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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session C

Poster I1 - Investigating the translation efficiency of modified mRNAs in different cell types

Friday, October 25, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Messenger RNA (mRNA) holds great promise as a gene therapy due to its fast but transient expression and the lack of risk of genome integration. However, mRNA is inherently immunogenic and extremely labile. Nucleotide modifications can alleviate these caveats, and have been implemented in FDA-approved vaccines against COVID-19 disease developed by Moderna and Pfizer/BioNTech. However, little is known on how the same nucleotide modifications affect different mRNA sequences and their translation efficiency in different cell types. The primary goal of this project was to investigate this question.

Materials and

Methods:  To generate mRNA, plasmids containing the T7 promoter and gene of interest were purchased from Addgene. First, a tail-PCR reaction was used to amplify the DNA fragment containing the T7 promoter and gene of interest whilst adding a polyT tail. Next, the PCR product was purified using a PureLink PCR Purification Kit and concentrated using the DNA concentrator Zymo Kit. In vitro transcription (IVT) was performed with the HiScribe T7 mRNA Kit with Clean Cap that enables the incorporation of a capping agent (CleanCap). Unmodified mRNAs contain natural nucleobases: adenine, cytosine, guanine, and uracil. In modified mRNAs, we substituted one or more of these bases by ethanol precipitation. To investigate resistance to enzymatic degradation, mRNAs were incubated with RNAses for different time points and analyzed by gel electrophoresis. To investigate translation efficiency, mRNAs and modified mRNAs were transfected into different cell lines, including human fibroblasts and cardiomyocytes, and mouse hepatocytes, using a commercially available transfection reagent (Lipofectamine or JetPEI). Expression levels were measured by immunohistochemistry and Western Blot.

Results, Conclusions, and Discussions: We generated mRNAs for a fluorescent reporter (mCherry) and five genes of interest. For each of them, we synthesized six sequences including unmodified mRNA (with natural bases) and the following five modifications: 5moU (5-methoxyluridine), m1ѰU (1-methylpsuedouridine), 5mC (5-methylcystosine), and the combinations of 5moU + 5mC, and m1ѰU + 5mC. All sequences were synthesized with high yield and purity. The RNAse degradation assay showed that the same nucleotide modification did not confer the same degree of enzymatic resistance to each of the five sequences. Next, transfection studies in human fibroblasts, human cardiomyocytes, and mouse hepatocytes showed that the transfection reagent needs to be carefully selected for each cell type. For example, Lipofectamine MessengerMax was able to efficiently transfect fibroblasts and cardiomyocytes but not hepatocytes, for which JetPEI had to be used. Results from the fluorescent signal of mCherry and Western Blot and immunofluorescence for genes of interest 1-4 confirmed that different nucleotide modifications had different effects on the transfection efficiency of each sequence. This finding is likely explained by different effects in mRNA folding and secondary structures, but requires further investigation. Whether these findings are consistent across a wide variety of cell lines from different origins is still under study. However, our work highlights the importance of individually optimizing each mRNA sequence to ensure optimal translation in the desired model.

Acknowledgements (Optional):  Ana Negrin Sanzhez was supported by an AHA Institutional Award for Undergraduate Student Training (24IAUST1187087). This research was funded by an AHA Early Faculty Independence Award (24SCEFIA1156995).