Testing the Limits of Antisense Oligonucleotide Treatment for Myotonic Dystrophy Type 1

Abstract

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biomedical Genetics, 2016.Myotonic dystrophy type 1 (DM1) is a dominantly-inherited muscular dystrophy that leads to progressive disease of skeletal muscle, the cardiac conduction system (CCS), and the brain. DM1 is caused by a microsatellite CTG repeat expansion in the 3`-untranslated region of DMPK. The mutant DMPK (mutDMPK) is transcribed, producing expanded CUG-repeat mRNAs that are retained in nuclear foci. The CUG-repeat RNA has gain-of-function properties and is considered a primary driver of disease in DM1. In an effort to accelerate decay of toxic CUG-repeat RNA, DMPK-targeting antisense oligonucleotides (ASOs) were developed, and show robust activity in cardiac and skeletal muscle. Indeed, proof-of-concept experiments in mouse models of CUG-repeat RNA toxicity showed biochemical and phenotypic reversal with systemic ASO treatment. However, as with any knockdown strategy for a dominant disease, a key question remains whether collateral silencing of wild-type DMPK will be tolerated. This is particularly important for DM1, as nuclear-retained mutDMPK transcripts are not translated, and DMPK protein levels are reduced by 50% in DM1 tissues. Importantly, it was previously suggested that the CCS is sensitive to DMPK dose, as both heterozygous and homozygous Dmpk knockout mice were reported to show CCS slowing. Furthermore, homozygous Dmpk knockout mice were reported to develop skeletal myopathy. This raises the possibility that CUG-repeat RNA may not be the only driver of disease in DM1, and the risk of DMPK reduction may be a contraindication for antisense knockdown therapy. To circumvent this concern, we first considered a method to selectively target mutDMPK mRNA over wild-type. We hypothesized that mutDMPK transcripts may be hyper-sensitive to ASO-targeting, due to their increased nuclear co-residence with the ASO-directed RNA endonuclease, RNase-H1. Using a transgenic mouse model that produces two alleles, one with an expanded CTG repeat (HSAXLR) and the other without (HSANR), we tested this hypothesis. Importantly, both alleles accumulate similar levels of mRNA in skeletal muscle, but only HSAXLR mice develop CUG-repeat RNA nuclear foci and DM1 biochemical and physiologic phenotypes. Nevertheless, subcutaneous injection of an ASO that targets sequence found in both alleles led to similar reductions of HSAXLR and HSANR mRNA in skeletal muscle. This suggests that selective-targeting of mutDMPK may not be feasible using the current highly-potent ASOs. Therefore, we focused on re-examining the pathophysiological role of DMPK loss in the CCS and skeletal muscle. Using Dmpk knockout mice we investigated the consequences of Dmpk deletion on cardiac and skeletal muscle physiologic function. Furthermore, to model the effects of ASO silencing in DM1 patients, we examined cardiac and skeletal muscle function in heterozygous Dmpk knockouts with long-term ASO treatment. Measurements of cardiac conduction intervals, cardiac contractile function, and skeletal muscle function were performed in mice on two genetic backgrounds, up to 18 months in age. Contrary to previous reports, we found that both complete absence of Dmpk protein or long-term reduction (>90%) by ASOs was well-tolerated, and did not significantly impact cardiac or skeletal muscle structure or function. These findings are encouraging for the development of ASO therapeutics for DM1, and suggest that collateral DMPK reduction will be tolerated. Lastly, the current antisense drugs for DM1 direct an RNase-H1 cleavage of DMPK upstream of the CUG repeat tract. Successful CUG-repeat RNA reduction, therefore, relies on the cellular RNA decay machinery to process through the expanded CUG repeat tract. It is unclear how efficient this clearance will be, and current methods to measure CUG-repeat RNA are limited due to background signal and incompatibility with variable repeat lengths. Therefore, we developed an assay to quantify CUG-repeat RNA mass, called Repeat Mass Assay (RMA), by reverse transcribing directly from the repeat tract. RMA shows excellent correlation (R2 = 0.99) with repeat RNA mass predictions in animal models. Furthermore, following ASO treatment, RMA measurements show excellent correlation (R2 = 0.99) with transgene knockdown measured by quantitative reverse transcriptase PCR. This validates the accuracy and utility of RMA, and suggests that in DM1 mouse models CUG-repeat decay is complete. In conclusion, while selective targeting of mutDMPK may not be feasible, ASO targeting is likely to lead to effective clearance of toxic CUG-repeat RNA. Additionally, the mouse CCS is not sensitive to Dmpk dose, and collateral reduction of DMPK protein is unlikely to exacerbate disease phenotypes. These findings are encouraging and give basic insight into DM1 mechanisms, and are critical for translation of gene-silencing therapy for DM1