Targeted Immunotherapy for Regulatory T Cells 2307761

Abstract Introduction Underperformance and dysfunction of CD4+ regulatory T cells (Tregs) has been implicated in the pathogenesis of many autoimmune diseases. As such, multiple approaches toward Treg immunotherapy are being developed, most of which require ex vivo expansion and/or manipulation of Tregs, and in vivo strategies remain challenging to accomplish. Here, we hypothesized that constitutively high expression of the high-affinity IL2 receptor on Tregs could be exploited to target selective uptake of mRNA-LNPs by Tregs, enabling the potential development of an in vivo immunotherapy platform. Methods IL2 or αCD25 was conjugated to LNPs containing N1-Methylpseudouridine-substituted mRNA for eGFP using a SATA-maleimide based strategy. In vivo experiments were done using 6-week-old female C57BL/6 mice given 5 µg of LNP intravenously. In vitro experiments were performed using normal donor human splenocytes or PBMCs. Targeting was assessed using eGFP expression and flow cytometry. Results To define the optimal method to target Tregs with mRNA-LNPs, we first compared αCD25 vs IL-2 conjugated LNPs. IL2-LNPs demonstrated superior targeting in vivo of splenic Tregs compared to αCD25-LNPs in both frequency (69.7% vs. 41.9% eGFP+ respectively, p = 0.03) and expression (1399 vs. 424 eGFP MFI, p = 0.01) 24 hours after delivery. IL2-LNP targeted Tregs expressing eGFP were present in spleen, lymph node, and blood 24 hours post-treatment (40-70% eGFP+) and remained detectable for at least 7 days. Little background uptake of IL2-LNPs was observed in other immune cell subsets. Finally, IL2-LNPs also delivered mRNA effectively to FoxP3+Tregs in vitro in human splenocytes in a dose-dependent manner with little uptake by other cell subsets. Conclusion Altogether, these data demonstrate that IL2-LNPs are an efficient and effective method of targeting Tregs in situ. Future studies will apply this powerful tool to transiently enhance and alter Treg function in vivo for interventional and therapeutic strategies in autoimmune disease models. Funding Source Breakthrough T1D (3-SRA-2024-1612-S-B) Topic Categories Therapeutic Approaches to Autoimmunity (THER)