CRISPR Cas Systems Delivered by Targeted Nanoparticles to Eradicate Herpesvirus Pathogens

Abstract Herpesviruses are chronic pathogens that infect for life there is no cure years since the discovery of the first tumor virus the herpesvirus Epstein Barr virus EBV there exist no specific antivirals for EBV during its latency phase and there are no vaccines The same holds true for most of the other eight human herpesviruses This proposal aims to develop novel tools to eradicate oncogenic gamma herpesviruses gHV in cancer cells We propose to adapt our existing nanoparticle based platform for the delivery of a DNA based CRISPR Cas system a powerful tool for targeted genome editing We will target and eliminate key viral genes and functional sequences of the herpesvirus genome to eradicate infection Our nanoparticle platform harnesses the remarkable multifunctional capabilities of the carbon nanoparticle oxidized graphene nanoribbons O GNRs Our recent in vitro results indicate that these graphene single sheet of graphite particles can serve as a versatile gene delivery platform to deliver DNA with low cytotoxicity and high transfection efficiency The overall objective of this project is to engineer an O GNR based platform that will deliver specific nucleic acids to targeted cells in order to cleave and inactivate a herpesvirus genome Importantly we will utilize the murine gHV pathogenesis system such that we can move quickly from cell culture based proof of principle experiments to in vivo validation of virus eradication in an accepted and validated animal model of gHV infection In Aim we will engineer the O GNR platform to deliver DNA to B cells In Aim we will Identify the guideRNAs that drive specific editing of the gamma herpesvirus genome Successful completion of the independent aims of this proposal will allow the consolidation of targeted gene delivery to B cells Aim with RNA guided editing of the virus genome to drive elimination of the oncogenic herpesvirus from the latency reservoir Aim The in vitro validation in phase will lead to highly tractable in vivo studies in an established animal pathogen system during phase Project Narrative Herpesviruses establish life long infections that can lead to significant disease and mortality especially in neonates the elderly and immunocompromised patients A major challenge for combating herpesvirus infections is the dormant phase of infection termed latency Since current antivirals only target productive lytic infection novel therapeutic interventions are needed We propose to apply gene editing technology to make precise cuts that inactivate the viral genome We will engineer and validate a nanoparticle platform for the delivery of nucleic acids that provide the instructions to eradicate a herpesvirus from an infected cell