Muscular miRNAs in SMA: use as biomarkers and therapeutics
Prof. Francesco Danilo Tiziano, Università Cattolica del Sacro Cuore; Dr Emanuela Abiusi, Università Cattolica del Sacro Cuore; Prof. Lucia Di Marcotullio, Università degli Studi di Roma "La Sapienza” ; Dr Paola Infante, Italian Institute of Technology
Università Cattolica del Sacro Cuore
Spinal Muscular Atrophy (SMA) is a relatively rare genetic disorder, characterized by muscle atrophy and degeneration of the spinal motor neurons. Despite a wide clinical variability ranging from very severe to very mild forms (type I-IV), all patients share the same genetic defect, the homozygous loss of the SMN1 gene. Unique to humans a hypomorphic allele of SMN1 (SMN2) is present in the same region. This copy gene produces insufficient levels of the SMN protein. The number of SMN2 genes is variable in patients and is inversely related to the clinical severity. Different therapeutic approaches to SMA are in the pipeline or have already been registered: most are based on SMN2 gene activity enhancement or gene therapy. The identification and validation of biomarkers is a critical issue in several conditions, including SMA. These tools are necessary for the objective evaluation of patients’ function, also in response to different therapeutics. MicroRNAs (miRs) can be stably detected in serum and are considered a promising class of disease biomarkers in several conditions.
We have determined by NGS the miRNome of muscle biopsies of 7 patients and 7 controls. About 100 miRs were differently expressed in muscle biopsies of patients. We have subsequently evaluated which muscular miRNAs are dosable and differently regulated in serum of patients, in order to evaluate their potential applicability as biomarkers. We have developed proprietary absolute real time PCR assays for the dosage of miRs in serum. The majority of miRs were not detectable and were not further studied. Three miRs were particularly promising and were tested in a wide cohort of patients and controls (51 vs. 40, respectively), since they were significantly increased in serum samples of patients and partially related to the clinical severity. These miRNAs are likely actively secreted in the blood flow by skeletal muscle, and these may act as a backward signaling system from skeletal muscle to spinal cord. To test this hypothesis, we injected SMA-like mice with 0.5nM of each of the 3 miR antagonists: one of the three antagomiR led to a significant increase in mice survival.
Our findings could be exploited to develop a commercial kit for the quantification of miRNA levels in biofluids, including serum samples but also biopsies or tissues pathogenically involved in a given condition. Due to the pleiotropy of miRNAs, our findings are likely not restricted to SMA only that, even though it is not ultra-rare, is a rare genetic disorder with a limited number of patients. Furthermore, differently from genomic biomarkers, that are tested once lifelong in patients, functional biomarkers, such as miRNAs, are tested repeatedly to monitor the prognosis and/or the therapeutic effects. Finally, if confirmed in a wider number of animal models, the modulation of the miRNAs we have identified might have a therapeutic role in SMA, in conjunction with treatments modulating the SMN2 gene expression, since they might delay or prevent neuron loss by a retrograde mechanism.
Our assay is not commercially available yet. We are validating our previous data in a wider cohort of patients, also undergoing to therapeutic interventions, in order to evaluate the miRs modulation during treatment. In pre-clinical models, at the same time, we are dissecting the molecular mode of action of miR modulation with respect to the survival of affected mice.
A priority Italian patent and a subsequent international PCT patent application have been filed .