768. The CRISPR/Cas9 System as an Anti-Viral Treatment to Prevent Primary Infection by HCMV Positive Hematopoietic Stem Cells

HCMV is a beta-herpes virus, which induces a life long latency in hematopoietic stem cells after an asymptomatic primary infection. In hematopoietic stem cell transplantation, donor hematopoietic stem cells will engraft and differentiate in the recipient. This differentiation induces the reactivation of HCMV in the myeloid compartment and the release of new virions from the donor cells. The HCMV spread leads a primary infection of the immunocompromised HCMV negative recipient and can cause severe end organ diseases. The common treatments of HCMV in immunosuppressed patients are viral DNA replication inhibitors, which cause strong side effects, such as inhibition of hematopoiesis, kidney toxicity, and are likely to promote resistant strain emergence. As a consequence, none of the available drugs can target the HCMV in latent state.To circumvent this, we aim to directly alter the HCMV genome by using the CRIPSR/Cas9 system to knock-out the immediate early gene (IE) encoding essential viral proteins for lytic replication as well as the end of latency. We transduced the low HCMV-permissive U373-MG cells with a lentiviral vector encoding the Cas9 and tree different single gRNA targeting the IE gene. We FACS sorted those cell lines based on their Cas-9-GFP expression and then infected them with a HCMV laboratory strain. We detected mutations at the target site in up to 70% of the viral genomes. We observed a concomitant reduction of 50% less infected cell by FACS staining of the IE protein. Moreover, we set up an assay to analyze the virion release of infected U373-MG cells, which will allow us to address the decrease or inability to release new virions form U373-MG-gRNA-Cas9 HCMV infected cells. We are currently investigating a multiplex strategy with 3 gRNA targeting different parts of the IE gene which are delivered within the same lentiviral vector. The multiplex strategy is auspicious to show higher efficiency than a single gRNA. This strategy will be also tested on HCMV latently infected CD34+ hematopoietic stem cells to prevent viral reactivation.The CRISPR/Cas9 system anti-HCMV is a promising tool to block viral replication. With this anti-HCMV tool we will be able to treat HCMV positive donor cells in order to prevent the primary infection of the immunosuppressed recipient.