Nucleoside-modified VEGFC mRNA induces organ-specific lymphatic growth and reverses experimental lymphedema

Lack or dysfunction of the lymphatics leads to secondary lymphedema formation that seriously reduces the function of the affected organs and results in degradation of quality of life. Currently, there is no definitive treatment option for lymphedema. Here, we utilized nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNPs) encoding murine Vascular Endothelial Growth Factor C (VEGFC) to stimulate lymphatic growth and function and reduce experimental lymphedema in mouse models. We demonstrated that administration of a single low-dose of VEGFC mRNA-LNPs induced durable, organ-specific lymphatic growth and formation of a functional lymphatic network. Importantly, VEGFC mRNA-LNP treatment reversed experimental lymphedema by restoring lymphatic function without inducing any obvious adverse events. Collectively, we present a novel application of the nucleoside-modified mRNA-LNP platform, describe a model for identifying the organ-specific physiological and pathophysiological roles of the lymphatics, and propose an efficient and safe treatment option that may serve as a novel therapeutic tool to reduce lymphedema.


Statistics
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Software and code
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Data analysis
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Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Zoltán Jakus, MD, PhD Norbert Pardi, PhD Feb 17, 2021 The following software programs were used to collect the data in this study: NIS-Elements Imaging Software (Nikon) (version: BR 4.60.00) BD CellQuest Pro (Becton Dickinson) (version: 6.1) LAS X Software (Leica) 7.4.2.18368 The following software programs were used to analyze the data in this study: NIS-Elements Imaging Software ( No statistical analysis was performed to predetermine sample sizes. Sample sizes were chosen in accordance with the standard protocols of the field. Groups were arranged to include at least 3 individual samples to analyze the phenotypes and perform statistical analysis. We used statistical analysis consistent with the sample size for each experiment and we found sufficient statistical power between groups with the noted number of samples.
No samples were excluded from the analysis.
Experiments were performed by using several mice of each condition. All experiments were repeated several times as it is indicated in the figure legends.
Mice were used from different cages in the same experimental group to assure randomization.
All investigators performing flow cytometry, Western Blot or ELISA assays were blinded for the sample origin.
Investigators performing microscopic imaging and quantification of microscopic images were blinded for treatment and sample origin.
In the genetic lymphedema model the thickness of the paw was assessed by spring loaded custom caliper (Kroeplin), and visible clinical signs of edema formation were scored on a 0 -10 scale by two investigators blinded for the treatment of the mice.
Investigators performing experiments monitoring lymphatic function in vivo were blinded for the treatment of the mice.

nature research | reporting summary
October 2018

Validation
The expressed amount of VEGFC was also determined with VEGFC Rat ELISA Kit (Invitrogen, BMS626-2).
The following dilutions were used during our study. For Western blot, we used 1:1 000 dilution for primary and 1:10 000 dilution for secondary antibodies. For immunhistochemistry and whole mount immunostaining we used 1:100 dilution for primary and 1:250 for secondary antibodies. For FACS analysis we used 1:200 dilution for all antibodies.
All primary antibodies were validated using appropriate controls.
The cell lines were not authenticated.
The cell lines were negative in mycoplasma testing.
None 6-12 week-old C57Bl/6 wild type mice were purchased from commercial sources (Envigo and the Hungarian National Institute of Oncology). 6-12 week-old Prox1GFP BAC lymphatic reporter animals obtained from the Mutant Mouse Regional Resource Centers (MMRRC) were maintained in heterozygous form. To eliminate the lymphatic endothelial cells in a genetic experimental lymphedema model the Flt4-CreERT2; iDTRflox/flox strain on the C57Bl/6 background was used (6-24 week-old animals). Experimental animals were housed in either specific pathogen free or conventional animal facilities. Both male and female mice were used. Experimental animals were housed in either specific pathogen free or conventional animal facilities between 18-22°C, 45% humidity and 12/12 hours dark-light cycles.
No wild animals were used in this study.
No field-collected samples were used in this study.
All animal experiments were approved by the Animal Experimentation Review Board of the Semmelweis University and the Government Office for Pest County (Hungary) under license number PE/EA/148-4/2018 and PEI/001/404-8/2015 with approval for the use of genetically modified organisms under license number TMF/305-63/2015 issued by the Ministry of Agriculture (Hungary).