Inhibition of HIV reactivation in brain cells by AAV-mediated delivery of CRISPR/Cas9.

Astrocytes are sites of virus persistence in the brain and may form virus reservoirs with latent HIV that can be reactivated in response to different stimuli. Silencing of HIV in astrocytes would be highly desirable to prevent production of neurotoxic HIV gene products and virus dissemination. However, astrocytic HIV reservoir cells in the brain are especially hard to target, since they are protected by the blood-brain-barrier. To identify viral vectors for transduction of astrocytes we screened a library of artificial AAV-capsids with a human neural stem cell line (HNSC.100 cells), maintained either as progenitor or as terminally differentiated astrocytic populations. This led to identification of the AAV9P1 vector, which was further validated for transduction of astrocytes with neural cells generated from human iPSCs (induced pluripotent stem cells). We demonstrate that AAV9P1 is able to introduce long-term transgene expression in differentiated astrocytes and can be used to deliver the genome editing tool CRISPR/Cas9 into its target cells. Guide RNAs, which direct the nuclease Cas9 to its target DNA, were designed to disrupt HIV expression. By using the HIV latency model HNSC.LatGFP1.2 (1), we could show that the AAV9P1-mediated delivery of CRISPR/Cas9 significantly inhibited reactivation of latent HIV-1 in astrocytic reservoirs cells