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

ABSTRACTMyotonic muscular dystrophyDMDis a genetic disorder characterized by muscle degeneration and weaknessIt is a common form of muscular dystrophy that generally begins in adulthoodUnfortunatelythere are no approved curative therapies for DMtreatments are largely palliativeThe more severe formmyotonic dystrophy typeDMis caused by theCTG n expansion in theUTR of the dystrophia myotonica protein kinase geneDMPKIn DMaffected cellsCUG n repeats in the DMPK mRNA specifically bind to splicing regulatory proteinsforming RNA protein complexes that accumulate within nucleus as foci that disrupt RNA splicing and ultimately lead to cellular dysfunctionTherapeutic strategies directly targeting expanded repeats in DMPK mRNAsuch as antisense oligonucleotidesASOor CUG array specific small moleculesthatreleasethe bound splicing factors have produced promising resultsHoweverdifficulties in ASO delivery and need for lifelong administration of the ASO therapeutic remain limiting factors for ASO based therapiesIn additionit appears that interfering the ability of CUG repeats to bind factors mitigates only a subset of the adverse consequences of the pathogenic DMexpanded mRNAIn this proposalwe propose to our Artificial SiteSpecific RNA EndonucleaseASREtechnology to finalize the design of CUG repeat specific RNA endonuclease that may selectively eliminate the pathogenic transcriptThe distinguishing feature of ASRE technology is the presence of PUF ribonucleotide binding domains that can be arranged in an array to recognize anytoribonucleotide sequenceIn Aimwe will finalize the design ofbase ASRE that specifically recognizes theCUG n repeatclone the ASREwith or without a nuclear localization sequence into a piggyBac cumate inducible transposon vectorand transduce the constructs into fibroblasts derived from patients affected by DMGMCoriell InstituteThis aim will seek to identify a lead ASRE candidate that can reverse the molecular phenotypes associated DMe gaccumulation of nuclear foci and aberrant splicing of muscle specific genesand validate that nuclear expression of the ASRE gene therapeutic is required for activityIn Aimwe will generate AAV vectors that express the lead ASRE candidate and validate that viral transduction can rescue the phenotypic anomalies associated with DMOnce feasibility is demonstratedPhase II studies will focus on the use of research grade AAV ASRE stocks for efficacy and safety studies in animal models of DMbefore progressing to production of clinical grade AAV for IND enabling safety and efficacy studies of this innovative curative gene therapeutic for DMNARRATIVENucleotide expansion disorders are a common cause of several degenerative diseases including myotonic dystrophyDMfor which there is no curative therapycurrent therapies only manage the symptoms of this genetic diseaseIn this proposalwe seek to use our patented RNA site specific targeting technology to finalize design of a gene therapeutic that will bind to the pathogenic RNA genetically responsible for this diseaseBy eliminating the pathogenic activity of the RNAour technology may provide a new therapeutic option that targets the underlying cause of the disorder