Genetically-edited induced pluripotent stem cells derived from HIV-1-infected patients on therapy can give rise to immune cells resistant to HIV-1 infection

Abstract

Objective: To assess the in-vitro CCR5---tropic and CXCR4---tropic HIV---1 infectivity of immune cells, particularly macrophages, derived from CCR5 gene---edited induced pluripotent stem cells (iPSCs) obtained from the peripheral blood mononuclear cells (PBMC) of HIV---infected patients on antiretroviral therapy (ART). Design: PBMC were obtained from six patients who had been HIV---infected for over 20 years and were on ART for 1---12 years prior to this study. Methods: The PBMC were derived into iPSCs and genetically edited with TALENs or CRISPR---cas9 endonucleases combined with PiggyBac technology to introduce the naturally occurring 32---bp deletion to the CCR5 gene. These iPSCs were differentiated into macrophages, and subsequently challenged with CCR5---tropic or CCR5/CXCR4 dual--- tropic HIV---1 strains. iPSC derivation, gene editing and immune cell differentiation were done in feeder---free, xeno---free in-vitro conditions. Results: Multiple unedited (wild---type) and CCR5 gene---edited (mutant) iPSCs were derived from patients’ PBMC. When differentiated into immune cells and HIV---1 challenged, mutant iPSC lines were resistant to CCR5---tropic and to some extent to CCR5/CXCR4 dual---tropic HIV---1 infection when compared to wild---type iPSC lines. Conclusion: Our study demonstrates that iPSC---derived, gene---edited immune cells are resistant to distinct HIV---1 strains. These findings have important implications for both in-vitro stem cell development and therapeutic approaches to cure HIV infection.

Contemporary Transposon Tools: A Review and Guide through Mechanisms and Applications of Sleeping Beauty, piggyBac and Tol2 for Genome Engineering

“…Indeed, PB was used for footprint-free correction of gene mutations responsible for epidermolysis bullosa in iPSCs [ 190 ]. In addition, in a novel approach using a combination of gene editing by designer nucleases and PB-mediated seamless excision, iPSCs reprogrammed from peripheral blood mononuclear cells of HIV-infected patients were edited to include the naturally occurring 32-bp deletion in the chemokine receptor 5 (CCR5) [ 191 ], known to hinder viral entry [ 192 ]. After differentiation into immune cells, resistance to CCR5-tropic and to some extent CCR5/CCRX4-dual-tropic HIV-1 infection was observed, providing exciting developments in potential future functional HIV cures.…”

Targeted Chromatinization and Repression of HIV-1 Provirus Transcription with Repurposed CRISPR/Cas9

“…With the development of versatile genome editing tools such as zinc finger nucleases, TAL effector nucleases, and RNA-guided CRISPR/Cas9 systems, it is possible to directly target and modify the HIV-1 genome [ 24 , 25 , 26 ]. Furthermore, these tools have been used to make cells resistant to HIV-1 infection by modifying HIV-1 receptors, CCR5, and/or CXCR4 [ 27 , 28 , 29 , 30 ]. In addition, CRISPR/Cas9-derived systems have been used to directly disrupt and excise integrated HIV-1 provirus from infected cells in vitro and in vivo [ 31 , 32 , 33 , 34 , 35 ].…”

Knowledge From London and Berlin: Finding Threads to a Functional HIV Cure

“…CRISPR/Cas9 was applied by Fernando et al using CCR5D32 genetically edited induced pluripotent stem cells combined with PiggyBac technology to obtain CD34+ HSCs that can offer resistance to the CCR5-tropic viruses and to some extent to CCR5/CXCR4 dual-tropic viruses (33). After transplanting the CRISPR/Cas9-modified HSPCs into NSG mice, the CCR5 deletions were validated by polymerase chain reaction analysis and the combination of CRISPR/Cas9 with a dual gRNA effectively ablated CCR5 in CD34+ HSPCs such that the biallelic inactivation frequencies reached 42%; additionally, the off-target mutation events accounted for approximately 0.6% of all captured off-target sites (34).…”