Compositions and methods for immunostimulatory rna oligonucleotides

Abstract

The present invention provides 4-nucleotide (4mer) RNA motifs that confer immunostimulatory activity, in particular, IFN-α-inducing activity to a RNA oligonucleotide. The present invention also provides RNA oligonucleotides, including siRNA, with high or low immunostimulatory activity. The present invention further provides the use of the RNA oligonucleotides of the invention for therapeutic purposes.

1 - 84 . (canceled) 
     
     
         85 . A pharmaceutical composition comprising an oligonucleotide and a pharmaceutically acceptable carrier,
 wherein the oligonucleotide comprises at least one of the 4-nucleotide (4-mer) motifs selected from the group consisting of: GUUC, GUCA, GCUC, GUUG, GUUU, GGUU, GUGU, GGUC, GUCU, GUCC, GCUU, UUGU, UGUC, CUGU, CGUC, UGUU, GUUA, UGUA, UUUC, UGUG, GGUA, GUCG, UUUG, UGGU, GUGG, GUGC, GUAC, GUAU, UAGU, GUAG, UUCA, UUGG, UCUC, CAGU, UUCG, CUUC, GAGU, GGUG, UUGC, UUUU, CUCA, UCGU, UUCU, UGGC, CGUU, CUUG, and UUAC,   wherein the nucleotide sequences of the motifs are 5′→3′,   wherein the oligonucleotide is between 6 and 64 nucleotides in length,   wherein:   (i) at least one strand of the RNA oligonucleotide has an IFN-α score of at least 23 when n=6; at least 26 when n=7; at least 28 when n=8; at least 30 when n=9; at least 1.4909×n+22.014 when n is greater than 9, wherein the IFN-α score is assigned according to a method comprising:
 (a) identifying all possible 3-nucleotide (3mer) motifs contained in the oligonucleotide; 
 (b) assigning an IFN-α point score for each individual 3mer motif:
 (i) for a 3mer motif which appears in Table 7, assigning an IFN-α point score according to Table 7; 
 (ii) for a 3mer motif which does not appear in Table 7, assigning an IFN-α point score of 0; and 
 
 (c) assigning the sum of the IFN-α point scores of individual 3mer motifs as the IFN-α score of the oligonucleotide; or 
   (ii) at least one strand of the RNA oligonucleotide has an IFN-α score of at least 0.58, wherein the IFN-α score is assigned according to a method comprising the steps of:
 (a) identifying all possible 3-nucleotide (3mer) motifs contained in the oligonucleotide; 
 (b) assigning an IFN-α point score for each individual 3mer motif according to Table 12A; and 
 (c) assigning the highest individual IFN-α point score as the IFN-α score of the oligonucleotide; 
   wherein n is length of the oligonucleotide; and   provided that the oligonucleotide is not 5′-UUGAUGUGUUUAGUCGCUA-3′,5′-GCACCACUAGUUGGUUGUC-3′,5′-GUUGUAGUUGUACUCCAGC-3′,5′-GCCCGUCUGUUGUGUGACUC-3′,5′-GUCUGUUGUGUG-3′, or 5′-GUUGUGGUUGUGGUUGUG-3′.   
     
     
         86 . The pharmaceutical composition of  claim 85 , wherein the 4-nucleotide motifs are selected from the group consisting of GUUC, GUCA, GCUC, GUUG, GUUU, and GGUU. 
     
     
         87 . The pharmaceutical composition  claims 86 , wherein spacer nucleotides between the 4-nucleotide motifs are identical, and the spacer nucleotide are selected from the group consisting of A, T, C, G, and variants and derivatives thereof. 
     
     
         88 . The pharmaceutical composition of  claim 85 , wherein the oligonucleotides does not have gene silencing activity for any known mammalian gene. 
     
     
         89 . A pharmaceutical composition comprising an oligonucleotide and a pharmaceutically acceptable carrier,
 wherein:   at least one strand of the oligonucleotide has an IFN-α score of at least 1.4909×n+31.014, wherein the IFN-α score is assigned according to a method comprising:
 (a) identifying all possible 3-nucleotide (3mer) motifs contained in the oligonucleotide; 
 (b) assigning an IFN-α point score for each individual 3mer motif:
 (i) for a 3mer motif which appears in Table 7, assigning an IFN-α point score according to Table 7; 
 (ii) for a 3mer motif which does not appear in Table 7, assigning an IFN-α point score of 0; and 
 
 (c) assigning the sum of the IFN-α point scores of individual 3mer motifs as the IFN-α score of the oligonucleotide; or 
   (ii) at least one strand of the RNA oligonucleotide has an IFN-α score of at least 0.58, wherein the IFN-α score is assigned according to a method comprising the steps of:
 (a) identifying all possible 3-nucleotide (3mer) motifs contained in the oligonucleotide; 
 (b) assigning an IFN-α point score for each individual 3mer motif according to Table 12A; and 
 (c) assigning the highest individual IFN-α point score as the IFN-α score of the oligonucleotide; and 
   wherein the oligonucleotide is an siRNA and n is between 14 and 25, or wherein the oligonucleotide is an antisense RNA and n is between 14 and 50.   
     
     
         90 . A pharmaceutical composition comprising an oligonucleotide and a pharmaceutically acceptable carrier,
 wherein:   (i) all strand(s) of the oligonucleotide has(have) an IFN-α score of at most 0.6075×−9.9484, wherein the IFN-α score is assigned according to a method comprising:
 (a) identifying all possible 3-nucleotide (3mer) motifs contained in the oligonucleotide; 
 (b) assigning an IFN-α point score for each individual 3mer motif:
 (i) for a 3mer motif which appears in Table 7, assigning an IFN-α point score according to Table 7; 
 (ii) for a 3mer motif which does not appear in Table 7, assigning an IFN-α point score of 0; and 
 
 (c) assigning the sum of the IFN-α point scores of individual 3mer motifs as the IFN-α score of the oligonucleotide; or 
   (ii) wherein all strand(s) of the oligonucleotide has(have) an IFN-α score of at most 0.11, wherein the IFN-α score is assigned according to a method comprising the steps of:
 (a) identifying all possible 3-nucleotide (3mer) motifs contained in the oligonucleotide; 
 (b) assigning an IFN-α point score for each individual 3mer motif according to Table 12A; and 
 (c) assigning the highest individual IFN-α point score as the IFN-α score of the oligonucleotide; and 
   wherein the oligonucleotide is an siRNA and n is between 19 and 25, or wherein the oligonucleotide is an antisense RNA and n is between 18 and 50.   
     
     
         91 . A pharmaceutical composition comprising an in vitro activated dendritic cell and a pharmaceutically acceptable carrier, wherein the in vitro activated dendritic cell is produced by a method comprising:
 (a) complexing an RNA oligonucleotide with a complexation agent;   (b) contacting dendritic cells isolated from a donor mammal with the complexed RNA oligonucleotide; and   (c) contacting the dendritic cells with an antigen,   wherein:wherein the oligonucleotide comprises at least one of the 4-nucleotide (4-mer) motifs selected from the group consisting of: GUUC, GUCA, GCUC, GUUG, GUUU, GGUU, GUGU, GGUC, GUCU, GUCC, GCUU, UUGU, UGUC, CUGU, CGUC, UGUU, GUUA, UGUA, UUUC, UGUG, GGUA, GUCG, UUUG, UGGU, GUGG, GUGC, GUAC, GUAU, UAGU, GUAG, UUCA, UUGG, UCUC, CAGU, UUCG, CUUC, GAGU, GGUG, UUGC, UUUU, CUCA, UCGU, UUCU, UGGC, CGUU, CUUG, and UUAC,   wherein the nucleotide sequences of the motifs are 5′→3′,   wherein the oligonucleotide is between 6 and 64 nucleotides in length,   wherein:   (i) at least one strand of the RNA oligonucleotide has an IFN-α score of at least 23 when n=6; at least 26 when n=7; at least 28 when n=8; at least 30 when n=9; at least 1.4909×n+22.014 when n is greater than 9, wherein the IFN-α score is assigned according to a method comprising:
 (a) identifying all possible 3-nucleotide (3mer) motifs contained in the oligonucleotide; 
 (b) assigning an IFN-α point score for each individual 3mer motif:
 (i) for a 3mer motif which appears in Table 7, assigning an IFN-α point score according to Table 7; 
 (ii) for a 3mer motif which does not appear in Table 7, assigning an IFN-α point score of 0; and 
 
 (c) assigning the sum of the IFN-α point scores of individual 3mer motifs as the IFN-α score of the oligonucleotide; or 
   (ii) at least one strand of the RNA oligonucleotide has an IFN-α score of at least 0.58, wherein the IFN-α score is assigned according to a method comprising the steps of:
 (a) identifying all possible 3-nucleotide (3mer) motifs contained in the oligonucleotide; 
 (b) assigning an IFN-α point score for each individual 3mer motif according to Table 12A; and 
 (c) assigning the highest individual IFN-α point score as the IFN-α score of the oligonucleotide; 
   wherein n is length of the oligonucleotide; and   provided that the oligonucleotide is not 5′-UUGAUGUGUUUAGUCGCUA-3′,5′-GCACCACUAGUUGGUUGUC-3′,5′-GUUGUAGUUGUACUCCAGC-3′,5′-GCCCGUCUGUUGUGUGACUC-3′,5′-GUCUGUUGUGUG-3′, or 5′-GUUGUGGUUGUGGUUGUG-3′.   
     
     
         92 . The pharmaceutical composition of  claim 91 , wherein the 4-nucleotide motifs are selected from the group consisting of GUUC, GUCA, GCUC, GUUG, GUUU, and GGUU. 
     
     
         93 . The pharmaceutical composition  claims 91 , wherein spacer nucleotides between the 4-nucleotide motifs are identical, and the spacer nucleotides are selected from the group consisting of A, T, C, G, and variants and derivatives thereof. 
     
     
         94 . The pharmaceutical composition of  claim 93 , wherein the oligonucleotide does not have gene silencing activity for any known mammalian gene.