Towards The Therapeutic Implementation of New CRISPR-Cas Systems

Development of clinical CRISPR Cas gene editing therapeutics requires a guide RNA with specificity to a region in the target geneand an endonuclease termed CasCRISPR associatedthat mediates RNA guided specific DNA cuttingEach CRISPR Cas system has specific functional requirements that limit their potential clinical utilityThe Casendonucleases derived from Spyogenes or Saureus have been utilized in preclinical studies and are being developed for clinical trials in humansHoweverboth Spyogenes and Saureus are common human pathogensresulting in potential adaptive immunity to CasThis pre existing immunity may limit the clinical efficacy of gene editing therapeutics based on Casin a subset of patientsespecially in situations involving viral vectors or repeated infusions of CasTheseand other limitations of specific Cas endonucleasesargue for the development of a diverse armamentarium of gene editing enzymesExcision BioTherapeutics is specialized in developing novel Cas therapeutics for targeting human viral pathogensRecentlyseveral novel Cas enzymestermed CasXCaseand CasYCasdwere identified in metagenomic sequencing data from environmental isolatesand are being actively developed by Excision BioTherapeutics for their unique potential for clinical applicationThese novel Casare from non pathogenic bacterial speciesare smaller than spCasand have distinct protospacer adjacent motifPAMrequirementsThereforein the current application we will leverage our expertise in CRISPR therapeutics to accelerate the pre clinical development of these therapeutically favorablenovel enzymesTo do sowe will synthesize engineered genes encoding CasX and CasY homologs that are optimized for both recombinant protein production and use in human cellsThrough an established recombinant Cas production pipelinewe will expresspurifyand validate the activity of CasX and CasY homologsIn vitro assays will be used to optimize gRNA format and cleavage conditionsUsing a combination of in vitro and in vivo assayswe will then perform a direct comparison of CasXand CasYwith Casin cleaving the HIVprovirusFinallynext generation sequencing approaches will be used to assess the relative efficiency and specificity of the enzymes in vitro and in HIVinfected human cellsUltimatelyour approaches will lead to the development of a panel of novelcompact CRISPR Cas therapeutics that can be administered to humans without potential interference from pre existing immunity NarrativeGene editing utilizing the CRISPR Cas system has emerged as a powerful means for targeting and destroying genes that cause diseaseincluding those derived from viral genomesSeveral extrinsic and intrinsic factors limit the potential clinical applications of widely used CRISPR Cas systemsThereforewe will accelerate the pre clinical development of a set of novel endonucleases that have significant potential for clinical development as next generation CRISPR Cas therapeutics