Double-stranded ribonucleic acids with rugged physico-chemical structure and highly specific biologic activity

Abstract

The invention relates to our discovery of a novel double-stranded ribonucleic acid (dsRNA) having specific biological activities, which includes acting as a selective agonist for activation of the Toll-like receptor 3. Its “rugged” molecular structure as measured by physico-chemical techniques is resistant to molecular unfolding (i.e., denaturation). This structure appears to be responsible for increased efficacy of dsRNA in therapeutic applications and improved biological activity (e.g., used as an immunoregulatory agent). Medicaments, processes for their manufacture, and methods for their use are provided herein.

1 . An isolated double-stranded ribonucleic acid (dsRNA) which is resistant to denaturation under conditions that are able to separate hybridized poly(riboinosinic acid) and poly(ribocytosinic acid) strands. 
   
   
       2 . The dsRNA of  claim 1  which contains only partially hybridized strands. 
   
   
       3 . The dsRNA of  claim 1 , wherein only a single strand of said dsRNA comprises one or more uracil or guanine bases that are not based paired to an opposite strand. 
   
   
       4 . The dsRNA of  claim 3 , wherein said single strand is partially hybridized to an opposite strand comprised of poly(riboinosinic acid). 
   
   
       5 . The dsRNA of  claim 3 , wherein said single strand is comprised of poly(ribocytosinic 4-29 uracilic acid). 
   
   
       6 . The dsRNA of  claim 5 , wherein said single strand is partially hybridized to an opposite strand comprised of poly(riboinosinic acid). 
   
   
       7 . The dsRNA of  claim 1 , wherein both strands of said dsRNA comprise one or more uracil or guanine bases that are not based paired to an opposite strand. 
   
   
       8 . The dsRNA of  claim 1 , wherein a strand of said dsRNA is comprised of ribo(I n ).ribo(C 4-29 U) n , in which ribo is a ribonucleotide and n is an integer from 40 to 40,000. 
   
   
       9 . The dsRNA of  claim 8 , wherein a strand of said dsRNA is comprised of ribo(I n ).ribo(C 11-14 U) n , in which ribo is a ribonucleotide and n is an integer from 40 to 40,000. 
   
   
       10 . The dsRNA of  claim 9 , wherein a strand of said dsRNA is comprised of ribo(I n ).ribo(C 12 U) n , in which ribo is a ribonucleotide and n is an integer from 40 to 40,000. 
   
   
       11 . The dsRNA of  claim 1  which has a molecular weight from about 250 Kda to about 320 Kda. 
   
   
       12 . The dsRNA of  claim 1  which has at least one strand of a length from about 380 bases to about 450 bases. 
   
   
       13 . The dsRNA of  claim 1  which has from about 30 to about 38 helical turns of duplexed RNA strands. 
   
   
       14 . A composition comprising one or more different dsRNAs as defined in  claim 1 . 
   
   
       15 . An isolated double-stranded ribonucleic acid (dsRNA) which is resistant to denaturation under conditions that are able to separate hybridized poly(riboinosinic acid) and poly(ribocytosinic acid) strands, wherein the isolated dsRNA:
 has an HPLC chromatogram substantially the same as the 5 minute peak of  FIG. 1B  or  FIG. 1C ;   is stable to exposure to thermal stress at 40° C.; and   has an increased bioactivity as evidenced by binding to receptor TLR3-ECD as compared to unselected poly(I):poly(C 12 U).   
   
   
       16 . The dsRNA of  claim 15  in which stability over time following thermal stress exposure at 40° C. substantially as shown in the HPLC purity peaks of  FIG. 20 . 
   
   
       17 . A method of treating a subject, said method comprising administration to the subject of at least the dsRNA defined in  claim 1  in a therapeutic amount. 
   
   
       18 . The method according to  claim 17 , wherein the therapeutic amount of at least said dsRNA or said composition is infused intravenously. 
   
   
       19 . The method according to  claim 17 , wherein the therapeutic amount is injected intradermally, subcutaneously, or intramuscularly; inhaled intranasally or intratracheally; or applied intranasally, intratracheally, oropharyngeally, or sublingually.