Introduction of Mutant APOBEC3G Into CD4+ T-Cells to Resist HIV-1

Abstract

Introduction: Human immunodeficiency virus 1 (HIV-1) is a potentially lethal retrovirus with high genetic diversity and replication ability. HIV-1 mainly targets human CD4+ T-cells, causing a rapid decline in CD4+ T-cell count after infection. Apolipoprotein B mRNA Editing Enzyme Catalytic Subunit 3G (APOBEC3G or A3G) is a human anti-viral enzyme that blocks early stages of HIV-1 replication by deaminating the cytosine residues to uracil in the viral minus-strand DNA during reverse transcription. Nevertheless, HIV-1 overcomes A3G anti-viral activity by employing Vif accessory protein to degrade A3G. Due to a proline/aspartate point mutation in residue 129 (P129D), an artificial A3G mutant, also known as A3G-P129D, was found to be resistant against diverse Vif variants, including HIV-1 Vif, thereby shedding light on a potential A3G-mediated gene therapy for HIV-1. Methods: We propose that, using CRISPR-Cas9 tools, the P129D mutation will be introduced into A3G expressed by human hematopoietic stem cells. Engineered stem cells will be transplanted into Non-Obese Diabetic Severe Combined Immunodeficient Gamma (NSG) mice, which will later be infected with HIV-1. Following infection, flow cytometry will be used to compare the CD4+ T-cell count in mice that express A3G-P129D versus control mice transplanted with unmodified human CD4+ T-cells. Results: We expect a notably higher CD4+ T-cell count in mice carrying the P129D mutation than in the control group, which would be attributed to the efficient inhibition of HIV-1 replication by the mutant A3G. Discussion: Transforming human A3G to A3G-P129D will prevent Vif-mediated degradation of A3G, allowing A3G to efficiently target HIV-1. Thus, this treatment would ultimately block HIV-1 replication and prevent CD4+ T-cell depletion. Conclusion: Overall, our novel gene therapy approach could open the door to a potential one-time treatment for HIV-1 infected patients.