Purification of recombinant adeno-associated virus by iodixanol gradient ultracentrifugation allows rapid and reproducible preparation of vector stocks for gene transfer in the nervous system
Purification of recombinant adeno-associated virus by iodixanol gradient ultracentrifugation allows rapid and reproducible preparation of vector stocks for gene transfer in the nervous system
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DOI：
10.1089/10430349950017563
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发表时间：
1999-07-20
影响因子：
4.2
通讯作者：
Verhaagen, J
中科院分区：
文献类型：
--
作者：
Hermens, WTJMC;Ter Brake, O;Verhaagen, J
重组腺相关病毒（rAAV）载体已成为有吸引力的体内基因转移工具。用于实验和治疗性基因转移的高滴度rAAV载体的生产和纯化继续得到改进，标准的rAAV载体纯化方案包括用氯化铯（CsCl）密度梯度离心纯化载体，然后通过透析对生理缓冲液进行广泛的脱盐以供体内使用。这些程序非常耗时，并经常导致传染媒介滴度的大量损失。作为CsCl的替代品，我们研究了使用氯二沙醇（一种x射线对比溶液）作为密度梯度介质。用碘沙醇纯化rAAV载体可将离心时间缩短至3小时，使rAAV载体的浓缩和纯化具有可重复性。我们发现，注射由氯地沙醇梯度衍生的rAAV可用于脑和脊髓的体内基因转移应用，而不会产生可检测到的细胞病变效应，并指导至少2个月的稳定转基因表达。
Recombinant adeno-associated virus (rAAV) vectors have become attractive tools for in vivo gene transfer. The production and purification of high-titer rAAV vector stocks for experimental and therapeutic gene transfer continue to undergo improvement, Standard rAAV vector purification protocols include the purification of the vector by cesium chloride (CsCl)-density gradient centrifugation followed by extensive desalination via dialysis against a physiological buffer for in vivo use. These procedures are extremely time consuming and frequently result in a substantial loss of the infectious vector titer. As an alternative to CsCl we have investigated the use of lodixanol, an X-ray contrast solution, as the density-gradient medium. Purification of rAAV vectors by Iodixanol shortened the centrifugation period to 3 hr and resulted in reproducible concentration and purification of rAAV-vector stocks. We show that injection of rAAV derived from an lodixanol gradient can be used for in vivo gene transfer applications in the brain and spinal cord without detectable cytopathic effects and directing stable transgene expression for at least 2 months.