Targeting DMPK with Antisense Oligonucleotide Improves Muscle Strength in Myotonic Dystrophy Type 1 Mice

Abstract

Myotonic dystrophy type 1 (DM1), a dominant hereditary muscular dystrophy, is caused by an abnormal expansion of a (CTG)_n trinucleotide repeat in the 3' UTR of the human dystrophia myotonica protein kinase (DMPK) gene. As a consequence, mutant transcripts containing expanded CUG repeats are retained in nuclear foci and alter the function of splicing regulatory factors members of the MBNL and CELF families, resulting in alternative splicing misregulation of specific transcripts in affected DM1 tissues. In the present study, we treated DMSXL mice systemically with a 2'-4'-constrained, ethyl-modified (ISIS 486178) antisense oligonucleotide (ASO) targeted to the 3' UTR of the DMPK gene, which led to a 70% reduction in CUG^exp RNA abundance and foci in different skeletal muscles and a 30% reduction in the heart. Furthermore, treatment with ISIS 486178 ASO improved body weight, muscle strength, and muscle histology, whereas no overt toxicity was detected. This is evidence that the reduction of CUG^exp RNA improves muscle strength in DM1, suggesting that muscle weakness in DM1 patients may be improved following elimination of toxic RNAs.

Keywords: gene therapy; muscle; muscular dystrophy; myotonic dystrophy; oligonucleotide.

Figure 1

In Vitro Evaluation of ISIS 486178 and ISIS 445569 ASOs in DM1 Muscle Satellite Cells (A) ASO targets on exon 15 of the DMPK mRNA transcript variant 1 (GenBank: NM_001081563). (B) Dose effect curves of ISIS 486178 and ISIS 445569 ASOs estimated by foci reduction in myotubes (n = 10). (C) FISH quantification of foci per nucleus reduction by ASO treatment in DM3200 myotubes (n = 6). (D) Northern blot shows expanded and normal DMPK mRNA after treatment by ASOs. Probes were hybridized and revealed one at a time on the same membrane. (E) Quantification of the reduction of DMPK mRNA from northern blot autoradiograms (n = 2). (F) Inclusion of mis-spliced events SORBS1, CAMK2G, DMD, PDLIM3, and TTN (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001 by unpaired two-tailed t test.

Figure 2

Evaluation of ISIS 486178 and ISIS 445569 ASOs upon Systemic Administration in DMSXL Mouse (A) FISH quantification of foci per nucleus in DMSXL mice quadriceps (n = 3; treated n = 9 per ASO; multiple sections throughout the muscle for each mice). **p < 0.01 by Kryskall Wallis test. (B) DMPK mRNA in transgenic DMSXL mice treated with ISIS 486178 (25 mg/kg) and ISIS 445569 (50 mg/kg) (DMSXL, n = 11; ISIS 486178, n = 17; 445569, n = 7). (C) Mice forelimb strength after treatment with by ASOs (wild-type [WT], n = 24; DMSXL, n = 15; ISIS 486178, n = 15; ISIS 445569, n = 7). (D) DMSXL mice serum chemistry after ISIS 486178 injection regiment. (E) Muscle fibers type 1/2a in mice soleus (WT, n = 3; DMSXL, n = 12; ISIS 486178, n = 5; ∼4,500 fibers/mouse). (F) Cell death, necrosis, and inflammation genes downregulated by a 2-fold change in DMSXL mice treated with ISIS 486178 determined by microarray (n = 6). *p < 0.05; **p < 0.01; ***p < 0.001 by unpaired two-tailed t test.