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.

中文翻译：

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AAV6 Vexosomes 在肝细胞癌的小鼠模型中介导稳健的自杀基因传递。

在重组腺相关病毒 (AAV) 生产过程中，大量的载体在培养上清液中释放，这些载体通常会被丢弃。已经表明，这些载体通常与泡状结构相关，例如外泌体。外泌体相关的 AAV（vexosomes）代表了一个额外的基因传递平台。这种 vexosomes 在自杀基因治疗中的效率尚未得到探索。在本研究中，我们通过基于微分超速离心的方案生成了含有诱导型半胱天冬酶 9 (iCasp9) 自杀基因的 AAV 血清型 6 vexosomes。我们进一步在体外和体内测试了这些 vexosomes 在人肝细胞癌 (HCC) 模型中的细胞毒性潜力。含有 vexosomes 的 AAV6-iCasp9，当用前药 (AP20187) 启动时，与模拟处理的 Huh7 细胞相比，细胞活力显着降低（57% ± 8% 对 100% ± 4.8%，p < 0.001）。在小鼠异种移植模型中肿瘤内施用 AAV6-iCasp9 vexosomes 和 AP20187 显示，与未治疗的动物相比，肿瘤消退增加了 2.3 倍。组织学分析和细胞凋亡测定进一步证实了这些发现。总之，我们的数据证明了 AAV6 vexosomes 在 HCC 异种移植模型中的治疗潜力。此外，vexosomes 的生产和分离的简单性应进一步促进其在其他恶性肿瘤中的应用。与未治疗的动物相比，肿瘤消退增加了 3 倍。组织学分析和细胞凋亡测定进一步证实了这些发现。总之，我们的数据证明了 AAV6 vexosomes 在 HCC 异种移植模型中的治疗潜力。此外，vexosomes 的生产和分离的简单性应进一步促进其在其他恶性肿瘤中的应用。与未治疗的动物相比，肿瘤消退增加了 3 倍。组织学分析和细胞凋亡测定进一步证实了这些发现。总之，我们的数据证明了 AAV6 vexosomes 在 HCC 异种移植模型中的治疗潜力。此外，vexosomes 的生产和分离的简单性应进一步促进其在其他恶性肿瘤中的应用。