PANORAMA: Neuromuscular Organoids for Refractory AChR+ Myasthenia Gravis (PANORAMA)

From PAtients to Neuromuscular Organoids in Refractory AChR+ Myasthenia grAvis: End-plate Dysfunction, Biomarker Discovery and Regeneration Strategies

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.

Study Overview

• Status: Recruiting
• Conditions: Myasthenia Gravis Generalized

Detailed Description

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.

• Study Type: Observational
• Enrollment (Estimated): 40

Contacts and Locations

• Study Contact: Name: Delia Gagliardi, MD, PhD; Phone Number: +39 02 5503 3802; Email: delia.gagliardi@policlinico.mi.it

Study Locations

• Italy
  • Milan, Italy, 20122
    • Recruiting
    • Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico
    • Contact: Delia Gagliardi, MD, PhD; Phone Number: +39 02 5503 3802; Email: delia.gagliardi@policlinico.mi.it
    • Contact: Mosè Parisi, MD; Email: mose.parisi@policlinico.mi.it
    • Principal Investigator: Delia Gagliardi, MD, PhD
    • Principal Investigator: Yuri M Falzone, MD

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult; Older Adult
• Accepts Healthy Volunteers: Yes

• Sampling Method: Non-Probability Sample

Study Population

Adults with acetylcholine receptor antibody-positive (AChR-positive) generalized myasthenia gravis, followed at two Italian tertiary referral centers for neuromuscular diseases, stratified by disease duration and response to conventional immunosuppressive therapy into treatment-naive, treatment-sensitive, and treatment-refractory subgroups. Both patients already followed at the participating centers (retrospective component) and newly evaluated patients (prospective component) are included. A control group of adults evaluated for neurological symptoms whose diagnostic work-up excluded neuromuscular disease is enrolled at the coordinating center only. Clinical data and residual (leftover) blood samples collected during routine clinical care are analyzed.

Description

Inclusion Criteria:

AChR-positive MG participants (all of the following):

• Age >= 18 years
• Established diagnosis of myasthenia gravis according to international criteria (fluctuating muscle weakness plus at least one of: positive anticholinesterase test; >10% decrement on repetitive nerve stimulation [SR-ENG]; or increased jitter on single-fiber EMG [SFEMG])
• Anti-AChR antibody positivity confirmed by serological testing
• Generalized MG stratified by disease duration and response to conventional immunosuppressive therapy into: treatment-naive MG (nMG); treatment-sensitive MG with stable disease and at least 12 months of follow-up (sMG); or treatment-refractory MG with at least 12 months of follow-up (rMG)
• Signed informed consent (prospective component)
• For the retrospective component, availability of biobanked biological samples with a signed specific informed consent

Control participants (all of the following):

• Age >= 18 years
• Absence of neuromuscular disease
• Absence of known autoimmune disease
• No ongoing immunosuppressive or immunomodulatory therapy
• Negative for anti-AChR and anti-MuSK antibodies
• Age- and sex-matched to the MG groups
• Signed informed consent

Exclusion Criteria:

• Seronegative (AChR-negative) generalized MG, or MG with anti-MuSK or other non-AChR antibodies
• Purely ocular myasthenia gravis
• Other concomitant neuromuscular disease (e.g., Lambert-Eaton myasthenic syndrome, polymyositis, muscular dystrophies)
• Pregnancy or breastfeeding
• Inability to provide informed consent

Study Plan

How is the study designed?

Number of groups / cohorts

4

Cohorts and Interventions

Group / Cohort
Treatment-naive AChR-positive generalized MG (nMG); Adults with acetylcholine receptor antibody-positive (AChR-positive) generalized myasthenia gravis with a recent diagnosis who have not yet started immunosuppressive therapy. Clinical data (disease duration, MGFA class, MG-ADL and QMG scores, antibody titers, treatment history) and residual (leftover) blood samples (serum, plasma, PBMC) collected during routine clinical care are analyzed. No study-specific intervention or extra blood draw is performed. Purified IgG from these participants is applied in vitro to human neuromuscular organoids to model antibody-mediated neuromuscular junction damage, and candidate circulating biomarkers (microRNAs, proteins) identified in the organoid models are validated in the participants' serum.
Treatment-sensitive AChR-positive generalized MG (sMG); Adults with AChR-positive generalized myasthenia gravis who respond to conventional immunosuppressive therapy and have stable disease, with at least 12 months of follow-up. Clinical data (disease duration, MGFA class, MG-ADL and QMG scores, antibody titers, treatment history) and residual (leftover) blood samples (serum, plasma, PBMC) collected during routine clinical care are analyzed. No study-specific intervention or extra blood draw is performed. Purified IgG from these participants is applied in vitro to human neuromuscular organoids to model antibody-mediated neuromuscular junction damage, and candidate circulating biomarkers (microRNAs, proteins) identified in the organoid models are validated in the participants' serum.
Treatment-refractory AChR-positive generalized MG (rMG); Adults with AChR-positive generalized myasthenia gravis who do not respond to appropriate conventional immunosuppressive therapy, with at least 12 months of follow-up and poor disease control. Clinical data (disease duration, MGFA class, MG-ADL and QMG scores, antibody titers, treatment history) and residual (leftover) blood samples (serum, plasma, PBMC) collected during routine clinical care are analyzed. No study-specific intervention or extra blood draw is performed. Purified IgG from these participants is applied in vitro to human neuromuscular organoids to model antibody-mediated neuromuscular junction damage, and candidate circulating biomarkers (microRNAs, proteins) identified in the organoid models are validated in the participants' serum.
Non-neuromuscular, AChR-negative controls; Adults without neuromuscular disease and negative for anti-AChR and anti-MuSK antibodies, age- and sex-matched to the MG groups and free of known autoimmune disease and ongoing immunosuppressive or immunomodulatory therapy. They are assessed for neurological symptoms with a diagnostic work-up that excludes neuromuscular disease. Demographic data and residual (leftover) blood samples (serum, plasma, PBMC) collected during routine clinical care are analyzed. No study-specific intervention or extra blood draw is performed. AChR-negative serum from these participants serves as the negative control in the in vitro human neuromuscular organoid experiments and as the reference group for immunological and biomarker comparisons.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Antibody-induced change in neuromuscular junction electrophysiological activity in a patient serum-derived organoid assay	Change in spontaneous electrophysiological activity, measured as firing rate (Hz) by high-density multielectrode array (HD-MEA), in human neuromuscular organoids exposed to purified IgG from participants' serum. The primary comparison is between organoids exposed to IgG from treatment-refractory MG participants versus organoids exposed to IgG from treatment-sensitive MG, treatment-naive MG, and AChR-negative control participants, at the day 14 (chronic) exposure timepoint. The measure is assessed on a specimen-derived in vitro assay using participant-purified IgG, not on the participants directly. Lower firing rate indicates greater antibody-mediated neuromuscular junction dysfunction.	Assessed in vitro at day 14 of organoid exposure to participant-derived IgG (chronic exposure timepoint)

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Antibody-induced change in neuromuscular junction morphology in a patient serum-derived organoid assay	Change in the alpha-Bungarotoxin/NF200 co-localization area, measured by confocal immunofluorescence and quantified with FIJI/ImageJ, in human neuromuscular organoids exposed to purified IgG from participants' serum. The comparison is between organoids exposed to IgG from treatment-refractory MG participants versus organoids exposed to IgG from treatment-sensitive MG, treatment-naive MG, and AChR-negative control participants, at the day 14 exposure timepoint. This measure is assessed on a specimen-derived in vitro assay using participant-purified IgG and supports the primary electrophysiological outcome. A reduced co-localization area indicates greater antibody-mediated neuromuscular junction damage.	Assessed in vitro at day 14 of organoid exposure to participant-derived IgG
Validation of candidate refractoriness biomarkers in participants' serum	Levels of candidate biomarkers, first identified in the serum-derived organoid models, measured in participants' serum to assess their association with a treatment-refractory disease course. Neuromuscular-enriched and inflammation-related microRNAs (such as miR-206, miR-133b, miR-1 and miR-499) are quantified by quantitative RT-PCR, and candidate proteins are quantified by immunoassay (Simple Plex/Ella or ELISA). Levels are compared across treatment-refractory, treatment-sensitive, and treatment-naive AChR-positive generalized MG participants and AChR-negative controls.	Baseline
Immunological profile across MG treatment-response subgroups	Characterization of the immunological profile in participants' blood, compared across treatment-refractory, treatment-sensitive, and treatment-naive AChR-positive generalized MG participants and AChR-negative controls. Assessments include anti-AChR antibody titer, complement factors, lymphocyte subpopulations by flow cytometry (T subsets including CD4+, CD8+ and regulatory T cells; B subsets including naive, memory and plasma cells), and circulating cytokines and chemokines measured by immunoassay (Simple Plex/Ella or ELISA). The aim is to identify immunological features that distinguish treatment-refractory disease.	Baseline
Correlation between clinical severity, immunological profile, and organoid alterations	Correlation between clinical severity of myasthenia gravis (MGFA classification, QMG score, MG-ADL score) and the immunological and molecular parameters measured in the study, including the immunological profile and the morphological and molecular alterations observed in the serum-derived organoid models. Associations are assessed using Spearman or Pearson correlation as appropriate, with multivariable regression to identify independent predictors of clinical severity and of a treatment-refractory course.	Baseline

Collaborators and Investigators

• Sponsor: Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico
• Collaborators: IRCCS Ospedale San Raffaele

Study record dates

Study Major Dates

• Study Start (Actual): June 4, 2026
• Primary Completion (Estimated): March 31, 2028
• Study Completion (Estimated): March 31, 2028

Study Registration Dates

• First Submitted: July 13, 2026
• First Submitted That Met QC Criteria: July 16, 2026
• First Posted (Actual): July 21, 2026

Study Record Updates

• Last Update Posted (Actual): July 21, 2026
• Last Update Submitted That Met QC Criteria: July 16, 2026
• Last Verified: June 1, 2026

More Information

Terms related to this study

• Keywords: biomarkers; refractory; myasthenia gravis; neuromuscular organoids; end-plate dysfunction; regeneration strategies; anti-AChR antibodies
• Additional Relevant MeSH Terms: Nervous System Diseases; Neoplasms by Site; Neoplasms; Neuromuscular Diseases; Autoimmune Diseases; Immune System Diseases; Autoimmune Diseases of the Nervous System; Neurodegenerative Diseases; Paraneoplastic Syndromes, Nervous System; Nervous System Neoplasms; Paraneoplastic Syndromes; Neuromuscular Junction Diseases; Myasthenia Gravis
• Other Study ID Numbers: 6999

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO
• IPD Plan Description: Individual participant data will not be shared. The protocol provides for publication of results in aggregate, anonymized form only, and the informed consent does not authorize the transfer of individual-level data to third parties. Data are owned by the Sponsor, and sharing of individual participant data is not planned in order to comply with the approved protocol, the informed consent, and applicable data protection regulation (EU Regulation 2016/679).

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No