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Catasus, N.

Publications and source records attributed to Catasus, N..

3 recordsLinked to original sources

NF2 lacking exon 11 isoform induced by antisense gene therapy is able to recover partially merlin deficiency on NF2-related Schwannomatosis iPSC-derived spheroid model

NF2-related Schwannomatosis (NF2-SWN) is an inherited autosomal dominant disorder resulting from loss-of-function mutations in the NF2 gene, for which no effective treatment is currently available. Furthermore, truncating variants in NF2 are associated with the severest phenotype compared to in-frame or missense variants. Previously, a shorter NF2 isoform with exon 11 skipped (merlin_e-11), induced through antisense morpholino oligomers (PMOs), was able to partially rescue the deleterious effect of nonsense variants located at that exon in patients primary fibroblast. To test the potential of this approach into Schwann cells, those responsible of NF2-SWN tumours, we developed an iPSC-based model carrying heterozygous and homozygous truncating variants on exon 11 of the NF2 gene and differentiated them to Schwann cells forming spheroids. After 3 days of treatment, merlin_e-11 expression was induced in NF2-deficient cell lines. Furthermore, key pathways associated with NF2-deficiency in schwannomas, such as PI3K/Akt/mTORC and YAP levels, were recovered without signs of toxicity. These results confirm that the PMO treatment induces effective skipping of exon 11 in Schwann cell spheroids, generating an hypomorphic merlin_e-11 that has the capacity to partially rescue merlin-deficiency in NF2-SWN spheroid cell model and it being a potential treatment of patients harbouring truncating variants located in exon 11.

genetics↗

Merlin-deficient iPSCs show altered pluripotency and constitute a potential in vitro model for NF2-related schwannomas

NF2-related schwannomatosis is an autosomal dominant syndrome that predisposes to the development of benign tumors of the nervous system. Schwannomas, particularly bilateral vestibular schwannomas (VS), are the most characteristic features of the disease. These tumors are caused by the bi-allelic inactivation of the NF2 gene in a cell of the Schwann cell lineage. Our current understanding of the molecular pathogenesis of the NF2 gene, as well as the development of new effective therapies is hampered by the absence of human non-perishable cell-based bearing distinct NF2 pathogenic variants. With this aim, we generated and characterized three isogenic paired induced pluripotent stem cell (iPSC) lines with single or bi-allelic inactivation of NF2 by combining the direct reprogramming of VS cells with the use of CRISPR/Cas9 editing. Our results show a critical function of NF2 for the maintenance of a stable pluripotent state. However, we were able to nudge them towards the Neural Crest-Schwann Cell (NC-SC) axis by applying a 3D Schwann cell differentiation protocol. NF2(+/-) and NF2(-/-) spheroids homogeneously expressed classical markers of the NC-SC lineage. In addition, NF2(-/-) SC-like spheroids showed dysregulation of multiple signaling pathways already described for merlin-deficient SC, and altered in human schwannomas. Therefore, NF2(+/-) and NF2(-/-) SC-like spheroids can represent a bona fide human in vitro cellular model to study the role of NF2 pathogenesis.

cell biology↗

Antisense oligonucleotides targeting exon 11 are able to partially rescue the Neurofibromatosis Type 2 phenotype in vitro

Neurofibromatosis type 2 (NF2) is an autosomal dominant condition caused by loss of function variants in the NF2 gene, which codes for the protein Merlin, and characterized by the development of multiple tumours of the nervous system. The clinical presentation of the disease is variable and related to the type of the inherited germline variant. Here, we tested if PMOs could be used to correct the splice signalling caused by variants at +/-13 within the intron-exon boundary region. Here we show that the PMOs designed for these variants do not constitute a therapeutic approach. Furthermore, we evaluated the use of phosphorodiamidate morpholino oligomers (PMOs) to reduce the severity of the effects of NF2 truncating variants with the aim of generating milder hypomorphic isoforms in vitro through the induction of the in-frame deletion of the exon-carrying variant. We were able to specifically induce the skipping of exons 4, 8 and 11 maintaining the NF2 gene reading frame at cDNA level. Only the skipping of exon 11 produced a hypomorphic Merlin (Merlin-e11), able to partially rescue the observed phenotype in primary fibroblast cultures from NF2 patients, being encouraging for the treatment of patients harbouring truncating variants located in exon 11.

genetics↗