Regenerative medicine

Lymphedema treatment using gene therapy

Regenerative medicine such as gene therapies and cell therapies are able to expand, repair, replace, or regenerate organs or poorly functioning organs, tissues, cells, genes, and metabolic processes in the body.
 Innovative approaches include the use of stem cells, tissue engineering, and gene therapy alone or in various combinations. Finally, gene therapy is emerging as a successful strategy not only in monogenic diseases, but also in multifactorial conditions.

Context

Lymphedema is a lymphatic vascular system disorder characterized by impaired lymphatic return and swelling of the extremities. Accumulation of undrained fluid/lymph results in fibrosis and adipose tissue deposition in the affected arm or leg. It can be inherited or occurs after cancer surgery and lymph node removal. Indeed, 10-15% of women develop lymphedema after surviving breast cancer. It is a common disease affecting more than 120 million people worldwide and causing a significant morbidity, however there is no curative treatment for lymphedema.

Objectives & Technical challenges

TheraLymph program: A Therapy to restore lymphatic flow lymphedema

The main objective of the TheraLymph project is to develop a treatment for Lymphedema using multi-gene delivery FlashRNA® technology in a non-integrative gene therapy approach. The project consortium’s translational research program brings together scientists and physicians from 5 European countries and is focused on patients who developed Lymphedema after breast cancer surgery. The project goal is to validate the best molecule combination for gene therapy and to finalize phase I/II clinical trials at the affiliated hospital.  For more information, please visit the Theralymph project website.

Timeline & Funding

The Theralymph project was launched in 2020 for the duration of 5 years and has received funding from the EU Horizon 2020 research and innovation program under Grant Agreement No 874708.

Methodology

Three main tasks constitute the project:

In vitro validation of therapeutic targets identified by the different partners, such as VEGFC and another factors to restore the lymphatic collecting network
In vivo determination of the efficacy of FlashRNA® particles as an RNA delivery compared to an integrative lentivector, in lymphedema mouse models
Treating patients using multi-gene delivery FlashRNA® technology.

Results

Preliminary findings demonstrate promising outcomes, with FlashRNA® particles showcasing efficacy in restoring lymphatic function in preclinical models. In vitro experiments validate the therapeutic potential of selected gene combinations, paving the way for clinical translation.

Perspective

As the project progresses, it holds the potential to revolutionize lymphedema treatment paradigms, offering hope to millions worldwide. Continued research efforts and clinical trials will be crucial in realizing the full therapeutic potential of gene therapy in regenerative medicine applications.

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Consortium members

University of Helsinki

This project will be carried out in the Biomedicum Helsinki Research Center, which is a unique environment, one of few in Europe where all fields of biomedical research are located in the Academic Medical Center Helsinki (AMCH). The Translational Cancer Biology group, led by Dr. Alitalo, investigates the induction of lymphatic vessel growth and its benefits in lymphedema, using viral vector transduction, as well as the organ-specific heterogeneity and functions of lymphatic vessels using a combination of state-of-the-art molecular, cellular and genetic in vivo methods.
The group also investigates the role of meningeal lymphatic vasculature in neuroinflammation and neurodegenerative diseases. The team further characterizes responses of lymphatic vessels to cancer therapies, with a focus on clinically relevant immunotherapy approaches.

University of Lausanne

Vascular and Tumor Biology Laboratory is part of the Department of Oncology of UNIL and is an adjunct member of Lausanne branch of Ludwig Institute for Cancer Research Lausanne, that is specializing in tumor immunology and immune therapy. Their main research interests are in the molecular mechanisms of lymphatic and blood vessel growth and remodeling, and their role in normal organ function and diseases, such as inflammation and cancer.

University of Uppsala

The Vascular Development Laboratory headed by Dr. Mäkinen develops and utilizes genetic mouse models in combination with molecular and cell biological approaches to study mechanisms that regulate the morphogenesis and functional specialization of the vasculature. The lab also investigates how regulators of developmental (lymph)angiogenesis impact on genetic human diseases such as lymphoedema and vascular malformations.

University of Liège

The Laboratory of Biology of Tumor and Development (LBTD) belongs to the GIGA-Cancer in the GIGA Research Center (ULg), a major multidisciplinary center of research in life sciences composed of >600 researchers. In this center, team members have access to fully equipped and staffed core facilities and platforms. One of the main topics of research include cancer and tumor microenvironment, with a particular interest on angiogenesis and lymphangiogenesis in the context of breast cancer.

De Duve Institute

The Laboratory of Human Molecular Genetics, headed by Prof Miikka Vikkula, MD, PhD, focuses on characterization of the underlying pathophysiology of vascular anomalies, such as lymphedema, as well as cleft lip and palate, and selected cancers. The long-term goal is to develop molecular precision therapies for these disorders. The lab is specialized in evaluating the contribution of genetic variation to human disease. This research is based on blood and tissue samples collected from patients in collaboration with clinicians and multidisciplinary centers worldwide.
The lab analyses patients’ genomes using highthroughput (NGS) sequencing, including targeted panels, WES (whole exome sequencing), WGS (whole genome sequencing) and RNAseq. With its full-time bioinformatician, the group has developed an in-house software called Highlander implementing specialized bioinformatic tools for NGS analyses.

Charles University

The Laboratory of Pathophysiology of Adipose Tissue presented a number of important contributions in studies focused at changes of metabolic, endocrine and immune characteristics of AT during lifestyle – diet and physical activity- interventions in obese population. The main aim of the present activity is to elucidate a role of lipogenic and adipogenic capacity and inflammatory state of AT in ethiopathogenesis of obesity, type 2 diabetes, lymphedema and in pathogenesis of metabolic disturbances associated with aging. Laboratory is focused on clinical and translational research combining clinical studies with patients and in vitro laboratory techniques to analyze processes that underlie, or mediate disturbances related to dysfunctional adipose tissue.

Inserm

The PI laboratory is located in the Institute of Cardiovascular and Metabolic Diseases (I2MC) created in 2011 by Inserm. The research activity focuses on metabolic, cardiovascular and renal diseases. The main feature of I2MC is the gathering of basic scientists together with clinicians working on metabolic risk factors (obesity, diabetes and dyslipidemia) and their cardiovascular complications (thrombosis, atherosclerosis, cardiac and renal failure).
The laboratory headed by Dr Garmy-Susini studies molecular regulations of (lymph)angiogenic factors in vascular pathologies. It developed research axes in the field of gene expression control in response to stress and in pathophysiology of the lymphatic system, connected to therapeutic axes of gene therapy of ischemic heart disease and lymphedema. The main objectives of the lab are to 1/identify the pathophysiology of the lymphatic system in lymphedema, 2/characterize regulation of lymphangiogenesis-related gene expression in stress conditions (hypoxia, ER stress…), and 3/develop innovative therapies to restore lymphatic flow to improve healing in ischemic heart and in lymphedema.

CNRS

The project is held by the Institute for Research on Cancer and aging of Nice (IRCAN). The team of Gilles Pagès focuses its research on the regulation of angiogenic factors and their role as markers of sensitivity or resistance to different cancers treatments. The first major paper of the team described the efficacy of Gleevec in chronic myeloid leukemia; (Legros, L et al Blood, 2004). More recently the team focused on renal cell carcinoma and published pivotal papers on the mechanisms of resistance to anti-angiogenic drugs.

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Revolutionizing Therapeutics with RNA delivery platform : FlashRNA®

FlashRNA® is a non-integrative viral RNA delivery system allowing transient, efficient and safe gene expression which marks a significant milestone in the field of gene and cell therapy. Its non-toxic, non-immunogenic nature, high cellular uptake, and ability to transduce various RNAs showcase its value. This versatile technology, compliant with Good Manufacturing Practices (cGMPs), creates new therapeutic possibilities with enhanced safety features.

Recent advances in gene and cell therapy highlight the complex challenge of delivering therapeutic agents tailored to specific diseases. While DNA-based therapies have gained notoriety (ILV & AAV administration), the focus is now shifting to RNA-based therapies as a versatile alternative, that can target undruggable pathways⁽¹⁾. RNA therapeutics are a rapidly expanding class of medicines that will change the standard of care for many diseases and bring personalized medicine up to date. It is a disruptive therapeutic technology. This shift is driven by the need for a universal delivery tool that can be used for a wide range of applications while minimising risks, particularly genotoxicity. RNA-based therapies thus offer the flexibility required to target specific therapeutic applications requiring transient expression, whether for gene modification, regenerative medicine or immuno-oncology. This field of application encompasses triggering cellular processes, modifying genetic sequences or directing cells along specific differentiation pathways. Once optimised, these genetically modified cells become effective medicines, illustrating an innovative approach in the landscape of next-generation 2.0 therapies⁽²⁾.

To overcome the safety limitations of AAV and lentiviral vectors, a revolutionary RNA delivery tool called FlashRNA® (formerly LentiFlash®) has been developed, providing safe and effective solution. This innovative bacteriophage-lentivirus chimera combines the RNA packaging system of bacteriophages with the structure of lentiviral particles, delivering several species of RNA in a single FlashRNA® particle⁽³⁾. This not only protects the RNAs from degradation but also enables them to be delivered efficiently into the cell cytoplasm, thanks to VSV-G pseudotyping that ensures broad tropism or alternatively use of particular pseudotyping to target specific cell types. RNA bioavailability and protein translation is fast, without any risk of integration into the host genome. As a result, transduction by FlashRNA® does not result in GMO generation. Furthermore, the packaged RNAs are devoid of any lentiviral sequence and are from biological origin: indeed, as they are synthesized in human producer cells, they undergo all cellular post-transcriptional modifications, thus preventing adverse immune responses upon delivery into target cells.

To highlight the three innovative therapeutic strategies – gene editing, regenerative medicine and immuno-oncology – we invite you to discover concrete examples that illustrate each of these approaches.

GENE EDITING

FlashRNA® delivers the CRISPR-Cas9 machinery in a single step in many types of primary cells, from stem cells to immune cells, as well as human induced pluripotent stem cells (iPSCs). This approach guarantees efficient gene editing without compromising cell viability or the differentiation capacity of stem cells. FlashRNA® was successfully used to generate knock-out and gene conversion in iPSCs in vitro ⁽⁴⁾ as well as to perform base editing in vivo ⁽⁵⁾.

REGENERATIVE THERAPY

Within the current Theralymph clinical project, FlashRNA® is being used to express two different mRNAs to restore lymphatic function in patients suffering from secondary lymphedema. Preclinical results obtained in mouse models demonstrated the potential to abolish lymphedema when 2 mRNAs are co-delivered, enabling lymphatic flow to be restored. This paves the way for a Phase I/II clinical trial, that will be conduced next year at the Toulouse University Hospital, France.

IMMUNO-ONCOLOGY

Through direct in vivo delivery of tumour antigen-carrying FlashRNA® or FlashRNA® modified-Dendritic cells, the immune system can then efficiently block tumour development in wild-type mice. This method allows the expression of multiple antigens, enhancing specific immune responses and minimising the risk of tumour relapse.

About FlashRNA® :

FlashRNA® is a non-integrative viral RNA delivery system allowing transient, efficient and safe gene expression which marks a significant milestone in the field of gene and cell therapy. This valuable technology is demonstrated by its non-toxic, non-immunogenic nature, combined with a high uptake due to its viral characteristics and its possibility to deliver multiple distinct RNAs. Its versatility, coupled with a production platform compliant with current cGMPs, opens new avenues for therapeutic approaches, providing additional safety considerations compared to other methods.