Restoration of HIV infected astrocytes functions by utilizing CRIPSR-Cas9

Purpose: HIV enters the brain, resides in neuroglia and invades immune cells. Antiretroviral therapy has been unsuccessful at reducing the proportion of HIV+ patients developing neurocognitive disorders, highlighting the necessity of eradicating HIV from the brain. As one of the most abundant neural cells, astrocytes provide physical and metabolic support to neurons and remain a significant HIV reservoir capable of reconstituting peripheral HIV infection in humanized mouse models. Current strategies aiming to eradicate the HIV genome from the brain have proved to be detrimental to astrocyte function. We hypothesize that successful HIV excision will improve astrocyte function and restore a neuro-supportive phenotype. Methods: Primary human astrocytes were infected with a dual red/green reporter HIV construct (R/G-HIV) to investigate the function of HIV+ astrocytes. CRISPR/Cas9/gRNAs were designed based on previous studies and evaluated for off target effects. The constructs will be cloned into a glial-targeting adenoviral vector to improve transfection efficiency and permit in vivo studies. Results: Pseudotyped HIV successfully integrated into the astrocyte genome and led to expression of construct reporters and HIV proteins. HIV+ astrocytes showed increased expression of proinflammatory mediators, reduced glutamate clearance and proliferation as compared to controls. The CRISPR/Cas9/gRNA construct will induce cuts in the 5' and 3' long terminal repeats to excise HIV. Conclusion: Successful completion of this study will identify HIV excision as an important tool capable of restoring the neuro-supportive phenotype of HIV(-) astrocytes, which could provide a worthy therapeutic strategy against HIV-associated neurocognitive disorders.