Preclinical evaluation for engraftment of CD34⁺ cells gene-edited at the sickle cell disease locus in xenograft mouse and non-human primate models.

Abstract

Sickle cell disease (SCD) is caused by a 20A > T mutation in the β-globin gene. Genome-editing technologies have the potential to correct the SCD mutation in hematopoietic stem cells (HSCs), producing adult hemoglobin while simultaneously eliminating sickle hemoglobin. Here, we developed high-efficiency viral vector-free non-footprint gene correction in SCD CD34⁺ cells with electroporation to deliver SCD mutation-targeting guide RNA, Cas9 endonuclease, and 100-mer single-strand donor DNA encoding intact β-globin sequence, achieving therapeutic-level gene correction at DNA (∼30%) and protein (∼80%) levels. Gene-edited SCD CD34⁺ cells contributed corrected cells 6 months post-xenograft mouse transplant without off-target δ-globin editing. We then developed a rhesus β-to-βs-globin gene conversion strategy to model HSC-targeted genome editing for SCD and demonstrate the engraftment of gene-edited CD34⁺ cells 10-12 months post-transplant in rhesus macaques. In summary, gene-corrected CD34⁺ HSCs are engraftable in xenograft mice and non-human primates. These findings are helpful in designing HSC-targeted gene correction trials.