Production of adeno-associated virus (AAV) serotypes by transient transfection of HEK293 cell suspension cultures for gene delivery.
Production of adeno-associated virus (AAV) serotypes by transient transfection of HEK293 cell suspension cultures for gene delivery.
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DOI：
10.1016/j.jviromet.2013.10.038
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发表时间：
2014-03
影响因子：
3.1
通讯作者：
Kamen AA
中科院分区：
文献类型：
--
作者：
Chahal PS;Schulze E;Tran R;Montes J;Kamen AA
HEK293 悬浮细胞的瞬时转染可有效产生 AAV 载体。生产了 9 种不同的 AAV 血清型，产量为 1E+13 Vg/L。在 3 L 生物反应器中生产的 AAV2 和 AAV6 的产量与摇瓶相当。该过程使用无血清培养基和 HEK293 主细胞库，与 cGMP 兼容。该工艺的工业化可用于生产 AAV 血清型。腺相关病毒（AAV）已成功用于基因治疗。不同血清型的AAV针对特定器官和组织具有高效率。大量生产用于临床前和临床试验的各种 AAV 血清型的需求不断增加。本研究描述了一种通用且可扩展的方法，通过转染在悬浮液和无血清培养基中生长的完全表征的 cGMP HEK293SF 细胞系，有效生产 AAV 血清型 (AAV1-9)。首先，使用 AAV2 作为模型血清型评估生产参数。其次，所有九种 AAV 血清型均已成功生产，细胞培养物产量为 1013 Vg/L。随后，AAV2 和 AAV6 血清型在 3 L 受控生物反应器中生产，产量高达 1013 Vg/L，与摇瓶中获得的产量相似。例如，对于 AAV2，在转染后 48 至 64 小时 (hpt) 之间测量了 1013 Vg/L 细胞培养物 (6.8 × 1011 IVP/L)。在此期间，获得了每个细胞 6800 Vg 和每个细胞 460 IVP 的平均细胞特异性 AAV2 产量，Vg 与 IVP 的比率小于 20。生物反应器中的成功操作证明了这种有效制造 AAV 血清型的通用工艺的规模化和工业化潜力。
Transient transfection of HEK293 suspension cells efficiently produce AAV vectors. Nine different AAV serotypes were produced with yields of 1E+13 Vg/L. AAV2 and AAV6 produced in 3-L bioreactors gave yields comparable to shake-flasks. The process is cGMP compatible using serum-free media and HEK293 master cell bank. Industrialization of the process is possible for manufacturing AAV serotypes. Adeno-associated virus (AAV) is being used successfully in gene therapy. Different serotypes of AAV target specific organs and tissues with high efficiency. There exists an increasing demand to manufacture various AAV serotypes in large quantities for pre-clinical and clinical trials. A generic and scalable method has been described in this study to efficiently produce AAV serotypes (AAV1-9) by transfection of a fully characterized cGMP HEK293SF cell line grown in suspension and serum-free medium. First, the production parameters were evaluated using AAV2 as a model serotype. Second, all nine AAV serotypes were produced successfully with yields of 1013 Vg/L cell culture. Subsequently, AAV2 and AAV6 serotypes were produced in 3-L controlled bioreactors where productions yielded up to 1013 Vg/L similar to the yields obtained in shake-flasks. For example, for AAV2 1013 Vg/L cell culture (6.8 × 1011 IVP/L) were measured between 48 and 64 h post transfection (hpt). During this period, the average cell specific AAV2 yields of 6800 Vg per cell and 460 IVP per cell were obtained with a Vg to IVP ratio of less than 20. Successful operations in bioreactors demonstrated the potential for scale-up and industrialization of this generic process for manufacturing AAV serotypes efficiently.
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影响因子：
12.4
作者：
Chadeuf, G;Ciron, C;Salvetti, A
通讯作者：
Salvetti, A
影响因子：
3.1
作者：
Durocher, Yves;Pham, Phuong Lan;Kamen, Amine
通讯作者：
Kamen, Amine
影响因子：
12.4
作者：
Gao, GP;Lu, FM;Wilson, JM
通讯作者：
Wilson, JM
影响因子：
4.2
作者：
Hauswirth, William W.;Aleman, Tomas S.;Jacobson, Samuel G.
通讯作者：
Jacobson, Samuel G.
影响因子：
4.2
作者：
Ellis, Brian L.;Potts, Patrick Ryan;Porteus, Matthew H.
通讯作者：
Porteus, Matthew H.