, for example, chemical synthesis using oligonucleotide synthesising machines) as are known in the art.

Preferably, the polyribonucleotide of the therapeutic agent of the invention comprises or consists of the sequence UUAGGG, or a fragment or variant thereof, which fragment or variant is capable of hybridising to the sequence UUAGGG.

By 'variant' we include polynucleotide sequences having insertions, deletions (e.g. truncations) and/or substitutions, either conservative or non-conservative, as compared to the polynucleotide of interest (for example, the polyribonucleotide of the therapeutic agent of the invention, which comprises or consists of the sequence UUAGGG). The variant may share at least 50% sequence identity with the polynucleotide sequence of interest, for example at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%. The percent sequence identity between two sequences may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to sequences which have been aligned optimally. The alignment may alternatively be carried out using the Clustal W program (as described in Thompson et al., 1994, Nuc. Acid Res. 22:4673-4680).

The parameters used may be as follows:

Fast pairwise alignment parameters: K-tuple(word) size; 1 , window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent.

Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05.

Scoring matrix: BLOSUM.

Alternatively, the BESTFIT program may be used to determine local sequence alignments.

Fragments and variants of a known polynucleotide sequence may be made using methods well known in the art (for example, as described in Molecular Cloning: A Laboratory Manual, 3rd edition, Sambrook & Russell, 2001 , Cold Spring Harbor Laboratory Press, the relevant disclosures in which document are hereby incorporated by reference). For example, sequence variation may be introduced using error prone PCR (Leung et al., Technique, 1 : 11-15, 1989), the GeneMorph II™ random mutagenesis kit (Stratagene) and other known methods of random mutagenesis, site-directed mutagenesis and other molecular biology techniques.

The prior art teaches a number of delivery strategies which can be used to efficiently deliver polynucleotides into a wide variety of cell types (for example, see Luft, 1998, J MoI Med 76:75-6; Kronenwett et al., 1998, Blood 91 :852-62; Rajur ef a/., 1997, Bioconjug Chem 8:935-40; Lavigne et al., 1997, Biochem Biophys Res Commun 237:566-71 ; Aoki et al., 1997, Biochem Biophys Res Commun 231 :540-5).

In addition, algorithms for identifying those polynucleotide sequences with the highest predicted binding affinity for their target sequence based on a thermodynamic cycle that accounts for the energetics of structural alternations in both the polynucleotide and its target are available (for example, see Walton et al., 1999, Biotechnol Bioeng 65:1-9). Several approaches for designing and predicting efficiency of specific oligonucleotides using an in vitro system are also known (for example, see Matveeva et al., 1998, Nature biotechnology 16: 1374-1375).

Several clinical trails have demonstrated safety, feasibility and activity of antisense oligonucleotides. For example, antisense oligonucleotides suitable for the treatment of cancer have been successfully used (Holmlund et al., 1999, Curr Opin MoI Ther 1 :372- 85; Gerwitz, 1999, Curr Opin MoI Ther 1 :297-306). More recently, antisense-mediated suppression of human heparanase gene expression has been reported to inhibit pleural dissemination of human cancer cells in a mouse model (Uno et al., 2001 , Cancer Res 61 :7855-60). Thus, persons skilled in the art are readily able to design and deliver the polyribonucleotide of the therapeutic agent of the invention.

It will be further appreciated by person skilled in the art that polynucleotide sequences are subject to degradation or inactivation by endogenous cellular nucleases. To counter that problem, it is possible to use modified polynucleotide molecules, for example, having altered intemucleotide linkages, in which the naturally-occurring phosphodiester linkages have been replaced with another linkage. For example, Agrawal et al (1988) Proc. Natl. Acad. ScL USA 85, 7079-7083 showed increased inhibition in tissue culture of HIV-1 using oligonucleotide phosphoramidates and phosphorothioates. Sarin et al (1988) Proc. Natl. Acad. Sci. USA 85, 7448-7451 demonstrated increased inhibition of HIV-1 using oligonucleotide methylphosphonates. Agrawal et a/ (1989) Proc. Natl. Acad. Sci. USA 86, 7790-7794 showed inhibition of HIV-1 replication in both early-infected and chronically infected cell cultures, using nucleotide sequence-specific oligonucleotide phosphorothioates. Leither et al (1990) Proc. Natl. Acad. Sci. USA 87, 3430-3434 report inhibition in tissue culture of influenza virus replication by oligonucleotide phosphorothioates.

Polynucleotides having artificial linkages have been shown to be resistant to degradation in vivo. For example, Shaw et al (1991) in Nucleic Acids Res. 19, 747-750, report that otherwise unmodified oligonucleotides become more resistant to nucleases in vivo when they are blocked at the 3' end by certain capping structures and that uncapped oligonucleotide phosphorothioates are not degraded in vivo. A detailed description of the H-phosphonate approach to synthesising oligonucleoside phosphorothioates is provided in Agrawal and Tang (1990) Tetrahedron Letters 31 , 7541- 7544, the teachings of which are hereby incorporated herein by reference. Syntheses of oligonucleoside methylphosphonates, phosphorodithioates, phosphoramidates, phosphate esters, bridged phosphoramidates and bridge phosphorothioates are known in the art. See, for example, Agrawal and Goodchild (1987) Tetrahedron Letters 28, 3539; Nielsen et al (1988) Tetrahedron Letters 29, 2911 ; Jager et a/ (1988) Biochemistry 27, 7237; Uznanski et al (1987) Tetrahedron Letters 28, 3401 ; Bannwarth (1988) HeIv. Chim. Acta. 71 , 1517; Crosstick and VyIe (1989) Tetrahedron Letters 30, 4693; Agrawal et al (1990) Proc. Natl. Acad. ScL USA 87, 1401-1405, the teachings of which are incorporated herein by reference. Other methods for synthesis or production also are possible.

The polyribonucleotide of the therapeutic agent of the invention useful in the invention preferably are designed to resist degradation by endogenous nucleolytic enzymes. In vivo degradation of oligonucleotides produces oligonucleotide breakdown products of reduced length. Such breakdown products are more likely to engage in non-specific hybridisation and are less likely to be effective, relative to their full-length counterparts. Thus, it is desirable to use the polyribonucleotide of the therapeutic agent of the invention that are resistant to degradation in the body and which are able to reach the targeted cells. The polyribonucleotide of the therapeutic agent of the invention can be rendered more resistant to degradation in vivo by substituting one or more internal artificial intemucleotide linkages for the native phosphodiester linkages, for example, by replacing phosphate with sulphur in the linkage. Examples of linkages that may be used include phosphorothioates, methylphosphonates, sulphone, sulphate, ketyl, phosphorodithioates, various phosphoramidates, phosphate esters, bridged phosphorothioates and bridged phosphoramidates. Such examples are illustrative, rather than limiting, since other intemucleotide linkages are well known in the art. The synthesis of the polyribonucleotide of the therapeutic agent of the invention having one or more of these linkages substituted for the phosphodiester intemucleotide linkages is well known in the art, including synthetic pathways for producing polynucleotides having mixed intemucleotide linkages.

Polynucleotides can be made resistant to extension by endogenous enzymes by "capping" or incorporating similar groups on the 5^' or 3' terminal nucleotides. A reagent for capping is commercially available as Amino-Link II™ from Applied BioSystems Inc, Foster City, CA. Methods for capping are described, for example, by Shaw et al (1991 ) Nucleic Acids Res. 19, 747-750 and Agrawal et al (1991 ) Proc. Natl. Acad. Sci. USA 88(17), 7595-7599. A further method of making polynucleotides resistant to nuclease attack is for them to be "self-stabilised" as described by Tang et al (1993) Nucl. Acids Res. 21 , 2729-2735. Self- stabilised polynucleotides have hairpin loop structures at their 3' ends, and show increased resistance to degradation by snake venom phosphodiesterase, DNA polymerase I and foetal bovine serum. The self-stabilised region of the polynucleotide does not interfere in hybridisation with complementary nucleic acids, and pharmacokinetic and stability studies in mice have shown increased in vivo persistence of self-stabilised oligonucleotides with respect to their linear counterparts.

In a preferred embodiment, the polyribonucleotide of the therapeutic agent of the invention comprises or consists of two or more copies of the UUAGGG sequence, or a fragment or variant thereof, which fragment or variant is capable of hybridising to the sequence UUAGGG. By "two or more copies of the UUAGGG sequence" we mean that the complete sequence of the polynucleotide or polyribonucleotide of the therapeutic agent of the invention contains two or more UUAGGG sequences.

Typically, the polyribonucleotide of the therapeutic agent of the invention comprises or consists of 3 or more; 4 or more; 5 or more; 6 or more; 7 or more; 8 or more; 9 or more; 10 or more; 20 or more; or 50 or more copies of the UUAGGG sequence, or a fragment or variant thereof, which fragment or variant is capable of hybridising to the sequence UUAGGG. For example, the polyribonucleotide of the therapeutic agent of the invention may comprise or consist of between 2-10; 10-20; 20-30; 30-40; or 40-50 copies of the UUAGGG sequence, or a fragment or variant thereof, which fragment or variant is capable of hybridising to the sequence UUAGGG.

In a preferred embodiment, the two or more copies of the UUAGGG sequence, or fragment or variant thereof, which fragment or variant is capable of hybridising to the sequence UUAGGG, are arranged in one or more tandem repeat.

The term "tandem repeat" includes a polynucleotide or polyribonucleotide molecule containing a number of copies of a particular sequence, which are arranged such that one copy of that sequence directly follows another copy of that sequence without any other intervening sequence (i.e. in tandem).

By "agent" we include a molecule which is purified and/or isolated, natural and/or chemically-synthesised, and salts (e.g. organic or inorganic acid addition salts), esters and/or solvates thereof. The term also includes a molecule which is conjugated and/or joined to one or more additional molecule, such as one or more polynucleotide, polypeptide and/or small chemical molecule, and which may be modified by the ionic and/or covalent addition of one or more chemical group. By "diagnostic agent" we include an agent capable for use in diagnosis; by "therapeutic agenf we include an agent capable for use in therapy.

It will be appreciated that the term "agent" further includes derivatives that have the same biological function and/or activity as the relevant agent and, for the purposes of this invention, prodrugs of the relevant agent (for example, esters). The term "prodrug" includes any composition of matter that, following oral or parenteral administration, is metabolised in vivo to form the relevant agent in an experimentally-detectable amount, and within a predetermined time of dosing.

The agent of the invention can comprise or consist of a polynucleotide or a polyribonucleotide that is isolated, purified and/or substantially purified.

The terms "purified" or "substantially purified" as used herein denotes that the indicated polynucleotide or polyribonucleotide is present in the substantial absence of other biological macromolecules, e.g., polynucleotides, proteins, and the like. In one embodiment, the polynucleotide or polyribonucleotide is purified such that it constitutes at least 95% by weight, more preferably at least 99% by weight, of the indicated biological macromolecules present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 Daltons, can be present).

The term "isolated" as used herein refers to a polynucleotide or polyribonucleotide separated from at least one other component (e.g., nucleic acid or polypeptide) present with the polynucleotide or polyribonucleotide in its natural source. In one embodiment, the polynucleotide or polyribonucleotide is found in the presence of (if anything) only a solvent, buffer, ion, or other component normally present in a solution of the same.

The terms "isolated" and "purified" do not encompass the polynucleotide or the polyribonucleotide of the invention when present in its natural source.

It is well known that a polynucleotide or polyribonucleotide can exist in a number of forms, including as a single-stranded or double-stranded molecule. Preferably, the polynucleotide of the invention or the polyribonucleotide of the therapeutic agent of the invention is single-stranded.

As discussed above, telomerases are intracellular enzymes comprising a reverse- transcriptase activity which permits synthesis of DNA sequence and facilitates the addition of telomeric repeats de novo to chromosome ends, thereby maintaining or extending the telomere length at the chromosome ends. Reverse transcription is an activity well-known in the art of molecular biology and includes the synthesis of a single- stranded DNA product from an RNA template.

Telomerases typically comprise a protein component (named Tert) which comprises a reverse-transcriptase activity and an RNA component (named Terc) which comprises a template for reverse-transcription and includes sequences complementary to the sequence repeats present in telomeres.

Thus, by "capable of inhibiting and/or reducing the activity of a telomerase" we include the meaning that the therapeutic agent and/or polyribonucleotide of the therapeutic agent of the invention is capable of inhibiting and/or reducing any in vitro and/or in vivo property or activity of a telomerase. For example, the therapeutic agent and/or polyribonucleotide of the therapeutic agent of the invention may prevent and/or reduce the ability of one or more component of telomerase to: physically associate with, or bind to, chromosome ends and/or telomeres; associate with other cellular structures or components; and/or perform its usual enzymatic and/or cellular functions (such as, for example, its ability to synthesise telomeric repeats by reverse-transcription).

For example, the therapeutic agent and/or polyribonucleotide of the therapeutic agent of the invention may be capable of reversibly or irreversibly binding to telomerase and/or the Terc component of telomerase, and thereby inhibit the ability of telomerase to synthesise telomeric repeats sequences on the chromosome. Such binding may be "selectively binding", by which we include the ability of the therapeutic agent and/or polyribonucleotide of the therapeutic agent of the invention to bind at least 10-fold more strongly to telomerase and/or the Terc component of telomerase than to another polypeptide or RNA molecule; preferably at least 50-fold more strongly and more preferably at least 100-fold more strongly. Preferably, the therapeutic agent and/or polyribonucleotide of the therapeutic agent of the invention bind to telomerase and/or the Terc component of telomerase under physiological conditions, for example, in vivo. Methods for measuring the binding or association of an agent or' polyribonucleotide with another molecule (such as a protein or polynucleotide) either in vivo or in vitro are well- known to those in the arts of biochemistry and cell biology and exemplary assays are described in the accompanying examples.

Biochemical assays for the cellular functions of telomerase are well known in the art. For example, assays for the ability of telomerase to bind to chromosome ends or telomeres and/or synthesise telomeric repeats by reverse transcription are well known. Exemplary assays are described in the accompanying examples.

Preferably, the therapeutic agent and/or polyribonucleotide of the therapeutic agent of the invention is capable of hybridising to the RNA component of a telomerase.

As discussed above, telomerase enzymes comprise an RNA component (named Terc) which contains the template for reverse-transcription and is complementary to the sequence repeats in the telomeres. For example, the Terc sequence in mammalian telomerase is complementary to the non-coding TTAGGG repeats in mammalian telomeres.

It is well known that certain nucleotide bases are capable of chemically linking with certain other nucleotide bases when present in polynucleotide molecules (also known as "base pairing"), which is responsible for holding together two polynucleotide strands (for example in a DNA double-helix). For example, the nucleotide base adenine pairs with thymine (or, in polyribonucleotides, uracil) and the nucleotide base cytosine pairs with guanine.

A polynucleotide which is "complementary" to a second polynucleotide has a nucleotide sequence which is capable of base-pairing with the second polynucleotide (such as, base-pairing identically to every nucleotide in the second polynucleotide).

In one embodiment, the invention provides a therapeutic agent and/or polyribonucleotide of the therapeutic agent wherein the reverse-transcriptase activity of telomerase is inhibited and/or reduced.

Preferably, the therapeutic agent and/or polyribonucleotide of the therapeutic agent comprises a detectable moiety, permitting detection of the therapeutic agent and/or polyribonucleotide of the therapeutic agent following administration to an individual. By a

"detectable moiety" we include the meaning that the moiety is one which, when located at the target site following administration of the compound of the invention into a patient, may be detected, typically non-invasively from outside the body and the site of the target located. Thus, the therapeutic agent and/or polyribonucleotide of the therapeutic agent are useful in imaging and diagnosis.

Preferably, the detectable moiety is selected from the group consisting of: a radioactive moiety or a fluorescent moiety.

Typically, the detectable moiety is or comprises a radioactive atom which is useful in imaging. Suitable radioactive atoms include ^99mTc and ¹²³I for scintigraphic studies.

Other readily detectable moieties include, for example, spin labels for magnetic resonance imaging (MRI) such as ¹²³I again, ¹³¹I, ¹¹¹In, ¹⁹F, ¹³C, ¹⁵N, ¹⁷O, gadolinium, manganese or iron. Clearly, the therapeutic agent and/or polyribonucleotide of the therapeutic agent of the invention must have sufficient of the appropriate atomic isotopes to be readily detectable.

In a further embodiment of the invention the radioactive atom is selected from the group consisting of technetium-99m, iodine-123, iodine-125, iodine-131 , indium-111 , fluorine- 19, carbon-13, nitrogen-15, oxygen-17, phosphorus-32, sulphur-35, deuterium, tritium, rhenium-186, rhenium-188 and yttrium-90.

The radio- or other labels may be incorporated in the therapeutic agent and/or polyribonucleotide of the therapeutic agent in known ways, for example, by incorporating one or more chemically-modified ribonucleotide comprising the radioactive label during synthesis of the therapeutic agent of the invention or the polyribonucleotide of the therapeutic agent of the invention.

Methods for detecting radioactive labels, and for detecting and/or measuring and/or visualising such labels in an individual, are known in the art.

In a further aspect, there is provided a therapeutic agent according to the invention for use in medicine. In one aspect, the invention provides an isolated or recombinant polynucleotide or polyribonucleotide as defined herein.

In a further aspect, the invention provides the use of an agent or an isolated or recombinant polynucleotide or polyribonucleotide according to the invention in the treatment of a proliferative disorder. In a still further aspect, the invention provides the use of an agent or an isolated or recombinant polynucleotide or polyribonucleotide according to the invention in the manufacture of a medicament for treating a proliferative disorder.

In a further aspect, the invention provides a pharmaceutical composition comprising or consisting of an effective amount of an agent of the invention or an effective amount of an isolated or recombinant polynucleotide or polyribonucleotide of the invention and a pharmaceutically-acceptable excipient, diluent or carrier.

A 'therapeutically effective amount', or 'effective amount', or 'therapeutically effective', as used herein, refers to that amount which provides a therapeutic effect for a given condition and administration regimen. Accordingly, we include an amount of the agent or polynucleotide or polyribonucleotide of the invention that is sufficient to reduce and/or alleviate and/or prevent symptoms associated with a proliferative disorder. This is a predetermined quantity of active material calculated to produce a desired therapeutic effect in association with the required additive and diluent, i.e. a carrier or administration vehicle. Further, it is intended to mean an amount sufficient to reduce and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in a host. As is appreciated by those skilled in the art, the amount of a compound may vary depending on its specific activity. Suitable dosage amounts may contain a predetermined quantity of active composition calculated to produce the desired therapeutic effect in association with the required diluent. In the methods and use for manufacture of compositions of the invention, a therapeutically effective amount of the active component is provided. A therapeutically effective amount can be determined by the ordinary skilled medical or veterinary worker based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, etc., as is well known in the art.

An effective amount may, for example, be determined by undertaking dose studies in animals (and, if possible, humans) for inhibition of proliferation and/or differentiation and/or metabolism of one or more cell, preferably in which one or more mutation has induced cellular changes characteristic of a proliferative disorder (such as unregulated proliferation and/or differentiation of that cell). An effective amount could also be determined in vitro using the methods described in the Examples (for example, the methods used to monitor antagonism of telomerase activity in a cell) or in vivo by monitoring the reduction and/or alleviation and/or prevention of symptoms in an individual (such as an animal) associated with a proliferative disorder, which symptoms will be know to those skilled in the relevant medical field.

The agents, polynucleotides, polyribonucleotides and pharmaceutical compositions of the present invention may be delivered using an injectable sustained-release drug delivery system. These are designed specifically to reduce the frequency of injections. An example of such a system is Nutropin Depot which encapsulates recombinant human growth hormone (rhGH) in biodegradable microspheres that, once injected, release rhGH slowly over a sustained period. Preferably, delivery is performed intra-muscularly (i.m.) and/or sub-cutaneously (s.c.) and/or intravenously (i.v.).

The agents, polynucleotides, polyribonucleotides and pharmaceutical compositions of the present invention can be administered by a surgically implanted device that releases the drug directly to the required site. For example, Vitrasert releases ganciclovir directly into the eye to treat CMV retinitis. The direct application of this toxic agent to the site of disease achieves effective therapy without the drug's significant systemic side-effects.

Electroporation therapy (EPT) systems can also be employed for the administration of the agents, polynucleotides, polyribonucleotides and pharmaceutical compositions of the invention. A device which delivers a pulsed electric field to cells increases the permeability of the cell membranes to the drug, resulting in a significant enhancement of intracellular drug delivery.

The agents, polynucleotides, polyribonucleotides and pharmaceutical compositions of the invention can also be delivered by electroincorporation (El). El occurs when small particles of up to 30 microns in diameter on the surface of the skin experience electrical pulses identical or similar to those used in electroporation. In El, these particles are driven through the stratum corneum and into deeper layers of the skin. The particles can be loaded or coated with drugs or genes or can simply act as "bullets" that generate pores in the skin through which the drugs can enter.

An alternative method of delivery of the agents, polynucleotides, polyribonucleotides and pharmaceutical compositions of the invention is the ReGeI injectable system that is thermo-sensitive. Below body temperature, ReGeI is an injectable liquid while at body temperature it immediately forms a gel reservoir that slowly erodes and dissolves into known, safe, biodegradable polymers. The active substance is delivered over time as the biopolymers dissolve.

The agents, polynucleotides, polyribonucleotides and pharmaceutical compositions of the invention can also be delivered orally. The process employs a natural process for oral uptake of vitamin B₁₂ and/or vitamin D in the body to co-deliver proteins and peptides. By riding the vitamin B₁₂ and/or vitamin D uptake system, the nucleic acids, molecules and pharmaceutical formulations of the invention can move through the intestinal wall. Complexes are synthesised between vitamin Bi₂ analogues and/or vitamin D analogues and the drug that retain both significant affinity for intrinsic factor (IF) in the vitamin B₁₂ portion/vitamin D portion of the complex and significant bioactivity of the active substance of the complex.

The molecules, polynucleotides, polyribonucleotides and pharmaceutical compositions of the invention can be introduced to cells by "Trojan peptides". These are a class of polypeptides called penetratins which have translocating properties and are capable of carrying hydrophilic compounds across the plasma membrane. This system allows direct targeting of oligopeptides to the cytoplasm and nucleus, and may be non-cell type specific and highly efficient. See Derossi et al. (1998), Trends Cell Biol 8, 84-87.

Preferably, the medicament and/or pharmaceutical composition of the present invention is a unit dosage containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of the active ingredient.

The agents, polynucleotides, polyribonucleotides and pharmaceutical compositions of the invention will normally be administered orally or by any parenteral route, in the form of a pharmaceutical composition comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated, as well as the route of administration, the compositions may be administered at varying doses.

In human therapy, the agents, polynucleotides, polyribonucleotides and pharmaceutical compositions of the invention can be administered alone but will generally be administered in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice. For example, the agents, polynucleotides, polyribonucleotides and pharmaceutical compositions of the invention can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications. The agents, polynucleotides, polyribonucleotides and pharmaceutical compositions of the invention may also be administered via intracavernosal injection.

Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose

(HPC)₁ sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the agents of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

The agents, polynucleotides, polyribonucleotides and pharmaceutical compositions of the invention can also be administered parenterally, for example, intravenously, intra- arterially, intraperitoneally, intra-thecally, intraventricular^, intrasternally, intracranially, intra-muscularly or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

Medicaments and pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes' which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The medicaments and pharmaceutical compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

For oral and parenteral administration to human patients, the daily dosage level of the agents, polynucleotides and pharmaceutical compositions of the invention will usually be from 0.1 to 100 mg per adult per day administered in single or divided doses.

Thus, for example, the tablets or capsules of the molecules of the invention may contain from 0.1 mg to 100mg of active agent for administration singly or two or more at a time, as appropriate. The physician in any event will determine the actual dosage which will be most suitable for any individual patient and it will vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited and such are within the scope of this invention.

The agents, polynucleotides, polyribonucleotides and pharmaceutical compositions of the invention can also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1 ,1 ,1 ,2-tetrafluoroethane (HFA 134A3 or 1 ,1 ,1 ,2,3,3,3- heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active agent, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a agent or polynucleotide of the invention and a suitable powder base such as lactose or starch. Aerosol or dry powder formulations are preferably arranged so that each metered dose or "puff¹ contains at least 0.1 mg of an agent or polynucleotide of the invention for delivery to the patient. It will be appreciated that he overall daily dose with an aerosol will vary from patient to patient, and may be administered in a single dose or, more usually, in divided doses throughout the day.

Alternatively, the agents, polynucleotides, polyribonucleotides and pharmaceutical compositions of the invention can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, gel, ointment or dusting powder. The agents, polynucleotides and pharmaceutical compositions of the invention may also be transdermal^ administered, for example, by the use of a skin patch. They may also be administered by the ocular route, particularly for treating diseases of the eye.

For ophthalmic use, the agents, polynucleotides, polyribonucleotides and pharmaceutical compositions of the invention can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.

For application topically to the skin, the agents, polynucleotides and pharmaceutical compositions of the invention can be formulated as a suitable ointment containing the active agent suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene agent, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol and water.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier. Generally, in humans, oral or parenteral administration of the agents, polynucleotides, polyribonucleotides and pharmaceutical compositions of the invention is the preferred route, being the most convenient.

For veterinary use, the agents, polynucleotides, polyribonucleotides and pharmaceutical compositions of the invention are administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal.

Conveniently, the formulation is a pharmaceutical formulation. Advantageously, the formulation is a veterinary formulation.

Methods for administering the agents, polynucleotides and/or polyribonucleotides of the invention are also well know in the art (see Dass, 2002, J. Pharm. Pharmacol. 54(1 ):3- 27; Dass, 2001 , Drug Deliv. 8(4):191-213; Lebedeva et al., 2000, Eur. J. Pharm. Biopharm. 50(1 ): 101 -19; Pierce et al., 2005, Mini Rev Med Chem. 5(1):41-55; Lysik & Wu-Pong, 2003, J. Pharm. ScL 2003 2(8): 1559-73; Dass, 2004, Biotechnol. Appl. Biochem. 40(Pt 2):113-22; Medina, 2004, Curr Pharm Des. 10(24):2981-9.

For example, the agents, polynucleotides and/or polyribonucleotides of the invention may be introduced into cells by methods involving retroviruses, so that the polynucleotide of the invention (or a polynucleotide encoding it) is inserted into the genome of the cell. For example, in Kuriyama et al (1991 ) Cell Struc. and Func. 16, 503-510 purified retroviruses are administered. Retroviral DNA constructs comprising a polynucleotide as described above may be made using methods well known in the art. To produce active retrovirus it is usual to use an ecotropic psi2 packaging cell line grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% foetal calf serum (FCS). Transfection of the cell line is conveniently by calcium phosphate co-precipitation, and stable transformants are selected by addition of G418 to a final concentration of 1 mg/ml (assuming the retroviral construct contains a neo^R gene). Independent colonies are isolated and expanded and the culture supernatant removed, filtered through a 0.45 μm pore-size filter and stored at -70^°C. For the introduction of the retrovirus into the tumour cells, it is convenient to inject directly retroviral supernatant to which 10 μg/ml Polybrene has been added. For tumours exceeding 10 mm in diameter it is appropriate to inject between 0.1 ml and 1 ml of retroviral supernatant; preferably 0.5 ml. Alternatively, as described in Culver et al (1992) Science 256, 1550-1552, cells which produce retroviruses are injected. The retrovirus-producing cells so introduced are engineered to actively produce retroviral vector particles so that continuous productions of the vector occurred within the tumour mass in situ. Thus, proliferating cells can be successfully transduced in vivo if mixed with retroviral vector-producing cells.

Targeted retroviruses are also available for use in the invention; for example, sequences conferring specific binding affinities may be engineered into pre-existing viral env genes (see Miller & Vile (1995) Faseb J. 9, 190-199 for a review of this and other targeted vectors for gene therapy).

Other methods involve simple delivery of the agents, polynucleotides and/or polyribonucleotides of the invention into the cell for expression therein either for a limited time or, following integration into the genome, for a longer time. An example of the latter approach includes liposomes (Nassander et al (1992) Cancer Res. 52, 646-653).

For the preparation of immuno-liposomes MPB-PE (N-[4-(p-maleimidophenyl)butyryl]- phosphatidylethanolamine) is synthesised according to the method of Martin & Papahadjopoulos (1982) J. Biol. Chem. 257, 286-288. MPB-PE is incorporated into the liposomal bilayers to allow a covalent coupling of the antibody, or fragment thereof, to the liposomal surface. The liposome is conveniently loaded with the agents, polynucleotides and/or polyribonucleotides of the invention (such as DNA or other genetic construct) for delivery to the target cells, for example, by forming the said liposomes in a solution of the agent, followed by sequential extrusion through polycarbonate membrane filters with 0.6 μm and 0.2 μm pore size under nitrogen pressures up to 0.8 MPa. After extrusion, entrapped polynucleotide is separated from free DNA construct by ultracentrifugation at 80 000 x g for 45 min. Freshly prepared MPB-PE-liposomes in deoxygenated buffer are mixed with freshly prepared antibody (or fragment thereof) and the coupling reactions are carried out in a nitrogen atmosphere at 4^'C under constant end over end rotation overnight. The immunoliposomes are separated from un-conjugated antibodies by ultracentrifugation at 80 000 x g for 45 min. Immunoliposomes may be injected intraperitoneally or directly into the tumour.

Other methods of delivery include adenoviruses carrying external DNA via an antibody- polylysine bridge (see Curiel Prog. Med. Virol. 40, 1-18) and transferrin-polycation conjugates as carriers (Wagner et al (1990) Proc. Natl. Acad. Sci. USA 87, 3410-3414).

In the first of these methods a polycation-antibody complex is formed with the agents, polynucleotides and/or polyribonucleotides of the invention, wherein the antibody is specific for either wild-type adenovirus or a variant adenovirus in which a new epitope has been introduced which binds the antibody. The polycation moiety binds the polynucleotide or polyribonucleotide via electrostatic interactions with the phosphate backbone. The adenovirus, because it contains unaltered fibre and penton proteins, is internalised into the cell and carries into the cell with it the agent, polynucleotide and/or polyribonucleotide of the invention. It is preferred if the polycation is polylysine.

The agent, polynucleotide and/or polyribonucleotide of the invention may also be delivered by adenovirus wherein it is present within the adenovirus particle, for example, as described below.

In an alternative method, a high-efficiency nucleic acid delivery system that uses receptor-mediated endocytosis to carry polynucleotides into cells is employed. This is accomplished by conjugating the iron-transport protein transferrin to polycations that bind nucleic acids. Human transferrin, or the chicken homologue conalbumin, or combinations thereof is covalently linked to the small DNA-binding protein protamine or to polylysines of various sizes through a disulfide linkage. These modified transferrin molecules maintain their ability to bind their cognate receptor and to mediate efficient iron transport into the cell. The transferrin-polycation molecules form electrophoretically stable complexes with polynucleotides independent of nucleic acid size (from short oligonucleotides to DNA of 21 kilobase pairs).

High-efficiency receptor-mediated delivery of the agents, polynucleotides and/or polyribonucleotides of the invention using the endosome-disruption activity of defective or chemically inactivated adenovirus particles produced by the methods of Cotten et al

(1992) Proc. Natl. Acad. Sci. USA 89, 6094-6098 may also be used. This approach appears to rely on the fact that adenoviruses are adapted to allow release of their DNA from an endosome without passage through the lysosome, and in the presence of, for example transferrin linked to the agents, polynucleotides and/or polyribonucleotides of the invention, the construct is taken up by the cell by the same route as the adenovirus particle.

This approach has the advantages that there is no need to use complex retroviral constructs; there is no permanent modification of the genome as occurs with retroviral infection; and the targeted expression system is coupled with a targeted delivery system, thus reducing toxicity to other cell types. It will be appreciated that "naked DNA" and DNA complexed with cationic and neutral lipids may also be useful in introducing the agents, polynucleotides and/or polyribonucleotides of the invention into cells of the individual to be treated. Non-viral approaches to gene therapy are described in Ledley (1995) Human Gene Therapy 6, 1129-1144.

Alternative targeted delivery systems are also known such as the modified adenovirus system described in WO 94/10323 wherein, typically, the polynucleotide is carried within the adenovirus, or adenovirus-like, particle. Michael et al (1995) Gene Therapy 2, 660- 668 describes modification of adenovirus to add a cell-selective moiety into a fibre protein. Mutant adenoviruses which replicate selectively in p53-deficient human tumour cells, such as those described in Bischoff et al (1996) Science 274, 373-376 are also useful for delivering the polynucleotide and/or polyribonucleotide of the invention to a cell. Thus, it will be appreciated that a further aspect of the invention provides a virus or virus-like particle comprising agents, polynucleotides and/or polyribonucleotides of the invention. Other suitable viruses or virus-like particles include HSV, AAV, vaccinia and parvovirus.

In a further aspect, the invention provides a method for treating a proliferative disorder in an individual comprising or consisting of the step of administering an effective amount of an therapeutic agent of the invention and/or an effective amount of an isolated or recombinant polyribonucleotide of the therapeutic agent invention and/or a pharmaceutical composition of the invention to an individual in need thereof.

As discussed above, in a preferred embodiment, the individual is a mammal, most preferably a human, or a non-human mammal such as a horse, pig, sheep, goat, dog, cat, rabbit, rat, mouse, or any other domesticated mammal, preferably of agricultural or commercial significance.

By "proliferative disorder" we include any condition or disorder associated with the unregulated and/or inappropriate growth, proliferation and/or differentiation of one or more cell in an individual, either when that one or more cell is present in its usual, physiological location in the body (for example, forming part of its usual tissue location) or following relocation to an unusual, non-physiological location in the body (for example, following metastasis in an individual). Preferably, the invention provides a use or method wherein the proliferative disorder is selected from the group comprising or consisting of: benign prostatic hyperplasia and cancer.

The agents, polynucleotides, polyribonucleotides and/or pharmaceutical compositions of the invention may be administered to treat proliferative disorders, such as cancer; for example, in therapeutically effective dosages alone or in combination with adjuvant cancer therapy such as surgery, chemotherapy, radiotherapy, thermotherapy, and laser therapy, and may provide a beneficial effect, e.g. reducing tumor size, slowing rate of tumor growth, inhibiting metastasis, or otherwise improving overall clinical condition, without necessarily eradicating the cancer.

The agents, polynucleotides, polyribonucleotides and/or pharmaceutical compositions of the invention can also be administered in therapeutically effective amounts as a portion of an anti-cancer cocktail. An anti-cancer cocktail is a mixture of the agent, polynucleotide, polyribonucleotides and/or pharmaceutical composition of the invention with one or more anti-cancer drugs in addition to a pharmaceutically acceptable carrier for delivery. The use of anti-cancer cocktails as a cancer treatment is routine. Anticancer drugs that are well known in the art and can be used as a treatment in combination with the polypeptide or modulator of the invention include: Actinomycin D, Aminoglutethimide, Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin (cis-DDP), Cyclophosphamide, Cytarabine HCI (Cytosine arabinoside), Dacarbazine, Dactinomycin, Daunorubicin HCI₁ Doxorubicin HCI, Estramustine phosphate sodium, Etoposide (V16-213), Floxuridine, 5-Fluorouracil (5-Fu), Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alpha-2a, Interferon Alpha-2b, Leuprolide acetate (LHRH-releasing factor analog), Lomustine, Mechlorethamine HCI (nitrogen mustard), Melphalan, Mercaptopurine, Mesna, Methotrexate (MTX), Mitomycin, Mitoxantrone HCI, Octreotide, Plicamycin, Procarbazine HCI, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Vincristine sulfate, Amsacrine, Azacitidine, Hexamethylmelamine, lnterleukin-2, Mitoguazone, Pentostatin, Semustine, Teniposide, and Vindesine sulfate.

In vitro models can be used to determine the effective doses of the agent, polynucleotide, polyribonucleotide or pharmaceutical composition of the invention as a potential cancer treatment. These in vitro models include: proliferation assays of cultured tumor cells; growth of cultured tumor cells in soft agar (see Freshney, (1987) Culture of Animal Cells: A Manual of Basic Technique, Wily-Liss, New York, NY Ch 18 and Ch 21 ); tumor systems in nude mice as described in Giovanella et al., J. Natl. Can. Inst, 52: 921-30 (1974); mobility and invasive potential of tumor cells in Boyden Chamber assays as described in Pilkington et al., Anticancer Res., 17: 4107-9 (1997); and angiogenesis assays such as induction of vascularization of the chick chorioallantoic membrane or induction of vascular endothelial cell migration as described in Ribatta et al., Intl. J. Dev. Biol., 40: 1189-97 (1999) and Li et al., CHn. Exp. Metastasis, 17:423-9 (1999), respectively. Suitable tumor cells lines are available, e.g. from American Type Tissue Culture Collection catalogs.

Cancer treatments promote tumor regression by inhibiting tumor cell proliferation, inhibiting angiogenesis (growth of new blood vessels that is necessary to support tumor growth) and/or prohibiting metastasis by reducing tumor cell motility or invasiveness. Therapeutic compositions of the invention may be effective in adult and pediatric oncology.

In a preferred embodiment, the cancer is selected from the group consisting or comprising of: solid phase tumours/malignancies, locally advanced tumours, human soft tissue sarcomas, metastatic cancer, including lymphatic metastases, blood cell malignancies including multiple myeloma, acute and chronic leukaemia's, and lymphomas, head and neck cancers including mouth cancer, larynx cancer and thyroid cancer, lung cancers including small cell carcinoma and non-small cell cancers, breast cancers including small cell carcinoma and ductal carcinoma, gastrointestinal cancers including esophageal cancer, stomach cancer, colon cancer, colorectal cancer and polyps associated with colorectal neoplasia, pancreatic cancers, liver cancer, urologic cancers including bladder cancer and prostate cancer, malignancies of the female genital tract including ovarian carcinoma, uterine (including endometrial) cancers, and solid tumor in the ovarian follicle, kidney cancers including renal cell carcinoma, brain cancers including intrinsic brain tumours, neuroblastoma, astrocytic brain tumours, gliomas, metastatic tumor cell invasion in the central nervous system, bone cancers including osteomas, skin cancers including malignant melanoma, tumor progression of human skin keratinocytes, squamous cell carcinoma, basal cell carcinoma, hemangiopericytoma and Karposi's sarcoma.

In a further aspect, the invention provides an in vitro method for inhibiting and/or reducing the activity of telomerase in a cell comprising or consisting of the step of treating the cell with an effective amount of a therapeutic agent according to the invention or an effective amount of an isolated or recombinant polyribonucleotide of the therapeutic agent according to the invention or a pharmaceutical composition according to the invention.

As discussed above, the activity exhibited by telomerase enzymes include reverse- transcriptase activity which permits synthesis of DNA sequence and facilitates the addition of telomeric repeats de novo to chromosome ends. Thus, by "capable of inhibiting and/or reducing the activity of a telomerase" we include the meaning that the agent and/or polynucleotide of the invention is capable of inhibiting and/or reducing any in vitro and/or in vivo property or activity of a telomerase. For example, an therapeutic agent or polyribonucleotide of the therapeutic agent of the invention may prevent and/or reduce the ability of telomerase to: physically associate with, or bind to, chromosome ends and/or telomeres; associate with other cellular structures or components; and/or perform its usual enzymatic and/or cellular functions (such as, for example, its ability to synthesise telomeric repeats by reverse-transcription).

In relation to this aspect of the invention "effective amount" includes an amount of the therapeutic agent or polyribonucleotide of the therapeutic agent or pharmaceutical composition of the invention that is sufficient to inhibit and/or reduce the activity of telomerase when used in that method of the invention. Methods for measuring and/or detecting one or more activity of telomerase are discussed above and in the accompanying examples, and would be known to those skilled in the art of molecular biology.

The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Preferred, non-limiting examples which embody certain aspects of the invention will now be described, with reference to the following figures:

Figure 1 : Transcription of (UUAGGG)_n repeat containing RNAs from mammalian telomeres is developmentally regulated and restricted to the nucleus.

(a) Total RNA prepared from mouse embryos and adult mouse tissues was subjected to Northern blotting and probed in parallel with ³²P-end labelled (CCCTAA)₄ or (TTAGGG)₄ oligonucleotide probes to detect r[UUAGGG]n and r[CCCUAA]n transcripts, respectively. In adult tissues the telomeric C-rich strand serves as a template for transcription. Different exposure times are indicated. The same blots were hybridized with a probe specific for major satellite and gapdh transcripts. Total RNA was stained with ethidium bromide after agarose gel eletrophoresis to control for loading and RNA integrity. Full- size Northern blots are shown in Figure 10a. (b) Northern blotting of total RNA and polyadenylated transcripts prepared from the indicated mouse and human cell lines using a [³²P] dCTP labelled 1 ,6kb telomeric probe. TeIRNAs show a broad size range reaching a maximum length of less than 6 kb. When indicated, total RNA was pre-treated with RNase. Hybridization with a gapdh probe was included as a control. Full-size Northern blots are shown in Figure 10b.

(c) TeIRNAs are unstable transcripts. U2OS cells were treated with Actinomycin D for the indicated periods to arrest global transcription. Total RNA was prepared and probed with ³²P-end labelled (CCCTAA)₄ or (TTAGGG)₄ oligonucleotide probes. TeIRNAs have a half-life of approximately 2,2 hours. Gapdh is a stable RNA with a half-life >6 hrs. Oligonucleotide-hybridizations were carried out in duplicates resulting in the given average value.

(d) [UUAGGG]_n transcripts are highly enriched in the nucleus. As reported, Gapdh and major satellite transcripts localize to the cytoplasma or nucleoplasma, respectively. Also shown is quantification of RNA dot blot analyses. Average values are derived from two independent oligonucleotide hybridizations. Where indicated, RNA was treated with RNase A.

Figure 2: TeIRNAs are polyadenylated RNA Polymerase Il transcripts.

(a) The indicated amount of polyadenylated RNA from two independent MEF cultures was subjected to dot blot analyses using ³²P-end labelled (CCCTAA)₄ or (TTAGGG)₄ probes to detect r[UUAGGG]n and r[CCCUAA]n transcripts, respectively. Gapdh, positive control. Enrichment of TeIRNAs in polyA RNA is also shown.

(b) TeIRNAs were decreased by inhibition of RNA Pol Il with α-amanitin. RNA Pol l-dependent transcription of the 28S ribosomal gene was not inhibited. Data are from two independent experiments.

(c) RNA Polymerase Il is associated with mammalian telomeres. Chromatin from the indicated cells was immunoprecipitated using antibodies against TRF1 , TRF2 and Pol II, blotted to nitrocellulose and probed with telomere, mouse major satellite or human alpha satellite probes. RNA Pol Il was immunoprecipitated with mouse major satellite or human chromosome 3 ϊ \ alphoid DNA probes. ChIP values (mean ± S. D.; n=3) for telomere and centromere repeats were normalized against the amount of telomere or centromere repeats in crosslinked chromatin (input).

(d) RNA Pol Il and TRF1 were immunoprecipitated from lysates from the indicated cell lines. Detection of TRF1 points to an interaction between TRF1 and RNA PoIII. Pl₁ pre-immune serum.

(e) TRF1 protein levels in mouse cells transfected with control siRNAs or siRNAs against TRF1. Actin protein and Ponceau-S stainings are shown as loading controls. Full-size Western blots are shown in Figure 10c.

(T) Decreased production of TeIRNAs in TRF1 knock-down cells (Northern blot). Where indicated, total RNA was treated with RNase A. Right panel: quantification of TeIRNA levels in control and TRF1 knock-down cells

(normalized against gapdh). Full-size Northern blots for TeIRNA and gapdh are shown in Figure 1Od.

(g) RNA Pol Il associates with mammalian telomeres in TRF1 knock down cells. Chromatin from mouse cells transfected with control siRNA or siRNA against

TRF1 was immunoprecipitated with antibodies against TRF1 , TRF2, H3K9me3, and RNA Pol II, blotted to nitrocellulose and probed with 1 ,6kb telomeric, mouse major satellite or human alpha satellite centromeric probes. Data for TRF1 and TRF2 were normalized against telomere DNA in crosslinked chromatin (input), H3K9me3 and H4K20me3 against telomere input and major satellite.

Figure 3: TeIRNAs are associated with telomeric DNA repeats, upregulated upon heat shock, and massively accumulated when expressed close to the inactive X chromosome.

(a) Telomeric transcripts were detected using RNA FISH in iMEFs, NS1 and U2OS cells. Localization of telomeric RNAs (red) was determined by co- staining using an anti mouse TRF1 antibody (green); DNA was stained using DAPI. Co-localization events are indicated by arrowheads. RNase treatment of permeabilized cells prior to fixation greatly reduces RNA signals without affecting localization of TRF1 to telomeric repeats. Scale bars represent 5 μm.

(b) Percentage of cells showing co-localization of TeIRNAs with TRF1 (mean ± S. E., n=3). Number of nuclei analysed (n) and total number of TRF1 and TeIRNA signals are indicated.

(c) TeIRNA FISH analyses was performed on cells incubated at 42°C for one hour. In parallel, cells were allowed to recover for one hour from heat shock. Untreated cells were included as a control. Control and experimental cells were fixed at the same time and TeIRNA FISH was performed. TeIRNA foci are shown in red; DNA in blue (DAPI). Scale bars represent 5 μm.

(d) Quantification of TeIRNA foci after heat shock treatment (TeIRNA in red, genomic DNA (DAPI) in blue. Number of analyzed cells (n), total number of

TeIRNA signals, mean number of TeIRNA foci per nucleus (mean ± S. D., n=3) and p values are indicated.

(e) Immortalized MEFs and mouse mammary epithelial cells (X3) carrying two inactive X chromosomes (2 Xi) were analyzed by RNA FISH - immunostaining for Telomeric RNA (red), Xist RNA (blue) and TRF1 (green);

DNA was stained with DAPI (white). Scale bars represent 5 μm. (V) Percentage (mean ± S. E., n=3) of Xist signals that are associated with accumulation of TeIRNAs (left graph) and of those aggregates that co-localize with TRF1 (right graph) in MEFs.

Figure 4: Epigenetic status of mammalian telomeres influences telomeric transcription

Total RNA was prepared from wild type cells and cells lacking major chromatin regulator and subjected to RNA dot blot analyses using ³²P-end labelled (CCCTAA)₄ or (TTAGGG)₄ oligonucleotide probes to detect r[UUAGGG]_n and r[CCCUAA]_n transcripts, respectively. Average values and s.d. were obtained from 2 independent oligonucleotide hybridisations.

(a) Mouse cells deficient for Suv39h or Suv4-20h HMTases display elevated levels of TeIRNAs presumably due to a more open telomeric chromatin structure.

(b) Mouse ES cells deficient for DNMT1, DNMT3a,b or Dicer with highly compact chromatin structure display reduced levels of r[UUAGGG]_n RNA.

Figure 5: r(UUAGGG)₃ RNA oligonucleotides block telomerase activity in vitro and are downregulated in advanced human cancer stages

Representative images of telomerase TRAP activity in mouse ES (a) and HeLa cells (b). To test a possible regulation of telomerase activity by UUAGGG repeat containing TeIRNAs, protein extracts were incubated with increasing amounts of (UUAGGG)₃ or (CCCUAA)₃ RNA oligonucleotides prior to the telomerase extension reaction. Controls were treated with RNase A to degrade Terc RNA. An internal control (IC) for PCR efficiency is also shown. A model for the function relevance of TeIRNAs for the regulation of mammalian telomeres is also shown (c). (d) Total RNA was prepared from different stages of the indicated human cancers using ³²P-end labelled (CCCTAA)₄ or (TTAGGG)₄ oligonucleotide probes to detect r[UUAGGG]_n and T[CCCUAA]_n transcripts, respectively. Note decreased abundance of TeIRNAs in advanced tumor stages. L, low grade tumor; H, high grade tumor.

Figure 6: Specificity of oligonucleotides

Total RNA prepared from mouse embryonic stem cells was used for northern analyses using [³²P]-labelled oligonucleotide probes that carry mutations of the original telomeric sequence. We found that only labelled (CCCTAA)₄ gives a specific signal.

Figure 7: Telomere transcription is conserved in distant vertebrate species such as zebra fish Total RNA prepared from iMEF and different developmental stages of zebra fish (Danio rerio) was subjected to Northern blotting and probed in parallel with ³²P-end labelled (CCCTAA)₄ or (TTAGGG)₄ oligonucleotide probes to detect T[UUAGGG]_n and r[CCCUAA]_n transcripts, respectively. In adult tissues the telomeric C-rich strand serves as a template for transcription. Total RNA was stained with ethidium bromide (EtBr) after agarose gel eletrophoresis to control for loading and RNA integrity. Full size Northern blots are shown.

Figure 8: Telomere length correlates with TeIRNA levels in human and mouse cell lines.

(a) Average telomere length of cell lines was determined by telomere restriction fragment (TRF) analysis. Terminal telomeric fragments were detected using a telomere specific ³²P-labelled probe. For additional information the original TRF without enhancement of telomere signals of human cell lines is provided in Figure 10(e).

(b) Total RNA was used to carry out RNA dot blot analysis to detect telomeric transcripts using ³²P-end labelled (CCCTAA)₄ or (TTAGGG)₄ oligonucleotide probes. RNase A pre-treatment greatly reduces the amount of (UUAGGG)_n repeat containing transcript. Gapdh hybridization is shown as a control. (c) Quantification of telomeric transcripts in human and mouse cell lines.

Absolute levels of (UUAGGG)_n RNA was determined from 2 independent hybridizations using oligonucleotide probes and a [³²P]-dCTP labelled 1 ,6kb telomeric probe to obtain the shown average values. Expression levels in NS1 cells was set "100".

(d) TeIRNA levels correlate with telomere length. A correlation between average telomere length and absolute transcript levels was determined by linear regression analysis. This correlation was highly significant when NS1 cells were removed from the analysis (right graph), probably due to the unusually short telomeres of this mouse cell line.

Figure 9: Reduced TeIRNA levels in T ere^1' mouse embryonic fibroblasts. Total RNA was prepared from immortalized mouse embryonic fibroblasts

(MEFs) derived from wild-type, second generation (F2) and forth generation (F4) and sixth generation (F6) telomerase-null mice (a) and primary MEFs prepared from wild type, first generation (F1 ) second generation (F2) and third generation (F3) of telomerase knock out mice (b). RNA was subjected to RNA dot blotting and probed with ³²P-end labelled (CCCTAA)₄ or

(TTAGGG)₄ oligonucleotide probes. In the absence of telomerase activity TeIRNA levels are reduced.

Figure 10: Full size images of Northern blots, Western blot and telomere restriction fragment (TRF) analysis.

(a) As additional information to Figure 1(a), the full size Northern blots monitoring expression levels of r[UUAGGG]_n, r[CCCUAA]_n, major satellite and gapdh RNAs in mouse embryos and mouse adult tissue are shown.

(b) As additional information to Figure 1(b), the full size Northern blots for TeIRNA (left panel) and gapdh (right panel) expression levels in mouse and human cell lines are shown. When indicated, total RNA was treated with RNase One, or the polyA RNA fraction was loaded on the gel.

(c) Additional information to Figure 2(e). Full-size Western blot of Figure showing reduced levels of TRF1 (left panel) in C2C12 cells transiently transfected with siRNAs against TRF1. Control siRNAs do not affect TRF1 protein levels. As loading control actin levels where monitored (right panel).

(d) Additional information to Figure 2(f). Full size Northern blot showing reduced TeIRNA levels upon TRF1 knockdown (siRNA TRF1 ) compared to control cells (siRNA control). As loading control gapdh RNA levels were determined (right panel). When indicated, total RNA was treated with RNase A.

(e) Additional information to Figure 8. Average telomere length of cell lines was determined by telomere restriction fragment (TRF) analysis. Terminal telomeric fragments were detected using a telomere specific ³²P-labelled probe. The original TRF without enhancement of telomere signals of human cell lines is shown. C2C12 cells where not considered in Figure 8.

EXAMPLE 1 - Experimental results

Overview of experiments

Mammalian telomeres consist of non-coding TTAGGG repeats that are bound by the multi-protein complex "shelterin", protecting chromosome ends from DNA repair mechanisms and degradation (1 ). Mammalian telomeric chromatin is enriched for the constitutive heterochromatin marks H3K9me3, H4K20me3 and HP1(2-7). Similar to pericentric heterochromatin, telomeric heterochromatin is thought to be fundamental for the maintenance of chromosomal integrity (8).

Here we report on the finding that telomeric repeats are transcribed by DNA dependent RNA polymerase II, which in turn interacts with the TRF1 shelterin protein. Telomere transcripts in human cells have also been reported by Azzalin et al. (28). Telomeric RNAs (TeIRNAs) contain UUAGGG repeats, are polyadenylated and transcribed from the telomeric C-rich strand. Transcription of mammalian telomeres is regulated by several mechanisms including developmental status, telomere length, cellular stress and chromatin structure. Using RNA-FISH we show that TeIRNAs are novel structural components of the telomeric chromatin. Importantly, we provide evidence that TeIRNAs block the activity of telomerase in vitro suggesting that TeIRNAs may regulate telomerase activity at chromosome ends. Our results indicate that TeIRNAs are novel components of mammalian telomeres, which are anticipated to be fundamental for understanding telomere biology and telomere-related diseases such as cancer and ageing.

Methods

Cells

Wild-type, G2 and G5 telomerase-null MEFs were of a mixed genetic background (129/SV and C57BL/6J) (15). MEFs were obtained from wild-type and 7erc-null embryos at embryonic day 13.5 as described (15). Mouse C2C12 cells were used for small interference experiments. Cell lines were cultivated under standard conditions. Cells deficient for Suv39h HMTases(29), Suv4-20h HMTases (7), Dicer (30), DNMT1 (31 ), DNM3a,b (32) were described elsewhere. To block transcription, cells were treated with 5 μg/ml Actinomycin D (Sigma) or 10 μg/ml α-amanitin (Sigma).

TRF analysis We prepared cells in agarose plugs and carried out TRF analysis as described previously (15).

RNA analysis

Northern analysis was performed using different amounts of RNA (Trizol; Invitrogen) as described previously (33). Total RNA from Danio rerio was obtained from M. L. Cayuela (Hospital Virgen de Ia Arrixaca, Murcia, Spain). PoIy-A RNA was prepared according to the manufacturers protocol (Invitrogen). For RNA dot blotting experiments RNA was transferred to a nitrocellulose membrane and probed with oligonucleotides end-labelled with Y³²P-ATP using T4 Polynucleotide kinase (New England Biolabs) or DNA probes labelled by random priming (Rediprime, GE healthcare). Oligo hybridisations were carried out using UltraHyb (Ambion) according to the manufacturer's suggestions. When indicated RNA samples where treated with RNaseA (Roche) or RNase One (Promega). Polyadenylated RNA was prepared using Oligotex beads (Qiagen).

Immunostaininqs and RNA FISH

Cells were attached to poly-l-lysine coated coverslips. Cell were permeabilized and fixed for 10 min at RT in 4% PFA in PBS. After 5 min at RT in 0.1% Na Citrate/0.1% Triton X- 100 cells were blocked for 60 min at RT in PBS containing 5% (wt/vol) BSA, 0.1 % Tween-20. Antibodies against mouse TRF1 (raised in our laboratory (6)) or against RNA Polymerase Il (Abeam, ab5408) where incubated 60 min at RT. Alexa Fluor 488 goat anti-rabbit (Invitrogen) or Alexa 680 goat anti-rabbit antibodies (Invitrogen) were used as secondary antibodies. PBS containing 5% (wt/vol) BSA, 0,1% Tween was used as washing solution. For combined TRF1-immunostaining/TelRNA FISH experiments, cells were fixed at RT for 10 min with 4% PFA after the washing step of the secondary antibody. Samples were dehydration and RNA FISH analysis was carried out as described (33). RNA FISH probes were generated by random priming (GE healthcare) using Cy3-dCTP (Amersham).

Telomere repeat amplification protocol (TRAP) Telomerase activity was measured with a modified telomere repeat amplification protocol (TRAP) as described (3). 1 μg of whole cell extract of HeIa and mouse ES cells was incubated with (UUAGGG)₃ or (CCCUAA)₃ RNA oligonucleotides for 5 min. on ice and 5 min at RT before starting the telomerase extension reaction. To degrade spiked RNA oligonucleotides prior to PCR amplification, samples where treated with 10 pg RNase A (Qiagen) at RT for 20 min. An internal control for PCR efficiency (IC) was included (TRAPeze kit Oncor, Gaithersburg, MD) (15).

Chromatin immunoprecipitation

Chromatin immunoprecipitation using anti-mouse TRF1 (6) (generated in our laboratory against the full-length mouse TRF1 protein), anti-human TRF2 (Upstate,05-521 ), trimethylated H3K9 (Upstate, 07-442) and RNA Pol Il antibodies (Abeam, ab5408) were carried out as described (6). Chromatin from mouse cells was immunoprecipitated with anti-mouse TRF1 (6) and anti-mouse TRF2 antibodies. Chromatin from human cells was immunoprecipitated with anti mouse TRF1 (6) and anti human TRF2 antibodies (Upstatβ.05-521 ).

lmmunoprecipitations lmmunoprecipitations using TRF1 (6) and RNA Pol Il antibodies (Abeam, ab5408) were carried out as previously described (6).

Small interference RNA siRNA oligonucleotides against mouse TRF1 (ON-TARGETplus smart pool, Dharmacon) and control oligonucleotides (ON-TARGETplus siControl, Dharmacon); were transiently transfected into mouse C2C12 cells using Dharmafect (Dharmacon according to the manufacturer's suggestions. 60 hrs post-transfection cells RNA, protein and material for chromatin immunoprecipitation was prepared.

Human tumor samples

Human tumor samples were provided by the Spanish National Tumour Bank Network coordinated by the Spanish National Cancer Centre (CNIO). Samples were collected following the technical and ethical procedures of the Network (http://www.cnio.es/ing/programas/progTumor01.asp), including anonimization processes.

Results and discussion

Chromatin at telomeric repeats shares several features of pericentric heterochromatin, such as the ability to silence nearby genes resulting in a phenomenon called telomere position effect (TPE) (9-13). Recent studies have provided new insights into the mechanisms of heterocriromatin formation, demonstrating a fundamental role of non- coding RNAs in the formation of heterochromatin in cis. This includes mechanisms like dosage compensation in mammals, imprinting, and RNAi mediated formation of heterochromatin (14).

In order to demonstrate a possible role for long non-coding RNAs in the regulation of telomeric heterochromatin, we carried out Northern blotting analysis of total RNA from mouse embryos and adult tissues using radiolabeled oligonucleotide probes specific for the C- or the G-rich telomeric strand. We were able to detect TeIRNAs consisting of UUAGGG repeats in all mouse adult tissues tested, with highest levels in thymus, kidney and spleen, while TeIRNAs were undetectable in mouse embryos (Fig. 1a). Telomeric transcripts consisting of CCCUAA repeats were expressed only at low or undetectable levels in all samples tested. This indicates that, in mice, the telomeric C-strand serves as a template for transcription giving rise to abundant transcripts containing UUAGGG repeats in a developmentally regulated manner (Fig. 1a). The specificity of the signals detected in the Northern experiments was tested using mutant oligonucleotide probes (Fig. 6), although we cannot rule out that a few RNAs containing the UUAGGG sequence may give some background hybridization signal. Interestingly, TeIRNAs were also detected in a distant vertebrate species such as Zebra fish (Danio renio; Fig. 7), suggesting that telomere transcription is a conserved feature of vertebrate telomeres.

In order to further characterize TeIRNAs, we carried out Northern blot (Fig. 16) and RNA dot blot (Fig. 8) analyses using total RNA from 4 mouse and 4 human cell lines that differ in the length of telomeric repeats from approximately 50 kb in mouse embryonic stem (ES) cells to 4 kb in human HeLa cells, as determined by telomeric restriction fragment analysis (Fig. 8a). The tested cell lines displayed variable levels of r[UUAGGG]n transcripts, highest in mouse ES cells and NS1 cells (Fig. 1b; Fig. Sb,c). In general, TeIRNAs were presented at lower levels in human compared to mouse cells (Fig. 1b; Fig. 8b, c). We found that telomere length and TeIRNA levels show a positive correlation, in a manner that cells having long telomeres display higher levels of r[UUAGGG)]n transcripts than cells with short telomeres (Fig. 8d). Northern blotting showed that TeIRNAs are heterogeneous in length reaching a maximum size of less than 6 kb both in human and mouse cells (Fig. 1b). The fact that length of TeIRNAs in human cell lines with short telomeres was similar to that in mouse cells indicates that the length of telomeric repeats does not determine the length of TeIRNAs. In contrast to the gapdh RNA, TeIRNAs were rapidly degraded with a half-life of approximately 2.2 hours when transcription was blocked by Actinomycin D (Fig. 1c). This suggests that the broad range of TeIRNAs observed in Northern blotting experiments may be due to degradation of telomeric RNAs. Finally, we found that r(UUAGGG)n TeIRNAs were highly enriched in the nuclear RNA fraction (Fig. 1cQ. As control, RNAs transcribed from the Gapdh housekeeping gene and major satellite repeats were localized to the cytoplasm and nucleoplasm, respectively (Fig. 1c/).

To further investigate the influence of telomere length on TeIRNA levels, we determined TeIRNA levels in immortalized (iMEFs) and primary mouse embryonic fibroblasts lacking telomerase activity (7erc^v')(15). We observed reduced levels of telomeric transcripts in both iMEFs and primary MEFs lacking Terc (Fig. 9). Although telomeres progressively shorten with increasing Terc'^' generations (G 1 to G6) (15), TeIRNA levels did not show a further decrease when comparing early to late generation cells (Fig. 9a,/?). This suggests that, beside the telomere length, other mechanisms can impact on the regulation of telomeric transcription. One of such mechanisms could be the activation of alternative pathways of telomere elongation (ALT) in late generation Terc-deficient cells (6), which in turn may influence telomere transcription.

Previous reports suggest that various long non-coding RNAs, involved in epigenetic gene regulation are polyadenylated at their 3^'-end and transcribed by DNA-dependent RNA Polymerase Il (RNA Pol II) (16). Consistent with this, we were able to enrich 8- to 10-fold for telomeric r(UUAGGG)n transcripts in purified polyadenylated RNA compared to total RNA (Fig. 2a). Moreover, we were able to detect TeIRNAs by Northern blotting using the polyA-enriched RNA fraction confirming the broad size range of telomeric transcripts going from 1 kb to less than 6 kb (Fig. 1b). As expected, the RNA encoded by the housekeeping gene Gapdh was also enriched in the poly-A RNA fraction; no telomeric r(CCCUAA)_n RNA was detectable in the polyadenylated RNA fraction (Fig. 2a). In support for an involvement of RNA Pol Il in telomere transcription, TeIRNAs levels were decreased in human U2OS cells and mouse ES cells upon treatment with the RNA Polymerase ll-specific inhibitor α-amanitin (Fig. 2b). As expected, α-amanitin treatment did not inhibit RNA Pol l-dependent transcription of the ribosomal 28S.

To further demonstrate that RNA Pol Il is responsible for transcription of telomeric DNA we carried out chromatin immunoprecipitations (ChIP) using anti-RNA Polymerase Il antibodies. We found that RNA Pol Il is associated to mammalian telomeres in both, mouse (skin keratinocytes, ES cells, and mammary epithelial X3 cells) and in human cells (HeLa and Helal.2.11 ), but also to pericentric repeats (major satellite repeats and alpha-satellite repeats in mice and humans, respectively), which where already reported to be transcribed by RNA Polymerase Il (17-19) (Fig. 2c). As expected, the TRF1 and TRF2 shelterin proteins were found to be associated to telomeric repeats but not to pericentric chromatin domains both in mouse and human cells (Fig. 2c).

In order to test whether shelterin proteins are able to interact with RNA Pol Il we carried out immunoprecipitation experiments with monoclonal antibodies raised against the CTD domain of RNA Pol II, using whole cell extracts prepared form a panel of mouse and human cells. RNA Pol Il antibodies are able to co-immunoprecipitate TRF1 in all mouse and human cells tested (Fig 2d), suggesting an interaction between RNA Pol Il and TRF1. Furthermore, this interaction seems to be important for telomere transcription, as the abundance of TeIRNAs was significantly decreased in cells where TRF1 had been knocked-down by siRNA (Figs. 2e,f). TRF1 , however, is not required to recruit RNA Pol Il to telomeres as indicated by the fact that TRF1 knocked down cells did not show decreased RNA Pol I! abundance at telomeres (Fig. 2g). All together, these results suggest a model in which a TRF1 containing "shelterin" complex may facilitate transcription of mammalian telomeric repeats in agreement with a role for TRF1 in alleviating TPE at telomeres (13).

A striking feature of some non-coding RNAs is their capacity to localize to chromatin, thereby controlling epigenetic gene regulation in cis (14,16). In mammals the non-coding Xist RNA spreads along one of the two female X chromosomes recruiting a machinery of heterochromatic proteins including Polycomb protein complexes, mediating silencing of one of the two female X chromosomes(20). In contrast to mammals, dosage compensation in Drosophila melanogaster is dependent on rox RNAs expressed from the male X chromosome, attaching to the single male X chromosome, triggering histone H4 hyperacetylation in cis (21-22).

To investigate a possible interaction of TeIRNAs with mammalian telomeric chromatin we have carried out RNA fluorescent hybridization (RNA-FISH) experiments in mouse and human cell using RNA-FISH probes consisting of Cy3 labelled CCCTAA repeats. We found that TeIRNAs were restricted to the nucleus and co-localized with TRF1 in a panel of mouse and human cells (Fig. 3a,b). This indicates that TeIRNAs are a common structural component of mammalian telomeres. Importantly, treatment with DNase free RNase before fixation dramatically reduced the RNA FISH signals without affecting TRF1 localization. This confirms that the observed signals are specific for TeIRNAs and not due to hybridisation of the RNA FISH probe into telomeric DNA sequences. Recently, it was demonstrated that pericentric heterochromatic acquire euchromatic features and give rise to polyadenylated RNAs containing Sat III repeats in response to thermal stress (17-19). To analyse whether heat shock modulates transcription at mammalian telomeres we performed TeIRNA - FISH experiments in immortalized MEFs subjected to heat shock. Incubation of cells at 42⁰C for one hour results in a highly significant increase in the number of TeIRNA foci per nucleus compared to cells grown at 37⁰C (Fig. 3c,d). When cells were shifted back to normal conditions after heat shock the number of TeIRNA foci was decreasing to normal levels, indicating that heat shock induced telomeric transcription is reversible (Fig. 3c,d). Based on the observation that transcription at mammalian telomeres can be modulated by exogenous stimuli we conclude that the generation of TeIRNA underlay tight transcriptional regulation.

Along the examination of TeIRNA localization patterns in different cell lines it became clear that, beside the typical focal co-localization pattern with TRF1 , TeIRNAs can occupy large subnuclear domains, highly reminiscent of the localization-pattern of the non-coding Xist RNA (23). This prompted us to carry out TeIRNA FISH studies combined with a RNA-FISH staining for mouse Xist RNA and immunostainings for TRF1 in iMEFs and in a mouse mammary gland epithelial cell line carrying two inactive X chromosomes (X3 cells). As expected, TRF1 shows focal co-localization with TeIRNA in both cell types (Fig. 3e,f). Importantly, we observed massive accumulation of TeIRNAs in the vicinity of, or partially overlapping with, the territory of the inactive X chromosome decorated by the Xist RNA (Fig. 3e,f). This observation opens the intriguing possibility that non-coding TeIRNAs may occupy large chromatin domains when they have access to a favourable chromatin environment.

Mammalian telomeres are enriched for the heterochromatic marks H3K9me3, H4K20me3 and HP1(2-8). Moreover, the DNA of mammalian subtelomeres is highly methylated (7). Loss of telomeric and subtelomeric heterochromatin causes increased frequency of telomere recombination (7) and is thought to result in elongated telomeres in cells deficient for the Suv39h, Suv4-20h HMTases, DNMT1, DNMT3a/b or Dicer activities (2-8,24). Consistent with a model of chromatin structure and transcription, we found increased levels of TeIRNAs in cells deficient for the Suv39h and Suv4-20h HMTases (Fig. 4a) that are characterized by a more "open" telomeric chromatin structure with decreased abundance of H3K9me3 and H4K20me3 histone trimethylation marks (5,7). In contrast, TeIRNA levels were slightly reduced in cells lacking DNMTase or Dicer activities (Fig. 4b) concomitant with an increased density of histone trimethylation marks in these cells (2,8,24). This indicates that the chromatin status at mammalian telomeres influences TeIRNA levels, which may further impact on telomere function.

Telomere length is maintained by the telomerase, a reverse transcriptase that adds telomeric repeats de novo to chromosome ends when expressed (25). Telomerase consists of a protein component known as Tert and of an RNA component or Terc, which contains the template for reverse transcription (25). Importantly, UUAGGG repeats present in TeIRNAs are complementary to the template sequence within Terc, opening the possibility that these TeIRNAs could inhibit telomerase activity by blocking the Terc template region. To test this possibility, protein extracts were pre-incubated with increasing amounts of (UUAGGG)₃ or (CCCUAA)₃ RNA oligonucleotides and subsequently subjected to a telomere repeat amplification protocol (TRAP) (3). Telomerase activity was completely abolished in mouse ES cells and human HeLa cells upon the addition of 2 pmol of (UUAGGG)₃ RNA oligonucleotides (Fig. 5a, b). In contrast, telomerase activity was not inhibited to the same extent when cell extracts were incubated with (CCCUAA)₃ RNA oligonucleotides. This suggests a speculative model in which TeIRNAs could potentially inhibit telomerase activity at telomeres, presumably by RNA duplex formation with the template region of Terc (Fig. 5c).

Interestingly, preliminary data suggests that TeIRNAs are significantly down-regulated in advanced stages of different types of human cancer compared to normal tissues (Fig. 5d), suggesting downregulation of these RNAs associated to undifferentiated cell stages (see also Fig. 1a showing that TeIRNAs are downregulated during embryonic development). All together, these results support the notion that TeIRNAs are tightly regulated by the cell. In addition, lower TeIRNA production associated to carcinogenesis may favour telomerase-mediated telomere elongation in cancer cells.

In summary, here we report on the discovery of transcription of telomeric RNAs (TeIRNAs) from mammalian telomeres. We show that telomeres are transcribed by the DNA-dependent RNA Polymerase Il giving rise to UUAGGG repeat containing telomeric transcripts with a maximum size of less than 6 kb. Interaction between TRF1 and RNA Pol II, together with the fact that TRF1 inhibition results in decreased TeIRNA abundance, suggest that a TRF1 -containing shelterin complex facilitates transcription through telomeric DNA repeats. TeIRNAs localize to telomeric chromatin, but also may accumulate in larger chromatin domains when expressed in silenced nuclear regions such as those occupied by the inactive X chromosome. Importantly we demonstrate for the first time that (UUAGGG)₃ RNA oligonucleotides can inhibit telomerase activity in vitro.

All together, these results suggest a speculative model where high amounts of TeIRNAs associated with long telomeres could locally inhibit telomerase activity by forming a RNA duplex with the template region of Terc, the RNA component of the telomerase (Fig. 5c). This model could explain the observation that shorter telomeres presenting TeIRNA at lower abundance are preferentially elongated by telomerase compared to longer telomeres expressing higher levels of TeIRNAs within the same cell (26). Furthermore, our observation that TRF1 facilitates TeIRNA production, which in turn could inhibit telomerase, may also explain the known role of TRF1 as a negative regulator of telomerase-dependent telomere elongation²⁷. In addition, consistent with the role of other long non-coding RNAs in important processes like dosage compensation and imprinting, TeIRNAs could also be essential for the recruitment of "heterochromatinizing" activities to the telomeres in cis (14) (Fig. 5c). This is supported by the fact that changes in the structure of telomeric heterochromatin due to abrogation of major chromatin regulators result in changes in the levels TeIRNAs. Telomeric heterochromatin could stabilize TeIRNAs at telomeres thereby contributing to the local control of telomerase activity.

Finally, it is foreseeable that the discovery of TeIRNAs will provide deeper insights into telomere function and foster our understanding of the role of telomere in fundamental processes like cancer and ageing.

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15. Blasco, M.A. et al. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell 91 , 25-34 (1997).

16. Mattick, J.S. & Makunin, I.V. Non-coding RNA. Hum. MoI. Genet. 15, R17-29 (2006).

17. Valgardsdottir, R. et al. Structural and functional characterization of noncoding repetitive RNAs transcribed in stressed human cells. MoI. Biol. Cell 16, 2597- 2604 (2005).

18. Rizzi, N. et al. Transcriptional activation of a constitutive heterochromatic domain of the human genome in response to heat shock. MoI. Biol. Cell 15, 543-551 (2004).

19. Jolly, C et al. Stress-induced transcription of satellite III repeats. J. Cell Biol. 164, 25-33 (2004).

20. Masui, O. & Heard, E. RNA and protein actors in X-chromosome inactivation. Cold Spring Harb Symp duant Biol 71 , 419-428 (2006). 21. Franke, A. & Baker, B. S. The rox1 and rox2 RNAs are essential components of the compensasome, which mediates dosage compensation in Drosophila. MoI. Cell 4, 117-122 (1999).

22. Kelley, R.L. et al. Epigenetic spreading of the Drosophila dosage compensation complex from roX RNA genes into flanking chromatin. Ce// 98, 513-522 (1999).

23. Cohen, H. R. & Panning, B. XIST RNA exhibits nuclear retention and exhibits reduced association with the export factor TAP/NXF1. Chromosoma 116, 373- 383 (2007).

24. Benetti, R. et al. Control of mammalian telomere length and telomere recombination by the Dicer-dependent miRNA network, submitted for publication

(2007).

25. Chan, S.W. & Blackburn, E.H. New ways not to make ends meet: telomerase, DNA damage proteins and heterochromatin. Oncogene 21 , 553-563 (2002).

26. Teixeira, M.T., Arneric, M., Sperisen, P. & Lingner, J. Telomere length homeostasis is achieved via a switch between telomerase- extendible and - nonextendible states. Ce// 117, 323-335 (2004).

27. Ancelin et al. Targeting assay to study the cis functions of human telomeric proteins: evidence for inhibition of telomerase by TRF1 and for activation of telomere degradation by TRF2. MoI Cell Biol. 22, 3474-3487 (2002). 28. Azzalin, CM., Reichenbach, P., Khoriauli, L, Giulotto, E., Lingner, J. Telomeric repeat containing RNA and RNA surveillance factors at mammalian chromosome ends. Science 318, 798-801 (2007). 29. Peters, A.H. et al. Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability. Ce// 107, 323-337 (2001). 30. Murchison, E.P., Partridge, J. F., Tarn, O. H., Cheloufi, S. & Hannon, GJ.

Characterization of Dicer-deficient murine embryonic stem cells. Proc. Natl. Acad.

ScL USA 102, 12135-12140 (2005). 31. Li, E., Bestor, T.H. & Jaenisch, R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69, 915-926 (1992). 32. Okano, M., Bell, D.W., Haber, D.A. & Li, E. DNA methyltransferases Dnmt3a and

Dnmt3b are essential for de novo methylation and mammalian development. Ce//

99, 247-257 (1999). 33. Wutz, A. & Jaenisch, R. A shift from reversible to irreversible X inactivation is triggered during ES cell differentiation. MoI. Cell 5, 695-705 (2000). EXAMPLE 2 - Exemplary pharmaceutical formulations

Whilst it is possible for an agent or polynucleotide of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. The carrier(s) must be "acceptable" in the sense of being compatible with the agent of the invention and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen-free.

The following examples illustrate medicaments and pharmaceutical compositions according to the invention in which the active ingredient is an agent or polynucleotide of the invention.

Preferably, the agent or polynucleotide of the invention is provided in an amount from 1 pg to 1g administered unit, depending on the patient, kind of disease and method of administration.

It will be appreciated that the following exemplary medicaments and pharmaceutical compositions may be prepared containing an amount of the agent or polynucleotide of the invention from 1 pg to 1g per administered unit depending on the patient, kind of disease and method of administration. For example, the agent or polynucleotide of the invention may be present in a 10^th or 100^th or 200^th or 500^th of the amount shown in the following exemplary medicaments and pharmaceutical compositions with the amounts of the remaining ingredients changed accordingly.

The agent or polynucleotide of the invention can be formulated at various concentrations, depending on the efficacy/toxicity of the agent or polynucleotide being used and the indication for which it is being used. Preferably, the formulation comprises the agent or polynucleotide of the invention at a concentration of between 0.1 μM and 1 mM or higher, depending on the patient, kind of disease and method of administration.

Example A: Tablet

Active ingredient 1 mg

Lactose 200 mg

Starch 50 mg

Polyvinylpyrrolidone 5 mg

Magnesium stearate 4 mg Tablets are prepared from the foregoing ingredients by wet granulation followed by compression.

Example B: Ophthalmic Solution

Active ingredient 1 mg

Sodium chloride, analytical grade 0.9 g

Thiomersal 0.001 g

Purified water to 100 ml pH adjusted to 7.5

Example C: Tablet Formulations

The following formulations A and B are prepared by wet granulation of the ingredients with a solution of povidone, followed by addition of magnesium stearate and compression.

Formulation A mq/tablet mq/tablet

(a) Active ingredient 1 1

(b) Lactose BP. 210 26

(c) Povidone B. P. 15 9

(d) Sodium Starch Glycolate 20 12

(e) Magnesium Stearate 5 3

251 51

Formulation B mq/tablet mq/tablet

(a) Active ingredient 1 1

(b) Lactose 150 -

(c) Avicel PH 101^® 60 26

(d) Povidone B. P. 15 9

(e) Sodium Starch Glycolate 20 12

(f) Magnesium Stearate 5 3

251 51 Formulation C mq/tablet

Active ingredient 1

Lactose 200 Starch 50

Povidone 5

Magnesium stearate 4

. 260

The following formulations, D and E, are prepared by direct compression of the admixed ingredients. The lactose used in formulation E is of the direction compression type.

Formulation D mq/capsule

Active Ingredient 1

Pregelatinised Starch NF15 150

151

Formulation E mq/capsule

Active Ingredient 1 Lactose 150

Avicel ^® 100

251

Formulation F (Controlled Release Formulation)

The formulation is prepared by wet granulation of the ingredients (below) with a solution of povidone followed by the addition of magnesium stearate and compression. mq/tablet

(a) Active Ingredient 1

(b) Hydroxypropylmethylcellulose 112

(Methocel K4M Premium)^®

(c) Lactose BP. 53

(d) Povidone B.P.C. 28

(e) Magnesium Stearate 7

201

Drug release takes place over a period of about 6-8 hours and was complete after 12 hours.

Example D: Capsule Formulations

Formulation A

A capsule formulation is prepared by admixing the ingredients of Formulation D in Example C above and filling into a two-part hard gelatin capsule. Formulation B (infra) is prepared in a similar manner.

Formulation B mq/capsule

(a) Active ingredient 1

(b) Lactose B. P. 143

(c) Sodium Starch Glycolate 25 (d) Magnesium Stearate 2

171

Formulation C mg/capsule

(a) Active ingredient 1

(b) Macrogol 4000 BP 350

351

Capsules are prepared by melting the Macrogol 4000 BP, dispersing the active ingredient in the melt and filling the melt into a two-part hard gelatin capsule. Formulation D mq/capsule

Active ingredient 1

Lecithin 100 Arachis Oil 100

201

Capsules are prepared by dispersing the active ingredient in the lecithin and arachis oil and filling the dispersion into soft, elastic gelatin capsules.

Formulation E (Controlled Release Capsule)

The following controlled release capsule formulation is prepared by extruding ingredients a, b, and c using an extruder, followed by spheronisation of the extrudate and drying. The dried pellets are then coated with release-controlling membrane (d) and filled into a two- piece, hard gelatin capsule.

mα/capsule

(a) Active ingredient 1

(b) Microcrystalline Cellulose 125

(c) Lactose BP 125

(d) Ethyl Cellulose 13

264

Example E: Injectable Formulation

Active ingredient 1 mg

Sterile, pyrogen free phosphate buffer (pH7.0) to 10 ml

The active ingredient is dissolved in most of the phosphate buffer (35-40^*C), then made up to volume and filtered through a sterile micropore filter into a sterile 10 ml amber glass vial (type 1 ) and sealed with sterile closures and overseals. Example F: Intramuscular injection

Active ingredient 1 mg

Benzyl Alcohol 0.10 g Glucofurol 75^® 1.45 g

Water for Injection q.s. to 3.00 ml

The active ingredient is dissolved in the glycofurol. The benzyl alcohol is then added and dissolved, and water added to 3 ml. The mixture is then filtered through a sterile micropore filter and sealed in sterile 3 ml glass vials (type 1 ).

Example G: Syrup Suspension

Active ingredient 1mg

Sorbitol Solution 1.500O g

Glycerol 2.0000 g

Dispersible Cellulose 0.0750 g

Sodium Benzoate 0.0050 g

Flavour, Peach 17.42.3169 0.0125 ml

Purified Water q.s. to 5.0000 ml

The sodium benzoate is dissolved in a portion of the purified water and the sorbitol solution added. The active ingredient is added and dispersed. In the glycerol is dispersed the thickener (dispersible cellulose). The two dispersions are mixed and made up to the required volume with the purified water. Further thickening is achieved as required by extra shearing of the suspension.

Example H: Suppository mq/suppository Active ingredient (63 μm)^* 1

Hard Fat, BP (Witepsol H15 - Dynamit Nobel) 1770

1771

^*The active ingredient is used as a powder wherein at least 90% of the particles are of 63 μm diameter or less. One fifth of the Witepsol H15 is melted in a steam-jacketed pan at 45^°C maximum. The active ingredient is sifted through a 200 μm sieve and added to the molten base with mixing, using a silverson fitted with a cutting head, until a smooth dispersion is achieved. Maintaining the mixture at 45"C, the remaining Witepsol H15 is added to the suspension and stirred to ensure a homogenous mix. The entire suspension is passed through a 250 μm stainless steel screen and, with continuous stirring, is allowed to cool to 4O⁰C. At a temperature of 38⁰C to 40^°C 2.02 g of the mixture is filled into suitable plastic moulds. The suppositories are allowed to cool to room temperature.

Example I: Pessaries mq/pessary

Active ingredient 1

Anhydrate Dextrose 380

Potato Starch 363 Magnesium Stearate 7

751

The above ingredients are mixed directly and pessaries prepared by direct compression of the resulting mixture.

EXAMPLE 3 - Treatment of a proliferative disorder using an agent of the invention

A patient with breast cancer is administered a formulation comprising 0.1 μM and 1 mM of the therapeutic agent of the invention having a polynucleotide sequence (UUAGGG)₄ by daily injection to the tumour of the affected area. Administration is repeated daily for a period of two weeks and the results of treatment determined.

EXAMPLE 4 - Detection of a proliferative disorder using an diagnostic agent of the invention

The diagnostic agent of the invention may be used to detect a proliferative disorder in the following manner:

1. Preparation of tissue or cells from patient using standard procedures known in the art. The preparation method will depend on the type of cell to be tested and the associated disease or disorder, and the sample type in which the cell or tissue is provided (for example blood or tissue biopsy).

2. Preparation of total RNA from the tissue or cells using standard procedures. For example, lyse cells (for example, using Tizol from Invitrogen), followed by the addition of 20% Chloroform (of lysate volume) and vigorous mixing. Following centrifugation for 20 min at 14.000 rpm at

4⁰C, transfer the transparent supernatant to a new tube, add an equal volume of isopropanol and put at -2O⁰C for 30 min to precipitate RNA; following centrifugation at 4⁰C for 30 min at 14000rpm, remove supernatant, wash RNA pellet with 70% ethanol and re-suspend the RNA pellet in RNase-free water.

3. Transfer total RNA to a nitrocellulose filter (i.e. by simple dot blotting), and cross-link RNA to the membrane by irradiation with U V-light.

4. Label the diagnostic agent or polynucleotide of the diagnostic agent of the invention (i.e. a DNA oligonucleotide comprising or consisting of the (CCCUAA)₄ sequence) with a detectable moiety. For example, labelling with gamma-³²P-ATP can be performed using T4 DNA polynucleotide kinase and standard procedures (T4 DNA polynucleotide kinase adds the radioactive ³²P moiety to the 5'-end of the oligonucleotide) and labelling with 16-biotin-dCTP can be performed by nick translation using standard procedures.

5. As control, a ³²P-labelled (TTAGGG)₄ oligonucleotide can be used - this oligonucleotide will not detect telomeric RNAs, but will however detect contaminating telomeric DNA repeats. 6. Pre-hybridise the membrane with an oligo-hybridisation solution (commercial, 42⁰C₁ 2 hrs).

7. Add the labelled probe to the membrane, and incubate under rotation, over-night (i.e. 16 hours) at 42⁰C.

8. Wash the membrane with: (i) a solution of 2xSSC, 0.1%SDS for 5 min 42⁰C; (ii) a solution of 0.2x SSC and 0.1 %SDS for 10 min at 42⁰C; and (iii) a solution of 0.1 x SSC and 0.1%SDS for 10 min at 42⁰C).

9. Detect the detectable moiety on the membrane using standard techniques (for example, by exposing the membrane over-night to X-Ray film and develop).

10. Re-hybridise the membrane with a radioactive probe against the 28S ribosomal RNA (generated using standard techniques, such as nick translation with alpha-³²P-dCTP); complete standard post-hybridisation washes, etc according to standard Northern blotting conditions.

11. Determine TeIRNA levels and normalize against 28S ribosomal RNA.

The result gives absolute TeIRNA levels normalized for the total RNA included in the measurement (28S) and levels of telomeric RNAs will identify proliferative disease, and also the tumour grade.

Claims

1. A diagnostic agent comprising a polynucleotide capable of hybridising to the sequence UUAGGG, or a fragment or variant thereof, which fragment or variant is capable of hybridising to the sequence UUAGGG.

2. The diagnostic agent according to Claim 1 wherein the polynucleotide is single- stranded.

3. The diagnostic agent according to Claim 1 or 2 wherein the polynucleotide comprises the sequence CCCTAA.

4. The diagnostic agent according to any of Claims 1 to 3 wherein the polynucleotide comprises the sequence (CCCTAA)₄.

5. The diagnostic agent according to any of Claims 1 to 4 comprising a detectable moiety.

6. The diagnostic agent according to Claim 5 wherein the detectable moiety is selected from the group consisting of: phosphorous-32 (³²P) and 16-biotin-dCTP.

7. A method for detecting the presence of a proliferative disorder in a test individual comprising the steps of:

(i) providing a test sample comprising one or more cell from an individual to be tested;

(ii) determining the amount and/or concentration in the test sample of a polyribonucleotide comprising or consisting of the sequence UUAGGG, or a fragment or variant thereof, which fragment or variant is capable of hybridising to the sequence UUAGGG;

(iii) detecting the presence of a proliferative disorder in the test individual if the amount and/or concentration of the polyribonucleotide in the test sample in step (ii) is lower than the amount and/or concentration of that polyribonucleotide in a control sample.

8. The method according to Claim 7 wherein the presence of a proliferative disorder is detected in the test individual if the amount and/or concentration of the polyribonucleotide in the test sample in step (ii) is equal to 60% or less than the amount and/or concentration of the test polyribonucleotide in the control sample.

9. The method according to Claim 8 wherein the presence of a proliferative disorder is detected in the test individual if the amount and/or concentration of the polyribonucleotide in the test sample in step (ii) is equal to 50% or less, or 40% or less, or 30% or less, or 20% or less, or 10% or less, than the amount and/or concentration of the test polyribonucleotide in the control sample.

10. The method according to any of Claims 7 to 9 wherein the amount and/or concentration of the polyribonucleotide comprising the sequence UUAGGG₁ or a fragment or variant thereof, which fragment or variant is capable of hybridising to the sequence UUAGGG, is detected using a diagnostic agent as defined in any of

Claims 1 to 9.

11. The method according to any of Claims 7 to 10 wherein the test individual is a mammal.

12. The method according to Claim 11 wherein the mammal is a human.

13. The method according to Claim 11 wherein the mammal is selected from the group comprising or consisting of: horse; pig; sheep; goat; dog; cat; rabbit; rat; mouse.

14. A therapeutic agent comprising a polyribonucleotide capable of inhibiting and/or reducing the activity of a telomerase.

15. The therapeutic agent according to Claim 14 wherein the polyribonucleotide is single-stranded.

16. The therapeutic agent according to Claim 14 or 15 wherein the polynucleotide is capable of hybridising to the Terc RNA component of a telomerase.

17. The therapeutic agent according to Claim 16 wherein the polyribonucleotide comprises the sequence UUAGGG, or a fragment or variant thereof, which fragment or variant is capable of hybridising to the sequence UUAGGG.

18. The therapeutic agent according to Claim 16 or 17 wherein the polyribonucleotide comprises two or more copies of the UUAGGG sequence, or a fragment or variant thereof, which fragment or variant is capable of hybridising to the sequence UUAGGG.

19. The therapeutic agent according to Claim 18 wherein the polyribonucleotide comprises 3 or more; 4 or more; 5 or more; 6 or more; 7 or more; 8 or more; 9 or more; 10 or more; 20 or more; or 50 or more copies of the UUAGGG sequence, or a fragment or variant thereof, which fragment or variant is capable of hybridising to the sequence UUAGGG.

20. The therapeutic agent according to Claim 18 wherein the polyribonucleotide comprises between 2-10; 10-20; 20-30; 30-40;or 40-50 copies of the UUAGGG sequence, or a fragment or variant thereof, which fragment or variant is capable of hybridising to the sequence UUAGGG.

21. The therapeutic agent according to any of Claims 18 to 20 wherein the two or more copies of the UUAGGG sequence, or fragment or variant thereof, are arranged in one or more tandem repeat.

22. The therapeutic agent according to any of Claims 14 to 21 wherein the reverse transcriptase activity of telomerase is inhibited and/or reduced.

23. The therapeutic agent according to any of Claims 14 to 22 further comprising a detectable moiety.

24. The therapeutic agent according to Claim 23 wherein the detectable moiety is a radioactive moiety.

25. A therapeutic agent as defined in any of Claims 14 to 24 for use in medicine.

26. An isolated or recombinant polyribonucleotide as defined in any of Claims 14 to ' 24.

27. A therapeutic agent as defined in Claim 25 or an isolated or recombinant polyribonucleotide as defined in Claim 26 for use in the treatment of a proliferative disorder.

28. Use of a therapeutic agent as defined in Claim 25 or an isolated or recombinant polyribonucleotide as defined in Claim 26 in the manufacture of a medicament for treating a proliferative disorder.

29. A pharmaceutical composition comprising an effective amount of a therapeutic agent as defined in Claim 25 or an effective amount of an isolated or recombinant polyribonucleotide as defined in Claim 26 and a pharmaceutically-acceptable excipient, diluent or carrier.

30. A method for treating a proliferative disorder in an individual comprising the step of administering an effective amount of a therapeutic agent as defined in Claim 25 and/or an effective amount of an isolated or recombinant polyribonucleotide as defined in Claim 26 and/or a pharmaceutical composition as defined in Claim 29 to an individual in need thereof.

31. A therapeutic agent according to Claim 27 or a use according to Claim 28 or a method according to Claim 30 wherein the proliferative disorder is selected from the group comprising: benign prostatic hyperplasia and cancer.

32. A therapeutic agent or use or method according to Claim 31 wherein the cancer is selected from the group comprising: lung cancer; squamous lung carcinoma; small cell lung cancer; colon cancer; colorectal cancer; stomach cancer; brain tumour; cancer of the liver; pancreatic cancer; breast cancer; gynaecological cancer; uterine cancer; prostate cancer; endometrial cancer; cervical cancer; ovarian cancer; melanoma; lymphoma; pancreatic cancer; gastric cancer; testicular cancer.

33. An in vitro method for inhibiting and/or reducing the activity of telomerase in a cell comprising or consisting of the step of treating the cell with an effective amount of a therapeutic agent according to Claim 25 or an effective amount of an isolated or recombinant polyribonucleotide as defined in Claim 26 or a pharmaceutical composition according to Claim 29.

34. A diagnostic agent or a polynucleotide substantially as described herein with reference to the accompanying description and/or one or more accompanying figures.

35. A therapeutic agent or a polyribonucleotide substantially as described herein with reference to the accompanying description and/or one or more accompanying figures.

36. A use or method substantially as described herein with reference to the accompanying description and/or one or more accompanying figures.