CRISPR-Cas9 mediated disruption of ALCAM gene in uninfected and HIV+ myeloid cells inhibits their adhesion to endothelial cells

Viral spread and seeding of different anatomical compartments within the body depends on cell-to-cell transmission and the ability of infected cells to disseminate by intra- and extravasation. ALCAM is a junctional protein over-expressed on activated T cells, monocytes, dendritic cells and also endothelium. Recent studies show increased levels of ALCAM on CD14+/CD16+ monocytes derived from HIV+ patients and a critical role in promoting transmigration of these cells across the blood brain barrier. Here we designed a pair of CRISPR guide RNAs targeting exon 1 of the human ALCAM gene. Successful cleavage of the target sites leads to deletion of the segment of DNA spanning the ALCAM start codon/signal peptide and to complete block of ALCAM expression. Using lentiviral delivery we created several single cell ALCAM knockout clones in pro-monocytic U937, and their latently infected with HIV-1 equivalent: U1 cells. CRISPR-Cas9 cleavage of ALCAM gene was verified by PCR based excision assay and Sanger sequencing of truncated, double cleaved/end-joined amplicons. The lack of ALCAM expression was confirmed by RT-qPCRs and flow cytometry. Next control and knockout cells were tested in adhesion assay using WT, and newly created ALCAM-/- cerebral endothelial cells, hCMEC/D3. All ALCAM knockout myeloid cell clones showed reduced adhesion to WT endothelial cells but not to ALCAM -/- ones. The approach presented here provides a new, gene editing based therapeutic avenue to treat any neuroinflammatory disorder with trafficking of immune cells component.