PROJECT SUMMARY Gene therapy has the potential to provide treatment options for most diseases, yet it has failed to live up to this promise in part because genome modification is difficult in the tissues and organs of living patients. This R15 AREA project aims to improve CRISPR/Cas genome editing by enhancing delivery of components using a novel cell penetrating peptide (CPP)-adaptor system. In most mammalian cell types, CPPs are capable of mediating penetration of the plasma membrane, allowing delivery of macromolecular cargos to the cell interior. Because of their general utility and low toxicity, CPPs are a potentially transformative technology. Our innovative approach uses high affinity but reversible noncovalent binding to attach cargo to CPPs, overcoming the serious limitation of endosomal escape. Our CPP-adaptor consists of the cell penetrating moiety of HIV transactivator of transcription (TAT) fused to calmodulin (CaM). TAT-CaM binds CaM binding- site (CBS)-containing cargos with nanomolar affinity in the presence of calcium but negligibly in its absence. Because mammalian cells typically maintain low resting concentrations of calcium, cargos dissociate from the CPP-adaptor once inside the cell. The largest technical hurdle to development of CPP therapeutics is failure to escape from endosomes and, as we have shown, reversible association solves this problem for many cargo proteins. We will adapt our CPPs to delivery of CRISPR/Cas complexes, effecting efficient editing in cultured cells including hepatocytes. Using parameters optimized in those experiments we will demonstrate proof-of-concept in vivo editing in a mouse model. We have chosen the common B form of hemophilia caused by mutations in the F9 gene encoding blood coagulation factor IX (FIX) to edit with our technology. In a previous study, injection of CRISPR plasmids in tail veins of hemophilia B mice produced almost an undetectable reversion of a deleterious mutations in hepatocytes but still restored coagulation. TAT-CaM-driven CRISPR internalization promises to be a relatively safe, effective method for treating diseases such as hemophilia B and holds promise to be a significant improvement over current CRISPR techniques. With established investigators, modern research facilities and cutting-edge instrumentation, we are positioned to accomplish the specific aims within the period of support. Success in these endeavors will not only significantly expand the utility of CRISPR/Cas but will also validate that our technology is an adaptable tool for delivery of a wide array of macromolecules, potentially improving the delivery of biomolecular therapeutics to ameliorate a variety of disorders as well as creating versatile research and diagnostic tools.

PROJECT NARRATIVE Currently limited by the need to transfect or transduce components into cells, this project seeks to enhance CRISPR/Cas gene editing technology by effecting efficient delivery of components to living cells using cell penetrating peptides attached to adaptor molecules that deliver and release cargos into the cell interior. Delivery will be optimized in cultured cells and then used to correct a defect in the F9 gene of a mouse hemophilia B model. Success will broaden the application of gene editing technology and further validate that our CPP-adaptors are effective tools for delivery of a wide array of macromolecules, potentially improving the delivery of cancer therapeutics, antivirals, and proteins to ameliorate a variety of disorders as well as creating versatile research and diagnostic tools.