Genome-wide knockout screen for dual-transduction inhibitors

Recombinant adeno-associated viral vectors (rAAV) are a powerful tool for gene delivery but have a limited DNA carrying capacity. Efforts to expand this genetic payload have focused on engineering the vector components, such as dual trans-splicing vectors which double the delivery size by exploiting the natural concatenation of rAAV genomes in host nuclei. We hypothesized that inefficient dual vector transduction could be improved by modulating host factors which affect concatenation. Since factors mediating concatenation are not well defined, we performed a genome-wide screen to identify host cell regulators. We discovered that Homologous Recombination (HR) is inhibitory to dual vector transduction. We demonstrate that depletion or inhibition of HR factors BRCA1 and Rad51 significantly increase reconstitution of a large split transgene by increasing both concatenation and expression from rAAVs. Our results define new roles for DNA damage repair in rAAV transduction and highlight the potential for pharmacological intervention to increase genetic payload of rAAV vectors.

重组腺相关病毒载体（recombinant adeno-associated viral vectors，rAAV）是一类极具应用前景的基因递送工具，但其天然DNA装载容量存在局限。为拓展该载体的遗传装载量，当前研究多聚焦于载体组件的工程化改造，例如双反式剪接载体——这类载体可利用宿主细胞核内rAAV基因组的天然串联特性，使可递送的遗传物质规模翻倍。我们提出假说：可通过调控影响基因组串联过程的宿主因子，改善双载体转导效率低下的问题。由于介导该串联过程的宿主因子尚未被明确界定，我们开展了全基因组筛选以鉴定相关调控因子。研究发现，同源重组（Homologous Recombination，HR）通路对双载体转导具有抑制作用。实验证实，对HR通路核心因子BRCA1与Rad51进行敲除或抑制，可通过同时提升rAAV基因组串联效率与载体表达水平，显著增强大型断裂转基因的重组重构效果。本研究明确了DNA损伤修复通路在rAAV转导过程中的全新功能，并揭示了通过药物干预提升rAAV载体遗传装载量的潜在应用价值。