DROSHA Knockout Leads to Enhancement of Viral Titers for Vectors Encoding miRNA-Adapted shRNAs.
DROSHA Knockout Leads to Enhancement of Viral Titers for Vectors Encoding miRNA-Adapted shRNAs.
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DOI：
10.1016/j.omtn.2018.07.002
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发表时间：
2018-09-07
期刊：
影响因子：
--
通讯作者：
Williams DA
中科院分区：
文献类型：
--
作者：
Park HH;Triboulet R;Bentler M;Guda S;Du P;Xu H;Gregory RI;Brendel C;Williams DA
利用miRNA适应的短发夹RNA（shRNAmiR）的基于RNAi的基因治疗是调节基因表达的有力方法。然而，我们已经观察到含有shRNAmiR的重组载体的低病毒滴度，并假设这可能是由于在病毒组装期间内源性微处理器复合物对病毒基因组RNA的切割。为了验证这一假设，我们靶向了微处理器复合体的核心组件DROSHA，并成功地产生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。通过聚合酶链反应（PCR）和蛋白质印迹分析验证DROSHA KO。我们生产了含有Venus的慢病毒载体，其具有或不具有shRNA发夹，并使用DROSHA KO包装细胞产生病毒上清液。我们观察到DROSHA KO生产细胞中含发夹的Venus转录物的荧光强度增加，这与编码mRNA转录物的微处理器切割减少一致，并且与不含发夹的构建体相比，含发夹的载体的病毒滴度恢复。我们通过北方印迹分析证实了shRNAmiR加工的缺乏，并表明这与全长载体基因组RNA量的增加相关。这些发现可能对未来生产基于RNAi的病毒shRNAmiR载体具有重要意义。
RNAi-based gene therapy using miRNA-adapted short hairpin RNAs (shRNAmiR) is a powerful approach to modulate gene expression. However, we have observed low viral titers with shRNAmiR-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 shRNAmiR 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 shRNAmiR-containing vectors for RNAi-based therapy.
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影响因子：
12.4
作者：
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影响因子：
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作者：
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Amatruda, James F.
影响因子：
10.5
作者：
Han, JJ;Lee, Y;Kim, VN
通讯作者：
Kim, VN