Periodic Reporting for period 5 - GENE FOR CURE (Expanding and extending gene therapy of monogenic diseases of the haematopoietic system)
Reporting period: 2022-10-01 to 2024-09-30
This project addresses the urgent need for curative therapies for severe monogenic immune and hematological disorders: Wiskott-Aldrich Syndrome (WAS), Artemis-deficient Severe Combined Immunodeficiency (SCID), Immune dysregulation Polyendocrinopathy X-linked (IPEX) syndrome, and Sickle Cell Disease (SCD). These life-threatening diseases, often appearing in early childhood, currently rely on limited treatments like chronic transfusions, immunosuppressants, or allogeneic hematopoietic stem cell transplantation (HSCT) which carries significant risks. Gene therapy (GT) offers a transformative alternative by correcting the genetic cause using the patient’s own cells, avoiding immune complications and donor-related risks.
The project aimed to develop, optimize, and evaluate GT and genome editing strategies through four work packages:
• WP1 assessed the long-term efficacy of a lentiviral-vector-based GT trial for WAS, focusing on immunological and hematological reconstitution, autoimmunity, and microthrombocytopenia over 10 years.
• WP2 focused on GT for Artemis-deficient SCID, which is characterized by T/B cell absence and radiation sensitivity. The objective of this WP2 was to launch and advance a Phase 1/2 clinical trial for Artemis-SCID, using autologous Hematopoietic Stem and Progenitor Cells (HSPC) transduced with a G2ARTE lentiviral vector.
• WP3 aimed at conducting the preclinical development of GT for IPEX, caused by loss of function mutation in FOXP3, impairing regulatory T cell (Treg) development and function. The goal was to design a lentiviral vector for CD4 T cells and develop a mouse model to validate in vivo the efficiency of the engineered Treg.
• WP4 explored innovative SCD treatments: a lentiviral β-globin GT trial and a genome editing approach to reactivate fetal hemoglobin, both showing therapeutic potential despite intrinsic challenges linked to HSPC biology.
The project aimed to develop, optimize, and evaluate GT and genome editing strategies through four work packages:
• WP1 assessed the long-term efficacy of a lentiviral-vector-based GT trial for WAS, focusing on immunological and hematological reconstitution, autoimmunity, and microthrombocytopenia over 10 years.
• WP2 focused on GT for Artemis-deficient SCID, which is characterized by T/B cell absence and radiation sensitivity. The objective of this WP2 was to launch and advance a Phase 1/2 clinical trial for Artemis-SCID, using autologous Hematopoietic Stem and Progenitor Cells (HSPC) transduced with a G2ARTE lentiviral vector.
• WP3 aimed at conducting the preclinical development of GT for IPEX, caused by loss of function mutation in FOXP3, impairing regulatory T cell (Treg) development and function. The goal was to design a lentiviral vector for CD4 T cells and develop a mouse model to validate in vivo the efficiency of the engineered Treg.
• WP4 explored innovative SCD treatments: a lentiviral β-globin GT trial and a genome editing approach to reactivate fetal hemoglobin, both showing therapeutic potential despite intrinsic challenges linked to HSPC biology.
The main results of this project are as follows:
- WP1 showed long-term safety and efficacy (up to 10 years) of lentiviral GT for WAS (NCT02333760), with sustained immune correction and clinical benefit. New statistical methods for clonal tracking revealed HSC heterogeneity (Six Blood 2020). Platelet function analyses indicated partial recovery due to reduced WASP expression. Autoimmunity improved, with WASP+ B cells gaining a selective advantage over time (Magnani Nat Med 2022). Overall, lentiviral GT offers sustained treatment, though further improvements in transduction protocols and conditioning regimens could enhance platelet recovery.
- WP2 allowed us to perform all the pre-clinical studies for GT of SCID caused by an Artemis mutation. This included the assessment of efficacy and genotoxicity of this gene therapy strategy, based on the ex vivo transduction of HSPC with G2ARTE lentiviral vector, expressing the DCLRE1C cDNA. The efficacy of this approach was tested in vitro and in vivo. The Phase 1/2 Open Label non-randomized study was authorized in 2022, and the clinical trial started in 2023 (NCT05071222). Five patients were enrolled and treated between 2023 and 2025. We also performed a comprehensive characterization of HSPCs from P1 and observed that transduced HSPCs initiate early T-cell rearrangements in vitro and the T-cell differentiation in vivo, demonstrating success of the ARTEGENE gene GMP transfer protocol. A longer follow-up confirm that HSPCs gene-corrected cells provide a stable engraftment & immune reconstitution with a polyclonal T and B cell repertoire and early antigen-specific response. Altogether, our data demonstrate that the ARTEGENE GT clinical trial is an effective treatment for restoring a functional T- and B-cell compartment in patients with Artemis-deficient SCID phenotypes.
WP3 designed a bidirectional lentiviral vector for efficient FOXP3 and ΔLNGFR co-expression (Patents EP2020052731, EP2019081820), advancing GT for IPEX syndrome. Using scurfy mice, we established a novel assay of Treg functionality (EP2020052162). And we showed that FOXP3-transduced CD4 T cells rescued autoimmune disease on the long-term (Delville Blood 2021), achieving preclinical proof-of-concept and establishing the basis for clinical translation for IPEX.
- WP4 allowed the development of two innovative GT trials for Sickle Cell Disease (SCD)
First, a lentiviral vector expressing anti-sickling HBB transgene was designed. Transduced HSPCs from SCD patients showed up to 60% anti-sickling HBB expression, reducing RBC sickling by 50% under hypoxia. The vector demonstrated robust efficacy in preclinical studies and has been used in the DREPAGLOBE clinical trial (NCT03964792).
Second, we have developed a genome editing CRISPR-Cas9 technology targeted fetal hemoglobin (HbF) silencers to mimic hereditary persistence of fetal hemoglobin (HPFH) mutations, mitigating SCD severity. We showed reactivation of HbF synthesis, reducing sickling phenotypes in SCD patient-derived HSPCs. In vivo studies confirmed high editing efficiency in repopulating HSPCs. (Antoniani Blood 2018 and Weber Sci Adv 2020). These findings identify HbF repressor sites as potential therapeutic targets for genome editing approaches in beta-hemoglobinopathies.
Achievements were shared in top journals and at key conferences (EBMT, ESGCT, ASH), showcasing clinical breakthroughs and helping standardize GT protocols worldwide.
- WP1 showed long-term safety and efficacy (up to 10 years) of lentiviral GT for WAS (NCT02333760), with sustained immune correction and clinical benefit. New statistical methods for clonal tracking revealed HSC heterogeneity (Six Blood 2020). Platelet function analyses indicated partial recovery due to reduced WASP expression. Autoimmunity improved, with WASP+ B cells gaining a selective advantage over time (Magnani Nat Med 2022). Overall, lentiviral GT offers sustained treatment, though further improvements in transduction protocols and conditioning regimens could enhance platelet recovery.
- WP2 allowed us to perform all the pre-clinical studies for GT of SCID caused by an Artemis mutation. This included the assessment of efficacy and genotoxicity of this gene therapy strategy, based on the ex vivo transduction of HSPC with G2ARTE lentiviral vector, expressing the DCLRE1C cDNA. The efficacy of this approach was tested in vitro and in vivo. The Phase 1/2 Open Label non-randomized study was authorized in 2022, and the clinical trial started in 2023 (NCT05071222). Five patients were enrolled and treated between 2023 and 2025. We also performed a comprehensive characterization of HSPCs from P1 and observed that transduced HSPCs initiate early T-cell rearrangements in vitro and the T-cell differentiation in vivo, demonstrating success of the ARTEGENE gene GMP transfer protocol. A longer follow-up confirm that HSPCs gene-corrected cells provide a stable engraftment & immune reconstitution with a polyclonal T and B cell repertoire and early antigen-specific response. Altogether, our data demonstrate that the ARTEGENE GT clinical trial is an effective treatment for restoring a functional T- and B-cell compartment in patients with Artemis-deficient SCID phenotypes.
WP3 designed a bidirectional lentiviral vector for efficient FOXP3 and ΔLNGFR co-expression (Patents EP2020052731, EP2019081820), advancing GT for IPEX syndrome. Using scurfy mice, we established a novel assay of Treg functionality (EP2020052162). And we showed that FOXP3-transduced CD4 T cells rescued autoimmune disease on the long-term (Delville Blood 2021), achieving preclinical proof-of-concept and establishing the basis for clinical translation for IPEX.
- WP4 allowed the development of two innovative GT trials for Sickle Cell Disease (SCD)
First, a lentiviral vector expressing anti-sickling HBB transgene was designed. Transduced HSPCs from SCD patients showed up to 60% anti-sickling HBB expression, reducing RBC sickling by 50% under hypoxia. The vector demonstrated robust efficacy in preclinical studies and has been used in the DREPAGLOBE clinical trial (NCT03964792).
Second, we have developed a genome editing CRISPR-Cas9 technology targeted fetal hemoglobin (HbF) silencers to mimic hereditary persistence of fetal hemoglobin (HPFH) mutations, mitigating SCD severity. We showed reactivation of HbF synthesis, reducing sickling phenotypes in SCD patient-derived HSPCs. In vivo studies confirmed high editing efficiency in repopulating HSPCs. (Antoniani Blood 2018 and Weber Sci Adv 2020). These findings identify HbF repressor sites as potential therapeutic targets for genome editing approaches in beta-hemoglobinopathies.
Achievements were shared in top journals and at key conferences (EBMT, ESGCT, ASH), showcasing clinical breakthroughs and helping standardize GT protocols worldwide.
The project has allowed progress beyond the state of the art as follows
- WP2 : In 2022, we secured the necessary legal authorization to initiate the ARTEGENE phase I/II clinical trial (NCT05071222), enabling us to enroll and treat five patients with Artemis mutations between 2023 and 2025. As Europe's first GT trial dedicated to Artemis deficiency, the ARTEGENE study marks a significant milestone, showcasing a robust restoration of T and B cell compartments. This achievement represents a major breakthrough in the treatment of this rare SCID disease.
- WP3 : The preclinical development of GT for IPEX patients using engineered CD4 T cells is providing a novel therapeutic strategy to restore Treg function in IPEX patients. The present work has allowed us to validate the efficiency of the approach and to process further with GMP lentiviral vector production which has been finalized in 2024. The IPEX GT clinical trial should be starting beginning of 2026 (EudraCT 2025-523305-15-00) and could pave the way for controlling autoimmune manifestations in other disease contexts.
- WP4 : Compared to previously published studies, we have developed a novel high-titer vector that demonstrated robust efficacy in preclinical studies. This vector has been used in a clinical trial conducted between 2019 and 2024 (NCT03964792) and demonstrated safety and efficacy. In parallel, we have developed a novel genome editing strategy and identified HbF repressor sites as potential therapeutic targets for genome editing approaches in SCD. This work paved new ways for developing CRISPR/Cas9 strategies for beta-hemoglobinopathies.
In conclusion, the project has provided strong evidence that GT and gene editing are viable and adaptable platforms for treating a range of severe genetic diseases, paving the way for broader clinical application and long-term benefit to patients and healthcare systems.
- WP2 : In 2022, we secured the necessary legal authorization to initiate the ARTEGENE phase I/II clinical trial (NCT05071222), enabling us to enroll and treat five patients with Artemis mutations between 2023 and 2025. As Europe's first GT trial dedicated to Artemis deficiency, the ARTEGENE study marks a significant milestone, showcasing a robust restoration of T and B cell compartments. This achievement represents a major breakthrough in the treatment of this rare SCID disease.
- WP3 : The preclinical development of GT for IPEX patients using engineered CD4 T cells is providing a novel therapeutic strategy to restore Treg function in IPEX patients. The present work has allowed us to validate the efficiency of the approach and to process further with GMP lentiviral vector production which has been finalized in 2024. The IPEX GT clinical trial should be starting beginning of 2026 (EudraCT 2025-523305-15-00) and could pave the way for controlling autoimmune manifestations in other disease contexts.
- WP4 : Compared to previously published studies, we have developed a novel high-titer vector that demonstrated robust efficacy in preclinical studies. This vector has been used in a clinical trial conducted between 2019 and 2024 (NCT03964792) and demonstrated safety and efficacy. In parallel, we have developed a novel genome editing strategy and identified HbF repressor sites as potential therapeutic targets for genome editing approaches in SCD. This work paved new ways for developing CRISPR/Cas9 strategies for beta-hemoglobinopathies.
In conclusion, the project has provided strong evidence that GT and gene editing are viable and adaptable platforms for treating a range of severe genetic diseases, paving the way for broader clinical application and long-term benefit to patients and healthcare systems.