Periodic Reporting for period 3 - TheraLymph (Gene Therapy to restore lymphatic flow lymphedema)
Periodo di rendicontazione: 2023-01-01 al 2024-06-30
Lymphedema is a disabling condition induced by the chronic accumulation of fluid and fat in the arms or in the legs. It is an untreatable disease that affects 4 million people in Europe and more than 250 million worldwide, and there are no curative treatments. It is handicapping, painful and impacts substantially the quality of life. In western countries, lymphedema is generally a consequence of cancer i.e. ten to fifteen percent of women will develop lymphedema after breast cancer. The main objective of Theralymph will be to establish a safe, non-integrative RNA-based gene therapy for patients who developed lymphedema after breast cancer.
The Theralymph translational research programme brings together bench scientists and physicians from six European countries to perform a Phase I/II trial. Our approach is based on combinatorial gene therapy targeting of both superficial lymphatic capillaries and deeper lymphatic collectors.
The project is structured into 7 Research and Innovation work packages (WP), each one managed by a WP leader from a participating centre, and aimed to:
• Determine risk factors and cartography the lymphatic network in lymphedema (WP1,2);
• Decipher whether lymphatic intrinsic molecules or microenvironmental peptides or lipids are modified in the lymphedema arm to promote lymphatic dysfunction (WP3,4);
• Identify therapeutic targets and validate the best molecular combination (VEGFC+identified target) to restores the lymphatic drainage in preclinical lymphedema models (WP5,6);
• Conduct a Phase I/II gene therapy clinical trial using the innovative LentiFlash® technology based on non-integrative lentiviral vectors that allow a transient and highly efficient in vivo gene delivery (WP7).
Two additional WPs concern the management and coordination through meetings with partners and scientific boards (WP8) and dissemination of the results and intellectual property management (WP9).
The Theralymph translational research programme brings together bench scientists and physicians from six European countries to perform a Phase I/II trial. Our approach is based on combinatorial gene therapy targeting of both superficial lymphatic capillaries and deeper lymphatic collectors.
The project is structured into 7 Research and Innovation work packages (WP), each one managed by a WP leader from a participating centre, and aimed to:
• Determine risk factors and cartography the lymphatic network in lymphedema (WP1,2);
• Decipher whether lymphatic intrinsic molecules or microenvironmental peptides or lipids are modified in the lymphedema arm to promote lymphatic dysfunction (WP3,4);
• Identify therapeutic targets and validate the best molecular combination (VEGFC+identified target) to restores the lymphatic drainage in preclinical lymphedema models (WP5,6);
• Conduct a Phase I/II gene therapy clinical trial using the innovative LentiFlash® technology based on non-integrative lentiviral vectors that allow a transient and highly efficient in vivo gene delivery (WP7).
Two additional WPs concern the management and coordination through meetings with partners and scientific boards (WP8) and dissemination of the results and intellectual property management (WP9).
The COVID-19 health crisis was a significant hurdle at the beginning of the project (2020). A 6-months extension to the original project duration had officially been granted by the Commission. Despite the difficulties faced, the project has made good progress towards its objectives.
WP1: The role of the main lymphangiogenic factor VEGF-C was evaluated in the aggressiveness of breast cancer (BC). Irradiation with X and P beams shapes a distinct secretory profile and induces endothelial-to-mesenchymal transition, particularly following X irradiation. We determined that chemotherapeutic paclitaxel stimulates VEGFC production without influencing the sensitivity to therapy. However, 5-FU was found to interfere with LEC metabolism.
WP2: Methods for the visualisation of collecting lymphatic vessels in mouse adipose tissue (AT) were established and single-cell transcriptomics of lymphatic endothelial cells (LEC) has identified signature genes for normal AT collecting lymphatic vessels . RNAseq analysis of tissues from human lymphedema patients revealed potential therapeutic targets that were studied in WP5-6 (Apelin, FGF1, SERCA2…).
WP3: Lipidomic analysis of the skin and AT from patients with lymphedema was performed and showed changes in the composition of lipid mediators when compared to healthy arm. Co-cultures of LECs and adipocytes revealed increased mRNA levels coding for proteins involved in fatty acid handling. We found that PPARα activity in LEC primarily supports fatty acid transport rather than their utilization
WP4: Eicosanoid profiles of lymphedematous skin demonstrated differential production of eicosanoid-derived lipids. Arachidonic Acid-derived metabolites generated by 15-lipoxygenase play a crucial role in lymphedema and suggest that lymphedema can be in part aggravated by a defect in the resolution of inflammation. We found that CD36 expression is tightly regulated by immune cells and flow in lymphoid tissues, suggesting a novel function of lymphatic CD36 in the regulation of immune responses.
WP5: A clone of NIH3T3 cells knock-out for VEGF-C was used to overproduce VEGF-C alone (rescue) or in combination with targets identified in WP2-4. The cellular tools are shared by several partners to evaluate the synergistic effect of different factor combinations on LEC functions in vitro.
WP6: Analysis of biodistribution of the lentiflash vector containing GFP- and luciferase-reporter genes has been performed using to determine the dose and site of injection. Combination of VEGF-C with potential targets have been evaluated in vivo. After validation using integrative lentivirus, targets alone or in combination were studied using RNA-delivery lentiflash vector.
WP7: Multidisciplinary meetings with methodologists, clinical staff, coordinator and scientists were organised in order to establish the design of the clinical trial. The first documents for the study have been initiated: information and consent form, synopsis and draft of the clinical protocol. These documents will be adjusted and completed according to the reglementary preclinical data.
WP8: An effective management framework ensures project progress. Virtual solutions are used for all consortium meetings, including biannual progress reviews and annual meetings. An independent ethical advisory board is regularly consulted on ethical and regulatory issues, particularly for the clinical trial.
WP9: The visual identity of the project was developed, i.e. design of the Theralymph logo, and the Theralymph website has been launched. A first version of the Data Management Plan, which covers all types of data to be generated during the Theralymph project, has been elaborated.
WP1: The role of the main lymphangiogenic factor VEGF-C was evaluated in the aggressiveness of breast cancer (BC). Irradiation with X and P beams shapes a distinct secretory profile and induces endothelial-to-mesenchymal transition, particularly following X irradiation. We determined that chemotherapeutic paclitaxel stimulates VEGFC production without influencing the sensitivity to therapy. However, 5-FU was found to interfere with LEC metabolism.
WP2: Methods for the visualisation of collecting lymphatic vessels in mouse adipose tissue (AT) were established and single-cell transcriptomics of lymphatic endothelial cells (LEC) has identified signature genes for normal AT collecting lymphatic vessels . RNAseq analysis of tissues from human lymphedema patients revealed potential therapeutic targets that were studied in WP5-6 (Apelin, FGF1, SERCA2…).
WP3: Lipidomic analysis of the skin and AT from patients with lymphedema was performed and showed changes in the composition of lipid mediators when compared to healthy arm. Co-cultures of LECs and adipocytes revealed increased mRNA levels coding for proteins involved in fatty acid handling. We found that PPARα activity in LEC primarily supports fatty acid transport rather than their utilization
WP4: Eicosanoid profiles of lymphedematous skin demonstrated differential production of eicosanoid-derived lipids. Arachidonic Acid-derived metabolites generated by 15-lipoxygenase play a crucial role in lymphedema and suggest that lymphedema can be in part aggravated by a defect in the resolution of inflammation. We found that CD36 expression is tightly regulated by immune cells and flow in lymphoid tissues, suggesting a novel function of lymphatic CD36 in the regulation of immune responses.
WP5: A clone of NIH3T3 cells knock-out for VEGF-C was used to overproduce VEGF-C alone (rescue) or in combination with targets identified in WP2-4. The cellular tools are shared by several partners to evaluate the synergistic effect of different factor combinations on LEC functions in vitro.
WP6: Analysis of biodistribution of the lentiflash vector containing GFP- and luciferase-reporter genes has been performed using to determine the dose and site of injection. Combination of VEGF-C with potential targets have been evaluated in vivo. After validation using integrative lentivirus, targets alone or in combination were studied using RNA-delivery lentiflash vector.
WP7: Multidisciplinary meetings with methodologists, clinical staff, coordinator and scientists were organised in order to establish the design of the clinical trial. The first documents for the study have been initiated: information and consent form, synopsis and draft of the clinical protocol. These documents will be adjusted and completed according to the reglementary preclinical data.
WP8: An effective management framework ensures project progress. Virtual solutions are used for all consortium meetings, including biannual progress reviews and annual meetings. An independent ethical advisory board is regularly consulted on ethical and regulatory issues, particularly for the clinical trial.
WP9: The visual identity of the project was developed, i.e. design of the Theralymph logo, and the Theralymph website has been launched. A first version of the Data Management Plan, which covers all types of data to be generated during the Theralymph project, has been elaborated.
We determine whether radiotherapy (photon versus proton) and chemotherapy for breast cancer can be a major risk factor for developing lymphedema. Then, we identified that adipose tissue is
We have determined that adipose tissue is a key player in lymphedema, not only because it houses lymphatic collecting vessels, but also because lipids play a crucial role in the function of these vessels. We have thus demonstrated that lipid mediators involved in the resolution of inflammation are important, paving the way for a new class of drugs.
The effect of VEGF-C combined with identified targets in WP2-4 was evaluated on LEC in vitro and in vivo. We decided to move forward with the combination of Apelin, a molecule that was described to have beneficial effect on both lymphatic vessels and adipocytes. The combination was produced in lentiflash vectors for a pre-clinical study to validate the combination that will be used in the clinical trial.
Lymphedema affects many patients treated for malignant gynecological tumors, with published rates reaching 70% in some populations. In a 2017 study, the cumulative incidence of lymphedema was 23.1% at 1 year, 32.8% at 3 years, and 47.7% at 10 years post-surgery. One in 5 women who survive breast cancer will develop lymphedema, equating to approximately 15,000 new cases of lymphedema per year in France (breast cancer incidence: 57,000 cases/year in France with a 5-year survival rate of 88% (Rochlin DH et al., JAMA Surg, 2023). The global lymphedema patient number is expected to grow significantly during the forecast period of 2022 to 2029.
The Theralymph clinical study will not only provide therapeutic solution for an unmet medical need that affects millions of patients, but will also considerably improve the quality of life and decrease the health care cost of this chronic incurable disease.
We have determined that adipose tissue is a key player in lymphedema, not only because it houses lymphatic collecting vessels, but also because lipids play a crucial role in the function of these vessels. We have thus demonstrated that lipid mediators involved in the resolution of inflammation are important, paving the way for a new class of drugs.
The effect of VEGF-C combined with identified targets in WP2-4 was evaluated on LEC in vitro and in vivo. We decided to move forward with the combination of Apelin, a molecule that was described to have beneficial effect on both lymphatic vessels and adipocytes. The combination was produced in lentiflash vectors for a pre-clinical study to validate the combination that will be used in the clinical trial.
Lymphedema affects many patients treated for malignant gynecological tumors, with published rates reaching 70% in some populations. In a 2017 study, the cumulative incidence of lymphedema was 23.1% at 1 year, 32.8% at 3 years, and 47.7% at 10 years post-surgery. One in 5 women who survive breast cancer will develop lymphedema, equating to approximately 15,000 new cases of lymphedema per year in France (breast cancer incidence: 57,000 cases/year in France with a 5-year survival rate of 88% (Rochlin DH et al., JAMA Surg, 2023). The global lymphedema patient number is expected to grow significantly during the forecast period of 2022 to 2029.
The Theralymph clinical study will not only provide therapeutic solution for an unmet medical need that affects millions of patients, but will also considerably improve the quality of life and decrease the health care cost of this chronic incurable disease.