Myotonic dystrophy type 1 (DM1), the most prevalent form of muscular dystrophy in adults, leads to progressive disability and premature death. No treatment that alters the course of DM1 is presently available. This disorder is caused by expansion of a CTG repeat in the 34 untranslated region of the DMPK gene. This unusual mutation leads to a novel, RNA-mediated disease process. Our working model for DM1 pathogenesis involves the following steps: (1) transcription of the mutant allele generates RNA with an expanded CUG repeat (CUGexp); (2) CUGexp RNA accumulates in the nucleus in discrete foci; (3) splicing factors in the muscleblind (MBNL) family, principally MBNL1, are sequestered in the nuclear foci; (4) loss of MBNL1 function leads to abnormal regulation of alternative splicing for a select group of pre-mRNAs; and (5) expression of incorrect splice isoforms leads to symptoms of DM1. A second type of myotonic dystrophy, DM2, has been identified, and it shares a similar RNA-mediated pathogenesis. Misregulated alternative splicing, or spliceopathy, is the major biochemical derangement that is currently recognized in DM1 or DM2. It is clear that spliceopathy can explain certain aspects of the phenotype, such as, myotonia, but the range of genes that are affected, and how the spliceopathy relates to the full spectrum of clinical features, is not clearly defined. To pursue this question, our first Aim is to use splicing sensitive microarrays to analyze spliceopathy in DM1 and DM2. We will obtain a comprehensive view of spliceopathy, and then apply this knowledge to create a custom, cost-effective array that is specifically designed to assess DM-related splicing changes. This tool will then be applied in a prospective study of DM1 and DM2 patients. This study will identify candidates for involvement in the muscle wasting of DM1. Furthermore, the validity of spliceopathy as a biomarker for disease severity and therapeutic response will be tested.
Our second aim i s to translate recent mechanistic insights into new treatments for patients with DM1. We propose a novel use of antisense oligonucleotides, namely, to bind to CUGexp RNA and displace sequestered proteins, thereby releasing these proteins to carry out their normal functions. Preliminary data indicate that a CAG-repeat oligonucleotide has this intended effect, and that physiological and biochemical signs of DM1 can be reversed by this strategy in a transgenic mouse model. To develop the therapeutic potential of CAG-repeat oligos, we will test oligonucleotides of different lengths and chemistries for their ability to interact with CUGexp RNA, to disrupt complexes of MBNL1-CUGexp in vitro, to inhibit the interaction of MBNL1 protein with CUGexp RNA in cells, and to reverse the spliceopathy and myotonia in a transgenic mouse model of DM. Methods for systemic delivery of these oligonucleotides will be tested, and potential off-target effects will be examined. This work may lead to the first treatment that is capable of reversing the symptoms of DM1. ? ? ? ?
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