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Rnai modulation of the bcr-abl fusion gene and uses thereof

Rnai modulation of the bcr-abl fusion gene and uses thereof description/claims

This application claims priority to U.S. Application Ser. No. 60/630,878, filed on Nov. 24, 2004, and to U.S. Ser. No. 60/632,403, filed on Dec. 1, 2004. The entire contents of these provisional applications are hereby incorporated by reference in the present application.

The invention relates to compositions and methods for modulating the expression of Bcr-Abl, and more particularly to the down-regulation of Bcr-Abl mRNA and Bcr-Abl protein levels by oligonucleotides via RNA interference, e.g., chemically modified oligonucleotides.

RNA interference or “RNAi” is a term initially coined by Fire and co-workers to describe the observation that double-stranded RNA (dsRNA) can block gene expression when it is introduced into worms (Fire et al., Nature 391:806-811, 1998). Short dsRNA directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function.

The discovery of the Philadelphia Chromosome (Ph) represented the first consistent chromosomal abnormality causing a specific human cancer (Nowell P C et al., 1960, Science 132:1467). The Ph Chromosome is generated by a reciprocal translocation between the long arms of Chromosome 9 and Chromosome 22 (Rowley J D, 1973, Nature, 243:290-293.). It occurs in almost all patients with chronic myelogenous leukemia (CML), in about 10-20% of the adults with acute lymphoblastic leukemia (ALL) (Westbrook C A et al., 1992 Blood, 80:2983) and about 2% of patients with acute myelogenous leukemia (AML). The t(9; 22) translocation fuses the Bcr gene from Chromosome 22 and the Abl gene from chromosome 9, resulting in the oncogenic Bcr-Abl fusion-gene (Heisterkamp N et al., 1983, Nature, 306:239). Variable breakpoints within the Bcr gene on chromosome 22 lead to the formation of different Bcr-Abl fusion gene variants which encode for different proteins p190Bcr-Abl (Mr 190,000), p210Bcr-Abl (Mr 210,000) and p230Bcr-Abl (Mr 230,000). In about 95% of the CML-patients the Bcr-Abl fusion transcripts e14a2 (former b3a2) and e13a2 (former b2a2) can be detected (reviewed in Barnes et al., 2002, Acta Haematologica, 108:180-202). The translated product is in each case a p210 kD Bcr-Abl protein. In patients with Ph+ ALL a shorter transcript version called Bcr-Abl-e1a2, predominates (reviewed in Faderl et al., 2003, Cancer, 98:1337). Translation of this variant results in the somewhat lighter p190Bcr-Abl protein. Both Bcr-Abl proteins p190Bcr-Abl and p210Bcr-Abl are characterised by a dramatically increased tyrosine-kinase activity, as compared to that of normal Abl protein, leading to aberrant phosphorylation of downstream target molecules.

The kinase activity of Bcr-Abl can be inhibited by a specific tyrosine kinase inhibitor, Imatinib mesylate (STI571, Glivec), which is effective for treatment of Ph+ leukemia (reviewed in Kurzrock et al., 2003, Ann. Intern. Med., 138:819). Nevertheless, both ALL and advanced CML patients frequently develop drug resistance after initial response predominantly caused by genetic abnormalities such as point mutations in the Bcr-Abl kinase domain or overexpression of Bcr-Abl (for review: Rothberg, 2003, Leukemia Res., 27:977). Therefore the development of alternative strategies to inhibit Bcr-Abl becomes increasingly important.

The breakpoint of the Bcr-Abl mRNA represents a unique and leukemia-specific nucleotide sequence. Such fusion transcripts encoding oncogenic proteins represent ideal targets for a disease-specific RNAi approach. The possibility to use RNAi for the specific degradation of the Bcr-Abl-e14a2 transcript variant, as well as other oncogenic fusion proteins, has been demonstrated recently (Wilda et al., Oncogene 2002, 21:5716; Scherr et al., Blood 2003, 101:1566; Heidenreich et al., Blood. 2003, 101:3157, Wohlbold et al., Blood 2003, 102:2236; Ritter U, et al., Oligonucleotides 2003, 13:365; Li et al., Oligonucleotides 2003, 13:401; Chen J, et al., J Clin Invest. 2004, 113:1784). The results presented are inconclusive, as Wilda et al. did not observe a sensitizing effect towards imatinib mesylate on Bcr-Abl-expressing cells by treatment with Bcr-Abl-specific siRNAs, whereas others did observe such effects. It was therefore unclear so far, whether the expression of two relevant Bcr-Abl transcripts other than the e14a2 transcript variant (e13a2 and e1a2) can be downregulated by an RNAi approach.

The present invention advances the art by providing methods and medicaments encompassing short dsRNAs leading to the down-regulation of p210Bcr-Abl and p190Bcr-Abl protein levels in murine 32D cells expressing the respective Bcr-Abl gene variants, in human leukemic MEG-01, K562 and SUP-B15 cells, and in cells freshly isolated from human subjects suffering from leukemia. These methods and medicaments may be used in research into, and in the treatment of, certain cancers.

The present invention is based on an investigation of the Bcr-Abl fusion gene using iRNA agents and further testing of the iRNA agents that target the fusion sites of Bcr-Abl breakpoint variants. Based on these findings, the present invention provides compositions and methods that are useful in reducing Bcr-Abl mRNA levels, Bcr-Abl fusion protein levels and undesirable cell proliferation in a subject, e.g., a mammal, such as a human.

The present invention specifically provides iRNA agents consisting of or comprising at least 15 contiguous nucleotides of one of the agents described in Table 1, agent numbers 1-6. The iRNA agent preferably comprises less than 30 nucleotides per strand, e.g., 21-23 nucleotides. The double stranded iRNA agent can either have blunt ends or more preferably have overhangs of 1-4 nucleotides from one or both 3′ ends of the agent.

Further, the iRNA agent can either contain only naturally occurring ribonucleotide subunits, or can be synthesized so as to contain one or more modifications to the sugar or base of one or more of the ribonucleotide subunits that is included in the agent. The iRNA agent can be further modified so at to be attached to a ligand that is selected to improve stability, distribution or cellular uptake of the agent, e.g. cholesterol. The agents can further be in isolated form or can be part of a pharmaceutical composition used for the methods described herein.

The present invention further provides methods for reducing the level of Bcr-Abl fusion mRNA in a cell. The present methods utilize the cellular mechanisms involved in RNA interference to selectively degrade Bcr-Abl fusion mRNA in a cell and are comprised of the step of contacting a cell with one of the iRNA agents of the present invention. Such methods can be performed directly on a cell or can be performed on a mammalian subject by administering to a subject one of the iRNA agents of the present invention. Reduction of Bcr-Abl fusion mRNA in a cell results in a reduction in the amount of Bcr-Abl fusion protein produced, and in an organism, may result in a decrease in undesirable cell proliferation, or it may sensitize proliferating cells towards the activity of another agent, e.g. a cytostatic or cytotoxic agent, e.g. imatinib mesylate or gamma radiation.

The methods and compositions of the invention, e.g., the methods and iRNA compositions can be used with any dosage and/or formulation described herein, as well as with any route of administration described herein.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from this description, the drawings, and from the claims. This application incorporates all cited references, patents, and patent applications by references in their entirety for all purposes.

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