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 beta-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 beta-globin sequence, achieving therapeutic-level gene correction at DNA (similar to 30%) and protein (similar to 80%) levels. Gene-edited SCD CD34(+) cells contributed corrected cells 6 months post-xenograft mouse transplant without off-target delta-globin editing. We then developed a rhesus beta-to-beta-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.