# US 12,005,074 B2 · Extrahepatic delivery

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- Number: US 12,005,074 B2
- Document ID: US12005074B2
- Status: Granted
- Kind: B2
- Application number: 18/311,484
- Filed: 2023-05-03
- Published: 2024-01-11
- Granted: 2024-06-11
- Earliest claimed: 2018-05-07
- Assignees: Alnylam Pharmaceuticals, Inc.
- Applicants: Alnylam Pharmaceuticals, Inc.
- Inventors: Jayaprakash K. Nair; Martin Maier; Vasant Jadhav; Stuart Milstein; Kirk Brown; Rubina G. Parmar; Kallanthottathil G. Rajeev; Muthiah Manoharan; Alexander V. Kel'In; Muthusamy Jayaraman; Klaus Charisse; Adam Castoreno; Christopher S. Theile; Kevin Fitzgerald
- Web page: https://patentgrep.com/patent/US12005074B2

## CPC

- A61K 31/713: Double-stranded nucleic acids or oligonucleotides
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)}
- A61K 9/0053: {Mouth and digestive tract, i.e. intraoral and peroral administration}
- A61K 47/543: {Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine}
- A61K 47/549: {Sugars, nucleosides, nucleotides or nucleic acids}
- A61K 47/55: the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug
- A61K 47/551: {one of the codrug's components being a vitamin, e.g. niacinamide, vitamin B3, cobalamin, vitamin B12, folate, vitamin A or retinoic acid}
- A61K 47/554: {the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid}
- A61K 47/65: Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
- 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 15/1135: {against oncogenes or tumor suppressor genes}
- C12N 15/1137: {against enzymes (viral enzymes C12N15/1131; receptors C12N15/1138)}
- C12N 15/1138: {against receptors or cell surface proteins}
- C12Y 115/01001: Superoxide dismutase (1.15.1.1)
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/312: Phosphonates
- C12N 2310/315: Phosphorothioates
- C12N 2310/321: 2'-O-R Modification
- C12N 2310/322: 2'-R Modification
- C12N 2310/3231: having an additional ring, e.g. LNA, ENA
- C12N 2310/33: of the base
- C12N 2310/344: Position-specific modifications, e.g. on every purine, at the 3'-end
- C12N 2310/3515: Lipophilic moiety, e.g. cholesterol
- C12N 2320/32: Special delivery means, e.g. tissue-specific
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

One aspect of the present invention relates to a double stranded iRNA agent comprising an antisense strand which is complementary to a target gene; a sense strand which is complementary to said antisense strand; and one or more lipophilic moieties conjugated to one or more internal positions on at least one strand, optionally via a linker or carrier. Another aspect of the invention relates to a method of gene silencing, comprising administering to a cell or a subject in need thereof a therapeutically effective amount of the lipophilic moieties-conjugated double-stranded iRNAs.

## Claims

### Claim 1

A double-stranded iRNA agent comprising:
an antisense strand which is complementary to a target mRNA;
a sense strand which is complementary to said antisense strand; and
one lipophilic moiety directly bonded to a 2′-O of a sugar moiety at an internal position of the sense strand, wherein:
the sense and antisense strands are each independently 19 to 25 nucleotides in length and form a double-stranded region 19-21 nucleotide pairs in length, having a blunt end at the 5′-end of the antisense strand;
the lipophilic moiety is a linear C16 alkyl chain;
the internal position is a position on the sense strand excluding the three terminal positions from each of the 5′-end and 3′-end of the sense strand and excluding positions 11 to 13, counting from the 3′-end of the sense strand; and
the double-stranded iRNA agent does not comprise a N-acetylgalactosamine (GalNAc) conjugate.

### Claim 2 (depends on 1)

The double-stranded iRNA agent of claim 1, wherein the internal positions exclude positions 9-12 counting from the 5′-end of the sense strand.

### Claim 3 (depends on 1)

The double-stranded iRNA agent of claim 1, wherein the sense and antisense strands are each independently 21 to 23 nucleotides in length, and the internal position is one of positions 4-8 and 13-18, counting from the 5′-end of the sense strand.

### Claim 4 (depends on 1)

The double-stranded iRNA agent of claim 1, wherein the internal position is position 5, counting from the 5′-end of the sense strand.

### Claim 5 (depends on 1)

The double-stranded iRNA agent of claim 1, wherein the internal position is position 6, counting from the 5′-end of the sense strand.

### Claim 6 (depends on 1)

The double-stranded iRNA agent of claim 1, wherein the internal position is position 7, counting from the 5′-end of the sense strand.

### Claim 7 (depends on 1)

The double-stranded iRNA agent of claim 1, wherein the sense strand and antisense strand are each 19 nucleotides in length, wherein the sense strand contains 2′-F modifications on the nucleotides at positions 7, 8, and 9 from the 5′-end of the sense strand.

### Claim 8 (depends on 7)

The double-stranded iRNA agent of claim 7, wherein the antisense strand contains 2′-O-methyl modifications on the nucleotides at positions 11, 12, and 13 from the 5′-end of the antisense strand.

### Claim 9 (depends on 1)

The double-stranded iRNA agent of claim 1, wherein the sense strand and antisense strand are each 20 nucleotides in length, wherein the sense strand contains 2′-F modifications on the nucleotides at positions 8, 9, and 10 from the 5′-end of the sense strand.

### Claim 10 (depends on 9)

The double-stranded iRNA agent of claim 9, wherein the antisense strand contains 2′-O-methyl modifications on the nucleotides at positions 11, 12, and 13 from the 5′-end of the antisense strand.

### Claim 11 (depends on 1)

The double-stranded iRNA agent of claim 1, wherein the sense strand and antisense strand are each 21 nucleotides in length, wherein the sense strand contains 2′-F modifications on the nucleotides at positions 9, 10, and 11 from the 5′-end of the sense strand.

### Claim 12 (depends on 11)

The double-stranded iRNA agent of claim 11, wherein the antisense strand contains of 2′-O-methyl modifications on the nucleotides at positions 11, 12, and 13 from the 5′-end of the antisense strand.

### Claim 13 (depends on 1)

The double-stranded iRNA agent of claim 1, wherein the sense strand is 21 nucleotides in length, and the antisense strand is 23 nucleotides in length and has a two nucleotide overhang at the 3′-end of the antisense strand, wherein:
the sense strand contains 2′-F modifications on the nucleotides at positions 9, 10, and 11 from the 5′-end of the sense strand; and
the antisense strand contains 2′-O-methyl modifications on the nucleotides at positions 11, 12, and 13 from the 5′-end of the antisense strand.

### Claim 14 (depends on 1)

The double-stranded iRNA agent of claim 1, wherein the first base pair at the 5′-end of the antisense strand is an AU base pair.

### Claim 15 (depends on 1)

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

### Claim 16 (depends on 15)

The double-stranded iRNA agent of claim 15, wherein the double-stranded iRNA agent comprises at least two phosphorothioate internucleotide linkages at the first five nucleotides on the antisense strand, counting from the 5′-end of the antisense strand.

### Claim 17 (depends on 15)

The double-stranded iRNA agent of claim 15, wherein the double-stranded iRNA agent comprises 8 phosphorothioate internucleotide linkages.

### Claim 18 (depends on 1)

The double-stranded iRNA agent of claim 1, wherein each of the sense and antisense strands contains at least two different modifications.

### Claim 19 (depends on 1)

The double-stranded iRNA agent of claim 1, wherein the double-stranded iRNA agent comprises a mismatch to the target RNA within the duplex region.

### Claim 20 (depends on 1)

The double-stranded iRNA agent of claim 1, wherein each of the sense and antisense strands is independently modified with one or more modifications selected from the group consisting of an acyclic nucleotide, LNA, HNA, CeNA, 2′-methoxyethyl, 2′- O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy, 2′-fluoro, 2′-O-N-methylacetamido (2′-O-NMA), a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE), 2′-O-aminopropyl (2′-O-AP), and 2′-ara-F.

### Claim 21 (depends on 20)

The double-stranded iRNA agent of claim 20, wherein one or more modification is acyclic nucleotide modification, and the acyclic nucleotide is an UNA or a GNA.

### Claim 22 (depends on 1)

The double-stranded iRNA agent of claim 1, further comprising a 2′-5′ internucleotide linkage.

### Claim 23 (depends on 1)

The double-stranded iRNA agent of claim 1, further comprising an overhang region at the 3′-end of the antisense strand containing two nucleotides and having a phosphorothioate or methylphosphonate internucleotide linkage between the two overhang nucleotides.

### Claim 24 (depends on 1)

The double-stranded iRNA agent of claim 1, further comprising an overhang region at the 3′-end of the antisense strand and two phosphorothioate internucleotide linkages between the terminal three nucleotides at the 3′-end of the antisense strand, in which two of the three nucleotides are the overhang nucleotides, and the third is the paired nucleotide next to the overhang nucleotide.

### Claim 25 (depends on 1)

The double-stranded iRNA agent of claim 1, further comprising a phosphate at the 5′-end of the antisense strand.

### Claim 26 (depends on 1)

The double-stranded iRNA agent of claim 1, further comprising a phosphate mimic at the 5′-end of the antisense strand.

### Claim 27 (depends on 26)

The double-stranded iRNA agent of claim 26, wherein the phosphate mimic is a 5′-vinyl phosphonate (VP).

### Claim 28 (depends on 1)

The double-stranded iRNA agent of claim 1, comprising
(a) a sense strand having:
(i) a length of 21 nucleotides;
(ii) 2′-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2′-F modifications at positions 7, and 9 to 11; and
(iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3, counting from the 5′-end of the sense strand; and
(b) an antisense strand having:
(i) a length of 23 nucleotides;
(ii) (1) 2′-OMe modifications at positions 1, 3 to 5, 7, 8, 10 to 13, 15, and 17 to 23, and 2′-F modifications at positions 2, 6, 9, 14, and 16, counting from the 5′-end of the antisense strand; or
(2) 2′-OMe modifications at positions 1, 3 to 5, 7, 10 to 13, 15, and 17 to 23, and 2′-F modifications at positions 2, 6, 8, 9, 14, and 16, counting from the 5′-end of the antisense strand; and
(iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, counting from the 5′-end of the antisense strand;
wherein the double-stranded iRNA agent has a two-nucleotide overhang at the 3′-end of the antisense strand, and a blunt end at the 5′-end of the antisense strand.