SMN Circular RNAs as Potential Biomarkers for the Therapeutic Response to Nusinersen in Spinal Muscular Atrophy Patients

Background/State of the art SMA represents the most frequent genetic cause of death in infancy with an incidence of ~1 in 10,000 live births . It is an autosomal recessive, neurodegenerative disorder caused by homozygous deletion or point mutation in the SMN1 gene, which encodes for SMN protein. Reduced SMN levels cause loss of motor neurons in the spinal cord and lead to progressive muscle weakness and atrophy in SMA patients. Notably, humans retain the almost identical SMN2 gene, as the complete absence of any form of SMN is embryonically lethal. Nevertheless, although SMN2 encodes a virtually identical protein, its expression levels are not sufficient to fully restore SMN activity because a translationally silent C-to-T transition at position +6 in exon 7 determines skipping of this exon from the mature mRNA. The resulting SMN7 protein displays a shorter C-terminal region and is highly unstable. At the moment Nusinersen is the only FDA-approved drug for treatment of SMA. It is an antisense oligonucleotide (ASO) that corrects SMN2 exon 7 splicing by masking a splicing silencer (ISS-N1) in intron 7 . Although intrathecal administration of Nusinersen ameliorates SMA phenotypes in patients, leading to substantial improvements in motor function, not all SMA patients respond equally well to this therapy. Thus, identification and development of combined and personalized therapies that take into account clinical variability observed between patients of the same genotype is necessary to improve clinical management of SMA. Due to their biochemical properties (stability, accumulation during aging, abundant secretion in body fluids) circular RNAs are now emerging as excellent and promising biomarkers for diagnosis and prognosis of numerous pathologies, such as cancer. Thousands of circRNAs have been discovered in eukaryotic cells and their expression is often regulated in a cell-type and stage-specific manner. Although the majority of circRNAs still lack functional annotations, recent observations revealed that they play some potentially important roles in gene regulation. Circular RNAs (circRNAs) originate from a back-splicing process of the pre-mRNA (i.e., the covalent joining of a downstream splice donor site with an upstream splice acceptor site from the same gene). Since canonical splicing and back-splicing utilize the same pre-mRNA and are both operated by the spliceosome, they likely compete with each other. The main mechanism favoring pre-mRNA circularization is the presence of repetitive sequences in inverted orientation, and in particular inverted Alu repeats.

Alu repeats belong to the primate-specific Short Interspersed Elements (SINE) family of retrotransposons. They are ~300 nucleotides long and account for up to 11% of the annotated human genome. Their presence in coding genes can modulate gene expression at multiple layers, including transcription, splicing, export and translation, thus profoundly impacting on their "host" gene regulation. SMN rank among the top human genes for Alu density, many of which are present in inverted orientation. Strikingly, Alus drive extensive and alternative circularization of the SMN pre-mRNA, thus negatively impacting on the protein-coding potential of the locus. Thus, based on the preliminary results, the propose will be to investigate whether cell-free SMN circRNAs circulating in body fluids of SMA patients represent biomarkers exhibiting diagnostic and prognostic power to predict the clinical response to Nusinersen therapy.

Hypothesis and significance:

The studies proposed in this project aim at investigating whether SMN circRNAs represent valuable biomarkers to predict the efficacy of Nusinersen in SMA patients that exhibit different degrees of disease severity. In particular, our work may help to better stratify the numerous SMA patients which do not respond as expected to treatment and/or for whom the SMN2 gene copy number does not correlate with phenotypic severity. Since extensive production of SMN circular transcripts, instead of the linear transcripts, in some patients could reduce the efficacy of Nusinersen, which selectively acts on the linear transcript, our study may also highlight the need for combined therapy to also limit pre- mRNA circularization in such patients. In the near future, the development of "personalized" therapies could circumvent such "individual" differences in the regulation of SMN expression, thus improving the clinical response of SMA patients to Nusinersen.

Preliminary data: