The current proposal entails a series of significant conceptual and technological advances. First, the foundation of this proposal is based on Xin Tang’s recent identification of the first collection of KCC2 expression-enhancing compounds (KEECs)¹, which provides the practical means to rigorously test the hypothesis that enhancing the expression of KCC2 (encoded by SLC12A5) is a broadly applicable therapeutic approach to treat autism spectrum disorder (ASD). Tang’s previous work has shown that KCC2 expression is reduced in human neurons differentiated from induced pluripotent stem cells (iPSCs) from patients with Rett syndrome² and in the brains of Mecp2 mutant mice³. He will further investigate whether a reduction in KCC2 gene expression is a common disease feature in a number of genetically defined cellular and mouse models of ASD and to test the therapeutic effectiveness of KEECs to restore KCC2 expression and rescue ASD phenotypes in preclinical models.

Second, this first generation of KEECs was discovered with a drug screening platform based on CRISPR/Cas9 gene-edited human stem cell-derived KCC2 expression reporter neurons. Tang targeted the endogenous KCC2 gene locus in human embryonic stem cells to insert a luciferase reporter gene and differentiated these stem cells into KCC2 reporter neurons, which allows for the convenient and faithful assessment of a drug’s effect on the expression level of the KCC2 gene in its original genomic and cellular context. Through large-scale unbiased drug screening, Tang and his lab discovered and validated a group of KEECs. Treatment of cellular and mouse models of Rett syndrome with the KEEC KW-2449 was shown to rescue neuronal functional deficits and physiological abnormalities¹. Tang now plans to adapt this proven drug discovery strategy to identify compounds that enhance the expression of other high-confidence ASD risk genes (including CHD8, PTEN, SHANK3 and SYNGAP1) for which haploinsufficiency reduces gene expression and causes ASD phenotypes.

A third advancement is a genome-scale CRISPR activation/inhibition screening platform that is currently under development in Tang’s lab. Tang and his team plan to conduct CRISPR screens to systematically identify genes that regulate KCC2 gene expression in neurons in order to help elucidate the molecular pathways that cause KCC2 dysregulation in ASD neurons and support the development of next-generation mechanism-guided KEECs. As a foundation for the screen, they have successfully generated iPSC-derived human neurons carrying puromycin resistance-green fluorescent protein dual reporters knocked into the KCC2 locus to enable the rapid isolation of neurons that have elevated KCC2 expression levels.

Findings from this project are expected to provide strong evidence to support the broad applicability of enhancing KCC2 expression as a therapeutic strategy to restore excitatory/inhibitory (E/I) balance and treat ASD, as well as yielding fundamental insights into molecular mechanisms that regulate KCC2 gene expression. Furthermore, Tang will expand the cutting-edge screening technologies he pioneered to discover drugs that stimulate the expression of other high-confidence ASD risk genes, with the ultimate goal of reversing ASD symptoms caused by gene haploinsufficiency after disease onset. The results and technologies from this project will be valuable to both basic science and translational research in the ASD field.

1. Tang X. et al. Sci. Transl. Med. 11, eaau0164 (2019) PubMed
2. Tang X. et al. Proc. Natl. Acad. Sci. U. S. A. 113, 751–756 (2016) PubMed
3. Banerjee A. et al. Proc. Natl. Acad. Sci. U. S. A. 113, E7287–E7296 (2016) PubMed