JP2002536967A - Ribozymes targeting the catalytic subunit of human telomerase (hTERT) - Google Patents

Abstract

(57) Abstract The present invention relates to anti-telomerase ribozymes and uses thereof. The ribozyme reduces the activity of the catalytic subunit of human telomerase (human telomerase enzyme reverse transcriptase; hTERT). Said ribozymes are utilized as telomerase inhibitors, limit the growth capacity and increase the sensitivity of tumor cells to cytostatics.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to anti-telomerase ribozymes and their uses. The ribozyme reduces the activity of the catalytic subunit of human telomerase (human telomerase enzyme reverse transcriptase; hTERT). They are used as telomerase inhibitors, limit their growth potential and increase the sensitivity of tumor cells to cytostatics.

[0002] Telomerase is a unique reverse transcriptase that, together with an internal RNA component (hTR), newly synthesizes a telomere sequence at the 3 'end of telomeric DNA. This opposes the progressive mitotic telomere shortening that ultimately causes replicative senescence in dying cells. The relevance of experimentally immortalized cells and other mechanisms of telomere elongation present in other species appears relatively small for human tumorigenesis. The vast majority of diverse human tumors exhibit high telomerase activity, whereas benign cells are most often characterized by low activity.

[0003] Telomerase activity can be reconstructed in vitro using only its RNA components and catalytic subunits. The catalytic subunit contains a telomerase-specific T motif in addition to the seven conserved sequence motifs for reverse transcriptase. Mutations in the conserved amino acids of the T motif result in severe loss of function in cell-free systems (Weinrich SL, Pruzan R, Ma L, Ouellette
M, Tesmer VM, Holt SH, Bodnar A, Lichtsteiner S, Kim NW, Trager JB, Tayl
or RD, Carlos R, Andrews WH, Wright W, Shay JW, Harley CB, Morin GB. Nat
ure Genetics 17, 1997, 498-502).

[0004] Inhibition of telomerase has previously been pointed out as "remortalising" and its highly specific potential for therapeutic control of tumor cells. However, it was clear that telomerase inhibition only resulted in a telomere-dependent cell cycle block after a considerable delay (Kipling D, Nature Genet.
ics 9, 1995, 104-105). Previous attempts at inhibiting telomerase activity have primarily been directed at the RNA component of the enzyme, as already described in 1995. Successful inhibition of telomerase has been demonstrated in HeLa cells, using the hTR antisense vector only once, leading to telomere shortening and senescence-like arrest of cell culture (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 269, 1995, 1236-124
1). Only recently, apoptosis-inducing or modified antisense RNs
A paper has been published reporting the inhibition of telomerase in glioma cells following differentiation by the use of A (Kondo Y, Kondo S, Li G, Silvermann RH, Cowell JK. Onc.
ogene 16, 1998, 3323-3330; Kondo Y, Kondo S, Tanaka Y, Haqqi T, Barna BP
, Cowell JK. Oncogene 16, 1998, 2243-2248). It is unclear how specific these effects are, but there is no telomere shortening, and in this case, probably not the cause of apoptosis. Since the RNA component is also expressed in normal cells, it may not be the best target for telomerase inhibition.

The well-known reverse transcriptase inhibitor “azidothymidine” and its analogs work well in cell-free systems, but show strong nonspecific side effects in cells (Strah
l C, Blackburn E, Mol Cell Biol 16, 1996, 53-65: Janta-Lipinski, Matthes
, Berlin-Buch, personal communication). As other techniques for inhibiting telomerase, for example, only the antisense method using peptide-linked nucleic acids or the technique using non-nucleosides have so far been tested in cell-free systems.

[0006] Hammerhead ribozymes are well-defined catalytic RNAs that specifically cleave RNA targets at the sequence of XUN (N = A, C, or U). The sequence of GUC is one of the best cleavable targets. Inhibition of telomerase by ribozymes targeting hTR and subsequent shortening of telomeres have been reported (Yokoyama Y, Takahashi Y, Shionohara A, Lian Z, Wan X, Niwa
K, Tamaya T. Cancer Res. 58, 1998, 5406-5410). Reliable inhibition of cell growth has not yet been achieved using anti-hTR ribozymes. hTERT
No ribozyme active against (mRNA of the catalytic subunit of human telomerase) has been reported so far.

The present specification is on the one hand the specific interaction between telomeres and telomerase, and on the other is the first indication on the DNA damage process. This means that, in addition to compensating for "normal" shortening of telomeres, telomerase may contribute to unrestricted growth of tumor cells. Telomeres are hypersensitive to oxidative stress (Petersen S, Saretzki G, von Zglinicki T. Exp Ce
ll Res 239, 1998, 152-160). Inhibition of telomerase in human glioblastoma cells results in the induction of apoptosis within a few days (ie, before significant shortening of telomeres can occur) (Kondo Y, Kondo S, Li G,
Silvermann RH, Cowell JK.Oncogene 16, 1998, 3323-3330), or sensitization to cisplatin (Kondo Y, Kondo S, Tanaka Y, Haqqi T, Barna BP, Co
well JK. Oncogene 16, 1998, 2243-2248). Telomerase inhibitors have been the subject of a number of patents. Many of the inhibitors described are RNAs of the enzyme
The components are targeted (eg, EP 666313, WO 97/37691, and WO 98/28442). U.S. Patents US Pat.
770613, US5703116, US5760062, and US56566
38 report an anticancer method using an inhibitor of telomerase. International patent application WO 98/14593 and the European (EP 841396) and German (DE 197 43 497) patent applications transferred therefrom relate to the catalytic subunit of human telomerase (hTERT). The polynucleotides and plasmids described therein are suitable for the diagnosis, prognosis, and treatment of human disease, and for altering the replication capacity of cells and organisms. WO 98/14593 and WO 98/14592 patent publications,
The present invention relates to an expression plasmid containing the T motif of hTERT and an antisense plasmid. Antisense plasmids can be used to inhibit telomerase.
German Patent Application DE19720151 describes chemically modified oligonucleotides which act as antisense to hTR and which react at the 5 'end with said hTERT catalytic subunit protein.

[0008] Ribozymes (ie, polyribonucleotides containing both a central RNA cleavage structure and an antisense flank) are not described in each of the aforementioned patent applications. There are no published results for specific inhibition of the catalytic subunit of telomerase.

An object of the present invention is to provide a human telomerase (human telomerase enzyme reverse transcriptase, hT
It is to reduce the activity of the catalytic subunit of ERT) and thus to obtain novel inhibitors of telomerase.

[0010] This object is achieved by developing a novel ribozyme targeting the human telomerase T motif. A ribozyme according to the invention comprises the following sequence: 5'-GCUCGACCUGAuGAGuCcGUGAgGaCGAAAC
GUACCA-3 ′ 5′-GCAGCUC CUGAuGAGuCcGUGAgGaCGAAAC
GACGUAC-3'5'-AAAGAAACUGAuGAGuCcGUGAgGaCGAAAC
CUGAGCA-3 '5'-UCUCCGU CUGAuGAGuCcGUGAgGaCGAA AC
AUAAAAG-3 '5'-UGCUCCA CUGAuGAGuCcGUGAgGaCGAA AC
ACUCUUC-3'5'-GACGGACGCUGAuGAGuCcGUGAgGaCGAA AC
ACACUCA-3 '5'-CUUUUGA CUGAuGAGuCcGUGAgGaCGAA AC
GUGGUCU-3 '5'-GCUUUGC CUGAuGAGuCcGUGAgGaCGAA AC
UUGCUCC-3'5'-AAGACUCUGAuGAGuCcGUGAgGaCGAA AG
CAGCUCG-3 ′ 5′-UGUUUUU CUGAuGAGuCcGUGAgGaCGAA AG
CCUGUC-3'5'-CAUAAAAACUGAuGAGuCcGUGAgGaCGAA AA
AGACCUG-3 ′ 5′-UUCUUU CUGAuGAGuCcGUGAgGaCGAA AA
ACGUGUGU-3 '5'-UCCGGUA CUGAuGAGuCcGUGAgGaCGAA AA
AAAGAGC-3 ′ (nucleotides listed in lower case can be chosen freely and they are complementary within the stem loop).

It has been discovered that a ribozyme according to the present invention cleaves the T motif of human telomerase (hTERT), as designed, at one of the sequences of GUC, GUA, GUU, CUC, or UUC.

[0012] The hammerhead ribozyme according to the present invention exhibits catalytic activity in vitro.
The ribozyme cleaves human telomerase (hTERT) mRNA within the T motif at the designed sequence. Telomerase inhibition is achieved by stably transfecting an expression vector containing the cloned ribozyme. It can also be achieved by other transfection methods (eg, transient transfection by infection with a recombinant virus). Ribozymes mutated in the catalytic center have no effect. Within the clones in which telomerase is inhibited by the present invention, telomere shortening and crisis occur.

The sensitivity to doxorubicin (determined by a colorimetric XTT assay as the concentration that kills 50% of cells [LD50]) increases by a factor of 2-3 in clones with reduced telomerase activity. Inhibition of telomerase by the ribozyme according to the present invention can be carried out by using the ribozyme alone, but can also be carried out in combination with irradiation and / or cytotoxic treatment. Such a combination promotes "typical" telomere shortening following telomerase inhibition, which leads to crisis and cell death after multiple cell divisions, and interferes with the independent repair properties of telomerase And, consequently, increase the sensitivity.

The essence of the invention lies in the combination of known elements and new solutions, which interact with each other, and which have applicable advantages and desirable successes, namely the reversal of human telomerase (human telomerase enzyme reversal). This results in the fact that inhibitors of the transcriptase (hTERT) catalytic subunit are now available.

The ribozymes according to the invention, alone or in combination with irradiation, cytotoxic therapy or inhibition of topoisomerase, are suitable as inhibitors of telomerase and for shortening of telomeres. Therefore, they are suitable for promoting telomere shortening due to DNA damage and reducing the repair properties of telomerase. The ribozymes according to the invention are suitable for treating tumors and for increasing the sensitivity of tumor cells to cytostatic and cytotoxic treatments. The following examples illustrate, but do not limit, the invention.

[0016]

【Example】

Example 1 << Ribozyme Design >> A ribozyme contains helix I and III that hybridize to a target sequence adjacent to a cleavage site, and a catalytic center and a stem loop (helix II) having a structure represented by CUGAuGAGuCcGUGAgGaCGAA. . Ribozymes can be produced as oligoribonucleotides. The sequence will be synthesized from G, C, A, and U ribonucleotides according to known methods. In principle, the 3 'and 5' ends can be stabilized by using modified nucleotide or cap structures. However, the effect of these modifications on the catalytic activity of the ribozyme must be tested in each case. The ribozyme can be used directly. Ribozyme cDNA can also be synthesized using G, C, A, and T deoxyribonucleotides. This form of the ribozyme can be prepared using a suitable promoter and transfection vector (
For example, an adenovirus vector or a retrovirus vector).

HTERT-T motif cDNA sequence (GenBank AF015950)
: The nucleotides outside the T motif are shown in italics. From this array,
Possible cleavage sites and sequences of the antisense helix for ribozymes 1-13 were derived. The cleavage site for the first ribozyme (R1) is shown in bold and its flanking sequence is underlined. The corresponding ribozymes are shown below.

(Equation 1)

[0018]

Example 2 Evidence for Catalytic Activity of Ribozymes 1-4 in vitro hTERT bound to a T7 promoter was transcribed in vitro to contain the T motif sequence and length of nucleotide (nt). RN of 224
A molecule was synthesized (target RNA). During the transfer, the RNA, radiolabeled with P ³² -UTP. Ribozymes were synthesized from ribonucleotides with a 2'-hydroxy group protected with Fpmp [11- (2-fluorophenyl) -4-methoxy-piperidin-1-yl] according to standard methods. They were purified by HPLC and deprotected before use. The target RNA was incubated for 60 or 180 minutes in the presence of ribozyme 1, 2, 3, or 4 or in the absence of ribozyme, and the reaction product was electrophoresed in a polyacrylamide gel. The gel was scanned in a phosphoimager and showed that ribozymes 3 and 4 were highly efficient and ribozymes 1 and 2 were less efficient, splitting the target RNA at the intended site. (FIG. 1).

[0019]

Example 3 << HBL100 (Immortal Breast Epithelial System with High Telomerase Activity) and MCF-
7 (Evidence of telomerase inhibition by stable transfection and expression of ribozyme 4 in breast tumor cell line) Ribozymes mutated in the catalytic center have no effect. Ribozyme 4 (R4
) Or a ribozyme mutR mutated in the catalytic center
Both strands of the cDNA encoding 4 were synthesized as oligodesoxyribonucleotides according to standard methods. They were expressed as expression plasmid pCDNA3.1
Cloned and inserted into MCF-7 (Figure 2A) or HBL-100 cells (
It was stably expressed in FIG. 2B). Telomerase activity in the numbered clones was measured by a semi-quantitative TRAP assay (telomerase repeat amplification protocol). The intensity of the ladder pattern (ladder pattern) in each lane was measured using the control band (
The telomerase activity is measured in comparison to the intensity at (triangle). HBL100 and MCF-7 clones transfected with a ribozyme mutated in the catalytic center (mutR4) showed telomerase activity similar to telomerase activity in parental cells (p = parent cell, R8 = positive control, NC = Negative control). Expression of R4 reduces telomerase activity to <1 and a value between about 30%.

[0020]

Example 4 Evidence for Telomere Shortening in Telomerase-Inhibited Clones Telomere length in MCF-7 (FIG. 3A) and HBL100 (FIG. 3B) cell lines was measured by Southern blot and "Petersen S, Saretzki G, von Zglini
cki T. Exp Cell Res 239, 1998, 152-160 ". The number "n" of clones tested and the corresponding telomerase activity (activity in parent cells / m
"%" of activity in utR4-transfected cells, x-axis). m
Telomere length in utR4 transfected clones or R4 transfected clones, which show only a slight decrease in telomerase activity, does not significantly deviate from telomere length in parental cells. However,
Telomere length in clones where telomerase is clearly inhibited is significantly shorter.

[0021]

Example 5 Evidence for Inhibition of Cell Growth in Telomerase-Inhibited MCF-7 Clones The net growth rate was established by counting cells in each channel. The number "n" of clones tested and the corresponding telomerase activity (activity in parent cells / "% of activity in mutR4 transfected cells", x-axis) are obtained.
In clones where telomerase activity is less than 25% of telomerase activity in parental cells, cell growth is inhibited (FIG. 4A).

<Evidence of Morphological Changes in MCF-7 Clones Inhibiting Telomerase> The morphology of living cells was measured using a reversed-phase contrast microscope (in
Control was performed in a verified phase contrast microscopy. Mutants stably transfected with mutR4 (left)
It shows normal fast growth and the same morphology as the parent cell. Clones in which telomerase is inhibited by R4 expression (right) show limited growth, and in most cases, cells, especially near the edge of relatively small colonies, show a characteristic senescence-like morphology. (The same magnification is used in the left and right figures) (FIG. 4B).

<Test of generation of senescent phenotype by infecting a retrovirus having ribozyme R4 in a large-scale culture of MCF-7. R4 was cloned into a retroviral expression vector (pBabe) and recombinant virus was generated by standard methods. 10 ⁵ each MCF-7 cells were infected with the unmodified vector (left) or R4-pBabe (right). Shown is a large culture two weeks after infection. R4-pBabe infection reduces telomerase activity and also blocks culture growth. Many cells are dead and you can see the cells rounded up without touching the dish. Few surviving cells show the senescent phenotype (left and right panels are at the same magnification) (FIG. 4C).

[0024]

Example 6 << The susceptibility of a clone in which telomerase is inhibited to doxorubicin is 2
HBL100 cells were transfected with R4 or mutR4, respectively. Telomerase activity was measured by TRAP assay. Clone
Treatment with doxorubicin at a concentration of 1000 ng / mL for 3 days. Next, XTT
The assay measured cell viability. As a result, the concentration of doxorubicin at which the XTT signal (colorimetric signal) was reduced by 50% was calculated (LD50). The number of clones tested "n" and the corresponding telomerase activity (activity in parental cells / mut)
"%" Of activity in R4-transfected cells, x-axis). Clones with telomerase activity less than 25% of the parental cells have about a 2-fold lower LD50.
(FIG. 5A).

Estimation of LD50 for doxorubicin in MCF-7 clones (see above) Clones with less than 25% telomerase activity are three to four times more sensitive to doxorubicin (FIG. 5B).

[0026]

Example 7 Evidence of Increased Sensitivity of Telomerase-Negative Cells to Topoisomerase-Inhibiting Cytotoxic Agents Sensitivity to the aforementioned cytotoxic agents was measured as LD50 using an XTT assay. The ratio of LD50 values of telomerase-positive clones to telomerase-negative clones is obtained. mutR4-transfected HBL
100-clones and MCF-7-clones versus R4-transfected HBL100-clones and MCF-7-clones, and hTERT-expressing fibroblasts versus parental BJ fibroblasts were tested (Bodnar AG, Ouellette M, Frolkis M, Ho
ltSE, Chiu CP, Morin GB, Harley CB, Shay JW, Lichtsteiner S, Wright W. S
cience 279, 1998, 349-352). Cells with low telomerase activity were identified as all topoisomerase inhibitors tested (mitozantrone, etoposide, and doxorubicin-
Rompp Chemie Lexikon 1995), which is more sensitive than syngeneic clones with active telomerase but has a different mechanism of action (eg, cisplatin or bleomycin) or the alkylating agent N-methyl. but not with respect to the oxidation challenge with respect -N'- nitro -N- nitrosoguanidine (MNNG), or hydrogen peroxide (H ₂ O ₂₎ (FIG. 6).

[0027]

[Sequence list]

[Procedural Amendment] Submission of translation of Article 34 Amendment

[Submission date] August 21, 2000 (2000.8.21)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Contents of correction]

[Claims]

2. The ribozyme according to claim 1, which cleaves the T motif of human telomerase (hTERT) in the sequence of GUC, GUA, GUU, CUC, or UUC.

3. (a) 5'-GCUCGAC CUGAuGAGuCcGU
GAgGaCGAA ACGUACACA-3 '(b) 5'-GCAGCUC CUGAuGAGuCcGUGAgGaCGAA
ACGACGGUAC-3 '(c) 5'-AAAGAAACUGAuGAGuCcGUGAgGaCGAA
ACCUGAGCA-3 '(d) 5'-UCUCCGU CUGAuGAGuCcGUGAgGaCGGAA
ACAUAAAAAG-3 '(e) 5'-UGCUCACUGAuGAGuCcGUGAgGaCGAA
ACACUCUC-3 '(f) 5'-GACGACCGCUGAuGAGuCcGUGAgGaCGGAA
ACACACUCA-3 '(g) 5'-CUUUGAGACUGAuGAGuCcGUGAgGaCGAA
ACGUGUGUCU-3 '(h) 5'-GCUUGGCCUGAuGAGuCcGUGAgGaCGAA
ACUUGCUCC-3 '(i) 5'-AAGACUCUGAuGAGuCcGUGAgGaCGAA
AGCAGCUCG-3 '(j) 5'-UGUUUU CUGAuGAGuCcGUGAgGaCGAA
AGCCUGUUC-3 '(k) 5'-CAUAAAAACUGAuGAGuCcGUGAgGaCGAA
AAAGACCUG-3 '(1) 5'-UUCUUU CUGAuGAGuCcGUGAgGaCGAA
AAACGUGGU-3 '(m) 5'-UCCGGUA CUGAuGAGuCcGUGAgGaCGAA
A hammerhead ribozyme having a sequence represented by AAAAAGAGC-3 ', the ribozyme according to claims 1 and 2.

4. A ribozyme according to claim 1-3, used as a telomerase inhibitor.

5. A ribozyme according to claim 1 to 3, used to telomere shortening.

6. cytostatic or cytotoxic agents, and / or the in combination with radiation, the ribozyme according to claim 1 to 3, used to telomere shortening.

The combination with inhibition of 7. Topoisomerase, ribozyme according to claim 1 to 3, used to telomere shortening.

8. Use according to claim 6 or 7 for promoting telomere shortening due to DNA damage and / or for removing repair-related properties of telomerase.

9. ribozyme according to claim 1-8, used to tumor therapy.

10. Use according to claim 9 , for increasing the sensitivity of tumor cells to cytostatic or cytotoxic agents.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 43/00 111 A61P 43/00 111 C12N 9/99 C12N 9/99 // C12N 15/09 15/00 A (72) Inventor Zaretzki Gabriel, Germany D-10318, Ahrlichstrasse 22 (72) Inventor Ludwig Anche, Germany, Berlin D-13059, Kreichzerstrasse 20 F-term (reference) 4B024 AA01 BA80 CA04 DA02 EA04 FA02 GA11 4B050 CC03 CC04 CC07 LL01 4C084 AA13 AA19 MA02 NA05 NA14 ZB21 ZB26 ZC02 4C086 AA01 AA02 AA03 AA04 EA16 MA01 MA02 MA04 NA05 NA14 ZB21 ZB26 ZC02

Claims (11)

[Claims] 1. A ribozyme which reduces the activity of the catalytic subunit of human telomerase hTERT (human telomerase enzyme reverse transcriptase). 2. The ribozyme according to claim 1, which targets the T motif of human telomerase. 3. The ribozyme according to claim 1, which cleaves a human telomerase T motif (hTERT) in a GUC, GUA, GUU, CUC, or UUC sequence. 4. (a) 5′-GCUCGAC CUGAuGAGuCcGU
GAgGaCGAA ACGUACACA-3 '(b) 5'-GCAGCUC CUGAuGAGuCcGUGAgGaCGAA
ACGACGGUAC-3 '(c) 5'-AAAGAAACUGAuGAGuCcGUGAgGaCGAA
ACCUGAGCA-3 '(d) 5'-UCUCCGU CUGAuGAGuCcGUGAgGaCGGAA
ACAUAAAAAG-3 '(e) 5'-UGCUCACUGAuGAGuCcGUGAgGaCGAA
ACACUCUC-3 '(f) 5'-GACGACCGCUGAuGAGuCcGUGAgGaCGGAA
ACACACUCA-3 '(g) 5'-CUUUGAGACUGAuGAGuCcGUGAgGaCGAA
ACGUGUGUCU-3 '(h) 5'-GCUUGGCCUGAuGAGuCcGUGAgGaCGAA
ACUUGCUCC-3 '(i) 5'-AAGACUCUGAuGAGuCcGUGAgGaCGAA
AGCAGCUCG-3 '(j) 5'-UGUUUU CUGAuGAGuCcGUGAgGaCGAA
AGCCUGUUC-3 '(k) 5'-CAUAAAAACUGAuGAGuCcGUGAgGaCGAA
AAAGACCUG-3 '(1) 5'-UUCUUU CUGAuGAGuCcGUGAgGaCGAA
AAACGUGGU-3 '(m) 5'-UCCGGUA CUGAuGAGuCcGUGAgGaCGAA
The ribozyme according to any one of claims 1 to 3, which is a hammerhead ribozyme having a sequence represented by AAAAAGAGC-3 '. 5. Use of the ribozyme according to claim 1 as a telomerase inhibitor. 6. Use of the ribozyme according to claim 1 for telomere shortening. 7. Use of the ribozyme according to claims 1 to 4 for shortening telomeres in combination with a cytostatic or cytotoxic agent and / or irradiation. 8. Use of a ribozyme according to claims 1-4 for telomere shortening in combination with topoisomerase inhibition. 9. Use according to claim 7 or 8 for promoting telomere shortening due to DNA damage and / or for removing the repair-related properties of telomerase. 10. Use of the ribozyme according to claim 1 for treating a tumor. 11. Use according to claims 1 to 4 for increasing the sensitivity of tumor cells to cytostatic or cytotoxic agents.