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Advancing Gene Delivery to Human Mesenchymal Stem Cells for Potential Therapeutic Applications
Human mesenchymal stem cells (hMSCs) are under study for applications in tissue engineering and regenerative medicine, but challenges remain before widespread clinical application of hMSCs can be realized. Engineering hMSCs with gene delivery could endow cells with enhanced therapeutic properties and advance hMSC clinical applications. While gene delivery with viral vectors is efficient, they present safety issues related to insertional mutagenicity. Nonviral methods are safer, but less efficient, especially in hMSCs. To better understand nonviral gene delivery mechanisms and to develop easily implemented protocols for more efficient transfection, our group has identified important transfection pathways and perturbed these pathways with pharmacological compounds to increase transfection efficiency, a strategy we call transfection ‘priming’. Specifically, priming hMSCs with glucocorticoid drugs (Gc) significantly increases transfection efficiency. Work presented in this dissertation investigates the mechanisms by which Gc priming enhances nonviral gene delivery to hMSCs. Our results suggest that specific pathways related to transfection-induced stress are modulated by Gc to enhance transgene expression, providing key insights towards developing more efficient nonviral gene delivery protocols for therapies. For example, enhanced transfection of hMSCs using priming strategies could be used for safe and effective delivery of CRISPR systems that can modulate gene expression (e.g. dCas9-p300 epigenome editing) to produce engineered hMSCs for therapies. Therefore, this dissertation also describes the use of Gc priming to develop a hMSC transfection protocol for nonviral delivery of a CRISPR epigenome editing system that enables precise control over gene expression, towards engineering hMSCs for potential therapies. Aside from engineering cells themselves, another application that could be advanced by gene delivery is the production of engineered exosomes. Recent research suggests that therapeutic properties of exosomes could be potentiated by inducing hMSCs to increase loading of specific therapeutic microRNAs (miRNAs) that have been shown to modulate many pathophysiological processes. Therefore, this dissertation also presents work towards developing a transgenic system that we show facilitates the active loading of exosomes with a chosen miRNA using cellular machinery.
Biomedical engineering|Cellular biology|Genetics
Hamann, Andrew, "Advancing Gene Delivery to Human Mesenchymal Stem Cells for Potential Therapeutic Applications" (2020). ETD collection for University of Nebraska-Lincoln. AAI28156003.