CRISPR-Cas9 mediated disruption of ALCAM gene inhibits adhesion and trans-endothelial migration of myeloid cells

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 leads to neuroinflammation, neuronal damage and sub- sequent HIV-induced central nervous system (CNS) dysfunction. These processes continue despite antiretroviral therapy (ART) due to limited pharmacological permeability of blood-brain barrier, the presence of re- sidual viral replication and reactivation of latent viruses. Activated leu- kocytes cell adhesion molecule (ALCAM/CD166) is a junctional protein elevated on activated T cells, monocytes, dendritic cells and endothelium. Recent studies identified ALCAM to be preferentially overexpressed on HIV-1 infected, mature CD14+CD16+ monocytes from people with HIV (PWH) on suppressive ART and critical for the transmigration ability of these cells. Furthermore, high throughput CRISPR screen identified ALCAM as one of the key HIV-1 host dependency factor (critical for virus propagation but non-essential for host cells). Here, we used a pair of CRISPR guide RNAs to excise exon 1 (spanning start codon and signal peptide region) and thus create inducible ALCAM gene knockout in myeloid cells. Using lentiviral delivery, we developed several knockout clones in pro-monocytic U937, and their latently infected with HIV-1 equivalent: U1 cells. Next, verified control and knockout cell clones were tested in adhesion and transmigration assays, using monolayers of cere- bral microvascular endothelial cells (hCMEC/D3). As expected, ALCAM-/- myeloid cells showed markedly reduced adhesion to and transmigration through endothelial cells. Next, using AAV6 delivery we replicated these results in primary human monocytes from three different healthy donors. In order to limit CRISPR-Cas9 editing to HIV-1 infected cells we placed Cas9 expression under the control of minimal Tat respon- sive HIV-1 LTR promoter (-80/+66). HIV-1BAL-GFP infection of AAV6-LTR-CRISPR-ALCAM treated CD4+ T cells and CD14+/ CD16+ monocytes, resulted in induction of Cas9 expression and CRISPR mediated cleavage of exon 1 of ALCAM gene in Tat expression dependent manner.