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Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9
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A simultaneous knockout knockin genome editing strategy in HSPCs potently inhibits CCR5- and CXCR4-tropic HIV-1

Amanda M Dudek1, William N Feist1, Elena J Sasu1

  • 1Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA; Institute for Stem Cell Biology & Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.

Cell Stem Cell
|April 5, 2024
PubMed

Insights

A new gene editing method using Cas9/AAV6 offers a potential cure for HIV-1 by modifying hematopoietic stem cells. This approach provides resistance to both CCR5- and CXCR4-tropic HIV strains for autologous transplants.

Area of Science:

  • Gene therapy
  • Virology
  • Hematopoietic stem cell transplantation

Background:

  • Allogeneic HSCT with CCR5 null cells can cure HIV-1 but has risks and limitations.
  • Existing treatments are not viable for most patients due to donor rarity and resistance to CXCR4-tropic HIV.

Purpose of the Study:

  • To develop a genome editing strategy for autologous HSCT to confer resistance to both CCR5- and CXCR4-tropic HIV-1.
  • To assess the safety and efficacy of edited HSPCs for HIV-1 treatment.

Main Methods:

  • Utilized a targeted Cas9/AAV6-based genome editing strategy.
  • Edited human hematopoietic stem and progenitor cells (HSPCs) for CCR5 and CXCR4 resistance.
  • Evaluated multi-lineage repopulation capacity in vivo and infection inhibition in vitro.

Main Results:

  • Edited HSPCs maintained repopulation capacity in vivo.
  • Edited T cells potently inhibited both CCR5- and CXCR4-tropic HIV-1 infection.
  • Achieved complete loss of CCR5-tropic replication and up to 2,000-fold reduction in CXCR4-tropic replication without CXCR4 locus disruption.

Conclusions:

  • This multi-factor editing strategy in HSPCs offers a promising approach for autologous HSCT.
  • It could provide a functional cure for both CCR5- and CXCR4-tropic HIV-1 infections.
  • This method broadens the scope of HSCT as a viable HIV-1 treatment option.

Keywords:
CCR5 knockoutCRISPR-Cas9HIVHIV restrictionautologous HSCTcell therapyfunctional curegene editinghematopoietic stem cell transplanthomology-directed repair

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