The recombinant adeno‐associated virus (rAAV) vectors used in gene therapy are usually produced by transfecting three different plasmids (Adenoviral helper plasmid (pHelper), AAV rep/cap plasmids (pRepCap), and Transgene plasmid (pAAV‐GOI)) into human embryonic kidney 293 (HEK293) cells. However, the high proportion of unwanted empty capsids generated during rAAV production is problematic. To simultaneously enhance the genome titer and full capsid ratio, the ratio of the three plasmids transfected into HEK293 cells was optimized using design‐of‐experiment (DoE). AAV2 and AAV9, which have different production kinetics, were selected as cell‐associated and secreted model AAVs, respectively. In 125 mL Erlenmeyer flasks, the genome titers of rAAV2 and rAAV9 at DoE‐optimized plasmid weight ratios (pHelper:pRep2Cap2:pAAV‐GOI = 1:3.52:0.50 for rAAV2 and pHelper:pRep2Cap9:pAAV‐GOI = 1:1.44:0.27 for rAAV9) were 2.23‐fold and 2.26‐fold higher than those in the widely used plasmid weight ratio (1:1:1), respectively. In addition, compared with the plasmid ratio of 1:1:1, the relative VP3 band intensities of rAAV2 and rAAV9, which represent the relative empty capsid ratios, were reduced by 26% and 25%, respectively, at the DoE‐optimized plasmid ratio. Reduced empty capsid ratios in the DoE‐optimized plasmid ratios were also confirmed using transmission electron microscopy (TEM). Taken together, regardless of the AAV serotype, DoE‐aided optimization of the triple plasmid ratio was found to be an efficient means of improving the production of rAAV with a high full capsid ratio.

中文翻译：

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通过三重质粒比例的实验设计优化，提高 HEK293 细胞中高全衣壳比例的腺相关病毒 (AAV)2 和 AAV9 的产量

用于基因治疗的重组腺相关病毒（rAAV）载体通常通过将三种不同的质粒（腺病毒辅助质粒（pHelper）、AAVrep/cap质粒（pRepCap）和转基因质粒（pAAV-GOI））转染到人体内来产生。胚胎肾 293 (HEK293) 细胞。然而，rAAV 生产过程中产生的大量不需要的空衣壳是有问题的。为了同时提高基因组滴度和完整衣壳比率，使用实验设计（DoE）优化了转染至 HEK293 细胞的三种质粒的比率。AAV2和AAV9具有不同的生产动力学，分别被选为细胞相关型和分泌型AAV。在 125 mL 锥形瓶中，rAAV2 和 rAAV9 在 DoE 优化的质粒重量比下的基因组滴度（rAAV2 的 pHelper:pRep2Cap2:pAAV-GOI = 1:3.52:0.50，rAAV2 和 pHelper:pRep2Cap9:pAAV-GOI = 1:1.44:0.27 rAAV9）分别比广泛使用的质粒重量比（1:1:1）高2.23倍和2.26倍。此外，与1:1:1的质粒比例相比，在DoE优化的质粒中，代表相对空衣壳比例的rAAV2和rAAV9的相对VP3条带强度分别降低了26%和25%比率。使用透射电子显微镜 (TEM) 也证实了 DoE 优化的质粒比例中空衣壳比例的降低。总而言之，无论 AAV 血清型如何，DoE 辅助的三重质粒比例优化被发现是提高具有高全衣壳比例的 rAAV 生产的有效方法。