The main advances of our study over current knowledge and published work are the following:
• The exploitation of a favorable window of opportunity opened at the peak of mobilization, when donor cells effectively compete with those in circulation to repopulate the depleted bone marrow niches, leading to establishment of stable chimerism (>20%). Importantly, these findings are initially explored in mice, where she shows their therapeutic potential by the rescue of a primary immunodeficiency disease model and are then reproduced and further optimized with human HSC in ad hoc designed in vivo human hematochimeric models, again reaching engraftment levels above the expected threshold for correction of several diseases.
• The uncovering of a competitive advantage conferred to HSC upon ex vivo culture, likely mediated by the recovered expression of surface molecules relevant for homing and engraftment, such as CXCR4, which are cleaved during in vivo G-CSF exposure. Furthermore, this ex vivo culture step was used to model an lentivirus-based gene replacement or CRISPR-Cas-based editing protocol for therapeutic purposes and is therefore immediately portable to currently established gene therapy strategies.
• Building on the above finding, the development of a novel mRNA-based strategy endowing HSPCs with enhanced but transient engraftment advantage, which can further increase the competitive advantage of donor cells and their chimerism level established in the recipients. We used an “mRNA-only” platform, allowing safe capture of powerful gain-of-function effectors for HSPC homing or retention, such as CXCR4, ITGA4, KIT and CD47, but our strategy might conceivably be extended to other genes involved in pathways such as self-renewal.
• The incorporation of mRNA-based delivery of engraftment enhancers into state-of-the-art ex vivo gene editing process, allowing to overcome detrimental impacts of the procedure on the cell homing ability and leading to seamless conditioning-free establishment of engineered human hematopoietic grafts.
• The feasibility and potential output of the mobilization-based HSCT in pediatric patients, estimated using data from ongoing HSC-GT clinical trial.
The project's findings have been disseminated through high-impact publications in journals like Cell and the British Medical Bulletin and presented at major international conferences (ASGCT and ISSCR). A patent was filed for the methods developed, showcasing the potential for commercial exploitation. The outcomes of the project also include significant advancements in non-genotoxic conditioning protocols, setting the stage for safer and more effective HSPC-GTs.
