PANORAMA: Neuromuscular Organoids for Refractory AChR+ Myasthenia Gravis

Overview

Myasthenia gravis (MG) is an autoimmune disease in which autoantibodies attack the neuromuscular junction, the site at which nerve cells communicate with muscle fibres, impairing signal transmission and causing fluctuating muscle weakness that worsens with sustained activity. In most patients this dysfunction is reversible and improves with treatments that suppress the immune response. About 10 to 15 percent of patients do not respond adequately to standard therapy, and the mechanisms of this refractory course remain unclear. The study is based on the hypothesis that in refractory patients the autoantibody attack causes irreversible damage to the neuromuscular junction, and that this damage sustains symptoms despite appropriate treatment. A further aim is to identify circulating biomarkers reflecting such damage that may help predict response to therapy. The study includes adults with generalised MG positive for antibodies against the acetylcholine receptor, stratified by disease duration and treatment response into treatment-naive, treatment-sensitive and treatment-refractory MG. Subjects without neuromuscular disease and negative for these antibodies serve as controls. Blood samples (serum, plasma and mononuclear cells) are obtained from material left over from blood draws performed as part of routine care, together with clinical data including disease duration, symptom severity measured with validated scales (MG-ADL and QMG), antibody titre and treatment history. No study-specific visit or blood draw is required. Antibodies purified from participants are applied to human neuromuscular organoids, three-dimensional models grown from stem cells of healthy donors that reproduce key features of the neuromuscular junction. Exposing these organoids to antibodies from patients at different disease stages reproduces the antibody-mediated attack under controlled laboratory conditions and allows the resulting structural and electrical changes to be measured. Molecules released by damaged organoids, including microRNAs and proteins, are identified and then measured in participants' blood. The immune profile of participants, including complement factors, lymphocyte subsets and cytokines, is characterised in parallel. The study will determine whether irreversible neuromuscular junction damage distinguishes treatment-refractory MG from treatment-responsive disease, and whether specific circulating biomarkers can identify a refractory course.

Experimental platform Neuromuscular organoids are generated from three independent induced pluripotent stem cell lines from healthy individuals, following the self-organising trunk protocol of Faustino Martins et al. (Cell Stem Cell 2020), which yields spinal motor neurons and skeletal muscle fibres forming functional neuromuscular junctions within a single self-patterning tissue. Most mechanistic work on antibody-mediated end-plate damage has relied on rodent experimental autoimmune myasthenia gravis or on myotube monolayers. Rodent junctions differ from human junctions in receptor subunit composition and in expression of membrane-bound complement regulators, while myotube monolayers lack presynaptic input and mature postsynaptic specialisations, so neither reports on the junction as an assembled human structure. Serum is centrifuged at 3000 g for 5 minutes at room temperature and IgG fractions are purified by protein G affinity chromatography, reconstituted in elution buffer and quantified by spectrophotometry, so that exposure is normalised to IgG concentration rather than to serum volume. Anti-AChR reactivity is determined by radioimmunoassay or cell-based assay. Organoids are exposed to purified IgG at 300 nM final concentration, supplemented with 2 percent human serum as a complement source, once neuromuscular junctions are established, at a stage of differentiation at which presynaptic and postsynaptic specialisations are mature. Applying the antibody challenge to a formed junction rather than during synaptogenesis ensures that the changes measured reflect damage to an established structure, consistent with the study hypothesis, rather than impaired junctional development. Readouts are acquired 3 days (acute exposure) and 14 days (chronic exposure) after the start of incubation, with organoids exposed to IgG from AChR-negative serum serving as negative control. Three technical replicates per iPSC line are analysed for each condition and timepoint. Morphological and functional readouts Morphological analysis combines immunofluorescence for spinal and muscle markers, alpha-bungarotoxin/SV2/neurofilament co-staining with stereological quantification of junction number, size and maturation, confocal imaging with two-photon or light-sheet acquisition for high-resolution three-dimensional reconstruction, and electron microscopy for ultrastructural assessment of synaptic components. Complement engagement is assessed by immunofluorescence for C5b-9 membrane attack complex deposition at the junction and by ELISA quantification of soluble C2, C3a and C5b-9 in organoid supernatant. Functional analysis is based on high-density multielectrode array recording, with 15-minute extracellular acquisition of spontaneous activity followed by pharmacological stimulation. Contractility is quantified by video recording as contraction frequency and amplitude in three distinct regions of each organoid, at baseline and after acetylcholine stimulation, with selective inhibitors separating muscle-specific from neuronal contributions. Calcium dynamics are visualised with Fluo-4AM and analysed for frequency, amplitude and propagation in both motor neurons and myofibres. Pharmacological challenge with neurotransmitter agonists and antagonists establishes whether the functional deficit is predominantly presynaptic or postsynaptic, a distinction not resolvable from the clinical phenotype. Molecular characterisation and biomarker pipeline Organoids showing the most pronounced neuromuscular abnormality within each clinical group are selected for molecular profiling. Spatial gene expression is assessed by digital spatial profiling, which maps expression within intact organoid tissue and allows regional and cell-type-specific comparison across conditions, with particular attention to genes encoding proteins involved in junctional homeostasis and in the immune response. Data are analysed through an integrated pipeline combining dedicated analysis software with custom R scripts based on Seurat and Monocle; candidate targets are prioritised by weighted gene co-expression network analysis and pathway enrichment, and validated by in situ hybridisation, immunofluorescence and Western blot. Organoid supernatant provides a compartment in which molecules released during junctional injury can be sampled without confounding by systemic sources. MicroRNA is extracted from supernatant and quantified by real-time PCR for neuromuscular-enriched species (miR-206, miR-133b, miR-1, miR-499) and for inflammation-related species previously associated with myasthenia gravis, while the proteome is characterised by liquid chromatography-mass spectrometry. Species showing the greatest dysregulation are subsequently quantified in participant serum by Simple Plex assays on the Ella platform or by ELISA. Candidates entering clinical validation are therefore anchored to a defined mechanism in a human model of the disease process, rather than selected by unsupervised association with clinical phenotype. Immunological profiling In parallel, circulating cytokines, chemokines and complement factors are quantified by Simple Plex or ELISA, alongside anti-AChR antibody titre. Statistical approach for the organoid component The unit of analysis for organoid data is the iPSC line, which constitutes the independent biological unit; technical replicates are nested within lines. To avoid pseudoreplication, the primary analytical method is a linear mixed-effects model with iPSC line as random intercept and experimental group and timepoint as fixed effects, with planned comparisons Bonferroni-corrected. One-way analysis of variance or Kruskal-Wallis testing with post hoc comparison is reported as a supplementary approach for aggregated data. Bootstrap simulation indicates power above 0.82 for a Cohen f of 0.45 and an intraclass correlation of 0.30 with three iPSC lines and three technical replicates per condition. For the serum biomarker panel, Benjamini-Hochberg control of the false discovery rate below 0.05 is the primary correction method, more appropriate than Bonferroni in an exploratory setting with correlated markers, with Bonferroni reported as a sensitivity analysis. No interim analysis is planned. Distribution of laboratory activities Organoid generation, morphological and functional characterisation, spatial transcriptomic and proteomic profiling, and serum validation of candidate biomarkers are centralised at the coordinating centre. Complement activity assessment on organoid supernatant and immunological profiling are centralised at the partner centre. Centralisation of each assay at a single facility avoids inter-site technical variability.