Investigate Proviral and Transgene Copy Numbers in Single Cells

Division Of Basic Sciences - Nci

Investigators

Abstract

Technologies developed in the Kearney lab are being used to determine HIV proviral copy numbers in single cells from donors before ART, on ART, and after experiencing virologic failure - especially with off-target mutations conferring antiviral resistance. It has been shown previously that some Env mutations confer resistance to multiple classes of HIV inhibitors in vitro. These mutants exhibit defects in cell-free infectivity and Env-mediated fusion but are highly proficient in cell-to-cell transmission. Integrase strand-transfer inhibitors (INSTIs) are particularly vulnerable to high-MOI infection, so we will focus these studies on INSTI virologic failure samples. Specifically, we want to answer whether the number of infected cells with multiple proviruses increases in association with INSTI virologic failure since it is hypothesized that the mechanism for resistance may be due to the very high efficiency of cell-cell viral transmission. In the proposed model, cells infected with mutant virus would carry more integrated proviral copies compared to cells infected with wild-type virus. If we find higher numbers of integrated proviruses in cells infected with the Env mutants compared to cells infected with the wild-type virus, we will conclude that resistance to ART may be due to the Env mutants facilitating multiple infection events in most target cells. Together, these findings may change our understanding of HIV replication, evolution, and drug resistance. Gateway mutations allowing low-level replication, and then selection for specific mutations, would also call for a change in the resistance assays now performed. Additionally, technologies in the Kearney lab are being modified to study integration sites and transgene genetic landscape in single engineered T cells in people being treated for cancer with CAR and TCR T-cell therapies, with the goal of understanding the persistence and retroviral mutagenesis of cellular immunotherapy. Gene therapy is an important tool for treating genetic disorders and cancers, often utilizing retroviral vectors for efficient transgene delivery. Despite their advantages, indiscriminate integration into the target cell genome may lead to unintended gene expression changes, loss of proliferative control, and rare oncogenesis. Moreover, the reverse transcriptase that converts RNA transgenes into DNA is prone to errors that can introduce point mutations or deletions, potentially impacting transgene expression and function. In this project, we will measure the frequency of transgenes in peripheral blood lymphocytes (PBL) after infusion with CAR and TCR T-cells, where they are integrated, if the cells that harbor transgenes clonally expand, and if the transgenes contain mutations or deletions.