Modified Short Interfering Nucleic Acid (siNA) Molecules and Uses Thereof

Abstract

Disclosed herein are short interfering nucleic acid (siNA) molecules comprising modified nucleotides and uses thereof. The siNA molecules may be double stranded and comprise modified nucleotides selected from 2′-O-methyl nucleotides and 2′-fluoro nucleotides. Further disclosed herein are siNA molecules comprising (a) a phosphorylation blocker, conjugated moiety, or 5′-stabilized end cap; and (b) a short interfering nucleic acid (siNA).

1 . A short interfering nucleic acid (siNA) molecule comprising:
 (a) a sense strand comprising a first nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to an RNA corresponding to a target gene, wherein the first nucleotide sequence:
 (i) is 15 to 30 nucleotides in length; and 
 (ii) comprises 15 or more modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein at least one modified nucleotide is a 2′-O-methyl nucleotide and the nucleotide at position 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and/or 19 from the 5′ end of the first nucleotide sequence is a 2′-fluoro nucleotide or wherein at least one modified nucleotide is a 2′-O-methyl nucleotide and at least one modified nucleotide is a 2′-fluoro nucleotide; and 
   (b) an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the RNA corresponding to the target gene, wherein the second nucleotide sequence:
 (iii) is 15 to 30 nucleotides in length; and 
 (iv) comprises 15 or more modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein at least one modified nucleotide is a 2′-O-methyl nucleotide and the nucleotide at position 2, 5, 6, 8, 10, 14, 16, 17, and/or 18 from the 5′ end of the second nucleotide sequence is a 2′-fluoro nucleotide or wherein at least one modified nucleotide is a 2′-O-methyl nucleotide and at least one modified nucleotide is a 2′-fluoro nucleotide; or 
   (b) a sense strand comprising a first nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to an RNA corresponding to a target gene, wherein the first nucleotide sequence:
 (i) is 15 to 30 nucleotides in length; and 
 (ii) comprises 15 or more modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein at least one modified nucleotide is a 2′-O-methyl nucleotide and at least one modified nucleotide is a 2′-fluoro nucleotide; and 
 an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the RNA corresponding to the target gene, wherein the second nucleotide sequence: 
 (iii) is 15 to 30 nucleotides in length; and 
 (iv) comprises 15 or more modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein at least one modified nucleotide is a 2′-O-methyl nucleotide and the nucleotide at position 2, 5, 6, 8, 10, 14, 16, 17, and/or 18 from the 5′ end of the second nucleotide sequence is a 2′-fluoro nucleotide. 
   
     
     
         2 . (canceled) 
     
     
         3 . The siNA of  claim 1 , wherein the first nucleotide sequence comprises 16, 17, 18, 19, 20, 21, 22, 23, or more modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide. 
     
     
         4 . The siNA of  claim 1 , wherein 70%, 75%, 80%, 85%, 90%, 95% or 100% of the nucleotides in the first nucleotide sequence are modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide. 
     
     
         5 . The siNA of  claim 1 , wherein:
 (i) at least 2, 3, 4, 5, or 6 modified nucleotides of the first nucleotide sequence are 2′-fluoro nucleotides;   (ii) no more than 10, 9, 8, 7, 6, 5, 4, 3, or 2 modified nucleotides of the first nucleotide sequence are 2′-fluoro nucleotides;   (iii) at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides; and/or   (iv) no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides.   
     
     
         6 . The siRNA of  claim 1 , wherein the second nucleotide sequence comprises 16, 17, 18, 19, 20, 21, 22, 23, or more modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide. 
     
     
         7 . The siNA of  claim 1 , wherein 70%, 75%, 80%, 85%, 90%, 95% or 100% of the nucleotides in the second nucleotide sequence are modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide. 
     
     
         8 . The siNA of  claim 1 , wherein:
 (i) at least 2, 3, 4, 5, or 6 modified nucleotides of the second nucleotide sequence are 2′-fluoro nucleotides;   (ii) less than or equal to 10, 9, 8, 7, 6, 5, 4, 3, or 2 modified nucleotides of the second nucleotide sequence are 2′-fluoro nucleotides;   (iii) at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides; and/or   (iv) less than or equal to 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides.   
     
     
         9 . The siNA of  claim 1  further comprising 1 or more phosphorothioate internucleoside linkage in the sense strand and/or antisense strand. 
     
     
         10 . The siNA of  claim 1  further comprising a phosphorylation blocker, a galactosamine, or 5′-stabilized end cap. 
     
     
         11 . The siNA of  claim 1 , wherein at least 1, 2, 3, 4, 5, 6, or 7 nucleotides at position 3, 5, 7, 8, 9, 10, 11, 12, and/or 17 from the 5′ end of the first nucleotide sequence is a 2′-fluoro nucleotide. 
     
     
         12 . The siNA of  claim 1 , wherein at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides at position 2, 5, 6, 8, 10, 14, 16, 17, and/or 18 from the 5′ end of the second nucleotide sequence is a 2′-fluoro nucleotide. 
     
     
         13 . The siNA of  claim 1 , wherein the nucleotides in the second nucleotide sequence are arranged in an alternating 1:3 modification pattern, and wherein 1 nucleotide is a 2′-fluoro nucleotide and 3 nucleotides are 2′-O-methyl nucleotides. 
     
     
         14 . The siNA of  claim 13 , wherein the alternating 1:3 modification pattern occurs 2-5 times. 
     
     
         15 . The siNA according to  claim 13 , wherein:
 (i) at least two of the alternating 1:3 modification pattern occur consecutively;   (ii) at least two of the alternating 1:3 modification pattern occurs nonconsecutively; and/or   (iii) at least 1, 2, 3, 4, or 5 alternating 1:3 modification pattern begins at nucleotide position 2, 6, 10, 14, and/or 18 from the 5′ end of the antisense strand.   
     
     
         16 . The siNA of  claim 1 , wherein:
 (i) the nucleotides in the second nucleotide sequence are arranged in an alternating 1:2 modification pattern, and   (ii) 1 nucleotide is a 2′-fluoro nucleotide and 2 nucleotides are 2′-O-methyl nucleotides.   
     
     
         17 . The siNA of  claim 16 , wherein the alternating 1:2 modification pattern occurs 2-5 times. 
     
     
         18 . The siNA according to  claim 16 , wherein:
 (i) at least two of the alternating 1:2 modification pattern occurs consecutively;   (ii) at least two of the alternating 1:2 modification pattern occurs nonconsecutively; and/or   (iii) at least 1, 2, 3, 4, or 5 alternating 1:2 modification pattern begins at nucleotide position 2, 5, 8, 14, and/or 17 from the 5′ end of the antisense strand.   
     
     
         19 . A short interfering nucleic acid (siNA) molecule represented by Formula (VIII): 
       
         
           
                 
                 
               
                     
                   5′-A n   1 B n   2 A n   3 B n   4 A n   5 B n   6 A n   7 B n   8 A n   9 -3′ 
                 
                     
                     
                 
                     
                   3′-C q   1 A q   2 B q   3 A q   4 B q   5 A q   6 B q   7 A q   8 B q   9 A q   10 B q   1l A q   12 -5′ 
                 
             
                
                
                
               
            
           
         
         wherein:
 the top strand is a sense strand comprising a first nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to an RNA corresponding to a target gene, wherein the first nucleotide sequence comprises 15 to 30 nucleotides; 
 the bottom strand is an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the RNA corresponding to the target gene, wherein the second nucleotide sequence comprises 15 to 30 nucleotides; 
 each A is independently a 2′-O-methyl nucleotide or a nucleotide comprising a 5′-stabilized end cap or a phosphorylation blocker; 
 B is a 2′-fluoro nucleotide; 
 C represents overhanging nucleotides and is a 2′-O-methyl nucleotide; 
 n 1 =1-4 nucleotides in length; 
 each n 2 , n 6 , n 8 , q 3 , q 5 , q 7 , q 9 , q 11 , and q 12  is independently 0-1 nucleotides in length; 
 each n 3  and n 4  is independently 1-3 nucleotides in length; 
 n 5  is 1-10 nucleotides in length; 
 n 7  is 0-4 nucleotides in length; 
 each n 9 , q 1 , and q 2  is independently 0-2 nucleotides in length; 
 q 4  is 0-3 nucleotides in length; 
 q 6  is 0-5 nucleotides in length; 
 q 8  is 2-7 nucleotides in length; and 
 q 10  is 2-11 nucleotides in length. 
 
       
     
     
         20 . A short interfering nucleic acid (siNA) molecule represented by Formula (IX): 
       
         
           
                 
                 
               
                     
                   5′-A 2-4 B 1 A 1-3 B 2-3 A 2-10 B 0-1 A 0-4 B 0-1 A 0-2 -3′ 
                 
                     
                     
                 
                     
                   3′-C 2 A 0-2 B 0-1 A 0-3 B 0-1 A 0-5 B 0-1 A 2-7 B 1 A 2-11 B 1 A 1 -5′ 
                 
             
                
                
                
               
            
           
         
         wherein:
 the top strand is a sense strand comprising a first nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to an RNA corresponding to a target gene, wherein the first nucleotide sequence comprises 15 to 30 nucleotides; 
 the bottom strand is an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the RNA corresponding to the target gene, wherein the second nucleotide sequence comprises 15 to 30 nucleotides; 
 each A is independently a 2′-O-methyl nucleotide or a nucleotide comprising a 5′-stabilized end cap or a phosphorylation blocker; 
 B is a 2′-fluoro nucleotide; 
 C represents overhanging nucleotides and is a 2′-O-methyl nucleotide. 
 
       
     
     
         21 . A short interfering nucleic acid (siNA) molecule comprising:
 (a) a sense strand comprising a first nucleotide sequence consisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are at positions 3, 7-9, 12, and 17 from the 5′ end of the first nucleotide sequence, and wherein 2′-O-methyl nucleotides are at positions 1, 2, 4-6, 10, 11, and 13-16 from the 5′ end of the first nucleotide sequence, and
 an antisense strand comprising a second nucleotide sequence consisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are at positions 2 and 14 from the 5′ end of the second nucleotide sequence, and wherein 2′-O-methyl nucleotides are at positions 1, 3-13, and 15-17 from the 5′ end of the second nucleotide sequence 
   (b) a sense strand comprising a first nucleotide sequence consisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are at positions 3, 7, 8, and 17 from the 5′ end of the first nucleotide sequence, and wherein 2′-O-methyl nucleotides are at positions 1, 2, 4-6, and 9-16 from the 5′ end of the first nucleotide sequence, and
 an antisense strand comprising a second nucleotide sequence consisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are at positions 2 and 14 from the 5′ end of the first nucleotide sequence; and wherein 2′-O-methyl nucleotides are at positions 1, 3-13, and 15-17 from the 5′ end of the first nucleotide sequence; 
   (c) a sense strand comprising a first nucleotide sequence consisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are at positions 3, 7-9, 12 and 17 from the 5′ end of the first nucleotide sequence, and wherein 2′-O-methyl nucleotides are at positions 1, 2, 4-6, 10, 11, and 13-16 from the 5′ end of the first nucleotide sequence, and
 an antisense strand comprising a second nucleotide sequence consisting of 17 to 23 nucleotides, wherein the nucleotides in the second nucleotide sequence are arranged in an alternating 1:3 modification pattern, and wherein 1 nucleotide is a 2′-fluoro nucleotide and 3 nucleotides are 2′-O-methyl nucleotides; 
   (d) a sense strand comprising a first nucleotide sequence consisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are at positions 5 and 7-9 from the 5′ end of the first nucleotide sequence, and wherein 2′-O-methyl nucleotides are at positions 1-4, 6, and 10-17 from the 5′ end of the first nucleotide sequence, and an antisense strand comprising a second nucleotide sequence consisting of 17 to 23 nucleotides, wherein the nucleotides in the second nucleotide sequence are arranged in an alternating 1:3 modification pattern, and wherein 1 nucleotide is a 2′-fluoro nucleotide and 3 nucleotides are 2′-O-methyl nucleotides;   (e) a sense strand comprising a first nucleotide sequence consisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are at positions 5 and 7-9 from the 5′ end of the first nucleotide sequence, and wherein 2′-O-methyl nucleotides are at positions 1-4, 6, and 10-17 from the 5′ end of the first nucleotide sequence, and an antisense strand comprising a second nucleotide sequence consisting of 17 to 23 nucleotides, wherein the nucleotides in the second nucleotide sequence are arranged in an alternating 1:2 modification pattern, and wherein 1 nucleotide is a 2′-fluoro nucleotide and 2 nucleotides are 2′-O-methyl nucleotides;   (f) a sense strand comprising a first nucleotide sequence consisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are at positions 5 and 7-9 from the 5′ end of the first nucleotide sequence, and wherein 2′-O-methyl nucleotides are at positions 1-4, 6, and 10-17 from the 5′ end of the first nucleotide sequence, and an antisense strand comprising a second nucleotide sequence consisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 6, 14, and 16 from the 5′ end of the second nucleotide sequence, and wherein 2′-O-methyl nucleotides are at positions 1, 3-5, 7-13, 15, and 17 from the 5′ end the second nucleotide sequence; or   (g) a sense strand comprising a first nucleotide sequence consisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are at positions 5, 9-11, and 14 from the 5′ end of the first nucleotide sequence, and wherein 2′-O-methyl nucleotides are at positions 1-4, 6-8, and 12-17 from the 5′ end of the first nucleotide sequence, and
 an antisense strand comprising a second nucleotide sequence consisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are at positions 2 and 14 from the 5′ end of the second nucleotide sequence, and wherein 2′-O-methyl nucleotides are at positions 1, 3-13, and 15-17 from the 5′ end the second nucleotide sequence. 
   
     
     
         22 - 58 . (canceled) 
     
     
         59 . The siNA of  claim 1 , wherein the sense strand further comprises TT sequence adjacent to the first nucleotide sequence. 
     
     
         60 . The siNA of  claim 1 , wherein the sense strand further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more phosphorothioate internucleoside linkages. 
     
     
         61 . The siNA of  claim 60 , wherein:
 (i) at least one phosphorothioate internucleoside linkage is between the nucleotides at positions 1 and 2 from the 5′ end of the first nucleotide sequence;   (ii) at least one phosphorothioate internucleoside linkage is between the nucleotides at positions 2 and 3 from the 5′ end of the first nucleotide sequence.   
     
     
         62 . The siNA of  claim 1 , wherein the antisense strand further comprises TT sequence adjacent to the second nucleotide sequence. 
     
     
         63 . The siNA of  claim 1 , wherein the antisense strand further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more phosphorothioate internucleoside linkages. 
     
     
         64 . The siNA of  claim 63 , wherein:
 (i) at least one phosphorothioate internucleoside linkage is between the nucleotides at positions 1 and 2 from the 5′ end of the second nucleotide sequence;   (ii) at least one phosphorothioate internucleoside linkage is between the nucleotides at positions 2 and 3 from the 5′ end of the second nucleotide sequence;   (iii) at least one phosphorothioate internucleoside linkage is between the nucleotides at positions 1 and 2 from the 3′ end of the second nucleotide sequence; and/or   (iv) at least one phosphorothioate internucleoside linkage is between the nucleotides at positions 2 and 3 from the 3′ end of the second nucleotide sequence.   
     
     
         65 . The siNA of  claim 1 , wherein:
 (i) the first nucleotide from the 5′ end of the first nucleotide sequence comprises a 5′ stabilizing end cap;   (ii) the first nucleotide from the 5′ end of the second nucleotide sequence comprises a 5′ stabilizing end cap;   (iii) the first nucleotide from the 5′ end of the first nucleotide sequence comprises a phosphorylation blocker; and/or   (iv) the first nucleotide from the 5′ end of the second nucleotide sequence comprises a phosphorylation blocker.   
     
     
         66 . The siNA of  claim 1 , wherein the first nucleotide sequence or second nucleotide sequence comprises at least one modified nucleotide selected from 
       
         
           
           
               
               
           
         
       
       where R is H or alkyl (or AmNA(N-Me)) when R is alkyl); 
       
         
           
           
               
               
           
         
       
       wherein B is a nucleobase. 
     
     
         67 . A short-interfering nucleic acid (siNA) molecule comprising:
 a. a phosphorylation blocker of Formula (IV):   
       
         
           
           
               
               
           
         
         wherein 
         R 1  is a nucleobase, 
         R 4  is —O—R 30  or —NR 31 R 32 , 
         R 30  is C 1 -C 8  substituted or unsubstituted alkyl; and 
         R 31  and R 32  together with the nitrogen to which they are attached form a substituted or unsubstituted heterocyclic ring; and 
         b. a short interfering nucleic acid (siNA). 
       
     
     
         68 . A short-interfering nucleic acid (siNA) molecule comprising:
 c. a 5′-stabilized end cap of Formula (Ia):   
       
         
           
           
               
               
           
         
         wherein 
         R 1  is a nucleobase, aryl, heteroaryl, or H, 
         R 2  is 
       
       
         
           
           
               
               
           
         
       
       —CH═CD-Z,-CD=CH—Z,-CD=CD-Z, —(CR 21 R 22 ) n —Z, or —(C 2 -C 6  alkenylene)-Z and R 20  is hydrogen; or
 R 2  and R 20  together form a 3- to 7-membered carbocyclic ring substituted with —(CR 21 R 22 ) n —Z or —(C 2 -C 6  alkenylene)-Z; 
 n is 1, 2, 3, or 4; 
 Z is —ONR 23 R 24 , —OP(O)OH(CH 2 ) m CO 2 R 23 , —OP(S)OH(CH 2 ) m CO 2 R 23 , —P(O)(OH) 2 , —P(O)(OH)(OCH 3 ), —P(O)(OH)(OCD 3 ), —SO 2 (CH 2 ) m P(O)(OH) 2 , —SO 2 NR 23 R 25 , —NR 23 R 24 , R 21  and R 22  are independently hydrogen or C 1 -C 6  alkyl; R 21  and R 22  together form an oxo group; 
 R 23  is hydrogen or C 1 -C 6  alkyl; 
 R 24  is —SO 2 R 25  or —C(O)R 25 ; or 
 R 23  and R 24  together with the nitrogen to which they are attached form a substituted or unsubstituted heterocyclic ring; 
 R 25  is C 1 -C 6  alkyl; and 
 m is 1, 2, 3, or 4; and 
 d. a short interfering nucleic acid (siNA). 
 
     
     
         69 . A short-interfering nucleic acid (siNA) molecule comprising:
 e. a 5′-stabilized end cap of Formula (Ib):   
       
         
           
           
               
               
           
         
         wherein 
         R 1  is a nucleobase, aryl, heteroaryl, or H, 
         R 2  is 
       
       
         
           
           
               
               
           
         
       
       
         
           
           
               
               
           
         
       
       —CH═CD-Z,-CD=CH—Z,-CD=CD-Z, —(CR 21 R 22 ) n —Z, or —(C 2 -C 6  alkenylene)-Z and R 20  is hydrogen; or
 R 2  and R 20  together form a 3- to 7-membered carbocyclic ring substituted with —(CR 21 R 22 ) n —Z or —(C 2 -C 6  alkenylene)-Z; 
 n is 1, 2, 3, or 4; 
 Z is —ONR 23 R 24 , —OP(O)OH(CH 2 ) m CO 2 R 23 , —OP(S)OH(CH 2 ) m CO 2 R 23 , —P(O)(OH) 2 , —P(O)(OH)(OCH 3 ), —P(O)(OH)(OCD 3 ), —SO 2 (CH 2 ) m P(O)(OH) 2 , —SO 2 NR 23 R 25 , —NR 23 R 24 , R 21  and R 22  are independently hydrogen or C 1 -C 6  alkyl; R 21  and R 22  together form an oxo group; 
 R 23  is hydrogen or C 1 -C 6  alkyl; 
 R 24  is —SO 2 R 25  or —C(O)R 25 ; or 
 R 23  and R 24  together with the nitrogen to which they are attached form a substituted or unsubstituted heterocyclic ring; 
 R 25  is C 1 -C 6  alkyl; and 
 m is 1, 2, 3, or 4; and 
 f. a short interfering nucleic acid (siNA). 
 
     
     
         70 . A short-interfering nucleic acid (siNA) molecule comprising:
 g. a 5′-stabilized end cap selected from the group consisting of Formula (1) to Formula (15), Formula (9X) to Formula (12X), and Formula (9Y) to Formula (12Y):   
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
       wherein R 1  is a nucleobase, aryl, heteroaryl, or H; and
 h. a short interfering nucleic acid (siNA). 
 
     
     
         71 . A short-interfering nucleic acid (siNA) molecule comprising:
 i. a 5′-stabilized end cap selected from the group consisting of Formulas (1A)-(15A), Formulas (9B)-(12B), Formulas (9AX)-(12AX), Formulas (9AY)-(12AY), Formulas (9BX)-(12BX), and Formulas (9BY)-(12BY):   
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         j. a short interfering nucleic acid (siNA). 
       
     
     
         72 - 73 . (canceled) 
     
     
         74 . A short interfering nucleic acid (siNA) molecule comprising:
 (a) a sense strand comprising a first nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to an RNA corresponding to a target gene, wherein the first nucleotide sequence comprises a nucleotide sequence of any one SEQ ID NOs: 1-56, 103-158, and 205-260; and   (b) an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the RNA corresponding to the target gene, wherein the second nucleotide sequence comprises a nucleotide sequence of any one of SEQ ID NOs: 57-102, 159-204, and 261-306.   
     
     
         75 . A interfering nucleic acid (siNA) molecule comprising:
 (a) a sense strand comprising a nucleotide sequence of any one of SEQ ID NOs: 307-362 and 415-444; and   (b) an antisense strand comprising a nucleotide sequence of any one of SEQ ID NOs: 363-409, 445-533, and 536-539.   
     
     
         76 . (canceled) 
     
     
         77 . The siNA according to  claim 10 , wherein the phosphorylation blocker has the structure of Formula (IV): 
       
         
           
           
               
               
           
         
       
       wherein
 R 1  is a nucleobase, 
 R 4  is —O—R 30  or —NR 31 R 32 , R 30  is C 1 -C 8  substituted or unsubstituted alkyl; and 
 R 31  and R 32  together with the nitrogen to which they are attached form a substituted or unsubstituted heterocyclic ring. 
 
     
     
         78 . The siNA of  claim 67 , wherein R 4  is —OCH 3  or —N(CH 2 CH 2 ) 2 O. 
     
     
         79 . The siNA according to  claim 10 , wherein: (i) the phosphorylation blocker is attached to the 5′ end of the sense strand; (ii) the phosphorylation blocker is attached to the 3′ end of the sense strand; (iii) the phosphorylation blocker is attached to the 5′ end of the antisense strand; or (iv) the phosphorylation blocker is attached to the 3′ end of the antisense strand. 
     
     
         80 . The siNA of  claim 78 , wherein:
 (i) the phosphorylation blocker is attached to the 5′ end of the sense strand via one or more linkers independently selected from a phosphodiester linker, phosphorothioate linker, and phosphorodithioate linker;   (ii) the phosphorylation blocker is attached to the 3′ end of the sense strand via one or more linkers independently selected from a phosphodiester linker, phosphorothioate linker, and phosphorodithioate linker;   (iii) the phosphorylation blocker is attached to the 5′ end of the antisense strand via one or more linkers independently selected from a phosphodiester linker, phosphorothioate linker, and phosphorodithioate linker; or   (iv) the phosphorylation blocker is attached to the 3′ end of the antisense strand via one or more linkers independently selected from a phosphodiester linker, phosphorothioate linker, and phosphorodithioate linker.   
     
     
         81 . The siNA of  claim 1 , wherein the siNA further comprises a galactosamine. 
     
     
         82 . The siNA of  claim 81 , wherein the galactosamine is N-acetylgalactosamine (GalNAc) of Formula (VII): 
       
         
           
           
               
               
           
         
         wherein each n is independently 1 or 2. 
       
     
     
         83 . The siNA of  claim 81 , wherein the galactosamine is N-acetylgalactosamine (GalNAc) of Formula (VI): 
       
         
           
           
               
               
           
         
       
       wherein
 m is 1, 2, 3, 4, or 5; 
 each n is independently 1 or 2; 
 p is 0 or 1; 
 each R is independently H; 
 each Y is independently selected from —O—P(═O)(SH)—, —O—P(═O)(O)—, —O—P(═O)(OH)—, and —O—P(S)S—; 
 Z is H or a second protecting group; 
 either L is a linker or L and Y in combination are a linker; and 
 A is H, OH, a third protecting group, an activated group, or an oligonucleotide. 
 
     
     
         84 . The siNA of  claim 83 , wherein: (i) A is an oligonucleotide;
 or (ii) A is 1-2 oligonucleotides.   
     
     
         85 . The siNA of any one of  claim 84 , wherein the oligonucleotide is dTdT. 
     
     
         86 . The siNA according to  claim 84 , wherein the galactosamine is attached to (i) the 3′ end of the sense strand; (ii) the 5′ end of the sense strand; (iii) the 3′ end of the antisense strand; or (iv) the 5′ end of the antisense strand. 
     
     
         87 . The siNA of  claim 86 , wherein the galactosamine is attached to:
 (i) the 3′ end of the sense strand via one or more linkers independently selected from a phosphodiester linker, phosphorothioate linker, or phosphorodithioate linker;   (ii) the 5′ end of the sense strand via one or more linkers independently selected from a phosphodiester linker, phosphorothioate linker, or phosphorodithioate linker;   (iii) the 3′ end of the atnisense strand via one or more linkers independently selected from a phosphodiester linker, phosphorothioate linker, or phosphorodithioate linker; or   (iv) the 5′ end of the atnisense strand via one or more linkers independently selected from a phosphodiester linker, phosphorothioate linker, or phosphorodithioate linker.   
     
     
         88 . The siNA according to  claim 1 , wherein the siNA further comprises a 5′-stabilized end cap. 
     
     
         89 . The siNA according to  claim 88 , wherein the 5′-stabilized end cap is a 5′ vinyl phosphonate or deuterated 5′ vinyl phosphonate. 
     
     
         90 . The siNA according to  claim 88 , wherein the 5′-stabilized end cap has the structure of Formula (Ia): 
       
         
           
           
               
               
           
         
       
       wherein
 R 1  is a nucleobase, aryl, heteroaryl, or H, 
 R 2  is 
 
       
         
           
           
               
               
           
         
       
       —CH═CD-Z,-CD=CH—Z,-CD=CD-Z, —(CR 21 R 22 ) n —Z, or —(C 2 -C 6 alkenylene)-Z and R 20  is hydrogen; or
 R 2  and R 20  together form a 3- to 7-membered carbocyclic ring substituted with —(CR 21 R 22 ) n —Z or —(C 2 -C 6  alkenylene)-Z; 
 
       n is 1, 2, 3, or 4;
 Z is —ONR 23 R 24 , —OP(O)OH(CH 2 ) m CO 2 R 23 , —OP(S)OH(CH 2 ) m CO 2 R 23 , —P(O)(OH) 2 , —P(O)(OH)(OCH 3 ), —P(O)(OH)(OCD 3 ), —SO 2 (CH 2 ) m P(O)(OH) 2 , —SO 2 NR 23 R 25 , —NR 23 R 24 , or —NR 23 SO 2 R 24 ; 
 R 21  and R 22  either are independently hydrogen or C 1 -C 6  alkyl, or R 21  and R 22  together form an oxo group; 
 R 23  is hydrogen or C 1 -C 6  alkyl; 
 R 24  is —SO 2 R 25  or —C(O)R 25 ; or 
 R 23  and R 24  together with the nitrogen to which they are attached form a substituted or unsubstituted heterocyclic ring; 
 R 25  is C 1 -C 6  alkyl; and 
 m is 1, 2, 3, or 4. 
 
     
     
         91 . The siNA according to  claim 88 , wherein the 5′-stabilized end cap has the structure of Formula (Ib): 
       
         
           
           
               
               
           
         
       
       wherein
 R 1  is a nucleobase, aryl, heteroaryl, or H, 
 R 2  is 
 
       
         
           
           
               
               
           
         
       
       —CH═CD-Z,-CD=CH—Z,-CD=CD-Z, —(CR 21 R 22 ) n —Z, or —(C 2 -C 6 alkenylene)-Z and R 20  is hydrogen; or
 R 2  and R 20  together form a 3- to 7-membered carbocyclic ring substituted with —(CR 21 R 22 ) n —Z or —(C 2 -C 6  alkenylene)-Z; 
 
       n is 1, 2, 3, or 4;
 Z is —ONR 23 R 24 , —OP(O)OH(CH 2 ) m CO 2 R 23 , —OP(S)OH(CH 2 ) m CO 2 R 23 , —P(O)(OH) 2 , —P(O)(OH)(OCH 3 ), —P(O)(OH)(OCD 3 ), —SO 2 (CH 2 ) m P(O)(OH) 2 , —SO 2 NR 23 R 25 , —NR 23 R 24 , or —NR 23 SO 2 R 24 ; 
 R 21  and R 22  either are independently hydrogen or C 1 -C 6  alkyl, or R 21  and R 22  together form an oxo group; 
 R 23  is hydrogen or C 1 -C 6  alkyl; 
 R 24  is —SO 2 R 25  or —C(O)R 25 ; or 
 R 23  and R 24  together with the nitrogen to which they are attached form a substituted or unsubstituted heterocyclic ring; 
 R 25  is C 1 -C 6  alkyl; and 
 m is 1, 2, 3, or 4. 
 
     
     
         92 . The siNA of  claim 90 , wherein R 1  is an aryl. 
     
     
         93 . The siNA of  claim 92 , wherein the aryl is a phenyl. 
     
     
         94 . The siNA according to  claim 88 , wherein the 5′-stabilized end cap is selected from the group consisting of Formula (1) to Formula (15), Formula (9X) to Formula (12X), and Formula (9Y) to Formula (12Y): 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
       wherein R 1  is a nucleobase, aryl, heteroaryl, or H. 
     
     
         95 . The siNA according to  claim 88 , wherein the 5′-stabilized end cap is selected from the group consisting of Formulas (IA)-(15A), Formulas (9B)-(12B), Formulas (9AX)-(12AX), Formulas (9AY)-(12AY), Formulas (9BX)-(12BX), and Formulas (9BY)-(12BY): 
     
     
         96 . The siNA according to  claim 88 , wherein the 5′-stabilized end cap has the structure of Formula (Ic): 
       
         
           
           
               
               
           
         
       
       wherein
 R 1  is a nucleobase, aryl, heteroaryl, or H, 
 R 2  is 
 
       
         
           
           
               
               
           
         
       
       —CH═CD-Z,-CD=CH—Z,-CD=CD-Z, —(CR 21 R 22 ) n —Z, or —(C 2 -C 6 alkenylene)-Z and R 20  is hydrogen; or
 R 2  and R 20  together form a 3- to 7-membered carbocyclic ring substituted with —(CR 21 R 22 ) n —Z or —(C 2 -C 6  alkenylene)-Z; 
 
       n is 1, 2, 3, or 4;
 Z is —ONR 23 R 24 , —OP(O)OH(CH 2 ) m CO 2 R 23 , —OP(S)OH(CH 2 ) m CO 2 R 23 , —P(O)(OH) 2 , —P(O)(OH)(OCH 3 ), —P(O)(OH)(OCD 3 ), —SO 2 (CH 2 ) m P(O)(OH) 2 , —SO 2 NR 23 R 25 , —NR 23 R 24 , or —NR 23 SO 2 R 24 ; 
 R 21  and R 22  either are independently hydrogen or C 1 -C 6  alkyl, or R 21  and R 22  together form an oxo group; 
 R 23  is hydrogen or C 1 -C 6  alkyl; 
 R 24  is —SO 2 R 25  or —C(O)R 25 ; or 
 R 23  and R 24  together with the nitrogen to which they are attached form a substituted or unsubstituted heterocyclic ring; 
 R 25  is C 1 -C 6  alkyl; and 
 m is 1, 2, 3, or 4. 
 
     
     
         97 . The siNA of  claim 96 , wherein R 1  is an aryl. 
     
     
         98 . The siNA of  claim 97 , wherein the aryl is a phenyl. 
     
     
         99 . The siNA according to  claim 88 , wherein the 5′-stabilized end cap is selected from the group consisting of Formula (21) to Formula (35): 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
       wherein R 1  is a nucleobase, aryl, heteroaryl, or H. 
     
     
         100 . The siNA according to  claim 88 , wherein the 5′-stabilized end cap is selected from the group consisting of Formulas (21A)-(35A), Formulas (29B)-(32B), Formulas (29AX)-(32AX), Formulas (29AY)-(32AY), Formulas (29BX)-(32BX), and Formulas (29BY)-(32BY): 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
       wherein R 1  is a nucleobase, aryl, heteroaryl, or H. 
     
     
         101 . The siNA according to  claim 1 , wherein the antisense strand and/or sense strand comprises at least one thermally destabilizing nucleotide selected from: 
       
         
           
           
               
               
           
         
       
     
     
         102 . The siNA according to  claim 10 , wherein the 5′-stabilized end cap is attached to (i) the 5′ end of the antisense strand; or (ii) the 5′ end of the sense strand. 
     
     
         103 . The siNA of  claim 102 , wherein the 5′-stabilized end cap is attached to:
 (i) the 5′ end of the antisense strand via one or more linkers independently selected from a phosphodiester linker, phosphorothioate linker, or phosphorodithioate linker; or 
 (ii) the 5′ end of the sense strand via one or more linkers independently selected from a phosphodiester linker, phosphorothioate linker, or phosphorodithioate linker. 
 
     
     
         104 . The siNA of  claim 1 , wherein;
 (i) the target gene is a viral gene;   (ii) the target gene is a gene is from a DNA virus;   (iii) the target gene is a gene from a double-stranded DNA (dsDNA) virus;   (iv) the target gene is a gene from a hepadnavirus;   (v) the target gene is a gene from a hepatitis B virus (HBV);   (vi) the target gene is a gene from a HBV of any one of genotypes A-J; or   (vii) the target gene is selected from the S gene or X gene of a HBV.   
     
     
         105 . The siNA according to  claim 1 , wherein:
 (i) the second nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to 15 to 30 nucleotides within positions 200-720 or 1100-1700 of SEQ ID NO: 410;   (ii) the second nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to 15 to 30 nucleotides within positions 200-280, 300-445, 460-510, 650-720, 1170-1220, 1250-1300, or 1550-1630 of SEQ ID NO: 410;   (iii) the second nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to 15 to 30 nucleotides within positions 200-230, 250-280, 300-330, 370-400, 405-445, 460-500, 670-700, 1180-1210, 1260-1295, 1520-1550, or 1570-1610 of SEQ ID NO: 410;   (iv) the second nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to 15 to 30 nucleotides starting at position 203, 206, 254, 305, 375, 409, 412, 415, 416, 419, 462, 466, 467, 674, 676, 1182, 1262, 1263, 1268, 1526, 1577, 1578, 1580, 1581, 1583, or 1584 of SEQ ID NO: 410;   (v) the first nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to 15 to 30 nucleotides within positions 200-720 or 1100-1700 of SEQ ID NO: 410;   (vi) the first nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to 15 to 30 nucleotides within positions 200-280, 300-445, 460-510, 650-720, 1170-1220, 1250-1300, or 1550-1630 of SEQ ID NO: 410;   (vii) the first nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to 15 to 30 nucleotides within positions 200-230, 250-280, 300-330, 370-400, 405-445, 460-500, 670-700, 1180-1210, 1260-1295, 1520-1550, or 1570-1610 of SEQ ID NO: 410; and/or   (viii) the first nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to 15 to 30 nucleotides starting at position 203, 206, 254, 305, 375, 409, 412, 415, 416, 419, 462, 466, 467, 674, 676, 1182, 1262, 1263, 1268, 1526, 1577, 1578, 1580, 1581, 1583, or 1584 of SEQ ID NO: 410.   
     
     
         106 . The siNA of  claim 1 , wherein:
 (i) the first nucleotide sequence comprises a nucleotide sequence of any one SEQ ID NOs: 1-56, 103-158, and 205-260;   (ii) the second nucleotide sequence comprises a nucleotide sequence of any one of SEQ ID NOs: 57-102, 159-204, and 261-306; (iii) the sense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 307-362 and 415-444; and/or   (iv) the antisense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 363-409, 445-533, and 536-539.   
     
     
         107 . The siNA of  claim 1 , wherein:
 (i) at least one end of the siNA is a blunt end;   (ii) at least one end of the siNA comprises an overhang, wherein the overhang comprises at least one nucleotide; or   (iii) both ends of the siNA comprise an overhang, wherein the overhang comprises at least one nucleotide.   
     
     
         108 . The siNA of  claim 1 , wherein the siNA is selected from ds-siNA-001 to ds-siNA-0178. 
     
     
         109 . The siNA of  claim 1 , wherein at least one 2′-fluoro nucleotide or 2′-O-methyl nucleotide is a 2′-fluoro or 2-O-methyl nucleotide mimic of Formula (V): 
       
         
           
           
               
               
           
         
       
       wherein
 R 1  is independently a nucleobase, aryl, heteroaryl, or H, Q 1  and Q 2  are independently S or O, 
 R 5  is independently-OCD 3 , —F, or —OCH 3 , and 
 R 6  and R 7  are independently H, D, or CD 3 . 
 
     
     
         110 . The siNA of  claim 109 , wherein the 2′-fluoro or 2′-O-methyl nucleotide mimic is a nucleotide mimic of Formula (16)-Formula (20): 
       
         
           
           
               
               
           
         
         wherein R 1  is a nucleobase and R 2  is independently F or —OCH 3 . 
       
     
     
         111 . The siNA of  claim 1 , wherein at least one 2′-fluoro nucleotide is a 2′-fluoro nucleotide mimic. 
     
     
         112 . The siNA according to  claim 111 , wherein:
 (i) at least 1, 2, 3, 4, 5, or more 2′-fluoro nucleotides on the antisense strand or the second nucleotide sequence is a 2′-fluoro nucleotide mimic;   (ii) the nucleotide at position 2, 5, 6, 8, 10, 14, 16, and/or 17 from the 5′ end of the antisense strand or the second nucleotide sequence is a 2′-fluoro nucleotide mimic;   (iii) at least 1, 2, 3, 4, 5, or more 2′-fluoro nucleotides on the sense strand or the first nucleotide sequence is a 2′-fluoro nucleotide mimic; and/or   (iv) the nucleotide at position 3, 5, 7, 8, 9, 10, 11, 12, 14, and/or 17 from the 5′ end of the sense strand or the first nucleotide sequence is a 2′-fluoro nucleotide mimic.   
     
     
         113 . The siNA according to  claim 111 , wherein:
 (i) at least 1, 2, 3, 4, 5, 6, or more 2′-fluoro nucleotide mimics is a f4P nucleotide   
       
         
           
           
               
               
           
         
         (ii) less than or equal to 10, 9, 8, 7, 6, 5, 4, 3, or 2 2′-fluoro nucleotide mimics is a f4P nucleotide 
       
       
         
           
           
               
               
           
         
         (iii) 1, 2, 3, 4, 5, 6, or more 2′-fluoro nucleotide mimics is a f2P nucleotide 
       
       
         
           
           
               
               
           
         
         (iv) less than or equal to 10, 9, 8, 7, 6, 5, 4, 3, or 2 2′-fluoro nucleotide mimics is a f2P nucleotide 
       
       
         
           
           
               
               
           
         
         (v) 1, 2, 3, 4, 5, 6, or more 2′-fluoro nucleotide mimics is a fX nucleotide 
       
       
         
           
           
               
               
           
         
       
       and/or
 (vi) less than or equal to 10, 9, 8, 7, 6, 5, 4, 3, or 2 2′-fluoro nucleotide mimics is a fX nucleotide 
 
       
         
           
           
               
               
           
         
       
     
     
         114 . The siNA of  claim 1 , wherein:
 (i) the first nucleotide from the 5′ end of the antisense strand or second nucleotide sequence is a d2vd3 nucleotide   
       
         
           
           
               
               
           
         
       
       and/or
 (ii) the first nucleotide from the 3′ end of the antisense strand or second nucleotide sequence is a d2vd3 nucleotide 
 
       
         
           
           
               
               
           
         
       
     
     
         115 . A composition comprising:
 (I) at least one siNA according to  claim 1 ; and (b) a pharmaceutically acceptable excipient.   
     
     
         116 . The composition of  claim 115 , comprising:
 (i) at least 1, 2, 3, 4, 5, or more siNAs that target an S gene of HBV; and/or   (ii) at least 1, 2, 3, 4, 5, or more siNAs that target an X gene of HBV.   
     
     
         117 . The composition according to  claim 115 , further comprising:
 (i) an additional HBV treatment agent;   (ii) an additional HBV treatment agent selected from a nucleotide analog, nucleoside analog, a capsid assembly modulator (CAM), a recombinant interferon, an entry inhibitor, a small molecule immunomodulator and oligonucleotide therapy;   (iii) an additional siNA;   (iv) an additional siNA selected from any of ds-siNA-001 to ds-siNA-0178;   (v) an oligonucleotide therapy selected from an antisense oligonucleotide (ASO), NAPs, or STOPs;   (vi) an ASO selected from ASO 1 or ASO 2; and/or   (vii) an additional HBV treatment agent selected from HBV STOPS™ ALG-010133, HBV CAM ALG-000184, ASO 1, recombinant interferon alpha 2b, IFN-a, PEG-IFN-a-2a, lamivudine, telbivudine, adefovir dipivoxil, clevudine, entecavir, tenofovir alafenamide, tenofovir disoproxil, NVR3-778, BAY41-4109, JNJ-632, JNJ-3989 (ARO-HBV), RG6004, GSK3228836, REP-2139, REP-2165, AB-729, VIR-2218, RG6346 (DCR-HBVS), JNJ-6379, GLS4, ABI-HO731, JNJ-440, NZ-4, RG7907, EDP-514, AB-423, AB-506, ABI-H03733 and ABI-H 2158 .   
     
     
         118 - 129 . (canceled) 
     
     
         130 . A method of treating hepatitis B virus (HBV), comprising administering to a subject with HBV at least one siNA according to  claim 1 .