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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session E

Poster I4 - The creation of interferon-reporter gene constructs for improved in vitro screening of RNA immunogenicity

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  An emerging field, RNA therapeutics accelerated upon the SARS CoV-2 pandemic proving its simplicity and effectiveness with quick turnaround times. However, there is no consensus on the relation between RNA therapies’ efficacy and immunogenicity. While there is some evidence that the cellular innate immune responses to exogenous RNAs hinder the RNA’s efficacy with effects like translational repression, RNA degradation, cell death, and systemic inflammation, the recognition by and activation of the innate immune pathway sometimes can also be correlated with increased efficacy. More studies on RNA immunogenicity are needed for the optimization and clinical translation of RNA therapeutics.

Cellular innate immune responses to RNA majorly involve type-1 interferon(IFN) pathways. The most common IFN screening methodology, enzyme-linked immunosorbent assays(ELISAs) are laborious, time-consuming and expensive. They quantify IFN amounts in cell media, which are population-based measurements of IFN–an initial molecule in the immune response pathway–with no insight into the single-cell level IFN production. An alternative method in vitro is reporter gene assays which involve IFN-sensitive cell lines with hybrid constructs of IFN-responsive promoters and reporter genes. This method is time- and cost-effective with direct and easy readouts. It allows cell-based censoring of downstream IFN-stimulated products; therefore, enabling a more accurate characterization of RNA-caused immune responses. Here, we develop luciferase- and green fluorescent protein(GFP)-based reporter plasmids and confirm the activity of the luciferase plasmid via transient transfection in C2C12 cells. Ultimately, we will create various stable cell lines for RNA immunogenicity reporter gene assays.

Materials and

Methods:  Both luciferase and GFP reporter constructs were generated as lentiviral transfer vectors. The coding sequences for IFN stimulated response element(ISRE)-minimal p promoter(minp)-luciferase, ISRE-minp, GFP, Human elongation factor 1 alpha(EF-1 alpha)-puromycin(puro) as lentiviral selection marker were amplified with PCR. After Gibson Isothermal Assembly, plasmids were cloned in competent Escherichia coli and purified using a ZymoPURE II Plasmid Miniprep Kit (Zymo Research). The sequences were verified with restriction enzyme test cuts.

C2C12 cells were grown in 10% FBS and 1% penicillin–streptomycin DMEM, plated at 10,000 cells/well in a 96-well clear plate and an opaque 96-well white microtitre plate, and allowed to adhere overnight. An mCherry transfection marker was co-transfected with the GFP plasmid using Lipofectamine 3000 according to the manufacturer’s protocol (Invitrogen) to assess transfection efficiency. The luciferase plasmid was transfected into white plate wells using Lipofectamine 3000 by the same protocol. After 18 hours, the C2C12 cells were treated with polyinosinic:polycytidylic acid(Poly(I:C)) (InvivoGen), an immunostimulant, at varying doses from 0 ng/mL to 1,000 ng/mL in triplicates. Appropriate controls were included (Figure 1.c).

After 24 hour incubation, for the clear plate, cells were PBS washed, trypsinized, and resuspended in a fluorescence-activated cell sorting buffer. Data were acquired using Attune NxT Flow Cytometer (Thermo Fisher Scientific). For the white plate, Bright-Glo Luciferase Assay System (Promega) was used to acquire data with SpectraMax L Microplate Reader for analysis using Prism (GraphPad). All incubations were done at 37°C 5% CO2.

Results, Conclusions, and Discussions: On average, 2-3% of cells expressed mCherry via flow cytometry. This demonstrated poor transfection efficiency justifying all sample wells having effectively zero GFP fluorescence. However, since luciferase bioluminescence has a higher sensitivity with a lower background compared to the fluorescence of GFP and the luciferase reporter plasmid was transfected at 2x the dose of the GFP reporter plasmid due to omission of the transfection marker, increasing the poly(I:C) concentration yielded an increasing trend in the luciferase bioluminescence output signal, demonstrating increasing luciferase reporter expression as expected (Figure 1.e). This indicates that more of the immunostimulant led to more representative activation of innate immunity for the transient luciferase reporter system. Further, the luciferase reporter activation followed the classical sigmoidal dose response curve (4 parameter nonlinear regression) (Figure 1.e). The large error bars in Figure 1.e can be explained by the poor and variable nature of the lipofectamine transfection requiring further future proof-of-concept and optimization experiments.

In future proof-of-concept experiments, an easier to transfect cell line HEK293 T could be used. This preliminary increasing trend innate immune response data for the transient C2C12 transfection of the luciferase reporter construct, after further validation with more transient transfection experiments,will justify moving forward with creating permanent cell lines through lentiviral transduction to create reporter gene assays as an easy, rapid, reliable, and cell-based research tool to screen RNA immunogenicity. Upon creation of the cell lines, validation experiments will be conducted to test for the specificity, accuracy, and robustness of the methodology. This tool is superior to purchasing commercially available interferon reporter cell lines in the field as it is customizable to test RNA therapies’ immunostimulation for any transfectable cell line in vitro. Further, after optimization and initial validation of the assay, the established cell line could potentially be used to assess the in vitro innate immune reaction to any RNA therapy. This would immensely speed up the in vitro RNA immunogenicity screening and characterization of RNA therapeutics with different sizes, types, structures, etc.

Acknowledgements (Optional):