MULTICISTRONIC siRNA CONSTRUCTS TO INHIBIT TUMORS

Abstract

Multicistronic short interfering RNA constructs targeting in various combinations a human urokinase-type plasminogen activator receptor (uPAR), human urokinase-type plasminogen activator (uPA), human matrix metalloprotease 9 (MMP-9) and cathepsin B (CB) inhibit tumors.

1 . A multicistronic short interfering RNA construct comprising at least a first and a second self-complementary sequence, wherein the construct is used to inhibit tumor formation and to regress pre-formed tumors. 
     
     
         2 . The multicistronic construct of  claim 1 , wherein the first self-complementary sequence comprises a nucleotide sequence of human urokinase-type plasminogen activator receptor (uPAR) and its complement, and the second self-complementary sequence comprises a nucleotide sequence of human urokinase-type plasminogen activator (uPA) and its complement. 
     
     
         3 . The multicistronic construct of  claim 2 , wherein the self complementary sequence of uPA is TGAGAGCCCTGCTGGCGCGCC-loop-GGCGCGCCAGCAGGGCTCTCA (SEQ ID NO: 1) and the self complementary sequence of uPAR is CTACAGCAGTGGAGAGCGATT-loop-AATCGCTCTCCACTGCTGTAG (SEQ ID NO: 2). 
     
     
         4 . The multicistronic construct of  claim 3 , wherein the self complementary sequences of uPAR and uPA are separated by an intervening sequence of length of about 22-35 base pairs. 
     
     
         5 . The multicistronic construct of  claim 4 , wherein the intervening sequence is AGCT TGGTACCGAG CTCG GATC (SEQ ID NO: 3). 
     
     
         6 . The multicistronic construct of  claim 1 , wherein the first self-complementary sequence comprises a nucleotide sequence of urokinase-type plasminogen activator receptor (uPAR) and its complement and the second self-complementary sequence comprises a nucleotide sequence of matrix metalloprotease 9 (MMP-9) and its complement. 
     
     
         7 . The multicistronic construct of  claim 6 , wherein the self complementary sequence of uPAR is CTACAGCAGTGGAGAGCGATT-loop-AATCGCTCTCCACTGCTGTAG (SEQ ID NO: 2) and the self complementary sequence of MMP-9 is CAAGTGGCACCACCACAACAA-loop-TTGTTGTGGTGGTGCCACTTG (SEQ ID NO: 4). 
     
     
         8 . The multicistronic construct of  claim 3 , wherein each loop comprises 9 nucleotides. 
     
     
         9 . The multicistronic construct of  claim 8 , wherein each loop is ATATATAAT. 
     
     
         10 . The multicistronic construct of  claim 1 , wherein the self complementary sequences are separated by an intervening sequence of about 22-68 base pairs in length. 
     
     
         11 . The multicistronic construct of  claim 10 , wherein the intervening sequence is GATCCA CTAGTAACGG CCGCCAGTGT GCTGG AATT (SEQ ID NO: 5). 
     
     
         12 . The multicistronic construct of  claim 1 , wherein the construct is a circular nucleic acid. 
     
     
         13 . A method of inhibiting tumor formation or regressing pre-formed tumors, the method comprising:
 (a) administering the short interfering RNA multicistronic construct of  claim 1  to the tumor; and   (b) reducing expression of a plurality of genes expressed in the tumor and targeted by the short interfering RNA construct, thereby inhibiting the tumor.   
     
     
         14 . The method of  claim 13 , wherein the short interfering RNA multicistronic construct targets urokinase-type plasminogen activator receptor (uPAR) and urokinase-type plasminogen activator (uPA). 
     
     
         15 . The method of  claim 14 , wherein the short interfering RNA multicistronic construct comprises TGAGAGCCCTGCTGGCGCGCC-loop-GGCGCGCCAGCAGGGCTCTCA-intervening sequence-CTACAGCAGTGGAGAGCGATT-loop-AATCGCTCTCCACTGCTGTAG (SEQ ID NOS 1 & 2, respectively). 
     
     
         16 . The method of  claim 1 , wherein the tumors are inhibited by reducing at least one of tumor cell proliferation, tumor cell invasion, tumor cell migration and angiogenesis. 
     
     
         17 . The method of  claim 1 , wherein the tumors are is selected from the group consisting of prostate cancer, glioma, meningioma, colon cancer, lung cancer, breast cancer, and melanoma. 
     
     
         18 . The method of  claim 17 , wherein the construct is administered through direct delivery. 
     
     
         19 . The method of  claim 13 , wherein the short interfering RNA multicistronic construct targets urokinase-type plasminogen activator receptor (uPAR) and matrix metalloprotease 9 (MMP-9). 
     
     
         20 . The method of  claim 13 , wherein the short interfering RNA multicistronic construct comprises CTACAGCAGTGGAGAGCGATT-loop-AATCGCTCTCCACTGCTGTAG-intervening sequence-CAAGTGGCACCACCACAACAA-loop-TTGTTGTGGTGGTGCCACTTG (SEQ ID NOS 2 & 4, respectively). 
     
     
         21 . A short interfering RNA molecule selected from the group consisting of RNA molecules targeted to:
 (a) urokinase-type plasminogen activator receptor (uPAR) and matrix metalloprotease 9 (MMP-9) the short interfering RNA molecule, comprising the nucleic acid sequence CUACAGCAGUGGAGAGCGAUU-loop-AAUCGCUCUCCACUGCUGUAG-intervening sequence —CAAGUGGCACCACCACAACAA-loop-UUGUUGUGGUGGUGCCACUUG (SEQ ID NOS 9 & 10, respectively); and   (b) urokinase-type plasminogen activator receptor (uPAR) and urokinase-type plasminogen activator (uPA), the short interfering RNA molecule comprising the nucleic acid sequence UGAGAGCCCUGCUGGCGCGCC-loop-GGCGCGCCAGCAGGGCUCUCA-intervening sequence —CUACAGCAGUGGAGAGCGAUU-loop-AAUCGCUCUCCACUGCUGUAG (SEQ ID NOS 11 & 9, respectively).   
     
     
         22 . A recombinant cell transformed with the multicistronic construct of  claim 1 . 
     
     
         23 . A recombinant virus transformed with the multicistronic construct of  claim 1 .