Targeted Degradation of CAG Repeat Expanded Huntingtin mRNA, Associated with Huntington's Disease, Using Artificial Site-Specific RNA Endonucleases

ABSTRACTTrinucleotide repeat expansions are a common cause of several neurodegenerative diseases including Huntington s diseaseHDHD is caused by expansion of CAG repeats in the first exon of huntingtinHTTthat is translated as a polyglutamine tractThe protein aggregates formed from the polyglutamine containing peptide are the main cause of neuronal cell deathalthough recent results have suggested that the RNA repeats itself may also be directly responsible for neurotoxicityTherapeutic strategies directly targeting mutated HTT mRNAsuch as antisense oligonucleotidesASOhave produced promising resultsHoweverdifficulties in ASO deliverythe unknown effects of ASO on structured CAG repeatsand possibility that ASOs may disrupt both expanded and normal transcripts remain unresolvedIn this proposalwe propose to use our Artificial SiteSpecific RNA EndonucleasesASREstechnology to design CAG repeat specific RNA endonuclease to destroy expanded pathogenic HTT RNAsASREs contain RNA binding domains isolated from PUF proteinswhich consist of a series ofamino acid modules that recognize one specific ribonucleotideIn a proof of concept studywe designed ASREs against expanded CAG repeatswhich are present in the DMPK genewhich is associated with theCUG n repeats disorderMyotonic Dystrophy typeDMWe demonstrated thatCUG n specific ASREs specifically degrade pathogenic DMPK mRNAs with minimal effect on wild type alleles in cells derived from DMpatients and corrected several molecular markers of DMdiseseWe seek to develop a protein based therapeutic approach for HD by designing ASREs that recognize CAG repeats in mutated HTT mRNAIn the long termcombined with gene delivery vectorsASREs may provide a new route for targeted therapyTo assess the feasibility of this approach we will first engineer ASREs that specifically recognizesnt ornt RNA sequences in three different frames of theCAG n repeatCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGand use a yeast three hybrid system to identify the PUF domain that has highest affinity forCAGrepeats with low affinity forCAGrepeatsPUF domains that selectively recognizesCAGat efficienciesfold higher thanCAGrepeats will be cloned into a piggyBac cumate inducible transposon vector to create aCAG n specific ASRE for cell studiesPB ASRE CAG nSecondwe will transduce PB ASRE CAG n into heterozygous knockin embryonic stemEScells that express endogenous levels of either human exoncontaining normalHttQor mutantHttQtalleles together with a normal mouseQ alleleHtt QUndifferentiated ES Cells and ES cell derived neurons will be cultured plus or minus cumate to quantitate the level of knockdown of the expanded mRNA relative to the normal allele by qPCR and quantitate the level of knockdown of the polyglutamine containing peptide relative to the normal peptide by Western blottingWe seek to demonstrate that induction of PB ASRE CAG n results in preferential downregulation of mutant RNA and proteinat least three fold preferential knockdown of the mutant RNA and proteinOnce feasibility is demonstratedPhasestudies will focus on the development of research grade adenoviral associated vectorsAAVthat constitutively express ASRE CAG n to develop gene delivery protocols and for initial efficacy and safety studies in animal models of HD before progressing to production of clinical grade AAV ASRE therapeutics for IND enabling safety and efficacy studies NARRATIVETrinucleotide repeat expansions are a common cause of several neurodegenerative diseases including Huntington s diseaseHDfor which there is no curative therapycurrent therapies only manage the symptomsIn this proposalwe seek to use our Artificial Site Specific RNA EndonucleaseASREtechnology to design CAG repeat specific RNA endonucleases that can destroy the expanded pathogenic RNAs associated with HDIf successfulour ASRE technology will provide a new therapeutic option that targets the underlying cause of the disorder