N31 - Microneedle array delivery of tolerizing immune cues to skin for countering autoimmunity

Introduction: : Autoimmunity occurs when the immune system attacks host-associated biomolecules, termed self-antigens, and damages self-tissue. For example, in multiple sclerosis the immune system attacks self-antigens in the central nervous system and damages neuronal circuitry. There is no cure for multiple sclerosis and current options deplete or broadly inhibit immunity, which leaves patients immunocompromised and at risk for deadly infections. This motivates the concept and development of antigen-specific, tolerizing immunotherapies to counter autoimmunity while leaving healthy immune functions intact. To pursue this impactful goal, we hypothesized that delivery of high-density tolerizing immune cues to the skin-immune niche could generate systemic antigen-specific tolerance. The rationale for our work is based on the high concentration and organization of antigen-presenting innate immune cells in skin. These antigen-presenting cells are critical to maintaining barrier immunity and regulating immunological balance. Thus, control of these unique and accessible immune cells could enable powerful therapeutic strategies. Here, to target skin we developed a platform that integrated nanotechnology-assembled immunotherapies and microfabricated microneedle arrays for skin delivery. Microneedle arrays are patches of tiny needles that deliver cargo to skin with precise spatial distribution. Microneedle arrays are also easily self-applied to skin, which could be transformative for multiple sclerosis patients with impaired motor function. We utilized the microneedle platform to deliver immunotherapies to skin and studied the mechanism of action using single-cell and spatial transcriptomics, showed prevention and reversal of paralysis in models of multiple sclerosis, and measured antigen-specific immune responses using adoptive transfer of transgenic immune cells.

Materials and

Methods: : We built self-assembling polyplexes (100 nm in diameter) based on peptide-nucleic acid electrostatic interactions (Figure 1A). In these polyplexes, the peptide was myelin self-antigen (myelin oligodendrocyte glycoprotein or proteolipid protein) modified with an increased cationic charge by adding arginine residues and the nucleic acid was an innate immune regulatory cue that antagonized toll-like receptor 9. Microneedle arrays were assembled through integration of manufacturing techniques at multiple length scales (2-photon polymerization, precision computer aided milling, soft lithography, and hot embossing) to produce hundreds of immunotherapy-loaded microneedle arrays in parallel (Figure 1B). The polyplexes were embedded into the tips of degradable microneedle arrays for delivery to skin (Figure 1C-D). Degradation was enabled using a fish gelatin matrix, which has been previously investigated for microneedle-based influenza vaccination. To analyze the skin immune response to polyplex-loaded microneedle arrays, we utilized single-cell RNA-seq. Briefly, microneedle arrays were applied to mouse dorsal flank skin at a controlled pressure and remained self-adherent for 4 hours. Single cells were enzymatically isolated from skin samples from untreated control and treated mice at 72 hours following treatment. The cells from biological replicates were pooled (n=10 for each group), encapsulated, barcoded, indexed, and sequenced. Analysis was performed using the Seurat R package and ShinyCell. Validation studies were carried out using RT-qPCR to compare the immune response to polyplexes, the microneedle matrix, and injury alone.

Results, Conclusions, and Discussions:: Our findings show that delivery of an antigen-specific immunotherapy to skin using microneedle arrays generates tolerogenic immune cells in the skin-immune niche. Single-cell transcriptomic data shows the biasing of skin from dendritic cells toward macrophage phenotypes following microneedle application. Skin from the group treated with polyplex-loaded microneedle arrays (Figure 1E) also contained a population of macrophages that expressed a high level of Arg1 (Arginase-I) transcripts (Figure 1F). RT-qPCR showed that this group was specifically associated with the delivery of polyplexes. This suggested that treatments in the skin were associated with a regenerative and anti-inflammatory phenotype, which has overlapping pathways with immune tolerance. Moreover, this result was only observed in the treatments containing self-antigen and the regulatory ligand, further validating that this response was not the result of microneedle application or the microneedle matrix degradation. Additional data from these unpublished studies show the therapeutic biodistribution and efficacy in mouse models of multiple sclerosis. Biodistribution analysis by IVIS indicates that the polyplexes reside in skin as depots of immune cues for weeks following microneedle application. In both the progressive and relapsing-remitting mouse models of multiple sclerosis we show that microneedle deliver of tolerizing cues to skin provide protection and reversal of disease severity (paralysis) without inhibiting protective immunity. Lastly, we conducted a spatial transcriptomic analysis of spinal cords from naïve and diseased mice with the EAE model of progressive multiple sclerosis (Figure 1G). This allowed us to align immunohistochemical analysis with transcriptomic data and revealed a dramatic downregulation of myelin generating transcripts in the spinal cord (Figure 1H). These regions were also associated with lesions that contained a high concentration of transcripts indicative of innate immune cell infiltration. Collectively, this data illustrates that multi-scale fabrication is a powerful strategy for engineering the skin-immune niche and provides proof-of-concept for an accessible, self-administrable, therapeutic option for autoimmunity.

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