McRae, Elisa; Solis Leal, Antonio; Giler, Noemi; Karlinsey, Dalton; Quaye, Abraham; Berges, Bradford (Brigham Young University)
Faculty Advisor: Berges, Bradford (Brigham Young University, Microbiology and Molecular Biology)
HIV-1 infects CD4 T-cells by inserting its genome into a cell's genetic sequence. CRISPR technology allows for gene editing within the cell, causing a break in DNA sequences targeted by specific guide RNAs. Plasmids encoding CRISPR and guide RNA (gRNA) genes, in the context of lentiviral delivery vectors, will be transfected to produce two lentiviral vectors. In vitro experiments include human T cells that will be transduced with the lentiviral vectors and analyzed with flow cytometry to determine cells that express CRISPR and gRNAs. These cells will then be sorted to create a population of cells that express both the CRISPR and gRNA genes and will then be infected with the NL4-3 strain of HIV. For in vivo experiments, human hematopoietic stem cells will be transduced with the lentivirus vectors, after which they will be transplanted into humanized mice, thus producing a human-like immune system for testing the efficacy of our anti-HIV approach. After the human immune system has sufficiently developed in the mice, HIV-1 will be introduced. We expect that human immune cells with CRISPRs will be protected against HIV infection and death due to the use of gRNAs. These cells are postulated to no longer be susceptible to HIV-1 infection, thus preventing further cell lineages from becoming infected. We will analyze data for three main endpoints: 1. Cell killing of HIV, 2. HIV rebound due to the high mutation rate of the virus, 3. Amount of HIV replication, examined by assessing the viral RNA outside of cells using Q-RT-PCR. Data from this project will support whether cells transfected with CRISPR and guide RNAs offer cell lineages that adequately disrupt the HIV-1 genome. Efforts of this study hope to address HIV infection in humans following trials with humanized mice.