Prospective study in myotonic dystrophy to determine extracellular RNA biomarkers

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Summary Myotonic dystrophy (dystrophia myotonica; DM) is an inherited neurodegenerative disease that causes progressive muscle weakness, cognitive dysfunction, and sleep disturbance. No effective treatments are available to slow or reverse symptoms. DM type 1 (DM1) is caused by an expanded CTG repeat in the DM protein kinase (DMPK) gene. Clinical features result from pathogenic effects of the DMPK mRNA that lead to deregulation of alternative pre-mRNA splicing of at least several dozen transcripts. In pre-clinical studies, alternative splicing outcomes in muscle tissue have been used as sensitive pharmacodynamic indicators of therapeutic antisense oligonucleotide (ASO) drug response. Many patients with DM1 also experience central sleep apnea, excessive daytime sleepiness, diminished working memory, impaired visuospatial skills, and deficits in problem-solving skills. These central nervous system (CNS) symptoms are associated with mis- regulated alternative splicing patterns and gene expression changes in post-mortem brain. Cerebrospinal fluid (CSF) surrounds and protects the brain and CNS from injury. Changes in the composition of CSF can serve as early indicators of changes in the molecular pathology of the CNS in living individuals. However, methods for the use of CSF to measure DM1-specific splicing patterns or gene expression changes in the CNS are unavailable. Extracellular RNA (exRNA) refers to mRNAs and noncoding RNAs that are released from and taken up by cells as a novel form of cell-to-cell communication. Our recent work suggests that splicing outcomes in urine exRNA have the capacity to serve as robust biomarkers of peripheral disease activity in DM1. In this prospective study, we will test the hypothesis that exRNA contains a representative transcriptome that is altered in DM1 and quantifiable in CSF. Splicing outcomes in CSF exRNA are expected to correlate with DM1 disease activity using clinical measures of CNS function. Aim 1 will identify and characterize the CSF exRNA profile in DM1 patients and unaffected control subjects. Droplet digital PCR (ddPCR), a state-of- the-art technology, will be used to quantify a panel of CSF exRNA splice events previously reported as altered in DM1 CNS. To identify new potential biomarkers, we will perform exRNA sequencing. Aim 2 will determine the test/re-test reliability of the exRNA profile by longitudinal monitoring in a prospective cohort of DM1 patients. Aim 3 will establish a relationship between the exRNA profile and quantitative clinical measures of CNS disease activity, including cognitive testing and brain MRI. Proposed context of use (COU): monitoring biomarker for the serial assessment of disease status and burden in DM1 patients. In future clinical trials, CSF exRNA biomarkers would enable patient stratification and convenient monitoring of molecular disease activity in the CNS during the course of drug treatment. The approach used in this proposal also will be applicable to DM type 2 (DM2) and to other neurodegenerative diseases more generally.