Optimized mRNA delivery for lymphocytes using jetMESSENGER® technology

Modifying gene-delivery approaches for lymphocytes is critical for both fundamental research and clinical immunotherapy. T cells, B cells, and natural killer (NK) cells are difficult to transfect, and physical and viral-based approaches demonstrate limited efficacy and less cell viability.

This study examines jetMESSENGER^® mRNA transfection reagent, a chemical-based reagent for mRNA transfection, as a potential option to conventional approaches.

jetMESSENGER^® reagent considerably outstrips Lipofectamine^™ 2000 transfection reagent in T cells, reaching more elevated GFP expression while preserving cell integrity. In B cells, it shows superior performance compared to Lipofectamine^™ MessengerMAX^™ reagent, providing a higher level of physiological and homogeneous expression.

For NK cells, jetMESSENGER^® reagent displays promising results, particularly with StemMACS^™ HSC Expansion Media, while NK MACS^® Medium enhances cell integrity.

In conclusion, jetMESSENGER^® reagent efficiently delivers gene delivery in lymphocytes that are difficult to transfect, representing a viable alternative to physical and viral-based approaches.

This enables potential use cases in a spectrum of medicinal domains for proof-of-concept research, like engineering T cells for chimeric antigen receptor (CAR) expression, improving B cells for autoimmune disease treatments, alongside improving NK cells as therapeutics for cancers and viral infections alike.

Introduction

Maximizing gene-delivery approaches for targeting lymphocytes with precision is a considerable problem in fundamental research concerning T cells, B cells, and natural killer (NK) cells, as well as in clinical immunotherapy studies.

T lymphocytes, considered hard-to-transfect cells, typically depend on electroporation for transfection, although this can lead to problems such as low efficiency and lower cell viability. Chemical transfection reagents that are widely utilized for mammalian cells also display constrained efficacy in T cells.¹

This comes as T cells are resistant to widespread commercially available chemical transfection reagents, which have a safety profile for numerous mammalian cells, like the Lipofectamine^™ 2000 transfection reagent.²

In spite of the potential cellular damage linked to the electroporation, it remains the predominant approach for transfecting B cells.³ In addition, in vitro delivery approaches for CRISPR/Cas9 into B cells typically depend on viral-based approaches that have constrained carrier ability and may lead to immunogenicity problems and potential insertional mutagenesis.⁴

Maximizing NK cells via traditional approaches such as electroporation, lipofection, or viral approaches is even more challenging than modifying T cells as NKs are considerably more resistant, leading to the evaluation of mRNA transfection as a different strategy.^5,6

To address these issues, modified non-viral gene delivery approaches that reduce cellular toxicity are needed for effective gene transfer to lymphocytes.

This research seeks to examine and maximize the transfection efficiency and cell viability performance of jetMESSENGER^® reagent, a chemical-based transfection reagent used to deliver mRNA into T lymphocytes, B lymphocytes, and NK cells. The results could elucidate whether jetMESSENGER^® reagent may be used for proof-of-concept research and make possible mediated expression of clinically relevant proteins in lymphocytes.

Methods

T cell isolation and activation

Human primary T cells from healthy donors were taken from peripheral blood by magnetic activated cell sorting (negative selection via CD14 Microbeads, Miltenyi Biotec,130-050-201) and then frozen. T cells were then thawed and subsequently washed when in X-VIVO^™ 15 medium (Lonza, BE02-060F) prior to being cultured in a medium supplemented with recombinant human IL-21 (10 ng/mL, PeproTech, 200-21) at a density of 1.5×10⁶ cells/mL.

Cells were set aside to rest for four hours prior to being activated with T Cell TransAct^™, human (10 µL/1.10⁶ cells, Miltenyi Biotec, 130-111-160). Cells were then activated for a period of 44 or 68 hours in a 37 °C, 5% CO₂ humidified incubator.

Comparing T cell transfection with jetMESSENGER^® and lipofectamine^™ 2000 transfection reagent

On the transfection day, T cells were washed on a singular occasion with 1 × PBS (5 min, room temperature) and suspended again in X-VIVO^™ 15 medium 30 minutes prior to transfection. The mRNA utilized was the CleanCap^® eGFP mRNA (5moU), L-7201 (TriLink).

In a 96-well plate, the conditions as follows were utilized for each well: for jetMESSENGER^® transfection reagent (Polyplus, 150-15), 125 ng mRNA was diluted in mRNA buffer (12.5 µL) and mixed by pipetting; the jetMESSENGER^® reagent (0.375 µL or 0.5 µL) was added to the diluted mRNA; for Lipofectamine^™ 2000 transfection reagent (Thermo Fisher, 11668027), 125 ng mRNA was diluted into 6.25 µL of Opti-MEM^™ serum (Gibco^™, 31985070) and added into the diluted transfection reagent (0.375 µL into 6.25 µL of Opti-MEM^™ serum).

Following incubation (15 min, room temperature), 12.5 µL of both transfection mixes were added per well. T cells in complete medium (62.5 µL at 3.10⁶ cells/mL) were additionally added to each well (187,500 cells per well). The plate was then lightly rocked back and forth and side to side before being incubated (4 hours, 37 °C, 5% CO₂).

Full medium (175 µL) with IL-21 (10 ng/mL) and T Cell TransAct^™ (10 µL/1.10⁶ cells) were added to each of the wells and cells were incubated (37 °C, 5% CO₂). Transfection efficiency was assessed 48 hours later by flow cytometry and fluorescence imaging.

Transfection efficiency evaluation

Cells were washed with 1 x PBS, resuspended in 200 µL of 1 x PBS with 2% FBS, and 2 mM EDTA with propidium iodide (1 µg/mL) was added. Cell fluorescence was assessed via a Guava^® easyCyte™ 5HT flow cytometer (Luminex, Merck Millipore) and outcomes were assessed with guavaSoft^™ and InCyte^™ Software. Cell images were additionally produced with a ZOE Fluorescent Cell Imager (Bio-Rad).

B cell isolation and expansion

Human primary B cells from healthy donors were taken from peripheral blood utilizing magnetic activated cell sorting (positive selection via CD19 Microbeads, Miltenyi Biotec, 130-050-301) and utilized in a direct manner.

B cells were cultivated in StemMACS^™ HSC Expansion Media XF (Miltenyi Biotec, 130-100-473) + 2 µL Human IL-4 (2.5 × 10⁵ IU/mL, Miltenyi Biotec, 130-093-922) + 5% AB serum (Sigma-Aldrich, H4522-100 ML) + 80 µL multimerized Human CD40-Ligand (Miltenyi Biotec, 130-098-776) at 0.2×10⁶ cells/mL (as recommended by Miltenyi Biotec). Cells were expanded for 13 or 14 days at 37 °C, 5% CO₂, and passaged once every 2 or 3 days.

Comparison of B cell transfection with jetMESSENGER^® reagent and lipofectamine^™ messengerMAX^™ reagent

Before transfection, B cells were washed on one occasion with 1 x PBS (5 min, room temperature) and suspended again in Opti-MEM^™ serum. Cells were transfected with CleanCap® eGFP mRNA (5moU) in 96-well plates utilizing the settings as follows: for jetMESSENGER^® transfection, 250 ng mRNA was diluted into mRNA buffer (12.5 µL) and blended by pipetting; the jetMESSENGER^® reagent (0.25 µL) was added to the diluted mRNA and combined by pipetting.

Following incubation (15 min, room temperature), the transfection mix (0.25 µL) was added to each of the wells. For Lipofectamine^™ MessengerMAX^™ reagent (Thermo Fisher, LMRNA001), transfection reagent (0.25 µL) was pre-diluted in Opti-MEM^™ (6.25 µL) and incubated for 10 minutes at room temperature, or 250 ng mRNA was diluted in Opti-MEM^™ (6.25 µL) and then added into the transfection reagent.

Following 5 minutes of incubation at room temperature, the transfection mix (12.5 µL) was added to each of the wells. B cells (62.5 µL at 3.10⁶ cells/ml) in complete medium were added to each of the wells (187,500 cells/well). The plate was lightly rocked back and forth and side to side and incubated at 37 °C, 5% CO₂.

Four hours later, the medium was substituted with 200 µL of fresh complete medium in each of the wells. Cells were incubated at 37 °C, 5% CO₂, and efficiency was assessed 24 hours later by flow cytometry and fluorescence imaging.

References and further reading

1. Wang, S. and Tian, D. (2022). High transfection efficiency and cell viability of immune cells with nanomaterials-based transfection reagent. BioTechniques. https://doi.org/10.2144/btn-2022-0024.
2. Zhao, N., et al. (2012). Transfecting the hard-to-transfect lymphoma/leukemia cells using a simple cationic polymer nanocomplex. Journal of Controlled Release, (online) 159(1), pp.104–110. https://doi.org/10.1016/j.jconrel.2012.01.007.
3. Chong, Z.X., Yeap, S.K. and Ho, W.Y. (2021). Transfection types, methods and strategies: a technical review. PeerJ, (online) 9. https://doi.org/10.7717/peerj.11165.
4. Keim, D., et al. (2021). Generation of Recombinant Primary Human B Lymphocytes Using Non-Viral Vectors. International Journal of Molecular Sciences, 22(15), p.8239. https://doi.org/10.3390/ijms22158239.
5. Allan, D.S.J., et al. (2021). Systematic improvements in lentiviral transduction of primary human natural killer cells undergoing ex vivo expansion. Molecular Therapy - Methods & Clinical Development, 20, pp.559–571. https://doi.org/10.1016/j.omtm.2021.01.008.
6. Carlsten, M. and Childs, R.W. (2015). Genetic Manipulation of NK Cells for Cancer Immunotherapy: Techniques and Clinical Implications. Frontiers in Immunology, 6. https://doi.org/10.3389/fimmu.2015.00266.
7. Lim, W.A. and June, C.H. (2017). The Principles of Engineering Immune Cells to Treat Cancer. Cell, (online) 168(4), pp.724–740. https://doi.org/10.1016/j.cell.2017.01.016.

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