# US 10,119,136 B2 · RNAi agents modified at the 4′-C position

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- Number: US 10,119,136 B2
- Document ID: US10119136B2
- Status: Granted
- Kind: B2
- Application number: 15/110,658
- Filed: 2015-01-09
- Published: 2017-02-02
- Granted: 2018-11-06
- Earliest claimed: 2014-01-09
- Assignees: Alnylam Pharmaceuticals, Inc.
- Applicants: Alnylam Pharmaceuticals, Inc.
- Inventors: Muthiah Manoharan; Shigeo Matsuda; Kallanthottathil G. Rajeev; Ivan Zlatev
- Web page: https://patentgrep.com/patent/US10119136B2

## CPC

- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/3125: Methylphosphonates
- C12N 2310/315: Phosphorothioates
- C12N 2310/32: of the sugar
- C12N 2310/321: 2'-O-R Modification
- C12N 2310/33: of the base
- C12N 2310/335: Modified T or U
- C12N 2310/341: Gapmers, i.e. of the type ===---===
- C12N 2310/343: having patterns, e.g. ==--==--==--
- C12N 2310/344: Position-specific modifications, e.g. on every purine, at the 3'-end
- C12N 2310/346: having a combination of backbone and sugar modifications
- C12N 2310/351: Conjugate
- C12N 2320/51: modulating the chemical stability, e.g. nuclease-resistance
- C12N 2320/53: reducing unwanted side-effects
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

One aspect of the present invention relates to double-stranded RNAi (dsRNA) duplex agent capable of inhibiting the expression of a target gene in vivo. The dsRNA duplex comprises one or more 4′-modifications in one or both strand. Other aspects of the invention relates to pharmaceutical compositions comprising these dsRNA agents suitable for in vivo therapeutic use, and methods of inhibiting the expression of a target gene by administering these dsRNA agents, e.g., for the treatment of various disease conditions.

## Claims

### Claim 1

A double-stranded RNAi agent capable of inhibiting the expression of a target gene, comprising a sense strand and an antisense strand, each strand having 12 to 30 nucleotides, wherein the duplex comprises at least one 4′-modified moiety, represented by one of the following formulas in group i) or group ii):
wherein:
R is independently H; OMe; F; OH; O-(CH2)2OMe; SMe, NMe2; NH2; Me; O-nPr; O-CH2C(O)NHMe; O-(CH2)2NMe2; O-(CH2)2O-(CH2)2 NMe2; O-alkyl; or O-alkylamino;
R′ is independently H or Me;
B is independently A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2-modified purines; N8-modified purines; phenoxazine; G-clamp; or non-canonical mono, bi and tricyclic heterocycles,
Y and Y′ are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH or CH2O, O, S, S(O), SO2, N(RN), C(R′)═C(R″), C≡C or C(O), NHCH(R)C(O), —C(O)—CH(R)—NH—, CO, CH═N—O,
or heterocyclyl; and
n is independently an integer from 0 to 14;
L represents, in each occurrence, a ligand selected from the group consisting of a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide, peptide, oligopeptide, RGD peptide, peptidoid, folate, cholesterol, lipid, and heterocycle; and
wherein the stereochemistry is R or S and combinations of R and S for the unspecified chiral centers;
or
wherein:
R is independently H; OMe; OEt, F; OTBDMS; O-(CH2)2OMe; SMe, NMe2; NPhth; Me; O-nPr; O-alkyl; or O-alkylamino;
R′ is independently H or Me; and
Bp and B are each independently ABz; CAc; 5-Me-CBz; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2-modified purines; N8-modified purines; phenoxazine; G-clamp; or non-canonical mono, bi and tricyclic heterocycles,
wherein the stereochemistry is R or S and combinations of R and S for the unspecified chiral centers.

### Claim 2 (depends on 1)

A double-stranded RNAi agent of claim 1, represented by formula (III):
sense: 5′np-Na-(XXX)i-Nb-YYY-Nb-(ZZZ)j-Na-nq3′
antisense: 3′np′-Na′-(X′X′X′)k-Nb′-Y′Y′Y′-Nb-(Z′Z′Z′)l-Na′-nq′5′ (III)
wherein:
j, k, and l are each independently 0 or 1;
p and q are each independently 0-6;
each Na and Na′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides each Nb and Nb′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof;
each np and nq independently represents an overhang nucleotide; and
XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides;
wherein at least one of n, N, X, Y, or Z is a 4′-modified nucleoside of group i) or group ii); and
wherein modifications on Nb is different than the modification on Y and modifications on Nb′ is different than the modification on Y′.

### Claim 3 (depends on 1)

The double-stranded RNAi agent of claim 1, wherein the duplex region is 17-30 nucleotide pairs in length.

### Claim 4 (depends on 1)

The double-stranded RNAi agent of claim 1, wherein the duplex region is 17-19 nucleotide pairs in length.

### Claim 5 (depends on 1)

The double-stranded RNAi agent of claim 1, wherein the duplex region is 27-30 nucleotide pairs in length.

### Claim 6 (depends on 1)

The double-stranded RNAi agent of claim 1, wherein each strand has 17-30 nucleotides.

### Claim 7 (depends on 1)

The double-stranded RNAi agent of claim 1, further comprising at least one ligand.

### Claim 8 (depends on 7)

The double-stranded RNAi agent of claim 7, wherein the ligand is a one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

### Claim 9 (depends on 8)

The double-stranded RNAi agent of claim 8, wherein the ligand is

### Claim 10 (depends on 7)

The double-stranded RNAi agent of claim 7, wherein the ligand is attached to the 3′ end of the sense strand.

### Claim 11 (depends on 1)

The double-stranded RNAi agent of claim 1, further comprising at least one phosphorothioate or methylphosphonate internucleotide linkage.

### Claim 12 (depends on 1)

The double-stranded RNAi agent of claim 1, wherein the nucleotide at the 1 position of the 5′-end of the duplex in the antisense strand is selected from the group consisting of A, dA, dU, U, and dT.

### Claim 13 (depends on 1)

The double-stranded RNAi agent of claim 1, wherein the base pair at the 1 position of the 5′-end of the duplex is an AU base pair.

### Claim 14 (depends on 2)

The double-stranded RNAi agent of claim 2, wherein the Y nucleotides contain a 2′-fluoro modification.

### Claim 15 (depends on 2)

The double-stranded RNAi agent of claim 2 wherein the Y′ nucleotides contain a 2′-O-methyl modification.

### Claim 16 (depends on 1)

A pharmaceutical composition comprising the double-stranded RNAi agent of claim 1, alone or in combination with a pharmaceutically acceptable carrier or excipient.

### Claim 17 (depends on 1)

A method for inhibiting the expression of a target gene comprising the step of administering the double-stranded RNAi agent of claim 1, in an amount sufficient to inhibit expression of the target gene.

### Claim 18 (depends on 17)

The method of claim 17, wherein the double-stranded RNAi agent is administered through subcutaneous or intravenous administration.