Removal of empty capsids from type 1 adeno-associated virus vector stocks by anion-exchange chromatography potentiates transgene expression
Removal of empty capsids from type 1 adeno-associated virus vector stocks by anion-exchange chromatography potentiates transgene expression
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DOI：
10.1016/j.ymthe.2005.11.024
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发表时间：
2006-04-01
影响因子：
12.4
通讯作者：
Ozawa, K
中科院分区：
文献类型：
--
作者：
Urabe, M;Xin, KQ;Ozawa, K
重组腺相关病毒（rAAV）的产生会产生大量的空衣壳或病毒样颗粒（VLP），即没有载体基因组的病毒蛋白壳。污染性 VLP 会通过竞争细胞表面受体来干扰转导，并且在体内施用时，会增加抗原负载，从而可能引发更强的免疫反应。密度梯度超速离心提供了一种将 VLP 与 rAAV 颗粒分离的方法，但对于大规模制备载体并不可行。由于单链DNA基因组的VLP和载体的组成不同，我们假设载体的等电点可能与VLP的不同。在尝试通过离子交换色谱法将 I 型 rAAV 颗粒与 VLP 分离时，我们测试了多种缓冲系统，发现硫酸三甲基铵或 [(CH3)(4)N](2)SO4 可以有效地将 rAAV1 颗粒与 VLP 分离。与 VLP 污染的 rAAV1-GFP 相比，从 VLP 中色谱分离的 rAAV1-GFP 在 HEK293 细胞中诱导更强的 GFP 表达。用从VLP分离的rAAV1-SEAP（碱性磷酸酶的分泌形式）转导小鼠肌肉也显示出比带有VLP的rAAV1 SEAP更高的血清SEAP水平。这些结果表明，从空衣壳中色谱分离 rAAV1 提高了 rAAV1 的功效。
Production of recombinant adeno-associated virus (rAAV) results in substantial quantities of empty capsids or virus-like particles (VLPs), virus protein shells without the vector genome. The contaminating VLPs would interfere with transduction by competing for cell-surface receptors and, when administered in vivo, contribute to antigen load, which may elicit a stronger immune response. Density-gradient ultracentrifugation provides a means to separate VLPs from rAAV particles, but is not feasible for large-scale preparations of vectors. Since the compositions of the VLP and vector differ by the single-stranded DNA genome, we hypothesized that the isoelectric point of the vector may differ from that of the VLP. In an attempt to separate type I rAAV particles from VLPs by ionexchange chromatography, we tested a number of buffer systems and found that trimethylammonium sulfate, or [(CH3)(4)N](2)SO4, effectively separated rAAV1 particles from VLPs. The rAAV1-GFP chromatographically separated from VLPs induced stronger GFP expression in HEK293 cells than rAAV1-GFP contaminated with VLPs. The transduction of mouse muscles with rAAV1-SEAP (secreted form of alkaline phosphatase) isolated from VLPs also showed higher serum SEAP levels than rAAV1 SEAP with VLPs. These results suggest that chromatographic separation of rAAV1 from empty capsids increased the efficacy of rAAV1.