2D and 3D iPSC-derived neuronal models for the study of the role of lncRNA PHOX2B-AS1 in the pathogenesis of Congenital Central Hypoventilation Syndrome

Paired-like homeobox 2B (PHOX2B) gene encodes for a master transcription factor essential for the development of the autonomic nervous system (ANS) and the central structures that participate in breathing control. Congenital Central Hypoventilation Syndrome (CCHS), a rare genetic neurodevelopmental disorder that affects the ANS and the central chemosensitivity, is caused by heterozygous mutations in PHOX2B. In vivo and in vitro studies suggest that a loss of function mechanism, combined with a dominant-negative effect and/or toxic gain of function of the mutated proteins, is responsible for the entire disease spectrum. Animal models with PHOX2B knockout are not viable, and the available conditional models only partially recapitulate the CCHS phenotype. We therefore generated induced pluripotent stem cells (iPSCs) from the fibroblasts of patients carrying different PHOX2B mutations. To investigate possible developmental defects at both central and peripheral level, we generated control and CCHS patient-derived 2D autonomic neurons and 3D cerebral organoids with cytoarchitectures resembling central chemoreceptors. PHOX2B-AS1, a natural antisense lncRNA mapping in the PHOX2B locus, was recently identified. A mainly cytoplasmic localization of both PHOX2B-AS1 and PHOX2B was observed by RNAscope experiments. Western blot analysis showed that the halving of PHOX2B-AS1, using a pool of three gapmers, does not affect the PHOX2B mRNA level, however there is a protein level reduction, suggesting that PHOX2B-AS1 favors PHOX2B translation. Moreover, an ectopic expression of PHOX2B and PHOX2B-AS1 was observed in some patient-derived iPSC lines, despite the expression of the pluripotency markers, suggesting that their dysregulation at earlier developmental stages may be involved in CCHS pathogenesis. These observations pave the way for a new therapeutic strategy based on the modulation of PHOX2B-AS1 expression and aimed at reducing the expression of mutant PHOX2B proteins.