# US 2025/0154504 A1 · Guide oligonucleotides for nucleic acid editing in the treatment of hypercholesterolemia

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- Number: US 2025/0154504 A1
- Document ID: US20250154504A1
- Status: Published application
- Kind: A1
- Application number: 18/838,201
- Filed: 2023-02-13
- Published: 2025-05-15
- Earliest claimed: 2022-02-14
- Assignees: ProQR Therapeutics II B.V.
- Applicants: ProQR Therapeutics II B.V.
- Inventors: Gerardus Johannes PLATENBURG; Bart KLEIN; Lenka VAN SINT FIET; Maarten HOLKERS
- Web page: https://patentgrep.com/patent/US20250154504A1

## CPC

- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- 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 31/7105: Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
- A61P 7/00: Drugs for disorders of the blood or the extracellular fluid
- C12N 15/86: Viral vectors
- C12N 2310/11: Antisense
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2310/315: Phosphorothioates
- C12N 2310/351: Conjugate
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- 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 guide oligonucleotides that can bring about specific changes to a target RNA or DNA molecule in a eukaryotic cell, preferably liver cells, for use in the treatment of hypercholesterolemia, cardiovascular disease, liver injury and/or alcohol-induced steatohepatitis in human subjects. More specifically, the invention relates to guide oligonucleotides and the use thereof in the editing of nucleic acids encoding an auto-cleavage site within the PCSK9 proprotein, thereby inhibiting, or inactivating the PCSK9 protein (or the matured PCSK9 complex) in its ability to cause LDL receptor protein degradation.

## Claims

### Claim 1

A guide oligonucleotide capable of inducing editing of a nucleic acid encoding a human Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) proprotein, wherein the nucleic acid editing decreases or prevents the ability of the PCSK9 proprotein from being processed by auto-cleavage of a proteolytic cleavage site.

### Claim 2 (depends on 1)

The guide oligonucleotide according to claim 1, wherein the proteolytic cleavage site is the P1 cleavage site.

### Claim 3 (depends on 1)

The guide oligonucleotide according to claim 1, wherein the nucleic acid is RNA or DNA.

### Claim 4 (depends on 3)

The guide oligonucleotide according to claim 3, wherein the nucleic acid is RNA and wherein the guide oligonucleotide can form a double stranded complex with a human PCSK9 pre-mRNA, mRNA, or a part thereof, wherein the guide oligonucleotide when complexed with the PCSK9 pre-mRNA, mRNA, or part thereof, is able to engage an endogenous human Adenosine Deaminases acting on RNA (ADAR) enzyme, thereby allowing the deamination of a target adenosine in the human PCSK9 pre-mRNA, mRNA, or part thereof.

### Claim 5 (depends on 4)

The guide oligonucleotide according to claim 4, wherein the target adenosine is the second nucleotide of the codon coding for the glutamine residue at position 152 in the PCSK9 proprotein.

### Claim 6 (depends on 4)

The guide oligonucleotide according to claim 4, wherein the guide oligonucleotide comprises an orphan nucleotide that is a cytidine, a cytidine analog, a uridine, or a uridine analog.

### Claim 7 (depends on 6)

The guide oligonucleotide according to claim 6, wherein the orphan nucleotide is a cytidine analog comprising a m 6-amino-5-nitro-3-(1′-P-D-2′-deoxyribofuranosyl)-2(1H)-pyridone base.

### Claim 8 (depends on 6)

The guide oligonucleotide according to claim 6, wherein the orphan nucleotide is a uridine analog and wherein the uridine analog is iso-uridine.

### Claim 9 (depends on 4)

The guide oligonucleotide according to claim 4, wherein the guide oligonucleotide consists of 20 to 50 nucleotides, and wherein the guide oligonucleotide is substantially complementary to a region within the human PCSK9 pre-mRNA, mRNA, or part thereof that comprises the codon coding for the glutamine residue at position 152 in the PCSK9 proprotein.

### Claim 10 (depends on 4)

The guide oligonucleotide according to claim 4, wherein the guide oligonucleotide is bound to a N-acetylgalactosamine (GalNAc) moiety.

### Claim 11 (depends on 4)

The guide oligonucleotide according to claim 4, comprising one or more nucleotides that comprise a modification of a nucleobase, a sugar and/or an inter-nucleosidic linkage.

### Claim 12 (depends on 11)

The guide oligonucleotide according to claim 11, wherein the guide oligonucleotide comprises:
(i) at least one non-naturally occurring inter-nucleosidic linkage modification selected from the group consisting of: phosphorothioate (PS), chirally pure PS, Rp PS, Sp PS, phosphorodithioate, phosphonoacetate, thiophosphonoacetate phosphonacetamide, thiophosphonacetamide, PS prodrug, S-alkylated PS, H-phosphonate, methyl phosphonate (MP), methyl phosphonothioate, methyl phosphate, methyl phosphorothioate, ethyl phosphate, ethyl PS, boranophosphate, boranophosphorothioate, methyl boranophosphate, methyl boranophosphorothioate, methyl boranophosphonate, methyl boranophosphonothioate, phosphorylguanidine, methyl sulfonylphosphoroamidate, phosphoramidite, phosphonamidite, N3′→P5′ phosphoramidate, N3′→P5′ thiophosphoramidate, phosphorodiamidate, phosphorothiodiamidate, sulfamate, dimethylenesulfoxide, sulfonate, triazole, oxalyl, carbamate, methyleneimino, thioacetamido, and their derivatives; and/or
ii at least one nucleotide that comprises a mono- or disubstitution at the 2′, 3′ and/or 5′ position of the sugar, selected from the group consisting of: —OH; —F; substituted or unsubstituted, linear or branched lower (C1-C10) alkyl, alkenyl, alkynyl, alkaryl, allyl, or aralkyl, that may be interrupted by one or more heteroatoms; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; O-, S-, or N-allyl; O-alkyl-O-alkyl; -methoxy; -aminopropoxy; -methoxyethoxy; -dimethylamino oxyethoxy; and -dimethylaminoethoxyethoxy, with the proviso that the orphan base is not modified with 2′-O-methyl or 2′-methoxyethoxy.

### Claim 13 (depends on 1)

A viral vector expressing a guide oligonucleotide of claim 1, wherein
(i) the guide oligonucleotide comprises an orphan nucleotide that is a cytidine or uridine,
(ii) the viral vector can treat, delay, or prevent cardiovascular disease, hypercholesterolemia, liver injury, and/or alcohol-induced steatohepatitis,
(iii) the oligonucleotide is capable of editing a nucleic acid encoding a human PCSK9 proprotein, and
(iv) the nucleic acid editing decreases or prevents the ability of the PCSK9 proprotein from being processed by auto-cleavage of a proteolytic cleavage site.

### Claim 14 (depends on 1)

A pharmaceutical composition comprising the guide oligonucleotide of claim 1 or a viral vector comprising said guide oligonucleotide, and a pharmaceutically acceptable carrier.

### Claim 15 (depends on 1)

A method for treating a human subject suffering from cardiovascular disease, hypercholesterolemia, liver injury, and/or alcohol-induced steatohepatitis, by decreasing or inhibiting an auto-cleaving ability of the PCSK9 proprotein in a liver cell of the subject, the method comprising administering to the subject the guide oligonucleotide of claim 1, or a viral vector or a pharmaceutical composition comprising said guide oligonucleotide, thereby editing a nucleic acid encoding the PCSK9 proprotein and thereby editing an auto-cleavage site of the PCSK9 proprotein.

### Claim 16 (depends on 15)

The method of claim 15, wherein the auto-cleavage site is the P1 cleavage site of PCSK9 and the nucleic acid is RNA or DNA.

### Claim 17 (depends on 16)

The method of claim 16, wherein
(i) the nucleic acid is RNA and the guide oligonucleotide can form a double stranded complex with a human PCSK9 pre-mRNA, mRNA, or a part thereof, and
(ii) the guide oligonucleotide is able to engage an endogenous human ADAR enzyme when complexed with the PCSK9 pre-mRNA, mRNA, or part thereof, thereby allowing the deamination of a target adenosine in the pre-mRNA, mRNA, or part thereof, at the P1 cleavage site.

### Claim 18 (depends on 17)

The method of claim 17, wherein the adenosine in the codon encoding glutamine at position 152 of the PCSK9 proprotein is deaminated to an inosine.

### Claim 19 (depends on 6)

The guide oligonucleotide according to claim 6, wherein the uridine analog is iso-uridine.

### Claim 20 (depends on 4)

The guide oligonucleotide according to claim 4, wherein the guide oligonucleotide consists of 23 to 27 nucleotides, and wherein the guide oligonucleotide is substantially complementary to a region within the human PCSK9 pre-mRNA, mRNA, or part thereof that comprises the codon coding for the glutamine residue at position 152 in the PCSK9 proprotein.