RNAi-based gene therapy using miRNA-adapted short hairpin RNAs (shRNA^miR) is a powerful approach to modulate gene expression. However, we have observed low viral titers with shRNA^miR-containing recombinant vectors and hypothesized that this could be due to cleavage of viral genomic RNA by the endogenous microprocessor complex during virus assembly. To test this hypothesis, we targeted DROSHA, the core component of the microprocessor complex, and successfully generated monoallelic and biallelic DROSHA knockout (KO) HEK293T cells for vector production. DROSHA KO was verified by polymerase chain reaction (PCR) and western blot analysis. We produced lentiviral vectors containing Venus with or without shRNA hairpins and generated virus supernatants using DROSHA KO packaging cells. We observed an increase in the fluorescence intensity of hairpin-containing Venus transcripts in DROSHA KO producer cells consistent with reduced microprocessor cleavage of encoded mRNA transcripts, and recovery in the viral titer of hairpin-containing vectors compared with non-hairpin-containing constructs. We confirmed the absence of significant shRNA^miR processing by northern blot analysis and showed that this correlated with an increase in the amount of full-length vector genomic RNA. These findings may have important implications in future production of viral shRNA^miR-containing vectors for RNAi-based therapy.

使用适合miRNA的短发夹RNA（shRNA ^miR）的基于RNAi的基因疗法是调节基因表达的有效方法。然而，我们已经观察到含有shRNA ^miR的重组载体的病毒滴度较低，并假设这可能是由于病毒组装过程中内源微处理器复合物切割了病毒基因组RNA。为了验证该假设，我们针对了DROSHA（微处理器复合体的核心组件），并成功生成了单等位基因和双等位基因DROSHA敲除（KO）HEK293T细胞用于载体生产。德罗莎KO通过聚合酶链反应（PCR）和Western印迹分析进行了验证。我们生产了带有或不带有shRNA发夹的维纳斯慢病毒载体，并使用DROSHA KO包装细胞生成了病毒上清液。我们观察到DROSHA KO生产细胞中含发夹的金星转录物的荧光强度增加，这与减少的微处理器对编码的mRNA转录物的裂解一致，并且与不含发夹的构建体相比，含发夹的载体的病毒滴度有所恢复。我们确认不存在显着的shRNA ^miR通过RNA印迹分析进行的加工，并表明这与全长载体基因组RNA的量增加有关。这些发现可能对未来用于基于RNAi的治疗的病毒shRNA ^miR载体的生产具有重要意义。