Currently incurable, Myotonic Dystrophy type 1 (DM1) is the most common inherited muscular dystrophy in adults. It is caused by the expansion of CTG trinucleotide repeats in the 3?-UTR of the dystrophia myotonica- protein kinase (DMPK) gene. These expanded repeats in turn lead to the accumulation of toxic ribonuclear foci and subsequent dysregulation of RNA binding proteins (RBPs) that are critical for alternative splicing. Although traditionally classified as a myopathy, DM1 is a multisystem disorder that also causes many different neurological deficits that greatly diminish the quality of life of patients. For example, individuals with DM1 experience cognitive impairments including deficits in executive function and attention, social impairments, and reduced IQ-values, suggestive of a frontal lobe dysfunction. Indeed, widespread toxic RNA foci along with the dysregulation of RBPs have been identified within the frontal cortex of patients with DM1. However, how CUG RNA toxicity causes these neurocognitive symptoms remains unclear, making an effective treatment strategy elusive. Interestingly, recent studies have identified altered expression and/or hyperphosphorylation of several synaptic vesicle proteins, along with the dysregulation of neurotransmitters within cortical tissue of both human patients and mouse models of DM1, that do not result from the missplicing of their coding transcripts. Together these data imply that CUG RNA toxicity causes neurocognitive dysfunction by disrupting synaptic transmission in the cortex through a mechanism independent of alternative splicing. It also highlights that the neuropathology underlying neurological symptoms of DM1 is complex with a multitude of pathological pathways occurring simultaneously. Thus, our central hypothesis is that therapeutic interventions for central nervous system (CNS)-specific symptoms of DM1 need to be centered on directly eliminating the primary toxicity underpinning this disorder. The overall objective of this proposal is to enhance our understanding of DM1 neuropathology in order to develop effective therapeutic approaches for neurological symptoms of DM1.
In Aim 1 we will determine if the dysregulation of RBPs in DM1 disrupts the stability of transcripts required for synaptic transmission in DM1 patient induced pluripotent stem cell-derived cortical organoids using crosslinking immunoprecipitation coupled with deep sequencing (eCLIP). This study will determine if mechanisms independent of missplicing contribute to DM1?s CNS-specific manifestations. It will also augment the molecular description of DM1 neuropathology revealing novel human-specific biomarkers that can be used to evaluate the utility of potential therapies or as therapeutic targets themselves. Further, in Aim 2 we will engineer a compact, cell-specific, low-immunogenicity RNA-targeting Cas system designed to eliminate CUG-expanded transcripts in the CNS. This study will result in a long-lasting gene therapy that safely and effectively eliminates toxic CUG-expanded transcripts in human neurons. Combined, our results will be the first step in achieving our long-term goal of developing an effective therapy for neurological symptoms of DM1.
Currently incurable, Myotonic Dystrophy type 1 (DM1) is the most common inherited muscular dystrophy in adults with an estimated prevalence of 1:8000 worldwide. Although traditionally classified as a myopathy, DM1 is a multisystem disorder that also causes many different neurological deficits that greatly diminish the quality of life of patients. Here we propose to use DM1 patient stem cell-derived models of the human brain to further characterize the human-specific molecular neuropathology of this disease and to evaluate the potential of our newly developed RNA-targeting CRISPR/Cas9 system as a therapeutic modality to treat neurological symptoms of this disorder.
