Evaluation of Morpholino oligomers therapeutic efficacy in C9orf72 ALS iPSC-derived lines

GGGGCC repeat expansions in C9ORF72 gene are the most common identified genetic cause of amyotrophic lateral sclerosis (ALS), a fatal disorder characterized by progressive degeneration of motor neurons (MNs). Many possible pathogenic mechanisms have been proposed, including loss of function of the C9Orf72 protein, gain of function from accumulation of RNA foci, and toxicity caused by dipeptide repeats proteins, however processes underlying C9-ALS are still largely unknown. Patient-specific induced pluripotent stem cells (iPSCs) and iPSC-derived MNs can provide fundamental insights to better understand C9-ALS pathogenesis. Antisense oligonucleotides (ASOs) are single-stranded nucleic acids designed to bind complementary mRNA and interfere with specific biological processes. We designed two different ASOs with Morpholino chemistry to target C9ORF72 expansion and C9ORF72 gene promoter. We aimed to characterize the pathological phenotype of the C9-ALS iPSC-derived lines and evaluate the therapeutic effect of ASOs administration on specific pathological markers. We reprogrammed iPSCs from C9-ALS patients and controls and differentiated them in motor neurons (MNs). We investigated the phenotype of the C9-ALS iPSC-derived lines compared to controls, evaluating cells survival, pluripotency and motor neuronal markers and identifying pathological features such as increase of DNA damage, R-loops accumulation, impairment of nucleolar size, reduced axonal elongation and impairment of expression of genes involved in axonal growth as Nfh, Stmn1, Stmn2 and Sept7. We transfected C9-ALS cells with different Morpholinos and we evaluated modification of the pathological markers: Morpholinos treatments could partially rescue the pathological phenotype observed in in vitro models. Our results suggest that patient specific iPSCs and iPSC-derived MNs are a valuable tool to deepen the knowledge of C9ORF72 pathogenic mechanisms, and that Morpholino-mediated approaches represent a promising therapeutic strategy that needs to be further validated.