Marwidi Yoko, Nguyen Hoang-Oanh B, Santos David, Wangzor Tenzin, Bhardwaj Sumita, Ernie Gabriel, Prawdzik Gregg, Lew Garrett, Shivak David, Trias Michael, Padilla Jada, Tran Hung, Meyer Kathleen, Surosky Richard, Ward Alex Michael
Sangamo Therapeutics, 501 Canal Boulevard, Richmond, CA 94804, USA.
Mol Ther Methods Clin Dev. 2024 Mar 4;32(2):101228. doi: 10.1016/j.omtm.2024.101228. eCollection 2024 Jun 13.
Manufacturing of adeno-associated viruses (AAV) for gene and cell therapy applications has increased significantly and spurred development of improved mammalian and insect cell-based production systems. We developed a baculovirus-based insect cell production system-the SGMO Helper-with a novel gene architecture and greater flexibility to modulate the expression level and content of individual Rep and Cap proteins. In addition, we incorporated modifications to the AAV6 capsid sequence that improves yield, capsid integrity, and potency. Production of recombinant AAV 6 (rAAV6) using the SGMO Helper had improved yields compared to the Bac-RepCap helper from the Kotin lab. SGMO Helper-derived rAAV6 is resistant to a previously described proteolytic cleavage unique to baculovirus-insect cell production systems and has improved capsid ratios and potency, and , compared with rAAV6 produced using Bac-RepCap. Next-generation sequencing sequence analysis demonstrated that the SGMO Helper is stable over six serial passages and rAAV6 capsids contain comparable amounts of non-vector genome DNA as rAAV6 produced using Bac-RepCap. AAV production using the SGMO Helper is scalable using bioreactors and has improved yield, capsid ratio, and potency. Our studies demonstrate that the SGMO Helper is an improved platform for AAV manufacturing to enable delivery of cutting-edge gene and cell therapies.
用于基因和细胞治疗的腺相关病毒（AAV）的生产显著增加，并推动了基于哺乳动物和昆虫细胞的改进生产系统的发展。我们开发了一种基于杆状病毒的昆虫细胞生产系统——SGMO辅助系统，它具有新颖的基因结构，在调节单个Rep和Cap蛋白的表达水平和含量方面具有更大的灵活性。此外，我们对AAV6衣壳序列进行了修饰，提高了产量、衣壳完整性和效力。与科廷实验室的Bac-RepCap辅助系统相比，使用SGMO辅助系统生产重组AAV 6（rAAV6）的产量有所提高。源自SGMO辅助系统的rAAV6对杆状病毒-昆虫细胞生产系统特有的一种先前描述的蛋白水解切割具有抗性，并且与使用Bac-RepCap生产的rAAV6相比，其衣壳比例和效力有所提高。下一代测序序列分析表明，SGMO辅助系统在连续六次传代中是稳定的，并且rAAV6衣壳中包含的非载体基因组DNA量与使用Bac-RepCap生产的rAAV6相当。使用SGMO辅助系统生产AAV可通过生物反应器进行扩大规模，并提高了产量、衣壳比例和效力。我们的研究表明，SGMO辅助系统是用于AAV生产的改进平台，能够实现前沿基因和细胞疗法的递送。
