Distribution of AAV8 particles in cell lysates and culture media changes with time and is dependent on the recombinant vector.
Distribution of AAV8 particles in cell lysates and culture media changes with time and is dependent on the recombinant vector.
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AAV8颗粒在细胞裂解物和培养基中的分布随时间变化，并取决于重组载体。
DOI：
10.1038/mtm.2016.15
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Meagher MM
中科院分区：
文献类型：
--
作者：
Piras BA;Drury JE;Morton CL;Spence Y;Lockey TD;Nathwani AC;Davidoff AM;Meagher MM
随着临床试验的进行，腺相关病毒（AAV）的有效临床生产以治疗大量患者仍然是一个挑战。我们在转染后第3、5、6和7天比较了用因子VIII（FVIII）包装的AAV 8在细胞培养基和裂解物中的分布，并且发现到第6天病毒产量增加，培养基中病毒颗粒的比例从第3天的76%增加到第7天的94%。与FVIII相比，用因子IX和保护蛋白/组织蛋白酶A载体包装的AAV 8显示从第3天至第6天从裂解物向培养基的更大转变，这意味着颗粒分布依赖于重组载体。更大规模的生产显示，在培养基和裂解物中，全与空AAV颗粒的比率是相似的，并且与在第3天收获的AAV相比，在转染后第6天收获的AAV在小鼠中提供等同的功能。这表明，可以通过延长转染后培养的持续时间来优化AAV 8生产，并且通过仅允许收获培养基来简化AAV 8生产，其中处理含有10%或更少的总载体产率的细胞。此外，不同表达盒的颗粒分布差异意味着重组载体依赖性加工机制，应在工艺开发期间予以考虑。
With clinical trials ongoing, efficient clinical production of adeno-associated virus (AAV) to treat large numbers of patients remains a challenge. We compared distribution of AAV8 packaged with Factor VIII (FVIII) in cell culture media and lysates on days 3, 5, 6, and 7 post-transfection and found increasing viral production through day 6, with the proportion of viral particles in the media increasing from 76% at day 3 to 94% by day 7. Compared to FVIII, AAV8 packaged with Factor IX and Protective Protein/Cathepsin A vectors demonstrated a greater shift from lysate towards media from day 3 to 6, implying that particle distribution is dependent on recombinant vector. Larger-scale productions showed that the ratio of full-to-empty AAV particles is similar in media and lysate, and that AAV harvested on day 6 post-transfection provides equivalent function in mice compared to AAV harvested on day 3. This demonstrates that AAV8 production can be optimized by prolonging the duration of culture post-transfection, and simplified by allowing harvest of media only, with disposal of cells that contain 10% or less of total vector yield. Additionally, the difference in particle distribution with different expression cassettes implies a recombinant vector-dependent processing mechanism which should be taken into account during process development.