Brimble Mark A, Cheng Pei-Hsin, Winston Stephen M, Reeves Isaiah L, Souquette Aisha, Spence Yunyu, Zhou Junfang, Wang Yong-Dong, Morton Christopher L, Valentine Marcus, Thomas Paul G, Nathwani Amit C, Gray John T, Davidoff Andrew M
Department of Surgery, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
Department of Haematology, University College London (UCL) Cancer Institute, London WC1E 6DD, UK.
Mol Ther Methods Clin Dev. 2022 Jan 19;24:280-291. doi: 10.1016/j.omtm.2022.01.008. eCollection 2022 Mar 10.
Recombinant adeno-associated virus (rAAV) vectors are increasingly being used for clinical gene transfer and have shown great potential for the treatment of several monogenic disorders. However, contaminant DNA from producer plasmids can be packaged into rAAV alongside the intended expression cassette-containing vector genome. The consequences of this are unknown. Our analysis of rAAV preps revealed abundant contaminant sequences upstream of the AAV replication (Rep) protein driving promoter, P5, on the Rep-Cap producer plasmid. Characterization of P5-associated contaminants after infection showed transfer, persistence, and transcriptional activity in AAV-transduced murine hepatocytes, in addition to evidence suggestive of integration. These contaminants can also be efficiently translated and immunogenic, revealing previously unrecognized side effects of rAAV-mediated gene transfer. P5-associated contaminant packaging and activity were independent of an inverted terminal repeat (ITR)-flanked vector genome. To prevent incorporation of these potentially harmful sequences, we constructed a modified P5-promoter (P5-HS), inserting a DNA spacer between an Rep binding site and an Rep nicking site in P5. This prevented upstream DNA contamination regardless of transgene or AAV serotype, while maintaining vector yield. Thus, we have constructed an rAAV production plasmid that improves vector purity and can be implemented across clinical rAAV applications. These findings represent new vector safety and production considerations for rAAV gene therapy.
重组腺相关病毒（rAAV）载体越来越多地用于临床基因转移，并已显示出治疗多种单基因疾病的巨大潜力。然而，来自生产质粒的污染性DNA可能会与预期的含表达盒载体基因组一起被包装到rAAV中。其后果尚不清楚。我们对rAAV制剂的分析显示，在Rep-Cap生产质粒上，AAV复制（Rep）蛋白驱动启动子P5上游存在大量污染序列。感染后对与P5相关的污染物进行表征，结果显示，除了有整合的证据外，这些污染物在AAV转导的小鼠肝细胞中还具有转移、持久性和转录活性。这些污染物还能被有效翻译并具有免疫原性，揭示了rAAV介导的基因转移以前未被认识到的副作用。与P5相关的污染物包装和活性与反向末端重复序列（ITR）侧翼的载体基因组无关。为了防止这些潜在有害序列的掺入，我们构建了一种改良的P5启动子（P5-HS），在P5的Rep结合位点和Rep切口位点之间插入一个DNA间隔区。这可以防止上游DNA污染，而不管转基因或AAV血清型如何，同时保持载体产量。因此，我们构建了一种rAAV生产质粒，提高了载体纯度，可应用于临床rAAV的各种应用。这些发现代表了rAAV基因治疗新的载体安全性和生产方面的考虑因素。
