IPSC-DERIVED SYMPATHETIC NEURONS AS A MODEL FOR STUDYING THE ROLE OF PHOX2B IN CONGENITAL CENTRAL HYPOVENTILATION SYNDROME

Congenital Central Hypoventilation Syndrome (CCHS) is a rare, life-threatening genetic neurodevelopmental disorder characterized by autonomic nervous system (ANS) dysfunctions. 90% of CCHS cases are due to heterozygous polyalanine expansion mutations in the 20 alanine stretch of PHOX2B gene, a master transcription factor of the ANS. Multiple 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. No pharmacological treatment is available. PHOX2B-AS1, the natural antisense lncRNA transcribed in the opposite direction and partially complementary to PHOX2B gene, acts by favouring PHOX2B translation, thus a modulation of its expression could represent a new therapeutic strategy aimed at reducing the expression of mutant PHOX2B protein. Due to the unavailability of suitable models, we generated autonomic sympathetic neurons (SNs) using induced pluripotent stem cells (iPSCs) derived from CCHS patients carrying different PHOX2B expansions, to investigate possible developmental defects. The differentiation process showed morphological abnormalities and alterations in the expression of multiple progenitor and neuronal markers. Furthermore, our preliminary data suggest a defect in migration and proliferation of sympathoadrenal progenitors, as well as in PHOX2B-mediated regulation of different ion channels in the patient lines. The generation of mutant iPSC-SNs also revealed a defective temporal expression of PHOX2B-AS1, along with the appearance of mutant lines-specific splicing variants in the mutant lines. Overall, our data shed new light on CCHS pathogenesis, paving the way for the identification of possible therapeutic targets.