Transgene Integration into the Human AAVS1 Locus Enhances Myosin II-Dependent Contractile Force by Reducing Expression of Myosin Binding Subunit 85
Transgene Integration into the Human AAVS1 Locus Enhances Myosin II-Dependent Contractile Force by Reducing Expression of Myosin Binding Subunit 85
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转基因整合到人类 AAVS1 位点中，通过减少肌球蛋白结合亚基 85 的表达来增强肌球蛋白 II 依赖性收缩力
DOI：
10.1016/j.bbrc.2015.08.018
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
and K. Kawabata
中科院分区：
文献类型：
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作者：
T. Mizutani;R. Li;H. Haga;and K. Kawabata
人类基因组中的腺相关病毒位点1(AAVS1)是通过将外源基因插入该基因治疗的有力候选者。AAVS1基因座包括肌球蛋白结合亚单位85(MBS85)的编码区。虽然MBS85的功能还不是很清楚，但MBS85的表达变化可能会影响肌球蛋白II依赖的收缩力量。在应用基因治疗之前，应检测MBS85表达改变对细胞收缩力的影响。在这项研究中，我们表明转基因整合到AAVS1中并随之而来的MBS85表达的降低改变了肌球蛋白II依赖的细胞收缩力量。我们利用CRISPR/Cas9基因组编辑系统建立了外源DNA敲除AAVS1(Ki细胞)的人成纤维细胞系。Western blotting分析显示，KI细胞显著降低了MBS85的表达。KI细胞也表现出比对照细胞更大的细胞收缩力量。肌球蛋白II调节轻链(MRLC)的磷酸化与肌缩力的增加有关。将MBS85表达载体导入KI细胞后，细胞的收缩能力和MRLC的磷酸化水平恢复到对照细胞的水平。这些数据表明，转基因整合到人AAVS1基因座可以诱导细胞收缩力量的增加，因此应该被认为是一种改变细胞收缩力量的基因治疗方法。
The adeno-associated virus site 1 (AAVS1) locus in the human genome is a strong candidate for gene therapy by insertion of an exogenous gene into the locus. TheAAVS1locus includes the coding region for myosin binding subunit 85 (MBS85). Although the function of MBS85 is not well understood, myosin II-dependent contractile force may be affected by altered expression of MBS85. The effect of altered expression of MBS85 on cellular contractile force should be examined prior to the application of gene therapy. In this study, we show that transgene integration intoAAVS1and consequent reduction of MBS85 expression changes myosin II-dependent cellular contractile force. We established a human fibroblast cell line with exogenous DNA knocked-in toAAVS1(KI cells) using the CRISPR/Cas9 genome editing system. Western blotting analysis showed that KI cells had significantly reduced MBS85 expression. KI cells also showed greater cellular contractile force than control cells. The increased contractile force was associated with phosphorylation of the myosin II regulatory light chain (MRLC). Transfection of KI cells with an MBS85 expression plasmid restored cellular contractile force and phosphorylation of MRLC to the levels in control cells. These data suggest that transgene integration into the humanAAVS1locus induces an increase in cellular contractile force and thus should be considered as a gene therapy to effect changes in cellular contractile force.