Biodistribution and cell-type affinity of myotropic extracellular vesicle candidates
Date
2023
Authors
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Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
Muscular pathologies represent a class of diseases and disorders comprising both inherited genetic disorders (i.e. muscular dystrophies) and secondary conditions (i.e. cachexia). The most promising therapeutics for these conditions are adeno-associated virus (AAV)-mediated gene therapies. This strategy has shown promise in clinical trials; however, immunogenicity and lack of specificity of the AAV are major hurdles for clinical approval. Extracellular vesicles (EVs), or cell-derived nanoparticles, have been proposed as alternative delivery vectors. By engineering targeting moieties on the surface of EVs, researchers have enhanced EV tropism to specific cell-types. There are a number of native proteins involved in myogenic fusion and adherence in skeletal muscle development, such as MyoMaker (MYMK), MyoMixer (MYMX) and M-Cadherin (M-Cad), as well as the synthetic myotropic peptides, ASSLNIA (MP1) and RRQPPRSISSHP (MP2), that could be displayed on the surface of EVs; however, no successful myotropic EV formulation has been generated to date. OBJECTIVES: 1) Examine the myotropic properties of EVs displaying the native myotropic protein candidate, MYMK, in vivo. 2) Determine the myotropic properties of EVs displaying chimeric myotropic protein candidates in vitro. HYPOTHESES: 1) MYMK-EVs will show greater affinity for skeletal muscle relative to non-engineered Human Embryonic Kidney (HEK293) cell-derived EVs in both C57/BL6 (1a) and mdx (1b) mouse models. 2) EVs displaying chimeric myotropic proteins will demonstrate selective delivery of cargo to muscle cells over non-muscle cell types. METHODS: HEK293 cells were transfected with lentivirus containing a MYMK transgene followed by selection using fluorescence-assisted cell sorting (FACS) to generate a stable MYMK-EV producer cell line. 6-11 week old male C57 and mdx mice received MYMK or control HEK293-EVs stained with either a lipophilic or amine-reactive dye. Select organs from the mice were harvested and analyzed via whole-organ fluorescence imaging, fluorescence microscopy and a fluorescent plate reader. For the in vitro experiments, plasmids encoding either MP-1 or MP-2 fused to the extracellular domain of prostaglandin F2 receptor inhibitor (PTGFRN) were transfected into HEK293 cells followed by antibiotic selection and FACS. EVs were labeled with an amine-reactive dye and incubated with muscle and non-muscle cell-types in vitro. The recipient cells were analyzed via fluorescence microscopy. RESULTS: There was no significant difference between the biodistribution of the MYMK-EVs and HEK293-EVs (p < 0.05) other than a significant decrease in the liver of mdx mice that received the MYMK-EVs as compared to the HEK293-EVs (p = 0.04). For both the MYMK (p = 0.004) and HEK293-EVs (p < 0.0001 ), a significant decline in signal was observed in the spleen of the mdx mice relative to the C57 mice. No signal above background was able to be measured in the tissues of mice that received EVs labeled with the amine-reactive dye. In vitro, the PTGFRN-EVs showed the highest affinity for the muscle cell-types as well as for HEK293 cells relative to non-engineered HEK293 EVs (p < 0.0001). The MP1-EVs also showed significant increases in delivery to these cell-types (HEK293: p < 0.0001, C2C12: p = 0.002, CM: p < 0.0001). The MP2-EVs showed enhanced delivery to the Kupffer cells (p = 0.003), but not to the muscle cell-types or HEK293 cells. CONCLUSION: MYMK did not significantly alter the biodistribution of EVs in vivo. PTGFRN-EV and MP1-EVs may represent promising myotropic EV candidates for future in vivo biodistribution experiments.
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Keywords
Biodistribution, Cellular tropism, Extracellular vesicles, Muscular dystrophy, Recombinant proteins, Gene therapies