Introduction

Telomerase is a ribonucleoprotein enzyme that maintains telomeres; the ends of eukaryotic chromosomes. Human telomerase synthesizes TTAGGG tandem repeat sequences at the 3' end of the chromosomes using part of its internal RNA moiety as a template.¹ The discovery that telomerase expression closely correlates with cell immortalisation and malignant progression has led to considerable interest. Telomerase activity has been found in the vast majority of human tumors (>85%) including the most prevalent cancers that include prostate, colon, lung, breast, ovary and pancreas.^2,3 Conversely, telomerase activity is below detection levels in most somatic cells with the exception of cells with high replication potential such as germ cells, epithelial cells, lymphocytes and stem cells.^2,3 By maintaining the telomere length of tumor cells, telomerase provides a mechanism for sustaining indefinite replicative capacity⁴ which is necessary to support tumour growth.

Identifying a universal target for cancer treatment is a significant challenge for modern medicine. Since telomerase appears to be a hallmark of the majority of cancerous cells, it has become a novel and potentially selective target for cancer chemotherapy. The rational for exploring telomerase as a therapeutic target is that disruption of telomerase activity would prevent cells from maintaining their telomeres and thus leads to telomere shortening. Critical telomere shortening would lead to cellular senescence and chromosome instability eventually resulting in cell death and tumor stasis or regression (Fig. 1).⁵

Figure 1

Effects of telomerase inhibition on human tumor cell growth. The possible activation of alternative pathways for telomere maintenance (see Chapter by Reddel) is indicated.

To exploit the potential of telomerase as a therapeutic target for chemotherapy, the development of potent and selective inhibitors is required. In the past decade, a variety of experimental approaches have been used to test and validate telomerase as an anti-cancer target. For example the dominant negative expression of human telomerase reverse transcriptase^6,7,8 (hTERT) and the expression of non-functional human telomerase RNA^9,10 (hTR) has been successfully exploited in various tumor cell lines. These studies resulted in inhibition of telomerase activity, telomere shortening and loss of cell viability. Various chemical approaches have also been explored. These have focused on three main areas as follows: the design and development of small molecules which inhibit the reverse transcriptase activity of telomerase^11,12 (i.e., azidothymidine and other nucleoside triphosphate analogues), small molecules that stabilise G-quadruplex structure of the telomeres,^13,14 and “antisense” inhibition of human telomerase by oligonucleotide analogues. It is the latter approach which is the focus of this Chapter.

The “Antisense” Approach to Telomerase Inhibition

The antisense approach normally refers to the inactivation of gene expression at the transcriptional level typically using a short oligonucleotide or mimic to target a complementary sequence of the corresponding messenger RNA (mRNA).^15,16 For telomerase, the “antisense” terminology has referred mainly to the targeting of defined sequences within the RNA subunit of human telomerase (hTR) by complementary oligonucleotides. “Normal” antisense targeting of telomerase (i.e., mRNA of hTERT as target) has been reported^17,18 but will not be featured in this article.

The RNA subunit of human telomerase is made up of 451 ribonucleotides and contains an 11 nucleotide (nt) single-stranded region known as the template region, of sequence 5'-CUAACCCUAAC-3' located from position 46 to 56.^9,19 The numbering of telomerase hTR used has been based on the sequence described by Feng et al.⁹ In the proposed model for DNA synthesis catalysed by telomerase, the template sequence has two roles (Fig. 2).^20,21 The alignment domain (nt 46 to 50) of the template hybridises to the 3' end of the telomere via Watson-Crick base pairing interactions. The telomere is subsequently extended by reverse transcription of the last six nucleotides of the template (nt 51 to 56), sometimes referred to as the elongation domain.²² The template region of telomerase is, therefore, accessible for hybridization with complementary nucleic acids and essential for templating telomeric DNA synthesis. Consequently, it has become the target region of choice for oligonucleotide-based inhibitors of human telomerase.

Figure 2

The mechanism of telomeric DNA synthesis catalysed by human telomerase.

Oligonucleotides with sequence complementarity to the template region of human telomerase can hybridise and thus compete with the 3' end of telomeric DNA for telomerase. By blocking access to the active site of the enzyme such molecules are capable of preventing telomere elongation (Fig. 2). An advantage of this approach is the potential affinity and selectivity that oligonucleotide mimics have for targeting nucleic acid. In the absence of significant structural information for telomerase, the primary structure of hTR from the work of Villeponteau and co-workers provides sufficient information to design oligonucleotide-based telomerase inhibitors.⁹

To be useful therapeutic agents, oligonucleotides must fulfill a number of criteria that include: high sequence specificity, in vivo stability, uptake and retention by target cells and a viable chemical synthesis on the required scale. Furthermore, anti-telomerase oligonucleotides that target the template region must bind with a very high affinity per base pair in order to compensate for the limited number of continuous bases accessible on the RNA template.^23,24 Corey and co-workers have evaluated the relative potency of a series of PNA oligomers overlapping both the template region of hTR and its 3' or 5' vicinity (spanning nt 36 to 67). These studies have shown that it is principally the template region and 4 nucleotides downstream and upstream of the template (nt 42 to 45 and 57 to 60 respectively) which are the most potent target sites for inhibition.^23,24 In particular, the four cytosines C₅₀-C_52, C₅₆ of the template sequence and to a lesser extent bases U₅₃, U₅₇, A₅₄ play an important role in the recognition and binding of complementary sequences.²⁴ In the same study, the authors also demonstrated that the potency of PNA oligomers complementary to the template region and its 3' vicinity increases with the length of the PNA inhibitors. Optimal inhibitory effects on telomerase activity were observed with PNA 11 to 13 bases in length of sequence 5'-TAGGGTTAGAC(AA)-3'.²⁴ These PNAs inhibited telomerase activity with a comparable IC₅₀ value of 10 nM. In contrast, shorter PNAs (6 to 10-mer) were typically 1,000 to 10,000-fold less potent than longer PNAs. Furthermore, the 15-mer of sequence 5'-GTTAGGGTTAGACAA-3', did not exert significantly stronger inhibitory effects.²⁴

Oligonucleotide Analogues as Human Telomerase Inhibitors

Natural oligonucleotides (DNA, RNA) have limited potential as therapeutic agents, the main reason being their poor in vivo stability as a result of the phosphodiester backbone's susceptibility to enzymatic cleavage by cellular nucleases.²⁵ To overcome this general problem in the antisense field, there have been significant advances in the discovery and development of analogues bearing structural features which improve the pharmacological properties of natural oligonucleotides. Several of these oligonucleotide analogues have been tested as telomerase inhibitors. The most potent include phosphorothioate DNA (PS-DNA), 2'-O-alkyl RNA and peptide nucleic acids (PNA) (Fig. 3).

Figure 3

Chemical backbone structure of DNA analogues that are the most potent inhibitors of telomerase. (Alkyl is methyl for 2'-OMe RNA and 2-methoxyethyl (C₂H₄OCH₃) for 2'-OMOE RNA). B= nucleobase (A, T, G, C).

PS-DNA is prepared by the modification of the phosphodiester backbone linkage of DNA by replacement of one non-bridging phosphate oxygen atom by sulphur.²⁶ Relative to DNA, PS-DNA offers several potential advantages including increased affinity and improved resistance to nuclease degradation.^27,28 However, PS-DNA can also interact with proteins in a non-specific manner with affinities typically in the micromolar to millimolar range,^29,30 which represents a possible limitation. One PS-DNA oligomer is currently used as an antisense drug to treat CMV retinitis³¹ and several other examples have entered clinical trials for other disease areas.^30,32 2'-O-alkyl RNA belongs to a class of oligonucleotide mimics with structural modification at the 2' position of the ribose sugar of RNA.³³ 2'-O-methyl RNA (2'-OMe RNA) and 2'-O-(2-methoxyethyl) RNA (2'-OMOE RNA) are examples of such a modification (Fig. 3). 2'-O-alkyl RNA binds complementary nucleic acid sequences with higher affinity than analogous DNA or RNA,^33,34 and have been shown to display greater oral bioavailability in vivo.³⁵ 2'-O-alkyl RNA oligomers are also currently being evaluated in clinical trials.³⁶ PNA is a peptide-nucleic acid hybrid in which the phosphate backbone has been replaced by an achiral and neutral backbone made from N-(2-aminoethyl)-glycine units (Fig. 3). The nucleobases A, G, T, C are attached to the amino nitrogen on the backbone via methylenecarbonyl linkages.³⁷ The use of PNA as antisense agents provides several benefits.^37,38 PNA oligomers bind complementary sequence with high affinity relative to analogous DNA or RNA.³⁸ PNA/DNA or PNA/RNA hybrids also exhibit higher thermal stability compared to the corresponding DNA/DNA or DNA/RNA duplex.³⁸ This is generally ascribed to the lack of electrostatic repulsion between the neutral PNA backbone and the negatively charged phosphodiester backbone of nucleic acids. Furthermore, PNA displays high sequence specificity.³⁸ PNA also possesses high chemical and biological stability, due to the unnatural amide backbone that is not recognized by nucleases or proteases.³⁹

Inhibition of Telomerase Activity by Antisense Oligonucleotides Directed Against the RNA Template

Several studies have shown that 2'-O-alkyl RNA, PS-DNA and PNA complementary to the template region of telomerase can disrupt human telomerase activity in vitro with IC₅₀ values in the low nanomolar range for the best inhibitors. These data have been summarized in Table 1.

Table 1

Reported oligonucleotide-based inhibition of human telomerase activity.

Natural oligonucleotides (DNA) have been shown to inhibit telomerase activity in ciliate,²⁰ mice⁴⁰ and human cells.⁴¹ However, high concentrations (typically micromolar) and long sequences (typically >18-mer) were required. Shorter oligonucleotides (13-mer) were found to be ineffective.⁴² In comparison, PS-DNA is a more potent inhibitor of human telomerase. However, independent studies by Corey et al and Lehmann et al have demonstrated that PS-DNA are 40 to 200 fold less potent inhibitors of telomerase activity than the analogous PNA (Table 1).^23,43 For instance the 13-mer PS-DNA 1 of sequence 5'-TAGGGTTAGACAA-3' overlapping the 11 nt template and 2 nt downstream possess an IC₅₀ value of 200 nM whereas the PNA analogue (PNA 2, Table 2) inhibits telomerase activity with a substantially lower IC₅₀ of 1 nM.⁴⁴ Stronger inhibitory effects were achieved with PS-DNA 15 nucleotides or longer.⁴³ For example, PS-DNA 6 (Table 2) has an IC₅₀ value of 0.5 nM. Inhibition of human telomerase by PS-DNA oligomers is also subject to lower sequence selectivity than PNA.^23,43 For instance the non-complementary 13-mer PS-DNA 3 and 15-mer PS-DNA 7 of scrambled sequence inhibited telomerase activity with IC₅₀ values only 2 to 3 fold higher (450 nM and 1.3 nM respectively, Table 2) than complementary PS-DNA 1 and PS-DNA 6 (200 nM and 0.5 nM, respectively).^23,43 The mode of action of PS-DNA still remains to be elucidated but their propensity to bind to proteins suggest hTERT or telomerase-associated proteins as putative targets. In contrast, PNA displays high sequence specificity. The 13-mer PNA 5 (Table 2) which contained 4 mismatches relative to the template is 1000-fold less active than complementary PNA 2 whereas PNA 4 of scrambled sequence showed no detectable inhibition.⁴⁴ These results demonstrate that the use of PNA provides important benefits over PS-DNA.

Table 2

Specificity and potency of human telomerase inhibition in vitro by template-targeting peptide nucleic acid (PNA) and phosphorothioate DNA (PS-DNA).

Only one class of oligonucleotide-based inhibitors of human telomerase with potencies superior to PNA has been published.^42,45 Corey and co-workers have made a direct comparison between the relative potency of 2'-O-alkyl RNA and PNA homologues to inhibit telomerase activity (Table 3).⁴² 13 base 2'-O-alkyl RNAs complementary to the template of telomerase and 2 nucleotides upstream (nt 46 to 58) inhibited telomerase with IC₅₀ values of 8 nM and 10 nM for 2'-OMe RNA 8 and 2'-OMOE RNA 9 respectively (Table 3). ^42,45 Inhibition by the analogous 13-mer PNA gave an IC₅₀ value of 20 nM (PNA 10, Table 3). 2'-O-alkyl RNA also shows high sequence selectivity. For example 2'-OMOE RNA 13 with 2 mismatches within the sequence was 1000 fold less active than complementary 2'-OMOE RNA 9 (Table 3).⁴⁵ The incorporation of 4 mismatches within the sequence completely abolishes the inhibitory effect (2'-OMe RNA 12, Table 3). It is interesting to note that 2'-O-alkyl RNA 8 and PNA 10 (Table 3) inhibit telomerase with IC₅₀ values of 8 nM and 20 nM respectively whereas the Tm values for target RNA are 58°C and 76°C respectively.⁴² This illustrates that for antisense inhibitors of telomerase, high target affinity does not necessarily correlate with higher potency.

Table 3

Specificity and potency of human telomerase inhibition in vitro by template-targeting 2'-OMe RNA, 2'-OMOE RNA, PNA and DNA.

PNA and 2'-OMeRNA are promising lead candidates for in vivo investigation, combining potency, sequence selectivity and minimized size. Despite having relatively poor sequence specificity, PS-DNA may still target telomerase selectively enough to be used as a lead compound. Although each of the above oligonucleotide mimics have significantly improved the properties of natural oligomers, none have entirely fulfilled the requisite criteria for therapeutics. Therefore a combination of chemical modification approaches may have to be used in order to optimise their benefits.⁴⁶ One form of modification that has been explored is the combination of 2'-O-alkyl RNA with PS-DNA linkages. The terminal phosphorothioate linkage has been used to cap 2'-O-alkyl RNA and generate improved inhibitors. The presence of phosphorothioate linkages at both end of 2'-O-alkyl RNA confer enhanced resistance to nucleases whilst maintaining the high specificity and potency of 2'-O-alkyl RNA (Table 3, 2'-OMe RNA 14, 2'-OMOE RNA 16). Mismatch sequences (2'-OMe RNA 15, 2'-OMOE RNA 17) still possess IC₅₀ values at least 1000-fold less potent than the corresponding matched sequences.^42,45 In contrast, the systematic substitution of all the phosphodiester linkages by phosphorothioates substantially reduces the sequence selectivity of these inhibitors.^42,45 The potency of these chimeras in vitro is improved with IC₅₀ values of 7 nM (i.e., 2'-OMOE RNA 20) but mismatch sequences are also potent inhibitors becoming only 25- to 100-fold less active than complementary and unmodified 2'-O-alkyl RNA (2'-OMe RNA 19, 2'-OMOE RNA 21, Table 3).

Inhibition of Telomerase by Non-Template Directed Oligonucleotides

Early in vitro studies suggested that PNA targeted to sequences of hTR outside the template region were relatively poor inhibitors of telomerase activity. More recently, however, Corey and co-workers demonstrated that PNA complementary to structurally and functionally important sequences of hTR represent a new and potentially valuable class of telomerase inhibitors.⁴⁷The ability to reconstitute telomerase activity from recombinant hTERT and hTR in the rabbit reticulocyte lysate enabled the authors to determine the effect of PNA inhibitors on the assembly pathway of telomerase. When added prior to the holoenzyme formation, IC₅₀ values for these non-template directed PNAs range from 0.01 to 15 μM, depending on the target sequence.⁴⁷ The most potent inhibitors were those complementary to specific sequences believed to be important either for hTR structure or binding to hTERT. For example, PNA 23 and PNA 22 in Table 4 inhibit telomerase activity with IC₅₀ values of 10 and 100 nM, respectively.⁴⁷

Table 4

Telomerase inhibition by non-template directed PNA (compiled from Hamilton et al).

Presumably, the mode of action of such PNA inhibitors is by trapping the RNA subunit of telomerase before it complexes with hTERT to form active telomerase. Alternatively, hybridisation of PNA to hTR may disrupt the RNA folding, distort the holoenzyme structure and adversely perturb telomerase activity. Interference in the formation of hTR RNA structure is expected by PNA 22 and PNA 23, affecting the pseudoknot domain of hTR adjacent to the template region (nt 65 to 144), and the P3 helix (nt 174 to 182), respectively.^19,48 PNA 24 complementary to sequence spanning nucleotides 291 to 303 in the H/ACA box domain of hTR is also a potent inhibitor with an IC₅₀ value of 50 nM. This region of hTR of conserved sequence is functionally important for binding hTERT as shown by Autexier et al⁴⁸and Collins et al.⁴⁹ When evaluated on a matured enzyme, the inhibitory effect of the non-template PNA inhibitors of telomerase decrease significantly with IC₅₀ values ranging from 2 μM to above 100 μM (Table 4) presumably because the sequences targeted are buried within the holoenzyme and not accessible for hybridisation.⁴⁷ Other approaches have been used to target non-template regions of hTR. The inhibitory effect of oligonucleotides conjugated with a 2',5'-oligoadenylate (2–5A) have been investigated.^50,51,52,53 The approach relies on the mediated degradation of single-stranded regions of hTR upon binding of the oligonucleotide and activation of endoribonuclease RNase L by the 2',5'-oligoadenylate segment (Fig. 4).

Figure 4

Schematic representation of RNase L-mediated cleavage of hTR by DNA-2',5'-oligoadenylate chimera.

The hammerhead ribozyme has also been explored for cleaving the RNA component of human telomerase.^54,55,56,57 This class of inhibitors consists of two flanking antisense sequences used to position the catalytic core of the ribozyme on the site of cleavage. Yokoyama and co-workers have qualitatively demonstrated that hammerhead ribozymes have the potential to cleave hTR and reduce telomerase activity.⁵⁴ However, detailed quantitative studies will be required to fully evaluate the scope of this strategy.

Cell Based Studies and in vivo Therapy

Most of the studies described above were carried out in a cell-free environment. However, a major issue concerning the development of anti-telomerase oligonucleotide inhibitors is the requirement for efficient cellular uptake and localisation. Duncan et al illustrated this with a potent PS-DNA inhibitor of telomerase in crude extract which had no inhibitory effect on telomerase in intact cells due to the oligonucleotides not being able to cross the cell membrane.⁵⁸ In the last decade, significant progress has been achieved with delivery methods for transporting oligonucleotides into cells.

Delivery of Antisense Agents into Cells

The inefficient transportation of oligonucleotides and PNA across cell membranes is a limiting factor for using anti-telomerase oligomers directly in therapy.^59,60 Various studies have demonstrated that the covalent conjugation of delivery vehicles to oligonucleotide and PNA can improve their uptake into living cells.^61,62 The efficacy of cell-penetrating peptides, such as Antennapedia, to internalise PNA has been demonstrated in human prostate tumor DU 145 cells and human melanoma cell lines JR8 and M14.^61,62 It was also demonstrated that the presence of cationic peptides (i.e., polylysine) and polyamine analogues at the termini of PNA and oligonucleotides facilitates transportation into mammalian cells.^63,64 The attachment of positively charged moieties to PNA also enhances the solubility of these otherwise hydrophobic molecules. Furthermore it was demonstrated by Harrison et al that the chemical conjugation of cationic peptides to PNA enhances the inhibition of telomerase activity.⁶⁵ Therefore, it is conceivable that such a modification may serve to have multiple benefits for the inhibition of telomerase in cells. The versatility of the PNA synthesis based on the well established solid phase peptide chemistry is particularly well suited for the rapid and easy development of such PNA chimeras.^61,66 Alternatively, Corey and co-workers have successfully used cationic lipids to “encapsulate” 2'-O-alkyl-RNA and phosphorothioate substituted derivatives for transfection into human prostate tumor DU 145 cells.^42,67 This simple lipid-mediated uptake technique is known as lipofection and it is widely used in the antisense field, but it fails to shuttle PNA into intact cells due to the neutral peptide-like backbone of the PNA which does not interact with the cationic lipids. This problem has been ingenuously overcome by using a “suicide” oligonucleotide whose function is merely to hybridise to the PNA and allow transfection by providing the negatively charged backbone required for interaction with the cationic lipids (Fig. 5).⁴⁷ Upon internalisation, the partially hybridized DNA strand is degraded by endogeneous nucleases and the PNA released into the cytoplasm. Once the PNA has been delivered inside cells, it has been demonstrated by Corey and co-workers using fluorescence labelling, that PNA is retained by cells 3 to 5 days after transfection and preferentially localized within the nucleus.^44,47 Such methodologies are being applied to evaluate the effects exerted by the most promising in vitro inhibitors of human telomerase in various human tumor cell lines.

Figure 5

Schematic illustration of cationic-lipid/DNA mediated lipofection of PNA.

Conclusion and Future Perspectives

The inhibition of telomerase by oligonucleotides and oligonucleotide mimics that target the RNA component (hTR) has been demonstrated in a number of studies by several groups. Cell-based studies using such inhibitors have provided support for a mechanistic hypothesis of telomerase-mediated telomere maintenance, although the time period required for some of the observable effects of such inhibitors does appear to vary between reports. Further work may elucidate more mechanistic details about the action of such compounds in cells, and the consequences of inhibiting telomerase activity. There is considerable scope to improve the efficacy of this class of inhibitors by using novel oligonucleotide analogues, and also the chemical derivatisation of such molecules, for example with peptide conjugates. Whilst it has already been demonstrated how valuable such molecules are as biological tools, there is more work to be done to prove their therapeutic potential. It is likely that the outcome of clinical studies of oligonucleotide inhibitors of telomerase will be reported during the next few years.

Abbreviations

C - cytosine base or cytidine nucleotide

G - guanine base or guanosine nucleotide

hTERT - human Telomerase Reverse Transcriptase

hTR - human Telomerase RNA

IC₅₀ - 50% inhibition coefficient

Kb - kilobase

Lys - lysine

nt - nucleotide

2'-OMe RNA - 2'-O-methyl RNA

2'-OMOE RNA - 2'-O-(2-methoxyethyl) RNA

PNA - peptide nucleic acid

PS-DNA - phosphorothioate DNA

T - thymine base or thymidine nucleotide

U - uracil base or uracil nucleotide

Acknowledgments

We thank Mark Farrow for proofreading the manuscript. We acknowledgethe support of the BBSRC, and the Cambridge European Trust.

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