UCLA

Gene delivery has widespread application in tissue regeneration and gene therapy. While non-viral gene delivery is less immunogenic as compared to viral gene delivery, it is hindered by its lack of efficiency. Studies aiming to improve the efficiency of non-viral gene delivery have mostly focused on improving the vector system. However, the cellular microenvironment where the cells reside is only beginning to be exploited as a means to enhance gene transfer. In this dissertation, the effect of different densities of extracellular matrix proteins namely collagen I (C I), vitronectin (Vt), laminin (Lm), collagen IV (C IV), fibronectin (Fn) and ECM gel (ECMg), and their combinations on gene transfer to mouse mesenchymal stem cells (mMSCs) were studied. Protein coatings that resulted in well spread cells such as Fn, ECMg and C IV, resulted in 14.6-, 7- and 6.1- fold increase in transgene expression, respectively, when compared to uncoated surfaces. The transgene expression was up to 90% inhibited on C I coated surface, which led to less spread cells. Interestingly, the same trend was not observed for polyplex internalization, where protein coats that resulted in less spread cells, such as C I and Vt, resulted in higher polyplex internalization. Subsequently, decreased transgene expression corresponded with inhibited trafficking of the internalized complexes to the nucleus. The effect of combining multiple ECM proteins on non-viral gene transfer was also investigated. Surfaces coated with combination including C I resulted in inhibition of transgene expression. In addition, surfaces coated with combination of Vt, C IV and Lm resulted in a statistically similar enhancement in transgene expression as compared to fibronectin. For all ECM combinations analyzed, the extent of cell spreading mediated by the ECM protein had a 70% correlation with the extent of overall gene transfer observed.

The role of different endocytic pathways and cytoskeletal components on gene transfer was later analyzed on collagen I (C I) and fibronectin (Fn), which had opposite influence on gene transfer. The mechanism by which these ECM proteins affect non-viral gene transfer involved the endocytosis pathway used for polyplex uptake and intracellular tension. Fn was found to promote internalization through clathrin-mediated endocytosis and this pathway brought about more efficient transfection than caveolae-mediated endocytosis and macropinocytosis. Likewise, the disruption of actin-myosin interactions resulted in an enhancement of gene transfer for cells plated on Fn coated surfaces, but not for cells plated on C I.

Furthermore, the molecular mechanism by which these ECM molecules affect the process of gene transfer is not completely understood. The Rho subfamily of GTPases regulates a number of cellular processes including cell morphology, cytoskeletal dynamics and uptake pathways and they are activated to different extents by different ECM proteins. The involvement of RhoGTPases in the ECM protein-mediated enhancement of non-viral gene transfer was studied. Fibronectin was used as the substrate for these studies. The interaction of mouse mesenchymal stem cells (mMSCs) with fibronectin was found to activate RhoGTPases (RhoA, Rac, and Cdc42). Inactivation of RhoGTPases using chemical inhibitor or expression of dominant negative genes resulted in significantly reduced transgene expression. However, the activation of RhoGTPases using chemical activators or expressing constitutively active genes did not further enhance transgene expression for cells plated on fibronectin. But, for cells plated on C I, which did not result in RhoGTPase activation, expression of constitutively active RhoA, Rac, Cdc42 genes resulted in enhanced non-viral gene transfer.

Cells in the body exist in a 3-D microenvironment. Cellular processes like proliferation, differentiation, apoptosis as well as cellular morphology have been shown to be significantly different in 2-D versus 3-D. In part, these differences are due to differential signaling as a consequent of different cytoskeletal assembly and cell-matrix adhesions in 2-D and 3-D. To fully understand the mechanism of gene transfer in cells, L-polyethylenimine (LPEI) mediated non-viral gene transfer mechanism constituting pathways of endocytosis, cytoskeletal dynamics and RhoGTPases, was studied in 3-D using hyaluronic acid hydrogels, in comparison with 2-D using tissue culture plates. Caveolae and clathrin mediated endocytosis were observed to regulate gene transfer in 2-D, while all three pathways of endocytosis namely clathrin and caveolae mediated endocytosis, and macropinocytosis regulated gene transfer in 3-D. Actin polymerization and RhoA mediated contractility highly contributed to efficient transgene expression in 3-D rather than 2-D. Furthermore, Rac and Cdc42 influenced internalization in 2-D but not in 3-D. In conclusion, endocytic pathways, cytoskeletal dynamics and RhoGTPase mediated signaling differentially modulated non-viral gene transfer in cells cultured in 2-D and 3-D. These results provide an understanding of the dimensionality of cell microenvironment to obtain efficient gene transfer for the purpose of tissue regeneration and gene therapy.

We believe that the cellular microenvironment can be engineered to enhance the ability of cells to become transfected, and through understanding of the mechanisms by which the ECM affects non-viral gene transfer, better materials and transfection protocols can be achieved.