Optimization of Lipoplexes Functionalized with a Sialic Acid Mimetic (F9-PEG) to Target the C1858T PTPN22 Variant for Preclinical Assessment of a Novel Immunotherapy in Endocrine Autoimmunity

The C1858T PTPN22 variant is strongly associated with Type 1 diabetes and autoimmune thyroid disease. Current treatment is the substitutive hormonal administration which does not target the disease pathogenetic mechanism. Background/Objectives: We previously implemented a novel immunotherapy, employing siRNA directed against the C1858T variant of PTPN22 delivered via functionalized lipoplexes in order to halt autoimmune disease progression. The objective of this study was to optimize lipoplexes formulations functionalized with F9-PEG (a Siglec-10's ligand) to facilitate targeted delivery, by testing their physical and chemical properties to warrant best performance in in vivo studies. Methods: The effectiveness of siRNA-liposome binding was evaluated by varying the relative lipid/siRNA charge ratio and analyzing the stability of the different formulations with respect to methods of F9-PEG addition and ATTO740 fluorescent labelling by electrophoresis, dynamic and dielectrophoretic light scattering (DLS, DELS), and high-performance liquid chromatography (HPLC). A preliminar investigation of lipoplexes biodistribution was conducted by fluorescent tagging in C57BL/6 mice. Results: The optimal charge ratio of +2 /-1 (lipid/siRNA) ensured greater stability of lipoplexes. Stability was improved by functionalization with F9-PEG further by the addition of ATTO740-dye. HPLC confirmed the integrity of siRNA after preparation. Injection of PKH26-labelled lipoplexes in mice showed selective accumulation in target tissues within 48 hours. Conclusions: The results of this study lead to formulations of F9-PEG lipoplexes with optimal properties that could be used for biodistribution and safety/efficacy studies in mice. Lipoplexes functionalized with F9-PEG could therefore represent a promising personalized nanotherapeutic platform for targeted siRNA delivery in endocrine C1858T patients.