# US 11,111,309 B2 · Method of reducing expression of DUX4 in a muscle cell by administering an anti-transferrin receptor antibody linked to an oligonucleotide targeting DUX4

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- Number: US 11,111,309 B2
- Document ID: US11111309B2
- Status: Granted
- Kind: B2
- Application number: 17/205,123
- Filed: 2021-03-18
- Published: 2021-07-08
- Granted: 2021-09-07
- Earliest claimed: 2018-08-02
- Assignees: Dyne Therapeutics, Inc.
- Applicants: Dyne Therapeutics, Inc.
- Inventors: Romesh R. Subramanian; Mohammed T. Qatanani; Timothy Weeden
- Web page: https://patentgrep.com/patent/US11111309B2

## CPC

- C07K 16/2881: {against CD71}
- A61K 47/6807: {the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense}
- A61K 47/6849: {the antibody targeting a receptor, a cell surface antigen or a cell surface determinant}
- A61K 47/6889: {Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment}
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- C07K 14/4705: {stimulating, promoting or activating activity}
- C12N 15/09: Recombinant DNA-technology
- 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)}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- A61K 2039/505: {comprising antibodies}
- C07K 2317/55: Fab or Fab'
- C07K 2317/77: Internalization into the cell
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C12N 2310/11: Antisense
- C12N 2310/3145: with the nitrogen in 3' or 5'-position
- C12N 2310/3233: Morpholino-type ring
- C12N 2310/3513: Protein; Peptide
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Aspects of the disclosure relate to complexes comprising a muscle-targeting agent covalently linked to a molecular payload. In some embodiments, the muscle-targeting agent specifically binds to an internalizing cell surface receptor on muscle cells. In some embodiments, the molecular payload inhibits expression or activity of DUX4. In some embodiments, the molecular payload is an oligonucleotide, such as an antisense oligonucleotide or RNAi oligonucleotide.

## Claims

### Claim 1

A method of reducing expression level of DUX4 in a muscle cell of a subject, the method comprising administering to the subject a complex that comprises an anti-transferrin receptor antibody covalently linked to an oligonucleotide that targets a DUX4 RNA,
wherein the anti-transferrin receptor antibody binds in the range of C89 to F760 of human transferrin receptor protein 1 (TfR1) having an amino acid sequence as set forth in SEQ ID NO: 1 and wherein the anti-transferrin receptor antibody does not specifically bind to the transferrin binding site of TfR1;
wherein the oligonucleotide is a single stranded oligonucleotide in the range of 15-35 nucleotides in length, wherein the oligonucleotide comprises a region of complementarity to the nucleotide sequence as set forth in SEQ ID NO: 46, wherein the region of complementarity is at least 15 nucleotides in length, and wherein the oligonucleotide is a phosphorodiamidate morpholino oligomer; and
wherein the complex is administered to the subject by infusion and the oligonucleotide brings about reduction of expression level of DUX4 in the muscle cell.

### Claim 2 (depends on 1)

The method of claim 1, wherein the region of complementarity is at least 20 nucleotides in length.

### Claim 3 (depends on 1)

The method of claim 1, wherein the oligonucleotide is 100% complementary to the nucleotide sequence of SEQ ID NO: 46.

### Claim 4 (depends on 1)

The method of claim 1, wherein the oligonucleotide comprises at least 20 consecutive nucleotides of SEQ ID NO: 45.

### Claim 5 (depends on 1)

The method of claim 1, wherein the oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 45.

### Claim 6 (depends on 1)

The method of claim 1, wherein the 5′ end of the oligonucleotide is covalently linked to a lysine in the anti-transferrin receptor antibody via a cleavable linker.

### Claim 7 (depends on 6)

The method of claim 6, wherein the cleavable linker comprises a valine-citrulline sequence.

### Claim 8 (depends on 6)

The method of claim 6, wherein the complex is obtained by a cycloaddition reaction between an azide and an alkyne to form a triazole.

### Claim 9 (depends on 8)

The method of claim 8, wherein prior to the cycloaddition reaction, the azide is located on the cleavable linker that is covalently linked to the 5′ end of the oligonucleotide and the alkyne is provided in a bicyclononyne moiety that further covalently links to the anti-transferrin receptor antibody.

### Claim 10 (depends on 7)

The method of claim 7, wherein the cleavable linker further comprises one or more polyethylene glycol units.

### Claim 11 (depends on 1)

The method of claim 1, wherein the anti-transferrin receptor antibody binds to human TfR1 with a KD in a range of 10−11 M to 10−8 M.

### Claim 12 (depends on 1)

The method of claim 1, wherein the anti-transferrin receptor antibody is in the form of a ScFv, Fab fragment, Fab′ fragment, F(ab′)2 fragment, or Fv fragment.

### Claim 13 (depends on 1)

The method of claim 1, wherein the anti-transferrin receptor antibody is in the form of a Fab fragment.

### Claim 14 (depends on 1)

The method of claim 1, wherein the subject has one or more deletions of D4Z4 repeats in chromosome 4.

### Claim 15 (depends on 14)

The method of claim 14, wherein the subject has 10 or fewer D4Z4 repeats.

### Claim 16 (depends on 1)

The method of claim 1, wherein the oligonucleotide brings about reduction of expression levels of one or more of ZSCAN1, MBDL3L2 and TRIM43 in the muscle cell.

### Claim 17 (depends on 1)

The method of claim 1, wherein the muscle cell is a skeletal muscle cell, cardiac muscle cell, or smooth muscle cell.

### Claim 18 (depends on 1)

The method of claim 1, wherein the subject is human.

### Claim 19 (depends on 1)

The method of claim 1, wherein the subject is cynomolgus.

### Claim 20 (depends on 14)

The method of claim 14, wherein the deletions of D4Z4 repeats in chromosome 4 is associated with facioscapulohumeral muscular dystrophy (FSHD).

### Claim 21 (depends on 1)

The method of claim 1, wherein the complex is administered to the subject by intravenous infusion.