PDF(Pubmed)
Abstract：
The widespread successful use of recombinant Adeno-associated virus (rAAV) in gene therapy has driven the demand for scale-up manufacturing methods of vectors with optimized yield and transduction efficiency. The Baculovirus/Sf9 system is a promising platform for high yield production; however, a major drawback to using an invertebrate cell line compared to a mammalian system is a generally altered AAV capsid stoichiometry resulting in lower biological potency. Here, we introduce a term of the structural and biological \"fitness\" of an AAV capsid as a function of two interdependent parameters: (1) packaging efficiency (yield), and (2) transduction efficiency (infectivity). Both parameters are critically dependent on AAV capsid structural proteins VP1/2/3 stoichiometry. To identify an optimal AAV capsid composition, we developed a novel Directed Evolution (DE) protocol for assessing the structural and biological fitness of Sf9-manufactured rAAV for any given serotype. The approach involves the packaging of a combinatorial capsid library in insect Sf9 cells, followed by a library screening for high infectivity in human Cre-recombinase-expressing C12 cells. One single DE selection round, complemented by Next-Generation Sequencing (NGS) and guided by in silico analysis, identifies a small subset of VP1 translation initiation sites (known as Kozak sequence) encoding \"fit\" AAV capsids characterized by a high production yield and superior transduction efficiencies.
摘要：
重组腺相关病毒（rAAV）在基因治疗中的广泛成功使用推动了对具有优化产量和转导效率的载体按比例放大制造方法的需求。杆状病毒/Sf9系统是高产量生产的有前途的平台；然而，与哺乳动物系统相比,使用无脊椎动物细胞系的主要缺点是AAV衣壳化学计量通常改变,导致较低的生物学效力。这里,我们引入了AAV衣壳的结构和生物学“适应性”的术语，作为两个相互依赖的参数的函数：（1）包装效率（产量），和(2)转导效率(感染性)。这两个参数都严重依赖于AAV衣壳结构蛋白VP1/2/3化学计量。为了确定最佳的AAV衣壳组成，我们开发了一种新的定向进化（DE）方案，用于评估Sf9制造的rAAV对任何给定血清型的结构和生物学适应性。该方法涉及在昆虫Sf9细胞中包装组合衣壳库，随后在表达人Cre重组酶的C12细胞中筛选高感染性的文库。一个单独的DE选择回合，以下一代测序（NGS）为补充，并以计算机分析为指导，鉴定了一小部分编码“fit”AAV衣壳的VP1翻译起始位点（称为Kozak序列），其特征在于高产量和优异的转导效率。
