Figure 1.

Representation of ex vivo HSCs gene editing, showing the crucial steps of the process. After harvesting the hematopoietic stem and progenitor cells (HSPCs) from mobilized peripheral blood or bone marrow, the CD34⁺ cells are enriched and cultured ex vivo in the presence of growth factors, which allows the maintenance and expansion of self-renewing stem cells, and are then subjected to gene editing tool transfer (e.g., meganucleases, ZFNs, TALENs, or CRISPR/Cas-derived nucleases). When the nuclease induces a standard DNA double-strand break (DSB) at the desired genomic loci, the homology-directed repair (HDR) machinery are recruited in order to repair the DNA, where a template donor DNA is supplied for the homologous recombination between the template and chromosomal DNA, or by non-homologous end-joining (NHEJ) without a homologous template DNA, resulting in small indels generation (insertions and deletions) if there is only one cut, or triggers large DNA deletions if two cuts. After the treatment, the patient receives a specific conditioning regimen that depletes endogenous HSPCs from the bone marrow and makes space for the ex vivo engineered cells to engraft. The gene-corrected cells are then reinfused intravenously and engraft in the bone marrow.