The CCR5 Gene Edited CD34+CD90+ Hematopoietic Stem Cell Population Serves as an Optimal Graft Source for HIV Gene Therapy

Abstract

Transplantation of allogenic hematopoietic stem and progenitor cells (HSPCs) with C-C chemokine receptor type 5 (CCR5) Δ32 genotype generates HIV-1 resistant immune cells. CCR5 gene edited autologous HSPCs can be a potential alternative to hematopoietic stem cell transplantation (HSCT) from HLA-matched CCR5 null donor. However, the clinical application of gene edited autologous HSPCs is critically limited by the quality of the graft, as HIV also infects the HSPCs. In this study, by using mobilized HSPCs from healthy donors, we show that the CD34+CD90+ hematopoietic stem cells (HSCs) express 7-fold lower CD4/CCR5 HIV receptors, higher levels of SAMHD1 anti-viral restriction factor, and possess lower susceptibility to HIV infection than the CD34+CD90- hematopoietic progenitor cells. Further, the treatment with small molecule cocktail of Resveratrol, UM729 and SR1(RUS) improved the in vivo engraftment potential of CD34+CD90+ HSCs. To demonstrate that CD34+CD90+ HSC population as an ideal graft for HIV gene therapy, we sort purified CD34+CD90+ HSCs, treated with RUS and then gene edited the CCR5 with single sgRNA. On transplantation, 100,000 CD34+CD90+ HSCs were sufficient for long-term repopulation of the entire bone marrow of NBSGW mice. Importantly, the gene editing efficiency of ~90% in the infused product was maintained in vivo, facilitating the generation of CCR5 null immune cells, resistant to HIV infection. Altogether, CCR5 gene editing of CD34+CD90+ HSCs provide an ideal gene manipulation strategy for autologous HSCT based gene therapy for HIV infection.

Xenotransplantation of Human Hematopoietic Stem Cells into NBSGW Mice: A Basic Model for Preclinical Development of Gene Therapy Approaches

“…Следует отметить, что на степень приживления ГСК человека у мышей NBSGW оказывают влияние многие факторы. Среди них можно указать пол животных-реципиентов, источник и дозу ГСК, способ мобилизации ГСК для выделения из периферической крови, индивидуальные особенности донора (возраст), присутствие в трансплантате примеси иммунных клеток донора и даже степень его обогащенности длительно репопулирующими ГСК [15][16][17][18][19][20][21][22]. Помимо учета перечисленных выше факторов разработка протоколов доклинической оценки безопасности продуктов генной и клеточной терапии на основе ГСК включает в себя оценку необходимого количества животных для исследования, распределение животных в группах по возрасту и полу.…”

Current approaches and potential challenges in the delivery of gene editing cargos into hematopoietic stem and progenitor cells

“…The development of mobilization-based chemotherapy-free HSPC transplantation and concomitant overexpression of homing/engraftment enhancers for early engraftment is expected to improve the success rates of HSPC gene editing ( Felker et al, 2022 ; Omer-Javed et al, 2022 ). The ex vivo culture-free HSPC gene editing and gene editing of a sub-population of HSPCs can potentially simplify the gene therapy approach ( Radtke et al, 2020 ; Karuppusamy et al, 2022 ; Venkatesan et al, 2023 ). Gene editing delivery tools have played a substantial role in the success of HSPC gene editing; however, they are also a cause of the high cost of HSPC gene therapy.…”

“…Cyclosporin A (CsA) and rapamycin cause a delay in proliferation, while PGE2 reduces the CD34 + CD90 + primitive cell population ( Wang et al, 2014 ; Petrillo et al, 2018 ; Schott et al, 2019 ). We have shown that culturing HSPCs with a cocktail of small molecules, resveratrol, UM171, and SR1 increases the frequency of gene-modified stem cells ( Christopher et al, 2022 ; Karuppusamy et al, 2022 ). Vesicular stomatitis virus envelope glycoprotein (VSV-G) pseudotypes from wild-type HIV provide a powerful tool for cargo delivery ( Duvergé and Negroni, 2020 ).…”

Recent advances in poor HIV immune reconstitution: what will the future look like?

“…HIV-1 infection can lead to impaired hematopoietic function of HSPCs in several ways. First, a large body of evidence indicates that low levels of CD4 receptors expressed by HSPCs can be targeted by HIV thereby leading to impaired function ( Sebastian et al, 2017 ; Chen, 2019 ; Karuppusamy et al, 2022 ). Second, HIV-1-produced Nef proteins expressed in HSPCs impeding the development and production of myeloid–erythroid cells and inducing apoptosis ( Zou et al, 2021 ).…”