Skip to main content

Gene Therapy to restore lymphatic flow lymphedema

Periodic Reporting for period 1 - TheraLymph (Gene Therapy to restore lymphatic flow lymphedema)

Reporting period: 2020-01-01 to 2021-06-30

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 people 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 gene therapy for this unmet medical need.
The Theralymph translational research programme brings together bench scientists and physicians from five European countries to perform a Phase I/II trial focusing on women who developed lymphedema after breast cancer. 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 for lymphedema and cartography the lymphatic network in lymphedema pathology (WP1,2);
• Decipher whether lymphatic intrinsic molecules or microenvironmental peptides or lipids are modified in the lymphedematous arm to promote lymphatic dysfunction (WP3,4);
• Identify therapeutic targets and validate the best molecular combination that restores the lymphatic drainage in preclinical lymphedema modelsstudies (WP5,6);
• Conduct a Phase I/II gene therapy clinical trial using the innovative LentiFlash® technology based on recently developed 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 ongoing COVID-19 health crisis has added a significant hurdle for the whole project. All Theralymph partners were severely impacted by temporary national lockdowns resulting in the closure of laboratories for several months. Moreover, severe restrictions to lab access were implemented in most partners’ institutions, which delayed staff recruitment and completion of some tasks that could not be done remotely. Consequently, most work packages were affected. A 6-months extension to the original project duration has 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 lymphangiogenesic factor VEGF-C, was evaluated in the aggressiveness of breast cancer (BC). We determined that chemotherapeutic paclitaxel stimulates VEGFC production without influencing the sensitivity to therapy.
WP2 – Visualisation of collecting lymphatic vessel and transcriptome analysis of lymphatic endothelial cells (LEC) from mouse adipose tissue (AT) have been initiated. RNAseq analysis of tissues from human lymphedema patients revealed potential therapeutic targets.
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 adipogenesis, RNA sequencing is ongoing to identify molecular regulations.
WP4 - Eicosanoid profiles of lymphedematous skin demonstrated differential production of eicosanoid-derived lipids. The effect of these lipid mediators was investigated on LEC proliferation, migration, tube formation. The results established a significant and distinct biological activity on lymphatic vessels.
WP5 - A clone of NIH3T3 cells knock-out for VEGF-C expression has been generated through CrispR/Cas9 technology and 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 8 potential targets have been cloned in multicistronic vectors to produce lentivectors. Targets alone or in combination were injected into lymphedema bearing mice. The first results indicate potential additive/synergistic activity of a combination that will be further tested in mouse models.
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 for the Theralymph consortium was set up to ensure the progress of the project towards the planned objectives. Due to the COVID 19 health crisis online and virtual solutions have been implemented for all consortium meetings including kick-off, regular work progress and annual meetings. The private project management platform has been activated. An independent external advisory board including an ethics advisor has been established.
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 investigate the expression level of VEGF-C in various stages of BC to evaluate the risk to develop lymphedema. VEGF-C knock-out cells were generated to determine the optimal treatment administration.
A single cell RNAseq (mouse) and bulk RNAseq (human) is performed to identify signature genes for collecting vessels from normal and lymphedematous AT, and thereby potential therapeutic targets.
The production of adipokines by the lymphedema-related AT is studied to identify adipokines affecting lymphedema.
We established that lipid mediators had distinct biological activity on LECs, which may be relevant for lymphatic vessel function in vivo and may contribute to the resolution of inflammation in lymphedema. These results will allow to better characterize the lymphedema pathology from molecular to microenvironmental aspects.
The effect of VEGF-C combined with identified targets in WP2-4 is evaluated on LEC proliferation, migration and tube forming to point out the best synergistic effects.
Lentivectors containing the combination of lymphatic regulators are generated to evaluate the effect of these molecules in mouse models of lymphedema. The best performers will be produced in lentiflash vectors for a pre-clinical study to identify the combination that will be used in the clinical trial.
The Theralymph clinical study will decipher the optimal therapeutic combination to cure lymphedema. This 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.
Logo of the Theralymph project