Myostatin Isoform

Abstract

The present invention provides splice variants of myostatin that promote muscle growth, and include polynucleotides and polypeptide sequences, constructs comprising the sequences and compositions for regulating muscle growth and treating diseases associated with muscle tissue. The splice variants include the consensus sequence X 1 I F L E X 2 X 3 X 4 Q X 5 C S I L X 6 X 7 X 8 X 9 X 10 wherein X 1 is I or L, X 2 is V or L, X 3 is Y, C, G or S, X 4 is I or F, X 5 is F or L, X 6 is G or E, X 7 is E or V, X 8 is A or T, X 9 is A or V and X 10 is absent, F or L. The present invention also provides for the use of the present sequences in identifying animals with altered muscle mass, and for use in selective breeding programs to produce animals with altered muscle mass.

1 . An isolated polypeptide comprising an amino acid sequence having the formula:
 X 1  I F L E X 2  X 3  X 4  Q X 5  C S I L X 6  X 7  X 8  X 9  X 10      
       wherein X 1  is I or L, X 2  is V or L, X 3  is Y, C, G or S, X 4  is I or F, X 5  is F or L, X 6  is G or E, X 7  is E or V, X 8  is A or T, X 9  is A or V and X 10  is absent, F or L. 
     
     
         2 . An isolated polypeptide that is selected from:
 (a) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOS: 48-95;   (b) a polypeptide that comprises a fragment or variant of a polypeptide in (a); and   (c) a polypeptide having at least 95%, 90%, 80% or 70% sequence identity with a polypeptide of (a), wherein the polypeptide is capable of promoting myoblast cell growth.   
     
     
         3 . (canceled) 
     
     
         4 . An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of:
 (a) a polynucleotide that encodes X 1  I F L E X 2  X 3  X 5  C S I L X 6  X 7  X 8  X 9  X 10  wherein X 1  is I or L, X 2  is V or L, X is Y, C, G or S, X is I or F, X 5  is F or L, X 6  is G or E, X 7  is E or V, X 8  is A or T, X 9  is A or V and X 10  is absent, F or L,   (b) a polynucleotide that encodes an isolated polypeptide that is selected from the group consisting of:
 (i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOS: 48-95; 
   (ii) a polypeptide that comprises a fragment or variant of a polypeptide in (a); and
 (iii) a polypeptide having at least 95%, 90%, 80% or 70% sequence identity with a polypeptide of (a), wherein the polypeptide is capable of promoting myoblast cell growth; 
   (c) a polynucleotide comprising a nucleotide sequence of any one of SEQ ID NOS: 1 to 5, 7 to 26, 28 to 47 or 96;   (d) a polynucleotide that comprises a fragment or variant of a polynucleotide in   (e) a polynucleotide having at least 95%, 90%, 80% or 70% sequence identity with a polynucleotide in (c):   (f) a polynucleotide which comprises a nucleotide sequence that is a complement of any one of (a) to (e);   (g) a reverse complement of any one of (a) to (e);   (h) a polynucleotide consisting of the sequence of SEQ ID NO: 6; and   (i) a polynucleotide consisting of the sequence of SEQ ID NO: 27.   
     
     
         5 . (canceled) 
     
     
         6 . (canceled) 
     
     
         7 . (canceled) 
     
     
         8 . (canceled) 
     
     
         9 . A vector comprising one or more of the polynucleotide sequences according to  claim 4 . 
     
     
         10 . A vector according to  claim 9 , wherein said vector is an expression vector comprising in operable linkage:
 (a) a promoter sequence;   (b) a polynucleotide according to  claim 4 ; and   (c) a gene termination sequence.   
     
     
         11 . (canceled) 
     
     
         12 . A vector according to  claim 10 , wherein the polynucleotide is in a sense orientation or an antisense orientation. 
     
     
         13 . (canceled) 
     
     
         14 . A host cell comprising a vector according to  claim 9 . 
     
     
         15 . A host animal comprising one or a plurality of host cells according to  claim 14 . 
     
     
         16 . (canceled) 
     
     
         17 . (canceled) 
     
     
         18 . (canceled) 
     
     
         19 . (canceled) 
     
     
         20 . A method for regulating muscle growth, comprising administering a composition that is selected from the group consisting of:
 (a) a polypeptide according to  claim 1  or  2 ;   (b) a polynucleotide according to  claim 4 ;   (c) a vector according to  claim 9 ; and   (d) a vector according to  claim 10 .   
     
     
         21 . A method for regulating muscle growth comprising administering a composition comprising a polynucleotide consisting of SEQ ID NO: 6 or SEQ ID NO: 27. 
     
     
         22 . A method for treating a disease associated with muscle tissue, comprising administering a composition that is selected from the group consisting of:
 (a) a polypeptide according to  claim 1  or  2 ;   (b) a polynucleotide according to  claim 4 ;   (c) a vector_according to  claim 9 ; and   (d) a vector according to  claim 10 .   
     
     
         23 . A method of treating a disease associated with muscle tissue, comprising administering a composition comprising a polynucleotide consisting of SEQ ID NO: 6 or SEQ ID NO: 27. 
     
     
         24 . The method of  claim 22 , wherein the disease associated with muscle tissue comprises a condition that is associated with muscle atrophy, selected from the group consisting of muscular dystrophy, muscle cachexia, atrophy, hypertrophy, amyotrophic lateral sclerosis (ALS), disease-associated muscle atrophy comprising muscle wasting associated with cancer or HIV/AIDS, and a disease associated with cardiac muscle. 
     
     
         25 . (canceled) 
     
     
         26 . (canceled) 
     
     
         27 . (canceled) 
     
     
         28 . The method according to  claim 24 , wherein the disease associated with cardiac muscle comprises infarct. 
     
     
         29 . A modulator of MSV gene expression comprising a composition that is able to specifically bind to a polynucleotide according to  claim 4 . 
     
     
         30 . A modulator of MSV gene expression comprising a composition that is able to specifically bind to a polynucleotide consisting of SEQ ID NO: 6 or SEQ ID NO: 27. 
     
     
         31 . (canceled) 
     
     
         32 . The modulator of MSV gene expression according to  claim 29 , wherein the compound is an RNAi or siRNA molecule. 
     
     
         33 . A method for treating a disease associated with muscle tissue in a patient, comprising administering to said patient a modulator according to  claim 29 . 
     
     
         34 . The method according to  claim 33 , wherein the disease associated with muscle tissue is a disease associated with muscle atrophy, selected from the group consisting of muscular dystrophy, muscle cachexia, atrophy, hypertrophy, amyotrophic lateral sclerosis (ALS), disease-associated muscle atrophy comprising muscle wasting associated with cancer or HIV/AIDS, and a disease associated with cardiac muscle. 
     
     
         35 . (canceled) 
     
     
         36 . (canceled) 
     
     
         37 . (canceled) 
     
     
         38 . The method according to  claim 34 , wherein the disease associated with cardiac muscle comprises infarct. 
     
     
         39 . A method for modulating MSV activity, comprising contacting a MSV propeptide with a propeptide convertase, under conditions and for a time sufficient for the convertase to alter proteolytic processing of the MSV propeptide into an active MSV peptide, and thereby modulating MSV activity. 
     
     
         40 . The method of  claim 39  wherein the contacting step comprises contacting the MSV propeptide with an agonist or antagonist of the convertase. 
     
     
         41 . The method of  claim 39 , wherein the propeptide convertase comprises a furin endoprotease. 
     
     
         42 . A method for treating a disease associated with muscle tissue, comprising administering a propeptide convertase to a subject having a disease associated with muscle tissue, said subject comprising a MSV propeptide, under conditions and for a time sufficient for the convertase to alter proteolytic processing of the MSV propeptide into an active MSV peptide, and thereby treating the disease. 
     
     
         43 . The method of  claim 42 , wherein the administering step further comprises contacting the MSV propeptide with an agonist or antagonist of the convertase. 
     
     
         44 . The method according to  claim 42 , wherein the propeptide convertase is furin endoprotease. 
     
     
         45 . The method according to  claim 42 , wherein the disease associated with muscle tissue is a disease associated with muscle atrophy, selected from the group consisting of muscular dystrophy, muscle cachexia, atrophy, hypertrophy, amyotrophic lateral sclerosis (ALS), disease-associated muscle atrophy comprising muscle wasting associated with cancer or HIV/AIDS, and a disease associated with cardiac muscle. 
     
     
         46 . (canceled) 
     
     
         47 . (canceled) 
     
     
         48 . (canceled) 
     
     
         49 . The method according to  claim 45 , wherein the disease associated with cardiac muscle comprises infarct. 
     
     
         50 . A method of regulating muscle growth of an animal comprising administering to said animal any one of: a composition according to  claim 16 , a modulator of MSV gene expression according to  claim 29  or an MSV propeptide that has been contacted with a propeptide convertase or an agonist or antagonist of a propeptide convertase. 
     
     
         51 . The method according to  claim 50 , wherein the propeptide convertase is a furin endoprotease. 
     
     
         52 . The method according to  claim 50 , wherein the animal has an increased muscle mass. 
     
     
         53 . A method of predicting muscle mass in an animal, comprising the steps of:
 i) obtaining a sample from the animal,   ii) determining a gene expression level from a polynucleotide having a sequence of any one of SEQ ID NOS: 1 to 47 or 96, a polynucleotide having 95%, 90%, 80% or 70% identity to SEQ ID NOS: 1 to 47 or 96 or a fragment or variant thereof,   (iii) comparing the gene expression level to an average; and   (iv) predicting the muscle mass of said animal.   
     
     
         54 . The method according to  claim 53 , wherein the level of gene expression is determined by a method comprising nucleic acid hybridization under stringent conditions to the polynucleotide. 
     
     
         55 . The method according to  claim 53 , wherein the level of gene expression is determined using RT-PCR or northern analysis. 
     
     
         56 . A method of predicting muscle mass in an animal, including the steps of:
 i) obtaining a sample from the animal,   ii) determining an amount of a polypeptide having a sequence of any one of SEQ ID NOS: 48 to 95, a polypeptide having 95%, 90%, 80% or 70% identity to any one of SEQ ID NOS: 48 to 95, or a fragment or variant thereof,   iii) comparing amount of polypeptide to an average; and   iv) predicting the muscle mass of said animal.   
     
     
         57 . The method according to  claim 56 , wherein the amount of polypeptide is determined by a method comprising detection of the polypeptide with an antibody that specifically binds to said polypeptide. 
     
     
         58 . The method according to  claim 56 , wherein the amount of polypeptide is determined using ELISA or western blot analysis. 
     
     
         59 . A method of increasing the muscle mass of one or more offspring of an animal comprising the steps of;
 i) selecting one or more animals predicted to have an increase in muscle mass by the method according to either of  claim 53  or  56 , and   ii) breeding the one or more animals selected to have an increased muscle mass to produce one or more offspring having increased muscle mass.   
     
     
         60 . The method according to  claim 59 , wherein the animal is selected from the group consisting of sheep, cattle, deer, poultry, turkey, pig, horse, mouse, rat and human. 
     
     
         61 . A mammalian antibody that specifically binds a polypeptide having
 (a) a sequence of any one of SEQ ID NOS: 48 to 95, or   (b) a sequence having 95%, 90%, 80% or 70% sequence identity to any one of SEQ ID NOS: 48 to 95.   
     
     
         62 . The antibody according to  claim 61  or a fragment or derivative thereof that is able to bind polypeptide having a sequence of any one of SEQ ID NOS: 47 to 95 or a polypeptide having 95%, 90% or 70% sequence identity to any one of SEQ ID NOS: 47 to 95. 
     
     
         63 . The method of  claim 20 , wherein said composition is an antisense polynucleotide that is capable of inhibiting or substantially impairing expression of a polypeptide product by a polynucleotide comprising a nucleotide sequence selected from the group consisting of:
 (a) a polynucleotide that encodes X 1  I F L E X 2  X 3  X 4  Q X 5  C S I L X 6  X 7  X 8  X 9  X 10  wherein X 1  is I or L, X 2  is V or L, X 3  is Y, C, G or S, X 4  is I or F, X 5  is F or L, X 6  is G or E, X 7  is E or V, X 8  is A or T, X 9  is A or V and X 10  is absent, F or L,   (b) a polynucleotide that encodes an isolated polypeptide that is selected from the group consisting of:
 (i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOS: 48-95; 
 (ii) a polypeptide that comprises a fragment or variant of a polypeptide in (a); and 
 (iii) a polypeptide having at least 95%, 90%, 80% or 70% sequence identity with a polypeptide of (a), wherein the polypeptide is capable of promoting myoblast cell growth; 
   (c) a polynucleotide comprising a nucleotide sequence of any one of SEQ ID NOS: 1 to 5, 7 to 26, 28 to 47 or 96;   (d) a polynucleotide that comprises a fragment or variant of a polynucleotide in (c);   (e) a polynucleotide having at least 95%, 90%, 80% or 70% sequence identity with a polynucleotide in (c);   (f) a polynucleotide which comprises a nucleotide sequence that is a complement of any one of (a) to (e);   (g) a reverse complement of any one of (a) to (e);   (h) a polynucleotide consisting of the sequence of SEQ ID NO: 6; and   (i) a polynucleotide consisting of the sequence of SEQ ID NO: 27.   
     
     
         64 . The method of  claim 22 , wherein said composition is an antisense polynucleotide that is capable of inhibiting or substantially impairing expression of a polypeptide product by a polynucleotide comprising a nucleotide sequence selected from the group consisting of:
 (a) a polynucleotide that encodes X 1  I F L E X 2  X 3  X 4  Q X 5  C S I L X 6  X 7  X 8  X 9  X 10  wherein X 1  is I or L, X 2  is V or L, X 3  is Y, C, G or S, X 4  is I or F, X 5  is F or L, X 6  is G or E, X 7  is E or V, X 8  is A or T, X 9  is A or V and X 10  is absent, F or L,   (b) a polynucleotide that encodes an isolated polypeptide that is selected from the group consisting of:
 (i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOS: 48-95; 
 (ii) a polypeptide that comprises a fragment or variant of a polypeptide in (a); and 
 (iii) a polypeptide having at least 95%, 90%, 80% or 70% sequence identity with a polypeptide of (a), wherein the polypeptide is capable of promoting myoblast cell growth; 
   (c) a polynucleotide comprising a nucleotide sequence of any one of SEQ ID NOS: 1 to 5, 7 to 26, 28 to 47 or 96;   (d) a polynucleotide that comprises a fragment or variant of a polynucleotide in (c);   (e) a polynucleotide having at least 95%, 90%, 80% or 70% sequence identity with a polynucleotide in (c);   (f) a polynucleotide which comprises a nucleotide sequence that is a complement of any one of (a) to (e);   (g) a reverse complement of any one of (a) to (e);   (h) a polynucleotide consisting of the sequence of SEQ ID NO: 6; and   (i) a polynucleotide consisting of the sequence of SEQ ID NO: 27.   
     
     
         65 . The modulator of  claim 29 , wherein said composition is:
 (1) an antisense polynucleotide that is capable of inhibiting or substantially impairing expression of a polypeptide product by a polynucleotide comprising a nucleotide sequence selected from the group consisting of:
 (a) a polynucleotide that encodes X 1  I F L E X 2  X 3  X 4  Q X 5  C S I L X 6  X 7  X 8  X 9  X 10  wherein X 1  is I or L, X 2  is V or L, X 3  is Y, C, G or S, X 4  is I or F, X 5  is F or L, X 6  is G or E, X 7  is E or V, X 8  is A or T, X 8  is A or V and X 10  is absent, F or L, 
 (b) a polynucleotide that encodes an isolated polypeptide that is selected from the group consisting of:
 (i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOS: 48-95; 
 (ii) a polypeptide that comprises a fragment or variant of a polypeptide in (a); and 
 (iii) a polypeptide having at least 95%, 90%, 80% or 70% sequence identity with a polypeptide of (a), wherein the polypeptide is capable of promoting myoblast cell growth; 
 
 (c) a polynucleotide comprising a nucleotide sequence of any one of SEQ ID NOS: 1 to 5, 7 to 26, 28 to 47 or 96; 
 (d) a polynucleotide that comprises a fragment or variant of a polynucleotide in (c); 
 (e) a polynucleotide having at least 95%, 90%, 80% or 70% sequence identity with a polynucleotide in (c); 
 (f) a polynucleotide which comprises a nucleotide sequence that is a complement of any one of (a) to (e); 
 (g) a reverse complement of any one of (a) to (e); 
 (h) a polynucleotide consisting of the sequence of SEQ ID NO: 6; and 
 (i) a polynucleotide consisting of the sequence of SEQ ID NO: 27, or 
   (2) an interfering RNA molecule.