# US 10,767,177 B2 · Sterol regulatory element binding protein (SREBP) chaperone (SCAP) iRNA compositions and methods of use thereof

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- Number: US 10,767,177 B2
- Document ID: US10767177B2
- Status: Granted
- Kind: B2
- Application number: 16/002,034
- Filed: 2018-06-07
- Published: 2018-09-27
- Granted: 2020-09-08
- Earliest claimed: 2015-12-10
- Assignees: Alnylam Pharmaceuticals, Inc.
- Applicants: Alnylam Pharmaceuticals, Inc.
- Inventors: Kevin Fitzgerald; Huilei Xu; Gregory Hinkle
- Web page: https://patentgrep.com/patent/US10767177B2

## 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)}
- A61K 31/713: Double-stranded nucleic acids or oligonucleotides
- A61K 47/549: {Sugars, nucleosides, nucleotides or nucleic acids}
- A61K 47/554: {the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid}
- A61P 1/16: for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
- A61P 3/06: Antihyperlipidemics
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/315: Phosphorothioates
- C12N 2310/318: where the PO2 is completely replaced, e.g. MMI or formacetal
- C12N 2310/346: having a combination of backbone and sugar modifications
- C12N 2310/351: Conjugate
- C12N 2310/3515: Lipophilic moiety, e.g. cholesterol
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

## Abstract

The invention relates to double stranded ribonucleic acid (dsRNAi) agents and compositions targeting the SCAP gene, as well as methods of inhibiting expression of a SCAP gene and methods of treating subjects having a SCAP-associated disorder, such as nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH), using such dsRNAi agents and compositions.

## Claims

### Claim 1

A double stranded ribonucleic acid (RNAi) agent for inhibiting expression of a sterol regulatory element binding protein (SREBP) chaperone (SCAP) gene, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, and
wherein the antisense strand comprises a region of complementarity to an mRNA encoding SCAP, wherein the region of complementarity comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from nucleotides 1132-1156 of SEQ ID NO:1.

### Claim 2 (depends on 1)

The double stranded RNAi agent of claim 1, wherein the double stranded RNAi agent comprises at least one modified nucleotide.

### Claim 3 (depends on 2)

The double stranded RNAi agent of claim 2, wherein at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, 2′-hydroxly-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, a nucleotide comprising a 5′-methylphosphonate group, a nucleotide comprising a 5′ phosphate or 5′ phosphate mimic, a nucleotide comprising vinyl phosphate, a nucleotide comprising adenosine-glycol nucleic acid (GNA), a nucleotide comprising thymidine-glycol nucleic acid (GNA) S-Isomer, a nucleotide comprising 2-hydroxymethyl-tetrahydrofurane-5-phosphate, a nucleotide comprising 2′-deoxythymidine-3′phosphate, a nucleotide comprising 2′-deoxyguanosine-3′-phosphate, and a terminal nucleotide linked to a cholesteryl derivative and a dodecanoic acid bisdecylamide group.

### Claim 4 (depends on 2)

The double stranded RNAi agent of claim 2, further comprising at least one phosphorothioate internucleotide linkage.

### Claim 5 (depends on 1)

The double stranded RNAi agent of claim 1, wherein the region of complementarity is at least 17 nucleotides in length; 19-23 nucleotides in length; or 19 nucleotides in length.

### Claim 6 (depends on 1)

The double stranded RNAi agent of claim 1, wherein each strand is no more than 30 nucleotides in length.

### Claim 7 (depends on 1)

The double stranded RNAi agent of claim 1, wherein at least one strand comprises a 3′ overhang of at least 1 nucleotide; or a 3′ overhang of at least 2 nucleotides.

### Claim 8 (depends on 1)

The double stranded RNAi agent of claim 1, wherein the double stranded RNAi agent further comprises a ligand.

### Claim 9 (depends on 8)

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

### Claim 10 (depends on 8)

The double stranded RNAi agent of claim 8, wherein the double stranded RNAi agent is conjugated to the ligand as shown in the following schematic
and, wherein X is O or S.

### Claim 11 (depends on 10)

The double stranded RNAi agent of claim 10, wherein the X is O.

### Claim 12 (depends on 8)

The double stranded RNAi agent of claim 8, wherein the ligand is a cholesterol.

### Claim 13

A double stranded ribonucleic acid (RNAi) agent for inhibiting expression of a sterol regulatory element binding protein (SREBP) chaperone (SCAP) gene, wherein said double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein said antisense strand comprises a region complementary to nucleotides 1132-1156 of SEQ ID NO:1, wherein each strand is about 14 to about 30 nucleotides in length, wherein said double stranded RNAi agent is represented by formula (III):
sense:5′np-Na-(XXX)i-Nb-YYY-Nb-(ZZZ)i-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 1 are each independently 0 or 1;
p, p′, q, 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, np′, nq, and nq′, each of which may or may not be present, independently represents an overhang nucleotide;
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;
modifications on Nb differ from the modification on Y and modifications on Nb′ differ from the modification on Y′; and
wherein the sense strand is conjugated to at least one ligand.

### Claim 14 (depends on 13)

The double stranded RNAi agent of claim 13, wherein formula (III) is represented by formula (Ma):
sense: 5′ np-Na-YYY-Na-nq3′
antisense: 3′ np′-Na′-Y′Y′Y′-Na′-nq′5′ (IIIa);
wherein formula (III) is represented by formula (IIIb):
sense: 5′np-Na-YYY-Nb-ZZZ-Na-nq3′
antisense: 3′np′-Na′-Na′-nq′5′ (IIIb)
wherein each Nb and Nb′ independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides;
wherein formula (III) is represented by formula (IIIc):
sense: 5′np-NaXXX-Nb-YYY-Na-nq3′
antisense: 3′ np′-Na′-X′X′X′-Nb′-Y′Y′Y′-Na′-nq′5′ (IIIc)
wherein each Nb and Nb′ independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides; or
wherein formula (III) is represented by formula (Ind):
sense: 5′np-Na-XXX-Nb-YYY-Nb-ZZZ-Na-nq3′
antisense: 3′np′-Na′-X′X′X′-Nb′-Y′Y′Y′-Nb′-Z′Z′Z′-Na′-nq′5′ (IIId)
wherein each Nb and Nb′ independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides and each Na and Na′ independently represents an oligonucleotide sequence comprising 2-10 modified nucleotides.

### Claim 15 (depends on 13)

The double stranded RNAi agent of claim 13, wherein the double stranded region is 15-30 nucleotide pairs in length; 17-23 nucleotide pairs in length; 17-25 nucleotide pairs in length; 23-27 nucleotide pairs in length; 19-21 nucleotide pairs in length; or 21-23 nucleotide pairs in length.

### Claim 16 (depends on 13)

The double stranded RNAi agent of claim 13, wherein the modifications on the nucleotides are selected from the group consisting of LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy, 2′-hydroxyl, and combinations thereof.

### Claim 17 (depends on 13)

The double stranded RNAi agent of claim 13, wherein the ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker; or a cholesterol.

### Claim 18 (depends on 13)

The double stranded RNAi agent of claim 13, wherein said agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

### Claim 19 (depends on 1, 13)

A cell containing the double stranded RNAi agent of claim 1 or 13.

### Claim 20 (depends on 1)

A pharmaceutical composition for inhibiting expression of a SCAP gene comprising the double stranded RNAi agent of claim 1.

### Claim 21 (depends on 1, 20)

A method of inhibiting for inhibiting expression of a sterol regulatory element binding protein (SREBP) chaperone (SCAP) gene in a cell, the method comprising:
(a) contacting the cell with the double stranded RNAi agent of claim 1 or the pharmaceutical composition of claim 20; and
(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a SCAP gene, thereby inhibiting expression of the SCAP gene in the cell.

### Claim 22 (depends on 21)

The method of claim 21, wherein said cell is within a subject.

### Claim 23 (depends on 22)

The method of claim 22, wherein the subject is a human.

### Claim 24 (depends on 23)

The method of claim 23, wherein the human subject suffers from a SCAP-associated disorder.

### Claim 25 (depends on 24)

The method of claim 24, wherein the SCAP-associated disease is nonalcoholic fatty liver disease (NAFLD; fatty liver (steatosis); or nonalcoholic steatohepatitis (NASH).

### Claim 26 (depends on 21)

The method of claim 21, wherein the SCAP expression is inhibited by at least about 30%.

### Claim 27 (depends on 1, 20)

A method of treating a subject having a disorder that would benefit from a reduction in SCAP expression, comprising administering to the subject a therapeutically effective amount of the double stranded RNAi agent of claim 1 or the pharmaceutical composition of claim 20, thereby treating said subject.

### Claim 28 (depends on 27)

The method of claim 27, wherein the subject suffers from a SCAP-associated disorder.

### Claim 29 (depends on 27)

The method of claim 27, wherein the subject is a human.

### Claim 30 (depends on 28)

The method of claim 28, wherein the SCAP-associated disease is nonalcoholic fatty liver disease (NAFLD); fatty liver (steatosis); nonalcoholic steatohepatitis (NASH).

### Claim 31 (depends on 27)

The method of claim 27, wherein the SCAP expression is inhibited by at least about 30%.

### Claim 32 (depends on 27)

The method of claim 27, further comprising administering an additional therapeutic agent to the subject.

### Claim 33 (depends on 27)

The method of claim 27, wherein the double stranded RNAi agent is administered at a dose of about 0.01 mg/kg to about 50 mg/kg.

### Claim 34 (depends on 27)

The method of claim 27, wherein the double stranded RNAi agent is administered to the subject subcutaneously.

### Claim 35 (depends on 27)

The method of claim 27, wherein the administration of the double stranded RNAi to the subject causes a decrease in one or more serum lipid and a decrease in SCAP protein accumulation; PNPLA3 protein accumulation; or SREBP double stranded RNAi accumulation.

### Claim 36 (depends on 1, 13)

A kit for inhibiting expression of a SCAP gene , comprising
a) the double stranded RNAi agent of claim 1 or 13, and
b) instructions for use, and
c) optionally, means for administering the double stranded RNAi agent to the subject.

### Claim 37 (depends on 1)

The double stranded RNAi agent of claim 1, wherein the antisense strand comprises the nucleotide sequence 5′-UAUGUAGGCAAACAAGAUGAUGU-3′ (SEQ ID NO:194).

### Claim 38 (depends on 1)

The double stranded RNAi agent of claim 1, wherein the antisense strand comprises the nucleotide sequence 5′-UAGAUGUAGGCAAACAAGAUGAU-3′ (SEQ ID NO:196).

### Claim 39 (depends on 1)

The double stranded RNAi agent of claim 1, wherein the antisense strand comprises the nucleotide sequence 5′-AGUAGAUGUAGGCAAACAAGAUG-3′ (SEQ ID NO:198).

### Claim 40 (depends on 37)

The double stranded RNAi agent of claim 37, wherein the sense strand comprises the nucleotide sequence 5′-AUCAUCUUGUUUGCCUACAUA-3′ (SEQ ID NO:193) and the antisense strand comprises the nucleotide sequence 5′-UAUGUAGGCAAACAAGAUGAUGU-3′ (SEQ ID NO:194).

### Claim 41 (depends on 38)

The double stranded RNAi agent of claim 38, wherein the sense strand comprises the nucleotide sequence 5′-CAUCUUGUUUGCCUACAUCUAA-3′ (SEQ ID NO:195) and the antisense strand comprises the nucleotide sequence 5′-UAGAUGUAGGCAAACAAGAUGAU-3′ (SEQ ID NO:196).

### Claim 42 (depends on 39)

The double stranded RNAi agent of claim 39, wherein the sense strand comprises the nucleotide sequence 5′-UCUUGUUUGCCUACAUCUACU-3′ (SEQ ID NO:197) and the antisense strand comprises the nucleotide sequence 5′-AGUAGAUGUAGGCAAACAAGAUG-3′ (SEQ ID NO:198).

### Claim 43 (depends on 8)

The double stranded RNAi agent of claim 8, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative conjugated to the 3′ end of the sense strand through a monovalent or branched bivalent or trivalent linker.

### Claim 44 (depends on 13)

A pharmaceutical composition for inhibiting expression of a SCAP gene comprising the double stranded RNAi agent of claim 13.

### Claim 45 (depends on 13, 44)

A method of inhibiting for inhibiting expression of a sterol regulatory element binding protein (SREBP) chaperone (SCAP) gene in a cell, the method comprising:
(a) contacting the cell with the double stranded RNAi agent of claim 13 or the pharmaceutical composition of claim 44; and
(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a SCAP gene, thereby inhibiting expression of the SCAP gene in the cell.

### Claim 46 (depends on 45)

The method of claim 45, wherein said cell is within a subject.

### Claim 47 (depends on 46)

The method of claim 46, wherein the subject is a human.

### Claim 48 (depends on 47)

The method of claim 47, wherein the human subject suffers from a SCAP-associated disorder.

### Claim 49 (depends on 48)

The method of claim 48, wherein the SCAP-associated disease is nonalcoholic fatty liver disease (NAFLD; fatty liver (steatosis); or nonalcoholic steatohepatitis (NASH).

### Claim 50 (depends on 45)

The method of claim 45, wherein the SCAP expression is inhibited by at least about 30%.

### Claim 51 (depends on 13, 44)

A method of treating a subject having a disorder that would benefit from a reduction in SCAP expression, comprising administering to the subject a therapeutically effective amount of the double stranded RNAi agent of claim 13 or the pharmaceutical composition of claim 44, thereby treating said subject.

### Claim 52 (depends on 51)

The method of claim 51, wherein the subject suffers from a SCAP-associated disorder.

### Claim 53 (depends on 51)

The method of claim 51, wherein the subject is a human.

### Claim 54 (depends on 52)

The method of claim 52, wherein the SCAP-associated disease is nonalcoholic fatty liver disease (NAFLD); fatty liver (steatosis); nonalcoholic steatohepatitis (NASH).

### Claim 55 (depends on 51)

The method of claim 51, wherein the SCAP expression is inhibited by at least about 30%.

### Claim 56 (depends on 51)

The method of claim 51, further comprising administering an additional therapeutic agent to the subject.

### Claim 57 (depends on 51)

The method of claim 51, wherein the double stranded RNAi agent is administered at a dose of about 0.01 mg/kg to about 50 mg/kg.

### Claim 58 (depends on 51)

The method of claim 51, wherein the double stranded RNAi agent is administered to the subject subcutaneously.

### Claim 59 (depends on 51)

The method of claim 51, wherein the administration of the double stranded RNAi to the subject causes a decrease in one or more serum lipid and a decrease in SCAP protein accumulation; PNPLA3 protein accumulation; or SREBP double stranded RNAi accumulation.