Compound and method for treating myotonic dystrophy
An antisense compound for use in treating myotonic dystrophy DM1 or DM2, a method of enhancing antisense targeting to heart and quadricep muscles, and a method for treating DM1 or DM2 in a mammalian subject are disclosed. The oligonucleotide has 8-30 bases, with at least 8 contiguous bases being complementary to the polyCUG or polyCCUG repeats in the 3′UTR region of dystrophia myotonica protein kinase (DMPK) mRNA in DM1 or DM2, respectively. Conjugated to the oligonucleotide is a cell-penetrating peptide having the sequence (RXRR(B/X)R) 2 XB, where R is arginine; B is β-alanine; and each X is —C(O)—(CH 2 ) n —NH—, where n is 4-6. The antisense compound is effective to selectively block the sequestration of muscleblind-like 1 protein (MBNL1) and/or CUGBP, in heart and quadricep muscle in a myotonic dystrophy animal model.
1. A peptide-antisense conjugate comprising a cell-penetrating peptide (CPP) covalently linked to a morpholino antisense oligomer, wherein:
the morpholino antisense oligomer comprises 12 to 30 morpholino subunits linked by phosphorus-containing linkages, wherein each morpholino ring supports a base pairing moiety, and wherein at least 12 contiguous base pairing moieties are complementary to a target muscle protein mRNA;
the target muscle protein is selected from the group consisting of dystrophin and dystrophia myotonica protein kinase;
the CPP is of the formula (RXRRBR) 2 (SEQ ID NO: 71) wherein R is arginine, X is 6-aminohexanoic acid, and B is 3-aminopropionic acid; and
the CPP is covalently linked to the 3′ or 5′ terminal morpholino subunit of the morpholino antisense oligomer at the CPP carboxy terminus by an XB linker.
2. The peptide-antisense conjugate of claim 1 , wherein the CPP is linked to the 3′ terminal morpholino subunit.
3. The peptide-antisense conjugate of claim 1 , wherein the CPP and linker taken together are of the formula:
wherein R′ is selected from H and acetyl.
4. The peptide-antisense conjugate of claim 3 , wherein the CPP is conjugated to the 5′ terminal morpholino subunit of the morpholino antisense oligomer, wherein the 5′ terminal morpholino subunit, CPP, and linker taken together are of the formula:
wherein:
R 1 is C 1 -C 6 alkyl, and
P j is a purine or pyrimidine base-pairing moiety.
5. The peptide-antisense conjugate of claim 4 , wherein R 1 is selected from methyl, ethyl, isopropyl, n-butyl, isobutyl, and t-butyl.
6. The peptide-antisense conjugate of claim 4 , wherein R 1 is methyl.
7. The peptide-antisense conjugate of claim 4 , wherein P j is selected from adenine, cytosine, guanine, uracil, thymine, inosine, hypoxanthine, and 5-methyl cytosine.
8. A peptide-antisense conjugate of formula:
or a pharmaceutically acceptable salt thereof, wherein:
R is selected from H and acetyl;
each R 1 is independently a C 1 -C 6 alkyl;
X is an integer from 3 to 19;
each P j is independently a purine or pyrimidine base-pairing moiety, wherein, taken together, the P j 's comprise a targeting sequence;
R 2 is selected from H and acetyl;
R 3 is selected from an electron pair and H;
the targeting sequence is complementary to a target skeletal muscle protein mRNA and/or a target cardiac muscle protein mRNA; and
the target muscle protein is selected from the group consisting of dystrophin and dystrophia myotonica protein kinase.
9. The peptide-antisense conjugate of claim 8 , wherein each R 1 is independently selected from methyl, ethyl, isopropyl, n-butyl, isobutyl, and t-butyl.
10. The peptide-antisense conjugate of claim 8 , wherein each R 1 is methyl.
11. The peptide-antisense conjugate of claim 8 , wherein each P j is independently selected from adenine, cytosine, guanine, uracil, thymine, inosine, hypoxanthine, and 5-methyl cytosine.