CRISPR/Cas9-MEDIATED PHOX2B FUNCTIONAL KNOCK-OUT IN IMR32 NEUROBLASTOMA CELLS IMPAIRS NEURONAL EXCITABILITY THROUGH DYSREGULATION OF ION CHANNELS GENES

Introduction: PHOX2B is a master transcriptional regulator of autonomic nervous system development whose mutations cause neurocristopathies including neuroblastoma and congenital central hypoventilation syndrome (CCHS). However, the identification and functional relevance of PHOX2B target genes remain incompletely defined, partly due to the lack of robust cellular models. Methods: Through CRISPR/Cas9-engineering we established a PHOX2B knockout IMR32 neuroblastoma model, and combined transcriptomic, chromatin-binding, electrophysiological, and live-cell imaging analysis to investigate PHOX2B-dependent pathways. We then assessed the developmental relevance of the obtained results in iPSC‑derived sympathetic neurons from CCHS patients carrying different PHOX2B mutations. Results: PHOX2B-knockout (PHOX2B-KO) cells exhibited morphological and molecular features indicative of enhanced neuronal maturation. RNA-seq and ChIP-seq analyses highlighted an unexpected direct role for PHOX2B in regulating the expression of ion channels, including KCNQ5, SCN3A and RYR2, primarily through transcriptional repression. Functionally, PHOX2B KO cells displayed significant alterations in intrinsic electrical properties, including depolarized resting membrane potential, reduced action potential amplitude, attenuated Ca2+ responses and profoundly altered K+ efflux dynamics. Functional rescue experiments confirmed that PHOX2B re-expression restores both transcriptional and electrophysiological phenotypes, thereby establishing a causal relationship between PHOX2B expression and the maintenance of intrinsic excitability homeostasis. Extending these findings to development, sympathetic neurons differentiated from CCHS patient-derived iPSCs displayed dysregulated maturation, transcriptional network rewiring, and altered expression of the same ion channel genes, supporting their relevance to disease. Discussion: Together, our data establish the PHOX2B knockout neuroblastoma model as a valuable platform for functional genomics, enabling systematic analysis of PHOX2B targets and highlightening its critical role in coordinating transcriptional programs that shape neuronal excitability and maturation.