Efficient Serotype-Dependent Release of Functional Vector into the Culture Medium During Adeno-Associated Virus Manufacturing
Efficient Serotype-Dependent Release of Functional Vector into the Culture Medium During Adeno-Associated Virus Manufacturing
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DOI：
10.1089/hum.2010.107
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发表时间：
2010-10-01
影响因子：
4.2
通讯作者：
Wilson, James M.
中科院分区：
文献类型：
--
作者：
Vandenberghe, Luk H.;Xiao, Ru;Wilson, James M.
基于腺相关病毒（AAV）的载体作为体内基因治疗的研究工具和试剂日益受到关注。目前该技术的一个限制是矢量的通用和可扩展制造。根据基于aav2的载体的经验，AAV载体颗粒仍然与细胞密切相关，通常从细胞裂解物中收获，并且必须广泛纯化才能使用。我们在此报告，基于其他AAV血清型的载体，包括AAV1、AAV8和AAV9，在有血清或不含血清的生产培养培养基中大量发现，并且可以从培养基中收获。对于AAV2，这种区隔化的差异很大程度上是由于AAV2颗粒对肝素的亲和力，因为肝素结合基序被切除的AAV2变体产量更高，并有效地从细胞中释放出来。从培养基中分离的载体颗粒在体外和体内的转导效率、免疫原性和组织亲和性方面似乎与从细胞裂解物中纯化的载体颗粒在功能上相当。我们的发现将直接导致提高载体产量和简化AAV载体生产过程的方法，这将有助于实验室规模的制备和大规模生产。
Vectors based on adeno-associated virus (AAV) are the subject of increasing interest as research tools and agents for in vivo gene therapy. A current limitation on the technology is the versatile and scalable manufacturing of vector. On the basis of experience with AAV2-based vectors, which remain strongly cell associated, AAV vector particles are commonly harvested from cell lysates, and must be extensively purified for use. We report here that vectors based on other AAV serotypes, including AAV1, AAV8, and AAV9, are found in abundance in, and can be harvested from, the medium of production cultures carried out with or without serum. For AAV2, this difference in compartmentalization is largely due to the affinity of the AAV2 particle for heparin, because an AAV2 variant in which the heparin-binding motif has been ablated gives higher yields and is efficiently released from cells. Vector particles isolated from the culture medium appear to be functionally equivalent to those purified from cell lysates in terms of transduction efficiency in vitro and in vivo, immunogenicity, and tissue tropism. Our findings will directly lead to methods for increasing vector yields and simplifying production processes for AAV vectors, which should facilitate laboratory-scale preparation and large-scale manufacture.