Exogenous loading of extracellular vesicles, virus-like particles, and lentiviral vectors with supercharged proteins

Abstract

Cell membrane-based biovesicles (BVs) are important candidate drug delivery vehicles and comprise extracellular vesicles, virus-like particles, and lentiviral vectors. Here, we introduce a non-enzymatic assembly of purified BVs, supercharged proteins, and plasmid DNA called pDNA-scBVs. This multicomponent vehicle results from the interaction of negative sugar moieties on BVs and supercharged proteins that contain positively charged amino acids on their surface to enhance their affinity for pDNA. pDNA-scBVs were demonstrated to mediate floxed reporter activation in culture by delivering a Cre transgene. We introduced pDNA-scBVs containing both a CRE-encoding plasmid and a BV-packaged floxed reporter into the brains of Ai9 mice. Successful delivery of both payloads by pDNA-scBVs was confirmed with reporter signal in the striatal brain region. Overall, we developed a more efficient method to load isolated BVs with cargo that functionally modified recipient cells. Augmenting the natural properties of BVs opens avenues for adoptive extracellular interventions using therapeutic loaded cargo.

Extracellular communication between brain cells through functional transfer of Cre mRNA

“…To detect the EV-mediated extracellular transfer of Cre and permanently register its activity in the genomic DNA (gDNA), we generated a reporter cell line based on the FLExNanoluc switch reporter, ( Breyne et al 2022 ). In the OFF-state, the FLExNanoluc system does not express Nanoluciferase (Nanoluc) as its coding sequence is flipped between LoxP regions ( Figure 1B ).…”

“…Due to its high sensitivity, the expression of Nanoluc in reporter cells is beneficial to detect a low number of Cre-mediated functional events in a limited timeframe. Indeed, our previously published FLExNanoluc system ( Breyne et al 2022 ) showed robustness in detecting Cre activity mediated by EV delivery through the expression of Nanoluc. In the brain, we took advantage of the well-established Flox-TdTomato Ai9 mouse model ( Madisen et al 2010 ), that expresses tdTomato upon Cre recombination, to track the uptake of bdEVs carrying functional Cre mRNA.…”

Bioengineered extracellular vesicles: future of precision medicine for sepsis

“…On the one hand, non-cell-based methods, also known as exogenous loading [77] (Fig. 1), involves direct filling of already isolated EVs with therapeutic agents.…”

Extracellular Vesicle-Based SARS-CoV-2 Vaccine

“…However, when using EVs as therapeutic DDS carriers, the vesicles need to be loaded with drugs to be delivered. There are two main methods for achieving this: pre-loading therapeutic nucleic acids, such as siRNA or miRNA, into the cells to be produced, and post-loading a drug externally into the produced EVs [ 137 , 138 , 139 , 140 , 141 , 142 , 143 , 144 , 145 ]. The former has drawbacks; for example, the mechanism by which contents such as nucleic acids are incorporated into EVs has not been fully elucidated.…”