Claims for Patent: 10,131,907

Summary for Patent: 10,131,907

Title:	Glycoconjugates of RNA interference agents
Abstract:	The present invention relates to agents, compositions and methods for inhibiting the expression of a target gene, comprising an RNAi agent bearing at least one galactosyl moiety. These are useful for delivering the gene expression inhibiting activity to cells, particularly hepatocytes, and more particularly in therapeutic applications.
Inventor(s):	Forst; Andrea (Bayreuth, DE), Hadwiger; Philipp (Kulmbach, DE), Vornlocher; Hans-Peter (Bayreuth, DE)
Assignee:	ALNYLAM PHARMACEUTICALS, INC. (Cambridge, MA)
Application Number:	14/500,356
Patent Claims:	1. An RNAi agent for inhibiting the expression of a target gene in a cell in vivo, wherein the RNAi agent consists essentially of two mutually complementary oligoribonucleotide strands of between 15 and 30 nucleotides in length, wherein at least one of the oligoribonucleotide strands is conjugated via a branched linker to an ASGPR ligand comprising at least two galactose moieties, wherein at least one of the oligoribonucleotide strands comprises at least two consecutive phosphorothioate modifications in the last 3 nucleotides of the oligonucleotide, wherein at least one of the oligoribonucleotide strands comprises at least one nucleotide with a 2'-modification, wherein at least one oligoribonucleotide strand is complementary to at least one portion of a mRNA corresponding to the target gene, and wherein the RNAi agent has the capability of inhibiting the expression of a target gene in a cell in vivo. 2. The RNAi agent of claim 1, wherein the ASGPR ligand conjugated to the oligoribonucleotide through the branched linker has a structure of formula (III) ##STR00021## wherein Z.sup.3 and Z.sup.4 are independently O or S; and the ASGPR ligand is linked to O via at least one linker group ##STR00022## wherein Z.sup.1 and Z.sup.2 are independently O or S, and n is 1-20. 3. The RNAi agent of claim 2, wherein at least one of Z.sup.3 and Z.sup.4 is S. 4. The RNAi agent of claim 2, wherein both of Z.sup.3 and Z.sup.4 are O. 5. The RNAi agent of claim 1, wherein the ASGPR ligand is linked to the branched linker through an intervening linker. 6. The RNAi agent of claim 5, wherein the ASGPR ligand linked to the branched linker through the intervening linker has a structure of formula (IV) ASGPR ligand-O--CH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.nOP(Z.sup.5)(Z.sup.6)-b- ranched-linker Formula (IV) wherein n is 1-20; and Z.sup.5 and Z.sup.6 are each independently O or S. 7. The RNAi agent of claim 6, wherein n is 3. 8. The RNAi agent of claim 6, wherein at least one of Z.sup.5 and Z.sup.6 is S. 9. The RNAi agent of claim 6, wherein both of Z.sup.5 and Z.sup.6 are O. 10. The RNAi agent of claim 1, wherein the galactose moieties are N-acetylgalactosamine moieties. 11. The RNAi agent of claim 10, wherein the distance between the N-acetylgalactosamine moieties is at least 4 .ANG.. 12. The RNAi agent of claim 11, wherein the RNAi agent is capable of inhibiting the expression of the target gene in the cell. 13. The RNAi agent of claim 12, wherein the cell harbors an asialoglycoprotein receptor on its surface. 14. The RNAi agent of claim 12, wherein the cell is a hepatocyte. 15. The RNAi agent of claim 1, wherein the 2'-modification comprises at least one of 2'-H, 2'-O-methyl, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-Fluoro, 2'-O--CH.sub.2--CO--NHMe, 2'-O--CH.sub.2CH.sub.2OCH.sub.2CH.sub.2N(Me).sub.2, 2'-4'-methylene (LNA), 2'-4'-ethylene (ENA), 2'-S-methyl, 2'-ara-fluoro, 2'-O-allyl, 2'-C-allyl, 2'-O--NH.sub.2, 2'-NH.sub.2 and 2'-ethynyl. 16. A pharmaceutical composition, comprising (i) an RNAi agent of claim 1; and (ii) a pharmaceutically acceptable excipient. 17. A method for the manufacture of an RNAi agent of claim 1, comprising the steps of (i) synthesizing two mutually complementary oligoribonucleotide strands of between 15 and 30 nucleotides in length, wherein at least one of the oligoribonucleotides is coupled to a ligand comprising a linker group and at least one galactose moiety; and (ii) effecting the hybridization of the at least two mutually complementary oligoribonucleotides. 18. The method of claim 17, further comprising the step of formulating the RNAi agent with a pharmaceutically acceptable excipient. 19. A method of treatment, comprising a step of administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition of claim 16. 20. The method of claim 19, wherein the subject is in need of a treatment for a disease or condition related to unwanted expression of a target gene in the liver. 21. The method of claim 19, wherein the subject is a vertebrate, mammal, or a human. 22. The RNAi agent of claim 10, wherein the distance between the N-acetylgalactosamine moieties is at least 10 .ANG.. 23. The RNAi agent of claim 10, wherein the distance between the N-acetylgalactosamine moieties is at least 15 .ANG.. 24. The RNAi agent of claim 10, wherein the distance between the N-acetylgalactosamine moieties is at least 20 .ANG.. 25. The RNAi agent of claim 1, wherein the ASGPR ligand comprises three or more galactose moieties. 26. The RNAi agent of claim 1, wherein the ASGPR ligand comprises four or more galactose moieties.