(M1430-06-37) Successful Transfection of Dendritic Cells by eGFP mRNA-Containing Lipid Nanoparticles

Purpose: Lipid nanoparticles (LNPs) are used to deliver small-molecule drugs or nucleic acids and usually composed of an ionizable lipid, a helper lipid, cholesterol, and a lipid containing polyethylene glycol (PEGylated lipid)¹. LNPs not only protect the nucleic acid cargo from degradation, but also enable its efficient delivery into the targeted cells². Before using the mRNA of interest as cargo, cell transfection can be confirmed with mRNA encoding for fluorescent proteins, such as luciferase or enhanced green fluorescent protein (eGFP). In this work, transfection of dendritic cells using eGFP mRNA LNPs was investigated to pave the way for future LNP delivery of therapeutic mRNA. To optimize LNP production, the identification of a low-cost model nucleic acid was desired, so MS2 RNA was selected as a candidate to be used as cargo and compared to eGFP and luciferase mRNA LNPs.

Methods: LNPs were produced by mixing D-Lin-MC3-DMA (AmBeed, Arlington, USA), DSPC (Avanti Polar Lipids Inc., Alabaster, USA), cholesterol (Sigma Aldrich, Saint Louis, USA) and DMG-mPEG-2000 (Sigma Aldrich, Darmstadt, Germany) (50:10:38.5:1.5) in ethanol with luciferase mRNA (CleanCap® FLuc mRNA (5moU), eGFP mRNA, CleanCap® EGFP mRNA (5moU), TriLink Biotechnologies, San Diego, USA) or MS2 RNA (Roche Diagnostics GmbH, Mannheim, Germany) in citric acid buffer (50 mM, pH 3) using the Automated Nanoparticle System (Unchained Labs, Royston, United Kingdom), with a total flow rate of 3 mL/min (eGFP mRNA) or 7 mL/min, a flow rate ratio (aqueous:ethanolic) of 3:1, a total lipid concentration of 20 mM and a N/P ratio of 6. Hydrodynamic diameter (HD) and polydispersity index (PDI) were measured by dynamic light scattering (Zetasizer Nano ZS, Malvern Panalytical GmbH, Kassel, Germany; in cumulants fit, backscatter mode (173 °C), 3 × 10 × 10 s at 25.0 °C. Encapsulation efficiency (EE) and total mRNA recovery (TmR) were obtained by fluorescence analysis with the Quant-iT™ Ribogreen™ RNA Assay Kit (Invitrogen/Thermo Fischer Scientific). Briefly, LNPs were incubated in 96-well plates at 37 °C for 10 minutes in 1x TE buffer, either pure or containing 1% Triton™ X-100 (Sigma-Aldrich, St. Louis, USA), to determine the fluorescence intensity of free mRNA (fmRNA) and total mRNA (tmRNA), respectively.
EE (%) = [(tmRNA - fmRNA)/tmRNA] * 100
TmR (%) = (tmRNA [µg/mL]/theoretical mRNA concentration [µg/mL]) * 100
For in vitro assays, human monocyte-derived dendritic cells (moDC) were derived from CD14+ monocytes, isolated from buffy coats obtained via the Red Cross Flanders3. Immature or mature moDC were incubated at 37 °C with eGFP mRNA LNPs in medium with PGE2 and TNFα and without serum for 2 hours, prior to the addition of RPMI 1640 containing 2,5% human serum albumin, penicillin, streptomycin, L-glutamine, and sodium-pyruvate. Electroporation using free eGFP mRNA was performed on a Gene Pulser Xcell device (Biorad)³. Fluorescence signal was measured by flow cytometry 48 hours post-transfection.

Results: Luciferase LNPs and MS2 LNPs were comparable regarding particle size distribution, EE and TmR (Figure 1). To confirm cell transfection of optimized LNPs, eGFP mRNA was delivered to moDCs by incubating them with in LNPs (HD = 83 nm, PDI = 0.18, encapsulated mRNA = 70 µg/mL and EE = 90%). The results were compared to the delivery of free eGFP by electroporation (EP) after 48 hours. The presence of fluorescence and eGFP positive cells for all used concentrations indicated successful cell transfection. An increase in expression of more than 40% was observed when incubated eGFP quantity was risen from 2 to 8 µg, but the mean fluorescence intensity of LNPs was considerably lower than that of EP for all samples (Figure 2). While the results showed good cell viability, no conclusions could be drawn regarding the difference in transfection of immature or mature moDCs.

Conclusion: MS2 showed results comparable to luciferase. Particle size was also similar, despite the length difference of both nucleic acids (3569 and 1929 bases, for MS2 and luciferase respectively). To our knowledge, this is the first time that this RNA is reported as LNP payload and presented it as a suitable model for production process development. Compared to EP, LNPs showed lower fluorescence intensity and expression, which differs from published results showing that, in the delivery of the same concentration of mRNA to human T cells, LNPs outperformed EP⁴. The cited study used a proprietary lipid composition, which was optimized for T cells, whereas the lipid formulation of our work was originally designed for uptake in hepatocytes by interaction with the low-density lipoprotein receptor (LDLR). Considering the high affinity of LDLR for apolipoprotein E⁵, supplementing the cells with this protein during incubation with LNPs might increase cell transfection, a step that was not performed in our investigations but was found to be essential for LNP mediated mRNA delivery in the mentioned study.

References: (1) Eygeris, Y.; Gupta, M.; Kim, J.; Sahay, G. Chemistry of Lipid Nanoparticles for RNA Delivery. Accounts of chemical research 2022, 55 (1), 2–12. DOI: 10.1021/acs.accounts.1c00544. Published Online: Dec. 1, 2021.
(2) Kon, E.; Elia, U.; Peer, D. Principles for designing an optimal mRNA lipid nanoparticle vaccine. Current opinion in biotechnology 2022, 73, 329–336. DOI: 10.1016/j.copbio.2021.09.016. Published Online: Oct. 26, 2021.
(3) Smits, E. L. J. M.; Stein, B.; Nijs, G.; Lion, E.; van Tendeloo, V. F.; Willemen, Y.; Anguille, S.; Berneman, Z. N. Generation and Cryopreservation of Clinical Grade Wilms' Tumor 1 mRNA-Loaded Dendritic Cell Vaccines for Cancer Immunotherapy. Methods in molecular biology (Clifton, N.J.) 2016, 1393, 27–35. DOI: 10.1007/978-1-4939-3338-9_3.
(4) Kitte, R.; Rabel, M.; Geczy, R.; Park, S.; Fricke, S.; Koehl, U.; Tretbar, U. S. Lipid nanoparticles outperform electroporation in mRNA-based CAR T cell engineering. Molecular Therapy - Methods & Clinical Development 2023, 31, 101139. DOI: 10.1016/j.omtm.2023.101139.
(5) Johnson, L. A.; Olsen, R. H. J.; Merkens, L. S.; DeBarber, A.; Steiner, R. D.; Sullivan, P. M.; Maeda, N.; Raber, J. Apolipoprotein E-low density lipoprotein receptor interaction affects spatial memory retention and brain ApoE levels in an isoform-dependent manner. Neurobiology of disease 2014, 64, 150–162. DOI: 10.1016/j.nbd.2013.12.016. Published Online: Jan. 9, 2014.

Acknowledgements: The presented work is part of the CanceRNA Consortium, publicly funded by the European Comission within the Horizon Europe Framework Programme (HORIZON) under the project ID 101057250. Izabella Lambart, Stefan Schiller and Simon Geissler are employed by EMD Serono (Merck Healthcare KGaA, Darmstadt, Germany). Karsten Mäder provides consulting for EMD Serono (Merck Healthcare KGaA, Darmstadt, Germany). The other authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript.


Figure 1. MS2 showed encapsulation efficiency and particle size comparable to luciferase. LNPs were produced with a 3:1 aqueous to ethanolic ratio, N/P ratio of 6 and total flow rate of 7 mL/min. Data is expressed as mean ± SD (n=3).


Figure 2. LNPs showed dose-dependent expression 48 hours post-transfection. After 48 hours, the amount of expressed eGFP within a cell (A) and the number of eGFP-expressing cells (B) was found to be dose-dependent. Although a high percentage of eGFP positive cells was achieved with 8 µg eGFP mRNA via LNP delivery, EP performed better at 2 µg eGFP mRNA. Data is expressed as mean ± SD for three (mature moDCs) or four (immature moDCs) donors.