# US 10,370,668 B2 · Manufacture of antisense oligomers and conjugates targeting PCSK9

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- Number: US 10,370,668 B2
- Document ID: US10370668B2
- Status: Granted
- Kind: B2
- Application number: 15/971,951
- Filed: 2018-05-04
- Published: 2018-11-01
- Granted: 2019-08-06
- Earliest claimed: 2013-06-27
- Assignees: Roche Innovation Center Copenhagen A/S
- Applicants: Roche Innovation Center Copenhagen A/S
- Inventors: Nanna Albæk; Maj Hedtjärn; Marie Wickstrom Lindholm; Niels Fisker Nielsen; Andreas Petri; Jacob Ravn
- Web page: https://patentgrep.com/patent/US10370668B2

## CPC

- C12N 15/1137: {against enzymes (viral enzymes C12N15/1131; receptors C12N15/1138)}
- A61K 47/545: {Heterocyclic compounds (A61K47/558 takes precedence)}
- 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 3/06: Antihyperlipidemics
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- 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)}
- A61K 31/712: Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
- C12N 2310/11: Antisense
- C12N 2310/3231: having an additional ring, e.g. LNA, ENA
- C12N 2310/341: Gapmers, i.e. of the type ===---===
- C12N 2310/351: Conjugate
- C12N 2310/3515: Lipophilic moiety, e.g. cholesterol
- C12N 2320/32: Special delivery means, e.g. tissue-specific
- C12N 2330/30: chemically synthesised
- 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 present invention relates to oligomeric compounds and conjugates thereof that target Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) PCSK9 mRNA in a cell, leading to reduced expression of PCSK9. Reduction of PCSK9 expression is beneficial for a range of medical disorders, such as hypercholesterolemia and related disorders.

## Claims

### Claim 1

A method of manufacturing a compound consisting of an antisense oligomer that is 16 contiguous nucleotides in length, the method comprising chemically synthesizing the compound using sequential solid phase oligonucleotide synthesis, wherein the sequence of the antisense oligomer is 100% complementary to the sequence of SEQ ID NO: 31, wherein the antisense oligomer is a gapmer comprising at least one LNA unit, and wherein the antisense oligomer targets mRNA encoding PCSK9.

### Claim 2 (depends on 1)

The method of claim 1, wherein the antisense oligomer is an intermediate.

### Claim 3 (depends on 1)

The method of claim 1, wherein the sequence of the antisense oligomer is SEQ ID NO: 26.

### Claim 4 (depends on 1)

The method of claim 1, wherein the LNA is oxy-LNA, thio-LNA, amino-5 LNA, 5′-methyl-LNA, ENA, cET, cMOE or a combination thereof.

### Claim 5 (depends on 1)

The method of claim 1, wherein the LNA is a stereoisomer in the beta-D-configuration or the alpha-L configuration.

### Claim 6 (depends on 1)

The method of claim 1, wherein the antisense oligomer comprises at least one cET unit.

### Claim 7 (depends on 1)

The method of claim 1, wherein the antisense oligomer comprises six or seven LNA units.

### Claim 8 (depends on 7)

The method of claim 7, wherein every LNA unit in the antisense oligomer is a beta-D-oxy LNA unit or every LNA unit in the antisense oligomer is an alpha-L-oxy-LNA unit.

### Claim 9 (depends on 7)

The method of claim 7, wherein every LNA unit in the antisense oligomer is a stereoisomer in the same configuration.

### Claim 10 (depends on 1)

The method of claim 1, wherein the sequence of the antisense oligomer comprises at least one phosphorothioate, phosphorodithioate, or boranophosphate internucleoside linkage.

### Claim 11 (depends on 10)

The method of claim 10, wherein all the internucleoside linkages are phosphorothioate.

### Claim 12 (depends on 10)

The method of claim 10, wherein one or more of the inernucleoside linkages comprises a chiral center in the R conformation and/or in the S conformation.

### Claim 13 (depends on 1)

The method of claim 1, wherein the sequence of the antisense oligomer is SEQ ID NO: 2.

### Claim 14 (depends on 1)

The method of claim 1, wherein the sequence of the antisense oligomer is SEQ ID NO: 3.

### Claim 15 (depends on 1)

The method of claim 1, wherein the compound and an oligomer of SEQ ID NO:31 can form a duplex with increased thermal stability with respect to a corresponding duplex comprising the corresponding antisense oligomer without LNA.

### Claim 16

A method of manufacturing an antisense oligonucleotide conjugate comprising covalently attaching at least one non-nucleotide or non-polynucleotide moiety covalently to an antisense oligomer that is 16 contiguous nucleotides in length directly or via a linker positioned between the antisense oligomer sequence and the non-nucleotide or non-polynucleotide moiety, wherein the sequence of the antisense oligomer is 100% complementary to the sequence of SEQ ID NO: 31, wherein the antisense oligomer is a gapmer comprising at least one LNA unit, and wherein the antisense oligonucleotide conjugate targets mRNA encoding PCSK9.

### Claim 17 (depends on 16)

The method of claim 16, wherein the non-nucleotide or non-polynucleotide moiety is a liver targeting moiety that is attached to the 5′-end or to the 3′-end of the antisense oligomer.

### Claim 18 (depends on 17)

The method of claim 17, wherein the liver targeting moiety is linked to the antisense oligomer via a linker.

### Claim 19 (depends on 18)

The method of claim 18, wherein the linker is a biocleavable linker.

### Claim 20 (depends on 17)

The method of claim 17, wherein the liver targeting moiety comprises a carbohydrate conjugate moiety comprising a carbohydrate or a carbohydrate group.

### Claim 21 (depends on 20)

The method of claim 20, wherein the carbohydrate is selected from the group consisting of galactose, lactose, N-acetylgalactosamine (GalNAc), mannose, mannose-6-phosphate, and combinations thereof.

### Claim 22 (depends on 20)

The method of claim 20, wherein the carbohydrate conjugate moiety is not a linear carbohydrate polymer.

### Claim 23 (depends on 20)

The method of claim 20, wherein the carbohydrate conjugate moiety is a carbohydrate group comprising 1 2, 3, or 4 carbohydrate moieties.

### Claim 24 (depends on 23)

The method of claim 23, wherein the carbohydrate moieties are identical or non-identical.

### Claim 25 (depends on 23)

The method of claim 23, wherein the carbohydrate moieties are non-identical.

### Claim 26 (depends on 20)

The method of claim 20, wherein the carbohydrate conjugate moiety comprises at least one asialoglycoprotein receptor targeting conjugate moiety.

### Claim 27 (depends on 26)

The method of claim 26, wherein the asialoglycoprotein receptor targeting conjugate moiety comprises a GalNAc cluster.

### Claim 28 (depends on 26)

The method of claim 26, wherein the asialoglycoprotein receptor targeting conjugate moiety comprises a monovalent, divalent, trivalent, or tetravalent GalNAc cluster.

### Claim 29 (depends on 27)

The method of claim 27, wherein the GalNAc cluster is a trivalent GalNAc cluster.

### Claim 30 (depends on 29)

The method of claim 29, wherein each GalNAc in the trivalent GalNAc cluster is attached to a branch point group via a spacer.

### Claim 31 (depends on 30)

The method of claim 30, wherein the branch point group comprises di-lysine.

### Claim 32 (depends on 30)

The method of claim 30, wherein the spacer comprises a PEG spacer.

### Claim 33 (depends on 18)

The method of claim 18, wherein the linker comprises a C6 to C12 amino alkyl group or a biocleavable phosphate nucleotide linker comprising between 1 to 6 nucleotides.

### Claim 34 (depends on 29)

The method of claim 29, wherein the trivalent GalNAc cluster comprises Conj 2a:

### Claim 35 (depends on 16)

The method of claim 16, wherein the non-nucleotide or non-polynucleotide moiety is covalently attached to the antisense oligomer via a covalent bond.

### Claim 36 (depends on 16)

The method of claim 16, wherein the antisense oligonucleotide conjugate is SEQ ID NO: 18.

### Claim 37 (depends on 16)

The method of claim 16, wherein the antisense oligonucleotide conjugate is SEQ ID NO: 19.

### Claim 38 (depends on 16)

The method of claim 16, wherein covalently attaching the at least one non-nucleotide or non-polynucleotide moiety to the antisense oligomer comprises:
(i) chemically synthesizing the antisense oligomer; and,
(ii) adding by chemical synthesis or conjugation the at least one non-nucleotide or non-polynucleotide moiety to the antisense oligonucleotide conjugate.

### Claim 39 (depends on 38)

The method of claim 38, wherein adding by chemical synthesis or conjugation the at least one non-nucleotide or non-polynucleotide moiety to the antisense oligonucleotide conjugate comprises:
(i) incorporating by chemical synthesis or conjugation at least one non-nucleotide or non-polynucleotide moiety to the antisense oligonucleotide conjugate;
(ii) incorporating by chemical synthesis or conjugation at least one linker to the antisense oligonucleotide conjugate;
(iii) incorporating by chemical synthesis or conjugation at least one branching point to the antisense oligonucleotide conjugate;
(iv) incorporating by chemical synthesis or conjugation at least one spacer to the antisense oligonucleotide conjugate; or,
(v) a combination thereof.

### Claim 40 (depends on 39)

The method of claim 39, wherein at least one linker is interposed between the antisense oligomer and a branching point.

### Claim 41 (depends on 39)

The method of claim 39, wherein at least one branching point is interposed between a linker and at least one non-nucleotide or non-polynucleotide moiety.

### Claim 42 (depends on 39)

The method of claim 39, wherein at least one, two, or three non-nucleotide or non-polynucleotide moieties are attached to a branching point.

### Claim 43 (depends on 39)

The method of claim 39, wherein at least one PEG spacer is interposed between each of the one, two, or three non-nucleotide or non-polynucleotide moieties and a branching point.

### Claim 44

A method of manufacturing an antisense oligonucleotide conjugate of SEQ ID NO: 18
wherein
a superscript L identifies a beta-D-oxy LNA unit,
a MeC identifies a 5-methylcytosine unit,
a subscript s identifies a phosphorothioate internucleoside linkage,
and wherein
is a Conj 2a asialoglycoprotein receptor targeting conjugate moiety
and wherein the antisense oligonucleotide conjugate targets mRNA encoding PCSK9, the method comprising covalently attaching the Conj 2a asialoglycoprotein receptor targeting conjugate moiety to the 5′ end of an antisense oligomer of SEQ ID NO: 2.

### Claim 45

A method of manufacturing an antisense oligonucleotide conjugate of SEQ ID NO: 19
wherein
a superscript L identifies a beta-D-oxy LNA unit,
a MeC identifies a 5-methylcytosine unit,
a subscript s identifies a phosphorothioate internucleoside linkage,
and wherein
is a Conj 2a asialoglycoprotein receptor targeting conjugate moiety
and wherein the antisense oligonucleotide conjugate targets mRNA encoding PCSK9, the method comprising covalently attaching the Conj 2a asialoglycoprotein receptor targeting conjugate moiety to the 5′ end of an antisense oligomer of SEQ ID NO: 3.