Selective activation and expansion of regulatory T cells using lipid encapsulated mRNA encoding a long-acting IL-2 mutein

Interleukin-2 (IL-2) is critical for regulatory T cell (Treg) function and homeostasis. At low doses, IL-2 can suppress immune pathologies by expanding Tregs that constitutively express the high affinity IL-2Rα subunit. However, even low dose IL-2, signaling through the IL2-Rβ/γ complex, may lead to the activation of proinflammatory, non-Treg T cells, so improving specificity toward Tregs may be desirable. Here we use messenger RNAs (mRNA) to encode a half-life-extended human IL-2 mutein (HSA-IL2m) with mutations promoting reliance on IL-2Rα. Our data show that IL-2 mutein subcutaneous delivery as lipid-encapsulated mRNA nanoparticles selectively activates and expands Tregs in mice and non-human primates, and also reduces disease severity in mouse models of acute graft versus host disease and experimental autoimmune encephalomyelitis. Single cell RNA-sequencing of mouse splenic CD4+ T cells identifies multiple Treg states with distinct response dynamics following IL-2 mutein treatment. Our results thus demonstrate the potential of mRNA-encoded HSA-IL2m immunotherapy to treat autoimmune diseases.


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For scRNASeq, EmptyDrops was used to distinguish droplets containing cells and droplets containing cell-free RNA (FDR threshold < .01, niter=100,000 for the Monte Carlo p-value calculation). Cells were further excluded if they were associated with low number of molecules (< 500 total molecules) or low number of genes detected (< 500 genes). Low count genes were excluded to retain genes detected in > 0.01% of the cells Main findings of scRNAseq were confirmed using other methods twice and were successful. All non-scRNAseq studies were repeated at least twice and the replication were succesful.
For the non human primate studies, animals were randomized into group while maintaining similar average body weight across groups. For all other studies, allocation was random.
The investigator in the EAE studies was blinded to the nature of the test articles. For all studies, the test articles were blinded to the animal facility personnel. During acquisition and data analysis, investigators were not blinded as no bias could be introduced. Validation of commercial antibodies was done by regular quality control for each lot by the manufacturer (e.g. Biolegend) The specificity and sensitivity of each antibody is thoroughly validated in the New Product Development stage. This is done by staining multiple target cells with either single-or multi-color analysis or by other testing approaches. The QC specifications and testing SOPs and gold standard for each product are then developed.
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Expi293F were purchased from ThermoFisher, Catalog Number A14527 No further authentication were performed beyond relying on manufacturer CoA. all cell lines tested negative for mycoplasma contamination Name any commonly misidentified cell lines used in the study and provide a rationale for their use.
The study did not involve wild animals study did not involve.
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