RNA-based drug discovery for spinal muscular atrophy: a story of small molecules and antisense oligonucleotides.

INTRODUCTION: Spinal Muscular Atrophy (SMA), the second most prevalent autosomal genetic disease affecting infants, is caused by the lack of SMN1, which encodes a neuron functioning vital protein, SMN. Improving exon 7 splicing in the paralogous gene SMN2, also coding for SMN protein, increases protein production efficiency from SMN2 to overcome the genetic deficit in SMN1. Several molecular mechanisms have been investigated to improve SMN2 functional splicing. AREAS COVERED: This manuscript will cover two of the three mechanistically distinct available treatment options for SMA, both targeting the SMN2 splicing mechanism. The first therapeutic, nusinersen (Spinraza®, 2017), is an antisense oligonucleotide (ASO) targeting the splicing inhibitory sequence in the intron downstream of exon 7 from SMN2, thus increasing exon 7 inclusion. The second drug is a small molecule, risdiplam (Evrysdi®, 2021), that enhances the binding of splice factors and also promotes exon 7 inclusion. Both therapies, albeit through different mechanisms, increase full-length SMN protein expression. EXPERT OPINION: Nusinersen and risdiplam have directly helped SMA patients and families, but they also herald a sea change in drug development for genetic diseases. This piece aims to draw parallels between both development histories; this may help chart the course for future targeted agents.

Identification and Optimization of RNA-Splicing Modulators as Huntingtin Protein-Lowering Agents for the Treatment of Huntington's Disease.

Huntington's disease (HD) is caused by an expanded CAG trinucleotide repeat in exon 1 of the huntingtin (HTT) gene. We report the design of a series of HTT pre-mRNA splicing modulators that lower huntingtin (HTT) protein, including the toxic mutant huntingtin (mHTT), by promoting insertion of a pseudoexon containing a premature termination codon at the exon 49-50 junction. The resulting transcript undergoes nonsense-mediated decay, leading to a reduction of HTT mRNA transcripts and protein levels. The starting benzamide core was modified to pyrazine amide and further optimized to give a potent, CNS-penetrant, and orally bioavailable HTT-splicing modulator 27. This compound reduced canonical splicing of the HTT RNA exon 49-50 and demonstrated significant HTT-lowering in both human HD stem cells and mouse BACHD models. Compound 27 is a structurally diverse HTT-splicing modulator that may help understand the mechanism of adverse effects such as peripheral neuropathy associated with branaplam.

Abrogation of complex glycosylation by swainsonine results in strain- and cell-specific inhibition of prion replication.

Neuroblastoma-derived N2a-PK1 cells, fibroblastic LD9 cells, and CNS-derived CAD5 cells can be infected efficiently and persistently by various prion strains, as measured by the standard scrapie cell assay. Swainsonine, an inhibitor of Golgi α-mannosidase II that causes abnormal N-glycosylation, strongly inhibits infection of PK1 cells by RML, 79A and 22F, less so by 139A, and not at all by 22L prions, and it does not diminish propagation of any of these strains in LD9 or CAD5 cells. Misglycosylated PrP(C) formed in the presence of swainsonine is a good substrate for conversion to PrP(Sc), and misglycosylated PrP(Sc) is fully able to trigger infection and seed the protein misfolding cyclic amplification reaction. Distinct subclones of PK1 cells mediate swainsonine inhibition to very different degrees, implicating misglycosylation of one or more host proteins in the inhibitory process. The use of swainsonine and other glycosylation inhibitors described herein enhances the ability of the cell panel assay to differentiate between prion strains. Moreover, as shown elsewhere, the susceptibility of prions to inhibition by swainsonine in PK1 cells is a mutable trait.

Cell expression of a four extra octarepeat mutated PrPC modifies cell structure and cell cycle regulation.

RK13 cell lines generated to express bovine PrP(C) with a four extra octarepeat insertional mutation (Bo-10ORPrP(C)) show partially insoluble PrP(C) and lower rates of cell growth when compared to either the same cells expressing wild type Bo-6ORPrP(C) or the original RK13 cell line. The expression of Bo-10ORPrP(C) in cell cultures was also associated with changes in cell size and reorganization of the actin cytoskeleton. This last process was reversed by Clostridium difficile toxin-B, a specific inhibitor of small GTPase proteins. Further, in clones expressing Bo-10ORPrP(C), increased proportions of cells at cell cycle stage G2/M were observed. Proteasome inhibitors caused a further expansion of G2/M-stage cells that was more marked in cell lines expressing Bo-10ORPrP(C) than those expressing Bo-6ORPrP(C), while this effect was minimal or null in the original RK13 cell line. Hence, the presence of Bo-10ORPrP(C) in RK13 cells promotes cell cycle arrest at G2/M, and the effect is amplified by proteasome inhibition. These findings suggest a role for PrP(C) in cell morphology and cell cycle regulation, and open new avenues for understanding the mechanisms underlying PrP mutation-associated diseases.

Cell-specific susceptibility to prion strains is a property of the intact cell.

Prions consist of PrP (Sc), a misfolded version of the cellular protein PrP (C). They occur in a variety of strains that share the amino acid sequence of PrP but differ in phenotypic properties, such as cell tropism and pathogenicity; strain-ness is attributed to the conformation of PrP (Sc). To gain insight as to how susceptibility of cells to a given prion strain comes about, we compared amplification of RML prions by PMCA, using cell lysates from related, RML-resistant and RML-susceptible cell lines as substrate. We found that both lysates supported amplification of RML PrP (Sc) equally well, despite a 280-fold difference in the susceptibility of the cells from which they were derived. Thus, susceptibility is an attribute of the intact cell.