# US 2023/0407315 A1 · Compositions and methods for treating non-age-associated hearing impairment in a human subject

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- Number: US 2023/0407315 A1
- Document ID: US20230407315A1
- Status: Published application
- Kind: A1
- Application number: 18/458,744
- Filed: 2023-08-30
- Published: 2023-12-21
- Earliest claimed: 2016-08-23
- Assignees: Akouos, Inc.
- Applicants: Akouos, Inc.
- Inventors: Emmanuel John Simons; Ellen Reisinger; Sebastian Kügler; Hanan Al-Moyed
- Web page: https://patentgrep.com/patent/US20230407315A1

## CPC

- C12N 15/52: Genes encoding for enzymes or proenzymes
- A01K 67/0276: Knock-out vertebrates
- A61K 38/1709: {from mammals}
- A61K 48/0075: {characterised by an aspect of the delivery route, e.g. oral, subcutaneous}
- A61P 27/16: Otologicals
- C07K 14/47: from mammals
- C12N 5/062: {Sensory transducers, e.g. photoreceptors; Sensory neurons, e.g. for hearing, taste, smell, pH, touch, temperature, pain}
- C12N 9/16: acting on ester bonds (3.1)
- C12N 15/65: using markers (enzymes used as markers C12N15/52)
- C12N 15/86: Viral vectors
- C12N 15/902: {using homologous recombination}
- A01K 2217/075: inducing loss of function, i.e. knock out
- A01K 2227/103: Ovine
- A01K 2267/03: Animal model, e.g. for test or diseases
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N 2830/008: cell type or tissue specific enhancer/promoter combination
- C12Y 301/03001: Alkaline phosphatase (3.1.3.1)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- 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
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Y: ENZYMES

## Abstract

Provided herein are compositions that include at least two different nucleic acid vectors, where each of the at least two different vectors includes a coding sequence that encodes a different portion of an otoferlin protein, and the use of these compositions to treat hearing loss in a subject.

## Claims

### Claim 1

A composition comprising two different nucleic acid vectors, wherein:
each of the two different vectors comprises a coding sequence that encodes a different portion of an otoferlin protein, each of the encoded portions being at least 30 amino acid residues in length;
no single vector of the at least two different vectors encodes a full-length otoferlin protein;
a first of the two different vectors comprises a promoter operably linked to a coding sequence that encodes an N-terminal portion of the otoferlin protein;
at least one of the coding sequences comprises a nucleotide sequence spanning two neighboring exons of otoferlin genomic DNA, and lacks an intronic sequence between the two neighboring exons; and
when introduced into a mammalian cell the at least two different vectors undergo concatemerization or homologous recombination with each other, thereby forming a recombined nucleic acid that encodes a full-length otoferlin protein.

### Claim at position 2

2-3. (canceled)

### Claim 4 (depends on 1)

The composition of claim 1, wherein each of the two different vectors is a viral vector selected from an adeno-associated virus (AAV) vector, an adenovirus vector, a lentivirus vector, or a retrovirus vector.

### Claim 5 (depends on 1)

The composition of claim 1, wherein each of the two different vectors is an AAV vector.

### Claim at position 5

6-14. (canceled)

### Claim 15 (depends on 1)

The composition of claim 1, wherein the N-terminal portion of the otoferlin protein is between 30 amino acids to 1600 amino acids in length.

### Claim 16

(canceled)

### Claim 17 (depends on 1)

The composition of claim 1, wherein the first vector further comprises a Kozak sequence.

### Claim 18 (depends on 1)

The composition of claim 1, wherein the promoter is an inducible promoter, a constitutive promoter, or a tissue-specific promoter.

### Claim 19 (depends on 1)

The composition of claim 1, wherein a second of the two different vectors comprises a coding sequence that encodes a C-terminal portion of the otoferlin protein.

### Claim 20 (depends on 19)

The composition of claim 19, wherein the C-terminal portion of the otoferlin protein is between 30 amino acids to 1600 amino acids in length.

### Claim 21

(canceled)

### Claim 22 (depends on 19)

The composition of claim 19, wherein the second vector further comprises a poly(dA) sequence.

### Claim 23 (depends on 1)

The composition of claim 1, further comprising a pharmaceutically acceptable excipient.

### Claim 24 (depends on 1)

A kit comprising a composition of claim 1.

### Claim 25 (depends on 24)

A kit of claim 24, further comprising a pre-loaded syringe comprising the composition.

### Claim at position 17

26-38. (canceled)

### Claim 39

A composition comprising two different nucleic acid vectors, wherein:
a first nucleic acid vector of the two different nucleic acid vectors comprises a promoter, a first coding sequence that encodes an N-terminal portion of an otoferlin protein positioned 3′ of the promoter, and a splicing donor signal sequence positioned at the 3′ end of the first coding sequence; and
a second nucleic acid vector of the two different nucleic acid vectors comprises a splicing acceptor signal sequence, a second coding sequence that encodes a C-terminal portion of an otoferlin protein positioned at the 3′ end of the splicing acceptor signal sequence, and a polyadenylation sequence at the 3′ end of the second coding sequence;
wherein each of the encoded portions is at least 30 amino acid residues in length,
wherein the amino acid sequences of the encoded portions do not overlap,
wherein no single vector of the two different vectors encodes a full-length otoferlin protein, and,
when the coding sequences are transcribed in a mammalian cell, to produce RNA transcripts, splicing occurs between the splicing donor signal sequence on one transcript and the splicing acceptor signal sequence on the other transcript, thereby forming a recombined RNA molecule that encodes a full-length otoferlin protein.

### Claim at position 19

40-46. (canceled)

### Claim 47 (depends on 39)

A kit comprising a composition of claim 39.

### Claim 48 (depends on 47)

A kit of claim 47, wherein the composition is pre-loaded in a syringe.

### Claim at position 22

49-63. (canceled)

### Claim 64 (depends on 1)

The composition of claim 1, wherein the promoter is a constitutive promoter.

### Claim 65 (depends on 1)

The composition of claim 1, wherein the promoter is a tissue-specific promoter.

### Claim 66 (depends on 1)

The composition of claim 1, wherein the otoferlin protein is an otoferlin isoform 5 protein.

### Claim 67 (depends on 1)

The composition of claim 1, wherein each of the two different vectors are encapsulated by AAV capsids and wherein the AAV capsids are AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, rh8, rh10, rh39, rh43, or Anc80.

### Claim 68 (depends on 39)

The composition of claim 39, wherein each of the two different vectors is a viral vector selected from an adeno-associated virus (AAV) vector, an adenovirus vector, a lentivirus vector, or a retrovirus vector.

### Claim 69 (depends on 39)

The composition of claim 39, wherein each of the two different vectors is an AAV vector.

### Claim 70 (depends on 39)

The composition of claim 39, wherein each of the two different vectors are encapsulated by AAV capsids and wherein the AAV capsids are AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, rh8, rh10, rh39, rh43, or Anc80.

### Claim 71 (depends on 39)

The composition of claim 39, wherein the N-terminal portion of the otoferlin protein is between 30 amino acids to 1600 amino acids in length.

### Claim 72 (depends on 39)

The composition of claim 39, wherein the first vector further comprises a Kozak sequence.

### Claim 73 (depends on 39)

The composition of claim 39, wherein the promoter is an inducible promoter, a constitutive promoter, or a tissue-specific promoter.

### Claim 74 (depends on 39)

The composition of claim 39, wherein the promoter is a constitutive promoter.

### Claim 75 (depends on 39)

The composition of claim 39, wherein the promoter is a tissue-specific promoter.

### Claim 76 (depends on 39)

The composition of claim 39, wherein the C-terminal portion of the otoferlin protein is between 30 amino acids to 1600 amino acids in length.

### Claim 77 (depends on 39)

The composition of claim 39, wherein the otoferlin protein is an otoferlin isoform 5 protein.

### Claim 78 (depends on 39)

The composition of claim 39, further comprising a pharmaceutically acceptable excipient.