Enhancing natural killer cells proliferation and cytotoxicity using imidazole-based lipid nanoparticles encapsulating interleukin-2 mRNA

Abstract

mRNA applications have undergone unprecedented applications-from vaccination to cell therapy. Natural killer (NK) cells are recognized to have a significant potential in immunotherapy. NK-based cell therapy has drawn attention as allogenic graft with a minimal graft-versus-host risk leading to easier off-the-shelf production. NK cells can be engineered with either viral vectors or electroporation, involving high costs, risks, and toxicity, emphasizing the need for alternative way as mRNA technology. We successfully developed, screened, and optimized novel lipid-based platforms based on imidazole lipids. Formulations are produced by microfluidic mixing and exhibit a size of approximately 100 nm with a polydispersity index of less than 0.2. They are able to transfect NK-92 cells, KHYG-1 cells, and primary NK cells with high efficiency without cytotoxicity, while Lipofectamine Messenger Max and D-Lin-MC3 lipid nanoparticle-based formulations do not. Moreover, the translation of non-modified mRNA was higher and more stable in time compared with a modified one. Remarkably, the delivery of therapeutically relevant interleukin 2 mRNA resulted in extended viability together with preserved activation markers and cytotoxic ability of both NK cell lines and primary NK cells. Altogether, our platforms feature all prerequisites needed for the successful deployment of NK-based therapeutic strategies.

Keywords: IL-2; KHYG-1 cells; LNP; MT: Delivery Strategies; NK cell therapy; NK-92 cells; interleukin-2; lipid nanoparticles; lipoplexes; messenger RNA; natural killer; non-modified mRNA.

Graphical abstract

Figure 1

Development of lipid-based systems for mRNA delivery in NK cells (A) Microfluidic formulation process of LNPs and liposomes, with handmade addition of mRNA to liposome leading to lipoplexes. (B) Schematic representation of lipoplex and LNP composition.

Figure 2

Lipoplexes transfection efficiency NK-92 cells were incubated at 37°C overnight with the indicated formulation containing eGFP mRNA. After 24 h, the transfection efficiency was measured by flow cytometry. Full bars, % of transfected cells; hatched bars, MFI; grey bars, % of dead cells. (A) Impact on transfection of the CL at different molar percentages (10%, 20%, 30%, 40%, and 50% for CL10, CL20, CL30, CL40, and CL50, respectively). (B) impact on transfection of cholesterol type and DMPE-PEG in liposomes containing 20% CL. iCD-Lx, cholesterol and DMPE-PEG; iCD-Lx (DMG-PEG), cholesterol and DMG-PEG; iCD-Lx (Sito), β-sitosterol and DMPE-PEG; iCD-Lx (DMG-PEG/Sito), β-sitosterol and DMG-PEG. (C) Impact on transfection of lipoplexes made with 20% CL at various N/P ratios [1/1, 2/1, and 3/1]. (D) Stability of liposomes (iCD-Lip) and (E) Lx (iCD-Lx) stored at 4°C. Data are presented as mean ± SD, ∗p < 0.03, ∗∗p < 0.002, ∗∗∗p < 0.001; ns, not significant, in comparison with iCD-Lx.

Figure 3

Screening of LNPs for NK-92 cell transfection (A) Transfection efficiency of various LNPs made with 0.5 μg of MNR (5-MoU) eGFP mRNA on NK-92 cells upon overnight incubation. (B) Size and PDI stability over weeks of storage of iD-LNPs in PBS at 4°C (n = 3 measurements). (C) Stability at 37°C of iD-LNPs in PBS or PBS with 10% serum. Data are presented as mean ± SD.

Figure 4

Impact of mRNA modifications on the transfection efficiency Cells were transfected overnight with ARCA UNR, cap1 UNR, MNR (5-MoU) or MNR (PsiU) mRNA encoding eGFP formulated as Lx or LNPs. (A) NK-92 cells and (B) KHYG-1 cells transfected with iCD-Lx. (C) NK-92 cells and (D) KHYG-1 cells were transfected with iD-LNPs. After 24 h, the transfection efficiency was measured by flow cytometry. Full bars, percentage of eGFP+ cells; hatched bars, MFI. (E) Fluorescence microscopy observations of NK-92 cells transfected with iCD-Lx or iD-LNP made with indicated mRNA types. Data are presented as mean ± SD, ∗p < 0.03, ∗∗p < 0.002, ∗∗∗p < 0.001; ns, not significant, in comparison with cap-1 UNR.

Figure 5

Kinetics of mRNA expression NK-92 cells were transfected overnight with UNR, MNR (5-MoU) or MNR (PsiU) cap-1 eGFP-mRNA formulated as (A and B) iCD-Lx or (C and D) iD-LNP. (A and C) Percentage of transfected cells and (B and D) MFI measured by flow cytometry every day for 1 week. Values are means ± SD of three independent measurements.

Figure 6

Transfection efficiency of iCD-Lx or iD-LNP compared with commercial mRNA transfecting reagents NK-92 cells (A) and KHYG-1 cells (B) were transfected with Screenfect, mRNAfect, LFM, iCD-Lx, or iD-LNP containing 0.5 μg cap1 UNR eGFP mRNA. Comparison between IL and other ionizable lipids. (C) Table showing the size, PDI, encapsulation efficiency, and concentration of LNPs made with exactly the same lipid composition except for the ionizable lipid (Imidazole-based), SM102, ALC-0315 and D-lin-MC3). (D) NK-92 cells were transfected overnight with indicated LNP encapsulating cap1 UNR eGFP. (E) Jurkat cells were transfected with either iCD-Lx or iD-LNP made of different ionizable lipids. Full blue bars, transfected cells (%); hatched blue bars, MFI; hatched black bars, cell mortality (%). Data are presented as mean ± SD, ∗p < 0.03, ∗∗p < 0.002, ∗∗∗p < 0.001; ns, not significant, in comparison with iD-LNPs.

Figure 7

Impact of IL-2 mRNA transfection on cell viability and cytotoxicity activity NK-92 cells were either incubated with or without IL-2 protein or transfected with cap1 UNR IL-2 mRNA formulated as iCD-Lx (A) or iD-LNP (B). (C) Cells were incubated with iCD-Lx containing 0.25 μg cap1 UNR eGFP and 0.25 μg IL-2 mRNA. The viability of the cells was measured every day by flow cytometry and expressed as a percentage relative to the total cells. Statistical analysis was done by comparison to IL-2 protein values. (D and E) NK-92 and K562 cells were co-cultured at diverse effector/target ratios for 4 h. The cytotoxicity was evaluated by measuring the expression of caspase 3/7 in K562 cells after NK-92 cells transfection with iD-LNP (D) and iCD-Lx (E). All data are represented by means ± SEM; n = 3 for each experiment. Paired Student t tests were used for statistical analysis.

Figure 8

Transfection efficiency of primary human NK cells with iCD-Lx IL-2 mRNA Primary human NK cells were obtained from at least three different donors. (A) The cells were transfected with iCD-Lx and iD-LNP containing eGFP mRNA. After 24 h the number of eGFP⁺ cells was determined by flow cytometry. (B) Viability of primary NK cells 48 h after eGFP mRNA transfection with iCD-Lx and iD-LNP measured by flow cytometry using live/dead dye and expressed as a percent relative to the total cells. (C–E) CD69, NKP30 and NKG2D expressions measured by flow cytometry after 48h of transfection with IL-2 mRNA iCD-Lx, IL-2 protein addition or starvation. (F) Evaluation of primary NK cells proliferation 48 h after transfection with IL-2 mRNA iCD-Lx, IL-2 protein addition or starvation. (G and H) primary NK cells and K562 cells were co-cultured for 4 h at various Effector/Target cell ratios. (G) The production of TNF-α in primary NK cells measured by flow cytometry. (H) Cytotoxicity measured by the expression of caspase 3/7 in K562 cells by flow cytometry. All data are represented by means ± SEM; n = 3 for each experiment. Each condition represents a donor. Paired Student t tests were used for statistical analysis.