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

endocrinopathies. The standard substitutive administration of the deficient hormonesdoes not halt the autoimmune process; therefore, development of immunotherapiesaiming to preserve the residual hormonal cells, is of crucial importance. PTPN22C1858T mutation encoding for the R620W lymphoid tyrosine phosphatase variant,plays a potential pathophysiological role in autoimmunity. The PTPN22 encodedprotein 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 avalid 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 measuredby CD spectroscopy and DLS. Lipoplexes internalization and toxicity evaluation wasassessed by confocal microscopy and flow cytometry analysis. Their effect on Lypexpression was evaluated by means of Western Blot and confocal microscopy.Functional assays through engagement of TCR signaling were established to evaluatebiological 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 incorporationwas visualized in CD3+ but also in CD3- peripheral blood immunotypes without signsof toxicity, damage or apoptosis. Efficacy in affecting Lyp protein expression wasdemonstrated in both transfected Jurkat T cells and PBMC. Moreover, impairment ofLyp inhibitory activity was revealed by increase of IL-2 secretion in culturesupernatants 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 ofautoimmune diseases based on the selective inhibition of variant PTPN22 allele usinglipoplexes of siRNA antisense oligomers.