Improved mRNA drug efficacy in hepatic cells through mannose functionalization of lipid nanoparticles

dc.contributor.authorBrännudd, Ingrid
dc.contributor.departmentChalmers tekniska högskola / Institutionen för biologi och biotekniksv
dc.contributor.examinerEsbjörner Winters, Elin
dc.contributor.supervisorvan Leeuwen, Daniël
dc.description.abstractThe recent development and worldwide use of messenger RNA (mRNA) vaccines against Coronavirus Disease 2019 (COVID-19) have shed light on the huge potential of mRNA therapeutics. Apart from viral vaccines, mRNA therapeutics can be utilized as treatment for abnormal gene expression, insufficient protein production and as targeted cancer drugs, addressing increasingly important medical needs in a world with an aging population. mRNA vaccines have a great potential of becoming the leading vaccine type due to their high safety and easily scalable production, but the current problem of low efficacy of cellular uptake and functional delivery (estimated to be 1.5-3.5 %) must first be tackled. In the COVID-19 mRNA vaccines, the mRNA is delivered through encapsulation in Lipid Nanoparticles (LNPs), which transport the mRNA into the cell and deliver it to the cytosol where it is translated into protein. In this Thesis, I have optimized LNP designs by introducing receptor-targeting ligands on their surface, in the form of the sugar moiety mannose, conjugated to a poly ethyleneglycol (PEG) lipid. The mannose conjugates were used to improve the efficacy of mRNA-LNPs, to ultimately decrease the proportion of mRNA-LNPs that are degraded and thus increase the fraction of functional delivery and release of mRNA into the cytosol per given dose. To achieve this, mRNA-encapsulating LNPs with varying ratios of incorporated mannose-PEG lipids were formulated and biophysically characterized, including controls for lipid and sugar specificity. Subsequently, live-cell confocal laser scanning microscopy, automated image analysis and flow cytometry techniques were used to study the mRNA uptake and functional delivery in human hepatic Huh7 cells and human neuronal SH-SY5Y cells. Interestingly, substitution of DMPE-PEG lipid in a standard LNP formulation for a DSPE-PEG-mannose lipid increased the mRNA uptake and functional delivery, presumably through targeting of mannose-receptors which could have subsequent redirection effects of the endocytic route. The mannosylated LNPs had an enhancing effect in Huh7 cells, but not in SH-SY5Y cells indicating that the sugar functionalization confers cell specificity. The optimal mannose-PEG lipid substitution for increased mRNA uptake and functional delivery was found to be 0.5 mol%. Improving mRNA uptake and functional delivery, as explored here, is essential to lower the dose of the mRNA therapeutic administered to patients. This is desirable since lower mRNA doses decrease the risk of potential drug side effects but also decrease expenses for both healthcare and patients. The findings in this Thesis, that mannose functionalization improves the mRNA delivery efficacy mediated by LNPs, represent a step forward in this respect, and may thereby serve as useful inspiration for future developments in LNP design. This work was performed at the Division of Chemical Biology in the Department of Biology and Biological Engineering at Chalmers, Gothenburg, and at the Advanced Drug Delivery department at AstraZeneca, Mölndal, within the Swedish industrial research centre for functional RNA delivery;
dc.subjectendosomal escapesv
dc.subjectlipid nanoparticlessv
dc.subjectHuh7 cellssv
dc.subjectSH-SY5Y cellssv
dc.subjectlive-cell confocal microscopysv
dc.subjectautomated image analysissv
dc.subjectflow cytometrysv
dc.titleImproved mRNA drug efficacy in hepatic cells through mannose functionalization of lipid nanoparticlessv
dc.type.degreeExamensarbete för masterexamensv
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