Herpesviruses are chronic pathogens that infect for life; there is no cure. 50 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/Cas9 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 1, we will engineer the O-GNR platform to deliver DNA to B cells. . In Aim 2, 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 1) with RNA-guided editing of the virus genome to drive elimination of the oncogenic herpesvirus from the latency reservoir (Aim 2). The in vitro validation in phase 1 will lead to highly tractable in vivo studies in an established animal pathogen system during phase 2.
Public Health Relevance Statement: 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.
Project Terms: abstracting; Animal Model; Animals; Antibodies; antibody conjugate; Antiviral Agents; B-Lymphocytes; base; Binding; bioimaging; Biological Assay; cancer cell; Carbon nanoparticle; Cell Culture Techniques; Cell Surface Receptors; Cell Survival; Cells; Chronic; Cleaved cell; Clustered Regularly Interspaced Short Palindromic Repeats; combat; combinatorial; CRISPR/Cas technology; cytotoxicity; Data; Disease; DNA; DNA delivery; Drug Kinetics; Elderly; Elements; endonuclease; Engineering; Epitopes; Exhibits; experimental study; Fluorescence; gammaherpesvirus; Gene Delivery; Genes; Genome; genome editing; Genomic Segment; graphene; Guide RNA; Half-Life; Herpesviridae; Herpesviridae Infections; Human; Human Herpesvirus 4; hydroxyl group; Immunocompromised Host; In Vitro; in vivo; Infection; Infection prevention; Instruction; interest; large scale production; Lead; Life; Lymphocyte; Lymphoma; Lytic Phase; Mediating; Monitor; mortality; Mus; nanoparticle; Natural graphite; neonate; novel; novel therapeutic intervention; Nucleic Acids; Oncogenic; Oncogenic Viruses; particle; pathogen; Pathogenesis; Phase; Property; Reporter; Specificity; Structural Genes; System; targeted delivery; Technology; Testing; tool; Toxic effect; Transfection; Vaccines; Validation; Viral Genes; Viral Genome; Virus; Water
