UCL Great Ormond Street Institute of Child Health


Great Ormond Street Institute of Child Health


Identification of enhancers of genetically manipulated hematopoietic stem and progenitor cells

Supervisors: Dr Alessia Cavazza, Dr Giorgia Santilli

Identification of enhancers of genetically manipulated hematopoietic stem and progenitor cells engraftment for therapeutic purposes

Precise targeting by means of gene editing has recently emerged as a promising technology to treat genetic disorders affecting the hematopoietic system. Indeed, autologous human hematopoietic stem and progenitor cells (HSPCs) can be collected from patients, genetically corrected using engineered nucleases and then infused back to the patients, where they will engraft into the bone marrow and give rise to healthy mature blood cells. One of the critical aspects of HSPC gene editing that requires thorough evaluation is the capability of corrected stem cells to efficiently engraft into the bone marrow upon transplantation, while preserving their long-term repopulating potential. While good engraftment rates of up to 70% have been observed following transplant of HSPCs electroporated with CRISPR/Cas into immunodeficient mice, the persistence of edited cells in the hematopoietic tissues decreases significantly within 8–16 weeks after transplant and in serial transplantation experiments1.The decline in the frequency of corrected cells in vivo could be due to the inefficient HDR-mediated editing in quiescent long-term repopulating HSCs, or their inability to self-renew upon their manipulation in vitro, including exposure to the editing reagents and culture conditions. Either case, the decreased frequency of engraftment of corrected HSPCs limits the therapeutic potential of this approach, especially for those blood disorders that require high chimerism post transplantation without the presence of a strong selective advantage of corrected cells. Thus, new strategies to enhance the engraftment of gene edited cells are urgently needed.

The aim of this project is to define new key players and regulators of HSPC engraftment for application in a gene editing clinical protocol, by taking advantage of CRISPR libraries able to selectively and transiently activate or inhibit genes in an unbiased and genome wide fashion.

Healthy donor HSPCs will be edited with CRISPR activator and CRISPR interfering libraries in parallel, leading to genome-wide selective activation or repression of single genes in a transient, inducible and traceable way2. Manipulated HSPCs will be then transplanted into immunodeficient mice, engrafted cells will be isolated 12-16 weeks post-transplant from the bone marrow of mice and serially transplanted into secondary recipients. 12-16 weeks after secondary transplant, engrafted cells will be isolated and sequenced to determine which activated/repressed genes are enriched in the engrafted pool compared to pre-transplant control cells. The top 5 genes defined by this in vivo screen will be then further validated separately, by single and specific activation/repression in HSPCs transplanted in vivo in immunodeficient mice and analysis of their enhanced engraftment potential. The most promising genes will be then activated/repressed in therapeutically gene edited HSPCs, to validate their use as enhancers of the engraftment of genetically manipulated HSPCs compared to unedited cells. Further experiments will be also designed to assess the mechanism by which the activated/repressed gene facilitates gene edited HSPC engraftment and to define possible therapeutic molecules/strategies that could be applied to  gene editing protocols currently used in pre-clinical studies to achieve such effects in a clinical setting.

1.  Rai R., Thrasher AJ., Cavazza A. (2020). Gene editing for the treatment of Primary Immunodeficiencies. HUMAN GENE THERAPY 2021; https://doi.org/10.1089/hum.2020.185.
2.  Sanjana NE. Genome-scale CRISPR pooled screens. Anal Biochem. 2017;532:95-99. doi:10.1016/j.ab.2016.05.014