(F-226) Developing a platform for induced pluripotent stem cell reprogramming through lipid nanoparticle-based mRNA delivery

Introduction:: Induced pluripotent stem cells (iPSCs) are derived from adult somatic cells and have shown potential in regenerative medicine since they can differentiate into a multitude of cell types and are less controversial than embryonic stem cells. However, iPSCs are difficult to transfect with foreign cargo, which is often needed for differentiation. The most common transfection methods are lipofection and electroporation, but these methods have low efficiency and high cell death rate. One novel method is utilizing lipid nanoparticles (LNPs), which are drug delivery vehicles that can have a wide multitude of cargo such as mRNA and siRNA. LNPs have been FDA-approved for COVID-19 vaccines as a method to deliver mRNA, and thus have high transfection potential in iPSCs. Still, they must be optimized for specific applications since existing formulations are not universal.


Materials and Methods:: LNPs were formulated by microfluidically mixing lipid and mRNA phases and dialyzing the product in 1x PBS buffer. The lipid phase is in ethanol and includes an ionizable lipid, a phospholipid, cholesterol, and a PEGylated lipid. The mRNA phase is composed of mRNA in citrate buffer. An LNP library was produced using nine different ionizable lipids, each synthesized via an SN2 reaction between a polyamine core and an epoxide, the latter with different lipid tail architectures: linear, isopropyl, tert-butyl, and sec-butyl branching. The LNPs were first formulated with luciferase mRNA. The four top candidates were identified through dose responses in which the luminescence signal was analyzed 24 hours after transfection. The lead LNP was then reformulated with mCherry mRNA. Flow cytometry was then performed by harvesting treated cells after 24 hours and analyzing the results with FACS. The results identified the percentage of cells successfully transfected and the maximum dosage possible before significant mortality.


Results, Conclusions, and Discussions:: The dose response results indicated that both isolated and clustered cells could be transfected. The results unexpectedly showed that the LNP containing the C12-200 ionizable lipid, which has broad transfection in a wide variety of cell lines, performed poorly in iPSCs. The results also indicated that ionizable lipid structure dramatically affected both efficacy and toxicity with branched lipids consistently performing better than linear lipids. The flow cytometry results indicate that the top LNP candidate transfected up to 70% of the iPSCs with little toxicity, with the upper limit being 1200 ng/90k. Additionally, the top candidate produced a consistent mCherry signal for 48 hours. This is significant since the mRNA would optimally not degrade within the iPSC differentiation time. We plan to use this lead candidate to induce iPSC differentiation into neurons using CRISPR/Cas9 for ex vivo therapy.


Acknowledgements (Optional): : I would like to thank the Penn Institute of Regenerative Medicine for collaborating with us. I would also like to thank the Jack Kent Cooke and Clark Scholars Foundations for supporting my undergraduate research.

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