WO2010027279A2 - Compositions and methods for the treatment and prevention of neoplastic disorders - Google Patents

Abstract

Compositions for the treatment and/or prevention of cancer in a mammal by means of RNA interference are provided, together with methods for the use of such compositions. The compositions comprise a small interfering nucleic acid molecule (siNA) that suppresses expression of a target gene that is involved in the biosynthesis of nucleotides within a tumor cell.

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT AND PREVENTION OF NEOPLASTIC DISORDERS

Field of the Invention

The present invention relates to the treatment of disorders such as cancer by means of RNA interference (RNAi). More specifically, the present invention relates to the targeted delivery of small nucleic acid molecules that are capable of mediating RNA Interference (RNAi) against ribonucleotide reductase subunit 1 which is involved in nucleotide synthesis.

Background of the Invention

Cancer, a group of diseases characterized by the uncontrolled growth and spread of abnormal cells, continues to be one of the leading causes of death throughout the world, claiming more than half a million lives every year in the US alone. Cancer cells evolve from normal cells and acquire traits that enable sustained proliferation, infinite replication, enhanced mobility, and invasiveness, as well as insensitivity to growth inhibition and programmed cell death cues. Such traits are often linked to the over-expression of genes that encode growth and survival promoting proteins.

Current treatments for cancer include chemotherapy, radiation treatment and/or surgical removal of tumors. There are significant limitations to all these techniques. For example, once a tumor has metastasized, it is almost impossible to surgically remove all cancerous cells. Radiation therapy can damage normal tissue surrounding a tumor, and also may not be sufficient to kill all the cancer cells. Similarly, chemotherapy often causes damage to non-cancerous, as well as cancerous, cells, leading to negative side effects. In addition, treatment of cancer with chemotherapeutic drugs often results in the emergence of drug-resistant cancer cells that no longer respond to chemotherapy. When the drug target is a protein, such as thymidylate synthase, resistance can result from increased expression of the target protein or from a mutation in the protein so that it is no longer a functional drug target. There thus remains a need for improved compositions and methods for the treatment of cancer. Deoxyribonucleotides, the precursors of DNA, are formed by the reduction of ribonucleotide diphosphates in the de novo synthesis pathway. These conversions are catalyzed by ribonucleotide reductase, an enzyme consisting of two subunits: RRMl, a 172 kDa dimmer; and RRM2, an 87 kDa dimer. The small subunit, RRM2, contains a stable tyrosyl radical that is essential for reduction of ribose, while RRMl has redox-active cysteines and allosteric sites to maintain balanced pools of nucleotides (Abid et al., J. Biol. Chem. 274:35991-35998, 1999). The reduction of ribonucleotides to deoxyribonucleotides is precisely controlled by allosteric control of the enzyme. Electrons are transferred from NADPH to sulfhydryl groups at the active sites of the enzyme by thioredoxin or glutaredoxin. A tyrosyl free radical that is generated by an iron center in the reductase participates in catalyzing the exchange of H for OH at the C-2 position of the ribose sugar.

RNA interference The treatment methods disclosed herein utilize the process of RNA interference (RNAi). RNAi is a post-transcriptional RNA silencing phenomenon used by most eukaryotic organisms as a defense mechanism against viral attack and transposable factors. This RNA silencing process was first identified in plants, where it is referred to as post-transcriptional gene silencing (PTGS), and was subsequently observed in the nematode C. elegans by Fire et al. {Nature 391 :806-811, 1998). RNAi involves the use of small interfering nucleic acid (siNA) or RNA molecules (siRNAs) that selectively bind with complementary mRNA sequences, targeting them for degradation and thus inhibiting corresponding protein production.

More specifically, in an initiation step double-stranded RNA (dsRNA) is digested by the enzyme Dicer (a member of the RNase III family of dsRNA-specific ribonucleases) into small interfering RNAs (siRNAs) of 19-25 nucleotides in length. Each siRNA consists of two separate, annealed single-stranded polynucleotides. Each strand may have a 2-3 nucleotide 3' overhang. In the effector step, siRNA duplexes interact with specific proteins to form the RNA-induced silencing complex (RISC). The RISC then targets the endogenous mRNA complementary to the siRNA within the complex, and cleaves the endogenous mRNA between nucleotides 10 and 1 1 from the 5' terminus of the antisense strand of the siRNA. Degradation of the endogenous mRNA is then completed by exonucleases.

Transfection of dsRNA duplexes greater than 30 base pairs into most mammalian cells causes nonspecific suppression of gene expression, as opposed to the gene-specific suppression seen in non-mammalian organisms. This is believed to be due to activation of an antiviral defense mechanism that includes the production of interferon, and that leads to a global shut-down of protein production. However, it has been shown that this pathway is not activated by dsRNAs less than 30 nucleotides in length, and that short dsRNAs of 21-23 nucleotides can be used to reduce specific gene expression in mammalian cells (Caplen et al, Proc. Natl. Acad. ScL USA 17:9742-9747, 2001 ; Elbashir et al, Nature 6836:494-498, 2001). Brummelkamp et al. have demonstrated that siRNAs targeting oncogenes are effective in reducing tumors in mice {Cancer Cell 2:243-247, 2002), while Zimmerman et al. have demonstrated effective RNAi-mediated gene silencing in non-human primates {Nature 441 : 1 1 1-1 14, 2006)

Summary of the Invention Briefly stated, the present invention provides compositions for the treatment and/or prevention of a cancer in a mammal by means of RNA interference, together with methods for the use of such compositions. The cellular target for siNA employed in the compositions is a gene that encodes an enzyme involved in the biosynthesis of nucleotides, such as ribonucleotide reductase subunit 1 (RRMl). The disclosed compositions comprise at least one small interfering nucleic acid molecule (siNA) that suppresses expression of the target gene within a target cell, and a pharmaceutically acceptable carrier.

In specific embodiments, isolated siNA are provided that are capable of reducing expression of RRMl in a cell by an RNA interference mechanism, wherein the siNA comprises (a) a first strand having a sequence corresponding to a cDNA sequence provided in SEQ ID NO: 1 -32, and (b) a second strand having a sequence corresponding to a DNA sequence that is sufficiently complementary to a sequence of SEQ ID NO: 1 -32 to anneal to a sequence of SEQ ID NO: 1-32. As will be appreciated by those of skill in the art, when the siNA is an siRNA molecule, the siRNA will contain ribonucleotides where the target cDNA sequence contains deoxyribonucleotides, and further that the siRNA will typically contain a uracil at positions where the target cDNA sequence contains thymidine.

In certain embodiments, the siNA is a double-stranded RNA or a short hairpin RNA. In certain embodiments, the disclosed siNA may have a length of 15 to 30 nucleotides.

In certain embodiments, the disclosed siNAs comprise at least one modified ribose or phosphate backbone moiety. Such modified siNAs have increased stability in vivo and/or in vitro, compared to the corresponding non-modified siNA. Methods for determining the stability of siNA are well known in the art and include, for example, those disclosed in Choung et al., Biochem. Biophys. Res. Commun. 342:919- 927, 2006 and Hoerter and Walter RNA 13: 1887-1893, 2007.

In certain embodiments, compositions are provided that comprise: (a) a first single-stranded siNA that has a sequence corresponding to a DNA sequence of SEQ ID NO: 1 -32; (b) a second single-stranded siNA that has a sequence corresponding to a DNA sequence that is sufficiently complementary to a sequence of SEQ ID NO: 1 - 32 to anneal to a sequence of SEQ ID NO: 1-32; and (c) a pharmaceutically acceptable carrier, wherein at least one of the first and second single-stranded siNAs comprises at least one nucleobase modification that prevents annealing of the first and second single-stranded nucleic acids prior to entry into a cell. In related embodiments, the first and second single-stranded siNAs have a length between 15 to 30 nucleotides, and may further comprise at least one ribose or phosphate backbone modification that protects against nuclease degradation. In certain embodiments, compositions are provided that comprise: (a) a binding agent that specifically binds to a target internalizable cell surface molecule, or antigen, that is expressed on the surface of a target cell; (b) at least one siNA that suppresses expression of a target gene within the target cell; and (c) a nucleic acid binding component, such as a cationic polymer or cationic lipid that condenses and protects the siNA, whereby, after binding to the target cell surface molecule, the binding agent and siNA are internalized into the cell, and the siNA released. In one embodiment, the compositions comprise a cationic lipid or cationic polymer covalently linked to a folic acid molecule or other folate-receptor binding ligand that binds to the folate receptor on cancer cells. Examples of folate-receptor binding ligands that may be employed in such compositions include, for example, those described in U.S. Patent 7,033,594.

In certain embodiments, the binding agent employed in the compositions is an antibody, or an antigen-binding fragment thereof. Other binding agents that may be effectively employed in the inventive compositions include cell-specific ligands, and peptides or small molecules that specifically bind to cell-specific receptors. Viral (capsid) proteins may also be employed as binding agents.

In one embodiment, the binding agent is linked to the siNA by means of a streptavidin-biotin linker as described below. In another embodiment, the siNA is complexed to a lipid carrier, such as a cationic or anionic lipid carrier, which in turn is linked to the binding agent. In a related embodiment, the siNA is encapsulated within a liposome, and the binding agent, or the antigen-binding portion thereof, is present on the surface of the liposome.

In certain of the disclosed compositions, the target cell surface molecule is an internalizable molecule that is expressed on the surface of a tumor cell, wherein binding of a complex to the cell surface molecule leads to internalization of the complex within the tumor cell. In certain embodiments the target cell surface molecule is selected from the group consisting of the receptors for: transferrin; endothelin I; and VEGF, including the VEGF 165b isomer. In a related aspect, methods are provided for the treatment and/or prevention of a cancer in a patient, and/or inhibiting tumor growth and/or neoplastic cell growth in a patient, comprising administering to the patient a composition disclosed herein. Cancers that may be treated using such methods include, but are not limited to, primary and metastatic tumors and carcinomas of the breast; colon; rectum; lung; oropharynx; hypopharynx; esophagus; stomach; pancreas; liver; gallbladder; bile ducts; small intestine; kidney; bladder; urothelium; cervix; uterus; ovaries; prostate; seminal vesicles; testes; endocrine glands, including thyroid, adrenal and pituitary; skin, including melanomas, sarcomas and Kaposi's sarcoma, head and neck cancer; tumors of the brain, nerves, eyes and meninges including astrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas, neuroblastomas; and solid tumors arising from hematopoietic malignancies such as leukemias, myelomas and lymphomas.

In other aspects, methods for inhibiting tumor growth and/or inhibiting neoplastic cell growth in a patient comprising: (a) administering to the patient a first composition comprising a first single-stranded siNA that has a sequence corresponding to a DNA sequence of SEQ ID NO: 1-32; and (b) administering to the patient a second composition comprising a second single-stranded siNA that has a sequence corresponding to a DNA sequence that is sufficiently complementary to a sequence of SEQ ID NO: 1-32 to anneal to a sequence of SEQ ID NO: 1-32, wherein at least one of the first and second single-stranded siNAs comprises at least one nucleobase modification that prevents annealing of the first and second single- stranded nucleic acids prior to entry into a cell.

In other aspects, methods for inhibiting or reducing the expression of RRMl in a cell are provided, such methods comprising contacting the cell with a composition disclosed herein.

In related aspects, methods for inhibiting or reducing the expression of RRMl in a cell are provided, such methods comprising (a) contacting the cell with a first composition comprising a first single-stranded siNA that has a sequence corresponding to a DNA sequence of SEQ ID NO: 1-32; and (b) contacting the cell with a second composition comprising a second single-stranded siNA that has a sequence corresponding to a DNA sequence that is sufficiently complementary to a sequence of SEQ ID NO: 1-32 to anneal to a sequence of SEQ ID NO: 1-32, wherein at least one of the first and second single-stranded siNAs comprises at least one nucleobase modification that prevents annealing of the first and second single- stranded nucleic acids prior to entry into a cell.

The compositions disclosed herein may include more than one siNA directed against one or more specific cancers. The compositions may be employed alone or in conjunction with known therapeutic methods and compositions currently employed in the treatment of cancer. In one embodiment, the compositions comprising an siNA targeted against a specific target gene that expresses a target protein are employed in conjunction with a known chemotherapeutic directed against the target protein. For example, compositions containing siNA targeted against RRMl may be employed in conjunction with gemcitabine. Cancer cells treated with the drugs gemcitabine or hydroxyurea, which target RRMl and RRM2, respectively, and that become drug resistant will still be susceptible to killing using siNA directed against RRMl or RRM2. The use of siNAs in combination with drugs that target the same cellular protein in a cancer cell may thus have benefits to a patient in terms of reducing the amounts of drugs required and in combating the emergence of drug-resistant cancer cells. In certain embodiments, the disclosed compositions comprise at least one siNA directed against a target gene, such as RRMl, in combination with at least one known chemotherapeutic agent, such as gemcitabine. Within related embodiments, combination therapeutic regimens for the treatment of disease are provided, such therapeutic regimens comprising the simultaneous and/or sequential administration of one or more siNA composition(s) in combination with at least one known chemotherapeutic modality.

These and other aspects of the present invention will become apparent upon reference to the following detailed description. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.

Brief Description of the Figures

Figs. IA and IB show siRNA-mediated growth inhibition of A549 cells measured 120 h and 240 h after transfection with 32 RRMl -specific siRNAs at 1 nM (Fig. IA) or 5 nM (Fig. I B), normalized against the average results obtained with two non-specific siRNA controls.

Fig. 2 shows siRNA-mediated reduction in RRMl mRNA levels by 8, 25, 74, 222, 667 or 2,000 pM siRNAl-1 , siRNAl-2 and siRNAl-3, respectively, expressed as a percentage of the knockdown with a non-specific control siRNA (81 Ctrl) tested in duplicate.

Fig. 3 shows the effect of in vitro knockdown on xenograft tumor growth in vivo in CD-I nude mice injected with A549 cells pre-transfected with 10 nM RR1-15 (diamonds) compared with the effect of control siRNA (triangles) or untransfected cells (circles). Fig. 4 shows the effect of in vitro knockdown on xenograft tumor growth in vivo in CD-I nude mice injected with A549 cells pre-transfected with 10 nM RRl -6 (filled squares) and RRM 1-19 (open triangles) compared with the effect of control siRNA (filled diamonds) or untransfected cells (open circles).

Fig. 5 shows eMBRACE assay detection of RR 15 siRNA cleavage product in cells transfected with 10 nM RRl -15 siRNA (filled squares) compared with untreated cells (open squares). The PCR cycle number is on the X-axis and fluorescence (465- 510 nm) on the Y-axis.

Detailed Description of the Invention As noted above, the present invention is generally directed to compositions and methods for the treatment of a cancer in a patient. In certain embodiments, the cancer is selected from the group consisting of: primary and metastatic tumors and carcinomas of the breast; colon; rectum; lung; oropharynx; hypopharynx; esophagus; stomach; pancreas; liver; gallbladder; bile ducts; small intestine; kidney; bladder; urothelium; cervix; uterus; ovaries; prostate; seminal vesicles; testes; endocrine glands, including thyroid, adrenal and pituitary; skin, including melanomas, sarcomas and Kaposi's sarcoma; tumors of the brain, nerves, eyes and meninges including astrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas, neuroblastomas, myelomas; head and neck cancers; and solid tumors arising from hematopoietic malignancies such as leukemias and lymphomas.

The compositions disclosed herein comprise at least one unmodified or modified small interfering nucleic acid molecule (siNA) directed against a target gene. In certain embodiments, compositions are provided that comprise a complex including: (a) at least one siNA, which may or may not be modified, specific for a target gene; and (b) a binding agent, such as an antibody, that specifically binds to a target antigen or cell surface molecule which is present on the surface of a target cell of interest. The target antigen recognizes and internalizes certain specific biological molecules, such that, on binding of the siNA-antibody complex to the target antigen, the complex is internalized into the target cell by endocytosis, the siNA is released from the complex, and the siNA reduces expression of the target gene by means of RNA interference.

As used herein, the term "target gene" refers to a polynucleotide that comprises a transcribed region that encodes an RNA or polypeptide of interest, and/or a polynucleotide region that regulates replication, transcription, translation or other processes important to expression of the polypeptide of interest.

As used herein, the term "small interfering nucleic acid molecule", or siNA, refers to any nucleic acid molecule that is capable of modulating the expression of a gene by RNA interference (RNAi), and thus encompasses: short interfering RNA (siRNA); double-stranded RNA (dsRNA); single-stranded siNA; complementary RNA/DNA hybrids; nucleic acid molecules containing modified (semi-synthetic) base/nucleoside or nucleotide analogues (which may or may not be further modified by conjugation to non-nucleic acid molecules); custom modified primary or precursor microRNA (miRNA); and short hairpin RNA (shRNA) molecules. In specific embodiments, the siNAs employed in the compositions and methods disclosed herein are targeted against human RRMl (accession number NM_001033; cDNA sequence provided in SEQ ID NO: 36). Examples of such siNAs include the siRNAs corresponding to the cDNA sequences provided in SEQ ID NO: 1-32 which are targeted against RRMl, as detailed below in Example 5. One of skill in the art will appreciate that, when comparing an RNA sequence to a DNA sequence, an RNA sequence will contain ribonucleotides where the DNA sequence contains deoxyribonucleotides, and further that the RNA sequence will typically contain a uracil at positions where the DNA sequence contains thymidine. In certain embodiments, the siNAs are double-stranded and are comprised of the listed sequence, or a modification thereof, together with its complement. In other embodiments, the compositions disclosed herein comprise a pair of single-stranded siNAs, with one of the single-stranded siNAs being an antisense sequence and the other single-stranded siNA being a sense sequence that is sufficiently complementary to bind to the antisense sequence. At least one of the sense or antisense strands includes one or more modifications that prevent formation of duplexes of the sense and antisense strands prior to entry into a target cell and that are removed following entry into the cell, thereby permitting formation of active double- stranded siNAs only after the single stranded siNAs have entered the target cell. The siNA may be targeted to the 5' untranslated region, the coding region, or the 3' untranslated region of the target gene or message. Additionally, regions of the promoter of a target gene, or regions usually upstream of a gene may be targeted for RNAi assisted heterochromatin formation.

An siNA can be unmodified or may be chemically-modified. For example, the siNAs may be protected against nuclease degradation by incorporating ribose modifications. Where the siNA is a synthetic duplex, this may have symmetrical 3' overhangs of 2-3 nt, asymmetrical overhangs at one or other end, or may be blunt- ended. Such siNA may be fully (i.e. at every nucleotide) or partially (at selected nucleotide positions) modified, and this modification may be on either the sense strand, the antisense strand, or both strands. Thus, for example, some or all of the nucleotides of an siNA may comprise modified nucleic acid residues, or analogs of nucleic acid residues. The ribose modifications may include, but are not limited to, 2'-deoxy, 2'-O-alkyl, 2'deoxy-2'fiuoro and 2'-O-methyl modifications. The modifications may be on alternating sugars such that the modifications are opposite each other in a dsNA molecule. In addition, or alternatively, the phosphate backbone of an siNA may be modified by incorporation of alternate linkages between the nucleosides. These linkages include, but are not limited to, phosphorothioate and boranophosphate linkages. Examples of modifications include those described, for example, in International Publication Nos. WO03/070970, WO03/074654 and WO03/064626; US Patent 7,459,547; and U.S. Published Patent Applications Nos. US2004/0014956, US2004/0192626, US2005/0282188, US2005/0233329, US2005/0020525, US2005/0266422, US2004/0171029, US2004/0171028, US2004/0203024, US2005/0037370, US2004/0171030, US2004/0161777, US2004/0146902, US2005/0119470, US2004/0147470, US2004/0161844, US2004/0171031, US2004/0171032, US2004/0147022, US2004/0147023, US2004/0171033, US2005/0053976, US2005/0042647, US2006/0287260, US2007/0160980 and US2007/0265220, the disclosures of which are hereby incorporated by reference. The hybridization characteristics of the modified siNA may be similar to or improved compared to the corresponding unmodified siNA. Such modifications can also improve the efficacy and safety of in vivo therapy by changing the stability, lifetime and circulation of the siNAs in the human body.

The siNA may be from 19 to 30 nucleotides in length (for example, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides), and comprises an antisense strand that is complementary to at least a portion of a nucleotide sequence, such as a mRNA sequence corresponding to a target DNA sequence. Methods for selecting suitable regions in a mRNA target are disclosed in the art (see, for example, Vickers et al., J. Biol. Chem. 278:7108-7118, 2003; Elbashir et al., Nature 411 :494-498, 2001; Elbashir et al., Genes Dev. 15:188-200, 2001) and are described below. In certain embodiments, the siNA is 19 to 23 or 25 to 30 nucleotides in length, such as, but not limited to, 21, 25 or 27 nucleotides in length. The sense and antisense strands may be separate, distinct polynucleotides, as in a dsRNA molecule, or may be linked as, for example, in a shRNA molecule. Those skilled in the art will appreciate that minor changes in the sequence of the siNAs directed against target sequences can yield siNAs that hybridize strongly and specifically to the target nucleic acid. For example, siNAs directed against target sequences that are shifted by one to four nucleotides 5' or 3' of the sequences disclosed herein may be effective. Assessing whether a gene has been down regulated, and the extent of down regulation, can be performed using, for example, real-time PCR, PCR, western blotting, flow cytometry or ELISA methods.

As noted above, in one embodiment, the disclosed methods for inhibiting expression of human RRMl in a target cell comprise separately contacting the cell with (or introducing into or delivering to the cell) an antisense single-stranded nucleic acid (ssNA) and a sense ssNA that are capable of forming a duplex that mediates silencing of human RRMl, wherein at least one of the antisense and sense ssNAs comprises at least one nucleobase modification that prevents annealing of the two ssNAs prior to entry into the cell. Once inside the cell, the nucleobase modifications are removed by the cellular machinery or chemical hydrolysis, and the antisense ssNA and sense ssNA anneal to form an active double- stranded nucleic acid (dsNA) duplex that initiates RNA interference thereby inhibiting expression of the target gene.

As noted, the sense and antisense ssNAs are sufficiently complementary to each other to anneal to form a double-stranded ribonucleotide duplex (dsRNA) following removal of the nucleobase modifications by the cellular machinery or chemical hydrolysis. In certain embodiments, the antisense single-stranded siNA and the sense single-stranded siNA contain between zero and 10 mismatches.

The sense and antisense ssNA strands may be provided in the same composition or in separate compositions. For example, the two ssNA strands may be delivered simultaneously, but in separate compositions. Alternatively, the two ssNAs may be delivered simultaneously and in the same composition, but, due to the presence of the nucleobase modifications, remain separate until they enter the cell and the modifications are removed. In both embodiments, the two ssNA strands enter the cell independently of each other and only anneal once both strands have entered the cell cytoplasm. As used herein, the term "binding agent", refers to a molecule that specifically binds to a target antigen expressed on the surface of a target cells, and includes, but is not limited to, antibodies, including monoclonal antibodies and polyclonal antibodies; antigen-binding fragments thereof, such as F(ab) fragments, F(ab')₂ fragments, variable domain fragments (Fv), small chain antibody variable domain fragments (scFv), and heavy chain variable domains (V_HH); small molecules; hormones; cytokines; ligands; peptides and viruses (either native or modified). Antibodies, and fragments thereof, may be derived from any species, including humans, or may be formed as chimeric proteins which employ sequences from more than one species. The term "binding agent" as used herein thus encompasses humanized antibodies and veneered antibodies.

A binding agent is said to "specifically bind," to a target antigen if it reacts at a detectable level (within, for example, an ELISA assay) with the target antigen, and does not react detectably with unrelated antigens under similar conditions.

Antibodies, and fragments thereof, may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies as described, for example, by Kohler and Milstein, Eur. J. Immunol. 6:51 1-519, 1976, and improvements thereto, via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies, or by protein synthesis.

In order to minimize any off-target effects, the binding agents employed in the disclosed compositions and methods are cell type-specific. Preferably the binding agent is specific for internalizable cell surface molecules or antigens found on tumor cells. Examples of such molecules include, but are not limited to, the receptors for transferrin, endothelin I and VEGF, including the VEGF- 165b isomer. Examples of binding agents that may be usefully employed in the disclosed compositions include: antibodies against transferrin and endothelin available from Abeam Inc. (Cambridge, MA), and antibodies against VEGF, available from Delta Biolabs, LLC (Campbell, CA), and antigen-binding fragments thereof.

In one embodiment, the compositions disclosed herein comprise a binding agent, such as an antibody, connected to an siNA by means of a streptavidin-biotin linkage. As used herein, the term "streptavidin" encompasses both streptavidin and avidin, and derivatives or analogues thereof that are capable of high affinity, multivalent or univalent binding of biotin. Techniques for the preparation of conjugates containing streptavidin-biotin linkages are well known in the art and include, for example, those described in U.S. Patent Nos. 6,287,792 and 6,217,869, the disclosures of which are hereby incorporated by reference. Biotin may be incorporated into the siNA using, for example Biotin-21-dUTP™ (BD Biosciences Clontech, Palo Alto, CA), which is a dTTP analog with biotin covalently attached to the pyrimidine ring through a 21 -atom spacer arm. The biotin-labeled siNA is then linked to the streptavidin-antibody conjugate via biotin-streptavidin binding, using techniques well known to those of skill in the art. In a further embodiment, complexes are provided that comprise a binding agent, such as an antibody, and a polynucleotide-binding component, such as a polycation, that is covalently bonded to the antibody through, for example, disulfide bonds. Polycations that may be employed as polynucleotide-binding components include, for example, polylysine, polyarginine, polyornithine, polyethylenimine, chitosan and basic proteins, such as histones, avidin and protamines. The polynucleotide-binding component is then attached to a siNA by means of electrostatic attraction between the opposite charges present on the siNA and the polynucleotide-binding component. The antibody is thus bound to the siNA without functionally altering either the siNA or the antibody. Both the bond between the antibody and the polynucleotide-binding components and that between the polynucleotide-binding component and the siNA are cleaved following internalization of the complex into the target cell. Such complexes may be prepared as described, for example, in U.S. Patent No. 5,166,320. Cleavable polymeric linkers which may be effectively employed to attach a genetic construct of the present invention to a binding agent are also described in U.S. Patent No. 6,627,616

In certain embodiments, the compositions disclosed herein comprise a siNA encapsulated in, or attached to, a delivery agent. For example, an siNA can be encapsulated in a liposome or polymer, or attached to a lipid or polymer carrier, which in turn can be attached to a binding agent, such as an antibody directed against the target antigen or a ligand for a cancer cell-specific receptor. Encapsulation of the siNA within a liposome protects the siNA from degradation by endonucleases. Methods for the encapsulation of biologically active molecules, such as nucleic acid molecules and proteins, within liposomes or polymers, and for the preparation of nucleic acid-lipid (lipoplex) and nucleic acid-polymer (polyplex) carrier complexes are well known in the art. See, for example, U.S. Patent Nos. 6,627,615, 4,241,046, 4,235,871 and 4,394,448; and Liposome Technology: Liposome Preparation and Related Techniques, ed. G. Gregoriadis, CRC Press, 1992. Liposome formulation, development and manufacturing services are available for example, from Gilead Liposome Technology Group (Foster City, CA). Lipids for the preparation of liposomes are available, for example from Avanti Polar Lipids, Inc. (Alabaster, AL).

The resulting liposome carrier containing the siNA of interest can then be conjugated to a binding agent, using methods well known in the art, such as those taught in U.S. Patent Nos. 5,210,040, 4,925,661, 4,806,466 and 4,762,915. Such methods include the use of linkers that fall into four major classes of functionality: conjugation through amide bond formation; disulfide or thioether formation; hydrazone formation; or biotin-streptavidin binding. In a preferred embodiment, the liposome is attached to the binding agent, such as an antibody, by means of a maleimide linker, as described, for example, in U.S. Patent No. 6,372,250, the disclosure of which is hereby incorporated by reference.

In one embodiment, the liposome employed in the disclosed compositions is a pegylated liposome, wherein the surface of the liposome is conjugated with multiple (up to several thousand) strands of poly(ethylene glycol) (PEG) of approx. 2000 Da. The binding agent is then conjugated to the tips of some of the PEG strands. The diameter of the liposome is preferably within the range of 100 run to 10 μm. The preparation of such pegylated liposomes and attachment of monoclonal antibodies to the liposomes is performed as described, for example, in Shi and Pardridge, Proc. Natl. Acad. ScL USA 97:7567-7572, 2000; and Shi et ai, Proc. Natl. Acad. Sci. USA 98: 12754-12759, 2000. Pegylation of the liposome increases the stability of the liposome and prevents non-specific attachment of cells, such as macrophages, and proteins to the liposome.

Other delivery agents that may be employed in the disclosed compositions include, for example, nanoparticles as described in U.S. Patent Publication no. US 2008/0095856; nanotransporters comprising a nanoparticle, or nanotube, core and at least one functional surface group as described for example, in PCT Patent Publication no. WO 2007/0869607; chitosan/RNA nanoparticles as described in PCT Patent Publication no. WO 2008/003329; and lipids as described in U.S. Patent Publication no. US 2007/0260055. In certain embodiments, the disclosed compositions include one or more siNA together with nanoparticles that are optically or magnetically detectable, as described, for example, in PCT Patent Publication no. WO 2007/067733, whereby uptake of the siNA may be monitored. Other nanoparticles-based methods for the delivery of siNA that may be used in conjunction with the disclosed compositions include, for example, those described in PCT Patent Publications no. WO 2007/016501 and WO 2008/073856, and in Suri et al, J. Occ. Med. Toxicol. 2: 16, 2007.

Alternative delivery agents that may be employed in, or in conjunction with, the disclosed compositions, include those described in Meade and Dowdy, Adv. Drug Deliv. Rev. 59: 134-140, 2007, and Akhtar and Benter, J Clin. Invest. 117:3623-3632, 2007.

The present invention further provides methods for the treatment of a cancer in a patient by administration of a therapeutically effective amount of a composition disclosed herein.

As used herein, a "patient" refers to any warm-blooded animal, including, but not limited to, a human. Such a patient may be afflicted with disease or may be free of detectable disease. In other words, the methods may be employed for the prevention or treatment of disease. As discussed above, the methods may be employed in conjunction with other known therapies currently employed for the treatment of cancer. For example, the disclosed compositions may be administered before, during or after, radiotherapy, chemotherapy, photodynamic therapy and/or surgery.

In general, the disclosed compositions may be administered by injection (e.g., intradermal, intramuscular, intravenous, intratumoral or subcutaneous), intranasally (e g-, by aspiration), orally, transdermally or epicutaneously (applied topically onto skin). In one embodiment, the compositions are in a form suitable for delivery to the mucosal surfaces of the airways leading to or within the lungs. For example, the composition may be suspended in a liquid formulation for delivery to a patient in an aerosol form or by means of a nebulizer device similar to those currently employed in the treatment of asthma.

For use in therapeutic methods, the disclosed compositions may additionally contain a physiologically acceptable carrier. While any suitable carrier known to those of ordinary skill in the art may be employed in the compositions, the type of carrier will vary depending on the mode of administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactic galactide) may also be employed as carriers for the compositions. Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075,109. Other components, such as buffers, stabilizers, biocides, etc., may be included in the disclosed compositions. The compositions may be provided in single dose or multi- dose containers, such as sealed ampoules and/or vials, and can be stored either frozen or freeze-dried.

The preferred frequency of administration and effective dosage, which will vary from one individual to another and will depend upon the particular disease being treated, may be determined by one skilled in the art, using known techniques. Preferably, siNA is administered at a dose of between 1 and 10 mg/kg. The compositions may be administered in a single dosage or in multiple, divided, dosages. In related embodiments, therapeutics, including combination therapeutics, are provided which employ one or more known chemotherapeutics in sequential and/or simultaneous combination with one or more therapeutic siRNA molecules. Such combination therapeutics provide an advantage over stand-alone chemotherapeutics by reducing drug toxicity and/or emergence of drug-resistant cells that no longer respond to the chemotherapeutic alone.

The following Examples are offered by way of illustration and not by way of limitation. Sequences referred to in the Examples below are given in both DNA and RNA nomenclature and represent the sequence of the antisense or guide strands of the siNAs. The final configuration of the strands within the duplexes will consist of a combination of ribonucleotides and deoxyribonucleotides. Further modifications of the phosphodiester backbone and nucleobases, often incorporated into siNAs, have not been explicitly listed, but are well-known in the art.

Example 1

PREPARATION OF ANTIBODY-CONJUGATED LIPOSOMES Preparation of pegylated liposomes, encapsulation of siNAs, and conjugation with monoclonal antibody may be carried out as follows. l-Palmitoyl-2-oleoyl-5«-glycerol-3-phosphocholine (POPC; Avanti Polar

Lipids, Alabaster AL; 19.2 μmol), didodecyldimethylammonium bromide (DDAB; Avanti Polar Lipids; 0.2 μmol), distearolyphosphatidylethanolamine ((DSPE)-PEG 2000; Shearwater Polymers, Huntsville, AL; 0.6 μmol) and DSPE-PEG 2000- maleimide (30 nmol) are dissolved in chloroform/methanol (2: 1 , vokvol) followed by evaporation. The lipids are dispersed in 1 ml 0.05 M Tris-HCl buffer (pH = 8.0) and sonicated for 10 min. siNA is added to the lipids and the liposome/siNA dispersion evaporated to a final concentration of 200 mM at a volume of 100 μl. The dispersion is frozen in ethanol/dry ice for 4-5 min and thawed at 4O⁰C for 1 -2 min. This freeze- thaw cycle is repeated 10 times. The liposome dispersion is then diluted to a lipid concentration of 40 mM, followed by extrusion 10 times each through two stacks of polycarbonate filter membranes. The mean vesicle diameters may be determined using a Microtrac Ultrafine Particle Analyzer (Leeds-Northmp, St. Petersburg, FL). siNA attached to the exterior of the liposomes is removed by nuclease digestion as described by Monnard et al. (Biochim. Biophys. Acta 1329:39-50, 1997). For digestion of the unencapsulated siNA, 5 units of pancreatic endonuclease I and 5 units of exonuclease II are added in 5 mM MgCl₂ and 0.1 mM DTT to the liposome/siNA mixture after extrusion. After incubation at 37⁰C for 1 h, the reaction is stopped by adding 7 mM EDTA. Monoclonal antibody specific for the target antigen is thiolated using a 40:1 molar excess of 2-iminothiolane (Traut's reagent) as described by Huwyler et al., Proc. Natl. Acad. Sci. USA 93:14164-14169, 1996. Thiolated antibody is then incubated with the liposomes overnight at room temperature, and the resulting immunoliposomes are separated from free monoclonal antibody by, for example, gel filtration chromatography.

Example 2 PREPARATION OF PEPTIDE-CONJUGATED POLYPLEXES

Conjugation of targeting peptides to polycations such as polyethylenimine (PEI), and preparation of peptide-targeted polyplexes may be carried out by the method of Schiffelers et al. {Nucleic Acids Res. 32:1-10, 2004) as follows.

NHS-PEG-VS is obtained from Nektar Therapeutics (San Carlos, CA). The targeting RGD peptide with the sequence H- ACD ARGD AFCG-OH (SEQ ID NO: 41) is synthesized, oxidized to form the intramolecular disulfide bridge, and purified by reverse-phase HPLC (Auspep Ltd., Parkeville, Australia). The resulting peptide (6 mg) is dissolved in DMSO (60 μl), neutralized with triethylamine (TEA, 2 mol/mol peptide), and coupled to NHS-PEG-VS (21 mg in 40 μl DMSO) for 4 hours at room temperature. The reaction is stopped by adding trifluoroacetic acid (TFA, equimolar to TEA), and the mixture is lyophilized. The intermediate RGD-PEG-VS is purified by dialysis against water, and the compound lyophilized to give a yield of 50-90%. Conjugation is confirmed by mass spectral analysis (matrix-assisted laser desorption ionization).

In the second step of synthesis, various amounts of the purified RGD-PEG-VS intermediate are dissolved in sodium carbonate buffer pH 9.0 (100 μl) and reacted with linear polyethylenimine at room temperature for 16 hours. The reaction is terminated by the addition of an excess of TFA and lyophilized. The product is purified by gel filtration on a Superdex Peptide column in 0.1% TFA, and lyophilized. The degree of conjugation of RGD-PEG to PEI is determined by proton NMR spectrometry on a 400 MHz spectrometer from the ratio of the areas under the peaks corresponding to the -CH₂- protons of PEI (2.8-3.1 ppm) and PEG (3.3-3.6 ppm).

Complexes are formed by mixing equal volumes of solutions of RGD-PEG- PEI and siNA in HEPES-buffered 5% glucose to give a molar ratio of PEI amine to RNA phosphate of 5: 1 to 10:1. The amount of free siNA is quantitated using the Pico Green assay (Invitrogen Corporation, Carlsbad, CA).

Example 3 DESIGN OF SIRNA OLIGONUCLEOTIDES

Potential target sites in the mRNA are identified based on rational design principles, which include target accessibility and secondary structure prediction. Each of these may affect the reproducibility and degree of knockdown of expression of the mRNA target, and the concentration of siRNA required for therapeutic effect. In addition, the thermodynamic stability of the siRNA duplex (e.g., antisense siRNA binding energy, internal stability profiles, and differential stability of siRNA duplex ends) may be correlated with its ability to produce RNA interference (Schwarz et al., Cell 1 15: 199-208, 2003; Khvorova et al, Cell 1 15:209-216, 2003). Empirical rules, such as those provided by the Tuschl laboratory (Elbashir et al., Nature 41 1 :494-498, 2001 ; Elbashir et al., Genes Dev. 15: 188-200, 2001) and the Morishita Laboratory (University of Tokyo; Ui-Tei et al., Nucleic Acids Research 32:936-948, 2004) are also used. Software and internet interactive services for siRNA design are available at the following websites: Ambion, Invitrogen, Deqor, Dharmacon, Emboss-2.9.0, Genscript, Cold Spring Harbor Laboratory (Jack Lin), Tuschl Laboratory (MPI), OptiRNA (Cui et al., Computer Methods and Programs in Biomedicine), Qiagen, siDirect and siRNA Design websites. Levenkova et al. describe a software system for design and prioritization of siRNA oligos (Bioinformatics 20:430-432, 2004). The Levenkova system is available on the internet and is downloadable freely for both academic and commercial purposes. The siRNA molecules disclosed herein were based on the Ambion, Invitrogen, Ui-Tei, Deqor, Elbashir and Levenkova recommendations. The selection of siRNA oligos disclosed in this application was based primarily on uniqueness vs. human sequences (i.e., a single good hit vs. human Unigene, and a big difference in hybridization temperature (T_m) against the second best hit) and on GC content (i.e., sequences with %GC in the range of 40-60%).

Optionally, for a more detailed picture on the potential hybridization of the oligos, RNA target accessibility and secondary structure prediction can be carried out using, for example, Sfold software (Ding Y and Lawrence, CE. (2004) Rational design of siRNAs with Sfold software. In: RNA Interference: from Basic Science to Drug Development. K. Appasani (Ed.), Cambridge University Press; Ding and Lawrence, Nucleic Acids Res. 29:1034-1046, 2001; Nucleic Acids Res. 31 :7280-7301, 2003). Sfold is available on the internet. RNA secondary structure determination is also described in Current Protocols in Nucleic Acid Chemistry, Beaucage et al., ed., 2000, at 1 1.2.1-1 1.2.10.

The targeted region is selected from a cDNA sequence, such as the cDNA sequence for ribonucleotide reductase subunit 1 (RRMl ; cDNA sequence provided in SEQ ID NO: 36). Potential target sequences and positions are typically identified by searching for specific 23 nucleotide (nt) motifs ("Tuschl patterns" such as AA(Nl 9)TT, where N is any nucleotide, and AA is referred to herein as the "target motif leader", NA(N21), or BA(N21), where B=C, G, U; Elbashir SM et al, Methods 26:199-213, 2002) in the cDNA sequence, starting at about 50-100 nt downstream of the start codon. The nt 22 and nt 23 need not be considered in searching for Tuschl patterns, since they are not involved in the base pairing between the mRNA target and the antisense siRNA strand. "Sense siRNA" is used herein to mean a target sequence without the NN leader. For example, the sequence of the sense siRNA corresponds to (N19)TT of the Tuschl pattern AA(N19)TT (positions 3-23 of the 23 nt motif).

The siRNAs may be designed with symmetric 3' overhangs in order to form a symmetric duplex (Elbashir et al, EMBO J. 20:6877-6888, 2001). For both sense and antisense siRNAs, either dTdT or UU are used as the 3' overhang. Thus for siRNAs with an AA target motif leader, the AA base pairs with the dTdT or UU overhang of the antisense siRNA. For BA leaders, the A pairs with the first dT or U of the overhang. It is known however, that the overhang of the sense sequence can be modified without affecting targeted mRNA recognition.

The antisense siRNA is synthesized as the complement to position 1-21 of the 23 nt motif. The 3' most nucleotide can be varied, but the nucleotide at position 2 of the 23 nt motif is selected to be complementary to the targeted sequence. These methods are well known in the art. For example, the siRNA may be selected corresponding to the target motif NAR (N 17) YNN, where R is (A,G) and Y is (C,U). The target sequence motifs are selected to have about 30-70% GC content, preferably 40-60% GC content. As used herein, the "% GC content" is calculated as: [the number of G or C nucleotides in the target sequence/ 21 for an AA target motif leader] x 100, [the number of G or C nucleotides in the target sequence/20 for a BA target motif leader] x 100, and [the number of G or C nucleotides in the target sequence/19 for an NB target motif leader] x 100. Following selection of siRNA duplexes from the target sequence, the thermodynamic properties of the sequences are determined, e.g., using the Sfold software referred to above. As used herein, "DSSE" refers to the differential stability of the siRNA duplex ends, i.e., the average difference between 5' antisense and 5' sense free energy values for the four nucleotide base pairs at the ends of the duplex. It has been shown that the 5' antisense region is less stable than the 5' sense terminus in functional siRNA duplexes and vice versa for nonfunctional siRNA duplexes (Khvorova et al, Cell 1 15:209-216, 2003). It is known that the siRNA duplex can be functionally asymmetric, in the sense that one of the two strands preferentially triggers RNAi (Schwartz et al., Cell 155: 199-208, 2003). As used herein, "AIS" refers to the average internal stability of the duplex at positions 9-14 from the 5' end of the antisense strand. Comparisons between functional and nonfunctional siRNA duplexes indicate that the functional siRNA has lower internal stability in this region. It is proposed that flexibility in this region may be important for target cleavage (the mRNA is cleaved between position 10 and 1 1) and/or release of cleaved products from RISC to regenerate RISC (Khvorova et al., Cell 115:209-216, 2003).

The siRNA sequences and their thermodynamic properties are further selected according to the following criteria: (a) 40% < GC content < 60%; (b) antisense siRNA binding energy < -15 kcal/mol; and (c) exclusion of target sequence with at least one of AAAA, CCCC, GGGG or UUUU. For siRNAs with NN dinucleotide leaders, two additional criteria are used: (d) DSSE > 0 kcal/mol (Zamore asymmetry rule); and (e) AIS > -8.6 kcal/mol (cleavage site instability rule). This is the midpoint between the minimum of -3.6 and maximum of -13.6 (Khvorova et al., Cell 1 15:209-216, 2003).

To increase the likelihood that only one gene will be targeted for degradation, the selected siRNA sequences are further checked for uniqueness against human and murine gene libraries (e.g., TIGR GI, ENSEMBL human genome), using Blast algorithms. Also, to increase the likelihood that the selected sequences will be active, sequences directed against targets having SNPs in the base pairing regions are excluded. The exemplary siRNAs provided in SEQ ID NO: 1-32 correspond to the target sequence provided in SEQ ID NO: 36.

Example 4 SYNTHESIS AND TESTING OF SIRNA DUPLEXES

SiRNA may be prepared by various methods, for example by chemical synthesis, or from suitable templates using in vitro transcription, siRNA expression vectors or PCR generated siRNA expression cassettes. Preferably, chemical synthesis is used. Methods for chemical synthesis of RNA are well known in the art and are described, for example, in Usman et al., J. Am. Chem. Soc. 109:7845-7854, 1987; Scaringe et al., Nucleic Acids Res. 18:5433-5441 , 1990; Wincott et al., Nucleic Acids Res. 23:2677-2684, 1995; and Wincott et al., Methods MoI. Biol. 74:59-68, 1997. Twenty-one nucleotide siRNAs may be synthesized, for example, using protected ribonucleoside phosphoramidites and a DNA/RNA synthesizer, and are commercially available from a number of suppliers, such as Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO), Perbio Science (Rockford, IL), Glen Research (Sterling, VA), ChemGenes (Ashland, MA), and Ambion Inc. (Austin, TX). The siRNA strands can then be deprotected, annealed and purified before use, if necessary. Annealing can be carried out, for example, by incubating single-stranded 21-nt RNAs in 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM Mg acetate, 1 min at 90°C, then 1 hr at 37°C. The solution is then stored frozen at -20°C. Useful protocols can be found in Elbashir et al., Methods 26: 199-213, 2002.

Example 5

SlRNA-MEDIATED INHIBITION OF RIBONUCLEASE REDUCTASE SUBUNIT 1 (RRMl)

In vitro growth inhibition

A549 cells (human lung cancer cell line; ATCC No. CCL- 185) were cultured in RPMI 1640 medium with 10% v/v heat-inactivated fetal calf serum (FCS). Cells were seeded at a density of 5x10⁴ cells/cm² in multi-well tissue culture plates. siRNA duplexes targeting human RRMl (SEQ ID NO: 1-32) are listed in Table 1 and are 19-

21-mers manufactured by Integrated DNA Technologies (IDT; Coralville IA). The siRNAs were reversed transfected at final concentrations of 1 and 5 nM into cells using Lipofectamine™ RNAiMAX transfection reagent (Invitrogen, Carlsbad CA). Stealth™ siRNA37-2 (SEQ ID NO: 35), with the same core sequence as siRRMl-2 (SEQ ID NO: 28), was used as positive control for growth inhibition and non-specific Stealth™ siRNAs (81 Ctrl and RRM-REV; SEQ ID NO: 33 and 34, respectively) were used as negative controls. RRM-REV has the same sequence as siRNA37-2 but with the proprietary Stealth™ modification on the antisense strand. The cellular DNA content was measured using a SYBR Green I-based fluorometric assay up to 10 days after transfection, as follows. Cells were harvested on days 5 and 10 following reverse transfection and frozen at -8O⁰C until processed. After the final time point, cells were thawed and cell lysis buffer (10 niM Tris-HCl pH 8.0 containing 2.5 mM EDTA, 1% Triton™ X-100) and SYBR Green I (l :10⁵ v/v; Invitrogen) was added to the wells and incubated overnight in the dark at 4⁰C. The following day, cell lysates were mixed thoroughly, and DNA fluorescence was measured with a Wallac Victor² plate reader (Turku, Finland) set at an excitation frequency of 485 nm and measuring emission at 535 nm.

Table 1

The inhibition of the growth of A549 cells transfected with 1 or 5 nM of the RRMl -specific siRNAs listed in Table 1 was measured at two different time points as shown in Fig. 1. Fig. IA shows at least 50% inhibition of cell growth by 28 of the 32 siRNAs at 1 nM at the 12O h timepoint, but the inhibitory effect was lost by 240 h, at which point only 6 of the 32 siRNAs were still causing 50% growth inhibition. When the final siRNA concentration was 5 nM, as shown in Fig. IB, 31 of the 32 siRNAs inhibited growth by more than 50% at 12O h and this level of inhibition remained high for 28 of the 32 siRNAs at the 240 h timepoint.

Effect of siRNA knockdown on RRMl mRNA level

To determine the effect of siRNA knockdown on RRMl mRNA levels, A549 cells were transfected with 8, 25, 74, 222, 667 or 2,000 pM RRMl -specific siRNAs (siRRMl-1, siRRMl-2 and siRRMl-3, for which the corresponding target sequences are given in SEQ ID NO: 27-29) or control siRNA (SEQ ID NO: 33) and relative mRNA levels quantified 24 h post-transfection. RNA was purified from cells using Trizol^® extraction as per the manufacturer's protocol (Invitrogen) and cDNA prepared by digesting approximately 200 ng purified RNA with DNAse I (Invitrogen) for 15 minutes at room temperature, followed by 15 minutes at 65⁰C in the presence of 25 mM EDTA. 30 ng random primers were added and incubated for 10 minutes at room temperature, followed by 1 minute on ice. Superscript^® III polymerase (Invitrogen) was added in the presence of 5 nM DTT (Sigma, St Louis, MO) and 1 mM deoxyribonucleotides (Invitrogen), and the reaction incubated at 25⁰C for 5 minutes, followed by an hour at 55⁰C. cDNA was diluted 1 :3 in 10 mM Tris pH 7.0, and quantitative RT-PCR carried out on a LightCycler^® 480 Real-Time PCR System (Roche Molecular Biochemicals, Basel, Switzerland) using LightCycler^® 480 SYBR" I Green Master (Roche) according to the manufacturer's instructions. Specific PCR primers for RRMl and the housekeeping gene lamin were used at 360 nM. The sequences of the PCR primer pairs used for RRMl and lamin are given in SEQ ID NO: 37 and 38, and 39 and 40, respectively.

As seen in Fig. 2, siRRMl-1, siRRMl-2 and siRRMl -3 effectively downregulated RRMl , with siRRMl-3 slightly more effective than siRRMl -2, which in turn induced more knockdown than siRRMl -1. All three siRNAs inhibited expression of RRMl by greater than 80% when used at a concentration of 222 pM. At lower concentrations, inhibition of RRMl expression is reduced, so that knockdown is less than 50% when the siRNAs were used at 8 pM.

Example 6

DETECTION OF SIRNA SPECIFIC RISC CLEAVAGE OF RRMl MRNA USING 5'RACE

AND 5'RACE COMBINED WITH MOLECULAR BEACON DETECTION.

5'RACE 5'RACE was performed using the GeneRacer™ kit (Life Technologies) with the manufacturer's instructions modified as follows. Total RNA was extracted using the PureLink™ 96 RNA Purification system (Life Technologies) from A549 cells transfected with RR- 15 (SEQ ID NO: 15) after 24 h as per the manufacturer's protocol. A549 cells were transfected with 1 nM RR- 15 using Lipofectamine™ RNAiMax. Purified RNA quality and concentration were assessed with a NanoDrop ND- 1000 spectrophotometer (Thermo Fisher Scientific, Waltham MA). 100 ng isolated total RNA was directly ligated to the RNA linker (SEQ ID NO: 42) without prior treatment. After phenol/ chloroform extraction and precipitation, first-strand cDNA was synthesized using a gene-specific primer, given in SEQ ID NO: 43. From this reaction, 1 μl was used in first-round 5'RACE reactions using the GeneRacer™ 5' primer and target gene-specific primer (SEQ ID NO: 44 and SEQ ID NO: 45) with cycling as described in the GeneRacer™ kit manual, but briefly one cycle of 94°C for 2 min, then five cycles of 94°C for 30 s and 72°C for 1 min, then five cycles of 94°C for 30 s and 70⁰C for 1 min, then 20 cycles of 94°C for 30 s and 68°C for 1 min.

eMBRACE

By combining standard 5'RACE with molecular beacon and light cycler technology, a rapid and specific method, termed eMBRACE, was developed to detect siRNA-induced cleavage of RRMl mRNA.

The first round 5'RACE reaction product synthesized as described above was used as a template for the eMBRACE reaction. The reaction contained 2 μl from the first-round RACE reaction diluted 1 :10 in RNase free water and 8 μl of the following Master mix: LightCycler Probes Master, 180 nM RR- 15 specific reverse primer (SEQ ID NO: 46), 3.6 μM RR-15 specific forward primer (SEQ ID NO: 47) and 250 nM Molecular Beacon probe (Integrated DNA Technologies Catalog No. 44164592; SEQ ID NO: 48). Reactions were run on a LightCycler® 480 with the following cycling conditions: 95°C for 10 min, then 55 cycles of 95°C for 10 s, 62°C for 30 s and 72 °C for 8 s, followed by 40 ⁰C for 30 s.

Results in Fig. 5 show that the molecular beacon detected the specific cleavage product when cDNA was prepared from A549 cells transfected with RR- 15 (SEQ ID NO: 15), a siRNA specific for RRMl, but not when the template was prepared from un-treated cells.

Effect of siRNA knockdown on RRM l protein levels

Cell lysates are prepared from A549 cells 48 and 72 h post-transfection with 1O nM RRMl -specific siRNA or control siRNA. Following Western blotting onto Immobilon-P PVDF filer (Millipore, Bedford MA) following standard protocols, membranes are probed with a goat anti-human polyclonal antibody specific for RRMl (Santa Cruz Biotechnology, Inc., Santa Cruz CA), at a concentration of 0.4 mg/ml. HRP-conjugated donkey anti-goat IgG (80 mg/ml; Santa Cruz) is used as a secondary antibody, and signal is detected with an ECL Plus Western blotting Detection System (GE Healthcare, UK) using a Typhoon Scanner (Molecular Dynamics, GE Healthcare, UK).

Effect of RRMl knockdown on tumor growth of cells transfected pre-implantation

To determine the effect of RRMl knockdown on tumor growth, A549 cells (human lung cancer cell line; ATCC No. CCL-185) were reverse-transfected prior to implantation in CD-I nude mice. In 175 cm² culture flasks, 1O x 10⁶ A549 cells (in RPMI medium supplemented with 10% fetal calf serum) were added to pre-made lipoplexes. To make lipoplexes, Lipofectamine™ RNAiMAX (Invitrogen) in serum- free RPMI medium was added to an equal volume of serum-free RPMI containing RRMl-specific siRNA RR-6 (SEQ ID NO: 6), RR-15 (SEQ ID NO: 15) and RR-19 (SEQ ID NO: 19), or control siRNA (SEQ ID NO: 33). After addition of cells, the final concentration of siRNA in the flask was 10 nM. Untransfected cells served as a further control. Flasks were incubated at 37°C for 24 h to allow transfection to proceed. The cells were harvested by trypsinization 24 h after the transfection, washed twice in PBS and viable cells counted. Equal numbers of cells (8 x 10⁶) were inoculated into CD-I nude mice (n=7-9). Briefly, 5 to 7 weeks old female nude mice were subcutaneously (s.c.) injected in the right flank with the prepared A549 cells in 100 μl PBS. Tumor growth was monitored three times per week by measuring tumor sizes using a digital caliper. Tumor volume was expressed in mm³ using the formula: V=α² x b x 0.52, where a and b represent the minimum and maximum tumor diameter, respectively. Mean tumor volumes calculated from each measurement were then plotted in a standard graph to compare the tumor growth of transfected cells to that of control. Results in Fig. 3 and Fig. 4 show that tumor growth inhibition was obtained with RR-6, RR-15, and RR-19 siRNA compared with the tumor growth rate in mice injected with cells containing the control siRNA or untransfected cells.

Treatment of established tumors with RRMl -targeting siRNA A549 cells (6-8 x 10⁶ cells) are inoculated s.c. into CD-I nude mice as described above. When tumors reach an average volume of approximately 100 mm³, the tumor-bearing mice are randomly assigned into three different treatment groups (RRMl siRNA, control siRNA and PBS) with 5 to 6 mice in each group so that there are no significant differences in tumor volumes among groups prior to the initiation of treatments. Mice are anesthetized by gaseous isoflurane and 50 μl siRNA in PBS or PBS only injected directly into tumors using a 30-gauge needle. Two experiments are conducted. In the first experiment, the animals receive a dose of 50 μg of siRNA in each intratumoral (i.t.) injection and the injections are performed 3 times per week for 2 weeks. In the second experiment, 25 μg siRNA is used and mice are injected 3 times per week for 3 weeks. Tumor volume is determined immediately before each injection by perpendicular measurements of the shortest and longest diameters as described above.

SEQ ID NO: 1-48 are set out in the attached Sequence Listing. The codes for polynucleotide and polypeptide sequences used in the attached Sequence Listing confirm to WIPO Standard ST.25 (1988), Appendix 2.

All references cited herein, including patent references and non-patent references, are hereby incorporated by reference in their entireties.

Claims

We Claim:

1. An isolated small interfering nucleic acid molecule (siNA) that is capable of reducing expression of ribonucleotide reductase subunit 1 (RRMl) in a cell by an RNA interference mechanism, wherein the siNA comprises a first strand that has a sequence corresponding to a DNA sequence of SEQ ID NO: 1-32 and a second strand that has a sequence corresponding to a DNA sequence that is sufficiently complementary to a sequence of SEQ ID NO: 1 -32 to anneal to the sequence of SEQ ID NO: 1 -32.

2. The isolated siNA of claim 1 , wherein the siNA comprises at least one modified ribose or phosphate backbone moiety.

3. The isolated siNA of any one of claims 1 or 2, wherein the siNA is 15 to 30 nucleotides in length.

4. The isolated siNA of any one of claims 1-3, wherein the isolated siNA is a double-stranded RNA.

5. The isolated siNA of any one of claims 1-3, wherein the isolated siNA is a short hairpin RNA.

6. A composition comprising a siNA of any one of claims 1 -5 and a pharmaceutically acceptable carrier.

7. The composition of claim 6, furthering comprising a chemotherapeutic agent.

8. The composition of claim 7, wherein the chemotherapeutic agent is gemcitabine.

9. A composition comprising:

(a) a first single-stranded siNA that has a sequence corresponding to a DNA sequence of SEQ ID NO: 1 -32;

(b) a second single-stranded siNA that has a sequence corresponding to a DNA sequence that is sufficiently complementary to a sequence of SEQ ID NO: 1-32 to anneal to a sequence of SEQ ID NO: 1 -32; and

(c) a pharmaceutically acceptable carrier, wherein at least one of the first and second single-stranded siNAs comprises at least one nucleobase modification that prevents annealing of the first and second single- stranded nucleic acids prior to entry into a cell.

10. The composition of claim 9, wherein the first and second single-stranded siNAs are between 15 to 30 nucleotides in length.

1 1. The composition of claim 9, wherein both the first and second single-stranded siNAs are modified.

12. The composition of claim 9, wherein at least one of the first and second single- stranded nucleic acids further comprises at least one ribose or phosphate backbone modification that protects against nuclease degradation.

13. A method for inhibiting expression of ribonucleotide reductase subunit 1 in a cell comprising contacting the cell with a composition of any one of claims 6-12.

14. The method of claim 13 further comprising contacting the cell with a chemotherapeutic agent.

15. A method for inhibiting tumor growth in a patient, comprising administering to the patient a composition of any one of claims 6-12.

16. A method for inhibiting neoplastic cell growth in a patient, comprising administering to the patient a composition of any one of claims 6-12.

17. The method of any one of claims 15 and 16 further comprising administering to the patient a chemotherapeutic agent.

18. A method for inhibiting tumor growth and/or inhibiting neoplastic cell growth in a patient comprising:

(a) administering to the patient a first composition comprising a first single-stranded siNA that has a sequence corresponding to a DNA sequence of SEQ ID NO: 1-32; and

(b) administering to the patient a second composition comprising a second single- stranded siNA that has a sequence corresponding to a DNA sequence that is sufficiently complementary to a sequence of SEQ ID NO: 1-32 to anneal to a sequence of SEQ ID NO: 1-32, wherein at least one of the first and second single-stranded siNAs comprises at least one nucleobase modification that prevents annealing of the first and second single- stranded nucleic acids prior to entry into a cell.

19. The method of claim 18, wherein the first and second single-stranded siNAs are between 15 to 30 nucleotides in length.

20. The method of claim 18, wherein both the first and second single-stranded siNAs are modified.

21. The method of claim 18, wherein at least one of the first and second single- stranded nucleic acids further comprises at least one ribose or phosphate backbone modification that protects against nuclease degradation.

22. A method for inhibiting expression of ribonucleotide reductase subunit 1 in a cell comprising:

(a) contacting the cell with a first composition comprising a first single-stranded siNA that has a sequence corresponding to a DNA sequence of SEQ ID NO: 1 -32; and

(b) contacting the cell with a second composition comprising a second single- stranded siNA that has a sequence corresponding to a DNA sequence that is sufficiently complementary to a sequence of SEQ ID NO: 1 -32 to anneal to a sequence of SEQ ID NO: 1 -32, wherein at least one of the first and second single-stranded siNAs comprises at least one nucleobase modification that prevents annealing of the first and second single- stranded nucleic acids prior to entry into a cell.