Exogenous DNA Loading into Extracellular Vesicles via Electroporation is Size-Dependent and Enables Limited Gene Delivery

Abstract

Extracellular vesicles (EVs) hold immense promise for utilization as biotherapeutics and drug delivery vehicles due to their nature as biological nanoparticles that facilitate intercellular molecular transport. Specifically, EVs have been identified as natural carriers of nucleic acids, sparking interest in their use for gene therapy and RNA interference applications. So far, small RNAs (siRNA and miRNA) have been successfully loaded into EVs for a variety of delivery applications, but the potential use of EVs for DNA delivery has scarcely been explored. Here, we report that exogenous linear DNA can be associated with EVs via electroporation in quantities sufficient to yield an average of hundreds of DNA molecules per vesicle. We determined that loading efficiency and capacity of DNA in EVs is dependent on DNA size, with linear DNA molecules less than 1000 bp in length being more efficiently associated with EVs compared to larger linear DNAs and plasmid DNAs using this approach. We further showed that EV size is also determinant with regard to DNA loading, as larger microvesicles encapsulated more linear and plasmid DNA than smaller, exosome-like EVs. Additionally, we confirmed the ability of EVs to transfer foreign DNA loaded via electroporation into recipient cells, although functional gene delivery was not observed. These results establish critical parameters that inform the potential use of EVs for gene therapy and, in agreement with other recent results, suggest that substantial barriers must be overcome to establish EVs as broadly applicable DNA delivery vehicles.

Nanoparticle orientation to control RNA loading and ligand display on extracellular vesicles for cancer regression

“…HEK293T EVs were used as they contain minimal intrinsic biological cargos compared to EVs generated by other cells. 5 As shown in Western blots (Fig. S1a), HEK293T isolated EVs showed negative staining for several common integrin markers as seen on EVs for cancerous origins, 6, 7 with only positive staining for TSG101.…”

Therapeutic and diagnostic applications of extracellular vesicles

“…Following this pioneering report, other research groups have shown comparable results for loading siRNA and even DNA strands up to a 1000 bp into EVs [164][165][166][167][168]. Besides nucleic acids, 5 nm superparamagnetic iron oxide particles and large proteins have been loaded in purified EVs using EP (table 2).…”

“…Since the publication of this report, several groups have tried to prevent this aggregate formation through the use of chelating agents (e.g. EDTA) [166] or membrane stabilizers (e.g. trehalose) [170,171].…”

Emerging potential of gene silencing approaches targeting anti-chondrogenic factors for cell-based cartilage repair

“…Mounting evidence suggests that extracellular vesicles (EV), particularly exosomes, play a crucial role in the transfer of RNA molecules between cells as part of cell communication processes. Different encapsulation approaches have been explored for loading exosomes with DNA or siRNA molecules [47,48]. Thus, exosomes represent interesting natural carriers potentially exploitable for the delivery of RNAi to a variety of cell types and tissues, including cartilage.…”