Inhibition of trans-endothelial migration of HIV-1 infected myeloid cells using virus expression dependent anti-ALCAM CRISPR-Cas9

Abstract

Abstract Migration of HIV-1 infected monocytes across the endothelial barrier plays an essential role in establishing and maintenance of viral reservoir in the brain and continues despite antiretroviral therapy (ART). Using the CRISPR-Cas9 gene-editing platform, we created an inducible knockout for activated leukocytes cell adhesion molecule gene (ALCAM/CD166), an HIV-1 host dependency factor shown to play a critical role in CNS entry of infected myeloid cells. First, using lentiviral delivery, we developed several ALCAM knockout clones in monocytic U937, and their latently infected with HIV-1 equivalent: U1 cells. As expected, ALCAM−/− myeloid cells showed markedly reduced adhesion to and transmigration through monolayers of endothelial cells. Next, using AAV6 delivery, we replicated these results in primary human monocytes from three different healthy donors. Finally, to limit CRISPR-Cas9 editing to HIV-1 infected cells, we placed Cas9 expression under the control of minimal HIV-1 Tat responsive LTR promoter (−80/+66). HIV-1BAL-GFP infection of AAV6/adeno-LTR-CRISPR-anti-ALCAM treated CD4+ T cells, monocytes, and monocyte-derived macrophages (MDMs) resulted in the induction of Cas9 expression and CRISPR mediated cleavage of exon 1 of ALCAM gene in Tat expression dependent manner and inhibition of their transendothelial migration. We demonstrated the feasibility of using CRISPR gene editing to disrupt a single CAM gene, i.e, ALCAM to disable adhesion and transmigration ability of HIV-1 infected myeloid cell line cells, primary monocytes, and macrophages. Moreover, we provided data proving the achievability of creating an HIV expression dependent CRISPR platform to restrict Cas9 cleavage activity only to HIV infected cells.