A groundbreaking advancement in in vivo genetic editing technology has been unveiled, demonstrating the potential to correct neurological disorders at their genetic roots. Utilizing a state-of-the-art dual adeno-associated virus (AAV) system equipped with a novel split base editor—TeABE—researchers have successfully targeted and restored normal gene function within the mouse brain. This innovative approach offers a promising pathway for treating inherited neurological disorders by enabling precise editing of disease-causing mutations directly within neurons.
The research team employed dual AAV vectors engineered with PHP.eB capsids, known for their remarkable ability to cross the blood-brain barrier and broadly transduce neural tissues. Administered via tail vein injection, these vectors delivered the split TeABE system alongside a neuron-specific green fluorescent protein (GFP) reporter under a human synapsin (hSyn) promoter. Immunofluorescence imaging revealed widespread GFP expression throughout multiple brain regions, confirming efficient and pervasive vector transduction across cortical and hippocampal areas.
To evaluate the efficiency of TeABE assembly and expression within the central nervous system, whole-brain lysates underwent immunoblot analysis using antibodies specific for the Cas9 N-terminus. At higher vector dosages, robust expression of both split fragments was evident, with intein-mediated protein splicing successfully reconstituting full-length Cas9. Impressively, approximately 80% of detected Cas9 signals corresponded to complete reconstitution, an unprecedented efficiency that validated the high-dose administration regimen for subsequent experimental phases.
The core therapeutic objective involved precise correction of a pathogenic mutation in the Chd3 gene, a critical chromatin remodeler implicated in neurodevelopmental disorders. Targeted next-generation sequencing (NGS) on dissected brain regions—including retrosplenial cortex (RSC), hippocampus, anterior cingulate cortex (ACC), olfactory bulb (OB), prefrontal cortex (PFC), and cerebellum (CB)—demonstrated substantive on-target editing efficiency. Specifically, the intended A11 nucleotide site underwent correction at rates ranging from 10 to 15%, while the adjacent bystander site A13 showed minimal editing (~2-3%), underscoring the editor’s high precision and specificity.
In addition to quantifying editing frequencies, allele outcome analysis revealed that over 80% of edited sequences displayed correction exclusively at the therapeutic target without concomitant bystander mutations. This selective editing pattern is critical for minimizing potential off-target consequences and for maximizing therapeutic benefits. Given that these analyses were conducted on bulk brain tissue containing both neurons and glia, and that the editor’s expression was driven by a neuron-specific promoter, the actual editing efficiency within neurons is estimated to be substantially higher.
Addressing concerns over off-target activity, the study employed GUIDE-seq methodology in the murine Neuro-2a neuronal cell line to identify potential off-target loci. Four candidate off-target sites—designated as mouse Ot1 through Ot4—were characterized and subjected to deep sequencing following in vivo delivery of TeABE. Across multiple brain regions in treated mice, off-target editing frequencies remained exceptionally low, consistently below 1%, contrasting sharply with the robust on-target correction at Chd3. These findings reaffirm the exceptional fidelity and safety profile of the TeABE system for in vivo therapeutic applications.
Immunohistochemical analyses further corroborated vector delivery efficiency and base editor expression. High levels of Cas9 protein were detected in key cortical areas (including RSC, parietal association cortex, and primary somatosensory cortex subregions) as well as hippocampal regions critical for cognitive processing. Correlating with Cas9 expression, restoration of CHD3 protein levels was observed, returning to near-normal levels in affected brain regions. Such biochemical and histological evidence establishes a direct link between gene editing and phenotypic correction.
To confirm these observations biochemically, Western blot analyses of whole brain and isolated brain regions showed significant elevation of CHD3 protein in mice receiving TeABE compared to both wild-type controls and animals given a non-targeting control editor. The improved CHD3 expression validates the therapeutic impact of the base editing approach in rescuing pathogenic deficiency caused by the Chd3^hR1025W mutation. These molecular corrections are fundamental to restoring neural function and ameliorating associated behavioral deficits.
The ability to deliver split-base editors systemically and achieve widespread neuronal targeting represents a significant advance over previous methods requiring direct intracranial injections. Through the use of engineered AAV-PHP.eB vectors and a neuron-selective promoter, this systemic approach enables efficient gene correction across broad neural circuits implicated in disease. The intein-mediated protein splicing mechanism permits assembly of the large Cas9-base editor complex from separate vector genomes, overcoming previous limitations in vector packaging capacity.
Notably, the authors emphasize the clinical implications of this technology for treating human neurodevelopmental disorders caused by dominant-negative mutations in chromatin remodeling genes such as CHD3. Precise in vivo genetic correction via minimally invasive systemic delivery could revolutionize therapeutic strategies for a range of heritable brain disorders. Moreover, the demonstrated high on-target editing frequency coupled with minimal off-target risks provides a compelling argument for translational efforts.
This innovative study not only highlights the feasibility of split Cas9-based systems for base editing in the adult brain but also opens avenues for addressing monogenic neurological diseases with complex spatial distributions. By combining viral vector engineering, protein chemistry for split editor reconstitution, and deep sequencing validation, the investigators have set a new gold standard for CNS gene editing therapies.
Future directions might involve optimizing vector dosages, improving neuronal subtype specificity, and expanding the editing toolkit to include prime editors or epigenetic modifiers. The integration of behavioral outcome assessments in treated animals will further elucidate the functional benefits of precise genetic restoration. Collectively, this work represents a transformative step towards safe, effective, and scalable gene therapy paradigms for brain disorders once deemed intractable.
In summary, the advent of highly efficient, neuron-specific, systemically deliverable base editors heralds a new era in neuroscience and therapeutic genome engineering. The successful in vivo reconstitution and targeted correction of pathogenic alleles in mouse brain using the TeABE system exemplify the convergence of molecular innovation and clinical need. As this technology advances towards human application, it promises to redefine treatment horizons for genetic brain diseases worldwide.
Subject of Research: In vivo base editing for correction of genetic mutations in the mouse brain, focusing on CHD3 mutation rescue.
Article Title: In vivo base editing of Chd3 rescues behavioural abnormalities in mice.
Article References:
Yang, K., Li, WK., Geng, YX. et al. In vivo base editing of Chd3 rescues behavioural abnormalities in mice. Nature (2026). https://doi.org/10.1038/s41586-026-10113-6
Image Credits: AI Generated
