(M1330-03-16) Mutant mRNA Encapsulated Aptamer Conjugated-Lipid Nanoparticles for Targeting Heart Cells

Purpose: Modified messenger RNA-based (modRNA) therapy holds great potential to treat various diseases including various cancers and viral infections. However, there is currently no data on the potential of modRNA therapy to treat heart disease and specifically target specific tissues such as the heart. Therefore, the purpose of the study is to develop an aptamer-conjugated modRNA lipid-nanoparticle to target specifically heart cells. For this purpose, we developed a lipid nanoparticle that is conjugated with an aptamer to target the heart’s cardiomyocytes. In our studies, we aimed to target mutant versions of two transcription factors, modRNA-YAP5SA and modRNA-STEMIN, that we previously showed to be important for the proliferation of cardiomyocytes (ref), by encapsulating these modRNAs into lipid nanoparticles.

Methods: Lipid nanoparticles (LNPs) were made using, cholesterol, DSPC, DMG-PEG, DBCO-PEG, DOTAP, and DLIN-MC3 and the molar percentage of these lipids were 19.3%, 5%, 0.72%, 0.08%, and 75%, respectively. The lipids were dissolved in ethanol and combined according to the desired percentage. A NanoSpark machine was used to synthesize the LNPs. The lipids were diluted in a sodium acetate (NaAc) buffer. The mRNA for the mCherry sequence was synthesized using HiScribe T7 RNA kits and the concentration was determined by Nanodrop. The size of the particle was determined by using Dynamic Light Scattering (DLS) instrumentation and the zeta potential was determined by Anton Paar's Zeta Potential instrumentation. The AC16 human ventricular cell line was used for the experiments.

Results: The size of the lipid nanoparticles was measured using DLS instrumentation and it varied between 88 to 120 nm. Zeta potential was measured as +70 mV using a zeta potential analyzer. AC16 cells were differentiated with insulin-containing horse serum, transfected with different doses of mCherry-labeled lipid nanoparticles (10 ,14 and 21 ul), and observed on a fluorescent microscope. It was observed that the AC16 heart cells produced red fluorescent. Therefore, we can deduce from this experiment that the nanoparticle went through the cells, and the encapsulated mRNA was released in the cytosol and then translated into protein.

Conclusion: In our lipid nanoparticle studies, the size of the particles is the most important since having a smaller particle size is easier to enter through the cells. The size of the synthesized particles used in our studies compares well to that of the literature, supporting that a smaller particle size results in optimal transfection.The transfection results showed that the aptamer-conjugated mCherry-mRNA lipid nanoparticle can effectively transfect AC16 cells. In future experiments, we are planning to use that lipid nanoparticle with other mutant mRNAs that may also have a proliferation effect on cardiomyocytes. In conclusion, aptamer-conjugated modRNA lipid nanoparticles may serve as a promising new drug carrier to target the heart.

References: Xiao S, Liang R, Lucero E, McConnell BK, Chen Z, Chang J, Navran S, Schwartz RJ, Iyer D. STEMIN and YAP5SA synthetic modified mRNAs regenerate and repair infarcted mouse hearts. J Cardiovasc Aging. 2022 Jul;2(3):31. doi: 10.20517/jca.2022.20. Epub 2022 Jun 15. PMID: 35891703; PMCID: PMC9311335.

Transfection AC16 differentiated cells with different doses of aptamer conjugated mcherry-LNP

Differentiated AC16 cells produced red fluorescent when transfected with aptamer-conjugated LNPs