Therapeutic Modulation of Myotonic Muscular Dystrophy

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 DESCRIPTION (provided by applicant): Myotonic dystrophy type 1 (DM1) results from a novel RNA-mediated disease process that is triggered by an unconventional mutation. The mutation is an unstable expansion of CTG repeats in the DM protein kinase (DMPK) gene, and the mechanism involves a toxic gain-of-function by transcripts containing an expanded CUG repeat. Studies indicate that symptom onset and progression of DM1 is driven by the age-dependent growth of the expanded CTG repeat in somatic cells. In an effort to attack DM1 at its root cause, we have partnered with colleagues in industry to develop antisense oligonucleotides (ASOs) targeting the toxic RNA. This approach was highly effective in mouse models and has recently entered clinical trials. In this proposal we seek to better define the therapeutic properties of these versatile new reagents. First we will determine whether ASOs may exhibit allelic selectively; that is, whether they preferentially target transcripts containing expanded CUG repeats (CUGexp). Our previous work suggested that CUGexp-containing transcripts are hypersensitive to ASO knockdown, owing to prolonged dwell time in the nucleus. We developed a new mouse model for testing the allelic discrimination of ASOs, based on the presence or absence of expanded CUG repeats in the target transcript. The sensitivity of expanded vs. non-expanded alleles to ASO/RNase H knockdown will be compared, and the therapeutic window for allelic selectivity will be determined for different dosing regimens. The results will guide th development of therapeutic strategies to maximize and preserve the residual function of the DM kinase. Next we will determine whether somatic instability of expanded repeats is reduced by DMPK-targeting ASOs. Previously we showed that CAG-repeat ASOs are able to stabilize CTG repeats in a DM1 model in vivo. However, as the preferred therapeutic strategy has now shifted to targeting outside of the repeat tract, we need to determine whether ASOs directed at the non-repetitive sequences of DMPK may also stabilize expanded repeats. If ASOs do in fact exhibit dual properties of mitigating RNA toxicity and stabilizing repeats, this will argue for early initiation of treatment. Next, we will examine the biochemical basis for the unusually prolonged duration of ASO activity in muscle cells. Using decoy oligonucleotides that are complementary to ASOs, coupled with quantitative assays of pre-mRNA, we will determine whether long-duration activity results from post-transcriptional silencing and depends on the continuous presence and action of the ASO. Finally, we will determine whether early initiation of ASO treatment can prevent dystrophic pathology, spliceopathy, and muscle weakness in a CUGexp-expressing, MBNL1-deficient model. Taken together, the results of the proposed studies will expand our understanding of the biological properties of ASOs in skeletal muscle, and provide important guidance for the optimal use of these reagents in therapeutic trials and clinical practice.