AAV6 Vexosomes Mediate Robust Suicide Gene Delivery in a Murine Model of Hepatocellular Carcinoma

Abstract

During recombinant Adeno-associated virus (AAV) production, a proportionately large amount of vectors is released in the culture supernatant, which is often discarded. It has been shown that these vectors often associate with vesiculated structures, such as exosomes. Exosome-associated AAV (vexosomes) represent an additional gene-delivery platform. The efficiency of such vexosomes in suicide gene therapy is unexplored. In the present study, we have generated AAV serotype 6 vexosomes containing an inducible caspase 9 (iCasp9) suicide gene by a differential ultracentrifugation-based protocol. We further tested the cytotoxic potential of these vexosomes in a human hepatocellular carcinoma (HCC) model in vitro and in vivo. The AAV6-iCasp9 containing vexosomes, when primed with a pro-drug (AP20187), demonstrated a significant loss in cell viability (57% ± 8% versus 100% ± 4.8%, p < 0.001) in comparison to mock-treated Huh7 cells. An intratumoral administration of AAV6-iCasp9 vexosomes and AP20187 in a murine xenograft model revealed a 2.3-fold increase in tumor regression in comparison to untreated animals. These findings were further corroborated by histological analysis and apoptosis assays. In conclusion, our data demonstrate the therapeutic potential of AAV6 vexosomes in a xenotransplantation model of HCC. Furthermore, the simplicity in production and isolation of vexosomes should further facilitate its application in other malignancies.

Therapeutic Application of Extracellular Vesicles-Capsulated Adeno-Associated Virus Vector via nSMase2/Smpd3, Satellite, and Immune Cells in Duchenne Muscular Dystrophy

“…In fact, clinical trials using genome editing have been discontinued because of suspicion of the development of chromosomal abnormalities. Therefore, as one of the novel gene therapy strategies, a drug delivery system for the AAV-encapsulating EV, which contains the AAV vector within the EV, was reported to be useful in the intervention of various target tissues in some diseases [189][190][191][192][193][194][195][196][197][198][199][200]. Furthermore, the effectiveness of gene therapy with AAV vectors is greatly affected by neutralizing antibodies [81,[201][202][203].…”

Targeting Non-coding RNA for Glioblastoma Therapy: The Challenge of Overcomes the Blood-Brain Barrier

“…None of the proposed viral vectors against GBM have been approved clinically ( 152 , 153 ). Nevertheless, a new category of AAV vector associated with exosomes, which are termed as vexosomes, has been reported ( 150 , 154 ) and their applications were studied recently ( 149 , 150 , 154 ). These vectors were efficiently transduced and were more resistant in neutralizing anti-AAV antibodies compared to conventionally purified AAV ( 149 ).…”

“…Although these vectors have not been investigated as delivery carriers for GBM treatment, they were very efficient in delivering DNA to the central nervous system (CNS) in mouse studies ( 150 ). Vexosomes were also used to deliver inducible caspase 9 (iCasp9) in Huh7 hepatic cancer cells and in hepatocellular carcinoma (HCC) xenograft mouse models ( 154 ). More exploratory studies are needed with the use of Vexosomes as drug delivery vehicles.…”

Current perspectives on exosomes in the diagnosis and treatment of hepatocellular carcinoma (review)

“…113 The modification of the exosomal membrane can increase the chemotaxis of exosomes to specific lesions. 114,115 Thus, exosomes can deliver anti-tumor drugs directly and effectively to the HCC tissues and prevent the progression of HCC. However, further studies are needed for the clinical application of exosomes for the diagnosis and treatment of HCC.…”