mRNA Expression of EGFP by lipid nanoparticles

Abstract

Two scientists, Katalin Karikó and Drew Weismann of Pennsylvania state university began research on mRNA as protein replacement therapeutics in the 90’s, but due to the mRNA’s immunogenicity it could not be used. However, in 2005 they found that the nucleotide uridine could be replaced with pseudouridine rendering the mRNA non immunogenic. When the pandemic hit, their work proved invaluable as many companies rushing to make the vaccines decided to use their technology. Their discoveries led to the first approved mRNA vaccines during the covid pandemic, saving many lives and earning them the Nobel prize in Physiology or Medicine 2023. Following this, RNA based therapeutics funding is now bigger than ever and researchers are hard at work seeking what other therapies RNA can be used for.

The technique used in the Sars cov 2 vaccine is cellular delivery by Lipid Nanoparticles (LNPs), a liposome like structure which can carry and deliver nucleic acids which are difficult to deliver by themselves. Among other factors the LNPs increase efficiency of delivery by increasing transfection, neither are they toxic to the body.

In this thesis we wanted to try to make these LNPs by ourselves and to see if we can express them in our chosen Human Colon Carcinoma cell line (HCT116). This was tested by seeding our cells with stained LNPs containing mRNA’s that code for Enhanced Green Fluorescent Protein (EGFP) and imaging them confocally.

Expression of EGFP was achieved testing for which concentration was optimal, and in the subsequent run of the experiment we tried out different methods of LNP preparation. The results of the experiments showed that expression is possible and can be quite high. They showed that N1-Methylpseudouridine mRNA gave better expression in vitro than did 5-Methoxyuridine and that acetate buffer during LNP preparation seems to increase mRNA expression compared to citrate buffer.

Two scientists, Katalin Karikó and Drew Weismann of Pennsylvania state university began research on mRNA as protein replacement therapeutics in the 90’s, but due to the mRNA’s immunogenicity it could not be used. However, in 2005 they found that the nucleotide uridine could be replaced with pseudouridine rendering the mRNA non immunogenic. When the pandemic hit, their work proved invaluable as many companies rushing to make the vaccines decided to use their technology. Their discoveries led to the first approved mRNA vaccines during the covid pandemic, saving many lives and earning them the Nobel prize in Physiology or Medicine 2023. Following this, RNA based therapeutics funding is now bigger than ever and researchers are hard at work seeking what other therapies RNA can be used for.

The technique used in the Sars cov 2 vaccine is cellular delivery by Lipid Nanoparticles (LNPs), a liposome like structure which can carry and deliver nucleic acids which are difficult to deliver by themselves. Among other factors the LNPs increase efficiency of delivery by increasing transfection, neither are they toxic to the body.

In this thesis we wanted to try to make these LNPs by ourselves and to see if we can express them in our chosen Human Colon Carcinoma cell line (HCT116). This was tested by seeding our cells with stained LNPs containing mRNA’s that code for Enhanced Green Fluorescent Protein (EGFP) and imaging them confocally.

Expression of EGFP was achieved testing for which concentration was optimal, and in the subsequent run of the experiment we tried out different methods of LNP preparation. The results of the experiments showed that expression is possible and can be quite high. They showed that N1-Methylpseudouridine mRNA gave better expression in vitro than did 5-Methoxyuridine and that acetate buffer during LNP preparation seems to increase mRNA expression compared to citrate buffer.