Non-viral Intracellular mRNA Delivery to Hard-to-transfect Cells using Synthetic Lipidoid Nanoparticles
Zhao, Xuewei.
2020
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Thesis (Ph.D.)--Tufts
University, 2020.
Submitted to the Dept. of Biomedical Engineering.
Advisor: Qiaobing Xu.
Committee: David Kaplan, James Deventer, and Bing Xu.
Keywords: Biomedical engineering, Nanotechnology, and Bioengineering.
mRNA-based therapeutics have become an emerging approach in vaccination, cancer immunotherapy and gene editing. ... read moreThe delivery of mRNA using nanoparticles is regarded as a safe and effective method with advantages include mRNA protection, low immunogenicity and adjustable tissue tropism. However, there are significant challenges in transfecting some specific types of cells ("hard-to-transfect cells"). Therefore, there remains a need for a material to effectively deliver mRNA into "hard-to-transfect cells", and express sufficient amount of proteins for the therapeutic or cell engineering purpose. Herein, we developed synthetic lipidoid nanoparticles to target two classes of "hard-to-transfect cells", including stem cells (mesenchymal stem cells, neural stem cells and induced pluripotent stem cells) and T cells (CD4+ and CD8+). In the stem cell delivery study, bioreducible lipidoids were identified and characterized for effective mRNA delivery and cytocompatibility. Formulation of the lipidoid nanoparticles with cholesterol, DOPE and DSPE-PEG improved the delivery efficacy to human mesenchymal stem cells (hMSCs), whereas non-formulated lipidoid performed better delivery to human neural stem cells (hNSCs) and human induced pluripotent stem cells (hiPSCs). The successful gene knockout in hMSCs was confirmed with T7E1 assay and TIDE analysis (86.1% editing) when Cas9 mRNA and sgRNA were co-delivered using lipidoid 400-O16B-3. Neural-like differentiation of hMSCs was induced through 400-O16B-3-mediated CRISPR knockout of neuron restrictive silencer factor (NRSF). In the T cell delivery study, lipidoids containing imidazole were identified for effective mRNA delivery through rational screening. A novel library of structurally similar lipidoids containing imidazole and imidazole analogue was constructed to identify a better structure. A novel tail library was also constructed for the structure-activity-relationship analysis. When imidazole-containing lipidoids were injected intravenously, lipidoid 93-O17S accumulated in the spleen and achieved 8.1% and 6.5% gene recombination in CD4+ and CD8+ T cells, respectively. Together, this work establishes a non-viral mRNA delivery system of "hard-to-transfect cells" both in vitro and in vivo. This elucidates an improved delivery platform to efficiently, safely and conveniently engineer stem cells and T cells.read less - ID:
- xs55ms84m
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