Periodic Reporting for period 1 - IMPROVING-GT (Innovative strategies to increase engraftment of engineered hematopoietic stem cells and bypass genotoxic conditioning, toward the next-generation gene therapy)
Période du rapport: 2022-01-01 au 2023-12-31
The IMPROVING-GT project was centered around the advancement of hematopoietic stem/progenitor cell gene therapy (HSPC-GT), a promising therapeutic approach for treating various genetic diseases. The administration of HSPC-GT involves the ex vivo genetic correction of HSPCs, which are then infused back into the patient after a myeloablative conditioning process to deplete the bone marrow and make space for the modified cells.
The issue being addressed by the project is two-fold: the challenges associated with the engraftment of these genetically corrected HSPCs, and the toxicities related to the conditioning regimes required for successful transplantation. These are significant obstacles in the field of gene therapy as they can limit the effectiveness of the treatment and pose substantial risks to patients.
Addressing these challenges is crucial for society as it has the potential to transform the treatment landscape for patients suffering from genetic diseases by improving the safety and efficacy of gene therapies. The ultimate goal is to fully leverage the life-saving capabilities of HSPC-GT by minimizing its associated risks and increasing its clinical success rate.
The overall objectives of the IMPROVING-GT project were:
* To maximize the engraftment of HSPCs and counteract the negative impacts of ex-vivo manipulation. This was pursued by enhancing human HSC engraftment through the transient overexpression of selected targets involved in various biological processes such as phagocytosis protection, homing, migration, retention, and self-renewal.
* To develop and optimize non-genotoxic conditioning protocols by exploiting enhanced mobilization reagents. This objective aimed to eliminate the need for harmful conditioning by using advanced mobilization agents that could lead to substantial, albeit temporary, emptying of bone marrow niches, thus creating a window for the engraftment of genetically modified cells.
The project successfully demonstrated a proof-of-principle for a novel, low-burden, genotoxic-free HSPCs transplant protocol that could potentially replace the current high-risk conditioning regimens with a safer alternative. This represents a substantial step toward reducing the toxicity associated with current HSPC-GT protocols, ultimately improving patient outcomes and expanding the applicability of gene therapies.
The issue being addressed by the project is two-fold: the challenges associated with the engraftment of these genetically corrected HSPCs, and the toxicities related to the conditioning regimes required for successful transplantation. These are significant obstacles in the field of gene therapy as they can limit the effectiveness of the treatment and pose substantial risks to patients.
Addressing these challenges is crucial for society as it has the potential to transform the treatment landscape for patients suffering from genetic diseases by improving the safety and efficacy of gene therapies. The ultimate goal is to fully leverage the life-saving capabilities of HSPC-GT by minimizing its associated risks and increasing its clinical success rate.
The overall objectives of the IMPROVING-GT project were:
* To maximize the engraftment of HSPCs and counteract the negative impacts of ex-vivo manipulation. This was pursued by enhancing human HSC engraftment through the transient overexpression of selected targets involved in various biological processes such as phagocytosis protection, homing, migration, retention, and self-renewal.
* To develop and optimize non-genotoxic conditioning protocols by exploiting enhanced mobilization reagents. This objective aimed to eliminate the need for harmful conditioning by using advanced mobilization agents that could lead to substantial, albeit temporary, emptying of bone marrow niches, thus creating a window for the engraftment of genetically modified cells.
The project successfully demonstrated a proof-of-principle for a novel, low-burden, genotoxic-free HSPCs transplant protocol that could potentially replace the current high-risk conditioning regimens with a safer alternative. This represents a substantial step toward reducing the toxicity associated with current HSPC-GT protocols, ultimately improving patient outcomes and expanding the applicability of gene therapies.
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.
• 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.
The IMPROVING-GT project has made remarkable strides in advancing the state of the art in HSPC-GT, which has historically been limited by engraftment challenges and conditioning-related toxicities.
The strategy developed here (mobilization based-HSCT) could be first tested and developed in the context of HSC-GT, given that autologous cells do not need to overcome immune barriers in the recipient and that a mixed chimerism ranging around 30% might be sufficient for therapeutic benefit in many diseases that are candidates for HSC-GT. These include most primary immunodeficiencies, but might also extend to hemoglobinopathies and some lysosomal storage disorders. If successful, the combination of mobilization and increased engraftment efficiency investigated in our study might provide an innovative way to entirely bypass the requirement for chemo/radiotherapy in HSPC-GT, conferring long-term therapeutic benefits with considerably less risk and long-term toxicity to patients.
Moreover, as new mobilization reagents become clinically applicable, they may achieve even higher niche depletion and, thus, even more effective exchange with exogenous cells. Furthermore, novel conditioning regimens based on selective immunodepletion might also fit her strategy, as matching engraftment enhancers could be used, such as a transiently expressed KIT mutants not recognized by the anti-KIT antibodies/immunotoxin used for depletion, similarly to what has been shown in the manuscript by testing a CXCR4 variant resistant to the antagonist used for mobilization but still highly responsive to its endogenous ligand, CXCL12.
These advancements not only demonstrate promising progress towards mitigating the toxicity associated with current HSPC-GT protocols but also have significant socio-economic and wider societal implications. By reducing the need for genotoxic conditioning, the improved protocol has the potential to make HSPC-GT accessible to a broader patient population, including those for whom traditional conditioning regimens pose too great a risk. This translates to improved patient outcomes, reduced healthcare costs associated with the management of conditioning-related complications, and overall, a transformative impact on the field of gene therapy, aligning with the societal push towards more humane and cost-effective medical treatments.
The strategy developed here (mobilization based-HSCT) could be first tested and developed in the context of HSC-GT, given that autologous cells do not need to overcome immune barriers in the recipient and that a mixed chimerism ranging around 30% might be sufficient for therapeutic benefit in many diseases that are candidates for HSC-GT. These include most primary immunodeficiencies, but might also extend to hemoglobinopathies and some lysosomal storage disorders. If successful, the combination of mobilization and increased engraftment efficiency investigated in our study might provide an innovative way to entirely bypass the requirement for chemo/radiotherapy in HSPC-GT, conferring long-term therapeutic benefits with considerably less risk and long-term toxicity to patients.
Moreover, as new mobilization reagents become clinically applicable, they may achieve even higher niche depletion and, thus, even more effective exchange with exogenous cells. Furthermore, novel conditioning regimens based on selective immunodepletion might also fit her strategy, as matching engraftment enhancers could be used, such as a transiently expressed KIT mutants not recognized by the anti-KIT antibodies/immunotoxin used for depletion, similarly to what has been shown in the manuscript by testing a CXCR4 variant resistant to the antagonist used for mobilization but still highly responsive to its endogenous ligand, CXCL12.
These advancements not only demonstrate promising progress towards mitigating the toxicity associated with current HSPC-GT protocols but also have significant socio-economic and wider societal implications. By reducing the need for genotoxic conditioning, the improved protocol has the potential to make HSPC-GT accessible to a broader patient population, including those for whom traditional conditioning regimens pose too great a risk. This translates to improved patient outcomes, reduced healthcare costs associated with the management of conditioning-related complications, and overall, a transformative impact on the field of gene therapy, aligning with the societal push towards more humane and cost-effective medical treatments.