Use of short interfering RNA delivered by cationic liposomes to enable efficient down-regulation of PTPN22 gene in human T lymphocytes

Abstract

Type 1 diabetes and thyroid disease are T cell-dependent autoimmune endocrinopathies. The standard substitutive administration of the deficient hormones does not halt the autoimmune process; therefore, development of immunotherapies aiming to preserve the residual hormonal cells, is of crucial importance. PTPN22 C1858T mutation encoding for the R620W lymphoid tyrosine phosphatase variant, plays a potential pathophysiological role in autoimmunity. The PTPN22 encoded protein Lyp is a negative regulator of T cell antigen receptor signaling; R620W variant, leading to a gain of function with paradoxical reduced T cell activation, may represent a valid therapeutic target. We aimed to develop novel wild type PTPN22 short interfering RNA duplexes (siRNA) and optimize their delivery into Jurkat T cells and PBMC by using liposomal carriers. Conformational stability, size and polydispersion of siRNA in lipoplexes was measured by CD spectroscopy and DLS. Lipoplexes internalization and toxicity evaluation was assessed by confocal microscopy and flow cytometry analysis. Their effect on Lyp expression was evaluated by means of Western Blot and confocal microscopy. Functional assays through engagement of TCR signaling were established to evaluate biological consequences of down-modulation. Both Jurkat T cells and PBMC were efficiently transfected by stable custom lipoplexes. Jurkat T cell morphology and proliferation was not affected. Lipoplexes incorporation was visualized in CD3+ but also in CD3- peripheral blood immunotypes without signs of toxicity, damage or apoptosis. Efficacy in affecting Lyp protein expression was demonstrated in both transfected Jurkat T cells and PBMC. Moreover, impairment of Lyp inhibitory activity was revealed by increase of IL-2 secretion in culture supernatants of PBMC following anti-CD3/CD28 T cell receptor-driven stimulation. The results of our study open the pathway to future trials for the treatment of autoimmune diseases based on the selective inhibition of variant PTPN22 allele using lipoplexes of siRNA antisense oligomers.

Fig 1. Circular dichroism spectroscopy.

(A) CD spectra of 1.3 μM siRNA in buffer solution (5 mM HEPES, 0.1 mM EDTA, pH 7.4). (B) CD spectra of 1.3 μM siRNA in the DMPC/1 formulation. (C) CD spectra of 1.3 μM siRNA in the DMPC/2 formulation. Spectra were recorded at 25°C.

Fig 2. Dynamic light scattering.

Hydrodynamic diameter distribution functions (averaged by number) obtained by CONTIN analysis of DLS data (scattering angle 90°, T = 25°C) for DMPC/1 liposomes (panel A); DMPC/2 liposomes (panel B); DMPC/1/siRNA lipoplexes (panel C); DMPC/2/siRNA lipoplexes (panel D).

Fig 3. Lyp protein expression in Jurkat T cells after siRNA s/a transfection using a commercial system.

(A) siRNA transfection with siRNA having higher affinity to the target mRNA sequence (siRNA1) resulted in a reduction of Lyp O.D. (optical density) percentages to untreated cells to 44%, 43% and 48% with the doses of 40-60-80 pmols respectively after 48 hours of transfection. O.D. values of Lyp and β-actin were obtained with ImageLab software. Lyp O.D. values for every treatment were normalized to the corresponding β-actin values. No reduction was obtained with siRNA molecule with lower affinity (siRNA2). * indicates p<0.05. (B) Representative WB image with all the corresponding experimental groups is shown. Lyp O.D. % with respect to untreated cells’ O.D. values calculated as percentage to untreated cells’ O.D. ‘Untreated’ represents the basal control level (100%) of Lyp protein expression in cells cultured in RPMI. The efficacy of transfection is calculated from the difference between 100 and the test O.D. percentage in each experiment.

Fig 4. Lyp protein levels in Jurkat T cells 48 hours after the beginning of O/N transfection with different doses of siRNA s/a in DMPC/1/siRNA lipoplexes.

Representative duplicate experiment among all replicas in S4 Fig. (A) Lyp expression in Jurkat T cells cultured in RPMI or after O/N transfection with DMPC/1, 20 pmols of siRNA and 20 pmols of siRNA in DMPC/1/siRNA lipoplexes (DMPC/1/siRNA20). 20 pmols of siRNA complexed with DMPC/1 resulted in a 31% reduction of Lyp expression. (B) Same experiment as in A using 60 pmols of siRNA complexed with DMPC/1 (DMPC/1/siRNA60). 39% reduction of Lyp expression was obtained. (C) Same experiment as in A using 100 pmols of siRNA complexed with DMPC/1 lipoplexes (DMPC/1/siRNA100). 47% reduction of Lyp expression was obtained. (D) Representative WB image within all experimental groups is shown. Under each blot Lyp O.D. values for every treatment are normalized over the corresponding β-actin values. All percentages were expressed relatively to untransfected cells (RPMI) that is considered the 100% of basal Lyp expression. Graphs A, B, C show the mean values and their standard deviations. In all experimental conditions (vide infra), a decrease in Lyp protein level was not observed in cells treated with the liposome alone or, more importantly, in cells treated with the correspondent dose of the siRNA s/a free molecule.

Fig 5. Lyp protein expression in Jurkat T cells 72 hours after the beginning of O/N transfection with different doses of siRNA s/a in DMPC/1/siRNA lipoplexes.

Representative duplicate experiment among all replicas in S4 Fig. (A) Lyp expression in Jurkat T cells after O/N transfection with DMPC/1 alone (DMPC/1), 20 pmols of siRNA alone (siRNA20) and 20 pmols of siRNA complexed with DMPC/1 (DMPC/1/siRNA20) or cultured in RPMI. 20 pmols of siRNA complexed with DMPC/1 resulted in a 15% reduction of Lyp expression. (B) Same experiment as in A using 60 pmols of siRNA (siRNA60) complexed with DMPC/1 (DMPC/1/siRNA60). 33% reduction of Lyp expression was obtained. (C) Same experiment as in A using 100 pmols of siRNA (siRNA100) complexed with DMPC/1 (DMPC/1/siRNA100). 45% reduction of Lyp expression was obtained. (D) Representative WB image within all experimental groups is shown.

Fig 6. Confocal microscopy analysis of lipoplexes incorporation in Jurkat T cells.

Data are shown after 60 minutes and 4 and half hours of incubation. Arrows indicate the localization of liposome fluorescence (red spots) compared to cell membrane (green) and nucleus (blue). Z-reconstructions obtained by confocal microscopy show the internalization of lipoplexes in Jurkat T cells in the X- and Y-axis projections. Plasma membrane is stained with wheat germ agglutinin (WGA), whereas nuclei are counterstained with Hoechst. Bar: 10 μm.

Fig 7. PBMC DMPC/2/siRNA internalization.

HD PBMC were administered with DMPC/2/siRNA formulation marked with rhodamine. After 4 and a half hours of treatment cells were fixed and stained for immunofluorescence with anti-CD3 antibody (white) to specifically label T cells, WGA to stain plasma membrane (green), and Hoechst to label nuclei acid (blue). (A) Confocal Z reconstructions show the presence of the lipoplexes (red dots, arrows) in the cytoplasm of CD3⁺ cells. Bar: 10μm. (B) Graphs show the analysis of the percentage of siRNA⁺ cells among CD3⁺ and CD3^- cells by scoring a total number of 1546 cells. *** indicates a p value <0.001 (p = 0.0007).

Fig 8. Confocal microscopy analysis of Lyp protein expression in Jurkat T cells after transfection with lipoplexes.

The comparison shown is between samples transfected with 60 and 100 pmols of Lipo/siRNA lipoplexes, and analyzed after 72 hours from the beginning of the O/N transfection. Control cells are untreated and cultured in RPMI (upper panels). Lyp protein expression is revealed by anti-mouse IgG conjugated to Alexa Fluor 555 (red signal). Cell morphology and DNA are detected by WGA (green) and Hoechst (blue) staining, respectively. Bar: 20 μm.

Fig 9. Confocal microscopy analysis of Lyp protein expression in human PBMC after transfection with lipoplexes.

The images show the concurrence of the lipoplexes (red dot/arrow) inside CD3⁺ (white) and CD3^- cells and the reduction in Lyp immunofluorescence signal (green). Cells nuclei are counterstained with Hoechst dye (blue) in comparison with untransfected cells. Bar: 20 μm.

Fig 10. Evaluation of cell death after lipoplexes internalization.

Flow cytometric analysis of HD PBMC (left) and Jurkat cells (right) treated with rhodamine-marked lipoplexes for 4 and a half hours. The histogram shows both the percentage of ‘transfected’ lymphocytes (rhodamine⁺ cells) and the percentage of dead cells among the transfected ones (DAPI⁺ and rhodamine ⁺ cells).

Fig 11. IL-2 detection ELISA assay of culture supernatants.

Histograms illustrate the increment of IL-2 upon 5 days of anti-CD3/CD28 activation on PBMC derived from two healthy volunteers HD1 (A) and HD2 (B) previously transfected O/N with DMPC/2/siRNA60. The amount of IL-2 was normalized to the total protein load of each sample (estimated by Thermo Scientific BCA colorimetric protein assay kit). US = control unstimulated cells.