Next generation CRISPR/Cas9-RNAi mouse models for accelerated drug discovery research

Abstract SignificanceNew approaches for rapid identification and early preclinical validation of novel therapeutic targets are crucial to make importantgo no godecisions and curb the cost of developing new cancer treatmentsGenetically engineered mouse modelsGEMMsare a powerful platform to study disease initiation and maintenancethe tumor microenvironment and the responsiveness of cancers to known or novel therapeuticshoweverthe long lead times and high costs required to developintercross and maintain models with various cancer predisposing gene combinations have limited their practical utility in the drug discovery processRecentlywe have shown RNA interferenceRNAiin mice can serve as a fast alterative to gene deletion and be exploited experimentally to silence nearly any gene targetby the expression of synthetic short hairpin RNAsshRNAsImportantlybecause it is reversiblegene silencing by RNAi better mimics the dynamics of small molecule inhibition than permanent genetic knockoutsFurthermorewith the advent of new genome editing techniquessuch as CRISPR Castechnologywe are able to introduce additional sensitizing lesions to induce disease pathogenesisIn synergy with RNAi technologycomplex multi allelic ESC based GEMMs can be generated without extensive intercrossingUsing this combination of CRISPR Casand RNAi technologieswe are able to not only model disease pathogenesisbut also mimic drug therapy in micegiving us unprecedented capabilities to perform preclinical studies in vivoHypothesisWe hypothesize that CRISPR CasRNAi GEMMs of cancer can be developed rapidly using new genome editing technologiesCRISPRsto introduce additional sensitizing lesions and recombinase mediated cassette exchangeRMCEfor precise integration of tetracycline inducible shRNAs to silence specific gene targetsPreliminary dataWe have previously used CRISRP Casand RMCE to generate RNAi GEMMs without any breedingSpecific AimsAs a proof of conceptwe will develop a model of lung adenocarcinoma by using the CRISPR Cassystem to introduce a conditional KrasGD allele into the endogenous locus and in situ delivery of sgRNAs targeting Trpwhich will be activated by a conditionally expressed CasalleleWe will further modulate mutant Kras or Mekactivity by introducing tetracycline inducible shRNAs to model therapeutic inhibitionFinallywe will expand our flexible platform by producing validatedoff the shelfviral vectors carrying combination sgRNAs targeting commonly altered genes in NSCLCTogetherthese studies will define a new paradigm and accelerate drug discovery research by creating a flexible platform for the generation of RNAiGEMMs that will serve as innovative research toolsguiding the development of novel and effective therapeutics