Optimized cell culture conditions promote ex-vivo manipulation and expansion of primitive hematopoietic stem cells for therapeutic gene editing

Abstract

During the last few years, gene editing has emerged as a powerful tool for the therapeutic correction of monogenic diseases. CRISPR/Cas9 applied to hematopoietic stem and progenitor cells (HSPCs) has shown great promise in proof-of-principle preclinical studies to treat haematological disorders, and clinical trials using these tools are now underway. Nonetheless, there remain important challenges that need to be addressed, such as the efficiency of targeting primitive, long-term repopulating HSPCs and expand them in vitro for clinical purposes. Here we have tested the effect exerted by different culture media compositions on the ability of HSPCs to proliferate and undergo homology directed repair-mediated knock-in of a reporter gene, while preserving their stemness features during ex-vivo culture. We tested different combinations of compounds and demonstrated that by supplementing the culture media with inhibitors of histone deacetylases, and/or by fine-tuning its cytokine composition it is possible to achieve high levels of gene targeting in long-term repopulating HSPCs both in vitro and in vivo, with a beneficial balance between preservation of stemness and cell expansion, thus allowing to obtain a significant amount of edited, primitive HSPCs compared to established, state-of-the-art culture conditions. Overall, the implantation of this optimized ex vivo HSPC culture protocol will improve the efficacy, feasibility and applicability of gene editing and will likely provide one step further to unlock the full therapeutic potential of such powerful technology.

Genome editing for primary immunodeficiencies: A therapeutic perspective on Wiskott-Aldrich syndrome

“…Indeed, it is well known that prolonged culture and stimulation of HSPCs, while being needed for efficient gene transfer, may adversely impact their engraftment and long-term repopulation capacity. Recent findings showed that addition to the culture medium of stemness preserving compounds, such as Stem Regenin-1, UM171, and 16,16-dimethyl prostaglandin E2 (dmPGE2), helps to maintain the long-term multilineage repopulation capacity of human corrected HSPCs transplanted in immunodeficient mouse models, partially overcoming the drawbacks of prolonged culture ( 96 ) and that fine-tuning of cytokine composition can lead to a beneficial balance between preservation of stemness and cell expansion ( 115 , 116 ). Another aspect that needs special attention is the tolerability of HSPCs to genetic modifications; indeed LT-HSCs, which require protection from mutational load over a lifetime, are more sensitive to DNA manipulation than differentiated cells, hence GE may trigger cellular responses that reduce their fitness and stemness.…”