DsRNA molecules comprising oligonucleotide analogs having modified intersubunit linkages and/or terminal groups

1. A conjugate, comprising a double-stranded ribonucleic acid (dsRNA) molecule linked to a peptide transport moiety, wherein:

said dsRNA molecule has a length of about 10 to 30 base pairs and comprises at least two sequences that are complementary to each other, wherein a sense strand comprises a first sequence and an antisense strand comprises a second sequence comprising a region of complementarity which is substantially complementary to at least a part of a target gene mRNA, wherein said region of complementarity is less than about 30 nucleotides in length, wherein one or both of the sense and antisense strands comprise an oligomer backbone having at least one morpholino subunit joined by intersubunit linkages, wherein each morpholino subunit supports a base-pairing moiety, and wherein the intersubunit linkages comprise a structure selected from the group consisting of (A) and (B) below:

(A) an intersubunit linkage having the following structure (A-I):

where

W is S or O,

X is NR 1 R 2 or OR 6 ;

Y is O or NR 7 , and

each said linkage (A-I) is selected from:

(a1) uncharged linkage (a1), where each of R 1 , R 2 , R 6 , and R 7 is independently selected from hydrogen and lower alkyl;

(b1) cationic linkage (b1), where X is NR 1 R 2 , Y is O, and NR 1 R 2 represents an optionally substituted piperazino group, such that R 1 R 2

is —CHRCHRN(R 3 )(R 4 )CHRCHR—, where:

each R is independently H or CH 3 ;

R 4 is H, CH 3 , or an electron pair; and

R 3 is selected from H, lower alkyl, C(═NH)NH 2 , Z-L-NHC(═NH)NH 2 , and [C(O)CHR′NH] m H, where Z is carbonyl (C(O)) or a direct bond, L is an optional linker up to 18 atoms in length having bonds selected from alkyl, alkoxy, and alkylamino, R′ is a side chain of a naturally occurring amino acid or a one- or two-carbon homolog thereof, and m is 1 to 6;

(b2) cationic linkage (b2), where X is NR 1 R 2 , Y is O, R 1 is H or CH 3 , and R 2 is LNR 3 R 4 R 5 , where L, R 3 , and R 4 are as defined above, and R 5 is H, lower alkyl, or lower (alkoxy)alkyl; and

(b3) cationic linkage (b3), where Y is NR 7 , X is OR 6 , and R 7 is LNR 3 R 4 R 5 , where L, R 3 , R 4 , and R 5 are as defined above, and R 6 is H or lower alkyl;

and at least one of said linkages (A-I) is selected from cationic linkages (b1), (b2), and (b3); and

(B) an intersubunit linkage having the following structure (B-I):

where each intersubunit linkage (B-I) is selected from:

(a2) linkage (a2) wherein:

W is S or O;

X is N(CH 3 ) 2 ; and

Y is O, and

(b4) linkage (b4) wherein:

W is S or O;

X is NR 11 R 12 or OR 13 ; and

Y is O or NR 14 ,

wherein each R 11 is independently hydrogen or optionally substituted C 2 -C 12 alkyl, each of R 12 , R 13 , and R 14 is independently hydrogen or optionally substituted C 1 -C 12 alkyl, or wherein each of R 11 , R 12 , R 13 , and R 14 may join with another one of R 11 , R 12 , R 13 or R 14 to form an optionally substituted 5-, 6- or 7-membered heterocycle comprising one or more N, O, or S atoms or combinations thereof,

wherein at least one of the intersubunit linkages has the structure (B-I), and

wherein at least one of the intersubunit linkages (B-I) comprises the following structure (B-II):

wherein Z represents a 5-, 6-, or 7-membered optionally substituted heterocycle comprising one or more N, O, or S atoms or combinations thereof; and

wherein at least one linkage (B-I) is:

(b5) linkage (b4), wherein X is 4-aminopiperidin-1-yl;

(b6) linkage (b4), wherein X is N-methyl-N-hexylamino;

(b7) linkage (b4), wherein X is 4-N—(N-ibu-Cys)piperazin-1-yl;

(b8) linkage (b4), wherein X is 4-N-(3,4,5-trimethoxybenzoyl)piperazin-1-yl;

(b9) linkage (b4), wherein X is 4-N-pyrrolidinylpiperidin-1-yl;

(b10) linkage (b4), wherein X is 4-N-(3-tetrazoylbenzoyl)piperazin-1-yl;

(b11) linkage (b4), wherein X is 4-N-succinamidopiperazin-1-yl;

(b12) linkage (b4), wherein X is 4-N-mercaptoacetylpiperazin-1-yl;

(b13) linkage (b4), wherein X is morpholino-4-yl;

(b14) linkage (b4), wherein X is S-2-methoxymethylpyrrolindin-1-yl;

(b15) linkage (b4), wherein X is R-2-methoxymethylpyrrolindin-1-yl;

(b16) linkage (b4), wherein X and Y may be taken together to form an optionally substituted heterocycle ring of 5, 6, or 7 members comprising one or more N, O, or S atoms or combinations thereof;

(b17) linkage (b4), wherein X is 4-trimethylaminopiperidin-1-yl; or

(b18) linkage (b4), wherein X is 4-guanidinylpiperidin-1-yl; and

the peptide transport moiety comprises 6 to 16 subunits selected from X′ subunits, Y′ subunits, and Z′ subunits, where

(i) each X′ subunit independently represents lysine, arginine or an arginine analog, said analog being a cationic α-amino acid comprising a side chain of the structure R 1 N═C(NH 2 )R 2 , where R 1 is H or R; R 2 is R, NH 2 , NHR, or NR 2 , where R is lower alkyl or lower alkenyl and may further include oxygen or nitrogen; R 1 and R 2 may together form a ring; and the side chain is linked to said amino acid via R 1 or R 2 ;

(ii) each Y′ subunit independently represents a neutral amino acid —C(O)—(CHR) n —NH—, where n is 2 to 7 and each R is independently H or methyl; and

(iii) each Z′ subunit independently represents an α-amino acid having a neutral aralkyl side chain;

and

wherein the peptide transport moiety is attached to a terminal residue of the antisense strand of the dsRNA oligomer.

2. The conjugate of claim 1 , wherein said morpholino subunits have the structure (i):

where each Pi is the same or different and independently a base-pairing moiety, and said intersubunit linkages connect the nitrogen atom of (i) to the 5′ carbon of an adjacent subunit.

3. The conjugate of claim 1 , wherein each of R 1 and R 2 , in linkages of type (a1), is methyl.

4. The conjugate of claim 1 , wherein said dsRNA oligomer includes at least two consecutive linkages of type (a1).

5. The conjugate of claim 1 , wherein the dsRNA oligomer has a length of about 15 to 25 base pairs.

6. The conjugate of claim 1 , wherein each base-pairing moiety is selected independently from the group consisting of adenine, guanine, uridine, cytosine, and analogues thereof that are capable of Watson-Crick base pairing to target-sequence RNA bases.

7. The conjugate of claim 1 , wherein the peptide transport moiety comprises a sequence consisting of at least two repeats of a single subsequence selected from (X′Y′X′), (X′X′Y′), (X′Y′), (X′Z′), and (X′Z′Z′).

8. The conjugate of claim 1 , wherein the peptide transport moiety comprises a sequence consisting of at least three repeats of a single subsequence selected from (X′Y′X′), (X′X′Y′), (X′Y′), (X′Z′), and (X′Z′Z′).

9. The conjugate of claim 1 , wherein said cationic linkages are of type (b1), where each R is H, R 4 is H, CH 3 , or an electron pair, and R 3 is selected from H, CH 3 , C(═NH)NH 2 , and C(O)-L-NHC(═NH)NH 2 .

10. The conjugate of claim 1 , wherein said cationic linkages are of type (b1), where each R is H, and each of R 3 and R 4 is independently H or CH 3 .

11. The conjugate of claim 10 , wherein each of R 3 and R 4 is H.

12. The conjugate of claim 1 , wherein said cationic linkages are of type (b2), where L is a linker up to 12 atoms in length having bonds selected from alkyl and alkylamino.

13. The conjugate of claim 1 , wherein W and Y are each O.

14. The dsRNA molecule of claim 1 , wherein linkage (b4) has one of the following structures (B-III), (B-IV), (B-V) or (B-VI):

wherein R 15 , R 16 , and R 17 are each independently optionally substituted C 1 -C 12 alkyl, amino, —C(═O)R 18 —, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycle, —SR 18 , or C 1 -C 12 alkoxy, wherein each R 18 is independently optionally substituted C 1 -C 12 alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycle, or C 1 -C 12 alkoxy.

15. The conjugate of claim 1 , wherein one of the sense and antisense strands has the following structure (C-I):

wherein, independently at each occurrence:

R 19 is absent, H, or C 1 -C 6 alkyl;

R 20 and R 21 independently are absent, H, a cell-penetrating peptide, or R 22 ;

R 22 is optionally substituted C 1 -C 30 alkyl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted aryl, optionally substituted C 1 -C 30 aralkyl, optionally substituted C 1 -C 30 alkylcarbonyl, optionally substituted C 3 -C 8 cycloalkylcarbonyl, optionally substituted C 3 -C 8 cycloalkylalkylcarbonyl, optionally substituted C 1 -C 30 arylcarbonyl, optionally substituted C 1 -C 30 aralkylcarbonyl, optionally substituted C 1 -C 30 alkyloxycarbonyl, optionally substituted C 3 -C 8 cycloalkyloxycarbonyl, optionally substituted C 1 -C 30 aryloxycarbonyl, or optionally substituted C 1 -C 30 aralkyloxycarbonyl;

W is S or O;

X is NR 23 R 24 or OR 25 ;

Y is O or NR 26 ;

B is a base-pairing moiety;

L′ is an optional linker up to 18 atoms in length comprising bonds selected from alkyl, hydroxyl, alkoxy, alkylamino, amide, ester, carbonyl, carbamate, phosphorodiamidate, phosphoroamidate, phosphorothioate, and phosphodiester; and

n is an integer of 8 or greater; and

wherein each of R 23 , R 24 , R 25 , and R 26 is independently hydrogen or optionally substituted C 1 -C 12 alkyl, and wherein each of R 23 , R 24 , R 25 , and R 26 may join with another of R 23 , R 24 , R 25 , or R 26 to form an optionally substituted 5-, 6-, or 7-membered heterocycle comprising one or more N, O, or S atoms or combinations thereof; and

wherein at least one of R 20 or R 21 is R 22 , and provided that both of R 19 and R 20 are not absent.

16. The conjugate of claim 1 , wherein at least 5% of intersubunit linkages are selected from the group consisting of (b1) to (b14).

17. The conjugate of claim 1 , wherein at least 5% of intersubunit linkages are (b1), b(2), or (b3).

18. The conjugate of claim 1 , wherein 5% of intersubunit linkages are cationic.

19. The conjugate of claim 1 , wherein at least two consecutive intersubunit linkages are uncharged linkages.