Cellular Cytoskeleton Dynamics Modulates Non-Viral Gene Delivery through RhoGTPases

Although it is well accepted that the constituents of the cellular microenvironment modulate a myriad of cellular processes, including cell morphology, cytoskeletal dynamics and uptake pathways, the underlying mechanism of how these pathways influence non-viral gene transfer have not been studied. Transgene expression is increased on fibronectin (Fn) coated surfaces as a consequence of increased proliferation, cell spreading and active engagement of clathrin endocytosis pathway. RhoGTPases mediate the crosstalk between the cell and Fn, and regulate cellular processes involving filamentous actin, in-response to cellular interaction with Fn. Here the role of RhoGTPases specifically Rho, Rac and Cdc42 in modulation of non-viral gene transfer in mouse mesenchymal stem (mMSCs) plated in a fibronectin microenvironment was studied. More than 90% decrease in transgene expression was observed after inactivation of RhoGTPases using difficile toxin B (TcdB) and C3 transferase. Expression of dominant negative RhoA (RhoAT19N), Rac1(Rac1T17N) and Cdc42 (Cdc42T17N) also significantly reduced polyplex uptake and transgene expression. Interactions of cells with Fn lead to activation of RhoGTPases. However, further activation of RhoA, Rac1 and Cdc42 by expression of constitutively active genes (RhoAQ63L, Rac1Q61L and Cdc42Q61L) did not further enhance transgene expression in mMSCs, when plated on Fn. In contrast, activation of RhoA, Rac1 and Cdc42 by expression of constitutively active genes for cells plated on collagen I, which by itself did not increase RhoGTPase activation, resulted in enhanced transgene expression. Our study shows that RhoGTPases regulate internalization and effective intracellular processing of polyplexes that results in efficient gene transfer.

尽管学界已普遍认可细胞微环境（cellular microenvironment）的组成成分可调控诸多细胞过程，包括细胞形态、细胞骨架动力学（cytoskeletal dynamics）及摄取通路（uptake pathways），但此类通路如何影响非病毒基因转染（non-viral gene transfer）的潜在机制尚未得到系统探究。
在纤连蛋白（fibronectin，Fn）包被的表面上，转基因表达（transgene expression）水平可因细胞增殖增强、细胞铺展以及网格蛋白内吞通路（clathrin endocytosis pathway）的主动激活而升高。RhoGTP酶（RhoGTPases）介导细胞与纤连蛋白之间的信号串扰（crosstalk），并响应二者的相互作用，调控涉及丝状肌动蛋白（filamentous actin）的细胞过程。
本研究针对RhoGTP酶（具体为Rho、Rac及Cdc42）在小鼠间充质干细胞（mouse mesenchymal stem cells，mMSCs）于纤连蛋白微环境中培养时对非病毒基因转染的调控作用展开探究。经艰难梭菌毒素B（difficile toxin B，TcdB）与C3转移酶（C3 transferase）处理以灭活RhoGTP酶后，转基因表达水平下降超过90%。显性负突变体RhoA（RhoAT19N）、Rac1（Rac1T17N）与Cdc42（Cdc42T17N）的表达，同样显著降低了多聚复合物（polyplex）的摄取量与转基因表达水平。细胞与纤连蛋白的相互作用可激活RhoGTP酶。
然而，对于在纤连蛋白表面培养的细胞，通过组成型激活基因（constitutively active genes）（RhoAQ63L、Rac1Q61L及Cdc42Q61L）进一步激活RhoA、Rac1与Cdc42，并未进一步提升转基因表达水平。与之相反，对于在I型胶原（collagen I）表面培养的细胞——I型胶原本身并不会增强RhoGTP酶的激活——通过组成型激活基因激活上述三种GTP酶，可显著提升转基因表达水平。
本研究表明，RhoGTP酶可调控多聚复合物的内化过程与有效细胞内加工（intracellular processing），从而实现高效的基因转染。