Ablation of the pathogenic RNA transcript associated with myotonic dystrophy, DM1

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ABSTRACT Myotonic muscular dystrophy (DMD) is a genetic disorder characterized by muscle degeneration and weakness. It is a common form of muscular dystrophy that generally begins in adulthood. Unfortunately, there are no approved curative therapies for DM1; treatments are largely palliative. The more severe form, myotonic dystrophy type 1 (DM1), is caused by the (CTG)n expansion in the 3 UTR of the dystrophia myotonica-protein kinase gene (DMPK). In DM1-affected cells, (CUG)n repeats in the DMPK mRNA specifically bind to splicing regulatory proteins, forming RNA-protein complexes that accumulate within nucleus as foci that disrupt RNA splicing and ultimately lead to cellular dysfunction. Therapeutic strategies directly targeting expanded repeats in DMPK mRNA, such as antisense oligonucleotides (ASO) or CUG-array specific small molecules, that “release” the bound splicing factors have produced promising results. However, difficulties in ASO delivery and need for lifelong administration of the ASO therapeutic remain limiting factors for ASO-based therapies. In addition, it appears that interfering the ability of CUG repeats to bind factors mitigates only a subset of the adverse consequences of the pathogenic DM1 expanded mRNA. In this proposal, we propose to our Artificial Site- Specific RNA Endonuclease (ASRE) technology to finalize the design of CUG repeat specific RNA endonuclease that may selectively eliminate the pathogenic transcript. The distinguishing feature of ASRE technology is the presence of PUF ribonucleotide binding domains that can be arranged in an array to recognize any 8- to 16-ribonucleotide sequence. In Aim 1, we will finalize the design of 10-base ASRE that specifically recognizes the (CUG)n repeat, clone the ASRE, with or without a nuclear localization sequence into a piggyBac cumate-inducible transposon vector, and transduce the constructs into fibroblasts derived from patients affected by DM1 (GM04602; Coriell Institute). This aim will seek to identify a lead ASRE candidate that can reverse the molecular phenotypes associated DM1 (e.g., accumulation of nuclear foci and aberrant splicing of muscle specific genes) and validate that nuclear expression of the ASRE gene therapeutic is required for activity. In Aim 2, we will generate AAV vectors that express the lead ASRE candidate and validate that viral transduction can rescue the phenotypic anomalies associated with DM1. Once feasibility is demonstrated, Phase II studies will focus on the use of research grade AAV ASRE stocks for efficacy and safety studies in animal models of DM1 before progressing to production of clinical grade AAV for IND enabling safety and efficacy studies of this innovative curative gene therapeutic for DM1.