Penaud-Budloo Magalie, François Achille, Clément Nathalie, Ayuso Eduard
INSERM UMR1089, University of Nantes, Centre Hospitalier Universitaire, Nantes, France.
Powell Gene Therapy, Department of Pediatrics, University of Florida, Gainesville, FL, USA.
Mol Ther Methods Clin Dev. 2018 Jan 8;8:166-180. doi: 10.1016/j.omtm.2018.01.002. eCollection 2018 Mar 16.
Recombinant adeno-associated viral (rAAV) vectors have been used in more than 150 clinical trials with a good safety profile and significant clinical benefit in many genetic diseases. In addition, due to their ability to infect non-dividing and dividing cells and to serve as efficient substrate for homologous recombination, rAAVs are being used as a tool for gene-editing approaches. However, manufacturing of these vectors at high quantities and fulfilling current good manufacturing practices (GMP) is still a challenge, and several technological platforms are competing for this niche. Herein, we will describe the most commonly used upstream methods to produce rAAVs, paying particular attention to the starting materials (input) used in each platform and which related impurities can be expected in final products (output). The most commonly found impurities in rAAV stocks include defective particles (i.e., AAV capsids that do contain the therapeutic gene or are not infectious), residual proteins from host cells and helper viruses (adenovirus, herpes simplex virus, or baculoviruses), and illegitimate DNA from plasmids, cells, or helper viruses that may be encapsidated into rAAV particles. Given the role that impurities may play in immunotoxicity, this article reviews the impurities inherently associated with each manufacturing platform.
重组腺相关病毒（rAAV）载体已在150多项临床试验中使用，具有良好的安全性，并且在许多遗传疾病中具有显著的临床益处。此外，由于其能够感染非分裂细胞和分裂细胞，并作为同源重组的有效底物，rAAV正被用作基因编辑方法的工具。然而，大量生产这些载体并符合现行良好生产规范（GMP）仍然是一个挑战，并且有几个技术平台正在争夺这一领域。在此，我们将描述生产rAAV最常用的上游方法，特别关注每个平台使用的起始材料（输入）以及最终产品（输出）中可能出现的相关杂质。rAAV库存中最常见的杂质包括缺陷颗粒（即不含治疗性基因或无感染性的AAV衣壳）、宿主细胞和辅助病毒（腺病毒、单纯疱疹病毒或杆状病毒）的残留蛋白，以及可能被包装到rAAV颗粒中的来自质粒、细胞或辅助病毒的非法DNA。鉴于杂质可能在免疫毒性中发挥的作用，本文综述了与每个生产平台固有相关的杂质。
