Dottorato in NEUROSCIENZE CLINICO-SPERIMENTALI E PSICHIATRIA

Titolo della tesi: PERSONALISED MEDICINE IN MYASTHENIA GRAVIS: ROLE OF NON-CODING RNAs AS BIOMARKERS OF TREATMENT RESPONSE

Myasthenia gravis (MG) is a rare T cell-dependent and B cell-mediated autoimmune disorder that affects the neuromuscular junction (NMJ). Pathogenic autoantibodies are directed towards different targets at the post-synaptic endplate, altering neuromuscular transmission with subsequent muscle fatigability and weakness. Current treatments for MG allow symptom control, however about ~ 80% of patients do not achieve complete stable remission, and the majority require lifelong immunosuppressive (IS) therapy with corticosteroids or non-steroidal immunosuppressants (NSISTs), which together are generally referred as conventional immunosuppressants (ISTs). Furthermore, about 10-15% of patients are treatment refractory or intolerant to conventional ISTs, with a great impact on their quality of life. Biological drugs, such as FcRn blockers and C5 inhibitors, represent a promising therapeutic alternative able to eliminate, or at least reduce, concomitant IS therapy. However, their use may be limited by various factors, including high costs and availability only in specialized neurological hub centers. Ineffective treatments, co-morbidities and inter-individual variation in efficacy and tolerability of IS drugs strongly highlight the need to identify biomarkers able to predict and monitor patient-specific response to therapies, ultimately leading to the development of more specific and effective personalized medicine (PM) approaches for MG. Non-coding RNAs (ncRNAs), such as microRNAs (miRNAs) and long ncRNAs (lncRNAs), are promising biomarkers for immune-mediated diseases, as they are able to regulate both immune response and drug metabolism. Various studies showed the involvement of miRNAs and lncRNAs in MG pathogenesis, however their role as biomarkers of drug response for individualized therapy in MG patients has been poorly explored yet. The aim of this study was to identify specific ncRNAs and their target mRNAs as biomarkers associated with response to conventional ISTs in MG patients. We initially stratified our cohort of MG patients into responder (R, n=23) and non-responder (NR, n=21) to conventional ISTs. We then performed whole miRNome analysis by microarrays on peripheral blood mononuclear cells (PBMCs) of patients and selected a set of miRNAs differentially expressed (DE) between R and NR patients for validation by real-time PCR: miR-21-5p, -29b-3p, -30e-5p, -125a-5p, -125b-5p, -145-5p, -150-5p, -192-5p and -423-5p. Selection of miRNA was based on fold change value and/or their role in regulating immune-related pathways. Notably, we found a significant reduction of miR-150-5p and miR-423-5p levels in PBMCs of NR compared to R patients, suggesting the ability of these miRNAs to discriminate between the two patients’ groups. MiR-150-5p is known to regulate T and B cell function and has previously been implicated in MG. MiRNA target prediction by bioinformatics tools revealed MYB and EEF2 as target genes of miR-150-5p and -423-5p respectively. Using real-time PCR, we observed higher mRNA levels of both MYB and EEF2 in NR versus R patients. Considering that these genes are involved in cell proliferation, our data indicate a relationship between dysregulated expression of miR-150-5p/MYB and miR-423-5p/EEF2 pairs, increased T and B cell proliferation and unresponsiveness to conventional ISTs in MG patients. As for lncRNAs, we focused on MALAT-1, considering its capacity to interact with miR-423-5p and regulate cell proliferation. Interestingly, MALAT-1 was overexpressed in PBMCs of NR compared to R patients, further correlating cell proliferation-associated ncRNAs to patient-specific response to ISTs. In conclusion, our study showed the potential of miR-150-5p, miR-423-5p and MALAT-1 as valid biomarkers of drug response in MG patients, allowing early identification of NR patients that would thus benefit from immediate treatment with biological drugs. These findings pave the way for the adoption of biomarker-guided PM approaches in MG, with the goal of improving therapeutic success, cost/effectiveness ratio of treatments and ultimately MG patients’ quality of life.