Engineered cell-derived extracellular vesicles (EVs) such as exosomes and microvesicles hold immense potential as safe and efficient drug carriers due to their lower immunogenicity and inherent homing capabilities to target cells. In addition to innate vesicular cargo such as lipids, proteins, and nucleic acids, EVs are also known to contain functional mitochondria/mitochondrial DNA that can be transferred to recipient cells to increase cellular bioenergetics. In this proof-of-concept study, we isolated naïve EVs and engineered EVs loaded with an exogenous plasmid DNA encoding for brain-derived neurotrophic factor (BDNF-EVs) from hCMEC/D3, a human brain endothelial cell line, and RAW 264.7 macrophages. We tested whether mitochondrial components in naïve or engineered EVs can increase ATP levels in the recipient brain endothelial cells. EVs (e.g., exosomes and microvesicles; EXOs and MVs) were isolated from the conditioned medium of either untreated (naïve) or pDNA-transfected (Luc-DNA or BDNF-DNA) cells using a differential centrifugation method. RAW 264.7 cell line-derived EVs showed a significantly higher DNA loading and increased luciferase expression in the recipient hCMEC/D3 cells at 72 h compared with hCMEC/D3 cell line-derived EVs. Naïve EVs from hCMEC/D3 cells and BDNF-EVs from RAW 264.7 cells showed a small, but a significantly greater increase in the ATP levels of recipient hCMEC/D3 cells at 24 and 48 h post-exposure. In summary, we have demonstrated (1) differences in exogenous pDNA loading into EVs as a function of cell type using brain endothelial and macrophage cell lines and (2) EV-mediated increases in the intracellular ATP levels in the recipient hCMEC/D3 monolayers.

Desmoplakin (DSP) is a large (~260 kDa) protein found in the desmosome, a subcellular complex that links the cytoskeleton of one cell to its neighbor. A mutation ‘hot-spot’ within the NH2-terminal third of the DSP protein (specifically, residues 299–515) is associated with both cardiomyopathies and skin defects. In select DSP variants, disease is linked specifically to the uncovering of a previously-occluded calpain target site (residues 447–451). Here, we partially stabilize these calpain-sensitive DSP clinical variants through the addition of a secondary single point mutation—tyrosine for leucine at amino acid position 518 (L518Y). Molecular dynamic (MD) simulations and enzymatic assays reveal that this stabilizing mutation partially blocks access to the calpain target site, resulting in restored DSP protein levels. This ‘molecular band-aid’ provides a novel way to maintain DSP protein levels, which may lead to new strategies for treating this subset of DSP-related disorders.

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