Search

EP-4735595-A1 - OLIGONUCLEOTIDES FOR TREATING NEUROMUSCULAR DISEASES

EP4735595A1EP 4735595 A1EP4735595 A1EP 4735595A1EP-4735595-A1

Abstract

The present invention relates to oligonucleotides that downregulate the expression of an allele carrying a dominant mutation in the COL6A1 , wherein the downregulation takes place through hybridization of said oligonucleotide to an RNA transcript of said allele at the site of the dominant mutation, and which either does not suppress the expression of the wild-type allele or does downregulate the expression of the wild-type allele to a lesser extent than it downregulates the expression of the allele carrying the dominant mutation. It is also related to the use of compositions to treat patients with muscular dystrophies, in particular collagen-VI related dystrophies.

Inventors

  • JIMENEZ MALLEBRERA, Cecilia
  • LÓPEZ MÁRQUEZ, Arístides
  • ZHOU, HAIYAN
  • MUNTONI, FRANCESCO
  • AGUTI, Sara

Assignees

  • Hospital Sant Joan de Deu

Dates

Publication Date
20260506
Application Date
20240626

Claims (17)

  1. Claim 1.- An oligonucleotide that downregulates the expression of a COL6A1 allele carrying a (CO/_6A1)c.877G>A mutation, wherein the oligonucleotide: is from 13 to 31 nucleotides long; is single-stranded; comprises both deoxyribonucleotides and ribonucleotides; and comprises a sequence CTCTGAG of deoxyribonucleotides.
  2. Claim 2.- The oligonucleotide according to claim 1, wherein the downregulation takes place through hybridization of said oligonucleotide to an RNA transcript of said allele at the site of the mutation.
  3. Claim 3.- The oligonucleotide according to any one of claims 1-2, wherein the downregulation takes place through hybridization of said oligonucleotide to an RNA transcript of said allele at the site of the mutation, and which either does not downregulate the expression of a COL6A1 wild-type allele or does downregulate the expression of the COL6A1 wild-type allele to a lesser extent than it downregulates the expression of the allele carrying the mutation.
  4. Claim 4.- The oligonucleotide according to any one of claims 1-3, which downregulates the expression by at least 25%.
  5. Claim 5.- The oligonucleotide according to any one of claims 1-4, which downregulates the expression by at least 30%.
  6. Claim 6.- The oligonucleotide according to any one of claims 1-5, which downregulates the expression by at least 40%.
  7. Claim 7.- The oligonucleotide according to any one of claims 1-6, which is arranged in a structure 5'-A-B-C-3', wherein “B” is from 7 to 13 deoxyribonucleotides long, and "A” and “C” are each from 3 to 9 ribonucleotides long; and wherein the oligonucleotide consists of: (a) a sequence {GGT}[ACCCAACAG](GTCTGAG)[GT*CCCCGGG]{TCT} (SEQ ID NO: 1), wherein - the nucleotides in brackets ( ) are deoxyribonucleotides, - the nucleotides in square brackets [ ] are deoxyribonucleotides or ribonucleotides, wherein T* is a thymine nucleotide when the nucleotide is a deoxyribonucleotide or an uracil nucleotide when the nucleotide is a ribonucleotide, and - the nucleotides in curly brackets { } are ribonucleotides, or (b) a fragment thereof lacking from 1 to 9 consecutive nucleotides from the 5'end and/or from 1 to 9 consecutive nucleotides from the 3'end.
  8. Claim 8.- The oligonucleotide according to any one of claims 1-7, which is from 17 to 20 nucleotides long.
  9. Claim 9.- The oligonucleotide according to any one of claims 1-8, which is arranged in a structure 5'-A-B-C-3', wherein - “B” is from 7 to 10 deoxyribonucleotides long, and - "A” and “C” are each 5 ribonucleotides long; and wherein the oligonucleotide consists of (a) a sequence {CCAAC}[AG](GTCTGAG)[G]{UCCCC} (SEQ ID NO: 41), wherein - the nucleotides in brackets ( ) are deoxyribonucleotides, - the nucleotides in square brackets [ ] are deoxyribonucleotides or ribonucleotides, and - the nucleotides in curly brackets { } are ribonucleotides; or (b) a fragment of (a) lacking from 1 to 2 consecutive nucleotides from the 5'end and/or 1 nucleotide from the 3'end.
  10. Claim 10. The oligonucleotide according to any one of claims 1-9, which comprises a sequence selected from the group consisting of a sequence [AACAG](GTCTGAG)[GUCCC] (SEQ ID NO: 14), a sequence [CAACA](GGTCTGAG)[GUCCC] (SEQ ID NO: 30), a sequence [CAACA](GGTCTGAGG)[UCCCC] (SEQ ID NO: 6) and a sequence [CCAAC](AGGTCTGAGG)[UCCCC] (SEQ ID NO: 37); wherein the nucleotides in brackets ( ) are deoxyribonucleotides and the nucleotides in square brackets [ ] are ribonucleotides.
  11. Claim 11. The oligonucleotide according to any one of claims 1-10, which comprises a sequence [AACAG](GTCTGAG)[GUCCC] (SEQ ID NO: 14).
  12. Claim 12.- The oligonucleotide according to any one of claims 1-11, wherein some or all of the deoxyribonucleotides have a phosphorothioated backbone and/or some or all of the ribonucleotides are 2'-O- Methyl (2'0-Me), 2'-MOE, locked nucleic acid (LNA) bases or 2'deoxy-2'-fluorobeta-D-arabinonucleic acid (2'FANA) RNA nucleotides.
  13. Claim 13.- The oligonucleotide according to any one of claims 1-12 wherein all the deoxyribonucleotides have a phosphorothioated backbone and all the ribonucleotides are selected from the group consisting of 2'-O- Methyl (2'0-Me) ribonucleotides and 2'-MOE ribonucleotides.
  14. Claim 14.- A composition comprising a delivery agent and an oligonucleotide as defined in any one of claims 1-13, wherein the oligonucleotide is associated to the delivery agent, in particular wherein the delivery agent is a nanovesicle that comprises a sterol and a non-lipid cationic surfactant, wherein the sterol comprises DC- cholesterol.
  15. Claim 15.- A pharmaceutical composition comprising a therapeutically effective amount of one or more oligonucleotides as defined in any one of claims 1-13, or the composition as defined in claim 14, together with a pharmaceutically acceptable excipient or carrier.
  16. Claim 16.- An oligonucleotide as defined in any one of claims 1-13, the composition as defined in claim 14, or the pharmaceutical composition as defined in claim 15, for use as a medicament.
  17. Claim 17.- An oligonucleotide as defined in any one of claims 1-13, the composition as defined in claim 14, or the pharmaceutical composition as defined in claim 15, for use in the treatment of a condition associated to the (CO/_6A1)c.877G>A mutation, wherein the condition is a collagen-VI related dystrophy.

Description

Oligonucleotides for treating neuromuscular diseases This application claims the benefit of European Patent Application 23382654.4 filed June 27th, 2023. Technical Field The present disclosure pertains to the field of medical treatment, in particular it refers to the treatment of neuromuscular diseases by gene therapy. Background Art Neuromuscular diseases are a heterogeneous group of rare disorders with prevalence between 1 and 10 per 100.000 in the population. There are several genes involved in these diseases, a fact that complicates the diagnosis. In addition, as there is a high variability in the phenotypes, the research and healthcare of the patients are complex. Congenital muscular dystrophies are a group of neuromuscular diseases caused by genetic alterations that occur at birth or in infancy. Among the different types of congenital muscular dystrophies, the collagen Vl-rel ated dystrophies (COL6-RDs) are one of the most common disorders, with prevalence below 1 per 100.000. Collagen type VI is a microfibrillar component of the extracellular matrix, synthesized in the interstitial fibroblasts (Zou, Zhang, Sabatelli, Chu, & Bbnnemann, 2008) and found in different tissues such as muscle, skin, tendon, cartilage, internal organs, and blood vessels. Collagen VI is involved in the structural and mechanical stabilization of the tissues, as well as in the interactions between the cells and the extracellular matrix; it links different components of the basement membranes and participates in cell adhesion and proliferation; and it stimulates the DNA synthesis of mesenchymal cells and the migration of the neural crest cells. In humans, there are three genes that encode for the three o-chains that compound the collagen VI: COL6A1 (MIM*120220), COL6A2 (MIM*120240), which are found in chromosome 21 and COL6A3 (MIM*120250) in chromosome 2. Collagen Vl-related dystrophies are congenital muscular dystrophies caused by mutations in the COL6A1, COL6A2 and COL6A3 genes. These patients present very variable phenotypes. The most severe is the Ullrich congenital muscular dystrophy and the milder is Bethlem myopathy. In between, there are intermediate clinical phenotypes. The clinical presentations of patients with intermediate clinical phenotypes are a mix between features of Ullrich Congenital Muscular Dystrophy and Bethlem myopathy, such as significant weakness in early childhood, distal laxity of the most distal interphalangeal joins, or contractures of the long finger flexors. Patients achieve ambulation, but they may lose it in the late teenage or early adult years. A typical feature of these patients is also progressive respiratory impairment. Curative treatments for the COL6-RDs are still in development and for the moment most of the patients only receive treatments to support the orthopedic and respiratory symptoms. Pharmacological treatments were first investigated, based on the cascade of events known to degenerate the myofibers in the collagen VI deficient skeletal muscle. Currently, as the causal mutations of the diseases have become better known over time, treatments based on genetic therapeutic approaches such as therapies focused on silencing gene expression are being investigated. The mutation (CO/_6A/)het.c.877G<A, p.Gly293Arg causes a COL6-RD with an intermediate phenotype. It is found in the position 19 of the exon 10 and it has a dominant negative effect, because it is a missense mutation that causes the substitution of a glycine from the triple helix domain by an arginine. Presently there is no treatment that can modify the course of the COL6-RD caused by this mutation, therefore there is a need in the art to provide therapeutical approaches for this disease. Summary of Invention Inventors have successfully developed a gene therapy that effectively downregulates the expression of the mutant allele (CO/_6A1)c.877G>A, which only presents a single mutation compared to the COL6A1 wild-type allele, without influencing the translation of this wild-type allele. Thus, a first aspect of the invention is an oligonucleotide that downregulates the expression of an allele carrying a mutation at position 877 in the COL6A1 coding sequence (CDS), wherein G is mutated to A compared to wild-type COL6A1.. In order to interact with the RNA targets, the oligonucleotides have to reach the intracellular space and, to that effect, delivery agents may be used to assist entering the cells. Thus, the invention also provides, as a second aspect, a composition comprising a delivery agent and an oligonucleotide as defined in the first aspect, wherein the oligonucleotide is associated to the delivery agent. As a third aspect, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of an oligonucleotides as defined in the first aspect, and/or the composition as defined in the second aspect, and a pharmaceutically acceptable excipient or carrier. A fourth aspect of the invention is an olig