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US-20260124274-A1 - METHODS OF INCREASING READTHROUGH OF A PREMATURE TERMINATION CODON

US20260124274A1US 20260124274 A1US20260124274 A1US 20260124274A1US-20260124274-A1

Abstract

Methods of increasing readthrough of a premature termination codon (PTC) by a readthrough-inducing agent in a cell comprising modulating translation in the cell are provided. Methods of treating a disease characterized by a pathogenic PTC in a disease-associated gene in a subject by performing a method of the invention are also provided, as are kits comprising a readthrough-inducing agent and a translation modulating agent.

Inventors

  • Rina Rosin-Arbesfeld
  • Amnon WITTENSTEIN
  • Michal Caspi

Assignees

  • RAMOT AT TEL-AVIV UNIVERSITY LTD.

Dates

Publication Date
20260507
Application Date
20251104

Claims (20)

  1. 1 . A method of increasing readthrough of a premature termination codon (PTC) by a readthrough-inducing agent in a cell, the method comprising modulating translation in said cell, wherein said modulating translation comprises at least one of: inhibiting cap-dependent translation initiation in said cell, accelerating translation elongation in said cell, and inhibiting translation termination in said cell, thereby increasing readthrough of a PTC by a readthrough-inducing agent.
  2. 2 . The method of claim 1 , wherein said readthrough-inducing agent is an antibiotic.
  3. 3 . The method of claim 2 , wherein said antibiotic is an aminoglycoside selected from the group consisting of: geneticin, gentamicin, paromomycin and tobramycin.
  4. 4 . The method of claim 2 , wherein said antibiotic is selected from the group consisting of: erythromycin, azithromycin, spiramycin, josamycin, and tylosin.
  5. 5 . The method of claim 1 , wherein said readthrough-inducing agent is selected from escin, ELX-02, NPC-14, 2,6-diaminopurine (DAP), CC-90009, amlexanox and ataluren.
  6. 6 . The method of claim 1 , further comprising contacting said cell with said readthrough-inducing agent, wherein said readthrough inducing agent is selected from the group consisting of: geneticin, gentamicin, paromomycin, tobramycin, erythromycin, azithromycin, spiramycin, josamycin, tylosin, escin, ELX-02, NPC-14, 2,6-diaminopurine (DAP), CC-90009, amlexanox and ataluren.
  7. 7 . The method of claim 1 , wherein said modulating translation comprises inhibiting cap-dependent translation initiation and comprises contacting said cell with a mammalian target of rapamycin (mTOR) inhibitor, a Eukaryotic translation initiation factor 4E (eIF4E) inhibitor, a Mitogen-activated protein kinase interacting kinase (MNK) inhibitor, or a combination thereof.
  8. 8 . The method of claim 7 , wherein said inhibiting cap-dependent translation initiation does not comprise administering a Ribosomal protein S6 kinase beta-1 (S6K1) inhibitor;
  9. 9 . The method of claim 1 , wherein said modulating translation comprises accelerating translation elongation and comprises contacting said cell with a eukaryotic elongation factor 2 kinase (eEF2K) inhibitor.
  10. 10 . The method of claim 1 , wherein said inhibiting translation termination comprises contacting said cell with a eukaryotic translation termination factor 1 (eRF1) inhibitor.
  11. 11 . The method of claim 7 , wherein at least one of: a. said mTOR inhibitor is selected from the group consisting of: everolimus, temsirolimus, ridaforolimus, umirolimus, zotarolimus, AZD8055, PP242, INK128, WYE-354, OSI-027 and KU-0063794, rapamycin and Torin-1; b. said eIF4E inhibitor is selected from the group consisting of: 4E1RCat, 4E2RCat, LY2275796, SBI-756 and 4EGI-1; and c. said MNK inhibitor is selected from the group consisting of: CGP57380, BAY1143269, ETC-206, MNK-11, MNK-12, MNK1/2-IN-7, SEL201, Cercosporamide, MNKI-8e and Tomivosertib.
  12. 12 . The method of claim 9 , wherein said eEF2K inhibitor is selected from NH125, rottlerin, and A-484954.
  13. 13 . The method of claim 10 , wherein said eRF1 inhibitor is selected from SRI-41315, Apidaecin, dimethyloxalylglycine (DMOG) and N-oxalylglycine (NOG).
  14. 14 . A method of treating a disease characterized by a pathogenic premature termination codon (PTC) in a disease-associated gene in a subject in need thereof, the method comprising increasing readthrough of said PTC by a readthrough-inducing agent in a cell of said subject by a method of any one of claim 1 , thereby treating a disease characterized by a pathogenic PTC.
  15. 15 . The method of claim 14 , further comprising selecting a subject suffering from a disease characterized by a pathogenic PTC in a disease-associated gene.
  16. 16 . The method of claim 14 , wherein a. said disease is cancer and said disease-associated gene is a tumor suppressor gene selected from the group consisting of: adenomatous polyposis coli (APC), Ataxia-telangiectasia mutated (ATM), Breast cancer type 1 susceptibility protein (BRCA1), BRCA2, cadherin-11 (CDH1), cyclin-dependent kinase inhibitor 2A (CDKN2A), Menin (MEN1), Neurofibromin (NF1), merlin (NF2), Protein patched homolog 1 (PTCH1), Phosphatase and tensin homolog (PTEN), retinoblastoma protein (RB1), SMAD family member 4, Mothers against decapentaplegic homolog 4 (SMAD4), Serine/threonine kinase 11 (STK11), p53 (TP53), tuberous sclerosis 1 (TSC1), TSC2, Von Hippel-Lindau tumor suppressor (VHL), and Wilms tumor protein (WT1); b. said disease is cystic fibrosis and said disease-associated gene is cystic fibrosis transmembrane conductance regulator (CFTR); c. said disease is muscular dystrophy and said disease-associate gene is dystrophin (DMD); d. said disease is nephropathic cystinosis and said disease-associated gene is cystinosin (CTNS); e. said disease is epidermolysis bullosa and said disease-associated gene is laminin beta 3 (LAMB3); f. said disease is hereditary hypotrichosis simplex and said disease-associated gene is Lysophosphatidic acid receptor 6 (LPAR6/P2RY5) or lipase H (LIPH); or g. said disease is adenomatous polyposis and said disease-associated gene is APC.
  17. 17 . The method of claim 16 , wherein said muscular dystrophy is Duchenne Muscular Dystrophy (DMD).
  18. 18 . The method of claim 14 , further comprising administering to said subject said readthrough-inducing agent, wherein said readthrough inducing agent is selected from the group consisting of: geneticin, gentamicin, paromomycin, tobramycin, erythromycin, azithromycin, spiramycin, josamycin, tylosin, escin, ELX-02, NPC-14, 2,6-diaminopurine (DAP), CC-90009, amlexanox and ataluren.
  19. 19 . A kit comprising: a. a readthrough-inducing agent, wherein said readthrough inducing agent is selected from the group consisting of: geneticin, gentamicin, paromomycin, tobramycin, erythromycin, azithromycin, spiramycin, josamycin, tylosin, escin, ELX-02, NPC-14, 2,6-diaminopurine (DAP), CC-90009, amlexanox and ataluren.; and b. an agent that modulates translation, wherein said agent that modulates translation is a mammalian target of rapamycin (mTOR) inhibitor, a Eukaryotic translation initiation factor 4E (eIF4E) inhibitor, a Mitogen-activated protein kinase interacting kinase (MNK) inhibitor, a eukaryotic elongation factor 2 kinase (eEF2K) inhibitor, a eukaryotic translation termination factor 1 (ERF1) inhibitor, or a combination thereof.
  20. 20 . The kit of claim 19 , wherein at least one of: a. said mTOR inhibitor is selected from the group consisting of: everolimus, temsirolimus, ridaforolimus, umirolimus, zotarolimus, AZD8055, PP242, INK128, WYE-354, OSI-027 and KU-0063794, rapamycin and Torin-1; b. said eIF4E inhibitor is selected from the group consisting of: 4E1RCat, 4E2RCat, LY2275796, SBI-756 and 4EGI-1; c. said MNK inhibitor is selected from the group consisting of: CGP57380, BAY1143269, ETC-206, MNK-I1, MNK-12, MNK1/2-IN-7, SEL201, Cercosporamide, MNKI-8e and Tomivosertib; d. said eEF2K inhibitor is selected from NH125, rottlerin, and A-484954; and e. said eRF1 inhibitor is selected from SRI-41315, Apidaecin, dimethyloxalylglycine (DMOG) and N-oxalylglycine (NOG).

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority of U.S. Provisional Patent Application No. 63/715,663, filed on Nov. 4, 2024, the contents of which are incorporated herein by reference in their entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (RMT-P-035-US.xml; Size: 4,380 bytes; and Date of Creation: Nov. 3, 2025) is herein incorporated by reference in its entirety. FIELD OF INVENTION The present invention is in the field of synthetic expression circuits. BACKGROUND OF THE INVENTION Nonsense mutations are single nucleotide substitutions in the coding regions that result in premature termination codons (PTCs) and produce truncated, mostly non-functional proteins. A meta-analysis based on the human gene mutation databases concluded that nonsense mutations are responsible for approximately 11% of all gene aberrations associated with inheritable diseases. Different compounds and small molecules can induce PTC readthrough, leading to misinterpretation of the PTC as a sense codon, thereby restoring protein translation. Genetic and biochemical studies have shown that these nonsense mutation readthrough agents act by binding a specific site on the rRNA, which causes the ribosome to introduce an amino acid instead of releasing the mRNA chain. Although little is known about the exact nature of the amino acid inserted or the precise readthrough mechanism, translation through the PTC often results in the expression of a full-length protein. Aminoglycoside antibiotics were the first drugs shown to induce PTC-readthrough, by enabling the misincorporation of near-cognate tRNA (nc-tRNA) at the A-site of the ribosome, leading to the expression of full-length proteins. Aminoglycosides function by a mechanism that competes with translation termination. However, the lack of specificity, modest readthrough effects, and toxicity of the aminoglycosides have led to the search for more efficient agents. Additional nonsense mutation readthrough-inducing compounds that increase protein production in several cell culture and animal disease models have been identified, but the readthrough levels were usually low, achieving no more than 5% of wild-type protein expression, and most compounds have not reached the clinic. Although nonsense mutation readthrough usually yields only a small percentage of the normal expression levels of the full-length protein, in some cases, such as in lysosomal storage disease, even 1% of normal protein function may restore a near-normal or clinically less severe phenotype, this threshold is disease and gene dependent as for cystic fibrosis (CF), it has been shown that 10-35% of CFTR activity might be needed to alleviate pulmonary morbidity significantly; in Duchenne muscular dystrophy (DMD)—1-30% of the full-length dystrophin protein is needed. It has also been demonstrated that readthrough activity can be chemically potentiated. As a large number of genetic diseases result from nonsense mutations, identifying and developing new therapeutic strategies by better understanding the mechanism that underlines induced nonsense mutation readthrough activity is of great interest. Adenomatous polyposis coli (APC) is a multifunctional tumor suppressor gene mutated in approximately 80% of sporadic and hereditary colorectal cancer (CRC) syndrome tumors. APC inhibits the activity of the oncogenic β-catenin protein as well as functions in cell cycle control, differentiation, and apoptosis. Mutations in APC are thought to be one of the key factors driving cancer initiation. A large number of the APC mutations are nonsense mutations, resulting in a truncated, unfunctional protein. Mechanisms that induce nonsense mutation suppression leading to restored expression of the full-length APC protein were therefore explored. Regulating protein translation is crucial for cell survival, and thus, translational dysregulation leads to aberrant growth and tumorigenicity. A new method of suppressing translation termination at PTCs is therefore greatly needed. SUMMARY OF THE INVENTION The present invention provides methods of increasing readthrough of a premature termination codon (PTC) by a readthrough-inducing agent in a cell comprising modulating translation in the cell are provided. Methods of treating a disease characterized by a pathogenic PTC in a disease-associated gene in a subject by performing a method of the invention are also provided, as are kits comprising a readthrough-inducing agent and a translation modulating agent. According to a first aspect, there is provided a method of increasing readthrough of a premature termination codon (PTC) by a readthrough-inducing agent in a cell, the method comprising modulating translation in the cell, wherein the modulating translation comprises at least one of: inhibiting cap-dependent translation initiation in the cell, accelerating translation elongation in the cell, and inhibiting translat