Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Clinic for Neurology with Experimental Neurology, Gene Therapy Group, Campus Benjamin Franklin, Hindenburgdamm27, 12203 Berlin, Germany.
Viruses. 2022 Dec 24;15(1):64. doi: 10.3390/v15010064.
The successful application of recombinant adeno-associated virus (rAAV) vectors for long-term transgene expression in clinical studies requires scalable production methods with genetically stable components. Due to their simple production scheme and the high viral titers achievable, first generation recombinant adenoviruses (rAdV) have long been taken into consideration as suitable tools for simultaneously providing both the helper functions and the AAV and genes for rAAV packaging. So far, however, such rAdV- vectors have been difficult to generate and often turned out to be genetically unstable. Through ablation of and inhibitory function in the AAV-2 genome we have succeeded in establishing separate and stable rAdVs for high-level AAV serotype 2 Rep and Cap expression. These allowed rAAV-2 production at high burst sizes by simple coinfection protocols after providing the AAV-ITR flanked transgene vector genome either as rAAV-2 particles at low input concentrations or in form of an additional rAdV. With characteristics such as the ease of producing the required components, the straightforward adaption to other transgenes and the possible extension to further serotypes or capsid variants, especially the rAdV-mediated rAAV amplification system presents a very promising candidate for up-scaling to clinical grade vector preparations.
成功应用重组腺相关病毒(rAAV)载体进行临床研究中的长期转基因表达需要具有遗传稳定性组件的可扩展生产方法。由于其简单的生产方案和可实现的高病毒滴度,第一代重组腺病毒(rAdV)长期以来一直被认为是同时提供辅助功能和 AAV 的合适工具,以及 rAAV 包装所需的基因。然而,到目前为止,这种 rAdV-载体难以生成,并且常常表现出遗传不稳定性。通过在 AAV-2 基因组中切除和抑制功能,我们成功地建立了用于高水平 AAV 血清型 2 Rep 和 Cap 表达的独立且稳定的 rAdV。通过简单的共感染方案,在提供侧翼 AAV-ITR 的转基因载体基因组作为低输入浓度的 rAAV-2 颗粒或以额外 rAdV 的形式提供后,这些 rAdV 允许 rAAV-2 以高爆发大小进行生产。具有易于生产所需组件、易于适应其他转基因以及可能扩展到其他血清型或衣壳变体等特点,特别是 rAdV 介导的 rAAV 扩增系统,为扩大到临床级载体制备提供了一个非常有前途的候选方案。