BACKGROUND Recombinant adeno-associated virus (rAAV) is widely used in the neuroscience field to manipulate gene expression in the nervous system. However, a limitation to the use of rAAV vectors is the time and expense needed to produce them. To overcome this limitation, we evaluated whether unpurified rAAV vectors secreted into the media following scalable PEI transfection of HEK293T cells can be used in lieu of purified rAAV. METHODS We packaged rAAV2-EGFP vectors in 30 different wild-type and mutant capsids and subsequently collected the media containing secreted rAAV. Genomic titers of each rAAV vector were assessed and the ability of each unpurified virus to transduce primary mixed neuroglial cultures (PNGCs), organotypic brain slice cultures (BSCs) and the mouse brain was evaluated. RESULTS There was ~ 40-fold wide variance in the average genomic titers of the rAAV2-EGFP vector packaged in the 30 different capsids, ranging from a low ~ 4.7 × 1010 vector genomes (vg)/mL for rAAV2/5-EGFP to a high of ~ 2.0 × 1012 vg/mL for a capsid mutant of rAAV2/8-EGFP. In PNGC studies, we observed a wide range of transduction efficiency among the 30 capsids evaluated, with the rAAV2/6-EGFP vector demonstrating the highest overall transduction efficiency. In BSC studies, we observed robust transduction by wild-type capsid vectors rAAV2/6, 2/8 and 2/9, and by capsid mutants of rAAV2/1, 2/6, and 2/8. In the in vivo somatic brain transgenesis (SBT) studies, we found that intra-cerebroventricular injection of media containing unpurified rAAV2-EGFP vectors packaged with select mutant capsids resulted in abundant EGFP positive neurons and astrocytes in the hippocampus and forebrain of non-transgenic mice. We demonstrate that unpurified rAAV can express transgenes at equivalent levels to lysate-purified rAAV both in vitro and in vivo. We also show that unpurified rAAV is sufficient to drive tau pathology in BSC and neuroinflammation in vivo, recapitulating previous studies using purified rAAV. CONCLUSIONS Unpurified rAAV vectors secreted into the media can efficiently transduce brain cells in vitro and in vivo, providing a cost-effective way to manipulate gene expression. The use of unpurified virus will greatly reduce costs of exploratory studies and further increase the utility of rAAV vectors for standard laboratory use.

中文翻译：

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利用最低限度纯化的分泌型 rAAV 快速且经济有效地操纵中枢神经系统中的基因表达。


背景技术重组腺相关病毒(rAAV)广泛应用于神经科学领域以操纵神经系统中的基因表达。然而，使用 rAAV 载体的一个限制是生产它们所需的时间和费用。为了克服这一限制，我们评估了 HEK293T 细胞可扩展 PEI 转染后分泌到培养基中的未纯化 rAAV 载体是否可以用来代替纯化的 rAAV。方法 我们将 rAAV2-EGFP 载体包装在 30 种不同的野生型和突变衣壳中，随后收集含有分泌的 rAAV 的培养基。评估了每种 rAAV 载体的基因组滴度，并评估了每种未纯化病毒转导原代混合神经胶质培养物 (PNGC)、器官型脑切片培养物 (BSC) 和小鼠大脑的能力。结果 包装在 30 种不同衣壳中的 rAAV2-EGFP 载体的平均基因组滴度存在约 40 倍的广泛差异，范围从 rAAV2/5-EGFP 的低约 4.7 × 1010 载体基因组 (vg)/mL 到rAAV2/8-EGFP 衣壳突变体的高约 2.0 × 1012 vg/mL。在 PNGC 研究中，我们在评估的 30 个衣壳中观察到了广泛的转导效率，其中 rAAV2/6-EGFP 载体表现出最高的总体转导效率。在 BSC 研究中，我们观察到野生型衣壳载体 rAAV2/6、2/8 和 2/9 以及 rAAV2/1、2/6 和 2/8 衣壳突变体的强转导。在体内体细胞脑转基因（SBT）研究中，我们发现脑室内注射含有未纯化的rAAV2-EGFP载体的培养基（包装有选定的突变衣壳），导致非转基因小鼠的海马和前脑中出现丰富的EGFP阳性神经元和星形胶质细胞。 我们证明未纯化的 rAAV 在体外和体内都可以以与裂解物纯化的 rAAV 相当的水平表达转基因。我们还表明，未纯化的 rAAV 足以驱动 BSC 中的 tau 病理学和体内神经炎症，概括了以前使用纯化的 rAAV 的研究。结论 分泌到培养基中的未纯化的 rAAV 载体可以在体外和体内有效地转导脑细胞，为操纵基因表达提供了一种经济有效的方法。使用未纯化的病毒将大大降低探索性研究的成本，并进一步提高 rAAV 载体在标准实验室用途中的效用。