Adeno-associated viruses (AAV) are promising vectors for gene therapy due to their efficacy in vivo. However, there is room for improvement to address key limitations such as the pre-existing immunity to AAV in patients, high-dose toxicity, and relatively low efficiency for some cell types. This study introduces a metabolic engineering approach, using knockout of the enzyme phosphatidylserine synthase 1 (PTDSS1) to increase the abundance of extracellular vesicle-enclosed AAV (EV-AAV) relative to free AAV in the supernatant of producer cells, simplifying downstream purification processes. The lipid-engineered HEK293T-ΔPTDSS1 cell line achieved a 42.7-fold enrichment of EV-AAV9 compared to free AAV9 in the supernatant. The rational genetic strategy also led to a 300-fold decrease of free AAV in supernatant compared to wild-type HEK293T. The membrane-engineered EV-AAV9 (mEV-AAV9) showed unique envelope composition alterations, including cholesterol enrichment and improved transduction efficiency in human AC16 cardiomyocytes by 1.5-fold compared to conventional EV-AAV9 and by 11-fold compared to non-enveloped AAV9. Robust in-vivo transduction four weeks after intraparenchymal administration of mEV-AAV9 was observed in the murine brain. This study shows promise in the potential of lipid metabolic engineering strategies to improve the efficiency and process development of enveloped gene delivery vectors.

中文翻译：

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代谢工程通过改变细胞外囊泡包封的 AAV 的脂质组成来提高转导效率和下游载体分离


腺相关病毒 （AAV） 因其体内疗效而成为有前途的基因治疗载体。然而，在解决关键限制方面仍有改进的空间，例如患者对 AAV 的免疫力、高剂量毒性以及某些细胞类型的效率相对较低。本研究介绍了一种代谢工程方法，利用磷脂酰丝氨酸合酶 1 （PTDSS1） 的敲除来增加生产细胞上清液中相对于游离 AAV 的细胞外囊泡包封的 AAV （EV-AAV） 的丰度，从而简化下游纯化过程。脂质工程改造的 HEK293T-ΔPTDSS1 细胞系的 EV-AAV9 富集程度是上清液中游离 AAV9 的 42.7 倍。与野生型 HEK293T 相比，合理的遗传策略还导致上清液中游离 AAV 减少了 300 倍。膜工程 EV-AAV9 （mEV-AAV9） 显示出独特的包膜组成改变，包括胆固醇富集和人 AC16 心肌细胞的转导效率，与传统 EV-AAV9 相比提高了 1.5 倍，与非包膜 AAV9 相比提高了 11 倍。在小鼠脑中观察到 mEV-AAV9 实质内给药 4 周后稳健的体内转导。本研究显示了脂质代谢工程策略在提高包膜基因递送载体的效率和工艺开发方面的潜力。