Recombinant adeno-associated virus (rAAV) is one of the prominent gene delivery vehicles that has opened promising opportunities for novel gene therapeutic approaches. However, the current major viral vector production platform, triple transfection in mammalian cells, may not meet the increasing demand. Thus, it is highly required to understand production bottlenecks from the host cell perspective and engineer the cells to be more favorable and tolerant to viral vector production, thereby effectively enhancing rAAV manufacturing. In this review, we provided a comprehensive exploration of the intricate cellular process involved in rAAV production, encompassing various stages such as plasmid entry to the cytoplasm, plasmid trafficking and nuclear delivery, rAAV structural/non-structural protein expression, viral capsid assembly, genome replication, genome packaging, and rAAV release/secretion. The knowledge in the fundamental biology of host cells supporting viral replication as manufacturing factories or exhibiting defending behaviors against viral production is summarized for each stage. The control strategies from the perspectives of host cell and materials (e.g., AAV plasmids) are proposed as our insights based on the characterization of molecular features and our existing knowledge of the AAV viral life cycle, rAAV and other viral vector production in the Human embryonic kidney (HEK) cells.

中文翻译：

---

解码重组腺相关病毒（rAAV）的细胞机制并改造宿主细胞工厂以强化病毒载体制造


重组腺相关病毒（rAAV）是重要的基因传递载体之一，为新型基因治疗方法带来了广阔的前景。然而，目前主要的病毒载体生产平台——哺乳动物细胞中的三重转染，可能无法满足日益增长的需求。因此，迫切需要从宿主细胞的角度了解生产瓶颈，并改造细胞使其更有利于和耐受病毒载体的生产，从而有效增强rAAV的生产。在这篇综述中，我们对 rAAV 生产中涉及的复杂细胞过程进行了全面的探索，包括各个阶段，如质粒进入细胞质、质粒运输和核递送、rAAV 结构/非结构蛋白表达、病毒衣壳组装、基因组复制、基因组包装和 rAAV 释放/分泌。每个阶段都总结了宿主细胞作为制造工厂支持病毒复制或表现出针对病毒生产的防御行为的基础生物学知识。基于分子特征的表征以及我们对 AAV 病毒生命周期、rAAV 和其他病毒载体在人类胚胎中生产的现有知识，我们提出了从宿主细胞和材料（例如 AAV 质粒）角度的控制策略。肾 (HEK) 细胞。