Modified iRNA agents

Patent metadata

Field	Value
Publication number	US-9290760-B2
Application number	US-201113822441-A
Country	US
Kind code	B2
Filing date	Sep 14, 2011
Priority date	Sep 15, 2010
Publication date	Mar 22, 2016
Grant date	Mar 22, 2016

Abstract

The present invention provides effective motifs for RNA agents conjugated to at least one ligand, which are advantageous for the in vivo delivery of iRNA duplex agents. Additionally, the present invention provides methods of making these compositions, as well as methods of introducing these iRNA duplex agents into cells using these compositions, e.g., for the treatment of various disease conditions.

First claim

We claim:

1. An iRNA duplex agent capable of silencing a target gene in vivo, comprising:
(a) a sense strand, wherein said sense strand comprises
(i) an alternating motif with at least 2 different chemically modified nucleotides; and
(ii) one or more carbohydrate ligand; and

(b) an antisense strand, wherein said antisense strand comprises
(i) an alternating motif with at least 2 different chemically modified nucleotides,

wherein the alternating motif is within the duplex region and the composition optionally further comprises one or more overhangs and/or capping groups.

2. The iRNA duplex agent of claim 1 , further comprising a phosphate or phosphate derivative at the 5′ carbon position of the antisense strand.

3. The iRNA duplex agent of claim 1 , further comprising at least one phosphorothioate internucleotide linkage; or at least one methylphosphonate internucleoside linkage.

4. The iRNA duplex agent of claim 1 , wherein the chemically modified nucleotide is selected from the group consisting of LNA, HNA, CeNA, 2′-methoxyethyl, 2′-OCH 3 , 2′-O-allyl, 2′-C- allyl, and 2′-fluoro.

5. The iRNA duplex agent of claim 1 , wherein the chemically modified nucleotides are 2′-OCH 3 and 2′-F.

6. The iRNA duplex agent of claim 1 , wherein the overhang is at least 2nucleotides in length and is selected from the group consisting of thymidine (T), 2′-O-methoxyethyl-5-methyluridine(Teo), 2′-O-methoxyethyladenosine (Aeo), 2-O-methoxyethyl-5-methylcytidine (m5Ceo), and combinations thereof, and optionally comprises a phosphorothioate between the two nucleotides, wherein the 2 nucleotides can be the same or different.

7. The iRNA duplex agent of claim 1 , wherein the overhang can form a mismatch with the target mRNA or it can be fully complemented with the target mRNA.

8. The iRNA duplex agent of claim 1 , wherein the duplex region is between 12-30 nucleotides in length.

9. The iRNA duplex agent of claim 1 , wherein the carbohydrate ligand is attached to the 3′ end of the sense strand.

10. The iRNA duplex agent of claim 1 ,
wherein said sense strand comprises
(i) an alternating 2′-fluoro modification, and
(ii) one or more carbohydrate ligand at the 3′-end; and

wherein said antisense strand comprises
(i) an alternating 2′-fluoro modification; and
(ii) a phosphorylated 5′ terminal antisense nucleotide.

11. The iRNA duplex agent of claim 10 , further comprising at least one phosphorothioate internucleotide linkage; or at least one methylphosphonate internucleoside linkage.

12. The iRNA duplex agent of claim 10 , wherein the chemically modified nucleotide is selected from the group consisting of LNA, HNA, CeNA, 2′-methoxyethyl, 2′-OCH 3 , 2′-O-allyl, 2′-C-allyl, and 2′-fluoro.

13. The iRNA duplex agent of claim 10 , wherein the chemically modified nucleotides are 2′-OCH 3 and 2′-F.

14. The iRNA duplex agent of claim 10 , wherein the overhang is at least 2 nucleotides in length and is selected from the group consisting of thymidine (T), 2′-O-methoxyethyl-5-methyluridine (Teo), 2′-O-methoxyethyladenosine (Aeo), 2′-O-methoxyethyl-5-methylcytidine (m5Ceo), and combinations thereof, and optionally comprises a phosphorothioate between the two nucleotides, wherein the 2 nucleotides can be the same or different.

15. The iRNA duplex agent of claim 10 , wherein the overhang can form a mismatch with the target mRNA or it can be fully complemented with the target mRNA.

16. The iRNA duplex agent of claim 10 , wherein the duplex region is between 12-30 nucleotides in length.

17. An iRNA duplex agent comprising a compound having the structure shown in formula (I′):

wherein:
A and B are each independently for each occurrence O, N(R N ) or S;
X and Y are each independently for each occurrence H, a protecting group, a phosphate group, a phosphodiester group, an activated phosphate group, an activated phosphite group, a phosphoramidite, a solid support, —P(Z′)(Z″)O-nucleoside, —P(Z′)(Z″)O-oligonucleotide, a lipid, a PEG, a steroid, a polymer, a nucleotide, annucleoside, —P(Z′)(Z″)O—R 1 -Q′-R 2 —OP(Z′″)(Z″″)O-oligonucleotide, or an oligonucleotide, —P(Z′)(Z″)-formula (I), -P(Z′)(Z″)— or -Q-R;
wherein the oligonucleotide comprises
(a) a sense strand, wherein said sense strand comprises
(i) an alternating 2′-fluoro modification and
(ii) at least one ligand; and

(b) an antisense strand, wherein said antisense strand comprises
(i) an alternating 2′-halogen modification; and
(ii) a first 5′ terminal antisense nucleotide, wherein said first 5′ terminal antisense nucleotide is phosphorylated at its 5′ carbon position;

R is L 1 or has the structure shown in formula (II)-(V)

q 2A , q 2B , q 3A , q 3B , q4 A , q 4B , q 5A , q 5B and q 5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different;
Q and Q′ are independently for each occurrence absent, —(P 7 -Q 7 -R 7 ) p -T 7 - or -T 7 -Q 7 -T 7′ -B-T 8′ -Q 8 -T 8 ;
P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C ,P 7 , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 5A , T 5B ,T 5C , T 7 , T 7′ , T 8 and T 8′ are each independently for each occurrence absent, CO, NH, O, S,OC(O), NHC(O), CH 2 , CH 2 NH or CH 2 O;
B is —CH 2 —N(B L )—CH 2 —;
B L is -T B -Q B -T B′ -R x ;
Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C , Q 7 , Q 8 and Q B are independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO 2 , N(R N ), C(R′)═C(R″), C≡C or C(O);
T B and T B′ are each independently for each occurrence absent, CO, NH, O, S, OC(O), OC(O)O, NHC(O), NHC(O)NH, NHC(O)O, CH 2 , CH 2 NH or CH 2 O;
R x is a carbohydrate;
R 1 , R 2 , R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C , R 7 are each independently for each occurrence absent, NH, O, S, CH 2 , C(O)O, C(O)NH, NHCH(R a )C(O), —C(O)—CH(R a )—NH—, CO, CH═N—O,

 or heterocyclyl;
L 1 , L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C are each independently for each occurrence a carbohydrate, e.g., monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide;
R′ and R″ are each independently H, C 1 -C 6 alkyl, OH, SH or N(R N ) 2 ;
R N is independently for each occurrence methyl, ethyl, propyl, isopropyl, butyl or benzyl;
R a is H or an amino acid side chain;
Z′, Z″, Z′″ and Z″″ are each independently for each occurrence O or S;
p represents independently for each occurrence 0-20.

18. The iRNA duplex agent of claim 17 , wherein the iRNA duplex agent further comprises at least one structure of formula (VI)

wherein X 6 and Y 6 are each independently H, a hydroxyl protecting group, a phosphate group, a phosphodiester group, an activated phosphate group, an activated phosphate group, a phosphoramidite, a solid support, —P(Z′)(Z″)O-nucleoside, —P(Z′)(Z″)O-oligonucleotide, a lipid, a PEG, a steroid, a polymer, —P(Z′)(Z″)O—R 1 -Q′-R 2 —OP(Z′

Assignees

• Rajeev Kallanthottathil G
• Zimmermann Tracy
• Manoharan Muthiah
• Maier Martin
• Fitzgerald Kevin
• Alnylam Pharmaceuticals Inc

Inventors

• Rajeev Kallanthottathil G
• Zimmermann Tracy
• Manoharan Muthiah
• Maier Martin
• Fitzgerald Kevin

Classifications

• A61P31/12

  Antivirals · CPC title

• A61P31/20

  for DNA viruses · CPC title

• A61P31/14

  for RNA viruses · CPC title

• C12N2310/322

  2'-R Modification · CPC title

• C12N2310/3515

  Lipophilic moiety, e.g. cholesterol · CPC title