Mesenchymal stem cell engineering by ARCA analog-capped mRNA

Anna Andrzejewska; Renata Grzela; Anna Stankiewicz-Drogon; Piotr Rogujski; Siranjeevi Nagaraj; Edward Darzynkiewicz; Barbara Lukomska; Miroslaw Janowski

doi:10.1016/j.omtn.2023.07.006

Mesenchymal stem cell engineering by ARCA analog-capped mRNA

Mol Ther Nucleic Acids. 2023 Jul 17:33:454-468. doi: 10.1016/j.omtn.2023.07.006. eCollection 2023 Sep 12.

Authors

Anna Andrzejewska^{1

2}, Renata Grzela^{3

4}, Anna Stankiewicz-Drogon³, Piotr Rogujski¹, Siranjeevi Nagaraj⁴, Edward Darzynkiewicz^{3

4}, Barbara Lukomska¹, Miroslaw Janowski^{2

5}

Affiliations

¹ NeuroRepair Department, Mossakowski Medical Research Institute, Polish Academy of Sciences, 02-106 Warsaw, Poland.
² Center for Advanced Imaging Research, Department of Diagnostic Radiology and Nuclear Medicine, University of Maryland, Baltimore, MD 21201, USA.
³ Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, 02-093 Warsaw, Poland.
⁴ Interdisciplinary Laboratory of Molecular Biology and Biophysics, Centre of New Technologies, University of Warsaw, 02-097 Warsaw, Poland.
⁵ Tumor Immunology and Immunotherapy Program, University of Maryland Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland, Baltimore, MD 21201, USA.

Abstract

We previously have shown that mRNA-based engineering may enhance mesenchymal stem cell (MSC) trafficking. However, optimal conditions for in vitro mRNA engineering of MSCs are unknown. Here, we investigated several independent variables: (1) transfection factor (Lipofectamine 2000 vs. TransIT), (2) mRNA purification method (spin column vs. high-performance liquid chromatography [HPLC] column), and (3) mRNA capping (ARCA vs. β-S-ARCA D1 and β-S-ARCA D2). Dependent variables included protein production based on mRNA template (measured by the bioluminescence of reporter gene luciferase over hours), MSC metabolic activity corresponding with their wellbeing measured by CCK-8 over days, and endogenous expression of genes by RT-qPCR related to innate intracellular immune response and decapping at two time points: days 2 and 5. We have found that Lipofectamine 2000 outperforms TransIT, and used it throughout the study. Then, we showed that mRNA must be purified by HPLC to be relatively neutral to MSCs in terms of metabolic activity and endogenous protein production. Ultimately, we demonstrated that β-S-ARCA D1 enables higher protein production but at the cost of lower MSC metabolic activity, with no impact on RT-qPCR results. Thus Lipofectamine 2000-based in vitro transfection of HPLC-purified and ARCA- or β-S-ARCA D1-capped mRNA is optimal for MSC engineering.

Keywords: ARCA-cap analogs; MT: oligonucleotides: therapies and applications; immune response; mRNA transfection; mesenchymal stem cells; metabolic activity.