# US 2025/0205272 A1 · Modified rna agents with reduced off-target effect

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- Number: US 2025/0205272 A1
- Document ID: US20250205272A1
- Status: Published application
- Kind: A1
- Application number: 19/025,271
- Filed: 2025-01-16
- Published: 2025-06-26
- Earliest claimed: 2016-11-23
- Assignees: Alnylam Pharmaceuticals, Inc.
- Applicants: Alnylam Pharmaceuticals, Inc.
- Inventors: Mark K. SCHLEGEL; Maja JANAS; Vasant R. JADHAV; Muthiah MANOHARAN; Kallanthottathil G. RAJEEV; Muthusamy JAYARAMAN; Alexander V. KEL'IN; Shigeo MATSUDA; Klaus CHARISSE; Jayaprakash K. NAIR; Martin A. MAIER; Alfica SEHGAL; Christopher BROWN; Christopher THEILE; Stuart MILSTEIN
- Web page: https://patentgrep.com/patent/US20250205272A1

## CPC

- A61K 31/713: Double-stranded nucleic acids or oligonucleotides
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- 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 2310/14: interfering nucleic acids [NA]
- C12N 2310/31: of the backbone
- C12N 2310/322: 2'-R Modification
- C12N 2310/351: Conjugate
- C12N 2310/3533: Halogen
- C12N 2320/53: reducing unwanted side-effects
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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 RNA (dsRNA) agent capable of inhibiting the expression of a target gene. The antisense strand of the dsRNA molecule comprises at least one thermally destabilizing nucleotide occurring at a seed region; the dsRNA comprises at least four 2′-fluoro modifications, and the sense strand of the dsRNA molecule comprises ligand, wherein the ligand is an ASGPR ligand. Other aspects of the invention relate to pharmaceutical compositions comprising these dsRNA molecules suitable for therapeutic use, and methods of inhibiting the expression of a target gene by administering these dsRNA molecules, e.g., for the treatment of various disease conditions.

## Claims

### Claim at position 1

-27. (canceled)

### Claim 28

A double-stranded RNA (dsRNA) capable of inhibiting the expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length, and wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference; and the antisense strand comprises one modification of the formula, at position 5, 6, 7, or 8, counting from the 5′-end of the antisense strand, wherein B is nucleobase.

### Claim 29 (depends on 28)

The dsRNA of claim 28, wherein the dsRNA contains one or more characteristic selected from the group consisting of, (i) the antisense strand comprises only 2, 3, 4, 5, or 6 2′-fluoro modifications; (ii) the antisense strand comprises only 1, 2, 3, or 4 phosphorothioate internucleotide linkages; (iii) the sense strand comprises only 2, 3, 4, or 5 2′-fluoro modifications; (iv) the sense strand comprises only 1, 2, 3, or 4 phosphorothioate internucleotide linkages; and (v) comprising a blunt end at 5′-end of the antisense strand.

### Claim 30 (depends on 28)

The dsRNA of claim 28, wherein there are no 2′-fluoro modifications at nucleotide positions 3-9 of the antisense strand.

### Claim 31 (depends on 28)

The dsRNA of claim 28, wherein, the sense strand is conjugated with a ligand.

### Claim 32 (depends on 31)

The dsRNA of claim 31, wherein the ligand is a cell or tissue targeting agent.

### Claim 33 (depends on 31)

The dsRNA of claim 31, wherein the ligand is an antibody.

### Claim 34 (depends on 31)

The dsRNA of claim 31, wherein the ligand is an alkyl or substituted alkyl group.

### Claim 35 (depends on 31)

The dsRNA of claim 31, wherein the ligand is an ASGPR ligand comprising one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

### Claim 36 (depends on 35)

The dsRNA of claim 35, wherein the ASGPR ligand is:

### Claim 37 (depends on 28)

The dsRNA of claim 28, wherein the thermally-destabilizing modification is located in position 6 of the antisense strand, counting from the 5′-end of the antisense strand.

### Claim 38 (depends on 28)

The dsRNA of claim 28, wherein the thermally-destabilizing modification is located in position 7 of the antisense strand, counting from the 5′-end of the antisense strand.

### Claim 39 (depends on 28)

The dsRNA of claim 28, wherein the sense strand has 21 nucleotides, and the antisense strand has 23 nucleotides.

### Claim 40 (depends on 28)

The dsRNA of claim 28, wherein the dsRNA has a melting temperature of from about 55° C. to about 67° C.

### Claim 41 (depends on 28)

The dsRNA of claim 28, wherein the antisense strand comprises 2′-fluoro modifications at positions 2, 14 and 16, counting from the 5′-end of the antisense strand.

### Claim 42 (depends on 41)

The dsRNA of claim 41, wherein the antisense strand comprises 2′-fluoro modifications at only positions 2, 6, 8, 9, 14 and 16, counting from the 5′-end of the antisense strand.

### Claim 43 (depends on 41)

The dsRNA of claim 41, wherein the antisense strand comprises 2′-fluoro modifications at only positions 2, 6, 14, and 16, counting from the 5′-end of the antisense strand.

### Claim 44 (depends on 41)

The dsRNA of claim 41, wherein the sense strand comprises 2′-F modifications at positions opposite or complimentary to (i) positions 11, 12 and 15 or (ii) positions 11, 12, 13 and 15, of the antisense strand counting from the 5′-end of the antisense strand.

### Claim 45 (depends on 41)

The dsRNA of claim 41, wherein the sense strand comprises a block of two, three, or four 2′-fluoro nucleotides.

### Claim 46 (depends on 28)

The dsRNA of claim 28, wherein the first base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair

### Claim 47 (depends on 28)

A method for silencing a target gene in a cell, the method comprising a step of introducing the dsRNA of claim 28 into the cell.

### Claim 48 (depends on 47)

The method of claim 47, wherein the dsRNA is administered through subcutaneous or intravenous administration.

### Claim 49 (depends on 28)

A method for suppressing off-target effects caused by the antisense strand of a dsRNA, the method comprising a step of introducing the dsRNA of claim 28 into a cell.