Morpholino oligomers ameliorates pathological hallmarks in C9orf72 cellular lines and mice

Introduction: Amyotrophic lateral sclerosis (ALS) is a fatal disorder characterized by progressive degeneration of motor neurons (MNs). GGGGCC repeat expansions in C9ORF72 gene are the most common identified genetic cause, and even if their pathogenic processes are still unknown, many possible mechanisms have been proposed, including loss of function of the C9Orf72 protein, gain of function from accumulation of RNA foci and sequestration of RNA binding proteins (RBPs), and toxicity caused by dipeptide repeats proteins (DPRs) produced by repeat-associated non-ATG (RAN) translation. One promising and reliable method to understand C9-ALS pathogenesis is represented by patient-specific induced pluripotent stem cells (iPSC)-derived lines and iPSC-derived MNs. Objectives: Our therapeutic approaches include the use of antisense oligonucleotides (ASOs) designed to bind complementary mRNA and interfere with specific biological processes. In our laboratory, two different ASOs with Morpholino chemistry have been designed: against the C9ORF72 expansion motif and against the whole C9ORF72 gene; our aim is to characterize the pathological phenotype of the C9-ALS iPSC-derived lines and evaluate the therapeutic effect of ASOs administration on specific pathological markers. Methods and Results: We reprogrammed iPSCs from C9-ALS patients and controls and differentiated them into MNs using a 14-days protocol. We investigated the phenotype of the C9-ALS lines compared to controls, evaluating cells survival, pluripotency and motor neuronal markers, STMN2 expression, defects in axonal elongation and nucleolar disfunctions. Next step was transfecting ALS-MNs with different Morpholinos and evaluating modification of the previously mentioned pathological markers. Interestingly, we identified in C9-ALS iPSC-derived lines pathological features such as accumulation DNA damage, minor axonal elongation, with decreased levels of Nfh, Stmn1 and Sept7 genes and Nefl and Nefh expression. After Morpholino treatments, we observed that ASO therapy could partially rescue the pathological phenotype. Then we translated the treatment with MoB to C9orf72 mice, and analysed brains in qPCR before and after the treatment for Nefh and Nefl expression: both markers seem to be restored by MoB. Conclusions: Our results suggest that patient specific iPSCs and iPSC-derived MNs, together with animal models, are a valuable tool to deepen the knowledge of C9ORF72 pathogenic mechanisms, and that Morpholino-mediated approaches represent a very promising therapeutic strategy that needs to be further validated.