Guda Swaroopa, Brendel Christian, Renella Raffaele, Du Peng, Bauer Daniel E, Canver Matthew C, Grenier Jennifer K, Grimson Andrew W, Kamran Sophia C, Thornton James, de Boer Helen, Root David E, Milsom Michael D, Orkin Stuart H, Gregory Richard I, Williams David A
Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts, USA.
Harvard Medical School, Boston, Massachusetts, USA.
Mol Ther. 2015 Sep;23(9):1465-74. doi: 10.1038/mt.2015.113. Epub 2015 Jun 17.
RNA interference (RNAi) technology using short hairpin RNAs (shRNAs) expressed via RNA polymerase (pol) III promoters has been widely exploited to modulate gene expression in a variety of mammalian cell types. For certain applications, such as lineage-specific knockdown, embedding targeting sequences into pol II-driven microRNA (miRNA) architecture is required. Here, using the potential therapeutic target BCL11A, we demonstrate that pol III-driven shRNAs lead to significantly increased knockdown but also increased cytotoxcity in comparison to pol II-driven miRNA adapted shRNAs (shRNA(miR)) in multiple hematopoietic cell lines. We show that the two expression systems yield mature guide strand sequences that differ by a 4 bp shift. This results in alternate seed sequences and consequently influences the efficacy of target gene knockdown. Incorporating a corresponding 4 bp shift into the guide strand of shRNA(miR)s resulted in improved knockdown efficiency of BCL11A. This was associated with a significant de-repression of the hemoglobin target of BCL11A, human γ-globin or the murine homolog Hbb-y. Our results suggest the requirement for optimization of shRNA sequences upon incorporation into a miRNA backbone. These findings have important implications in future design of shRNA(miR)s for RNAi-based therapy in hemoglobinopathies and other diseases requiring lineage-specific expression of gene silencing sequences.
利用通过RNA聚合酶（pol）III启动子表达的短发夹RNA（shRNA）的RNA干扰（RNAi）技术已被广泛用于调节多种哺乳动物细胞类型中的基因表达。对于某些应用，例如谱系特异性敲低，需要将靶向序列嵌入到由pol II驱动的微小RNA（miRNA）结构中。在这里，我们以潜在的治疗靶点BCL11A为例，证明与在多种造血细胞系中由pol II驱动的miRNA适配的shRNA（shRNA(miR)）相比，由pol III驱动的shRNA导致显著增强的敲低效果，但同时也增加了细胞毒性。我们表明，这两种表达系统产生的成熟引导链序列相差4个碱基对的移位。这导致了不同的种子序列，从而影响了靶基因敲低的效果。将相应的4个碱基对的移位引入shRNA(miR)的引导链中，可提高BCL11A的敲低效率。这与BCL11A的血红蛋白靶点——人γ-珠蛋白或小鼠同源物Hbb-y的显著去抑制有关。我们的结果表明，在将shRNA序列整合到miRNA骨架中时需要对其进行优化。这些发现对未来设计用于血红蛋白病和其他需要谱系特异性表达基因沉默序列的疾病的基于RNAi治疗的shRNA(miR)具有重要意义。
