AAV8 transduction capacity is reduced by prior exposure to endosome-like pH conditions.
AAV8 transduction capacity is reduced by prior exposure to endosome-like pH conditions.
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通过事先暴露于内体样pH条件来降低AAV8转导能力。
DOI：
10.4103/1673-5374.299272
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发表时间：
2021-05
影响因子：
6.1
通讯作者：
Lemmon VP
中科院分区：
文献类型：
--
作者：
Lowell JA;Mah KM;Bixby JL;Lemmon VP
腺相关病毒(AAV)是神经科学家工具包中的一种重要工具，它允许传递DNA来提供荧光蛋白或遗传指令的标记，以调节基因表达。在神经再生领域，神经元的转导可以观察和调节轴突的生长和再生，未来很可能成为促进中枢神经系统损伤后发芽和再生的分子治疗机制。传统的AAV制剂允许在生理条件下进行有效的病毒转导，但对AAV转导机制局限性的更好理解可能有助于为研究和治疗目的生产更具弹性的AAV毒株。我们在AAV8血清型(AAV8)先前暴露于内体吞噬过程中遇到的环境pH范围(pH 7.4至pH 4.4)的背景下研究了AAV的转导，在此期间，低pH触发了病毒衣壳的结构和自身蛋白分解变化，被认为是内体逃逸和病毒脱壳所必需的。由于这些过程的基本性质，我们假设AAV8颗粒过早暴露在酸性pH下会在体外减少HT1080细胞的病毒转导，通过高含量成像分析的荧光报告基因表达来衡量。我们发现，日益酸性的孵育条件伴随着转导效率的降低，转导细胞中报告基因表达的数量水平也同样下降。转导的最大降幅出现在pH 7.4和pH 6.4之间，这表明在该范围内可能同时发生与pH相关的事件和病毒灭活。综上所述，这些发现表明，AAV8暴露在酸性pH下短短1小时对转导能力是有害的。未来的研究是必要的，以了解与pH相关的致病机制。这项研究于2018年7月12日由美国迈阿密大学机构动物保护和使用委员会(#18-108-LF议定书)批准。
Adeno-associated virus (AAV) is an essential instrument in the neuroscientist’s toolkit, which allows delivery of DNA to provide labeling with fluorescent proteins or genetic instructions to regulate gene expression. In the field of neural regeneration, the transduction of neurons enables the observation and regulation of axon growth and regeneration, and in the future will likely be a mechanism for delivering molecular therapies to promote sprouting and regeneration after central nervous system injury. Traditional formulations of AAV preparations permit efficient viral transduction under physiologic conditions, but an improved understanding of the mechanistic limitations of AAV transduction may facilitate production of more resilient AAV strains for investigative and therapeutic purposes. We studied AAV transduction in the context of prior exposure of AAV serotype 8 (AAV8) to environmental pH within the range encountered during endosomal endocytosis (pH 7.4 to pH 4.4), during which low pH-triggered structural and autoproteolytic changes to the viral capsid are believed to be necessary for endosome escape and virus uncoating. Due to the fundamental nature of these processes, we hypothesized that premature exposure of AAV8 particles to acidic pH would decrease viral transduction of HT1080 cells in vitro, as measured by fluorescent reporter gene expression using high-content imaging analysis. We found that increasingly acidic incubation conditions were associated with concomitant reductions in transduction efficiency, and that quantitative levels of reporter gene expression in transduced cells were similarly decreased. The biggest decrease in transduction occurred between pH 7.4 and pH 6.4, suggesting the possible co-occurrence of a pH-associated event and viral inactivation within that range. Taken together, these findings indicate that exposure of AAV8 to acidic pH for as little as 1 hour is deleterious to transduction ability. Future studies are necessary to understand the pH-associated causative mechanisms involved. This study was approved by the University of Miami Institutional Animal Care and Use Committee, USA (Protocol #18-108-LF) on July 12, 2018.
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