# US 2023/0272384 A1 · Methods and compositions for the adar-mediated editing of retinochisin 1 (rs1)

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- Number: US 2023/0272384 A1
- Document ID: US20230272384A1
- Status: Published application
- Kind: A1
- Application number: 17/985,978
- Filed: 2022-11-14
- Published: 2023-08-31
- Earliest claimed: 2020-05-15
- Assignees: Korro Bio, Inc.
- Applicants: Korro Bio, Inc.
- Inventors: Matthew Jarpe; Mallikarjuna Reddy Putta; Andrew Fraley
- Web page: https://patentgrep.com/patent/US20230272384A1

## 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 38/50: acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
- C12Y 305/04004: Adenosine deaminase (3.5.4.4)
- C12N 2310/315: Phosphorothioates
- C12N 2310/318: where the PO2 is completely replaced, e.g. MMI or formacetal
- C12N 2310/319: linked by 2'-5' linkages, i.e. having a free 3'-position
- C12N 2310/321: 2'-O-R Modification
- C12N 2310/3231: having an additional ring, e.g. LNA, ENA
- C12N 2310/3233: Morpholino-type ring
- C12N 2310/3235: having the O of the ribose replaced by another atom
- C12N 2310/332: Abasic residue
- C12N 2320/34: Allele or polymorphism specific uses
- 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)
- C12Y: ENZYMES

## Abstract

The present invention relates to methods and compositions for editing an RS1 polynucleotide, e.g., an RS1 polynucleotide comprising a SNP associated with X-linked retinoschisis (XLRS). The invention also relates to methods and compositions for treating or preventing XLRS in a subject.

## Claims

### Claim 1

A method of editing an RS1 polynucleotide comprising a single nucleotide polymorphism (SNP) associated with X-linked retinoschisis (XLRS), the method comprising contacting the RS1 polynucleotide with a guide oligonucleotide capable of effecting an adenosine deaminase acting on RNA (ADAR)-mediated adenosine to inosine alteration of the SNP associated with XLRS, thereby editing the RS1 polynucleotide.

### Claim 2

(canceled)

### Claim 3 (depends on 1)

The method of claim 1, wherein the cell endogenously expresses ADAR.

### Claim at position 4

4-9. (canceled)

### Claim 10

A method of treating XLRS in a subject in need thereof, the method comprising
contacting the RS1 polynucleotide in a cell of the subject with a guide oligonucleotide capable of effecting an adenosine deaminase acting on RNA (ADAR)-mediated adenosine to inosine alteration of the SNP associated with XLRS, thereby treating the subject.

### Claim 11

A method of treating XLRS in a subject in need thereof, the method comprising
contacting the RS1 polynucleotide in a cell with a guide oligonucleotide capable of effecting an adenosine deaminase acting on RNA (ADAR)-mediated adenosine to inosine alteration of the SNP associated with XLRS, and
administering the cell to the subject, thereby treating the subject.

### Claim at position 7

12-13. (canceled)

### Claim 14 (depends on 1)

The method of claim 1, wherein the guide oligonucleotide comprises a nucleic acid sequence complementary to an RS1 mRNA sequence comprising the SNP associated with XLRS.

### Claim 15 (depends on 1)

The method of claim 1, wherein the oligonucleotide further comprises one or more adenosine deaminase acting on RNA (ADAR)-recruiting domains.

### Claim 16 (depends on 1)

The method of claim 1, wherein the RS1 polynucleotide encodes an RS1 protein comprising a pathogenic amino acid comprising a lysine at position 72, and/or a glutamine at position 102 resulting from the SNP.

### Claim 17 (depends on 16)

The method of claim 16, wherein the adenosine to inosine alteration substitutes the pathogenic amino acid with a wild type amino acid, wherein the wild type amino acid at position 72 is a glutamic acid, and/or the wild type amino acid at position 102 is an arginine.

### Claim 18

(canceled)

### Claim 19 (depends on 1)

The method of claim 1, wherein the guide oligonucleotide comprises the structure:
[Am]-X1-X2-X3-[Bn]
wherein each of A and B is a nucleotide;
m and n are each, independently, an integer from 5 to 40;
X1, X2, and X3 are each, independently, a nucleotide, wherein at least one of X1, X2, or X3 is an alternative nucleotide.

### Claim 20 (depends on 1)

The method of claim 1, wherein the guide oligonucleotide comprises the structure:
[Am]-X1-X2-X3-[Bn]
wherein each of A and B is a nucleotide;
m and n are each, independently, an integer from 5 to 40;
X1, X2, and X3 are each, independently, a nucleotide, wherein at least one of X1, X2, or X3 has the structure of any one of Formula I-IV:
wherein N1 is hydrogen or a nucleobase;
R1 is hydroxy, halogen, or C1-C6 alkoxy;
R2 is hydrogen, hydroxy, halogen, or C1-C6 alkoxy; and
R3 is hydrogen, hydroxy, halogen, or C1-C6 alkoxy.

### Claim at position 15

21-27. (canceled)

### Claim 28 (depends on 20)

The method of claim 20, wherein at least one of X1, X2, and X3 has the structure of Formula I, wherein R1 is fluoro, hydroxy, or O-methyl, and N1 is a nucleobase.

### Claim at position 17

29-102. (canceled)

### Claim 103 (depends on 1)

The method of claim 1, wherein the guide oligonucleotide comprises the structure:
[Am]-X1-X2-X3-[Bn]
wherein each of A and B is a nucleotide;
m and n are each, independently, an integer from 5 to 40;
X1, X2, and X3 are each, independently, a nucleotide, wherein at least one of X1, X2, and X3 has the structure of any one of Formula XII-XV:
wherein N1 is hydrogen or a nucleobase;
R6 is hydrogen, hydroxy, or halogen;
R7 is hydrogen, hydroxy, halogen, or C1-C6 alkoxy;
R8 is hydrogen or halogen;
R9 is hydrogen or hydroxy, halogen, or C1-C6 alkoxy;
R10 is hydrogen or halogen; and
R11 is hydrogen or hydroxy, halogen, or C1-C6 alkoxy.

### Claim at position 19

104-106. (canceled)

### Claim 107 (depends on 103)

The method of claim 103, wherein at least one of X1, X2, and X3 has the structure of Formula XIII, in which each of R8 and R9 is hydrogen.

### Claim at position 21

108-128. (canceled)

### Claim 129 (depends on 19)

The method of claim 19, wherein each of [Am] and [Bn] comprises at least four terminal phosphorothioate linkages.

### Claim at position 23

130-137. (canceled)

### Claim 138 (depends on 19)

The method of claim 19, wherein at least 20% of the nucleotides of [Am] and [Bn] combined are 2′-O-methyl-nucleotides.

### Claim at position 25

139-144. (canceled)

### Claim 145 (depends on 20)

The method of claim 20, wherein at least one of X1, X2, and X3 has the structure of Formula I, wherein R1 is fluoro, hydroxy, or methoxy and N1 is a nucleobase, each of X1, X2, and X3 that does not have the structure of Formula I is a ribonucleotide; [Am] and [Bn] each comprise at least five terminal 2′-O-methyl-nucleotides and at least four terminal phosphorothioate linkages; and at least 20% of the nucleotides of [Am] and [Bn] combined are 2′-O-methyl-nucleotides.

### Claim 146

(canceled)

### Claim 147 (depends on 103)

The method of claim 103, wherein at least of X1, X2, and X3 has the structure of Formula XIII, wherein R8 and R9 are each hydrogen, and each of X1, X2 and X3 that does not have the structure of Formula XII is a ribonucleotide; [Am] and [Bn] each include at least five terminal 2′-O-methyl-nucleotides and at least four terminal phosphorothioate linkages; and at least 20% of the nucleotides of [Am] and [Bn] combined are 2′-O-methyl-nucleotides.