A research team in Korea created an in vitro, or laboratory, model to screen and validate medicines that aim to treat spinal muscular atrophy (SMA) by promoting protein production by the SMN2 gene.
The system is based on the use of a special cell line as well as stem cells derived from patients, and the scientists’ work found a small-molecule candidate compound called rigosertib that showed promise as a potential treatment using this screening platform.
SMA, a neurodegenerative disorder characterized by the gradual loss of motor neurons — the nerve cells responsible for controlling voluntary muscles — in the spinal cord, leading to muscle weakness, is normally caused by mutations in the SMN1 gene, which encodes for the SMN protein that is essential for motor neuron survival.
However, the SMN2 gene can also produce the SMN protein, and it usually remains unaffected in SMA patients. SMN2 is known to influence the course of the disease, depending on the number of copies present.
But unlike SMN1, SMN2 rarely encodes the full-length protein (only in about 10% of all cases). Instead, it tends to produce shorter, unstable versions of SMN lacking exon 7 through a process called alternative splicing. (An exon is the coding sequence of a gene that provides instructions to make proteins; alternative splicing is the process of creating different proteins from the same gene.)
“Although the antisense oligonucleotide nusinersen [Spinraza, by Biogen] has recently been approved for use by the FDA, there is still much progress to be made and other new and promising approaches to explore,” wrote the researchers, with the University of Science and Technology (UST) in Daejeon. Spinraza increases the ability of the SMN2 gene to produce a full-length SMN protein.
The team created a new system for drug screening based on the splicing status of SMN2, and then validated it using an in vitro model of SMA based on induced pluripotent stem cells (iPSCs) derived from SMA type 1 patients’ and fibroblasts (connective tissue cells) from healthy people serving as controls.
iPSCs are derived from either skin or blood cells that have been programmed back into a stem cell-like state, which allows for the development of an unlimited source of any type of human cell needed for therapeutic purposes.
The SMN2 splicing reporter cell line that the researchers created contains a smaller version of the SMN2 gene that lacks exon 7, fused with the firefly luciferase gene (a light-emitting enzyme from fireflies) to act as a tracer.
“In this assay system, any compound that enhances the inclusion of exon 7 would increase FLuc [firefly luciferase] activity because exon 7-included mRNAs [messenger RNAs] would produce a larger protein fused with FLuc protein,” the researchers wrote. This means that cells with larger SMN proteins would emit higher amounts of green fluorescent light, visible under the microscope or through specialized laboratory assays.
Using this approach, scientists identified a small molecule called rigosertib, an inhibitor of the Polo-like kinase (PLK, an enzyme involved in cell-cycle regulation), that boosted the production of the full-length SMN protein by SMN2.
To assess its therapeutic potential, investigators tested rigosertib in motor neuron progenitors obtained from SMA patient-derived iPSCs (SMA iPSC-pMNs).
Results showed that rigosertib successfully corrected SMN2 splicing and significantly increased the levels of the full-length SMN protein, improving outcomes linked to SMA, including motor neuron survival and neurite (nerve segments) outgrowth.
“Our combined screening platform representing a pMN [motor neuron progenitors] model of human SMA provides an efficient and reliable drug screening system and is a promising resource for drug evaluation and the exploration of drug modes of action,” the researchers wrote.
“[We believe] our findings may contribute to the development of effective therapies for SMA and other neuromuscular diseases,” they added.
Rigosertib is in clinical trials, with the compound largely being tested as a treatment for various cancers.