# US 2025/0084136 A1 · Molecules for organelle-specific protein delivery

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- Number: US 2025/0084136 A1
- Document ID: US20250084136A1
- Status: Published application
- Kind: A1
- Application number: 18/886,511
- Filed: 2024-09-16
- Published: 2025-03-13
- Earliest claimed: 2020-03-26
- Assignees: Larimar Therapeutics, Inc.
- Applicants: Larimar Therapeutics, Inc
- Inventors: Joan David BETTOUN; Rebecca Wissner
- Web page: https://patentgrep.com/patent/US20250084136A1

## CPC

- A61P 25/28: for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
- C07K 14/47: from mammals
- A61K 47/64: Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent (peptidic linkers A61K47/65)
- A61P 25/16: Anti-Parkinson drugs
- C07K 14/005: from viruses
- C12N 9/104: {Aminoacyltransferases (2.3.2)}
- C12Y 203/02: Aminoacyltransferases (2.3.2)
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C07K 2319/01: containing a localisation/targetting motif
- C07K 2319/02: containing a signal sequence
- C07K 2319/07: containing a mitochondrial localisation signal
- C07K 2319/095: containing a nuclear export signal
- C07K 2319/50: containing protease site
- C12N 2740/16322: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- 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)
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

The present disclosure provides a fusion protein useful for treating a non-nuclear organelle associated disorder, such as a genetic disorder, e.g., Friedrich's Ataxia. The fusion protein may comprise a protein of interest to be delivered to a non-nuclear organelle; an organelle targeting sequence (OTS); a cell penetrating peptide (CPP); and a target enhancing sequence (TES); wherein the CPP is capable of interference with delivery of the protein of interest to the non-nuclear organelle; and wherein the TES prevents said interference by the CPP. The fusion protein may also comprise a protein of interest to be delivered to a non-nuclear organelle; a CPP and a TES. The present disclosure also provides methods for treating a non-nuclear organelle associated disorder by administering the fusion protein to a subject in need thereof.

## Claims

### Claim 1

A fusion protein, comprising:
a protein of interest to be delivered to a cell;
a cell penetrating peptide (CPP); and
a target enhancing sequence (TES);
wherein:
the protein of interest is Frataxin (FXN) comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1;
the CPP is selected from the group consisting of an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOS: 12-17; and
the TES is a protease-sensitive peptide.

### Claim 2 (depends on 1)

The fusion protein of claim 1, wherein the fusion protein comprises, starting at the N-terminus:
CPP;
TES; and
a protein of interest; or
wherein the fusion protein comprises, starting at the N-terminus:
a protein of interest;
TES; and
CPP.

### Claim 3 (depends on 1)

The fusion protein of claim 1, wherein the CPP is located at the N-terminus of the fusion protein and wherein the TES is fused at the C-terminus of the CPP or wherein the CPP is located at the C-terminus of the fusion protein and wherein the TES is fused at the N-terminus of the CPP.

### Claim 4 (depends on 1)

The fusion protein of claim 1, wherein the protein of interest comprises an amino acid sequence of SEQ ID NO: 1.

### Claim 5 (depends on 1)

The fusion protein of claim 1, wherein the CPP comprises an amino acid sequence of any one of SEQ ID NOS: 12-17.

### Claim 6 (depends on 1)

The fusion protein of claim 1, wherein the TES is selected from the group consisting of a ubiquitin-like modifier, a caspase cleavage domain, a calpain cleavage domain, SUMO1, SUMO2, SUMO3, SUMO4, ISG15, Atg8, Atg12 and NEDD8.

### Claim 7 (depends on 1)

The fusion protein of claim 1, wherein the TES comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-35 and 84.

### Claim 8

A fusion protein, comprising:
a protein of interest to be delivered to a cell;
a cell penetrating peptide (CPP); and
a target enhancing sequence (TES);
wherein:
the protein of interest is Frataxin (FXN) comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1;
the CPP is selected from the group consisting of an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOS: 12-17; and
the TES is a nuclear export sequence (NES).

### Claim 9 (depends on 8)

The fusion protein of claim 8, wherein the protein of interest comprises an amino acid sequence of SEQ ID NO: 1.

### Claim 10 (depends on 8)

The fusion protein of claim 8, wherein the CPP comprises an amino acid sequence of any one of SEQ ID NOS: 12-17.

### Claim 11 (depends on 8)

The fusion protein of claim 8, wherein the NES is selected from the group consisting of NES1, NES2, NES3, NES4, NES5, NES6, NES7 and NES8.

### Claim 12 (depends on 8)

The fusion protein of claim 8, wherein the NES comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 36-43.

### Claim 13 (depends on 1)

A conjugate for intracellular delivery of proteins to non-nuclear organelles comprising the fusion protein of claim 1 and a moiety linked to said fusion protein, wherein said moiety is selected from a group consisting of a radioactive label, a fluorescent label, a small molecule, and a polymeric molecule.

### Claim 14 (depends on 1)

A cell comprising the fusion protein of claim 1.

### Claim 15 (depends on 1)

A pharmaceutical composition comprising the fusion protein of claim 1.

### Claim 16 (depends on 1)

A method of delivering a protein of interest to a cell, said method comprising contacting said cell with the fusion protein of claim 1.

### Claim 17 (depends on 1)

A method of intracellular delivery of a protein of interest to a non-nuclear organelle in a cell, said method comprising contacting said cell with the fusion protein of claim 1.

### Claim 18 (depends on 17)

The method of claim 17, wherein said non-nuclear organelle is mitochondria.

### Claim 19 (depends on 1)

A method for treating a non-nuclear organelle associated disorder, said method comprising administering to a subject in need thereof the fusion protein of claim 1.

### Claim 20 (depends on 19)

The method of claim 19, wherein the non-nuclear organelle associated disorder is Friedreich's Ataxia (FRDA).

### Claim 21 (depends on 19)

The method of claim 19, wherein said subject is a human.

### Claim 22 (depends on 8)

A conjugate for intracellular delivery of proteins to non-nuclear organelles comprising the fusion protein of claim 8 and a moiety linked to said fusion protein, wherein said moiety is selected from a group consisting of a radioactive label, a fluorescent label, a small molecule, and a polymeric molecule.

### Claim 23 (depends on 8)

A cell comprising the fusion protein of claim 8.

### Claim 24 (depends on 8)

A pharmaceutical composition comprising the fusion protein of claim 8.

### Claim 25 (depends on 8)

A method of delivering a protein of interest to a cell, said method comprising contacting said cell with the fusion protein of claim 8.

### Claim 26 (depends on 8)

A method of intracellular delivery of a protein of interest to a non-nuclear organelle in a cell, said method comprising contacting said cell with the fusion protein of claim 8.

### Claim 27 (depends on 26)

The method of claim 26, wherein said non-nuclear organelle is mitochondria.

### Claim 28 (depends on 8)

A method for treating a non-nuclear organelle associated disorder, said method comprising administering to a subject in need thereof the fusion protein of claim 8.

### Claim 29 (depends on 28)

The method of claim 28, wherein the non-nuclear organelle associated disorder is Friedreich's Ataxia (FRDA).

### Claim 30 (depends on 28)

The method of claim 28, wherein said subject is a human.