The effect of cassette order on gene expression knockdown by triple tRNA‐shRNA constructs designed for anti‐HIV gene therapy (749.6)

Abstract

Gene therapy utilizing RNA interference (RNAi) directed against HIV‐1 is being studied as an alternative to small‐molecule antiretroviral drugs. Because HIV‐1 is a rapidly mutating virus, it is advantageous to utilize multiple gene therapies at once to effectively prevent the virus from replicating and infecting new cells. Previous research has shown that it is possible to fuse a tRNA together with a short‐hairpin RNA (shRNA) to create a tRNA‐shRNA chimera capable of knocking down the expression of target genes. In this project, triple chimera constructs were created consisting of three different tRNA‐shRNA chimera cassettes on a single plasmid. The effect of differing the order of the tRNA‐shRNA chimeras on the plasmid was investigated using a dual‐luciferase assay with HCT116 cells and the relative knockdown efficiency was compared for each target. The long‐term goal of this project is to optimize a multiplexing strategy for anti‐HIV gene therapy.