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Alternative splicing of neuronal genes is controlled in part by the coordinated action of the polypyrimidine tract binding proteins (PTBP1 and PTBP2). While PTBP1 is ubiquitously expressed, PTBP2 is predominantly neuronal, controlling the expression of such targets as DLG4, which encodes PSD95, a protein important in synaptic function whose deficiency causes neurodevelopmental disorders. Here, we fully define the PTBP2 footprint in the human transcriptome using both human brain tissue and neurons derived from human induced pluripotent stem cells (iPSC-neurons). We identify direct PTBP2 binding sites and define PTBP2-dependent alternative splicing events, finding novel targets such as STXBP1 and SYNGAP1, which are synaptic genes also associated with neurodevelopmental disorders. The resultant PTBP2 binding and splicing maps were used to test if PTBP2 binding could be manipulated to increase gene expression in PTBP-targeted genes that cause disease when haploinsufficient. We find that PTBP2 binding to SYNGAP1 mRNA promotes alternative splicing and non-sense mediated decay. Antisense oligonucleotides that disrupt PTBP binding sites on SYNGAP1 redirect splicing and increase gene and protein expression. Collectively, our data provide a comprehensive view of PTBP2-dependent alternative splicing in human neurons and human cerebral cortex, guiding the development of novel therapeutic tools that may benefit a range of neurodevelopmental disorders.

To date, gene therapies for human application rely on engineered promoters that cannot be finely controlled. Here, we report a universal switch element that allows precise control for gene silencing or gene replacement after exposure to a small molecule. Importantly, these small molecule inducers are in human use, are orally bioavailable when given to animals or humans, and can reach both peripheral tissues and the brain. Moreover, the switch system (Xon) does not require the co-expression of any regulatory proteins. Using Xon, translation of desired elements for gene knockdown or gene replacement occurs after a single oral dose, and expression levels can be controlled by drug dose or in waves with repeat drug intake. This universal switch can provide temporal control of gene editing machinery and gene addition cassettes that can be adapted to cell biology applications and animal studies. Additionally, due to the oral bioavailability and safety of the drugs employed, the Xon switch provides an unprecedented opportunity to refine gene therapies for more appropriate human application.