Ex vivo culture resting time impacts transplantation outcomes of genome-edited human hematopoietic stem and progenitor cells in xenograft mouse models.

Ex vivo resting culture is a standard procedure following genome editing in hematopoietic stem and progenitor cells (HSPCs). However, prolonged culture may critically affect cell viability and stem cell function. We investigated whether varying durations of culture resting times impact the engraftment efficiency of human CD34+ HSPCs edited at the BCL11A enhancer, a key regulator in the expression of fetal hemoglobin. We employed electroporation to introduce CRISPR-Cas9 components for BCL11A enhancer editing and compared outcomes with nonelectroporated (NEP) and electroporated-only (EP) control groups. Post-electroporation, we monitored cell viability, death rates, and the frequency of enriched hematopoietic stem cell (HSC) fractions (CD34+CD90+CD45RA- cells) over a 48-hour period. Our findings reveal that while the NEP group showed an increase in cell numbers 24 hours post-electroporation, both EP and BCL11A-edited groups experienced significant cell loss. Although CD34+ cell frequency remained high in all groups for up to 48 hours post-electroporation, the frequency of the HSC-enriched fraction was significantly lower in the EP and edited groups compared to the NEP group. In NBSGW xenograft mouse models, both conditioned with busulfan and nonconditioned, we found that immediate transplantation post-electroporation led to enhanced engraftment without compromising editing efficiency. Human glycophorin A+ (GPA+) red blood cells (RBCs) sorted from bone marrow of all BCL11A edited mice exhibited similar levels of γ-globin expression, regardless of infusion time. Our findings underscore the critical importance of optimizing the culture duration between genome editing and transplantation. Minimizing this interval may significantly enhance engraftment success and minimize cell loss without compromising editing efficiency. These insights offer a pathway to improve the success rates of genome editing in HSPCs, particularly for conditions like sickle cell disease.

Gene therapies on the horizon for sickle cell disease: a clinician's perspective.

INTRODUCTION: Sickle cell disease (SCD) is a monogenic disorder that exerts several detrimental health effects on those affected, ultimately resulting in significant morbidity and early mortality. There are millions of individuals globally impacted by this disease. Research in gene therapy has been growing significantly over the past decade, now with two FDA approved products, aiming to find another cure for this complex disease. AREAS COVERED: This perspective article aims to provide a clinician's insight into the current landscape of gene therapies, exploring the novel approaches, clinical advances, and potential impact on the management and prognosis of SCD. A comprehensive literature search encompassing databases such as PubMed, Web of Science and Google Scholar was employed. The search covered literature published from 1980 to 2024, focusing on SCD and curative therapy. EXPERT OPINION: After careful evaluation of the risks and benefits associated with the use of gene therapy for affected patients, the need for a cure outweighs the risks associated with treatment in most cases of SCD. With advances in current technologies, gene therapies can increase access to cures for patients with SCD.