Cell-penetrating peptides for enhanced delivery of CRISPR/Cas

PROJECT SUMMARYNew Echota Biotechnology proposes to wed two highly promising technologiesCRISPR Cas genome editing and cell penetrating peptidesCPPsto enhance genome editing in a wide array of cell linestissues and organismsCPPs are capable of mediating penetration of the plasma membraneallowing delivery of macromolecular cargos to the cell interiorCPP delivery of cargos to the interior compartments of cells is a potentially transformative platform technology for the development of therapeuticsdiagnostics and research toolsThis SBIR Phaseapplication proposes to develop an enhanced CRISPR Casproduct based on our novel CPP adaptor technologyOur innovative approach to cargo delivery is the use of high affinity but reversible noncovalent binding to attach cargo to CPPsWe have designed CPP adaptor fusion proteins consisting of the cell penetrating moiety from HIV transactivator of transcription or other CPP fused to calmodulinCaMor other EF hand proteinsOur prototypes bind CaM binding siteCBScontaining cargos with nanomolar affinity in the presence of calcium but negligibly in its absenceBecause mammalian cells typically maintain low resting concentrations of calciumcargos dissociate from the CPP adaptor once inside the cellThe largest technical hurdle to development of CPP therapeutics is failure to escape from endosomesour technology solves this problemWe will deliver active CassgRNA complexes to the nuclei of mammalian cells using our proprietary CPPadaptor technologyand using an established CRISPR assayoptimize delivery for efficiency of easily assayable editingWe will then use the optimized conditions to perform proof of concept experimentsknocking out Fwhich encodes blood coagulation factor IXFIXdefects in which cause hemophilia BWe will also design and execute a small mouse study to demonstrate efficacy of our approach in animalsmodeling from a previous studies in which editing was effected via injection of CRISPR plasmids in tail veinsNew Echota Biotechnology sNEBsystem has speedsafety and efficiency advantages over competing methods and ease of use and dosing advantages over other CPP methodsThe significance of this application is that success will enable utilization of CPP adaptors to enhance CRISPR technology in a way that overcomes currently intractable limitations PROJECT NARRATIVE This project seeks to develop its core technology to enhance the delivery of CRISPR Cas gene editing components to living cells using cell penetrating peptides attached to adaptor molecules that bind payloads with high affinity and release them in the cell interiorCRISPR Cas is currently limited by the need to transfect or transduce components into cells which is often problematicSuccess will broaden the application of gene editing technology and further validate that our technology is an adaptable tool for delivery of a wide array of macromoleculespotentially improving the delivery of cancer therapeuticsantiviralsand proteins to ameliorate a variety of disorders as well as creating versatile research and diagnostic tools