CCR5 gene knock-out mediated TALEN technique and its effects against HIV infection on lymphocytes and macrophages

Abstract

Most strains of HIV-1 use CD4 and CCR5 as receptors for cell entry. A naturally occurring CCR5 gene mutation known as CCR5-Δ32 results in CCR5 dysfunction and makes the cells resistant to HIV-1 infection. Previously, two patients who received hematopoietic stem cell transplantation which carried homozygous CCR5-Δ32 have had their HIV-1 infection functionally cured, suggesting that gene-editing targeting CCR5 could make it possible for curing HIV-1 infection. Currently, the transcription activator-like effector nucleases (TALEN) system provides the highest accuracy on gene editing with good efficiency. In this study, I aim to establish a TALEN-based gene-editing platform targeting CCR5 for HIV-1 cure. Previous publication has indicated that mRNA has more advantages than DNA when delivered to the cells. Thus, firstly in this study, I optimized electroporation delivery of mRNAs to the cells and then the protocol of in vitro transcription of mRNA. After transfecting primary T cells with TALEN mRNAs, the expression of surface CCR5 was downregulated. The disruption of the CCR5 gene was confirmed on the genomic level. After HIV-1 challenge, the CCR5 gene-edited primary T cells showed increased HIV-1 resistance. Macrophages also express CD4 and CCR5, which makes them another target of HIV-1 besides CD4+ T cells. Moreover, HIV-1-infected macrophages are more resistant to immune attack and are a key reservoir of HIV-1. Thus, I tested this TALEN system on monocytes-derived-macrophages (MDMs). After transfection of the TALEN mRNAs, the surface CCR5 expression on the MDMs was also downregulated. The CCR5 gene knock-out was confirmed on the genomic level as well. The TALEN-treated MDMS showed increased resistance to HIV-1 infection compared with the un-edited controls after HIV-1 challenge. The result of this study provides evidence and support for using TALEN to knock out CCR5 as a method for curing HIV-1 infection.