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Cellular cytoskeleton dynamics modulates non-viral gene delivery through RhoGTPases
Anandika Dhaliwal1, Maricela Maldonado, Clayton Lin
1Biomedical Engineering Interdepartmental Program, University of California Los Angeles, Los Angeles, California, United States of America.
Plos One
|April 18, 2012
Summary
Rho GTPases regulate non-viral gene transfer by controlling polyplex uptake and intracellular processing. Inactivating these proteins significantly decreases transgene expression, highlighting their crucial role in gene delivery efficiency.
Area of Science:
- Cell Biology
- Biotechnology
- Molecular Biology
Background:
- Cellular microenvironment significantly influences cellular processes like morphology and uptake pathways.
- Fibronectin (Fn) enhances transgene expression via increased proliferation, cell spreading, and clathrin-mediated endocytosis.
- Rho GTPases mediate cell-Fn interactions, regulating actin dynamics and cellular responses.
Purpose of the Study:
- To investigate the role of Rho GTPases (Rho, Rac, Cdc42) in non-viral gene transfer within a fibronectin microenvironment.
- To determine how Rho GTPase activity affects polyplex uptake and transgene expression in mouse mesenchymal stem cells (mMSCs).
Main Methods:
- Inactivation of Rho GTPases using Clostridium difficile toxin B (TcdB) and C3 transferase.
- Expression of dominant-negative (RhoAT19N, Rac1T17N, Cdc42T17N) and constitutively active (RhoAQ63L, Rac1Q61L, Cdc42Q61L) Rho GTPase variants.
- Culturing mMSCs on fibronectin-coated and collagen I-coated surfaces.
Main Results:
- Inactivating Rho GTPases decreased transgene expression by over 90%.
- Dominant-negative Rho GTPases significantly reduced polyplex uptake and transgene expression.
- While fibronectin activates Rho GTPases, further activation did not enhance transgene expression on Fn.
- Activating Rho GTPases on collagen I, however, enhanced transgene expression.
Conclusions:
- Rho GTPases are critical regulators of non-viral gene transfer.
- These proteins modulate polyplex internalization and intracellular processing for efficient gene delivery.
- The microenvironment, specifically fibronectin, influences Rho GTPase-mediated gene transfer efficiency.
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