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JP-7856428-B2 - Muscle targeting complexes and their use for treating facultiform, scapular, and brachial muscular dystrophy

JP7856428B2JP 7856428 B2JP7856428 B2JP 7856428B2JP-7856428-B2

Inventors

  • スブラマニアン，ロメシ，アール．
  • カタナニ，モハメド，ティー．
  • ウィーデン，ティモシー

Assignees

  • ダイン セラピューティクス，インコーポレーテッド

Dates

Publication Date
20260511
Application Date
20190802
Priority Date
20180802

Claims (17)

  1. A complex comprising an anti-transferrin receptor antibody covalently linked to an oligonucleotide targeting DUX4, The oligonucleotides are 18 , 19, 20, 21, 22, 23, 24, or 25 nucleotides in length and include a region complementary to the DUX4 sequence represented by Genbank accession number NM_001306068.2 , the complementary region being at least 12 nucleotides in length; Oligonucleotides mediate the degradation of DUX4 RNA; The oligonucleotide is covalently linked to the anti-transferrin receptor antibody via a cleavable linker, the cleavable linker containing a valine-citrulline sequence ; and the anti-transferrin receptor antibody binds to the C89-F760 region of human transferrin receptor protein 1 (TfR1) having the amino acid sequence represented by SEQ ID NO: 1 , and the anti-transferrin receptor antibody does not specifically bind to the transferrin binding site of TfR1 ; The aforementioned composite.
  2. Anti-transferrin receptor antibodies: (a) In the form of ScFv, Fab fragment, Fab' fragment, F(ab') 2 fragment, or Fv fragment; (b) Conjugates to human TfR1 at a KD of 10-11 M to 10-6 M; and/or (c) comprises a humanized antibody; The composite according to claim 1.
  3. The complex according to claim 1 or 2, wherein the oligonucleotide comprises a chain complementary to the 5' or 3' UTR sequence of DUX4.
  4. The complex according to any one of claims 1 to 3, wherein the oligonucleotide comprises one or more modified nucleosides, wherein optionally, one or more modified nucleosides are one or more 2'-modified nucleosides selected from the group consisting of 2'-O-methyl, 2'-fluoro, 2'-O-methoxyethyl, and 2',4'-bridged nucleosides.
  5. The complex according to any one of claims 1 to 4, wherein the oligonucleotide comprises one or more modified nucleoside linkages, and optionally, the oligonucleotide comprises one or more phosphorothioate linkages.
  6. The complex according to any one of claims 1 to 5, wherein the oligonucleotide comprises a phosphorodiamidite tomorpholino oligomer (PMO).
  7. Oligonucleotides: (a) comprising a region complementary to at least 15 consecutive nucleotides of SEQ ID NO: 46; or (b) comprising at least 15 consecutive nucleotides of SEQ ID NO: 45; The composite according to any one of claims 1 to 6.
  8. The complex according to any one of claims 1 to 7 , wherein an anti-transferrin receptor antibody is covalently linked to an oligonucleotide via conjugation to a lysine residue or cysteine residue of the anti-transferrin receptor antibody.
  9. The complex according to any one of claims 1 to 8 , wherein the complex is configured to promote the internalization of oligonucleotides into muscle cells mediated by transferrin receptors.
  10. The complex according to any one of claims 1 to 9 , wherein the oligonucleotide results in a reduction in the expression level of DUX4 in muscle cells.
  11. The complex according to any one of claims 1 to 10 , wherein the oligonucleotide comprises or consists of an antisense oligonucleotide.
  12. The complex according to any one of claims 1 to 10 , wherein the oligonucleotide comprises siRNA.
  13. The complex according to claim 12 , wherein the siRNA comprises one or more 2'-modified nucleosides selected from the group consisting of 2'-O-methyl and 2'-fluoro.
  14. The complex according to claim 12 or 13 , wherein the siRNA comprises a sense strand and an antisense strand.
  15. The composite according to claim 14 , wherein the sense chain and the antisense chain are of different lengths.
  16. A complex according to any one of claims 1 to 15 for use in a method of treating a disease or illness that can be improved or prevented by reducing the expression of DUX4 in muscle cells, wherein the method comprises bringing muscle cells into contact with the complex.
  17. A complex according to any one of claims 1 to 15 for use in a method for treating facultivic, scapulohumeral, or brachial muscular dystrophy (FSHD) in subjects having one or more deletions of the D4Z4 repeat in chromosome 4, wherein the method comprises administering an effective amount of the complex to the subject.

Description

This application claims the benefits as of the filing date of U.S. Provisional Application No. 62/713,933, titled "MUSCLE TARGETING COMPLEXES AND USES THEREOF FOR TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY," filed on 2 August 2018; its contents are incorporated herein by reference in their entirety. Field of the Invention This application relates to targeted complexes for delivering molecular payloads (e.g., oligonucleotides) to cells, and their uses, specifically for the treatment of diseases. Reference to Sequence Listing This application has been filed electronically with a sequence listing. The sequence listing is provided as a file titled D082470001WO00-SEQ.txt, created on July 31, 2019, and measuring 60 kilobytes. The information in the electronic sequence listing is incorporated herein by reference in its entirety. Background of the Invention Muscular dystrophy (MD) is a group of diseases characterized by progressive weakness and loss of muscle mass. These diseases are caused by mutations in genes that code for proteins necessary for forming healthy muscle tissue. Facial-scapular-brachial muscular dystrophy (FSHD) is a dominant inherited type of MD that primarily affects the muscles of the face, scapula, and upper arm. Other symptoms of FSHD include weakness of the abdominal muscles, retinal abnormalities, hearing loss, and joint pain and inflammation. FSHD is the most prevalent of the nine types of MD that affect both adults and children, with an incidence of approximately 1 in 8,300 worldwide. FSHD is caused by the abnormal production of a protein whose function is unknown, double homeobox 4 (DUX4). The DUX4 gene, which encodes the DUX4 protein, is located within the D4Z4 repeat region on chromosome 4 and is typically expressed only during fetal development. Subsequently, the DUX4 gene is suppressed by hypermethylation of the D4Z4 repeats, which surround and compact the DUX4 gene. Two types of FSHD have been described: type I and type II. Type I, which accounts for approximately 95% of cases, is associated with deletions of the D4Z4 repeat on chromosome 4. While unaffected individuals generally have more than 10 repeats arranged in the subtelomeric region of chromosome 4, the most common form of FSHD (FSHD1) is caused by a reduction in the array to fewer than 10 repeats, associated with reduced epigenetic suppression in skeletal muscle and altered expression of DUX4. Type II FSHD, which accounts for approximately 5% of cases, is associated with mutations in the SMCHD1 gene on chromosome 18. Aside from supportive care and treatments to address the symptoms of the disease, there is no effective treatment for FSHD. Summary of the Invention In several aspects, this disclosure provides a complex that targets muscle cells for the purpose of delivering a molecular payload to those cells. In some embodiments, the complex provided herein is specifically useful for delivering a molecular payload that inhibits the expression or activity of DUX4 in subjects having or suspected of having fascio-scapulobrachial muscular dystrophy (FSHD), for example. Consequently, in some embodiments, the complex provided herein comprises a muscle targeting agent (e.g., a muscle-targeting antibody) that specifically binds to a receptor on the surface of muscle cells for the purpose of delivering the molecular payload to muscle cells. In some embodiments, the complex is taken up into the cell via receptor-mediated internalization, and the molecular payload can then be released into the cell to perform its function. For example, a complex modified to deliver an oligonucleotide may release the oligonucleotide so that the oligonucleotide can inhibit the expression of the DUX4 gene in muscle cells. In some embodiments, the oligonucleotide is released by endosomal cleavage of a covalent linker connecting the oligonucleotide and the muscle targeting agent of the complex. Several aspects of this disclosure involve a complex comprising a muscle targeting agent covalently linked to a molecular payload configured to inhibit the expression or activity of DUX4, wherein the muscle targeting agent specifically binds to an internalized cell surface receptor on muscle cells. In some embodiments, the muscle targeting agent is a muscle targeting antibody. In some embodiments, the muscle targeting antibody is an antibody that specifically binds to an extracellular epitope of the transferrin receptor (for example, an epitope of the apical domain of the transferrin receptor). The muscle targeting antibody may specifically bind to an epitope of a sequence in the range C89–F760 of SEQ ID NOs. 1–3. In some embodiments, the equilibrium dissociation constant (Kd) of the binding of the muscle targeting antibody to the transferrin receptor is in the range of 10⁻¹¹ M to 10⁻⁶ M. In some embodiments, the muscle-targeting antibody of the complex competes with the antibodies listed in Table 1 for specific binding to the transferrin receptor epitope (e.g., c