Antisense composition and method for treating muscle atrophy
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.
1. An antisense morpholino oligonucleotide of 15-40 bases, comprising a base sequence that is complementary to at least 12 contiguous bases of SEQ ID NO: 6, wherein the antisense morpholino oligonucleotide inhibits the expression of human myostatin in a cell, and wherein the antisense morpholino oligonucleotide comprises phosphorus-containing intersubunit linkages, in accordance with the structure:
wherein each intersubunit linkage is independently selected from the group where Y 1 is O, Z is 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, alkyl amino, dialkylamino, or 1-piperazine.
2. The antisense morpholino oligonucleotide of claim 1 , wherein the antisense morpholino oligonucleotide is conjugated to an arginine-rich peptide.
3. The antisense morpholino oligonucleotide of claim 1 , wherein at least 2 and no more than half of the total number of phosphorus-containing intersubunit linkages are positively charged at physiological pH.
4. The antisense morpholino oligonucleotide of claim 3 , wherein
X, for any uncharged linkages, is independently alkyl, alkoxy, thioalkoxy, or an alkyl amino of the form NR 2 , where each R is independently hydrogen or methyl, and X, for any positively charged linkages, is 1-piperazine.
5. The antisense morpholino oligonucleotide of claim 2 , wherein the arginine rich peptide is selected from SEQ ID NOS: 7-9.
6. The antisense morpholino oligonucleotide of claim 2 , wherein the arginine-rich peptide is covalently coupled at its C terminus to the 5′ end of the antisense morpholino oligonucleotide.
7. The antisense morpholino oligonucleotide of claim 1 , wherein the base sequence is complementary to at least 12 contiguous bases of SEQ ID NO: 10.
8. The antisense morpholino oligonucleotide of claim 7 , wherein the base sequence is SEQ ID NO: 1.
9. The antisense morpholino oligonucleotide of claim 1 , wherein the base sequence is complementary to at least 12 contiguous bases of a splice site in a preprocessed human myostatin transcript.
10. The antisense morpholino oligonucleotide of claim 9 , wherein the splice site in the preprocessed myostatin transcript has a sequence selected from SEQ ID NOS: 11-14.
11. The antisense morpholino oligonucleotide of claim 10 , wherein the base sequence is selected from SEQ ID NOS: 2-5.