Antisense composition and method for treating muscle atrophy

Abstract

A method and compound for treating skeletal muscle mass deficiency in a human subject are disclosed. The composition is an oligomer of morpholino subunits and phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, contains between 10-40 nucleotide bases, has a base sequence effective to hybridize to an expression-sensitive region of processed or preprocessed human myostatin RNA transcript, identified, in its processed form, by SEQ ID NO:6, and is capable of uptake by target muscle cells in the subject. In practicing the method, the compound is administered in an amount and at a dosage schedule to produce an overall reduction in the level of serum myostatin measured in the patient, and preferably to bring the myostatin level within the a range determined for normal, healthy individuals.

It is claimed: 
     
         1 . A method of treating skeletal muscle mass deficiency in a human subject comprising
 (a) measuring blood or tissue levels of myostatin in the subject,   (b) administering to the subject, a myostatin-expression-inhibiting amount of a substantially uncharged antisense compound (i) composed of morpholino subunits and phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit. (ii) capable of uptake by target muscle cells in the subject, (iii) containing between 10-40 nucleotide bases, and (iv) having a base sequence effective to hybridize to an expression-sensitive region of processed or preprocessed human myostatin RNA transcript, identified, in its processed form, by SEQ ID NO:6,   (c) by said administering, forming within target muscle cells in the subject, a base-paired heteroduplex structure composed of human myostatin RNA transcript and the antisense compound and having a Tm of dissociation of at least 45° C., thereby inhibiting expression of myostatin in said cells,   (d) at a selected time following said administering, measuring a blood or tissue level of myostatin in the subject, and   (e) repeating said administering, using the myostatin levels measured in (d) to adjust the dose or dosing schedule of the amount of antisense compound administered, if necessary, so as to reduce measured levels of myostatin over those initially measured and to maintain such levels of myostatin measured in step (d) within a range determined for normal, healthy individuals.   
     
     
         2 . The method of  claim 1 , wherein the myostatin value measured in step (a) is above a selected threshold for normal healthy people. 
     
     
         3 . The method of  claim 1 , wherein the morpholino subunits in the compound administered are joined by phosphorodiamidate linkages, in accordance with the structure: 
       
         
           
           
               
               
           
         
         where Y 1 =O, Z=O, Pj is a purine or pyrimidine base-pairing moiety effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide, and X is alkyl, alkoxy, thioalkoxy, amino or alkyl amino, including dialkylamino. 
       
     
     
         4 . The method of  claim 1 , wherein the compound is administered as a conjugate of the compound and an arginine-rich polypeptide effective to promote uptake of the compound into muscle target cells. 
     
     
         5 . The method of  claim 2 , in which at least 2 and no more than half of the total number of intersubunit linkages are positively charged at physiological pH. 
     
     
         6 . The method of  claim 2 , wherein said morpholino subunits are joined by phosphorodiamidate linkages, in accordance with the structure: 
       
         
           
           
               
               
           
         
         where Y 1 =O, Z=O, Pj is a purine or pyrimidine base-pairing moiety effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide, and X for the uncharged linkages is alkyl, alkoxy, thioalkoxy, or an alkyl amino of the form wherein NR 2 , where each R is independently hydrogen or methyl, and for the positively charged linkages, X is 1-piperazine. 
       
     
     
         7 . The method of  claim 4 , wherein the arginine rich peptide has one of the sequences identified as SEQ ID NOS:7-9, and the arginine-rich peptide is covalently coupled at its C terminus to the 5′ end of the antisense compound. 
     
     
         8 . The method of  claim 1 , wherein said antisense compound is effective to hybridize to a target region at or adjacent the start site of the processed human myostatin transcript, the compound has a base sequence that is complementary to a target region containing at least 12 contiguous bases in a processed human myostatin transcript identified by SEQ ID NO:10, and formation of the heteroduplex in step (c) is effective to block translation of said processed transcript. 
     
     
         9 . The method of  claim 8 , wherein the antisense compound includes a base sequence identified by the sequence SEQ ID NO:1. 
     
     
         10 . The method of  claim 1 , wherein the antisense compound is effective to hybridize to a splice site of preprocessed human myostatin transcript, and has a base sequence that is complementary to at least 12 contiguous bases of a splice site in a preprocessed human myostatin transcript, and formation of the heteroduplex in step (c) is effective to block processing of a preprocessed myostatin transcript to produce a full-length, processed myostatin transcript. 
     
     
         11 . The method of  claim 10 , wherein the splice site in the preprocessed myostatin transcript has a sequence selected from the group consisting of: SEQ ID NOS: 11-14. 
     
     
         12 . The method of  claim 11 , wherein the antisense compound includes a base sequence selected from the group consisting of: SEQ ID NOS: 2-5. 
     
     
         13 . The method of  claim 1 , wherein said administering and measuring is carried out for a selected period of at least 2 weeks. 
     
     
         14 . The method of  claim 1 , wherein said administering is by oral administration. 
     
     
         15 . An antisense composition for use in increasing skeletal muscle mass in a human subject comprising a substantially uncharged antisense compound
 (i) composed of morpholino subunits and phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit.   (ii) capable of uptake by target muscle cells in the subject,   (iii) containing between 10-40 nucleotide bases, and   (iv) having a base sequence effective to hybridize to an expression-sensitive region of processed or preprocessed human myostatin RNA transcript, identified, in its processed form, by SEQ ID NO:6.   
     
     
         16 . The composition of  claim 15 , wherein the morpholino subunits in the compound are joined by phosphorodiamidate linkages, in accordance with the structure: 
       
         
           
           
               
               
           
         
         where Y 1 =O, Z=O, Pj is a purine or pyrimidine base-pairing moiety effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide, and X is alkyl, alkoxy, thioalkoxy, amino or alkyl amino, including dialkylamino. 
       
     
     
         17 . The composition of  claim 15 , which includes said compound conjugated to an arginine-rich polypeptide effective to promote uptake of the compound into target muscle cells. 
     
     
         18 . The composition of  claim 15 , in which a least 2 and no more than half of the total number of intersubunit linkages are positively charged at physiological pH. 
     
     
         19 . The composition of  claim 18 , wherein said morpholino subunits are joined by phosphorodiamidate linkages, in accordance with the structure: 
       
         
           
           
               
               
           
         
         where Y 1 =O, Z=O, Pj is a purine or pyrimidine base-pairing moiety effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide, and X for the uncharged linkages is alkyl, alkoxy, thioalkoxy, or an alkyl amino of the form wherein NR 2 , where each R is independently hydrogen or methyl, and for the positively charged linkages, X is 1-piperazine. 
       
     
     
         20 . The composition of  claim 17 , wherein the arginine rich peptide has one of the sequences identified as SEQ ID NOS: 7-9. 
     
     
         21 . The composition of  claim 17 , arginine-rich peptide is covalently coupled at its C terminus to the 5′ end of the antisense compound. 
     
     
         22 . The composition of  claim 15 , wherein said antisense compound is effective to hybridize to a target region at or adjacent the start site of the processed human myostatin transcript, the compound has a base sequence that is complementary to a target region containing at least 12 contiguous bases in a processed human myostatin transcript identified by SEQ ID NO:10, and formation of the heteroduplex in step (c) is effective to block translation of said processed transcript. 
     
     
         23 . The composition of  claim 22 , wherein the antisense compound includes a base sequence identified by the sequence SEQ ID NO: 1. 
     
     
         24 . The composition of  claim 15 , wherein the antisense compound is effective to hybridize to a splice site of preprocessed human myostatin transcript, and has a base sequence that is complementary to at least 12 contiguous bases of a splice site in a preprocessed human myostatin transcript, and formation of the heteroduplex in step (c) is effective to block processing of a preprocessed myostatin transcript to produce a full-length, processed myostatin transcript. 
     
     
         25 . The composition of  claim 24 , wherein the splice site in the preprocessed myostatin transcript has a sequence selected from the group consisting of: SEQ ID NOS: 11-14. 
     
     
         26 . The composition of  claim 25 , wherein the antisense compound includes a base sequence selected from the group consisting of: SEQ ID NOS: 2-5. 
     
     
         27 . A method of measuring myostatin expression levels in a mammalian subject, comprising
 (a) administering to the subject, a myostatin-expression-inhibiting amount of a substantially uncharged antisense compound (i) composed of morpholino subunits and phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit. (ii) capable of uptake by target muscle cells in the subject, (iii) containing between 10-40 nucleotide bases, (iv) having a base sequence effective to hybridize to a region of processed or preprocessed human myostatin RNA transcript, identified, in its processed form, by SEQ ID NO:6, and (v) capable of forming with a such myostatin RNA transcript, a base-paired heteroduplex structure composed of human myostatin RNA transcript and the antisense compound and having a Tm of dissociation of at least 45° C.,   (b) within about 8-72 hours following said administering, analyzing a body-fluid sample obtained from the subject for the presence of a heteroduplex composed of said antisense compound and a complementary region of said myostating RMA transcript, to determine the concentration of transcript in said sample.