Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors
Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors
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DOI：
10.1016/s1046-2023(02)00220-7
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发表时间：
2002-10-01
期刊：
影响因子：
4.8
通讯作者：
Snyder, RO
中科院分区：
文献类型：
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作者：
Zolotukhin, S;Potter, M;Snyder, RO
基于血清型2的重组腺相关病毒（rAAV）载体目前正在动物和人类临床试验中进行最广泛的评估。在动物模型中，由其他AAV血清型（scrota pes 1、3、4、5和6）构建的rAAV载体可以比rAAV 2载体更有效地和以不同的特异性感染某些组织。本文描述了用于生产和纯化用AAV 1或AAV 5衣壳假型化的含有AAV 2反向末端重复序列的载体的试剂和方法。为了便于假型化，在腺病毒质粒骨架中构建AAV 2 rep/AAV 1cap和AAV 2 rep/AAV 5cap辅助质粒。得到的质粒pXYZ 1和pXYZ 5.用于产生rAAV 1和rAAV 5载体。分别通过瞬时转染。由于AAV 5和AAV 1均不与用于纯化rAAV 2载体的肝素亲和层析树脂结合，因此基于阴离子交换层析开发纯化方案。纯化的载体储备液纯度为99%，滴度为1 × 10（12）至1 × 10（13）载体基因组/ml。(C)2002 Elsevier Science（美国）。All rights reserved.
Recombinant adeno-associated viral (rAAV) vectors based on serotype 2 are currently being evaluated most extensively in animals and human clinical trials. rAAV vectors constructed from other AAV serotypes (scrota pes 1, 3, 4, 5 and 6) can transduce certain tissues more efficiently and with different specificity than rAAV2 vectors in animal models. Here, e describe reagents and methods for the production and purification of AAV2 inverted terminal repeat-Containing vectors pseudotyped with AAV1 or AAV5 capsids. To facilitate pseudotyping, AAV2rep/AAV1cap and AAV2rep/AAV5cap helper plasmids were constructed in an adenoviral plasmid backbone. The resultant plasmids, pXYZ1 and pXYZ5. were used to produce rAAV1 and rAAV5 vectors. respectively, by transient transfection. Since neither AAV5 nor AAV1 binds to the heparin affinity chromatography resin used to purify rAAV2 vectors, purification protocols were developed based on anion-exchange chromatography. The purified vector stocks are 99% pure with titers of 1 X 10(12) to 1 X 10(13) vector genomes/ml. (C) 2002 Elsevier Science (USA). All rights reserved.