WO2003040182A1

WO2003040182A1 - Antisense oligonucleotides modulating bcl-2 expression

Info

Publication number: WO2003040182A1
Application number: PCT/EP2002/012502
Authority: WO
Inventors: Sergio Capaccioli; Laura Papucci; Nicola Schiavone; Martino Donnini; Andrea Lapucci; Alessio Tempestini; Rosario Brancato
Original assignee: Visufarma S.R.L.
Priority date: 2001-11-09
Filing date: 2002-11-08
Publication date: 2003-05-15
Also published as: ITMI20012367A1

Abstract

Antisense oligonucleotides targeting the ARE region of bcl-2 mRNA, pharmaceutical compositions containing the same and uses thereof as therapeutic agents.

Description

I ANTISENSE OLIGONUCLEOTIDES MODULATING BCL-2 EXPRESSION

The present invention regards antisense oligonucleotides and their use as modulators of bcl-2 gene expression. More specifically, the invention provides "RNA-mimetic" oligonucleotides complementary to the Adenine and Uracil Rich Element (A + U-rich Element, ARE) in the 3 ' untranslated region (3'UTR) of bcl-2 messenger RNA and pharmaceutical compositions containing the same for the treatment or prevention of apoptosis-related diseases.

Background of the invention

The bcl-2 gene encodes for the multifunctional BCL-2 protein known to be involved in cell growth and differentiation and in apoptosis prevention (Tsujimoto and Shimizu, 2000). According to its functional role, up- and down-modulation of bcl-2 expression has important effects on the cell. The increase of BCL-2 levels induced by cytokines, such as interleukin 5 (IL-5), IL-6, EL-7, interferon-α (IFN-α) (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 signaling molecules, such as bFGF and TNF-α (Konig et al., 1997; Genestier et al., 1995), is associated with increased cell survival and protection from apoptosis in haemopoietic cell lines. Expression of viral and cellular genes such as LMP-1 of EBV, c-myb and K-ras also protects cell from death by increasing BCL-2 levels (Henderson S. et al., 1991 ; Salomon P. et al., 1997; Fan J. and Bertino J. R., 1997). Conversely, removal of IL-2 or IL-3 from medium of cytokine- dependent cell lines, as well as Fas-receptor signal pathway activation or oncosuppressor p-53 gene overexpression, cause the decrease of BCL-2 levels (Suzuki A. et al., 1996; Miyashita T. et al, 1994). Up and down-modulation of BCL-2 is always associated with apoptosis and/or increased susceptibility to apoptotic agents. A number of scientific articles report that decreased BCL-2 levels are associated with apoptotic cell death in response to a variety of injurious stimuli in vitro and in vivo.

Modulation of bcl-2 expression has been the object of a number of studies demonstrating that it occurs at transcriptional (Young R. L. et al.,

1993; Miyashita T. et al., 1994), translational (Harigai M. et al., 1996) and post-translational levels (Blagosklonny M. V. et al., 1997; Haldar S. et al.,

1996).

A number of experimental evidences indicate that bcl-2 expression may be modulated either at transcriptional or at post-transcriptional level and that the post-transcriptional modulation may be exerted both at the mRNA and protein levels. Besides the transcriptional regulation operated by the promoter (Young et al., 1993), recently two estrogen responsive element have been characterized in the coding region of bcl-2 (Perillo et al., 2000) and a mechanism of post-transcriptional regulation mediated by BCL-2 phosphorylation at different aminoacidic positions has been described (Breitschopf et al., 2000; Huang et al., 1999).

A new mechanism of post-transcriptional regulation of bcl-2 expression has been recently identified (Schiavone et al., 2000; Donnini et al. 2001, Lapucci et al. 2002, Luzi et al, 2002 in press). This mechanism involves an Adenine- and Uracil-rich element (A+U rich element, ARE) in the 3' untranslated region (3 '-UTR) of bcl-2 mRNA (named "bctt ARE") with destabilizing activity (Schiavone et al., 2000). In comparison with AREs of other genes, such as c-fos or GM-CSF (Winstall et al., 1995), the destabilizing activity of bcl-2 ARE is relatively scarce in basal condition but dramatically increases in response to apoptotic stimuli: this leads to increased bcl-2 mRNA degradation and, consequently, bcl-2 gene downregulation (Schiavone et al., 2000). In addition, targeting the bcl-2 ARE with a synthetic hammerhead ribozyme was shown to downregulate bcl-2 expression in lymphoma cells (Luzi et al., 2002 in press).

Given the key role exerted by BCL-2 protein in apoptosis prevention, a number of studies have been focused on the modulation of bcl-2 expression by the antisense technology. Experiments of bcl-2 inactivation by antisense oligonucleotides have demonstrated that the decrease of bcl-2 expression under a critical threshold is able to induce apoptosis (Ziegler A. et al., 1997). Prior art US 6,291,668 proposes the use of oligonucleotides targeting bcl-2 mRNA, precisely the terminal segment of its coding region between nucleotides 1880 and 2194, for treatment of pathological conditions related to excessive biosynthesis of BCL-2 protein. W096/27663 describes a chimeric antisense transcript complementary to the bcl-2/lgH hybrid pre-mRNA. This transcript contributes to bcl-2 gene overexpression in follicular lymphoma cells with 14; 18 translocation. In the same patent publication, synthetic oligonucleotides targeting the bcl-2/ϊgK antisense transcript and inhibiting its bcl-2- upregulating function are described. Description of the invention

It has now been found that bcl-2 gene expression can be modulated by specific, chemically-modified antisense oligonucleotides targeting the Adenine and Uracil-rich Element (ARE) in the 3' untranslated region (3'- UTR) of bcl-2 mRNA (bcl-2 ARE). In particular, bcl-2 ARE-targeting oligonucleotides with suitable chemical modifications were found to significantly increase bcl-2 mRNA and BCL-2 protein levels elevating the apoptotic threshold with consequent benefit for all pathological conditions that are related to excessive apoptosis.

As used herein, the expression "bcl-2 ARE-targeting oligonucleotides" means oligonucleotides that are able to match either the entire ARE region or ARE segments of various length.

The antisense oligonucleotides of this invention chemically are 2'-0- (Cι -C₃)alkyloligoribonucleotides, where nucleotides carry a (C₁-C₃)alkyl group, preferably a methyl group, linked by an ether bond to the hydroxyl at ribose-C2 (Johansson H. E. et al. 1994). Alternatively, the oligonucleotides sequences may contain either 2'-0-(C₁-C₃)alkylribonucleotides or 2'- deoxyribonucleotides, thus generating 2'-0-methyl/deoxy gapmers (Cramer H. et al., 2000). In a further alternative, the 2'-0-(C C₃)alkyloligoribonucleotides can be modified by addition of transplatin (Boudvillain M. et al., 1997). According to a preferred embodiment, 2'-0- methyloligoribonucleotides are used (Figure 1).

The oligonucleotides of the invention are known as RNA-mimetic oligodeoxiribonucleotides. Common characteristics of this class of oligonucleotides are high resistance to degradation by endo- and eso- nucleases, high affinity for the targeted region, absence of translation inhibition of the targeted mRNA, high uptake by cells and, in contrast to normal antisense deoxyribonucleotides, incapacity to induce cleavage of complemented mRNA by activation of RNAse H (Beban M. et al., 2000; Iribarren A. M. et al., 1994; Iribarren A. M. et al., 1990; Kuznetsova S. A. et al.,1996; Lapham J. et al., 1997; Schmitz J. C. et al., 2001; Shirohzu H. et al., 2000; Sproat B. S. 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; al., Beban M. et al., 1994). According to the invention, the bcl-2 mRNA region (bcl-2 ARE) targeted by oligonucleotides extends from nucleotide 936 to 1050 of bcl-2 cDNA sequence (GenBank accession number M13994 - see also Tsujimoto and Croce, 1986; and Schiavone et al., 2000). In addition, ARE flanking regions can also be targeted by the oligonucleotides for a maximal extension of 100 nucleotides up- and downstream of the ARE region. In this case the oligonucleotides are complementary to a bcl-2 mRNA sequence including at least an ARE segment and a tract of the flanking sequence.

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

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

1. 5 ' -TGTCTT A A AT AA AT AAATCTTTTTTTC-3 '

2. 5 ' -TTAATAATGTAAAAAATAAATGATAT-3 ' 3. 5 ' -TTCCCTTTGGC AGT AAATAGCTGATT-3 '

4. 5'-GATTTCCTTTGACGGTGTTTTGATGGGTC-3 '.

In addition to the chemical-modifications described above, single nucleotides may present different modifications in the sugar moiety to improve the in vitro/in vivo stability and activity of the oligonucleotides without impairing their biological efficacy. For example, the (oligo)nucleotides may contain phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, alkyl phosphonates, phosphinates, phosphoroamidates, thionophosphoroamidates, thionoalkylphosphonates or phosphotriesters groups. Other modifications may regard the nucleotidic backbone, particularly the sugar moiety, the internucleosidic bond or the purine or pyrimidine bases, e.g. by introducing purines and pyrimidines variously substituted on the heterocyclic rings, for example by alkyl, hydroxy- or halo-alkyl, halogen, hydroxyl, sulfur, amino or aza groups. Moreover, the oligonucleotides of the invention can be conjugated with different groups or functionalities able to increase their activity, distribution or cellular uptake. Such groups or functionalities include lipids, aliphatic chains, poliethilenglycol chains, polyamines and phospholipids. According to a preferred embodiment, the oligonucleotides are conjugated with lipids, as described in O90/10448, in order to enhance their transport through membranes and to enable subsequent release of active molecules by means of cytoplasmic enzymes.

The RNA-mimetic oligonucleotides carrying a 2 '-modified ribose can be prepared as described in Cramer H. et al. (2000) and Dominski Z. et al. (1996). Methods and procedures generally utilized for oligonucleotides synthesis are 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).

In a further embodiment, the invention is directed to pharmaceutical compositions containing one or more oligonucleotides herein provided. The oligonucleotides, free or salified with metal cations such as sodium, potassium, magnesium, calcium, or with organic bases, preferably ammines, are suitably formulated with pharmaceutically acceptable vehicles and excipients. The pharmaceutical preparations are preferably administered by the oral or parenteral routes in the form of solutions, suspensions, powders, granules, capsules, tablets, and can be prepared with techniques known to one skilled in the art (see for example Remington's Pharmaceutical Science Handbook (Mack Pub. Co. XVII ed. NY, USA). According to a preferred embodiment, the oligonucleotides are formulated with liposomes, in particular with cationic lipids such as DOTAP, DOGS, DOTMA, DOPE (Misterova J. et al., 2001), optionally with agents that enhance cellular uptake such as fatty acids, mono- and diglicerides, acylcarnitine, acylcoline. The oligonucleotide compositions can be used in the treatment of pathological conditions that involve excessive apoptosis or that are related to a low bcl-2 expression. Diseases that may benefit from such a treatment include hypoxic or toxic conditions consequent to cytotoxic treatments (Lasorella A., 1995), some neurological pathologies such as ischemic damage, Alzheimer disease, Parkinson disease, Huntington chorea, lateral amiotrophic sclerosis (Yuan J. and Yankner B. A., 2000; Sastry P. S. et al., 2000) and several ophthalmologic diseases (McKinnon S. J., 1997; Capaccioli S. et al., 1998; Carella G. et al., 1998; Spaeth G. L. et al., 1998; Reme C. E. et al., 1998; Nickells R. W. et al., 1999; Osborne N. N. et al., 1999; Farkas R. H. et al., 2001; Wilson S. E. et al., 1999).

The therapeutic compositions may contain from 0,1 to 150 mg of one or more oligonucleotides and will be generally administered from 1 to 4 times a day, depending on the specific disease under treatment and on the selected administration route.

In a further embodiment, the antisense oligonucleotides of the invention are used as research tools for in vitro modulation of bcl-2 expression. The following examples illustrate the invention. EXAMPLE 1 - Oligonucleotide synthesis and conjugation with DOTAP

Using a DNA synthesizer, the following 2'-0-methylribonucleotides were prepared:

1 ) ODN 1 5 ' -TGTCTT AAAT AAAT AAATCTTTTTTTC-3 '

2) ODN2 5 ' -TT AAT AATGT AAA A A AT AAATG ATAT-3 ' 3) ODN3 5'-TTCCCTTTGGCAGTAAATAGCTGATT-3'

4) ODN4 5 ' -GATTTCCTTTGACGGTGTTTTGATGGGTC-3 ' .

The 2'-0-methylribosilic residues were prepared according to Zbigniew D. et al., 1996. The ODNs were purified by HPLC, preincubated at 37°C for 15 min in the presence of DOTAP (13 μM final) and applied to the culture medium at final concentration of 5 μM each, i.e. 20 μM total. EXAMPLE 2 - Treatment of cell cultures with ODNs

SH-SY5Y neuroblastoma cells (Primm - Milan) were cultured in Ham's F12:MEM 1 : 1 medium supplemented with 15% fetal bovine serum, 1% NEAA, 1% glutamine, in a 5% C0₂/95% air atmosphere, in 10cm plates. The acquisition of the differentiated neuronal phenotype, which is characterized by decrease of the pyrenophore diameter, increase of neurofilament number and length, arrest or reduction of cellular proliferation, is associated to an increase of neuron-specific enolase (NSE), growth-associated protein-43 (GAP-43), neuropeptide Y and vesicular proteins (sinaptine/sinaptophysine, secretogranine II, SV2).

A mixture of the oligonucleotides ODN1 + ODN2 + ODN3 + ODN4 conjugated to DOTAP (Example 1) was added to the culture medium at a final concentration of 5 μM for each molecular species (20 μM total). Plates were incubated at 37°C for 5 days. According to the relevant scientific literature (Beban M. et al., 2000; Iribarren A. M. et al., 1994; Iribarren A. M. et al., 1990; Kuznetsova S. A. et al., 1996; Lapham J. et al., 1997; Schmitz J. C. et al., 2001; Shirohzu H. et al., 2000; Sproat B. S. 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), ODNs were administered as follows: a first application 24 h after seeding (time 0) and a second application on the third day using half of the initial dose (final concentration of 2,5 μM for each ODN). The following controls were set up:

• SH-SY5Y cells treated with 10 μM all-trans retinoic acid, which induces neuronal differentiation determining bcl-2 upregulation (Lasorella A. Cancer Research 55: 4711-4716, 1995);

• SH-SY5Y cells treated with fully degenerated 30mer oligonucleotides conjugated with DOTAP 13 μM, at 20 μM concentration.

At various times after oligonucleotide administration, the cells were analyzed for the following parameters: • cell morphology at light microscopy (detection of differentiated phenotype);

• cell growth;

• number of apoptotic events under basal conditions; • number of apoptotic events following application of the cytotoxic stimuli cis-platin, antimycin A or glucose-deprivation (Locke medium);

• bcl-2 mRNA cellular levels evaluated by quantitative RT-PCR;

• BCL-2 protein cellular levels evaluated by Western blotting. Methods Analysis of bcl-2 mRNA levels by quantitative RT-PCR.

The bcl-2 mRNA levels, either in basal conditions or after the above ODN treatment, were determined by quantitative RT-PCR as previously described (Quattrone et al., 1995; Schiavone et al., 2000) using a Storm Phosphorlmager and the Image QuaNT software (Molecular Dynamics). Briefly, total RNA (1 μg) extracted from untreated cells, or from cells treated with ODNs conjugated to the cationic lipid DOTAP, was reverse-transcribed into cDNA in the presence of random hexamers. Increasing amounts of cDNA were amplified by PCR (denaturation at 94°C for 2 min, annealing at 58°C for 1 min, extension at 72°C for 2 min for 25 cycles in 50μl final volume). The β- actin housekeeping gene was used as internal standard. The PCR amplification products were analyzed on agarose gel. Values within the linear interval of cDNA volume/ amplification product were used to obtain quantitative data. Analysis of BCL-2 protein by Western blotting.

Bcl-2 protein levels, either in basal conditions or after RNA mimetic ODN treatment, were determined by Western blot using an anti-Bcl-2 monoclonal antibody (Santa Cruz Biotechnology Inc.).

Living cell count, cell morphology analysis, apoptotic and mitotic cumulative events analysis. Living cells were counted by the Trypan blue exclusion test. Cell morphology was analyzed by an inverted-phase Zeiss microscope with a 10X lens. Cumulative apoptotic events were evaluated by dynamic examination of cellular cultures by Time-lapse videomicroscopy using an inverted-phase Zeiss microscope with a 10X lens, a Panasonic CCD videocam and a time- lapse JVC BR9030 recorder. Each apoptotic event was counted as previously reported (Schiavone N. et al., 2000), when the cells imploded and detached from the bottom of the plate started producing cytoplasmic vesicles and subcellular fragments (apoptotic bodies) typical of apoptosis (Evan et al., 1992). Results

The following effects were observed treating SH-SY5Y cells with a mixture of 2'-0-methyloligonucleotides (ODNl+ODN2+ODN3+ODN4):

1. increased bcl-2 mRNA levels evaluated by quantitative RT-PCR (Figure 2).

2. increased BCL-2 protein levels by Western blotting analysis (Figure 3).

3. induction of a neuronal differentiated phenotype (Figure 4).

4. reduction of cell proliferation (Figure 5).

5. decrease of the number of apoptotic events scored by Time-lapse Videomicroscopy in response to three apoptotic stimuli: cis-platin, hypoxia and glucose-deprivation (Locke medium - Figure 6).

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Claims

1. Antisense oligonucleotides selected from 2'-0-(Cι-C₃)alkyl- oligoribonucleotides and 2'-0-methyl/deoxy-gapmers, which are complementary to a portion of the Adenine and Uracil-rich Element in the 3 ' untranslated region of bcl-2 mRNA (bcl-2 ARE).

2. Antisense oligonucleotides according to claim 1, which are 2'-0- methyloligoribonucleotides.

3. Antisense 2'-0-(C₁-C₃)alkyl-oligoribonucleotides according to claim 1, which are complexed with transplatin.

4. Antisense oligonucleotides according to claims 1-3, wherein said bcl-2 ARE spans nucleotides 936 to 1050 of the sequence deposited at GenBank accession number M13994.

5. Antisense oligonucleotides according to claim 4, targeting upstream and downstream of said bcl-2 ARE for a maximum of 100 nucleotides.

6. Antisense oligonucleotides according to claims 1-5, containing from 10 to 50 nucleotides.

7. Antisense oligonucleotides according to claim 6, containing from 13 to 30 nucleotides.

8. Antisense oligonucleotides according to claim 7, which are selected from:

A) 5 ' -TGTCTT AA AT AA AT A A ATCTTTTTTTC-3 '

B) 5'-TTAATAATGTAAAAAATAAATGATAT-3 '

C) 5 ' -TTCCCTTTGGC AGTAAATAGCTGATT-3 ' D) 5'-GATTTCCTTTGACGGTGTTTTGATGGGTC-3'.

9. Antisense oligonucleotides according to claims 1-8, for use as medicament.

10. A pharmaceutical composition containing an oligonucleotide according to claims 1-8.

11. A pharmaceutical composition according to claim 10, which is in the form of liposomial preparation.

12. A composition according to claim 11, where liposomes contain cationic lipids.

13. A composition according to claim 12, where the cationic lipid is selected from DOTAP, DOGS, DOTMA, DOPE.

14. The use of an antisense oligonucleotide according to claims 1-8 for the preparation of a therapeutic agent for treating pathologies involving a decreased BCL-2 protein expression.

15. Use of oligonucleotides according to claim 14 for the treatment of ophthalmologic pathologies, toxicity by cytotoxic agents, hypoxia damage, Alzheimer disease, Parkinson disease, Huntington corea, lateral amiotrophic sclerosis.

16. The use of an antisense oligonucleotide according to claims 1-8 for the preparation of a research tool for in vitro modulation of bcl-2 expression.