ITMI20012367A1 - ANTISENSE OLIGONUCLEOTIDES THAT REGULATE THE EXPRESSION OF THE ANTIAPOPTOTIC GENE BCL-2 - Google Patents

Description

Description of the patent for industrial invention entitled:

"ANTISENSE OLIGONUCLEOTIDES THAT REGULATE THE EXPRESSION OF THE ANTI-APOPTOTIC GENE BCL-2"

The present invention refers to chemically modified antisense oligonucleotides and their use in modulating the expression of the antiapoptotic gene bcl-2. More precisely, the invention provides RNA-mimetic oligonucleotides complementary to the region rich in adenine and uracil (ARE) present in the bcl-2 messenger RNA (Schiavone et al., 2000).

The bcl-2 gene encodes the multifunctional protein BCL-2 known to be involved in the control of cell growth and differentiation and in the prevention of death by apoptosis (Tsujimoto and Shimizu, 2000). In line with its functional role, the modulation of bcl-2 expression, both in the sense of an increase and in the sense of a decrease, has impressive effects on the fate of the cell. An increase in BCL-2 levels induced by cytokines, such as interleukin 5 (IL-5), IL-6, IL-7, interferon a (IFNa) (Ochiai K. et al., 1997; Teague T.K. et al ., 1997; Hernandez-Caselles T. et al.

1995; Jewell A. P. et al., 1994) and other molecules signaled such as bFGF and TNF-α (Konig et al., 1997; Genestier et al. 1995) is associated with an increase in cell survival and protection from apoptosis in hematopoietic cell lines . Similarly, the expression of viral and cellular genes, such as EBV LMP-1, c-myb, and K-ras, protects the cell from death by increasing BCL-2 levels (Henderson S. et al., 1991; Salomoni P. et al., 1997; Fan J. and Bertino J.R., 1997). Conversely, the removal of IL-2 or IL-3 from the medium of cytokine-dependent cell lines, as well as the activation of the signal translated by the Fas receptor or the over-expression of the tumor suppressor gene p53 lead to a lowering of BCL-2 levels ( Suzuki A. et al., 1996; Miyashita T. et al., 1994). This is invariably associated with apoptosis and increased susceptibility to apoptotic agents. The link between lowering of BCL-2 levels and cell death due to apoptosis is supported by numerous scientific articles that indicate how a variety of damaging stimuli exert their effect in vitro and in vivo through this mechanism.

The control of bcl-2 expression has been the subject of numerous studies that have revealed regulatory mechanisms operating at the transcriptional level (Young R.L. et al., 1993; Miyashita T. et al., 1994), translational (Harigai M. et al., al., 1996) and post-translational (Blagosklonny M.V. et al., 1997; haldar S. et al., 1996).

Numerous experimental evidences indicate that the expression of bcl-2 can be modulated at both the transcriptional and post-transcriptional level and that post-transcriptional control can affect both messenger RNA and protein. In addition to the transcriptional regulation exerted at the promoter level (Young et al., 1993), two estrogen responsive elements have recently been characterized which are located within the coding region of the gene (Perillo et al., 2000) and a mechanism of post-transcriptional regulation mediated by phosphorylation of the BCL-2 protein in different amino acid positions (Breitschopf et al., 2000; Huang et al., 1999).

A new mechanism for post-transcriptional regulation of bcl-2 expression has been identified in our laboratory (Schiavone et al., 2000). This is based on a negative regulatory cis element rich in Adenine and Uracili (A + U rich element, ARE) located in the 3 'non-coding region (3'-UTR) of the bcl-2 mRNA (Schiavone et al. , 2000). Compared to ARE of other genes such as c-fos or GM-CSF (Chen CYA and Shyu A-.B., 1995), TARE of bcl-2 possesses relatively modest basal destabilizing activity but which increases dramatically in response to apoptotic stimuli (Schiavone et al., 2000).

Given the importance that the BCL-2 protein plays in the control of apoptotic mechanisms, numerous studies have been directed to the modulation of its expression using antisense technology. Bcl-2 inactivation experiments using antisense oligonucleotides have shown that reducing the expression of this gene below a critical threshold is in itself capable of inducing apoptosis (Ziegler A. et al., 1997). US 6291668 proposes the use of oligonucleotides directed against the bcl-2 mRNA, in particular against the terminal coding region between nucleotides 1880 and 2194, for the treatment of pathological states associated with the biosynthesis of the BCL-2 protein. In patent publication WO96 / 27663 a chimeric antisense transcript is described which hybridizes with the pre-mRNA of the bcl-2 hybrid gene! IgH. This transcript contributes to the overexpression of the bcl-2 gene in follicular lymphoma cells carrying the t (14; 18) translocation. The same publication describes complementary oligonucleotides to the antisense transcript bcl-2! IgH, able to inhibit its function.

It has now been found that the expression of the BCL-2 protein can be modulated using particular antisense oligonucleotides directed against the adenine and uracil rich region (ARE) located in the non-coding 3 '(3'-UTR) portion of the mRNA of bcl-2. In particular, it has been seen that complementary oligonucleotides to the ARE of bcl-2, bearing appropriate chemical modifications, determine a significant increase in the levels of the mRNA of bcl-2, and therefore of the corresponding protein, thus determining an increase in the apoptotic threshold. , with consequent benefit for all pathological conditions related to an excess of apoptotic phenomena. As used herein, the expression "complementary oligonucleotides to the ARE of bcl-2" <1> indicates oligonucleotides that can pair up to the entire ARE region or to a portion of it of variable length.

These oligonucleotides, object of the present invention, are chemically modified by introducing a C1-C3 alkyl group, preferably a methyl group, in position 2 ', i.e. in substitution of the hydrogen atom present in the hydroxyl linked to the C2 of the furan ring (Johansson HE et al., 1994). These 2'-0-alkyloligonucleotides can in turn be modified by adding transplatin (Boudvillain M et al., 1997). Alternatively, the oligonucleotides of the invention can have both 2'-deoxy- and 2'-methyl-modified residues in their sequence, thus generating 2'-0-methyl / deoxy haparmers (Cramer H et al., 2000) . According to a preferred embodiment, 2'-0-methyloligoribonucleotides are used (Figure 2).

The 2-modified oligonucleotides according to the invention are known as RNA-mimetic oligodeoxyribonucleotides. Characteristics common to this class of oligoneucleotides are the high resistance to the degradation of the corresponding mRNA by endo and exonucleases, the high affinity for the target region, the absence of inhibition of the translation of mRNA into protein, the high capacity to penetrate inside the cell and, unlike normal antisense deoxyribonucleotides, the inability to induce cleavage of complemented mRNA following activation of Rnase H (Beban M. et al., 2000; Iribarren AM et al., 1994; Iribarren AM et al., 1990; Kuznetsova SA et al., 1996; Lapham J. Et al., 1997; Schmitz JC et al., 2001; Shirohzu H. et al., 2000; Sproat BS et al., 1989; Zbigniew D. et al., 1996: Dominski Z. et al., 1996; Majlessi M et al., 1998; Shohami E. et al., 2000; Ushijima K et al, 1999; Beban M et al, 2000 ).

The target region of the bcl-2 mRNA (ARE) affected by the binding of the oligonucleotides according to the invention corresponds to the section between nucleotides 936 and 1021 of the bcl-2 cDNA sequence published in GenBank under access number M 13994 (Figure 1 - see also Tsujimoto and Croce, 1986, and Schiavone et al., 2000), where "mRNA region corresponding to a tract of the bcl-2 cDNA" obviously means the sequence of ribonucleic acid transcribed by cDNA in question. In addition, even the regions flanking the ARE may be involved in the binding of oligonucleotides, for a maximum extension of 100 nucleotides starting from the extremes of the AER indicated above. Therefore, the oligonucleotides of the invention may occupy a bcl-2 mRNA sequence comprising at least one fragment of the ARE and a portion of the flanking sequence.

Preferably, the oligonucleotides according to the invention have a length ranging from 10 to 50 nucleotides, more preferably from 13 to 30.

According to a preferred embodiment, such RNA-mimetic oligonucleotides are selected from the group comprising:

p

nucleotide units may have others, in different functional domains of the molecule, in order to improve the stability or in vivo activity of the oligonucleotides, without affecting their biological effectiveness. For example, oligonucleotides bearing phosphorothioate, phosphorodithioate, phosphotriesters, aminoalkylphosphotriesters, alkyl phosphonates, phosphates, phosphoroamidates, thionophosphoroamidates, thionoalkylphosphonates or phosphotriesters groups can be provided. Other modifications can affect the nucleotide skeleton, i.e. the sugar part and the intemucleoside bond, or the nucleobases, by introducing purines or pyrimidines variously substituted on the heterocyclic rings, for example with alkyl, hydroxy or halo-alkyl, halogen, hydroxyl groups , sulfide, amino, aza and others. Furthermore, the oligonucleotides of the invention can be conjugated to different groups or functions capable of increasing their activity, distribution or cell uptake. Such groups or functions can be lipids, aliphatic chains, polyethylene glycol chains, polyamines and phospholipids. According to a preferred embodiment, the oligonucleotides will be conjugated with a lipid, as described in W090 / 10448, in order to facilitate their transport through the membranes and allow the subsequent release of the active molecule by the cytoplasmic enzymes.

RNA-mimetic oligonucleotides modified in position 2 of ribose can be prepared as described in Cramer H. et al. (2000) and Dominski Z, et al. (1996). The methods and procedures generally adopted for the synthesis of oligonucleotides are instead described in Narang A. (Tetrahedron 39: 3, 1983), Itakura K. (Synthesis and use of synthetic oligonucleotides. Ann Rev Biochem 53: 323, 1984) and in “Oligonucleotides Synthesis; A Practical Approach ”(Gait M. J. Ed. IRL Press, Oxford, UK, 1984).

Another aspect of the invention relates to pharmaceutical compositions containing one or more oligonucleotides of the invention. The latter, in free or salified form with metal cations such as sodium, potassium, magnesium, calcium, or with organic bases, preferably amines, will be suitably formulated using pharmaceutically acceptable carriers and excipients. Preferred pharmaceutical forms are those for oral and parenteral administration and include solutions, suspensions, powders, granules, capsules, tablets. These and others can be prepared with techniques known to the expert in the field, for example as described in Remington's Pharmaceutical Sciences Handbook (Mack Pub. Co. XVII ed. NY, USA). According to a preferred embodiment, the oligonucleotides are formulated with liposomes, in particular with cationic liposomes such as DOTAP, DOGS, DOTMA, DOPE (Misterova J. et al., 2001), possibly with the use of agents that favor cellular penetration such as acids fats, mono- and diglycerides, acylcamitine, acylcholine.

The compositions of the invention are used in the treatment of pathological processes in which an excess of apoptosis is the main pathogenetic mechanism, as well as other pathological processes related to low levels of bcl-2 expression. These processes include, for example, states of hypoxia or toxicity in response to cytotoxic treatments (Lasorella A, 1995), or certain neurological pathologies, such as ischemic damage, Alzheimer's disease, Parkinson's disease, Huntington's chorea, lateral amyotrophic sclerosis (Yuan J and Yankner B.A., 2000 Sastry PS t al., 2000; Yuan J and Yankner BA, 2000) and ophthalmological (McKinnon SJ. 1997; Capaccioli S. et al., 1998; Carella G. et al. , 1998; Spaeth GL. Et al., 1998; Reme CE et al. 1998; Nickells RW. Et al., 1999; Osbome NN. Et al., 1999; Farkas RH. Et al., 2001; Wilson SE. Et al., 1999).

The compositions of the invention can contain from 0.1 to 100 mg of one or more oligonucleotides and can be administered from 1 to 4 times a day, depending on the specific pathology, the general health conditions of the patient and other factors depending on the doctor's judgment.

The invention is illustrated by the following examples.

EXAMPLE 1 - Synthesis of oligonucleotides and their conjugation with DOTAP

Oligonucleotides having the following sequence were synthesized using a DNA synthesizer.

These oligonucleotides consist of 2'-0-methyldeoxyribosyl residues and are prepared as indicated in Zbigniew D. et al., 1996. The three ODNs are purified in HPLC, pre-incubated at 37 ° C for 15 minutes in the presence of DOTAP (13 μΜ final) and applied in culture medium in final concentrations of 5 μΜ each ODN, i.e. 15 μΜ total.

EXAMPLE 2 - Treatment of cell cultures with the oligonucleotides of the invention

Neuronal cells from a neuroblastoma cell line (SH-SY5Y) were used. These cells are commercially available from the Primm Company (Milan). The cells are grown in Ham's F12 culture medium: MEM 1: 1, with the addition of fetal bovine serum at 15%, NEAA 1%, glutamine 1%, in an atmosphere of 5% C02 / 95% air. These cells are characterized by the fact that the acquisition of the differentiated phenotype (reduction of the diameter of the pyrenophore, increase in the number and length of neurofilaments, block or reduction of cell proliferation) is associated with the modification of the following quantifiable biochemical parameters: 1) increase in neuron-specific enolase (NSE) 2) increase in growthassociated protein-43 (GAP-43) 3) increase in neuropeptide Y levels 4) increase in vesicular proteins (synaptin / synaptophysin, secretogranin II, SV2).

The cells were seeded in 10cm diameter plates containing the above mentioned culture medium. The ODN1 ODN2 ODN3 complex conjugated with DOTAP as indicated in example 1 was added to the culture medium in order to obtain a final concentration of 5 μΜ for each molecular species (15 μΜ total). The plates were incubated at 37 ° C for 5 days. Considering some articles related to the administration of 2'-0-methyl ribonucleotides (Beban M. et al., 2000; we administered our ODNs according to the following scheme: a first application 24 hours after sowing (time 0) and a second application on the third day using half of the initial dose (ie at the final concentration of 2.5 μΜ each).

The following controls have been set up:

- SH-SY5Y cells treated with 10 μΜ trans-retinoic acid, known inducer of neuronal differentiation associated with upregulation of bcl-2 (Lasorella A. Cancer Research 55: 471 1-4716, 1995);

- SH-SY5Y cells treated with the oligonucleotide of the completely degenerated sequence reported above and at a concentration of 15 μΜ conjugated with DOTAP 13 μΜ.

At various times from the application of the oligonucleotides, the cells were analyzed for the following parameters:

- morphology under the optical microscope (for the detection of the differentiated state);

- growth rate;

- number of apoptotic events in basal conditions;

- number of apoptotic events in response to cytotoxic stimuli (cisplatin and antimycin A);

- cellular levels of bcl-2 mRNA by quantitative RT-PCR; - cellular levels of BCL-2 protein by Western blotting.

Methods

Quantitative RT-PCR mediated analysis of bcl-2 mRNA levels.

The levels of bcl-2 mRNA, both in basal conditions and after treatment with the protective ODNs, were determined by a quantitative RT-PCR method already described in our other works (Quattrone et al. 1995; Schiavone et al., 2000 ) using Storm Phosphorlmager and Image QuaNT (Molecular Dynamics) software. Briefly, total TRNA (1 pg) extracted from untreated cells or cells treated with protective ODNs carried by the cationic lipid DOTAP was back-transcribed to cDNA in the presence of random hexamers. Increasing quotas of cDNA were amplified by PCR (denaturation at 94 ° for 2 min, annealing at 58 ° for 1 min, extension at 72 ° for 2 min. For 25 cycles in 50 μΐ of final volume. The gene was used as internal standard. housekeeping of β-actin. The PCR amplifiers were analyzed on agarose gel. To obtain the quantitative values, only the bands included in the linearity range between the volume of cDNA and the amount of amplified were used.

Analysis of BCL-2 protein levels by Western blotting.

The bcl-2 mRNA levels, both in basal conditions and after treatment with the protective ODNs, were determined by the classic Western blotting method using an anti-Bcl-2 monoclonal antibody (Santa Cruz Biotechnology Ine.).

Counting of live cells, analysis of cell morphology and cumulative apoptotic and mitotic events. .

Live cells were counted by the trypan blue exclusion test. Cell morphology was analyzed by Zeiss inverted phase microscope with 10X objective. Cumulative apoptotic events were assessed by dynamic examination of cell cultures at time-lapse videomicroscopy using a Zeiss inverted phase microscope with 10X objective, a Panasonic CCD video camera and a JVC BR9030 time-lapse recorder. Each apoptotic event was counted as previously reported (Schiavone N. et al., 2000), i.e. at the moment when the cell, detached from the bottom of the plate and imploded into itself, was forming cytoplasmic vesicles and was dividing into subcellular fragments (apoptotic bodies) characteristic of apoptosis (Evan et al. 1992).

Results

- Treatment of SH-SY5Y cells with the three 2'-0-methyloligonucleotides (ODN1 + ODN2 + ODN3) complementary to the bcl-2 ARE increases the bcl-2 mRNA levels evaluated by quantitative RT-PCR (Figure 3 ).

- The treatment of SH-SY5Y cells with the three 2'-0-methyloligonucleotides (ODN1 + ODN2 + ODN3) complementary to the ARE of bcl-2 increases the levels of BCL-2 protein evaluated by Western blotting (Figure 4).

- The treatment of SH-SY5Y cells with the three 2 '-O-methyloligonucleotides (ODN1 + ODN2 + ODN3) complementary to the ARE of bcl-2 induces the appearance of differentiated neuronal phenotype (Figure 5). - The treatment of SH-SY5Y cells with the three 2'-0-methyloligonucleotides (ODN1 + ODN2 + ODN3) complementary to the ARE of bcl-2 slows down cell proliferation (Figure 6).

The treatment of SH-SY5Y cells with the three 2'-0-methyloligonucleotides (ODN1 + ODN2 + ODN3) complementary to the ARE of bcl-2 lowers the number of apoptotic events detectable in Time-lapse Videomicroscopy in response to treatment with apoptotic stimuli (cis-Platinum and hypoxia).

Claims (12)

CLAIMS 1. Antisense oligonucleotides selected from 2'-0-aIchyl (Cl-C3) oligoribonucleotides and 2'-0-methyl / deoxy haemers complementary to the bcl-2 mRNA in the region between nucleotides 936 and 1021 of the cDNA published in GenBank at access number M 13994. 2. Antisense oligonucleotides according to claim 1, which are 2'-0-methyloligoribonucleotides. 3. Oligonucleotides according to claims 1-2, extended to the regions supporting said sequence interval up to a maximum of 100 nucleotides from the indicated extremes. 4. Oligonucleotides according to claims 1-3, containing from 10 to 50 nucleotides. 5. Oligonucleotides according to claim 4, containing 13 to 30 nucleotides. 6. Oligonucleotides according to claims 2 and 5, selected from the group comprising: 7. Oligonucleotides according to claims 1-6, for use as a medicament. 8. Pharmaceutical composition containing an oligonucleotide according to claims 1-6. 9. Pharmaceutical composition according to claim 8, in liposomal form. 10. Composition according to claim 9, wherein the liposome is selected from DOTAP, DOGS, DOTMA, DOPE. 11. Use of an oligonucleotide according to claims 1-7 for the preparation of a therapeutic agent for the treatment of pathologies in which a reduced expression of the BCL-2 protein is involved. Use according to claim 11 for the treatment of ophthalmic diseases, toxicity due to cytotoxic agents, damage due to hypoxia, Alzheimer's disease, Parkinson's disease, Huntington's chorea, amyotrophic lateral sclerosis.