Cellular Cytoskeleton Dynamics Modulates Non-Viral Gene Delivery through RhoGTPases

Abstract

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.

Temporal endogenous gene expression profiles in response to polymer-mediated transfection and profile comparison to lipid-mediated transfection

“…One potential explanation of the altered transfection is that a similar mechanism may be at play as in a recent study showing that activation of regulators of G‐protein signaling (RGSs) prevent cyclic adenosine monophosphate and protein kinase A activity , and protein kinase A has been recently shown to modulate intracellular routing of polyplexes . Another potential mechanism of altered transfection is that RGS1 is a GAP that would alter cellular cytoskeleton dynamics via RhoGTPase signal transduction and therefore alter internalization and intracellular processing of polyplexes . In either event, altering the expression level of RGS1 can alter nonviral transfection possibly via G‐coupled signal transduction (GTPases Rho, Rac and CDC42) ending in processes known to affect transfection, such as endocytosis , cell spreading, migration, stress fibers, focal adhesion and cell motility (Figure ).…”

“…Another potential mechanism of altered transfection is that RGS1 is a GAP that would alter cellular cytoskeleton dynamics via RhoGTPase signal transduction and therefore alter internalization and intracellular processing of polyplexes . In either event, altering the expression level of RGS1 can alter nonviral transfection possibly via G‐coupled signal transduction (GTPases Rho, Rac and CDC42) ending in processes known to affect transfection, such as endocytosis , cell spreading, migration, stress fibers, focal adhesion and cell motility (Figure ).…”

“…SNX24 , identified as differentially expressed, may be involved in endocytic uptake of polyplexes because, in activating (by 4‐hydroxytamoxifen, binds to estrogen receptor and alters expression of estrogen‐dependent genes) or inhibiting (by benzoic acid, which alters glycine‐dependent metabolism) SNX24 , we saw a 5.3‐fold increase or 4.0‐fold decrease in transfection, respectively (Table ). Taken together, given the downregulation of so many genes at the 2‐h time point, the cell may act to shutdown certain processes to prevent further uptake of polyplexes such as filopodia ( ARHGAP24 , GSN , IGF2BP1 , and PDZD2 ), GTPases ( ARHGAP24 , GSN , IGF2BP1 , RGS1 ) and endocytic uptake or escape ( PDZD2 and SNX24 ) (Figure ). Such a shutdown has been reported after viral infection, where host‐cell transcription and nucleocytoplasmic trafficking is halted , although the event has not been reported for nonviral transfection.…”

Transfection in the third dimension

“…In relation to the cell itself, cell proliferation, cell area, internalization, intracellular trafficking and integrin expression modulate gene transfer efficiency. Furthermore, for cells plated in 2-D on ECM proteins, the gene transfer is modulated by internalization pathways, cytoskeletal dynamics and RhoGTPases mediated signalling 16, 17 . Most of the studies have focused on studying the parameters influencing non-viral gene delivery in cells plated in 2-D, while little is known about 3-D.…”

It’s All in the Delivery: Designing Hydrogels for Cell and Non-viral Gene Therapies

“…Such methods often involve incubating a porous scaffold in a solution rich in condensed DNA before washing the scaffold to remove unbound particles [142][143][144][145] or hydrating a lyophilized hydrogel in a condensed DNA solution. [146][147][148][149] Via the first method, Saul et al 138 reported a maximal loading capacity of approximately 44 mg complexed DNA per 8-mm diameter by 2-mm thickness porous fibrin gel. 142 The load amount varies with the DNA:PEI ratio used to form the polyplexes, which controls the surface charge of the polyplexes.…”