A Simplified Baculovirus-AAV Expression Vector System Coupled With One-step Affinity Purification Yields High-titer rAAV Stocks From Insect Cells
A Simplified Baculovirus-AAV Expression Vector System Coupled With One-step Affinity Purification Yields High-titer rAAV Stocks From Insect Cells
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DOI：
10.1038/mt.2009.128
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发表时间：
2009-11-01
影响因子：
12.4
通讯作者：
Kotin, Robert M.
中科院分区：
文献类型：
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作者：
Smith, Richard H.;Levy, Justin R.;Kotin, Robert M.
随着重组腺相关病毒（rAAV）介导的基因治疗领域接近临床，重组腺相关病毒（rAAV）生产的可扩展方法最近获得了很多兴趣。特别地，通过使用重组杆状病毒技术在昆虫细胞中生产rAAV载体已被证明是rAAV生产的有效且可扩展的手段。在这里，我们描述了一种方法，用于生产rAAV血清型1和2在昆虫细胞中使用一个简化的杆状病毒-AAV表达载体系统，再加上通过亲和层析颗粒纯化。通过遗传修饰AAV rep基因以允许从单个mRNA物种表达AAV编码的复制酶Rep 78和Rep 52，并将修饰的rep基因与AAV cap基因表达盒组合在单个杆状病毒构建体中，减少了rAAV生产所需的单独杆状病毒构建体的数量。此外，我们描述了裂解，结合，和洗脱条件与市售的亲和介质（AVB琼脂糖凝胶高性能），用于纯化rAAV颗粒在一个单一的色谱步骤接近同质兼容。使用所描述的方法，我们从重组杆状病毒感染的昆虫细胞的悬浮培养物中获得了每个细胞7 × 10（4）个纯化rAAV颗粒的平均产量（范围：3.7 × 10（4）至9.6 × 10（4））。
Scalable methods of recombinant adeno-associated virus (rAAV) production have gained much recent interest as the field of rAAV-mediated gene therapy approaches the clinic. In particular, the production of rAAV vectors in insect cells via the use of recombinant baculovirus technology has proven to be an efficient and scalable means of rAAV production. Here, we describe a method for the production of rAAV serotypes 1 and 2 in insect cells using a simplified baculovirus-AAV expression-vector system coupled with particle purification via affinity chromatography. The number of separate baculovirus constructs required for rAAV production was reduced by genetically modifying the AAV rep gene to allow expression of the AAV-encoded replication enzymes, Rep78 and Rep52, from a single mRNA species and combining the modified rep gene with an AAV cap gene expression cassette in a single baculovirus construct. Additionally, we describe lysis, binding, and elution conditions compatible with a commercially available affinity medium (AVB Sepharose High Performance) used to purify rAAV particles to near homogeneity in a single chromatography step. Using the described method, we obtained an average yield of 7 x 10(4) purified rAAV particles per cell (range: 3.7 x 10(4) to 9.6 x 10(4)) from suspension cultures of recombinant baculovirus-infected insect cells.