EP1149161A2 - Ribozymes directed against the catalytic subunit of the human telomerase (htert) - Google Patents

Abstract

The invention relates to anti-telomerase ribozymes and to the use thereof. The ribozymes diminish the activity of the catalytic subunit of the human telomerase (human Telomerase Enzyme Reverse Transcriptase, hTERT). They serve as telomerase inhibitors, limit the proliferative capacity and increase the sensitivity of tumor cells with regard to cytostatic agents.

Description

Ribozymes directed against the catalytic subunit of human telomerase (hTERT)

description

The invention relates to anti-telomerase ribozymes and their use. The ribozymes reduce the activity of the catalytic subunit of human telomerase (human telomerase enzyme reverse transcriptase, hTERT). They serve as telomerase inhibitors, limit the proliferative capacity and increase the sensitivity of tumor cells to cytostatics.

Telomerase is a unique reverse transcriptase that uses an internal RNA component to attach telomer sequences de novo to the 3 'DNA end of the telomeres. It thus counteracts a progressive mitotic telomere shortening, the result of which leads to proliferative senescence in mortal somatic cells. Although there are alternative options for maintaining telomere length, especially in experimentally immortalized cells and in other species, their importance for tumor development in humans is apparently rather small. The vast majority of different human tumors have a high telomerase activity, while starting cells and non-invasive tumor precursors usually have only a low activity. The telomerase activity can be reconstituted in vitro solely from the RNA component and the catalytic subunit. In addition to 7 conserved sequence motifs for reverse transcriptases, a telomerase-specific T motif was detected in the catalytic subunit. Mutations in conserved amino acids of the T motif lead to a drastic loss of function in the cell-free system (Weinrich SL, Pruzan, R .. Ma, L., Oulette, M., Tesmer, VM, Holt, SH, Bodnar, A., Lichtsteiner, S., Kim, NW, Trager, JB, Taylor, RD, Carlos, R., Andrews, WH, Wright, W., Shay, JW, Harley, CB, Morin, GB, Nature Genetics, 17, 1997, 498- 502).

Inhibition of telomerase was proposed very early on as a potentially highly specific possibility for "remortalization" and therapeutic control of tumor cells. However, it was clear that telomerase incorporation would only lead to telomer-dependent cell cycle blockade after a considerable time delay (Kipling, D., Nature Genet 9, 1995: 104-105).

The previous efforts to switch off the activity of telomerase have primarily been directed against the RNA component of the enzyme, since this has been known since 1995. A successful telomerase inhibition by hTR antisense vectors, which leads to a telom lead shortening and senescence of the corresponding culture has been demonstrated only once in HeLa cells (Feng, J., Funk, W., Wang, SS, Weinrich, SL,. Avilion, AA, Chiu, CP, Adams, RR, Chang , E., Allsopp, RC, Yu, J., Le, S., West, MD, Harley, CB, Andrews, WH, Greider, CW, Villeponteau, B, Science 1995, 269: 1236-1241). Only recently have a group of works published that report telomerase inhibition in glioma cells with subsequent induction of apoptosis or differentiation by using modified antisense RNA (Kondo, Y., Kondo, S., Li, G., Silvermann, RH, Cowell, JK, Oncogene 1998, 16: 3323-3330; Kondo, Y., Kondo, S., Tanaka, Y., Haqqi, T., Barna, BP, Cowell, JK, Oncogene 1998, 16: 2243- 2248). However, the specificity of these effects is unclear, telomeric erosion has not been demonstrated and is probably not the cause of apoptosis induction here. The RNA component may not be the best target for telomerase introduction because it is also expressed in normal cells.

Nucleoside analogs, e.g. the known inhibitor of reverse transcriptases azidothymidine, work well in the cell-free system, but show strong non-specific side effects in cells (Ray, C, Blackburn, E., Mol Cell Biol 16, 1996, 53-65; Janta-Lipinski, Matthes, MDC Book, personal communication). Other strategies to inhibit telomerase such as B. Antisense technology with peptide-coupled nucleic acids or the use of non-nucleosides have so far only been tested in the cell-free system. Hammerhead ribozymes are catalytic RNAs with a well-defined structure that specifically cleave RNA targets with the sequence XUN (N = A, C or U). The GUC sequence is one of the most easily cleavable targets. Inhibition of telomerase and subsequent telomere shortening by ribozymes against hTR have been described (Yokoyama, Y., Takahashi, Y., Shinohara, A., Lian, Z., Wan, X., Niwa, K., Tamaya, T., Cancer Res., 58, 1998: 5406-5410). So far, no reliable inhibition of cell growth has been achieved with ribozymes against hTR. Ribozymes with activity against hTERT, the mRNA of the catalytic subunit of telomerase, have not previously been described.

Recently there have been first indications of specific interrelations between telomeres and telomerase on the one hand and DNA damaging processes on the other. This means that telomerase could not only contribute to the unlimited proliferation of tumor cells by compensating for "normal" telomer erosion. Telomeres are hypersensitive to oxidative stress (Petersen, S., Saretzki, G., von Zglinicki, T. Exp. Cell Res. 1998, 239: 152-160). Inhibition of telomerase in human glioblastoma cells leads to the induction of apoptosis (Kondo, Y., Kondo, S., Li, G., Silvermann, RH, Cowell, JK, Oncogene 1998, 16: 3323-3330) or to sensitization to cis- Platinum (Kondo, Y., Kondo. S., Tanaka, Y., Haqqi, T., Barna, BP, Cowell, JK, Oncogene 1998, 16: 2243-2248) within a few days. ie before a significant telomere shortening can occur.

Telomerase inhibitors are described several times in the patent literature. They are directed primarily against the RNA component of the enzyme (e.g. EP 666313 WO 97/37691 and WO 98/28442). Methods for cancer treatment using telomerase inhibitors are reported in US Patents US 5767278, US 5770613, US 57031 16, US 5760062 and 5656638. The catalytic subunit of human telomerase (hTERT) has the international patent application WO 98/14593 and the resulting European (EP 841396) or German patent application (DE 19743497) to the content. The polynucleotides and plasmids described therein are suitable for the diagnosis, prognosis and treatment of human diseases and for changing the proliferation capacity of cells and organisms. The patent specifications WO 98/14593 and WO 98/14592 relate to plasmids, including those which span the region of the T motif of hTERT, both expression plasmid (s) and antisense plasmids. Antisense plasmids can be used to inhibit telomerase. German patent application DE 19720151 describes a chemically modified oligodeoxynucleotide which on the one hand exerts an antisense effect against hTR and on the other hand reacts at its 5 'end with the protein of the catalytic subunit hTERT. Ribozymes, i.e. However, polyribonucleotides which connect antisense flanks with a central RNA-cutting structure are described in the abovementioned. Patents not panned. There are no published results on the specific inhibition of the catalytic subunit of telomerase.

The object of the invention was to reduce the activity of the catalytic subunit of human telomerase (human telomerase enzyme reverse transcriptase, hTERT) and thus to make new telomerase inhibitors available.

The task was solved by new ribozymes, which are directed against the T motif of human telomerase. The ribozymes according to the invention contain the following sequences: 1. 5 ^' -GCUCGAC CUGAuGAGuCcGUGAgGaCGAA ACGUAC AC A-3 ^Λ 2. 5 ^' -GCAGCUC CUGAuGAGuCcGUGAgGaCGAA ACGACGUAC-3 '

3.5 ^' -AAAGAAA CUGAuGAGuCcGUGAgGaCGAA ACCUGAGCA-3'

4.5 ^' -UCUCCGU CUGAuGAGuCcGUGAgGaCGAA ACAUAAAAG-3'

5. S ^' -UGCUCCA CUGAuGAGuCcGUGAgGaCGAA ACACUCUUC-3 ^'

6.5 ^' -GACGACG CUGAuGAGuCcGUGAgGaCGAA ACACACUCA-3 ^' 7.5 '-CUUUUGA CUGAuGAGuCcGUGAgGaCGAA ACGUGGUCU-3'

8. 5 '-GCUUUGC CUGAuGAGuCcGUGAgGaCGAA ACUUGCUCC-3 ^'

9.5 'AAGACCU CUGAuGAGuCcGUGAgGaCGAA AGCAGCUCG-3'

10. 5'-UGUUUUU CUGAuGAGuCcGUGAgGaCGAA AGCCUGUUC-3 '11. 5' -CAUAAAA CUGAuGAGuCcGUGAgGaCGAA AAAGACCUG-3 ^'

12. 5'-UUCUUUU CUGAuGAGuCcGUGAgGaCGAA AAACGUGGU-3 ^'

13. 5'-UCCGGUA CUGAuGAGuCcGUGAgGaCGAA AAAAAGAGC-3 '(small letters denote freely selectable nucleotides in the context of the complementarity of the nucleotides in the stem loop). It has been found that the ribozymes according to the invention cleave the T motif of human telomerase (hTERT) as designed on one of the sequences GUC, GUA, GUU, CUC or UUC.

The hammerhead ribozymes according to the invention have catalytic activity in vitro. They cleave the mRNA for human telomerase (hTERT) in the T motif on the sequences planned by the design. The inhibition of telomerase in cells is achieved by stable transfection of an expression vector into which the ribozyme has been cloned. It can also be achieved with other transfection methods (e.g. transient transfection or infection with a recombinant virus). A ribozyme mutated in the catalytic center has no effect. According to the invention, telomere shortening and crisis occur in clones with inhibited telomerase.

The clones with inhibited telomerase increase the sensitivity to doxorubicin (measured using the XTT assay - colorimetric assay - as a concentration that kills 50% of the cells - LD50) by a factor of 2-3. The inhibition of telomerase by the ribozymes according to the invention is achieved both in sole use and in combination with cytostatic administration and / or radiation. This accelerates both the "classic" telomere shortening after inhibition of telomerase, which ultimately leads to the crisis and cell death in the course of several cell divisions, and the rate of telomere shortening due to DNA damage, or it eliminates independent repair properties of telomerase, which increases the rate Sensitivity causes.

The essence of the invention lies in a combination of known elements and new approaches that mutually influence each other and in their new overall effect give a benefit in use and the desired success, which lies in the fact that now inhibitors of catalytic subunit of human telomerase (human telomerase enzyme reverse transcriptase, hTERT) are available.

The ribozymes according to the invention are suitable for use as telomerase inhibitors and for shortening telomeres, also in combination with cytostatic administration and / or radiation or with topoisomerase inhibition and thereby for accelerating the rate of telomeres shortening due to DNA damage and for switching off the repair properties of telomerase . The ribozymes according to the invention are suitable for the treatment of tumors and for increasing the sensitivity of tumor cells to cytostatics.

The following examples serve to illustrate the invention without restricting it to these examples.

embodiments

Example 1: Design of the Ribozymes

Ribozymes consist of helices I and III, which hybridize with the target sequences flanking the interface, as well as the catalytic core and the stem loop (Helix II) with the structure CUGAuGAGuCcGUGAgGaCGAA. The ribozymes can be produced, for example, as an oligoribonucleotide. For this purpose, the sequence is synthesized from the ribonucleotides G, C, A and U as described by known methods. In principle, stabilization of the 3 'and 5 ^' ends by means of modified nucleotides or cap structures is possible; the influence of these modifications on the catalytic activity of the ribozyme must be tested in individual cases. These ribozymes can be used directly. Another possibility is the synthesis of the cDNA for the ribozyme from the deoxyribonucleotides G, C, A and T. This form of the ribozymes can be used in connection with suitable promoters and transfection vectors (eg adeno- or retroviral vectors).

cDNA sequence of the T motif from hTERT (GenBank AF015950): Nucleotides not belonging to the T motif are shown in italics. The possible interfaces and the sequences of the antisense helices for the possible ribozymes 1-13 result from the sequence. The interface of the first ribozyme (Rl) is shown in bold and the flanking sequences are underlined once or twice. Below is the associated ribozyme with the corresponding representation: 1681 caagttcctg cαetggctga tgagtgtgta cgtcgtcgag ctgctcaggt ctttctttta 1741 tgtcacggag accacgtttc aaaagaacag gctctttttc taccggaaga gtgtctggag 1801 caagttgcaa agcattcggggcgcgggggggcgcgggggggggggggggggggggggggcggggg

5 -GCUCGAC CUGAuGAGuCcGUGAgGaCGAA ACGUACACA-3 ^'

Example 2: Detection of the catalytic activity of the ribozymes 1-4 in vitro A 224 nucleotide (nt) long RNA, which includes the sequence of the T motif, was obtained by in vitro transcription from the cDNA of hTERT linked to a T7 promoter produced (target RNA). During the transcription, the radioactive labeling was carried out using P ^j2 -UTP. Ribozymes were synthesized by standard methods from ribonucleotides with 2'-hydroxyl groups protected by Fpmp (1 l- (2-fluorophenyl) -4-methoxy-piperidin-l-yl) and purified by HPLC. They were deprotected before use. The target RNA was incubated with ribozyme 1, 2, 3 or 4 or without ribozyme (/) for 60 and 180 min and the reaction products were separated electrophoretically in the polyacrylamide gel. The gel was evaluated in the phosphoimager and shows cleavage of the target RNA at the designated sites by the ribozymes 3 and 4 with high efficiency and low effectiveness of the ribozymes 1 and 2 (FIG

1).

Example 3:

Detection of inhibition of telomerase by stable transfection and expression of ribozyme 4 in HBL100, an immortal mammary epithelial cell line with high telomerase activity, and in MCF-7, a breast tumor cell line. A ribozyme imitated in the catalytic center has no effect.

Both strands of the cDNA coding for the ribozyme R4 (4th ribozyme in the list) and for the ribozyme mutR4 mutated in the catalytic center were synthesized as oligodeoxyribonucleotides by standard methods. They were cloned into the expression vector (plasmid) pCDNA3.1 and stably expressed in MCF-7 (FIG. 2a) or HBL-100 cells (FIG. 2b). The telomerase activity in the numbered clones was measured using a semiquantitative TRAP assay (Telomerase Repeat Amplification Protocol). The intensity of the conductor pattern in the respective track in relation to the intensity of the control band (triangle) is a measure of the activity of the telomerase. The HBL-100 and MCF-7 clones that were transfected with a ribozyme mutated in the catalytic center (mut R4) show approximately the same telomerase activity as the parental cells (p = parental cells, R8 = positive con trolls, NC = negative control). Expression of R4 reduces telomerase activity to values between <1 and about 30%.

Example 4: Detection of telomere shortening in clones with inhibited telomerase The telomere length in the cell lines MCF-7 (FIG. 3A) and HBL-100 (FIG. 3B) was measured in the Southern blot and quantified as described (Petersen, S., Saretzki, G., by Zglinicki, T. Exp. Cell Res. 1998, 239: 152-160). The number of clones n examined and the respective telomerase activity (in% of the activity in parental / mutR4-transfected cells, X axis) is indicated. The telomeric length in mutR4-transfected clones (mutR4) or in the R4-transfected clones, which show only a slight reduction in telomerase activity, does not differ significantly from that in parental cells. The telomere length in the clones with clearly inhibited telomerase is, however, significantly shorter.

Example 5: Detection of inhibition of cell growth in MCF-7 clones with inhibited telomerase

Net proliferation rates were determined by cell counting in each passage. The number of examined clones n and the respective telomerase activity (in% of the activity in parental / mutR4-transfected cells, X axis) is indicated. In clones with a telomerase activity below 25% of the starting cells, cell growth is inhibited (FIG. 4A). Evidence of an altered morphology in telomerase-incorporated MCF-7 clones. The morphology of living cells was checked in the phase contrast reversal microscope. Clones stably transfected with mutR4 (left) show normal, rapid growth and the same morphology as parental cells. Clones with telomerase inhibited by R4 (right) show restricted growth and for the most part, especially on the periphery of the resulting, comparatively small colonies, cells with characteristic senescent morphology (same magnification left and right) (FIG. 4B).

Evidence of the generation of a senescent phenotype by retroviral infection with the ribozyme R4 in a mass culture of MCF-7. R4 was cloned into a retroviral expression vector (pBabe) and recombinant viruses were generated according to the standard procedure. 10 MCF-7 cells were infected either with the unmodified vector (left) or with R4-pBabe (right). The mass cultures are shown 2 weeks after infection. Infection with R4-pBabe reduces telomerase activity and blocks growth in the culture. Numerous cells die and detach from the culture dish. The few over- living cells show a senescent phenotype (same magnification left and right) (Figure 4C).

Example 6: Detection of a sensitivity of clones with inhibited telomerase to doxorubicin increased by a factor of 2-3

HBL-100 cells were transfected with R4 or mutR4. Telomerase activity was measured using a TRAP assay. The clones were treated with doxorubicin in concentrations between 10 and 1000 ng / ml for three days and then cell survival was measured with an XTT assay. The doxorubicin concentration resulting in a 50% reduced XTT signal (colorimetric signal) (LD50) was calculated. The number of clones n examined and the respective telomerase activity (in% of the activity in parental / mutR4-transfected cells, X axis) is indicated. Clones with a telomerase activity <25% of the starting cells have an LD50 reduced by a factor of 2 (FIG. 5A). Determination of the LD50 for doxorubicin in MCF-7 clones (see above). Clones with a telomerase activity below 25% are three to four times more sensitive to doxorubicin (Figure 5B).

Example 7:

Evidence of the increased sensitivity of telomerase-negative cells to topoisomerase-inhibiting cytostatics

The sensitivity to the specified cytostatics was measured as LD50 using the XTT assay. The ratio of the LD50 of the telomerase-positive to the telomerase-negative clones is given. MutR4-transfected vs. R4-transfected HBL-100 and MCF-7 clones and hTERT-expressing vs parental BJ fibroblasts (Bodnar, AG, Oulette, M., Frolkis, M., Holt, SE, Chiu, CP Morin, GB, Harley, CB, Shay, JW, Lichtsteiner, S., Wright, W. Science 1998, 279: 349-352). Cells with reduced telomerase activity are more sensitive than the isogenic clones with active telomerase to all investigated topoisomerase inhibitors (mitoxantrone, etoposide and doxorubicin - Römpp Chemie Lexikon 1995), but not to the cytostatic agents cis-platinum and bleomycin or the alkylating agent N-methyl -N'-nitro-N-nitrosoguanidine (MN G) or oxidative exposure using hydrogen peroxide (H ₂ O9) (Figure 6).

Claims

claims 1. Ribozymes which reduce the activity of the catalytic subunit of human telomerase hTERT (human telomerase enzyme reverse transcriptase). 2. Ribozymes according to claim 1, characterized gekemizeiclmet. that they are directed against the T motif of human telomerase. 3. Ribozymes according to claims 1 and 2, characterized in that they cut the T motif of human telomerase (hTERT) on the sequences GUC, GUA, GUU, CUC or UUC. 4. Ribozymes according to claim 1 to 3, characterized in that it is hammerhead ribozymes with the following sequences: a) 5'-GCUCGAC CUGAuGAGuCcGUGAgGaCGAA ACGUACACA-3 b) 5'-GCAGCUC CUGAuGAGuCcGUGAgGaCGAA 3CGAAAUACA) CUGAuGAGuCcGUGAgGaCGAA ACCUGAGCA-3 d) 5'-UCUCCGU CUGAuGAGuCcGUGAgGaCGAA ACAUAAAAG-3 e) 5'-UGCUCCA CUGAuGAGuCcGUGAgGaCGAA ACACUCUUC-3 f) 5-GACGACG CUGAuGAGuCcGUGAgGaCGAA ACACACUCA-3 g) of 5'-CUUUUGA CUGAuGAGuCcGUGAgGaCGAA ACGUGGUCU-3 h) 5'-GCUUUGC CUGAuGAGuCcGUGAgGaCGAA ACUUGCUCC-3 i) 5'-AAGACCU CUGAuGAGuCcGUGAgGaCGAA AGCAGCUCG-3 j) 5'-UGUUUUU CUGAuGAGuCcGUGAgGaCGAA AGCCUGUUC-3 k) 5'-CAUAAAA CUGAGGAGCCCGAAAGAGC 1) 5'-UUCUUUU CUGAuGAGuCcGUGAgGaCGAA AAACGUGGU-3 m) 5'-UCCGGUA CUGAuGAGuCcGUGAgGaCGAA AAAAAGAGC-3 5. Use of ribozymes according to claims 1 to 4 as telomerase inhibitors. 6. Use of ribozymes according to claim 1 to 4 for telomere shortening. 7. Use of ribozymes according to claim 1 to 4 in combination with cytostatic administration and / or radiation to shorten telomeres. 8. Use of ribozymes according to claim 1 to 4 in combination with topoisomerase inhibition for telomere shortening. 9. Use according to claim 7 or 8 for accelerating the rate of telomere shortening by DNA damage and for eliminating the repair properties of the telomerase. 10. Use of ribozymes according to claim 1 to 9 for the treatment of tumors. 11. Use according to claim 1 to 4 for increasing the sensitivity of tumor cells to cytostatics.