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US-20260124308-A1 - OLIGONUCLEOTIDES CONJUGATED TO OLEIC ACID AND USES THEREOF

US20260124308A1US 20260124308 A1US20260124308 A1US 20260124308A1US-20260124308-A1

Abstract

The invention provides oligonucleotide and/or oligonucleotide analogue molecules that are antagonists of a microRNA, preferably antagonists of human microRNAs hsa-miR-23b-3p and hsa-miR-218-5p, that comprise a mixture of phosphorothioate and phosphodiester linkages, and that are conjugated to at least one oleic acid molecule. Inhibiting these microRNAs allows to increase the endogenous levels of the corresponding proteins MBNL1 and/or MBNL2. The present invention further provides compositions comprising said oligonucleotides and/or oligonucleotide analogue molecules and their uses for the treatment and prevention of DM in a subject in need thereof.

Inventors

  • Rubén ARTERO ALLEPUZ
  • Nerea MORENO CERVERA
  • Irene GONZÁLEZ MARTÍNEZ
  • Estefanía CERRO HERREROS
  • Eric G. Marcusson
  • María Beatriz LLAMUSÍ TROISI

Assignees

  • UNIVERSITAT DE VALÈNCIA
  • ARTHEX BIOTECH S.L.

Dates

Publication Date
20260507
Application Date
20230523
Priority Date
20220523

Claims (20)

  1. 1 . A conjugate comprising: (i) an oleic acid molecule, which functions as a pharmaceutically acceptable vehicle or carrier; and (ii) an oligonucleotide molecule; wherein the oligonucleotide molecule is an active ingredient for treatment of a disease, wherein the oleic acid molecule is conjugated to the 3′ and/or 5′ end of the oligonucleotide molecule.
  2. 2 . The conjugate according to claim 1 , wherein the oligonucleotide molecule comprises a mixture of phosphorothioate and phosphodiester linkages chemically linking the nucleotides, and wherein the number of nucleotides that are chemically linked by a phosphorothioate linkage is greater than the number of nucleotides that are chemically linked by a phosphodiester linkage.
  3. 3 . The conjugate according to claim 1 , wherein the oligonucleotide molecule is an antagonist of a microRNA.
  4. 4 . The conjugate according to claim 3 , wherein the microRNA is human hsa-miR-23b-3p or human hsa-miR-218-5p.
  5. 5 . A method of delivering an oligonucleotide molecule to muscular and/or central nervous system (CNS) cells in a subject in need thereof, comprising administering the conjugate of claim 1 to the subject in need thereof, wherein the oleic acid functions as a vehicle to delivers the oligonucleotide molecule to muscular and/or CNS cells in the subject in need thereof, and wherein the conjugate is administrated via intravenous, intraarterial, or subcutaneous route.
  6. 6 . A method of preventing or treating muscular diseases, nervous system diseases, and/or RNAopathies, comprising administering the conjugate of claim 1 to a subject in need thereof.
  7. 7 . The method of claim 6 , wherein the disease is myotonic dystrophy.
  8. 8 . The method of claim 7 , wherein the myotonic dystrophy is myotonic dystrophy type 1.
  9. 9 . A composition comprising one or more oligonucleotide molecules, wherein, the one or more oligonucleotide molecules is conjugated at its 3′ and/or 5′ ends to at least one oleic acid molecule, wherein the one or more oligonucleotide molecules is: (i) an antagonist of human hsa-miR-23b-3p or human hsa-miR-218-5p, and (ii) 10 to 30 nucleotides in length, and (iii) comprises at least 15 consecutive nitrogen bases of nucleotides with at least 80% identity to the sequence of a region present in SEQ ID NO: 1 (antimiR-218-5p) or 2 (antimiR-23b-3p), or SEQ ID NO: 52-110.
  10. 10 . The composition of claim 9 , further comprising a mixture of phosphorothioate and phosphodiester linkages chemically linking the nucleotides, wherein the number of nucleotides that are chemically linked by a phosphorothioate linkage is greater than the number of nucleotides that are chemically linked by a phosphodiester linkage.
  11. 11 . The composition of claim 9 , wherein the one or more oligonucleotide molecules comprises at least 15 consecutive nitrogen bases of nucleotides that are identical to the sequence of a region present in SEQ ID NO: 1 (antimiR-218-5p) or 2 (antimiR-23b-3p).
  12. 12 . The composition of claim 9 , wherein the one or more oligonucleotide molecules is at least 80% identical to SEQ ID NOs: 22, 23 or 25, and wherein the spacer molecule defined in said SEQ ID NOs: 22, 23 or 25 is selected from the group consisting of NHC3, NHC5, NHC6, and threoninol.
  13. 13 . The composition of claim 9 , wherein the one or more oligonucleotide molecules is at least 80% identical to SEQ ID NOs: 22, 23 or 25, wherein the spacer molecule defined in said SEQ ID NOs: 22, 23 or 25 is selected from the group consisting of NHC3, NHC5, NHC6, and threoninol, and wherein the one or more oligonucleotide molecules is capable of increasing the endogenous levels of MBNL1 and/or MBNL2 proteins.
  14. 14 . The composition of claim 9 , wherein the nucleotide sequence of the one or more oligonucleotide molecules consists of SEQ ID NOs: 3, 4, or 7.
  15. 15 . A pharmaceutical composition comprising, the composition of claim 9 and a carrier and/or one or more pharmaceutically acceptable excipients.
  16. 16 . A method of targeting muscular cells and/or CNS cells in a subject in need thereof comprising administering the pharmaceutical composition of claim 15 to a subject in need thereof.
  17. 17 . The method according to claim 16 , wherein the pharmaceutical composition is administrated via intravenous, intraarterial or subcutaneous route.
  18. 18 . A method of treating or preventing muscular diseases, nervous system diseases, and/or RNAopathies in a subject in need thereof, comprising administering the pharmaceutical composition of claim 15 , to the subject in need thereof.
  19. 19 . The method of claim 18 , wherein the muscular disease is myotonic dystrophy.
  20. 20 . The method of claim 19 , wherein the myotonic dystrophy is myotonic dystrophy type 1.

Description

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (920235_401USPC_SL.xml; Size: 824,336 bytes; and Date of Creation: Nov. 18, 2024) is herein incorporated by reference in its entirety. TECHNICAL FIELD OF THE INVENTION The present invention relates to the field of medicine. More specifically, the invention relates to oligonucleotide antagonists of endogenous microRNAs, particularly hsa-miR-218-5p and hsa-miR-23b-3p, that are conjugated to oleic acid, and their uses. BACKGROUND OF THE INVENTION Myotonic dystrophy type 1 (DM1) is a rare genetic disease with no current effective treatment. DM1 is associated with a substantial disease burden resulting in impairment across many different patient systems and tissues. Muscle weakness and fatigue constitute the two most common disease manifestations, reported by 93% and 90% of patients, respectively, followed by muscle locking (73%). Other phenotypes include cardiac dysfunctions, cataracts, insulin resistance, and cognitive impairment. DM1 disease is based on CTG repeat expansions occurring in the DM1 protein kinase (DMPK) gene, which are transcribed into pathogenic mRNAs. It is currently well established that CUG expansions bind with high affinity to the Muscleblind-like (MBNL1, 2, and 3) family of proteins, thereby inhibiting their normal function, but other alterations may contribute to MBNL1 and MBNL2 depletion. In skeletal muscle and brain, MBNL1 and MBNL2, respectively, are preferentially expressed, whereas MBNL3 is expressed primarily during embryonic development and adult tissue regeneration. MBNL1 and MBNL2 proteins control alternative splicing and polyadenylation of several transcripts, specifically by causing a shift from foetal to adult patterns, and act antagonistically to CUGBP Elav-like family member 1 (CELF) proteins in splice regulation, which are found upregulated and mislocated in DM1. Further, it has been shown that there is genetic redundancy between MBNL1 and 2 genes, as the deletion of only one resulted in the upregulation of the other and occupancy of its binding sites in target RNAs (see Lee 2013. Compound loss of muscleblind-like function in myotonic dystrophy. EMBO Mol Med 5:1887-900.) The depletion of MBNL1 protein function has been shown to be a critical factor in the course of the disease. Indeed, MBNL1 loss of function accounts for more than 80% of mis-splicing events and nearly 70% of expression defects. MBNL genes and/or MBNL protein upregulation in DM1 mice and patient-derived fibroblasts is well tolerated and rescues several symptoms, such as myotonia and mis-splicing events, as well as the reduction of foci formation, opening the path for the development of therapeutic approaches aimed at increasing the expression of these genes. MBNL1 and MBNL2 depletion also impinge on several other gene expression processes, for example impairing trafficking of membrane-associated mRNAs or miRNA biogenesis. MicroRNAs (also referred herein as “miRNA” or “miRs”) are a class of small non-coding RNAs that play important roles in regulating gene expression, particularly in gene silencing. In human cells, the expression of hsa-miR-23b-3p and hsa-miR-218-5p has been shown to regulate MBNL1 and MBNL2 transcripts directly by luciferase reporter assay (Cerro-Herreros et al. 2018 Nat. Commun. 9, 2482). Silencing of hsa-miR-23b-3p and hsa-miR-218-5p increases Muscleblind-like protein expression and alleviates myotonic dystrophy phenotypes in mammalian models. On the other hand, antimiRs are a class of oligonucleotides that prevents other molecules, such as microRNAs, from binding to a target site on an RNA, particularly in messenger RNA (mRNA) molecules. The use of regular antimiRs as therapeutic molecules has limitations in their development as drug candidates, including a short life span due to degradation in the cellular environment, poor cellular intake from extracellular media, and limited therapeutic window expressed as the ratio of the concentrations at which a compound reaches median toxicity and efficacy (TC50/EC50) so that the higher the ratio, the better. Thus, methods aimed at increasing antimiRs stability, potency, tissue-specific uptake, and therapeutic window, among other pharmacological parameters, need to be further developed in order to exploit the full potential of antimiRs in inhibiting their target hsa-miR-218-5p and hsa-miR-23b-3p. On the one hand, albumin is one of the most abundant proteins in plasma and provides the transport of fatty acids, drugs, ions and other metabolites. Conjugation of the oligonucleotides with fatty acids may increase the albumin binding affinity of the oligonucleotides, enhancing their ability to cross the endothelial barrier and improving their functional uptake into muscles, thereby increasing the oligonucleotide potency in vivo. However, the wide variety of saturated and unsaturated fatty acids that differ in their structure may, in turn, influence protein binding