Investigating the molecular basis of microRNA as biomarker and disease modifier

Investigating the molecular basis of microRNA as biomarker and disease modifier in spinal muscular atrophy

Background:
Spinal Muscular Atrophy (SMA) is the second most common genetic condition and the leading genetic cause of infant mortality. It is caused by mutations in the survival of motor neuron gene 1 (SMN1) and characterized by progressive muscle atrophy and paralysis resulted from motor neuron degeneration in the spinal cord. A breakthrough in the therapy for SMA was recently achieved with various SMN-restoring approaches developed by our group and others.1-4 Antisense oligonucleotide nusinersen (Spinraza) is the first drug approved for treatment in SMA patients. Alongside drug development, there are increasing demands on studies of putative biomarkers for treatment, as patients do not respond conformably to nusinersen treatment.

Specific microRNA (miRNA) signatures in bio-fluids can provide useful non-invasive tools to monitor disease progression and predict drug response. My group has previously reported the potential of serum miRNAs as non-invasive and informative biomarkers of disease progression and response to therapeutic antisense treatment in SMA transgenic mice.5 More recently, we have conducted a deeper miRNA discovery profiling in serum and cerebral spinal fluid (CSF) from SMA patients, and have identified a list of miRNAs with great promise as biomarker to monitor patients’ response to nusinersen. Moreover, some miRNAs show close association between the baseline levels and the response to drug treatment, indicating their potential as predictive biomarker for nusinersen.

Aims/Objectives:
This PhD project aims to:
1) explore the underlying molecular mechanism of the lead microRNAs in SMA disease pathogenesis, and to unveil their biological significance as biomarkers or disease modifier in nusinersen-treated patients;
2) to investigate the therapeutic potential of miRNAs and their synergistic effect with the existing SMN-restoring treatment, in order to further improve the current therapeutic regimen. 

Methods:
1. To identify miRNA targets: two lead miRNAs identified as biomarkers in CSF and blood from nusinersen-treated patients will be selected for further studies. We will use both IP-Ago and in silico methods to explore the target genes of these two miRNAs. Human iPSC-derived neurons and myoblasts will be used as the cellular models. The predicted target genes will also be validated at protein levels in cells.
2. To validate the target genes at protein levels in: 1) patients’ CSF and blood samples in response to nusinersen treatment, measured by Simoa assay, ELISA or western blotting; 2) in organs from SMA transgenic mice treated with SMN-restoring antisense drug.
3. To evaluate microRNAs as novel therapeutic target in SMA: synthetic miR-mimics and scAAV-miR will be tested in SMA mice on survival and motor functions. This will also be investigated in combination with SMN-restoring antisense therapy.

Timeline:
Month 1-12: Identification of the target genes of the two lead miRNAs in vitro.
Month 13-18: Validation of the target proteins in SMA patients and transgenic mice.
Month 19-30: Validation of the therapeutic potential of the two miRNAs in SMA mice.
Month 31-36: Data analysis, manuscript submission and thesis write-up.

References:
1.   Sardone V et al. Molecules. 2017 Apr 5;22(4) pii: E563.
2.   Zhou H et al. Hum Gene Ther. 2013;24(3):331-42.
3.   Zhou H et al. Hum Mol Genet. 2015;24(22):6265-77.
4.   Zhou H et al. J Cachexia Sarcopenia Muscle. 2020. doi:10.1002/jcsm.12542.
5.   Catapano F et al. Mol. Ther. Nucleic Acids. 2016;5:e331.