# US 10,119,138 B2 · iRNA agents with biocleavable tethers

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- Number: US 10,119,138 B2
- Document ID: US10119138B2
- Status: Granted
- Kind: B2
- Application number: 15/260,803
- Filed: 2016-09-09
- Published: 2016-12-29
- Granted: 2018-11-06
- Earliest claimed: 2003-04-17
- Assignees: Alnylam Pharmaceuticals, Inc.
- Applicants: Alnylam Pharmaceuticals, Inc.
- Inventors: Muthiah Manoharan; Kallanthottathil G. Rajeev
- Web page: https://patentgrep.com/patent/US10119138B2

## 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 2310/323: modified ring structure
- C12N 2310/3511: intercalating or cleaving agent
- C12N 2320/51: modulating the chemical stability, e.g. nuclease-resistance
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention relates to iRNA agents, which preferably include a monomer in which the ribose moiety has been replaced by a moiety other than ribose that further includes a tether having one or more linking groups, in which at least one of the linking groups is a cleavable linking group. The tether in turn can be connected to a selected moiety, e.g., a ligand, e.g., a targeting or delivery moiety, or a moiety which alters a physical property. The cleavable linking group is one which is sufficiently stable outside the cell such that it allows targeting of a therapeutically beneficial amount of an iRNA agent (e.g., a single stranded or double stranded iRNA agent), coupled by way of the cleavable linking group to a targeting agent—to targets cells, but which upon entry into a target cell is cleaved to release the iRNA agent from the targeting agent.

## Claims

### Claim 1

A modified RNA agent comprising a sense strand and an antisense strand, wherein one or more ribose replacement modification subunit (RRMS) comprising a ligand is incorporated into at least one of said strands via a phosphatase cleavable linking group, and wherein the RRMS is a cyclic carrier.

### Claim 2 (depends on 1)

The modified RNA agent of claim 1, wherein the cyclic carrier is a carbocyclic ring system or a heterocyclic ring system.

### Claim 3 (depends on 1)

The modified RNA agent of claim 1, wherein the cyclic carrier is selected from the group consisting of hydroxyproline, piperidine, morpholine, piperazine, and decalin.

### Claim 4 (depends on 1)

The modified RNA agent of claim 1, wherein the phosphatase cleavable linking group is a phosphate-based linking group.

### Claim 5 (depends on 4)

The modified RNA agent of claim 4, wherein the phosphate-based linking group is selected from the group consisting of —O—P(O)(ORk)—O—, —O—P(S)(ORk)—O—, —O—P(S)(SRk)—O—, —S—P(O)(ORk)—O—, —O—P(O)(ORk)—S—, —S—P(O)(ORk)—S—, —O—P(S)(ORk)—S—, —S—P(S)(ORk)—O—, —O—P(O)(Rk)—O—, —O—P(S)(Rk)—O—, —S—P(O)(Rk)—O—, —S—P(S)(Rk)—O—, —S—P(O)(Rk)—S—, and —O—P(S)(Rk)—S—, wherein Rk at each occurrence is, independently, H, C1-C20 alkyl, C1-C20 haloalkyl, C6-C10 aryl, or C7-C12 aralkyl.

### Claim 6 (depends on 4)

The modified RNA agent of claim 4, wherein the phosphate-based linking group is selected from the group consisting of —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—, and —O—P(S)(H)—S—.

### Claim 7 (depends on 1)

The modified RNA agent of claim 1, wherein the RRMS is incorporated into the sense strand.

### Claim 8 (depends on 7)

The modified RNA agent of claim 7, wherein the RRMS is incorporated into the 3′ end of the sense strand.

### Claim 9 (depends on 8)

The modified RNA agent of claim 8, wherein the RRMS is placed within 1, 2, or 3 positions of the 3′ end of the sense strand.

### Claim 10 (depends on 1)

The modified RNA agent of claim 1, wherein the RRMS is incorporated into the antisense strand.

### Claim 11 (depends on 10)

The modified RNA agent of claim 10, wherein the RRMS is incorporated into the 3′ end of the antisense strand.

### Claim 12 (depends on 1)

The modified RNA agent of claim 1, wherein the sense strand and the antisense strand are independently 17 to 25 nucleotides in length.

### Claim 13 (depends on 1)

The modified RNA agent of claim 1, wherein the modified RNA agent includes a duplex region between 17 and 23 pairs in length.

### Claim 14 (depends on 1)

The modified RNA agent of claim 1, wherein the modified RNA agent includes at least one 3′ overhang of 2-3 nucleotides in length.

### Claim 15 (depends on 1)

The modified RNA agent of claim 1, wherein the ligand is selected from the group consisting of a folic acid radical; a steroid radical; a carbohydrate radical; a vitamin A radical; a vitamin E radical; and a vitamin K radical.

### Claim 16 (depends on 15)

The modified RNA agent of claim 15, wherein the ligand is a carbohydrate radical.

### Claim 17 (depends on 1)

The modified RNA agent of claim 1, wherein the ligand is a targeting group selected from the group consisting of mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, multivalent fucose, glycosylated polyaminoacids, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, and an RGD peptide.

### Claim 18 (depends on 1)

The modified RNA agent of claim 1, wherein at least two RRMS subunits are incorporated into at least one of said strands.

### Claim 19 (depends on 18)

The modified RNA agent of claim 18, wherein at least two RRMS subunits are incorporated into the sense strand.

### Claim 20 (depends on 18)

The modified RNA agent of claim 18, wherein at least two RRMS subunits are incorporated into the antisense strand.