Methods and agents for maintaining muscle mass and for preventing muscle atrophy and biomarkers for monitoring same

• US 20060003959A1
• Filed: 06/10/2005
• Published: 01/05/2006
• Est. Priority Date: 06/10/2004
• Status: Abandoned Application

• Current Assignee: University of York
• Original Assignee: University of York
• Inventors: Littman, Daniel, Burden, Steven
• Application Number:
  US11/150,888
• Publication Number:
  US 20060003959A1
• Time in Patent Office: Days
• US Class Current: 514/44.R
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A01K 2217/20 Animal model comprising regulated expression system
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/03 Animal model, e.g. for test or diseases
• CPC Class Codes: A01K 2267/035 Animal model for multifactorial diseases
• CPC Class Codes: A01K 67/0276 Knock-out vertebrates
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• CPC Class Codes: C07K 14/51 Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 2830/008 cell type or tissue specific enhancer/promoter combination
• CPC Class Codes: G01N 33/6887 from muscle, cartilage or connective tissue

Abstract

The present invention relates to methods for stimulating muscle growth and/or for inhibiting muscle atrophy in a subject suffering from or predisposed to having a muscle wasting disease by administering an agent that increases the expression of the runx1 gene or gene product in an individual. The invention further provides a biomarker for a muscle wasting disease or condition and methods for screening or diagnosing a subject having, or predisposed to having, a muscle wasting disease by measuring the level of expression of the runx1 gene, or its target genes or gene products in an individual. The invention further relates to the preparation of pharmaceutical compositions using agents that may aid in muscle growth and that may prevent or inhibit muscle atrophy by increasing the expression and/or activity of the runx1 gene and/or gene product.

Claims

1. A method of stimulating muscle growth in a mammal in need thereof, the method comprising administering to the mammal an effective amount of an agent capable of stimulating runx1 gene expression or activity in a muscle cell.

3. The method of either of claims 1 or 2, wherein the mammal is a human.

4. The method of claim 1, wherein the muscle cell is a myoblast or a satellite cell.

5. The method of claim 1, wherein the muscle cell is a skeletal muscle cell, a cardiac muscle cell, or a smooth muscle cell.

6. The method of claim 1, wherein the muscle growth results from an increase in size of muscle fibers present in the mammal.

7. The method of either of claims 1 or 2, wherein the mammal suffers from a condition involving muscle damage or muscle atrophy/wasting.

8. The method of claim 7, wherein the condition is a skeletal muscle disease.

9. The method of claim 8, wherein the skeletal muscle disease is a myopathy or a dystrophy.

10. The method of claim 8, wherein the skeletal muscle disease results from a neural condition or an injury.

11. The method of claim 10, wherein the injury is a nerve injury.

12. The method of claim 8, wherein the skeletal muscle disease results from a neuropathy.

13. The method of either one of claims 1 or 2, wherein the muscle cell is in a patient suffering from a cardiac muscle disorder.

14. The method of claim 13, wherein the cardiac muscle disorder is selected from the group consisting of a cardiomyopathy, ischemic damage, a degenerative congenital disease and cardiac trauma.

15. The method of either of claims 1 or 2, wherein the muscle cell is found in a patient suffering from a smooth muscle disorder.

16. The method of claim 15, wherein the smooth muscle disorder is selected from the group consisting of arterial sclerosis, a vascular lesion, and a congenital vascular disease.

17. The method of claim 16, wherein the congenital vascular disease is characterized by muscular damage.

18. The method of either of claims 1 or 2, wherein the mammal is a patient who is incapacitated as a result of injury or age, and experiences a decreased level of the runx1 gene or runx1 gene expression compared to an individual who is not incapacitated.

19. The method of either of claims 1 or 2, wherein the muscle cell is found in a patient suffering from myasthenia gravis or a congenital myasthenia gravis syndrome.

20. The method of either of claims 1 or 2, for the prophylaxis or treatment of a mammal having, or at risk for having, a condition involving decreased muscle fiber size, or muscle atrophy, or muscle cell autophagy, the method comprising administering to the mammal an effective amount of an agent that stimulates runx1 gene expression.

2. A method of inhibiting muscle atrophy or muscle wasting in a mammal in need thereof, the method comprising administering to the mammal an effective amount of an agent capable of stimulating runx1 gene expression or activity in a muscle cell.

21. A method for identifying a candidate compound for regulating skeletal muscle mass or function, comprising:

a. contacting a test compound with a cell containing the runx1 gene, or a target of the runx1 gene;

b. determining whether the test compound results in upregulation/activation of a runx1 gene, or a target of a runx1 gene, or a corresponding gene product;

c. selecting a compound that upregulates/activates the runx1 gene, or the target of the runx1 gene or corresponding gene product and further determining whether the test compound increases muscle mass or function in a skeletal muscle atrophy model system in innervated or denervated muscle; and

d. identifying a test compound that modulates muscle mass or function as a candidate compound for regulating skeletal muscle mass or function.

22. The method for identifying candidate compounds according to claim 21, wherein the runx1 gene has a nucleotide sequence that is greater than 80% identical to the sequence of SEQ ID NO:

1.

23. The method for identifying candidate compounds according to claim 21, wherein the runx1 gene has a nucleotide sequence that is greater than 90% identical to the sequence of SEQ ID NO:

1.

24. The method for identifying candidate compounds according to claim 21, wherein the runx1 gene has a nucleotide sequence corresponding to the nucleic acid sequence of SEQ ID NO:

1.

25. The method of claim 21, wherein the cell containing the runx1 gene is a eukaryotic cell.

26. The method of claim 25, wherein the eukaryotic cell is a muscle cell or muscle cell line, or any other eukaryotic cell which contains runx1, or has been genetically engineered to express runx1.

27. The method for identifying candidate compounds according to claim 26, wherein the cell further comprises a reporter gene operatively associated with a runx1 responsive element and wherein measuring cellular runx1 level involves measuring expression of the reporter gene.

28. The method of claim 21, wherein the determining is accomplished by performing an assay for measuring runx1 RNA or protein expression, and wherein the assay selected from the group consisting of an immunoassay, an RNase protection assay and an assay wherein a nucleic acid probe is used for detection of the runx1 gene.

29. A method of determining if a subject is at risk for developing a muscle wasting disease, the method comprising:

(I) measuring an amount of a runx1 gene or a target gene of runx1 or a corresponding gene product in a tissue sample derived from the subject, wherein the runx1 gene or the target of the runx1 gene or corresponding gene product is;

(a) a DNA corresponding to SEQ ID NO;

1, or to any one of SEQ ID NOS;

3-31 or a nucleic acid derived therefrom;

(b) a protein comprising SEQ ID NO;

2, or a protein encoded by the nucleic acid sequence of any one of SEQ ID NOS;

3-31;

(c) a nucleic acid comprising a sequence hybridizable to SEQ ID NO;

1, or to any one of SEQ ID NOS;

3-31 or their complements under conditions of high stringency, or a protein comprising a sequence encoded by the hybridizable sequence;

(d) a nucleic acid at least 90% homologous to SEQ ID NO;

1, or to any one of SEQ ID NOS;

3-31 or their complements as determined using the NBLAST algorithm;

or a protein encoded thereby; and

(II) comparing the amount of the gene product of runx1 or at least one of its target genes in the subject with the amount of gene product of runx1 or at least one of its target genes present in a normal tissue sample obtained from a subject who does not have a muscle wasting disease or in a predetermined standard, wherein a decrease in the amount of the gene product of runx1 or at least one of its target genes in the subject compared to the amount in the normal tissue sample or pre-determined standard indicates a risk of developing a tissue wasting disease in the subject.

30. A method for screening, diagnosing or prognosing of a disease in a subject, wherein the disease is characterized by muscle atrophy selected from the group consisting of myopathies, dystrophies, myoneural conductive diseases, traumatic muscle injury, nerve injury, cardiomyopathies, ischemic damage, congenital disease, and other conditions resulting in muscle wasting, the method comprising:

(I) measuring an amount of a runx1 gene or target gene for runx1 or their corresponding gene product in a tissue sample derived from the subject, wherein the runx1 gene or runx1 target gene or corresponding gene product is;

(a) a DNA corresponding to SEQ ID NO;

1, or to any one of SEQ ID NOS;

3-31 or a nucleic acid derived therefrom;

(b) a protein comprising SEQ ID NO;

2, or a protein encoded by any one of the nucleic acid sequences of SEQ ID NOS;

3-31;

(c) a nucleic acid comprising a sequence hybridizable to SEQ ID NO;

1, or to any one of SEQ ID NOS;

3-31 or their complements under conditions of high stringency, or a protein comprising a sequence encoded by the hybridizable sequence;

(d) a nucleic acid at least 90% homologous to SEQ ID NO;

1, or to any one of SEQ ID NOS;

3-31 or their complement as determined using the NBLAST algorithm;

or a protein encoded thereby; and

(II) comparing the amount of the runx1 gene product or the target of the runx1 gene in the subject with the amount of runx1 gene product or the target of the runx1 gene present in a normal tissue sample obtained from a subject who does not have a muscle wasting disease or in a predetermined standard, wherein a decrease in the amount of the runx1 gene product or the runx1 target gene in the subject compared to the amount in the normal tissue sample or pre-determined standard indicates the presence of a tissue wasting disease in the subject.

31. A method for determining a predisposition to, onset of, or presence of a muscle wasting disease in a subject, comprising detecting in the subject the existence of a change in the level of a runx1 gene, or a runx1 target gene or corresponding gene product, as set forth in SEQ ID NOs:

1-31.

32. The method of claim 31, comprising the steps of:

a) obtaining a tissue biopsy from the subject;

b) permeabilizing the cells in the tissue biopsy;

c) incubating the tissue biopsy or cells isolated from the tissue biopsy with one of the following;

i) an antibody specific for the runx1 gene product, or for the gene product of the runx1 target gene;

ii) a nucleic acid probe specific for the runx1 gene, or for the runx1 target gene;

d) detecting and quantifying the amount of antibody or nucleic acid probe bound; and

e) comparing the amount of antibody or nucleic acid probe bound in the biopsy sample in the subject to the amount of antibody or nucleic acid probe bound in a normal tissue or cellular sample;

wherein the amount of labeled antibody or nucleic acid probe bound correlates inversely with the predisposition, the onset or the presence of a muscle wasting disease in a subject.

33. A biomarker associated with and/or predictive of a muscle wasting disease or condition comprising a runx1 gene or gene product, a runx1 target gene or its corresponding gene product, or a nucleic acid sequence hybridizable to a runx1 gene or a runx1 target gene or their corresponding gene products, under conditions of high stringency.

34. The biomarker of claim 33, wherein the runx1 gene comprises the nucleic acid sequence as set forth in SEQ ID NO:

1, and wherein a runx1 target gene is selected from the group consisting of any one of SEQ ID NOS;

3-31, or combinations thereof.

35. The biomarker of claim 33, wherein the runx1 gene encodes a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO. 2.

36. The biomarker of claim 33, wherein the muscle wasting disease or condition is a skeletal muscle wasting disease or condition.

37. The biomarker of claim 36, wherein the skeletal muscle wasting disease is a myopathy or a dystrophy.

38. The biomarker of claim 36, wherein the skeletal muscle wasting disease or condition is the result of a neural disorder, a neuropathy or a nerve injury.

39. The biomarker of claim 36, wherein the skeletal muscle wasting disease or condition is the result of an accident or injury.

40. The biomarker of claim 33, wherein the muscle wasting disease or condition is characterized by muscle damage, muscle atrophy or muscle cell autophagy.

41. The biomarker of claim 40, wherein the muscle damage, muscle atrophy or muscle cell autophagy occurs in skeletal muscle, cardiac muscle or smooth muscle.

42. The biomarker of claim 41, wherein the muscle damage, muscle atrophy, or muscle cell autophagy occurs in skeletal muscle and is the result of a muscle wasting disease or condition, a muscle injury, or a result of aging.

43. The biomarker of claim 41, wherein the muscle damage, muscle atrophy, or muscle cell autophagy occurs in cardiac muscle and is the result of a cardiomyopathy, an ischemic event, a degenerative congenital disease or any cardiac trauma.

44. The biomarker of claim 41, wherein the muscle damage, muscle atrophy, or muscle cell autophagy occurs in smooth muscle and is the result of arterial sclerosis, a vascular lesion, or a congenital vascular disease.