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Multi-factor genome editing offers improved outcomes for HIV-1 treament

Allogeneic hematopoietic stem and progenitor cell transplantation (HSCT) of CCR5 null (CCR5Δ32) cells is a potentially curative procedure for patients infected with type 1 human immunodeficiency virus (HIV-1). However, it has only limited utility as matched bone marrow donors are rare, the risk from associated toxicities is high, and CCR5Δ32 transplant does not protect against the CXCR4-tropic virus. To address these shortcomings, researchers from Stanford University School of Medicine, CA, and Aarhus University, Denmark, have developed a targeted genome editing strategy using human hematopoietic stem and progenitor cells (HSPCs) and primary human T cells. Their data, published in Cell Stem Cell, show the edited HSPCs to maintain multi-lineage repopulation capacity in vivo, while the edited primary human T cells inhibit both CCR5- and CXCR4-tropic replication in vitro. It is suggested that this approach could help make autologous HSCT a viable option for HIV-1 patients. 

 

 

A simultaneous knockout knockin genome editing strategy 

Multiple pathways of the HIV-1 lifecycle must be inhibited to maintain viral suppression. For this reason, Dudek et al. combined Cas9/RNP mediated knockout of CCR5, the major co-receptor for HIV-1 entry to CD4+ T cells, with AAV6 knockin of a transmembrane-anchored fusion inhibitor (C46) and/or a restriction factor (TRIM5α) at the CCR5 locus. TriLink provided the single guide RNAs (sgRNAs) used for CCR5 editing. Initial testing showed that one of the start site sgRNAs induced knockout INDEL formation in over 85% of CCR5 alleles in both umbilical cord blood derived CD34+ HSPCs and primary human CD4+ T cells, with no evidence of off-target INDELs. 

Edited HSPCs retain multi-lineage repopulation capacity in vivo 

To assess the multi-lineage repopulation capacity of the edited HSPCs, Dudek et al. injected the cells into sub-lethally irradiated NSG-SGM3 mice and performed a series of measurements at 14 weeks post-transplant. Key findings included the observation that all three constructs produced a similar distribution of progenitor-derived colonies to control samples, with the engrafted lineages being capable of mobilizing to sites of maturation. It was also found that knockout INDEL frequencies of greater than 80% total alleles were maintained at the engraftment endpoint and that knockin was detected in all mice at similar levels. Additional testing confirmed there were no major defects in in T cell subset distribution or functionality. 

Multi-factor editing inhibits infection by both CCR5- and CXCR4-tropic HIV-1 in vitro 

An in vitro infection model was used to evaluate treatment efficacy against both CCR5- and CXCR4-tropic HIV-1. Briefly, CD4+ T cells isolated from healthy donors were edited prior to challenge with replication-competent CCR5-tropic or CXCR4-tropic HIV-1, then flow cytometric analysis was performed, virus concentration was quantified by p24 ELISA, and genomic DNA was extracted for analysis. All editing conditions showed virtually complete loss of surface CCR5, while the percentage of cells expressing CXCR4 remained consistent after editing. Upon challenge with CCR5-tropic HIV-1, both knockout and knockin completely suppressed viral replication in undiluted culture supernatant until the infection endpoint. Responses to the CXCR4-tropic virus varied between donors, but significant inhibition of replication was achieved in all cases. 

Conclusion 

Although allogeneic HSCT has cured HIV-1 infection in a small number of patients, it is not feasible in the majority of cases. The multi-factor genome editing strategy developed by Dudek et al. promises to help make autologous HSCT a viable option, both for treating infectious diseases like HIV and for resolving genetic deficiencies, provided there is continued progress in the field. 

 

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Article reference: Dudek A.M., Feist W.N., Sasu E.J., et al. A simultaneous knockout knockin genome editing strategy in HSPCs potently inhibits CCR5- and CXCR4-tropic HIV-1 infection. Cell Stem Cell. 2024;31(4):499-518.e6. http://dx.doi.org/10.1016/j.stem.2024.03.002