Inhibitors of ribonucleotide reductase subunit 2 and uses thereof

Abstract

The present application relates to inhibitors of ribonucleotide reductase subunit 2 (R2), and methods and compositions related to the R2 inhibitors. In certain embodiments, the R2 inhibitors include nucleic acids, such as for example siRNAs.

1 . A nucleic acid comprising:
 (i) a first strand of 15 to 30 nucleotides in length having a sequence set forth in SEQ ID NOs: 5, and   (ii) a second strand of 15 to 30 nucleotides in length,   wherein said nucleic acid optionally comprises one or more modified backbone or base moieties.   
     
     
         2 . The nucleic acid of  claim 1 , wherein the nucleic acid is a double-stranded RNA. 
     
     
         3 . The nucleic acid of  claim 1 , wherein the nucleic acid is a hairpin RNA. 
     
     
         4 . The nucleic acid of  claim 3 , wherein the loop region of the hairpin RNA is from 4 to 10 nucleotides in length. 
     
     
         5 . The nucleic acid of  claim 1 , wherein the double-stranded portion of the RNA is from about 15 to about 30 nucleotides in length. 
     
     
         6 . The nucleic acid of  claim 1 , wherein the first strand is a DNA polynucleotide and the second strand is an RNA polynucleotide. 
     
     
         7 . The nucleic acid of  claim 1 , wherein the first and/or second strand further comprises a 3′ overhang region, a 5′ overhang region, or both 3′ and 5′ overhang regions. 
     
     
         8 . The nucleic acid of  claim 7 , wherein the overhang region is from 1 to 10 nucleotides in length. 
     
     
         9 . (canceled) 
     
     
         10 . The nucleic acid of  claim 1 , wherein the modified backbone or base moieties are one or more of the following: alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxylmethyl esters, carbonates, and phosphate triesters. 
     
     
         11 . The nucleic acid of  claim 1 , wherein the nucleic acid comprises at least one 2′-O-alkylated ribonucleotide. 
     
     
         12 . The nucleic acid of  claim 1 , wherein the first strand comprises a sequence selected from SEQ ID NOs: 9, 17, 19, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 87, 89, 91, 93, and 95. 
     
     
         13 . The nucleic acid of  claim 1 , wherein the second strand comprises a sequence selected from SEQ ID NO:10, 18, 20, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 88, 90, 92, 94, and 96. 
     
     
         14 . The nucleic acid of  claim 1 , wherein the nucleic acid inhibits R2 expression in cells by 50% or greater, when contacted with the cells under physiological conditions at a concentration of 10 nanomolar. 
     
     
         15 . An isolated nucleic acid comprising a sequence that hybridizes to a region of an R2 transcript corresponding to nucleotides 616-667 of SEQ ID NO: 1 under physiological conditions and decreases the expression of R2 in a cell, wherein said nucleic acid optionally comprises one or more modified backbone or base moieties. 
     
     
         16 . The isolated nucleic acid of  claim 15 , wherein the nucleic acid comprises a sequence that hybridizes to a region of an R2 transcript corresponding to nucleotides 626-657 of SEQ ID NO: 1. 
     
     
         17 . The isolated nucleic acid of  claim 15 , wherein the nucleic acid comprises a sequence that hybridizes to a region of an R2 transcript corresponding to nucleotides 631-652 of SEQ ID NO: 1. 
     
     
         18 . The nucleic acid of  claim 15 , wherein the nucleic acid comprises at least 10 consecutive nucleotides that are complementary to one of said regions of R2. 
     
     
         19 . The nucleic acid of  claim 15 , wherein the nucleic acid is from about 14 to about 50 nucleotides in length. 
     
     
         20 . The nucleic acid of  claim 15 , wherein the nucleic acid is single-stranded. 
     
     
         21 . The nucleic acid of  claim 15 , wherein the nucleic acid is double-stranded. 
     
     
         22 . The nucleic acid of  claim 15 , wherein the nucleic acid is a DNA molecule. 
     
     
         23 . The nucleic acid of  claim 15 , wherein the nucleic acid is a RNA molecule. 
     
     
         24 . The nucleic acid of  claim 15 , wherein the nucleic acid comprises a DNA strand and an RNA strand. 
     
     
         25 . The nucleic acid of  claim 15 , wherein the nucleic acid is an RNAi construct. 
     
     
         26 . The nucleic acid of  claim 25 , wherein the RNAi construct is a dsRNA. 
     
     
         27 . The nucleic acid of  claim 25 , wherein the RNAi construct is a hairpin RNA. 
     
     
         28 . The nucleic acid of  claim 25 , wherein the duplex portion of the RNAi construct is from about 15 to about 30 nucleotides in length. 
     
     
         29 . The nucleic acid of  claim 25 , wherein the RNAi construct comprises a sequence as set forth in SEQ ID NO: 5. 
     
     
         30 . The nucleic acid of  claim 25 , wherein the RNAi construct comprises a sequence selected from of SEQ ID NOs: 9, 10, 17-20, 45-64, 87-96. 
     
     
         31 . (canceled) 
     
     
         32 . The nucleic acid of  claim 15 , wherein the nucleic acid comprises at least one internucleotide linkage selected from alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxylmethyl esters, carbonates, and phosphate triesters. 
     
     
         33 . The nucleic acid of  claim 15 , wherein the nucleic acid comprises at least one 2′-O-alkylated ribonucleotide. 
     
     
         34 . The nucleic acid of  claim 15 , wherein the nucleic acid is an enzymatic nucleic acid. 
     
     
         35 - 36 . (canceled) 
     
     
         37 . The nucleic acid of  claim 15 , wherein the nucleic acid inhibits R2 expression in cells by 50% or greater, when contacted with the cells under physiological conditions at a concentration of 10 nanomolar. 
     
     
         38 . A pharmaceutical composition comprising the nucleic acid of  claim 1  or  15 , and a pharmaceutically acceptable carrier. 
     
     
         39 . The pharmaceutical composition of  claim 38 , wherein the pharmaceutically acceptable carrier includes a cationic polymer. 
     
     
         40 . The pharmaceutical composition of  claim 38 , wherein the pharmaceutically acceptable carrier includes a cyclodextrin polymer. 
     
     
         41 . The pharmaceutical composition of  claim 40 , wherein the cyclodextrin structure is im-CDP as illustrated in  FIG. 27 . 
     
     
         42 . The pharmaceutical composition of  claim 38 , comprising a particle that includes a cyclodextrin polymer and the nucleic acid of  claim 1  or  15  and is PEGylated. 
     
     
         43 . The pharmaceutical composition of  claim 42 , wherein the particle further comprises adamantane. 
     
     
         44 . The pharmaceutical composition of  claim 38 , further comprising a ligand that targets a particular tissue or cell type. 
     
     
         45 . The pharmaceutical composition of  claim 44 , wherein the ligand comprises galactose. 
     
     
         46 . The pharmaceutical composition of  claim 38 , comprising nanoparticles, wherein the nanoparticles are from 10 to 100 nm in diameter. 
     
     
         47 . The pharmaceutical composition of  claim 46 , wherein the nanoparticles are about 50 to 70 nm in diameter. 
     
     
         48 . The pharmaceutical composition of  claim 47 , wherein the nanoparticles are about 50 nm in diameter. 
     
     
         49 . The pharmaceutical composition of  claim 38 , wherein the pharmaceutically acceptable carrier comprises:
 an imidazole-modified cyclodextrin-containing cationic polymer, and   a targeting moiety comprising adamantane-PEG-ligand,   wherein the polymer and targeting moiety form nanoparticles that encapsulates the nucleic acid.   
     
     
         50 . The pharmaceutical composition of  claim 49 , wherein the nanoparticles have a diameter of about 50 to 120 nm. 
     
     
         51 . The pharmaceutical composition of  claim 50 , wherein the nanoparticles have a diameter of about 50 to 100 nm. 
     
     
         52 . The pharmaceutical composition of  claim 51 , wherein the nanoparticles have a diameter of about 50 to 70 nm. 
     
     
         53 . The pharmaceutical composition of  claim 49 , wherein the nanoparticles have a diameter of about 50 nm. 
     
     
         54 . The pharmaceutical composition of  claim 49 , wherein the targeting ligand comprises galactose. 
     
     
         55 . The pharmaceutical composition of  claim 49 , wherein the targeting ligand comprises transferrin. 
     
     
         56 . Use of the nucleic acid of  claim 1  or  15  in the manufacture of a medicament for the treatment of a disease or condition associated with unwanted proliferation of cells. 
     
     
         57 . The use of  claim 56 , wherein the cells are cancerous or tumor cells. 
     
     
         58 . The use of  claim 56 , wherein the cells are pathogen cells. 
     
     
         59 . The use of  claim 56 , wherein the cells are normal cells, the unwanted proliferation of which leads to the disease of condition. 
     
     
         60 . A method for treating a patient having a cancer comprising administering to the patient a therapeutically effective amount of the double-stranded nucleic acid of  claim 1  or  15 . 
     
     
         61 . The method of  claim 60 , wherein the nucleic acid is formulated with a pharmaceutically acceptable carrier. 
     
     
         62 . The method of  claim 60 , wherein the nucleic acid is formulated with a ligand targeting the cancer cell. 
     
     
         63 . The method of  claim 62 , wherein the ligand is transferrin. 
     
     
         64 . The method of  claim 62 , wherein the cancer cell is a hepatocyte and the ligand comprises galactose. 
     
     
         65 . The method of  claim 60 , wherein the nucleic acid is formulated as a component of a polymeric nanoparticle. 
     
     
         66 . The method of  claim 65 , wherein the nanoparticle is from 10 to 120 nm in diameter. 
     
     
         67 . The method of  claim 65 , wherein the nanoparticle is from 50 to 120 nm in diameter. 
     
     
         68 . The method of  claim 67 , wherein the nanoparticle is from 50 to 100 nm in diameter. 
     
     
         69 . The method of  claim 65 , wherein the nanoparticle is 50 nm in diameter. 
     
     
         70 . The method of  claim 60 , wherein the route of administration is systemic. 
     
     
         71 . The method of  claim 60 , wherein the route of administration is intrahepatic arterial administration. 
     
     
         72 . The method of  claim 71 , wherein the cancer is liver cancer. 
     
     
         73 . The method of  claim 60 , wherein the cancer cell expresses a higher level of R2 compared to a noncancerous cell from a comparable tissue. 
     
     
         74 . The method of  claim 60 , further including at least one additional anti-cancer chemotherapeutic agent that inhibits cancer cells in an additive or synergistic manner with the nucleic acid. 
     
     
         75 . The method of  claim 74 , wherein the chemotherapeutic agent is fluorouracil (5FU). 
     
     
         76 - 84 . (canceled)