Diagnosis Of MuSK Antibodies In Myasthenia Gravis Patients And Establishment Of The EAMG Animal Model

Background: Myasthenia gravis(MG)is an autoimmune disease caused by the pathogenic antibodies against the neuromuscular junctions(NMJs)proteins including acetylcholine receptor(AChR),muscle-specific kinase(MuSK),lipoprotein receptor-related protein 4(Lrp4)and agrin.Around 80% of patients have antibodies to AChR,followed by antibodies to MuSK.The pathogenic mechanism of MuSK-MG is that the binding of autoantibodies to endogenous MuSK proteins suppresses signal transduction by MSK and prevents ACR clustering,causing weakened endplate potential at the NMJ.Current symptomatic treatments have been used to treat AChR-MG patients with success,but only showed limited effect on patients with MuSK-MG.Known as refractory myasthenia gravis,MuSK-MG has been problematic in clinical treatment.Adeno-associated virus(AAV)has been a promising tool for gene therapy because of its safety and relatively high efficacy.We examined major binding sites of MuSK autoantibodies,and explored the possibility of recombinant AAV to treat MuSK-induced experimental autoimmune myasthenia gravis(MuSK-EAMG)mouse model.Objective: To identify the major binding sites of MuSK binding sites in MuSK-MG patients;To generate the MuSK-EAMG mouse model;and to explore the feasibility of using recombinant AAV to treat MuSK-EAMG.Methods: Making MuSK extracellular region expression construct by molecular cloning;Expression of the MuSK extracellular region by transfection in cultured cells and purification of MuSK extracellular region;Detection of anti-MuSK autoantibodies and identification of the major binding sites by ELISA;Generation of MuSK-EAMG model by immunization with purified MuSK extracellular region and examination by blood and behavior tests;Exploration of possibility of rAAV9 as a tool to effectively infect mouse skeletal muscles.Results:(1)Expression and purification of the MuSK extracellular region;(2)Detection of 22 anti-MuSK autoantibodies positive patients in 85 MG patients,and identification of the major binding sites of MuSK autoantibodies as the Ig1 domain of MuSK extracellular region;(3)Generation of MuSK-EAMG model by immunization with purified MuSK extracellular region;(4)Confirmation of rAAV9 as an efficient tool to locally infect mouse skeletal muscles.Conclusions: We found roughly 26% MuSK autoantibody positive in MG patients examined,and these autoantibodies mainly recognized the Ig1 domain of MuSK;Application of recombinant AAV in MuSK-EAMG mouse may specifically block the interaction between autoantibodies and endogenous proteins,thus provides potential antigen-specific treatment for MuSK-MG.