Microvesicle-Mediated Delivery of Minicircle DNA Results in Effective Gene-Directed Enzyme Prodrug Cancer Therapy

Abstract

An emerging approach for cancer treatment employs the use of extracellular vesicles, specifically exosomes and microvesicles, as delivery vehicles. We previously demonstrated that microvesicles can functionally deliver plasmid DNA to cells and showed that plasmid size and sequence, in part, determine the delivery efficiency. In this study, delivery vehicles comprised of microvesicles loaded with engineered minicircle (MC) DNA that encodes prodrug converting enzymes developed as a cancer therapy in mammary carcinoma models. We demonstrated that MCs can be loaded into shed microvesicles with greater efficiency than their parental plasmid counterparts and that microvesicle-mediated MC delivery led to significantly higher and more prolonged transgene expression in recipient cells than microvesicles loaded with the parental plasmid. Microvesicles loaded with MCs encoding a thymidine kinase (TK)/nitroreductase (NTR) fusion protein produced prolonged TK-NTR expression in mammary carcinoma cells. In vivo delivery of TK-NTR and administration of prodrugs led to the effective killing of both targeted cells and surrounding tumor cells via TK-NTR-mediated conversion of codelivered prodrugs into active cytotoxic agents. In vivo evaluation of the bystander effect in mouse models demonstrated that for effective therapy, at least 1% of tumor cells need to be delivered with TK-NTR-encoding MCs. These results suggest that MC delivery via microvesicles can mediate gene transfer to an extent that enables effective prodrug conversion and tumor cell death such that it comprises a promising approach to cancer therapy.

Extracellular Vesicle–Mediated In Vitro Transcribed mRNA Delivery for Treatment of HER2+ Breast Cancer Xenografts in Mice by Prodrug CB1954 without General Toxicity

“…That EVs might be superior is widely recognized, as shown in numerous studies involving si-and miRNA delivery. Examples are, let 7ainordertodeliverlet-7a (let7a) to treat breast cancer in mice (36); plasma siRNAs to blood cells for selective gene silencing (37); siRNA to the brain to treat opiate addiction (38); miR-26 to treat liver cancer (39); miR-16-based mimics to treat malignant pleural mesothelioma (40); and use of DNA minicircle for prodrug treatment (41).…”

Characterizing Extracellular Vesicles and Their Diverse RNA Contents

“…EVs are particularly interesting for novel therapeutic strategies. Because EVs have a suspected mechanism for targeting and delivering their cargoes to certain cell populations (Hyenne et al, 2019 ; Verweij et al, 2019 ), several groups are investigating EV-syndicated pharmacologic strategies to effectively increase drug potency and decrease systemic toxicity (De Jong et al, 2019 ; Kanada et al, 2019 ). Kanada et al showed that minicircle DNA encoding a prodrug converting enzyme was delivered by EVs to tumors in vivo , and prodrug administration led to tumor death when at least 1% of the tumor cells in a given tumor received the minicircle DNA.…”

Cell-Based Tracers as Trojan Horses for Image-Guided Surgery

“…EVs can be divided into three subgroups: (i) exosomes, which are particles with a lipid bilayer that are released from cells and are found in most fluids of the body (diameter: 40–100 nm; Figure 2 and Table 1 ), (ii) microvesicles (MV), also known as shedding vesicles and ectosomes (diameter: 50 nm–1 μm), which are formed through outward budding and membrane shedding, especially from transformed or injured cells, and (iii) apoptotic bodies (diameter: 50 nm–5 μm), which are parts of the cellular content of apoptotic cells, containing DNA, RNA, and histone proteins [ 117 , 118 , 119 ]. Given their natural functionality of cell-to-cell transfer of biomolecules, EVs have been modified for their use as drug delivery vehicles: for example, for the administration of anti-cancer agents in mice [ 120 , 121 ]. With respect to imaging applications, the focus of current research lies primarily on exosomes.…”