PRODUCTS AND METHODS FOR PROMOTING MYOGENESIS

20240335505

18/271503

February 10, 2022

The present invention relates generally to the field of long non-coding RNAs and transcription factors and modulation of their expression for use in medicine and agriculture, such as the treatment and prevention of diseases associated with muscle atrophy and the production of livestock. More particularly, the invention provides agonists of Cytor, a long non-coding RNA, e.g. agents that increase the amount of Cytor RNA in skeletal muscle as well as antagonists for Teadl, a transcription factor, e.g. agents that decrease the amount of Teadl RNA or protein in skeletal muscle and their use in therapy.

1. A method of: a) treating a subject having or being at risk of developing skeletal muscle atrophy, starvation, sarcopenia, cachexia (e.g. cancer cachexia or AIDS cachexia), sepsis, diabetes (particularly type I diabetes), muscular dystrophy (e.g. Duchenne Muscular Dystrophy, Becker's MD, Congenital MD, Myotonic dystrophy (e.g. type I and type II), Limb-girdle MD, Emery-Dreifuss MD, Distal MD and Facioscapulohumeral Muscular Dystrophy), Chronic Heart Failure (e.g. diaphragm muscle atrophy resulting from Chronic Heart Failure), Chronic Obstructive Pulmonary Disease (e.g. diaphragm muscle atrophy resulting from Chronic Obstructive Pulmonary Disease), Amyotrophic Lateral Sclerosis (ALS), Spinal Muscular Atrophy, Guillain-Barré syndrome, intensive care unit acquired weakness, immobilization resulting from bone fractures (e.g. hip (femur neck) fractures) or immobilization resulting from an infection; b) promoting myogenesis in skeletal muscle of a subject, wherein the subject has or is at risk of developing skeletal muscle atrophy, starvation, sarcopenia, cachexia (e.g. cancer cachexia or AIDS cachexia), sepsis, diabetes (particularly type I diabetes), muscular dystrophy (e.g. Duchenne Muscular Dystrophy, Becker's MD, Congenital MD, Myotonic dystrophy (e.g. type I and type II), Limb-girdle MD, Emery-Dreifuss MD, Distal MD and Facioscapulohumeral Muscular Dystrophy), Chronic Heart Failure (e.g. diaphragm muscle atrophy resulting from Chronic Heart Failure), Chronic Obstructive Pulmonary Disease (e.g. diaphragm muscle atrophy resulting from Chronic Obstructive Pulmonary Disease), Amyotrophic Lateral Sclerosis (ALS), Spinal Muscular Atrophy, Guillain-Barré syndrome, intensive care unit acquired weakness, immobilization resulting from bone fractures (e.g. hip (femur neck) fractures) or immobilization resulting from an infection; c) increasing skeletal muscle mass in a subject, wherein the subject has or is at risk of developing skeletal muscle atrophy, starvation, sarcopenia, cachexia (e.g. cancer cachexia or AIDS cachexia), sepsis, diabetes (particularly type I diabetes), muscular dystrophy (e.g. Duchenne Muscular Dystrophy, Becker's MD, Congenital MD, Myotonic dystrophy (e.g. type I and type II), Limb-girdle MD, Emery-Dreifuss MD, Distal MD and Facioscapulohumeral Muscular Dystrophy), Chronic Heart Failure (e.g. diaphragm muscle atrophy resulting from Chronic Heart Failure), Chronic Obstructive Pulmonary Disease (e.g. diaphragm muscle atrophy resulting from Chronic Obstructive Pulmonary Disease), Amyotrophic Lateral Sclerosis (ALS), Spinal Muscular Atrophy, Guillain-Barré syndrome, intensive care unit acquired weakness, immobilization resulting from bone fractures (e.g. hip (femur neck) fractures) or immobilization resulting from an infection; d) improving skeletal muscle function in a subject, wherein the subject has or is at risk of developing starvation, sarcopenia, cachexia (e.g. cancer cachexia or AIDS cachexia), sepsis, diabetes (particularly type I diabetes), muscular dystrophy (e.g. Duchenne Muscular Dystrophy, Becker's MD, Congenital MD, Myotonic dystrophy (e.g. type I and type II), Limb-girdle MD, Emery-Dreifuss MD, Distal MD and Facioscapulohumeral Muscular Dystrophy), Chronic Heart Failure (e.g. diaphragm muscle atrophy resulting from Chronic Heart Failure), Chronic Obstructive Pulmonary Disease (e.g. diaphragm muscle atrophy resulting from Chronic Obstructive Pulmonary Disease), Amyotrophic Lateral Sclerosis (ALS), Spinal Muscular Atrophy, Guillain-Barré syndrome, intensive care unit acquired weakness, immobilization resulting from bone fractures (e.g. hip (femur neck) fractures) or immobilization resulting from an infection; or e) preventing or treating skeletal muscle atrophy and/or skeletal muscle dysfunction in a subject, optionally wherein the subject has or is at risk of developing starvation, sarcopenia, cachexia (e.g. cancer cachexia or AIDS cachexia), sepsis, diabetes (particularly type I diabetes), muscular dystrophy (e.g. Duchenne Muscular Dystrophy, Becker's MD, Congenital MD, Myotonic dystrophy (e.g. type I and type II), Limb-girdle MD, Emery-Dreifuss MD, Distal MD and Facioscapulohumeral Muscular Dystrophy), Chronic Heart Failure (e.g. diaphragm muscle atrophy resulting from Chronic Heart Failure), Chronic Obstructive Pulmonary Disease (e.g. diaphragm muscle atrophy resulting from Chronic Obstructive Pulmonary Disease), Amyotrophic Lateral Sclerosis (ALS), Spinal Muscular Atrophy, Guillain-Barré syndrome, intensive care unit acquired weakness, immobilization resulting from bone fractures (e.g. hip (femur neck) fractures) or immobilization resulting from an infection; or by administering to the subject a Cytor agonist and/or a Tead1 antagonist, wherein the Cytor agonist (i) comprises RNA comprising a nucleotide sequence as set forth in any one of SEQ ID NOs: 1-15 or a nucleotide sequence with at least 80% sequence identity to a sequence as set forth in any one of SEQ ID NOs: 1-15, wherein the RNA promotes myogenesis in skeletal muscle; (ii) comprises a nucleic acid molecule encoding Cytor RNA or an orthologue thereof or a functionally equivalent fragment or variant of said Cytor RNA or orthologue, wherein the nucleic acid molecule comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 20-34 or a nucleotide sequence with at least 80% sequence identity to a sequence as set forth in any one of SEQ ID NOs: 20-34, wherein the nucleic acid molecule encodes an RNA that promotes myogenesis in skeletal muscle; or (iii) comprises a nucleic acid molecule encoding a protein that induces expression of endogenous Cytor RNA, wherein the protein comprises a domain that binds to the Cytor promoter operably linked to a transcriptional activator, and wherein the protein comprising a domain that binds to the Cytor promoter is a CRISPR associated protein (e.g. a nuclease-deficient CRISPR associated protein, e.g. dCas9) and the agonist further comprises a guide RNA capable of hydridising with the Cytor promoter nucleic acid; and/or wherein the Tead1 antagonist (i′) is verteporfin; (ii′) is an inhibitor, wherein the inhibitor is an aptamer, a ribozyme, a siRNA, a shRNA, an antisense oligonucleotide, an antibody or an antibody mimetic, wherein the antibody mimetic is preferably selected from affibodies, adnectins, anticalins, DARPins, avimers, nanofitins, affilins, Kunitz domain peptides, Fynomers, trispecific binding molecules and probodies; or (iii′) is a nucleotide based inhibitor comprising (a) a nucleic acid sequence which comprises or consists of a nucleic acid sequence being complementary to at least 12 continuous nucleotides of a nucleic acid sequence selected from SEQ ID NOs: 39 to 42, (b) a nucleic acid sequence which comprises or consists of a nucleic acid sequence which is at least 70% identical to the complementary strand of one or more nucleic acid sequences selected from SEQ ID NOs: 39 to 42, (c) a nucleic acid sequence which comprises or consists of a nucleic acid sequence according to (a) or (b), wherein the nucleic acid sequence is DNA or RNA, or (d) an expression vector expressing the nucleic acid sequence as defined in any one of (a) to (c), preferably under the control of a skeletal muscle-specific promoter.

2-5. (canceled)

6. The method of claim 1, wherein the subject has or is at risk of skeletal muscle atrophy.

7. The method of claim 1, wherein the subject is heterozygous or homozygous for the G allele of cis-eQTL rs74360724.

8. The method of claim 1, wherein the subject (i) is at least 45 years old, (ii) is inactive or immobile, and/or (iii) has or is at risk of developing sarcopenia.

9. The method of claim 1, wherein the agonist comprises a nanoparticle containing the RNA or the nucleic acid molecule as defined in claim 1, item (ii).

10. (canceled)

11. (canceled)

12. The method of claim 1, wherein the agonist increases the amount of Cytor RNA in skeletal muscle directly.

13. (canceled)

14. The method of claim 1, wherein the agonist comprises RNA comprising a nucleotide sequence as set forth in any one of SEQ ID NOs:1-15 or a nucleotide sequence with at least 80% sequence identity to a sequence as set forth in any one of SEQ ID NOs:1-15.

15. The method of claim 9, wherein the nanoparticle is a liposome.

16-19. (canceled)

20. The method of claim 1, wherein the nucleic acid molecule comprises a nucleotide sequence as set forth in any one of SEQ ID NOs:20-34 or a nucleotide sequence with at least 80% sequence identity to a sequence as set forth in any one of SEQ ID NOs:20-34.

21. The method of claim 9, wherein the nanoparticle is a viral vector.

22-24. (canceled)

25. The method of claim 1, wherein the agonist inhibits degradation of endogenous Cytor RNA.

26. The method of claim 1, wherein the agonist that inhibits degradation of endogenous Cytor RNA is an RNA binding protein.

27. The method of claim 1, wherein the agonist is a small molecule.

28. The method of claim 1, wherein the antagonist inhibits the expression and/or the activity of Tead1.

29-31. (canceled)

32. A modified farmed animal or farmed fish which has an increased amount of Cytor RNA or a decreased amount of or no Tead1 RNA in its skeletal muscle in comparison to a corresponding unmodified non-human animal, preferably wherein the animal has been genetically-modified to increase the expression of Cytor RNA and/or to decrease or prevent the expression of Tead1 RNA.

33. The modified farmed animal or farmed fish of claim 32, wherein the farmed animal is a poultry, pig, cattle, sheep or goat or the farmed fish is a salmon, tilapia or tuna.

34-36. (canceled)

37. A method for producing the modified farmed animal or farmed fish of claim 32, the method comprising: (i) providing a cell of a farmed animal or farmed fish which has been genetically-modified such that myoblast cells derived from said cell express an increased amount of Cytor RNA and/or a decreased amount of or no Tead1 RNA or protein compared to unmodified myoblast cells; and (ii) generating a genetically-modified farmed animal or farmed fish from said genetically-modified cell of a farmed animal or farmed fish.

38. An in vitro method for: a) producing muscle fibres comprising culturing modified myoblasts which have increased amounts of Cytor RNA and/or a decreased amount of or no Tead1 RNA or protein in comparison to unmodified myoblasts under conditions suitable to produce muscle fibres; b) determining the risk of developing skeletal muscle atrophy (e.g. sarcopenia) in a subject comprising determining the genotype of the subject at the cis-eQTL rs74360724, wherein when the subject is heterozygous or homozygous for the G allele they have an increased risk of developing muscle atrophy (e.g. sarcopenia) compared to a subject that is homozygous for the A allele, and/or at the cis-eQTL rs79200838, wherein when the subject is heterozygous or homozygous for the T allele they have an decreased risk of developing muscle atrophy (e.g. sarcopenia) compared to a subject that is homozygous for the C allele; c) predicting the performance of a subject in an activity associated with fast-twitch muscle (e.g. an athlete, e.g. a sprinter) comprising determining the genotype of the subject at the cis-eQTL rs74360724, wherein when the subject is heterozygous or homozygous for the A allele they have an increased likelihood of outperforming a subject that is homozygous for the G allele, and/or at the cis-eQTL rs79200838, wherein when the subject is heterozygous or homozygous for the C allele they have an decreased of outperforming a subject that is homozygous for the T allele; d) predicting the capability of a subject to produce fast-twitch muscle comprising determining the genotype of the subject at the cis-eQTL rs74360724, wherein when the subject is heterozygous or homozygous for the A allele they have an increased capability of producing fast-twitch muscle compared a subject that is homozygous for the G allele, and/or at the cis-eQTL rs79200838, wherein when the subject is heterozygous or homozygous for the C allele they have an decreased capability of producing fast-twitch muscle compared a subject that is homozygous for the T allele.

39. The in vitro method of claim 38, wherein the myoblasts have been genetically-modified to increase the expression of Cytor RNA and/or to decrease the expression of Tead1 RNA or protein.

40. The in vitro method of claim 38, wherein the myoblasts comprise a nucleic acid molecule encoding Cytor RNA operably linked to a heterologous promoter and/or a mutated or deleted gene encoding Tead1 RNA.

41-46. (canceled)

61; 01; 61; 61; 61; 12; 12; 12; 12

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