Preclinical Evaluation for Engraftment of CD34 Cells Gene-edited at the Sickle Cell Disease Locus in Xenograft Mouse and Non-human Primate Models

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.