Behavioral Improvement Through In Vivo Base Editing in a Mouse Model of Snijders Blok-Campeau Syndrome

Behavioral Improvement Through In Vivo Base Editing in a Mouse Model of Snijders Blok-Campeau Syndrome | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Biological Sciences - Article Behavioral Improvement Through In Vivo Base Editing in a Mouse Model of Snijders Blok-Campeau Syndrome Kan Yang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5690634/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Feb, 2026 Read the published version in Nature → Version 1 posted You are reading this latest preprint version Abstract Snijders Blok-Campeau Syndrome (SNIBCPS) is a rare neurodevelopmental disorder caused by mutations in the CHD3 gene. Here, we report a de novo single-nucleotide variant (c.C3073T, p.R1025W) in CHD3 identified in a child with SNIBCPS, which leads to accelerated degradation of the CHD3 protein. Using a Chd3 R1025W/+ knock-in mouse model, we observed impaired vocalization, cognition, and autism-like behaviors. To address these deficits, we developed an improved TadA-embedded adenine base editor (TeABE) and delivered into the mouse brain via Adeno-associated virus (AAV). Base editing in vivo significantly restored CHD3 protein levels in the mouse brain and ameliorated various behavioral abnormalities. Furthermore, we validated the AAV-mediated delivery efficacy of TeABE in nonhuman primate, highlighting its translational potential. These findings establish in vivo base editing as a promising therapeutic strategy for SNIBCPS and pave the way for clinical applications targeting brain disorders. Health sciences/Health care/Therapeutics/Gene therapy/Targeted gene repair Health sciences/Diseases/Neurological disorders/Neurodevelopmental disorders/Autism spectrum disorders Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Snijders Blok-Campeau Syndrome (SNIBCPS, OMIM #618205) is a rare autosomal dominant genetic disorder caused by pathogenic mutations in the CHD3 ( Chromodomain Helicase DNA-binding Protein 3 ) gene. First described by Lot Snijders Blok in 2018 1 , over a hundred cases have since been reported 2 . The primary clinical features of SNIBCPS include global developmental delay, speech delay, intellectual disability, hypotonia, abnormal facial features, and structural brain anomalies, frequently accompanied by autism spectrum disorders (ASD) 1-6 . While most cases are caused by single-nucleotide variations (SNVs), one instance involving a complete deletion and duplication of the CHD3 gene has been documented 5 . Despite these findings, the pathogenesis of SNIBCPS remains poorly understood. The CHD protein family is critical for chromatin remodeling, relying on ATP hydrolysis to regulate chromatin structure and gene transcription 7,8 . Members of this family are implicated in various neurodevelopmental disorders, including CHD2 in epilepsy 9 , CHD7 in CHARGE syndrome 10 , and CHD8 in ASD 11 . CHD3, along with CHD4 and CHD5, forms a core component of the nucleosome remodeling and deacetylase (NuRD) complex, which modulates chromatin structure and DNA accessibility, playing vital roles in cell cycle regulation and embryonic stem cell differentiation 12-17 . CHD3 is essential for cerebral cortex development, as its deficiency results in defects in cortical neuron differentiation and migration 18 . Furthermore, CHD3 mutations have been linked to childhood apraxia of speech 19,20 . Most pathogenic SNVs in CHD3 occur within the helicase ATP-binding and C-terminal domains, which are critical for its function. While mutations in the ATP-binding domain may disrupt ATP hydrolysis and chromatin remodeling, this mechanism does not fully explain the diverse effects of all known SNVs associated with SNIBCPS 1 . The advent of CRISPR-Cas9 technology has revolutionized gene editing 21,22 . However, the potential for DNA double-strand breaks to induce genomic damage poses limitations. Base editing, an optimized approach, allows precise base transformations without cutting DNA 23-25 . Cytosine base editors (CBEs) and adenine base editors (ABEs) can convert C•G to T•A and A•T to G•C, respectively 24,25 . This technology has shown promise in animal models of various diseases 26,27 . In humans, gene therapy has achieved remarkable success in treating autosomal recessive deafness 9 and inherited retinal dystrophies 28,29 . Additionally, preclinical studies have demonstrated the efficacy of gene therapy in addressing neurological disorders such as Angelman syndrome, spinal muscular atrophy, and ASD in mouse models 30-32 . Here, we report a de novo single-nucleotide variant (SNV) in the CHD3 gene (c.C3073T, NM_001005271.3; p.R1025W, NP_001005271.2) associated with Snijders Blok-Campeau Syndrome (SNIBCPS) in a child. The R1025W mutation results in accelerated degradation of the CHD3 protein. To investigate the underlying mechanisms, we generated a Chd3 R1025W/+ knock-in mouse model, which exhibited deficits in vocalization, cognition, and autism-like behaviors. To correct the genetic mutation, we developed a TadA-embedded adenine base editor (TeABE) to edit the mutated A·T base pair in Chd3 R1025W/+ in the mouse brain. Using a blood-brain-barrier-crossing AAV viral delivery system, TeABE was administered via intravenous injection. Remarkably, this treatment significantly restored CHD3 protein levels in the brain and ameliorated behavioral abnormalities resembling those observed in SNIBCPS patients. Moreover, we demonstrated the transduction efficacy of TeABE in the brain of nonhuman primate, validating its clinical applicability. These findings establish in vivo base editing as a viable approach to mitigate behavioral abnormalities in SNIBCPS and highlight the potential of TeABE for clinical therapeutic applications. Results A de novo SNV in the CHD3 gene associated with SNIBCPS In recent works, encompassing over one hundred persons with SNIBCPS, researchers identified a series of de novo and inherited variants in CHD3 33 . A schematic illustration of several locations of genetic mutations in the CHD3 protein is shown (Fig.1a). In whole-exome sequencing using the genome DNA of one child with SNIBCPS and unaffected parents obtained at the Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, we identified a de novo SNV in the CHD3 gene (c.C3073T, NM_001005271.3; p.R1025W, NP_001005271.2), validated using Sanger sequencing (Fig.1b). The amino acid change (R1025W) caused by the de novo variant is located between the Helicase ATP-binding domain and the Helicase C-terminal domain of the CHD3 protein (Fig.1a). This variant ( CHD3 , chromosome 17:7803967) is absent in East Asian populations in the gnomAD database (http://gnomad.broadinstitute.org), suggesting that it is a rare variant. We obtained the mRNA expression profile of CHD3 from the BrainSpan (Atlas of Developing Human Brain, http://www.brainspan.org/rnaseq/), covering from embryonic development to adulthood. We found that CHD3 is highly expressed in the striatum (STR), anterior cingulate (ACC), hippocampus (HIP), cerebellar cortex (CBC), medial prefrontal cortex (mPFC) and primary motor cortex (M1C) (Extended Data Fig.1a). The protein level of CHD3 in the mouse brain gradually decline from embryonic development to adulthood (Extended Data Fig.1b). To determine whether the R1025W mutation impacts the normal function of the CHD3 protein, we examined the expression level of FLAG-tagged human wild-type CHD3-WT and CHD3-R1025W mutant protein in mouse cortical neurons and HEK293 cells. Surprisingly, the protein level of human CHD3-R1025W was strikingly lower than that of human CHD3-WT (Fig.1c and Extended Data Fig.1c). However, the mRNA levels of transfected human WT and R1025W CHD3 remained the same (Fig.1c and Extended Data Fig.1c and d), indicating that the R1025W mutation affects the level of CHD3 protein. To further investigate the importance of R1025 for CHD3 protein stability, we determined the impact of all aromatic amino acids, namely tryptophan (Trp, W), phenylalanine (Phe, F) and tyrosine (Tyr, Y) (Fig.1d). The Alphafold3-predicted structure revealed that the R1025W, R1025F and R1025Y mutants exhibit more compact conformations with a reduced spatial distance between the key Helicase ATP-binding domain and Helicase C-terminal domain compared to the WT CHD3 (Extended Data Fig.1e). We found that R1025W, R1025F and R1025Y all led to decreased levels of CHD3 protein (Extended Data Fig.1f). Similarly, we found substitution of aromatic amino acids in R1025 showed drastic decrease of immunofluorescent intensity of CHD3 protein in the nucleus of HEK293 cells (Fig.1e and f), indicating that R1025 is critical for CHD3 protein stability, while having no influence in mRNA level (Extended Data Fig.1f). After cycloheximide (CHX) treatment, an inhibitor of protein translation, the levels of CHD3-R1025W protein decreased faster than CHD3-WT, suggesting that the R1025W mutation accelerated CHD3 protein degradation (Fig.1g). Treatment with the proteasome inhibitor bortezomib (BTZ) restored the levels of CHD3-R1025W protein, indicating that degradation of CHD3-R1025W protein was mediated by a ubiquitin-dependent pathway (Fig.1h). CHD3-R1025W leads to abnormal dendritic morphology of mouse cortical neurons To further explore the influence of CHD3-R1025W on neuronal morphology, we constructed short hairpin RNAs (shRNAs) specifically targeting the mouse Chd3 gene (Extended Data Fig.2a). The knockdown efficiency of three designed shRNAs was tested by measuring endogenous Chd3 mRNA levels in mouse cortical neurons (Extended Data Fig.2b). The expression of endogenous CHD3 protein was effectively reduced by any of the shRNAs (Extended Data Fig.1c, d). By applying cultured mouse cortical neuron, we found that knockdown of CHD3 led to decreased dendritic length and branch numbers at both 3 and 7 days in vitro (DIV), while having no significant impact on the axonal length and branch numbers (Extended Data Fig.2e-k). These abnormalities were restored by co-transfection of a shRNA-resistant CHD3-WT construct but not CHD3-R1025W (Extended Data Fig.2e-k). These results indicate that CHD3 is critical for neuronal development and that the R1025W mutation hampers dendritic morphology. Chd3 R1025W/+ mice exhibited decreased CHD3 level and abnormal behaviors To investigate the role of CHD3-R1025W in the pathogenesis of SNIBCPS, we constructed Chd3 R1025W/+ knock-in mice with human allele substitutions utilizing CRISPR/Cas9-mediated gene targeting (Fig.2a and Extended Data Fig.3a). The CHD3 protein levels of Chd3 R1025W/+ mice were largely decreased in a variety of brain regions compared to WT mice (Fig.2b and Extended Data Fig.3b). As CHD3 is widely expressed in the cortex and hippocampus, immunohistochemical staining was performed to examine the expression of CHD3 in the brain of Chd3 R1025W/+ mice. We found that the fluorescence intensity of CHD3 signals in the Chd3 R1025W/+ micewas reduced in retrosplenial cortex (RSC), parietal association cortex (PtA), primary somatosensory cortex trunk area (S1Tr), primary somatosensory cortex barrel field area (S1BF), CA1, CA2, CA3 and dentate gyrus (DG) compared to WT mice (Extended Data Fig.3c). As nearly 90% of individuals with SNIBCPS present with speech disorders, we conducted infant ultrasonic vocalization (USV) tests on Chd3 R1025W/+ newborn pups following maternal separation. Consistent with other mouse models of speech disorders, Chd3 R1025W/+ pups vocalized less frequently and for shorter durations compared to their WT littermates (Fig. 2c). Additionally, these pups produced fewer U-shaped inverted and complex vocalizations, indicative of a dysphonia-like disorder (Fig. 2c). Given that over 70% of individuals with SNIBCPS experience intellectual disabilities, we assessed whether the Chd3-R1025W mutation impaired cognitive functions in mice. In the novel object recognition test, Chd3 R1025W/+ mutant mice spent significantly less time exploring novel objects and exhibited a reduced recognition preference index, highlighting impaired cognitive recognition abilities (Fig. 2d). Since over 30% of individuals with SNIBCPS exhibit traits associated with autism spectrum disorder (ASD), we further examined autistic-like behaviors in Chd3 R1025W/+ mutant mice, with a focus on social interactions and repetitive stereotypical behaviors. In the three-chamber test, mutant mice showed a significant reduction in social novelty preference, indicating impaired social behaviors. Additionally, marble-burying and self-grooming tests revealed markedly increased repetitive stereotyped behaviors compared to their WT littermates (Fig. 2e, f). Collectively, Chd3 R1025W/+ mice recapitulated core behavioral abnormalities observed in SNIBCPS, including dysphonia, intellectual disability, and autistic-like traits. Design of the TeABE To screen various TeABE-sgRNA combinations and identify the most efficient tool for correcting Chd3-R1025W , we established a human Chd3 R1025W/R1025W cell line using AeCBE-mediated base editing 32 . Initially, we designed two candidate sgRNAs (sgRNA-C10, sgRNA-C11) (Fig. 3a) and two AeCBEs (Fig. 3b), using cytidine deaminases APOBEC3A(Y130F) and LpCDA1L1_1 34,35 respectively. These combinations were transfected into HEK293 cells, followed by selection of positive monoclonal colonies using puromycin resistance (Fig. 3c). The successful construction of the point mutation cell line was confirmed via Sanger sequencing (Fig. 3d), and a CHD3 R1025W/R1025W homozygous cell line was identified (Fig. 3e) that showed no detectable bystander editing effects. Subsequently, we designed multiple sgRNAs (sgRNA-A4 to sgRNA-A15, Fig. 3f) and developed several TeABEs by inserting TadA constructs—­comprising a loss-of-function TadA as the scaffold and TadA-F148A as the functional unit—at various internal positions within spCas9 36 (Fig. 3g). These sgRNA and ABE combinations were transfected into the Chd3 R1025W/R1025W cell line, and double-positive cells were sorted using FACS 72 hours post-transfection (Fig. 3h). Among the tested combinations, TeABE-1249 (TadA inserted after the 1249 th amino acid of Cas9) paired with sgRNA-A11 exhibited the highest on-target base editing efficiency at the A11 site (Fig. 3i). However, this combination also showed over 30% editing efficiency at a bystander site (A13), raising concerns about potential side effects of base editing (Fig. 3i). To minimize bystander editing, we systematically evaluated the editing efficiency at A11 and A13 across a series of TeABE variants (Fig. 3j). Notably, TeABE-1248 (Δ1249-1263) achieved the highest efficacy at A11 while significantly reducing editing at A13 (Fig. 3j). This improvement is likely attributed to the removal of flexible sequences around the 1249 insertion site, which otherwise increase TadA access to mismatched off-target sites. To assess the off-target potential of TeABE variants, we performed Guide-seq analysis and identified three potential off-target sites (Fig. 3k). Further analysis revealed that TeABE-1248 (Δ1249-1263) greatly minimized off-target editing, particularly at OT3 (Fig. 3l). Based on these findings, we selected TeABE-1248 (Δ1249-1263) as the optimized tool for subsequent in vivo genetic correction experiments in the Chd3 R1025W/+ mouse model. In vivo base editing by TeABE in Chd3 R1025W/+ mice We first evaluated the delivery efficiency of AAV-PHP.eB vectors using AAV-hSyn-GFP administered via tail intravenous injection. Widespread expression of AAV-PHP.eB vectors was observed throughout the mouse brain (Fig. 4a). To assess in vivo on-target base editing efficiency, next-generation sequencing was performed on cerebral cortical tissues from Chd3 R1025W/+ mice treated with either GFP or TeABE. Following TeABE injection, a significantly higher proportion of reads with target base editing was detected in the cerebral cortex compared to GFP-treated controls, indicating successful base correction in the Chd3 R1025W/+ brain (Extended Data Fig. 4a). Immunofluorescent staining of the SpCas9 confirmed efficient expression of the dual AAV-TeABE system in cortical regions, including the retrosplenial cortex (RSC), parietal association cortex (PtA), primary somatosensory cortex trunk (S1Tr), and barrel field (S1BF), as well as hippocampal areas such as CA1, CA2, CA3, and the dentate gyrus (DG) (Fig. 4b). Restoration of CHD3 protein levels in these brain regions was confirmed, consistent with the SpG expression results (Fig. 4c). Immunoblot analysis of WT mice injected with AAV-GFP and Chd3 R1025W/+ mutant mice treated with GFP or TeABE further demonstrated a significant increase in CHD3 protein levels after TeABE delivery (Fig. 4d). These findings confirm the effectiveness of AAV-PHP.eB-mediated Cas9 transduction and in vivo base editing for correcting Chd3-R1025W in the mouse brain. TeABE improves the behavioral abnormalities of Chd3 R1025W/+ mice To evaluate the therapeutic effects of TeABE treatment in Chd3 R1025W/+ mutant mice, we conducted a series of behavioral experiments. Intellectual disability, a hallmark symptom of SNIBCPS, was assessed using the three-chamber novel object recognition test and the Barnes maze test to measure cognitive and spatial learning abilities. In the novel object recognition test, treated mice exhibited a restored preference for novel objects and a significant increase in the recognition preference index, which was absent in untreated Chd3 R1025W/+ mice (Fig. 5a, Extended Data Fig. 5a). Similarly, in the Barnes maze test, treated mice demonstrated a shorter latency to locate the target hole during test 1 on day 6 and test 2 on day 13, reflecting enhanced spatial learning and memory abilities following treatment (Fig. 5b). Autistic-like phenotypes, including social impairments and repetitive stereotyped behaviors, are common in SNIBCPS. To evaluate social competence, we employed the three-chamber social interaction test and the social intruder test. In the three-chamber test, treated mice spent significantly more time sniffing stranger mice compared to untreated Chd3 R1025W/+ mice, with a concurrent rise in the social preference index, indicating amelioration of social deficits (Fig. 5c, Extended Data Fig. 5b). Similar improvements were observed in the social intruder test, where treated mice exhibited increased interaction times during both the T1 and T5-T4 trials, reflecting enhanced sociability and social novelty recognition (Fig. 5d). While untreated mutant mice lacked social familiarity and novelty processes (e.g., changes in sniffing time), these processes were restored in treated mice (Fig. 5e). To assess repetitive stereotyped behaviors, we performed marble-burying and self-grooming experiments. Treated mice displayed a significant reduction in the number of buried marbles and self-grooming duration compared to untreated mice, indicating improved stereotyped behaviors (Fig. 5f). Anxiety levels were examined using the elevated plus maze and open field tests. No significant differences were observed among the groups in terms of time spent in open arms, entries to open arms, or exploratory activity (Extended Data Fig. 5c, d), suggesting that the Chd3-R1025W mutation does not impact anxiety. In summary, TeABE treatment effectively alleviated autistic-like behaviors, including social deficits and stereotyped behaviors, in Chd3 R1025W/+ mutant mice. Hypotonia, a common symptom of SNIBCPS, is characterized by involuntary, sustained muscle contractions that result in abnormal posture and movement disorders, including gait abnormalities, altered stride, and reduced grip strength in mice. In the tail suspension experiment, Chd3 R1025W/+ mutant mice were evaluated for limb tightness and scored for clasping (Fig. 6a,). Treated mice exhibited significantly lower scores compared to untreated mutants, indicating a reduction in involuntary muscle contractions following treatment (Fig. 6a). Additionally, SNIBCPS is frequently associated with joint abnormalities. Joint laxity scores revealed that treated mice displayed improved joint stability, accompanied by a significant reduction in both redness and swelling of the joints compared to untreated mutant mice (Fig. 6b). Gait analysis further assessed locomotor abnormalities associated with hypotonia. Treated mice demonstrated improvements, including a reduced hindlimb foot angle, decreased hindlimb spacing, and increased stride length, with values approaching those of WT controls (Fig. 6c, d). These results suggest that TeABE treatment enhanced locomotor coordination and normalized gait patterns. Grip strength was evaluated using the rotarod test, which measures the ability of mice to maintain their grip on a rotating rod as the speed increases. Untreated Chd3 R1025W/+ mutant mice fell at lower speeds, whereas treated mice remained on the rod for longer durations, covering greater distances and enduring higher rotational speeds, reflecting significantly improved grip strength (Fig. 6e, Extended Data Video 1). Collectively, these findings demonstrate that TeABE treatment effectively alleviates hypotonia-related phenotypes in Chd3 R1025W/+ mice. Efficient assembly of TeABE in the monkey brain To evaluate the feasibility of TeABE via the dual AAV strategy in clinical applications, we utilized a nonhuman primate model to examine the efficacy of delivery and assemble of TeABE in the primate brain. Dual vectors of TeABE using AAV9 capsid was administered through intrathecal injection to facilitate expression of the CHD3 base editor across brain regions (Fig. 7a). Immunoblot analysis demonstrated that the assemble efficiency of the base editor reached approximately 70% in various parts of the monkey brain (Fig. 7b). Furthermore, immunofluorescence analysis of the monkey brain revealed widespread expression of the base editor, with successful transduction observed in over 30% of neurons (Fig. 7c, d). These results provide compelling evidence for the safety and efficacy of TeABE in a nonhuman primate model, paving the way to application of TeABE in clinical trials. This study represents a significant advancement in gene therapy for brain disorders and highlights the transformative potential of base editing in treating neurological diseases. Discussion Snijders Blok-Campeau Syndrome (SNIBCPS) is a rare autosomal genetic disorder characterized by language and intellectual development impairments, along with other symptoms 1,5 . This condition imposes a significant burden on affected children and their families. Despite the severity of the disorder, no effective treatment targeting SNIBCPS has been reported. The primary association of SNIBCPS with specific mutations in the CHD3 gene highlights gene therapy as a promising therapeutic strategy 1,2 . In this study, we developed and applied a base editor to repair the c.C3073T point mutation and assessed its therapeutic potential in Chd3 R1025W/+ mutant mice. Behavioral experiments demonstrated significant improvements in key symptoms, including cognitive deficits, spatial learning and memory impairments, autistic-like behaviors, and hypotonia, underscoring the efficacy of our approach. Our investigation into the pathogenic mechanism of the Chd3-R1025W mutation revealed that it accelerates the degradation of CHD3 protein, leading to reduced protein levels and associated neuronal abnormalities. While our findings provide insights into the molecular impact of this mutation, further mechanistic studies are necessary to elucidate how the mutation affects CHD3's chromatin remodeling functions and contributes to disease pathology. By employing base editing, we successfully corrected the single-nucleotide variant (SNV), restoring CHD3 protein levels both in vitro and in vivo . These results emphasize the potential of gene editing as a precise therapeutic strategy for addressing genetic defects and mitigating disease symptoms. Compared to the CRISPR-Cas9 system, base editing offers a safer alternative as it avoids inducing double-strand DNA breaks 23-25 . Base editing has already demonstrated success in clinical trials for repairing genetic defects in various organs 26,27,37,38 . However, applying this technology to the central nervous system (CNS), particularly the brain, poses unique challenges due to its complexity and the risks associated with intervention 39 . While base editing has shown promise in nonhuman primate models for non-CNS applications 40,41 , its use in the CNS has been limited to mouse models. These models are valuable for evaluating phenotype improvements but fall short of providing guidance on clinical dosing or application in humans. Bridging the gap between preclinical research and clinical translation remains a significant challenge. Given the large size of the base editor used in this study, we employed a dual-vector delivery strategy for assembly within cells. However, this approach occasionally resulted in unsuccessful splicing, thereby reducing efficiency. In our nonhuman primate model, we observed a splicing efficiency of approximately 70%. To address this limitation, optimizing the viral delivery system is critical. High-capacity adenoviral vectors (HCAds), which offer larger packaging capacities 42-44 , or the development of smaller Cas homologs 45 , could enable single-vector packaging, eliminating the need for splicing and enhancing the system’s overall efficacy. In conclusion, this study demonstrates the feasibility and therapeutic potential of base editing for SNIBCPS and highlights critical areas for future optimization. These findings represent a significant step toward developing effective gene therapy strategies for brain disorders and pave the way for broader clinical applications of base editing technologies. Declarations Acknowledgements We thank the families with SNIBCPS patients for their participation and support in this study. We thank Yonghong Wang, Linxiao Yue, and Song Guo for excellent technical assistance. We thank Yandong Li for consulting on veterinarian regulations. K.Y. was funded by the Youth Innovation Promotion Association Chinese Academy of Sciences (2022269), the Youth Science Fund of State Key Laboratory of Neuroscience (SKLN-2022B006) and the National Natural Science Foundation of China (NSFC, 82001211). W-K.L was funded by NSFC (32400933). F.L. was funded by NSFC (82430104, 82125032, 81930095), the China Brain Initiative Grant (STI2030-Major Projects 2021ZD0200800), the Science and Technology Commission of Shanghai Municipality (19410713500, 23Y21900500 and 2018SHZDZX01), the Shanghai Municipal Commission of Health and Family Planning (GWVI-11.1-34, 2020CXJQ01, 2018YJRC03), Innovative research team of high-level local universities in Shanghai (SHSMU-ZDCX20211100), and the Guangdong Key Project (2018B030335001). T-L.C. was funded by NSFC (32371144). Z-L.Q. was funded by NSFC (82430046). Author contributions K.Y., T-L.C. and Z-L.Q. designed this study. K.Y. and W-K.L. performed the plasmid construction. K.Y., Z-K.X., R-C.X., C-X.L., J.W., R.Z. and Y-X.G. carried out primary neuronal culture and molecular experiments. Y-B.C., Y-T.Y., Y-F.Z. and K.Y. performed construction, identification and management of point mutant mice. W-K.L., X-Y.Z., J.L., S-Q.Z. and T-L.C. performed the editor design and in vitro efficiency verification. K.Y., Y-X.G., Y-T.Y. and W-K.L. performed mouse virus injection and in vivo editing efficiency verification. K.Y., T-Y.Z., Z-H.L., W-X.W. and J-W.W. performed animal behavioral experiments. Z-Y.Y., L.G., W.Z., S-H.W., W-X.Y., J-S.W., K.Y. and Y-X.G. performed intrathecal injection and expression efficiency verification in monkeys. F.L., Z-L.Q., K.Y., T-L.C. and W-K.L. provided the experimental funds. K.Y., Y-X.G. and Z-L.Q. wrote the manuscript. Z-L.Q., T-L.C., F.L. and J-S.L. revised the manuscript. References Snijders Blok, L. et al. CHD3 helicase domain mutations cause a neurodevelopmental syndrome with macrocephaly and impaired speech and language. 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Supplementary Files CHD3supplementaryinformation.docx Supplementary information CHD3ExtendedDataVideoRotarod.mp4 Extended Data Video_Rotarod Cite Share Download PDF Status: Published Journal Publication published 18 Feb, 2026 Read the published version in Nature → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5690634","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":406745045,"identity":"50652731-1e7d-4a67-9b22-e709b02d4939","order_by":0,"name":"Kan Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYBACCSgtB2MxNhCrxZh0LYkNRGuRbD97TOLnjtr0+bPbn27mYbCR3XCA+dkDfFqkefLSJHvPHM/dcOeM2W0ehjTjDQfYzA3waZFjyDGT4G07lrtBIocNqOVw4oYDPGwSeLXwvzGT/Nt2LF1+RvozoJb/hLVIS+SYSfO21SQw3EgAOewAYS2SM94YW8u2HTDccCPH7OYcg2TjmYfZzPBqkTifY3jzbVudPMhhN95U2Mn2HW9+hlcLELAAFRyGskFBxUxAPUjJBwaGOsLKRsEoGAWjYOQCAAZsSadhVIUkAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-1563-1406","institution":"Xinhua Hospital, Shanghai Jiao Tong University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Kan","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2024-12-21 16:30:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5690634/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5690634/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41586-026-10113-6","type":"published","date":"2026-02-18T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":74920437,"identity":"5e1a787b-1405-48b2-a8f4-fcf0010b13d1","added_by":"auto","created_at":"2025-01-28 10:37:52","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1153322,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ede novo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutation in the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCHD3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene associated with SNIBCPS causes rapid degradation of the CHD3 protein. a, \u003c/strong\u003eSchematic view of reported \u003cem\u003ede novo\u003c/em\u003e mutations and the R1025W variant in CHD3 (NP_001005271.2). \u003cstrong\u003eb, \u003c/strong\u003eIllustration of the SNIBCPS family and validation using Sanger sequencing (red arrow).\u003cstrong\u003e c, \u003c/strong\u003eImmunoblotting of human CHD3-WT/R1025W FLAG expressed in primary neurons and quantitative analysis of protein (n=6) and mRNA (n=4). GAPDH, glyceraldehyde 3-phosphate dehydrogenase. \u003cstrong\u003ed, \u003c/strong\u003eArginine (Arg,R) replaced by three aromatic amino acid, tryptophan (Trp, W), phenylalanine (Phe, F) and tyrosine (Tyr, Y).\u003cstrong\u003ee, \u003c/strong\u003eFluorescence imaging and intensity analysis (orange dashed line) of human CHD3-WT/R1025W/R1025F/R1025Y. \u003cstrong\u003ef,\u003c/strong\u003e Quantitative analysis of mean fluorescence intensity shown in \u003cstrong\u003ee\u003c/strong\u003e.\u003cstrong\u003eg,\u003c/strong\u003e Immunoblotting and quantitative analysis of human CHD3-WT/R1025W expressed in HEK293 cells, with CHX (20 μg/ml) for the indicated time (two-way analysis of variance). To equate the initial signals for a comparative analysis of degradation, exposure time of\u003cem\u003e \u003c/em\u003eCHD3-R1025W was increased. \u003cstrong\u003eh,\u003c/strong\u003eImmunoblotting and quantitative analysis of human CHD3-WT/R1025W expressed in HEK293 cells, with BTZ (100 nM) or CHX (20 μg/ml) treatment for 12 h. \u003cem\u003en\u003c/em\u003e represents biologically independent experiments. Statistical values represent the mean ± SD *P \u0026lt; 0.05, **P \u0026lt; 0.01, ****P \u0026lt; 0.0001 by Student’s unpaired two-sided t-test or two-way analysis of variance.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5690634/v1/e7f18ceda00706eb3f965477.jpg"},{"id":74919848,"identity":"0b471349-045e-4cd7-b635-6f4265fc1542","added_by":"auto","created_at":"2025-01-28 10:29:52","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1537407,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eChd3\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003eR1025W/+\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e mutant mice were constructed and recapitulated key behavioral abnormalities of SNIBCPS. a, \u003c/strong\u003ePrinciple of construction of \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e mice. The human mutation-containing allele was used for replacement. Red arrows point to the mutation site, and black arrows point to other bases on the human allele that are not consistent with the mouse gene. \u003cstrong\u003eb, \u003c/strong\u003eImmunoblotting and quantitative analysis of CHD3 protein levels in \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e mice (n=4). PFC, Prefrontal Cortex; ACC, anterior cingulate cortex; RSC, retrosplenial cortex; Hip, Hippocampus. \u003cstrong\u003ec, \u003c/strong\u003eResults of ultrasonic vocalizations (USVs) experiments in \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e pups and statistical analysis of differences in number, duration and type of calls (n=7). USVs were induced due to maternal isolation.\u003cstrong\u003e d, \u003c/strong\u003eThe model and representative locomotion heatmaps in the novel object recognition test. The duration of touching familiar object or novel object was statistically analyzed in \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e mice (n=12).\u003cstrong\u003e e, \u003c/strong\u003eThe model and representative locomotion heatmaps in the three-chamber social novelty test. Quantification of time spent in sniffing in \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e mice (n=12). O1, object 1; No, novel object. \u003cstrong\u003ef, \u003c/strong\u003eRepresentative images showing the marble burying test and quantitative analysis of the number of buried marbles within 10 min (n=12) and the during time of self-grooming (n=12). \u003cem\u003en\u003c/em\u003e represents the number of mice. Statistical values represent the mean ± SD *P\u0026nbsp;\u0026lt;\u0026nbsp;0.05, **P\u0026nbsp;\u0026lt;\u0026nbsp;0.01, ***P\u0026nbsp;\u0026lt;\u0026nbsp;0.001, ****P\u0026nbsp;\u0026lt;\u0026nbsp;0.0001 by Student’s unpaired two-sided t-test.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5690634/v1/214c7abb6c297e33665be337.jpg"},{"id":74919850,"identity":"a6784d96-b2a7-4c41-aa76-7e62675c1477","added_by":"auto","created_at":"2025-01-28 10:29:52","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1503438,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDesign of TeABE. a,\u003c/strong\u003e Design of candidate sgRNAs (sgRNA-C10, sgRNA-C11) for \u003cem\u003eCHD3\u003c/em\u003e point mutation cell line generation. Arg (CGG), targeting amino acid. \u003cstrong\u003eb,\u003c/strong\u003eIllustration of the candidate sgRNAs and CBEs for constructing the \u003cem\u003eCHD3\u003c/em\u003epoint mutation cell line. \u003cstrong\u003ec,\u003c/strong\u003eWorkflow for the screening of the point mutation cell line. \u003cstrong\u003ed,\u003c/strong\u003e Sanger sequencing chromatogram of the HEK293 cell genome after transient transfection with sgRNA and CBEs. \u003cstrong\u003ee,\u003c/strong\u003e Sanger sequencing chromatogram of the HEK293 cell line genome of two representative monocolony after 4 days of puromycin treatment. The red box (C\u0026gt;T) in the figure indicates the target base position (d and e). \u003cstrong\u003ef,\u003c/strong\u003e Design of candidate gRNAs for correcting \u003cem\u003eCHD3-R1025W \u003c/em\u003emutation. \u003cstrong\u003eg,\u003c/strong\u003e Schematic diagram of the candidate sgRNA and adenine base editor for repairing the \u003cem\u003eCHD3-R1025W\u003c/em\u003e point mutation. \u003cstrong\u003eh,\u003c/strong\u003e A flowchart for screening the base editing efficiency of multiple sgRNA and ABE combinations. \u003cstrong\u003ei,\u003c/strong\u003e Statistical analysis of on-target base editing efficiency and by-stander effects for different candidate sgRNAs. \u003cstrong\u003ej,\u003c/strong\u003e Statistical analysis of the on-target editing (A11) and bystander editing (A13) of the various TeABE editors with different TadAs embedding sites. \u003cstrong\u003ek,\u003c/strong\u003eGuide-seq of TeABE to identify the potential off-targeting sites in the genome. \u003cstrong\u003el,\u003c/strong\u003e Statistical analysis of on-target editing and off-targeting effects for TeABEs (1249 and 1248(D1249-1263)).\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5690634/v1/3011842612d2083ea636ba99.jpg"},{"id":74920439,"identity":"9b286c90-185f-43b8-a24b-6be6803697ed","added_by":"auto","created_at":"2025-01-28 10:37:52","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2108534,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRestoration of CHD3 protein levels in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eChd3\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003eR1025W/+\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e mice after TeABE editing. a, \u003c/strong\u003eIllustration of the dual AAV strategy and representative immunofluorescence for GFP (green) and DAPI (blue) in the brain via tail intravenous injection (TIV). \u003cstrong\u003eb, \u003c/strong\u003eRepresentative immunofluorescence staining and quantification of the average fluorescence intensity for spCas9 (red) and DAPI (blue) in the brain in CHD3 WT mice treated with AAV-GFP, and \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e mutant mice injected with either GFP or TeABE (n=12). \u003cstrong\u003ec, \u003c/strong\u003eRepresentative immunofluorescence staining and quantification of the average fluorescence intensity for CHD3 (red) and DAPI (blue) in the brain in CHD3 WT mice treated with AAV-GFP, and \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e mutant mice injected with either GFP or TeABE (n=12). \u003cstrong\u003ed, \u003c/strong\u003eRepresentative immunoblotting and quantification of the immunoblotting of the brain, RSC and Hip extracted from\u003cem\u003e Chd3\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e mice treated with AAV-GFP, and \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e mutant mice injected with either GFP or TeABE (n=3 mice). \u003cem\u003en\u003c/em\u003e represents biologically independent experiments. Statistical values represent the mean ± SD *P\u0026nbsp;\u0026lt;\u0026nbsp;0.05, **P\u0026nbsp;\u0026lt;\u0026nbsp;0.01, ***P\u0026nbsp;\u0026lt;\u0026nbsp;0.001, ****P\u0026nbsp;\u0026lt;\u0026nbsp;0.0001 by Student’s unpaired two-sided t-test.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5690634/v1/c601b15953591d1891233949.jpg"},{"id":74920438,"identity":"3edf04cf-ef8b-4b13-bb3b-471c84eaefef","added_by":"auto","created_at":"2025-01-28 10:37:52","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1989213,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAmelioration of intellectual disability and autistic-like behaviors in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eChd3\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003eR1025W/+\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e mutant mice with TeABE editing \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. a, \u003c/strong\u003eThe model and representative locomotion heatmaps in the three-chamber novel object recognition test of \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e (GFP), \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+ \u003c/em\u003e\u003c/sup\u003e(GFP) and \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e (TeABE) mice. Quantification of time spent in sniffing (n=7). O1, object 1; NO, novel object. \u003cstrong\u003eb, \u003c/strong\u003eThe model and quantification of latency time before hiding in the target hole in the Barnes maze test of \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e (GFP), \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003e(GFP) and \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e (TeABE) mice (n=7, two-way analysis of variance). \u003cstrong\u003ec, \u003c/strong\u003eThe model and representative locomotion heatmaps in the three-chamber social novelty test of\u003cem\u003e Chd3\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e (GFP), \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003e(GFP) and \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e (TeABE) mice. Quantification of time spent in sniffing (n=7). \u003cstrong\u003ed, \u003c/strong\u003eThe model of social intruder test and statistical results of sniffing time at T1 and Social novelty index (T5–T4) of \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e (GFP), \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003e(GFP) and \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e (TeABE) mice (n=7, two-way analysis of variance). \u003cstrong\u003ee, \u003c/strong\u003eSniffing time of\u003cem\u003e Chd3\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e (GFP), \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003e(GFP) and \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e (TeABE) mice.\u003cstrong\u003e \u003c/strong\u003eMean cumulative sniffing time (black) and individual mice (gray) in the social intruder test for \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e (GFP), \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003e(GFP) and \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e (TeABE) mice (n=7, two-way analysis of variance). \u003cstrong\u003ef, \u003c/strong\u003eRepresentative images showing the marble burying test and quantitative anlysis of the number of buried marbles within 10 min (n=7) and the during time of self-grooming for \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e (GFP), \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003e(GFP) and \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e (TeABE) mice (n=7). \u003cem\u003en\u003c/em\u003e represents the number of mice. Statistical values represent the mean ± SD *P\u0026nbsp;\u0026lt;\u0026nbsp;0.05, **P\u0026nbsp;\u0026lt;\u0026nbsp;0.01, ***P\u0026nbsp;\u0026lt;\u0026nbsp;0.001, ****P\u0026nbsp;\u0026lt;\u0026nbsp;0.0001 by one-way analysis of variance or two-way analysis of variance.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5690634/v1/45e695ba53dfc03fbb69ded5.jpg"},{"id":74919851,"identity":"67df8846-e4c0-46c4-a789-9157496a9247","added_by":"auto","created_at":"2025-01-28 10:29:52","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1032515,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAmelioration of hypotonia in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eChd3\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003eR1025W/+\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e mice with TeABE editing \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. a, \u003c/strong\u003eRepresentative images showing the tail suspension test and quantitative anlysis of the clasping score for \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e (GFP), \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003e(GFP) and \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e (TeABE) mice (n=7). The blue arrow points to the limbs clasping. \u003cstrong\u003eb, \u003c/strong\u003eQuantitative anlysis of joint laxity score for \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e (GFP), \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003e(GFP) and \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e (TeABE) mice (n=7).\u003cstrong\u003e c, \u003c/strong\u003eRepresentative images showing the gait analysis and quantitative anlysis of the hindlimb foot angle and hindlimb spacing for \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e (GFP), \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003e(GFP) and \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e (TeABE) mice (n=7). The Angle refers to the intersection of the extension lines of the middle finger of the hindlimb of the mouse (red angle). The hindlimb spacing refers to the distance between the center points of the two hind feet (blue line segment). \u003cstrong\u003ed, \u003c/strong\u003eRepresentative images showing the stride analysis and quantitative anlysis of the stride for \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e (GFP), \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003e(GFP) and \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e (TeABE) mice (n=7). Blue ink marks the hindlimbs and red ink marks the forelimbs.\u003cstrong\u003e \u003c/strong\u003eThe distance between two adjacent blue markers on the same side is the stride (bule line segment).\u003cstrong\u003e e, \u003c/strong\u003eRepresentative images showing the rota-rod test and quantitative anlysis of distance, speed to fall and time to fall for \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e (GFP), \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003e(GFP) and \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e (TeABE) mice (n=7). Blue arrows point to fallen mice. \u003cem\u003en\u003c/em\u003e represents the number of mice. Statistical values represent the mean ± SD *P\u0026nbsp;\u0026lt;\u0026nbsp;0.05, **P\u0026nbsp;\u0026lt;\u0026nbsp;0.01, ***P\u0026nbsp;\u0026lt;\u0026nbsp;0.001, ****P\u0026nbsp;\u0026lt;\u0026nbsp;0.0001 by one-way analysis of variance.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5690634/v1/7274e68aad6dee2d0bd6e1f5.jpg"},{"id":74919853,"identity":"15d8ea11-0657-40fd-b031-76bb8339d13c","added_by":"auto","created_at":"2025-01-28 10:29:53","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2052872,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eValidation of TeABE editing efficiency in monkeys. a, \u003c/strong\u003eIllustration of the dual AAV strategy via spinal cord injection. \u003cstrong\u003eb, \u003c/strong\u003eImmunoblotting and quantitative analysis of full-Cas9 and Cas9 N-terminal in two monkeys’ brains when 42 days after AAV9-TeABE delivery. Statistical results of Cas9-TeABE assembly efficiency in different regions of the brain (n=4). The monkeys was injected at 4 years. #9402, the monkey with the high dose (1500 μl of 1.13 × 10\u003csup\u003e14 \u003c/sup\u003evg ml\u003csup\u003e−1 \u003c/sup\u003eAAV9-hSyn-nCas9-1-1029 + 1500 μl of 1.49 × 10\u003csup\u003e14 \u003c/sup\u003evg ml\u003csup\u003e−1 \u003c/sup\u003eAAV9- hSyn -nCas9-1030-1248-TadA-F148A-nCas9-1264-1368-U6-sgRNA); #9500, the monkey with the low dose (1500 μl of 3.00 × 10\u003csup\u003e13 \u003c/sup\u003evg ml\u003csup\u003e−1 \u003c/sup\u003eAAV9-hSyn-nCas9-1-1029 + 1500 μl of 3.00 × 10\u003csup\u003e13 \u003c/sup\u003evg ml\u003csup\u003e−1 \u003c/sup\u003eAAV9- hSyn-nCas9-1030-1248-TadA-F148A-nCas9-1264-1368-U6-sgRNA) \u003cstrong\u003ec, \u003c/strong\u003eRepresentative immunofluorescence staining for Cas9 (red), NeuN (green) and DAPI (blue) in the monkey brain with the high dose. \u003cstrong\u003ed,\u003c/strong\u003e The neuronal transduction rate of Cas9 (n=4). The neuronal transduction rate was obtained by counting Cas9-expressing cells in NeuN-stained cells. \u003cem\u003en\u003c/em\u003e represents biologically independent experiments. Statistical values represent the mean ± SD *P\u0026nbsp;\u0026lt;\u0026nbsp;0.05, **P\u0026nbsp;\u0026lt;\u0026nbsp;0.01, ***P\u0026nbsp;\u0026lt;\u0026nbsp;0.001 by Student’s unpaired two-sided t-test.\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5690634/v1/ce87fe09a53abd005ab82509.jpg"},{"id":102977808,"identity":"0d0df7b4-5135-4563-8ca8-93ec4bc9fae1","added_by":"auto","created_at":"2026-02-19 08:14:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12423128,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5690634/v1/3f3a63b7-dd99-4bce-b65f-394df815e6ed.pdf"},{"id":74919846,"identity":"a45e9623-1bc9-42ff-9111-6e23903c56e3","added_by":"auto","created_at":"2025-01-28 10:29:52","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1585666,"visible":true,"origin":"","legend":"Supplementary information","description":"","filename":"CHD3supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5690634/v1/849a51e6b795d220dcf70f7e.docx"},{"id":74919854,"identity":"242de15e-a17a-4ea8-b42d-1c7f14ed1d63","added_by":"auto","created_at":"2025-01-28 10:29:53","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":21589300,"visible":true,"origin":"","legend":"Extended Data Video_Rotarod","description":"","filename":"CHD3ExtendedDataVideoRotarod.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5690634/v1/161cbde5b685f5625bbf24d4.mp4"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Behavioral Improvement Through In Vivo Base Editing in a Mouse Model of Snijders Blok-Campeau Syndrome","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSnijders Blok-Campeau Syndrome (SNIBCPS, OMIM #618205) is a rare autosomal dominant genetic disorder caused by pathogenic mutations in the \u003cem\u003eCHD3\u003c/em\u003e (\u003cem\u003eChromodomain Helicase DNA-binding Protein 3\u003c/em\u003e) gene. First described by Lot Snijders Blok in 2018\u003csup\u003e1\u003c/sup\u003e, over a hundred cases have since been reported\u003csup\u003e2\u003c/sup\u003e. The primary clinical features of SNIBCPS include global developmental delay, speech delay, intellectual disability, hypotonia, abnormal facial features, and structural brain anomalies, frequently accompanied by autism spectrum disorders (ASD)\u003csup\u003e1-6\u003c/sup\u003e. While most cases are caused by single-nucleotide variations (SNVs), one instance involving a complete deletion and duplication of the \u003cem\u003eCHD3\u003c/em\u003e gene has been documented\u003csup\u003e5\u003c/sup\u003e. Despite these findings, the pathogenesis of SNIBCPS remains poorly understood.\u003c/p\u003e\n\u003cp\u003eThe CHD protein family is critical for chromatin remodeling, relying on ATP hydrolysis to regulate chromatin structure and gene transcription\u003csup\u003e7,8\u003c/sup\u003e. Members of this family are implicated in various neurodevelopmental disorders, including \u003cem\u003eCHD2\u003c/em\u003e in epilepsy\u003csup\u003e9\u003c/sup\u003e, \u003cem\u003eCHD7\u003c/em\u003e in CHARGE syndrome\u003csup\u003e10\u003c/sup\u003e, and \u003cem\u003eCHD8\u003c/em\u003e in ASD\u003csup\u003e11\u003c/sup\u003e. CHD3, along with CHD4 and CHD5, forms a core component of the nucleosome remodeling and deacetylase (NuRD) complex, which modulates chromatin structure and DNA accessibility, playing vital roles in cell cycle regulation and embryonic stem cell differentiation\u003csup\u003e12-17\u003c/sup\u003e. CHD3 is essential for cerebral cortex development, as its deficiency results in defects in cortical neuron differentiation and migration\u003csup\u003e18\u003c/sup\u003e. Furthermore, \u003cem\u003eCHD3\u003c/em\u003e mutations have been linked to childhood apraxia of speech\u003csup\u003e19,20\u003c/sup\u003e. Most pathogenic SNVs in \u003cem\u003eCHD3\u003c/em\u003e occur within the helicase ATP-binding and C-terminal domains, which are critical for its function. While mutations in the ATP-binding domain may disrupt ATP hydrolysis and chromatin remodeling, this mechanism does not fully explain the diverse effects of all known SNVs associated with SNIBCPS\u003csup\u003e1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe advent of CRISPR-Cas9 technology has revolutionized gene editing\u003csup\u003e21,22\u003c/sup\u003e. However, the potential for DNA double-strand breaks to induce genomic damage poses limitations. Base editing, an optimized approach, allows precise base transformations without cutting DNA\u003csup\u003e23-25\u003c/sup\u003e. Cytosine base editors (CBEs) and adenine base editors (ABEs) can convert C•G to T•A and A•T to G•C, respectively\u003csup\u003e24,25\u003c/sup\u003e. This technology has shown promise in animal models of various diseases\u003csup\u003e26,27\u003c/sup\u003e. In humans, gene therapy has achieved remarkable success in treating autosomal recessive deafness 9 and inherited retinal dystrophies\u003csup\u003e28,29\u003c/sup\u003e. Additionally, preclinical studies have demonstrated the efficacy of gene therapy in addressing neurological disorders such as Angelman syndrome, spinal muscular atrophy, and ASD in mouse models\u003csup\u003e30-32\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eHere, we report a \u003cem\u003ede novo\u003c/em\u003e single-nucleotide variant (SNV) in the \u003cem\u003eCHD3\u003c/em\u003e gene (c.C3073T, NM_001005271.3; p.R1025W, NP_001005271.2) associated with Snijders Blok-Campeau Syndrome (SNIBCPS) in a child. The R1025W mutation results in accelerated degradation of the CHD3 protein. To investigate the underlying mechanisms, we generated a \u003cem\u003eChd3\u003csup\u003eR1025W/+\u003c/sup\u003e\u003c/em\u003e knock-in mouse model, which exhibited deficits in vocalization, cognition, and autism-like behaviors. To correct the genetic mutation, we developed a TadA-embedded adenine base editor (TeABE) to edit the mutated A·T base pair in \u003cem\u003eChd3\u003csup\u003eR1025W/+\u003c/sup\u003e\u0026nbsp;\u003c/em\u003ein the mouse brain. Using a blood-brain-barrier-crossing AAV viral delivery system, TeABE was administered via intravenous injection. Remarkably, this treatment significantly restored CHD3 protein levels in the brain and ameliorated behavioral abnormalities resembling those observed in SNIBCPS patients. Moreover, we demonstrated the transduction efficacy of TeABE in the brain of nonhuman primate, validating its clinical applicability. These findings establish \u003cem\u003ein vivo\u003c/em\u003e base editing as a viable approach to mitigate behavioral abnormalities in SNIBCPS and highlight the potential of TeABE for clinical therapeutic applications.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eA \u003cem\u003ede novo\u003c/em\u003e SNV in the \u003cem\u003eCHD3\u003c/em\u003e gene associated with SNIBCPS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn recent works, encompassing over one hundred persons with SNIBCPS, researchers identified a series of \u003cem\u003ede novo\u003c/em\u003e and inherited variants in \u003cem\u003eCHD3\u003c/em\u003e\u003csup\u003e33\u003c/sup\u003e. A schematic illustration of several locations of genetic mutations in the CHD3 protein is shown (Fig.1a). In whole-exome sequencing using the genome DNA of one child with SNIBCPS and unaffected parents obtained at the Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, we identified a \u003cem\u003ede novo\u003c/em\u003e SNV in the \u003cem\u003eCHD3\u003c/em\u003e gene (c.C3073T, NM_001005271.3; p.R1025W, NP_001005271.2), validated using Sanger sequencing (Fig.1b). The amino acid change (R1025W) caused by the \u003cem\u003ede novo\u003c/em\u003e variant is located between the Helicase ATP-binding domain and the Helicase C-terminal domain of the CHD3 protein (Fig.1a). This variant (\u003cem\u003eCHD3\u003c/em\u003e, chromosome 17:7803967) is absent in East Asian populations in the gnomAD database (http://gnomad.broadinstitute.org), suggesting that it is a rare variant.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe obtained the mRNA expression profile of \u003cem\u003eCHD3\u003c/em\u003e from the BrainSpan (Atlas of Developing Human Brain, http://www.brainspan.org/rnaseq/), covering from embryonic development to adulthood. We found that \u003cem\u003eCHD3\u003c/em\u003e is highly expressed in the striatum (STR), anterior cingulate (ACC), hippocampus (HIP), cerebellar cortex (CBC), medial prefrontal cortex (mPFC) and primary motor cortex (M1C) (Extended Data Fig.1a). The protein level of CHD3 in the mouse brain gradually decline from embryonic development to adulthood (Extended Data Fig.1b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo determine whether the R1025W mutation impacts the normal function of the CHD3 protein, we examined the expression level of FLAG-tagged human wild-type CHD3-WT and CHD3-R1025W mutant protein in mouse cortical neurons and HEK293 cells. Surprisingly, the protein level of human CHD3-R1025W was strikingly lower than that of human CHD3-WT (Fig.1c and Extended Data Fig.1c). However, the mRNA levels of transfected human WT and R1025W CHD3 remained the same (Fig.1c and Extended Data Fig.1c and d), indicating that the R1025W mutation affects the level of CHD3 protein.\u003c/p\u003e\n\u003cp\u003eTo further investigate the importance of R1025 for CHD3 protein stability, we determined the impact of all aromatic amino acids, namely tryptophan (Trp, W), phenylalanine (Phe, F) and tyrosine (Tyr, Y) (Fig.1d). The Alphafold3-predicted structure revealed that the R1025W, R1025F and R1025Y mutants exhibit more compact conformations with a reduced spatial distance between the key Helicase ATP-binding domain and Helicase C-terminal domain compared to the WT CHD3 (Extended Data Fig.1e). We found that R1025W, R1025F and R1025Y all led to decreased levels of CHD3 protein (Extended Data Fig.1f). Similarly, we found substitution of aromatic amino acids in R1025 showed drastic decrease of immunofluorescent intensity of CHD3 protein in the nucleus of HEK293 cells (Fig.1e and f), indicating that R1025 is critical for CHD3 protein stability, while having no influence in mRNA level (Extended Data Fig.1f). After cycloheximide (CHX) treatment, an inhibitor of protein translation, the levels of CHD3-R1025W protein decreased faster than CHD3-WT, suggesting that the R1025W mutation accelerated CHD3 protein degradation (Fig.1g). Treatment with the proteasome inhibitor bortezomib (BTZ) restored the levels of CHD3-R1025W protein, indicating that degradation of CHD3-R1025W protein was mediated by a ubiquitin-dependent pathway (Fig.1h).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCHD3-R1025W leads to abnormal dendritic morphology of mouse cortical neurons\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further explore the influence of CHD3-R1025W on neuronal morphology, we constructed short hairpin RNAs (shRNAs) specifically targeting the mouse \u003cem\u003eChd3\u003c/em\u003e gene (Extended Data Fig.2a). The knockdown efficiency of three designed shRNAs was tested by measuring endogenous \u003cem\u003eChd3\u003c/em\u003e mRNA levels in mouse cortical neurons (Extended Data Fig.2b). The expression of endogenous CHD3 protein was effectively reduced by any of the shRNAs (Extended Data Fig.1c, d). By applying cultured mouse cortical neuron, we found that knockdown of CHD3 led to decreased dendritic length and branch numbers at both 3 and 7 days \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003e(DIV), while having no significant impact on the axonal length and branch numbers (Extended Data Fig.2e-k). These abnormalities were restored by co-transfection of a shRNA-resistant CHD3-WT construct but not CHD3-R1025W (Extended Data Fig.2e-k). These results indicate that CHD3 is critical for neuronal development and that the R1025W mutation hampers dendritic morphology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eChd3\u003csup\u003eR1025W/+\u003c/sup\u003e\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;mice exhibited decreased CHD3 level and abnormal behaviors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the role of CHD3-R1025W in the pathogenesis of SNIBCPS, we constructed \u003cem\u003eChd3\u003csup\u003eR1025W/+\u003c/sup\u003e\u003c/em\u003e knock-in mice with human allele substitutions utilizing CRISPR/Cas9-mediated gene targeting (Fig.2a and Extended Data Fig.3a). The CHD3 protein levels of \u003cem\u003eChd3\u003csup\u003eR1025W/+\u003c/sup\u003e\u003c/em\u003e mice were largely decreased in a variety of brain regions compared to WT mice (Fig.2b and Extended Data Fig.3b). As CHD3 is widely expressed in the cortex and hippocampus, immunohistochemical staining was performed to examine the expression of CHD3 in the brain of \u003cem\u003eChd3\u003csup\u003eR1025W/+\u003c/sup\u003e\u003c/em\u003e mice. We found that the fluorescence intensity of CHD3 signals in the \u003cem\u003eChd3\u003csup\u003eR1025W/+\u003c/sup\u003e\u0026nbsp;\u003c/em\u003emicewas reduced in retrosplenial cortex (RSC), parietal association cortex (PtA), primary somatosensory cortex trunk area (S1Tr), primary somatosensory cortex barrel field area (S1BF), CA1, CA2, CA3 and dentate gyrus (DG) compared to WT mice (Extended Data Fig.3c).\u003c/p\u003e\n\u003cp\u003eAs nearly 90% of individuals with SNIBCPS present with speech disorders, we conducted infant ultrasonic vocalization (USV) tests on \u003cem\u003eChd3\u003csup\u003eR1025W/+\u003c/sup\u003e\u003c/em\u003e newborn pups following maternal separation. Consistent with other mouse models of speech disorders, \u003cem\u003eChd3\u003csup\u003eR1025W/+\u003c/sup\u003e\u003c/em\u003e pups vocalized less frequently and for shorter durations compared to their WT littermates (Fig. 2c). Additionally, these pups produced fewer U-shaped inverted and complex vocalizations, indicative of a dysphonia-like disorder (Fig. 2c).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGiven that over 70% of individuals with SNIBCPS experience intellectual disabilities, we assessed whether the \u003cem\u003eChd3-R1025W\u003c/em\u003e mutation impaired cognitive functions in mice. In the novel object recognition test, \u003cem\u003eChd3\u003csup\u003eR1025W/+\u003c/sup\u003e\u003c/em\u003e mutant mice spent significantly less time exploring novel objects and exhibited a reduced recognition preference index, highlighting impaired cognitive recognition abilities (Fig. 2d). Since over 30% of individuals with SNIBCPS exhibit traits associated with autism spectrum disorder (ASD), we further examined autistic-like behaviors in \u003cem\u003eChd3\u003csup\u003eR1025W/+\u003c/sup\u003e\u003c/em\u003e mutant mice, with a focus on social interactions and repetitive stereotypical behaviors. In the three-chamber test, mutant mice showed a significant reduction in social novelty preference, indicating impaired social behaviors. Additionally, marble-burying and self-grooming tests revealed markedly increased repetitive stereotyped behaviors compared to their WT littermates (Fig. 2e, f). Collectively, \u003cem\u003eChd3\u003csup\u003eR1025W/+\u003c/sup\u003e\u003c/em\u003e mice recapitulated core behavioral abnormalities observed in SNIBCPS, including dysphonia, intellectual disability, and autistic-like traits.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDesign of the TeABE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo screen various TeABE-sgRNA combinations and identify the most efficient tool for correcting \u003cem\u003eChd3-R1025W\u003c/em\u003e, we established a human \u003cem\u003eChd3\u003csup\u003eR1025W/R1025W\u003c/sup\u003e\u003c/em\u003e cell line using AeCBE-mediated base editing\u003csup\u003e32\u003c/sup\u003e.\u0026nbsp;Initially, we designed two candidate sgRNAs (sgRNA-C10, sgRNA-C11) (Fig. 3a) and two AeCBEs (Fig. 3b), using cytidine deaminases APOBEC3A(Y130F) and LpCDA1L1_1\u003csup\u003e34,35\u003c/sup\u003e respectively. These combinations were transfected into HEK293 cells, followed by selection of positive monoclonal colonies using puromycin resistance (Fig. 3c). The successful construction of the point mutation cell line was confirmed via Sanger sequencing (Fig. 3d), and a \u003cem\u003eCHD3\u003csup\u003eR1025W/R1025W\u003c/sup\u003e\u003c/em\u003ehomozygous cell line was identified (Fig. 3e) that showed no detectable bystander editing effects.\u003c/p\u003e\n\u003cp\u003eSubsequently, we designed multiple sgRNAs (sgRNA-A4 to sgRNA-A15, Fig. 3f) and developed several TeABEs by inserting TadA constructs—­comprising a loss-of-function TadA as the scaffold and TadA-F148A as the functional unit—at various internal positions within spCas9\u003csup\u003e36\u003c/sup\u003e (Fig. 3g). These sgRNA and ABE combinations were transfected into the \u003cem\u003eChd3\u003csup\u003eR1025W/R1025W\u003c/sup\u003e\u003c/em\u003e cell line, and double-positive cells were sorted using FACS 72 hours post-transfection (Fig. 3h). Among the tested combinations, TeABE-1249 (TadA inserted after the 1249\u003csup\u003eth\u003c/sup\u003e amino acid of Cas9) paired with sgRNA-A11 exhibited the highest on-target base editing efficiency at the A11 site (Fig. 3i). However, this combination also showed over 30% editing efficiency at a bystander site (A13), raising concerns about potential side effects of base editing (Fig. 3i).\u003c/p\u003e\n\u003cp\u003eTo minimize bystander editing, we systematically evaluated the editing efficiency at A11 and A13 across a series of TeABE variants (Fig. 3j). Notably, TeABE-1248 (Δ1249-1263) achieved the highest efficacy at A11 while significantly reducing editing at A13 (Fig. 3j). This improvement is likely attributed to the removal of flexible sequences around the 1249 insertion site, which otherwise increase TadA access to mismatched off-target sites. To assess the off-target potential of TeABE variants, we performed Guide-seq analysis and identified three potential off-target sites (Fig. 3k). Further analysis revealed that TeABE-1248 (Δ1249-1263) greatly minimized off-target editing, particularly at OT3 (Fig. 3l). Based on these findings, we selected TeABE-1248 (Δ1249-1263) as the optimized tool for subsequent \u003cem\u003ein vivo\u003c/em\u003e genetic correction experiments in the \u003cem\u003eChd3\u003csup\u003eR1025W/+\u003c/sup\u003e\u003c/em\u003e mouse model.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vivo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;base editing by TeABE in \u003cem\u003eChd3\u003csup\u003eR1025W/+\u003c/sup\u003e\u003c/em\u003e mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe first evaluated the delivery efficiency of AAV-PHP.eB vectors using AAV-hSyn-GFP administered via tail intravenous injection. Widespread expression of AAV-PHP.eB vectors was observed throughout the mouse brain (Fig. 4a). To assess \u003cem\u003ein vivo\u003c/em\u003e on-target base editing efficiency, next-generation sequencing was performed on cerebral cortical tissues from \u003cem\u003eChd3\u003csup\u003eR1025W/+\u003c/sup\u003e\u003c/em\u003e mice treated with either GFP or TeABE. Following TeABE injection, a significantly higher proportion of reads with target base editing was detected in the cerebral cortex compared to GFP-treated controls, indicating successful base correction in the \u003cem\u003eChd3\u003csup\u003eR1025W/+\u003c/sup\u003e\u003c/em\u003e brain (Extended Data Fig. 4a). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eImmunofluorescent staining of the SpCas9 confirmed efficient expression of the dual AAV-TeABE system in cortical regions, including the retrosplenial cortex (RSC), parietal association cortex (PtA), primary somatosensory cortex trunk (S1Tr), and barrel field (S1BF), as well as hippocampal areas such as CA1, CA2, CA3, and the dentate gyrus (DG) (Fig. 4b). Restoration of CHD3 protein levels in these brain regions was confirmed, consistent with the SpG expression results (Fig. 4c). Immunoblot analysis of WT mice injected with AAV-GFP and \u003cem\u003eChd3\u003csup\u003eR1025W/+\u003c/sup\u003e\u003c/em\u003e mutant mice treated with GFP or TeABE further demonstrated a significant increase in CHD3 protein levels after TeABE delivery (Fig. 4d). These findings confirm the effectiveness of AAV-PHP.eB-mediated Cas9 transduction and \u003cem\u003ein vivo\u003c/em\u003e base editing for correcting \u003cem\u003eChd3-R1025W\u003c/em\u003e in the mouse brain.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTeABE improves the behavioral abnormalities of \u003cem\u003eChd3\u003csup\u003eR1025W/+\u003c/sup\u003e\u003c/em\u003e mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the therapeutic effects of TeABE treatment in \u003cem\u003eChd3\u003csup\u003eR1025W/+\u003c/sup\u003e\u003c/em\u003e mutant mice, we conducted a series of behavioral experiments. Intellectual disability, a hallmark symptom of SNIBCPS, was assessed using the three-chamber novel object recognition test and the Barnes maze test to measure cognitive and spatial learning abilities. In the novel object recognition test, treated mice exhibited a restored preference for novel objects and a significant increase in the recognition preference index, which was absent in untreated \u003cem\u003eChd3\u003csup\u003eR1025W/+\u003c/sup\u003e\u003c/em\u003e mice (Fig. 5a, Extended Data Fig. 5a). Similarly, in the Barnes maze test, treated mice demonstrated a shorter latency to locate the target hole during test 1 on day 6 and test 2 on day 13, reflecting enhanced spatial learning and memory abilities following treatment (Fig. 5b).\u003c/p\u003e\n\u003cp\u003eAutistic-like phenotypes, including social impairments and repetitive stereotyped behaviors, are common in SNIBCPS. To evaluate social competence, we employed the three-chamber social interaction test and the social intruder test. In the three-chamber test, treated mice spent significantly more time sniffing stranger mice compared to untreated \u003cem\u003eChd3\u003csup\u003eR1025W/+\u003c/sup\u003e\u003c/em\u003e mice, with a concurrent rise in the social preference index, indicating amelioration of social deficits (Fig. 5c, Extended Data Fig. 5b). Similar improvements were observed in the social intruder test, where treated mice exhibited increased interaction times during both the T1 and T5-T4 trials, reflecting enhanced sociability and social novelty recognition (Fig. 5d). While untreated mutant mice lacked social familiarity and novelty processes (e.g., changes in sniffing time), these processes were restored in treated mice (Fig. 5e).\u003c/p\u003e\n\u003cp\u003eTo assess repetitive stereotyped behaviors, we performed marble-burying and self-grooming experiments. Treated mice displayed a significant reduction in the number of buried marbles and self-grooming duration compared to untreated mice, indicating improved stereotyped behaviors (Fig. 5f). Anxiety levels were examined using the elevated plus maze and open field tests. No significant differences were observed among the groups in terms of time spent in open arms, entries to open arms, or exploratory activity (Extended Data Fig. 5c, d), suggesting that the \u003cem\u003eChd3-R1025W\u003c/em\u003e mutation does not impact anxiety. In summary, TeABE treatment effectively alleviated autistic-like behaviors, including social deficits and stereotyped behaviors, in \u003cem\u003eChd3\u003csup\u003eR1025W/+\u003c/sup\u003e\u003c/em\u003e mutant mice.\u003c/p\u003e\n\u003cp\u003eHypotonia, a common symptom of SNIBCPS, is characterized by involuntary, sustained muscle contractions that result in abnormal posture and movement disorders, including gait abnormalities, altered stride, and reduced grip strength in mice. In the tail suspension experiment, \u003cem\u003eChd3\u003csup\u003eR1025W/+\u003c/sup\u003e\u003c/em\u003e mutant mice were evaluated for limb tightness and scored for clasping (Fig. 6a,). Treated mice exhibited significantly lower scores compared to untreated mutants, indicating a reduction in involuntary muscle contractions following treatment (Fig. 6a). Additionally, SNIBCPS is frequently associated with joint abnormalities. Joint laxity scores revealed that treated mice displayed improved joint stability, accompanied by a significant reduction in both redness and swelling of the joints compared to untreated mutant mice (Fig. 6b). Gait analysis further assessed locomotor abnormalities associated with hypotonia. Treated mice demonstrated improvements, including a reduced hindlimb foot angle, decreased hindlimb spacing, and increased stride length, with values approaching those of WT controls (Fig. 6c, d). These results suggest that TeABE treatment enhanced locomotor coordination and normalized gait patterns.\u003c/p\u003e\n\u003cp\u003eGrip strength was evaluated using the rotarod test, which measures the ability of mice to maintain their grip on a rotating rod as the speed increases. Untreated \u003cem\u003eChd3\u003csup\u003eR1025W/+\u003c/sup\u003e\u003c/em\u003e mutant mice fell at lower speeds, whereas treated mice remained on the rod for longer durations, covering greater distances and enduring higher rotational speeds, reflecting significantly improved grip strength (Fig. 6e, Extended Data Video 1). Collectively, these findings demonstrate that TeABE treatment effectively alleviates hypotonia-related phenotypes in \u003cem\u003eChd3\u003csup\u003eR1025W/+\u003c/sup\u003e\u003c/em\u003e mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEfficient assembly of TeABE in the monkey brain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the feasibility of TeABE via the dual AAV strategy in clinical applications, we utilized a nonhuman primate model to examine the efficacy of delivery and assemble of TeABE in the primate brain. Dual vectors of TeABE using AAV9 capsid was administered through intrathecal injection to facilitate expression of the CHD3 base editor across brain regions (Fig. 7a). Immunoblot analysis demonstrated that the assemble efficiency of the base editor reached approximately 70% in various parts of the monkey brain (Fig. 7b). Furthermore, immunofluorescence analysis of the monkey brain revealed widespread expression of the base editor, with successful transduction observed in over 30% of neurons (Fig. 7c, d).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese results provide compelling evidence for the safety and efficacy of TeABE in a nonhuman primate model, paving the way to application of TeABE in clinical trials. This study represents a significant advancement in gene therapy for brain disorders and highlights the transformative potential of base editing in treating neurological diseases.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSnijders Blok-Campeau Syndrome (SNIBCPS) is a rare autosomal genetic disorder characterized by language and intellectual development impairments, along with other symptoms\u003csup\u003e1,5\u003c/sup\u003e. This condition imposes a significant burden on affected children and their families. Despite the severity of the disorder, no effective treatment targeting SNIBCPS has been reported. The primary association of SNIBCPS with specific mutations in the \u003cem\u003eCHD3\u003c/em\u003e gene highlights gene therapy as a promising therapeutic strategy\u003csup\u003e1,2\u003c/sup\u003e. In this study, we developed and applied a base editor to repair the c.C3073T point mutation and assessed its therapeutic potential in \u003cem\u003eChd3\u003csup\u003eR1025W/+\u003c/sup\u003e\u003c/em\u003e mutant mice. Behavioral experiments demonstrated significant improvements in key symptoms, including cognitive deficits, spatial learning and memory impairments, autistic-like behaviors, and hypotonia, underscoring the efficacy of our approach.\u003c/p\u003e\n\u003cp\u003eOur investigation into the pathogenic mechanism of the \u003cem\u003eChd3-R1025W\u003c/em\u003e mutation revealed that it accelerates the degradation of CHD3 protein, leading to reduced protein levels and associated neuronal abnormalities. While our findings provide insights into the molecular impact of this mutation, further mechanistic studies are necessary to elucidate how the mutation affects CHD3's chromatin remodeling functions and contributes to disease pathology. By employing base editing, we successfully corrected the single-nucleotide variant (SNV), restoring CHD3 protein levels both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. These results emphasize the potential of gene editing as a precise therapeutic strategy for addressing genetic defects and mitigating disease symptoms.\u003c/p\u003e\n\u003cp\u003eCompared to the CRISPR-Cas9 system, base editing offers a safer alternative as it avoids inducing double-strand DNA breaks\u003csup\u003e23-25\u003c/sup\u003e. Base editing has already demonstrated success in clinical trials for repairing genetic defects in various organs\u003csup\u003e26,27,37,38\u003c/sup\u003e. However, applying this technology to the central nervous system (CNS), particularly the brain, poses unique challenges due to its complexity and the risks associated with intervention\u003csup\u003e39\u003c/sup\u003e. While base editing has shown promise in nonhuman primate models for non-CNS applications\u003csup\u003e40,41\u003c/sup\u003e, its use in the CNS has been limited to mouse models. These models are valuable for evaluating phenotype improvements but fall short of providing guidance on clinical dosing or application in humans. Bridging the gap between preclinical research and clinical translation remains a significant challenge.\u003c/p\u003e\n\u003cp\u003eGiven the large size of the base editor used in this study, we employed a dual-vector delivery strategy for assembly within cells. However, this approach occasionally resulted in unsuccessful splicing, thereby reducing efficiency. In our nonhuman primate model, we observed a splicing efficiency of approximately 70%. To address this limitation, optimizing the viral delivery system is critical. High-capacity adenoviral vectors (HCAds), which offer larger packaging capacities\u003csup\u003e42-44\u003c/sup\u003e, or the development of smaller Cas homologs\u003csup\u003e45\u003c/sup\u003e, could enable single-vector packaging, eliminating the need for splicing and enhancing the system’s overall efficacy.\u003c/p\u003e\n\u003cp\u003eIn conclusion, this study demonstrates the feasibility and therapeutic potential of base editing for SNIBCPS and highlights critical areas for future optimization. These findings represent a significant step toward developing effective gene therapy strategies for brain disorders and pave the way for broader clinical applications of base editing technologies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the families with SNIBCPS patients for their participation and support in this study. We thank Yonghong Wang, Linxiao Yue, and Song Guo for excellent technical assistance. We thank Yandong Li for consulting on veterinarian regulations. K.Y. was funded by the Youth Innovation Promotion Association Chinese Academy of Sciences (2022269), the Youth Science Fund of State Key Laboratory of Neuroscience (SKLN-2022B006) and the National Natural Science Foundation of China (NSFC, 82001211). W-K.L was funded by NSFC (32400933). F.L. was funded by NSFC (82430104, 82125032, 81930095), the China Brain Initiative Grant (STI2030-Major Projects 2021ZD0200800), the Science and Technology Commission of Shanghai Municipality (19410713500, 23Y21900500 and 2018SHZDZX01), the Shanghai Municipal Commission of Health and Family Planning (GWVI-11.1-34, 2020CXJQ01, 2018YJRC03), Innovative research team of high-level local universities in Shanghai (SHSMU-ZDCX20211100), and the Guangdong Key Project (2018B030335001). T-L.C. was funded by NSFC (32371144). Z-L.Q. was funded by\u0026nbsp;NSFC (82430046).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK.Y., T-L.C. and Z-L.Q. designed this study. K.Y. and W-K.L. performed the plasmid construction. K.Y., Z-K.X., R-C.X., C-X.L., J.W., R.Z. and Y-X.G. carried out primary neuronal culture and molecular experiments. Y-B.C., Y-T.Y., Y-F.Z. and K.Y. performed construction, identification and management of point mutant mice. W-K.L., X-Y.Z., J.L., S-Q.Z. and T-L.C. performed the editor design and \u003cem\u003ein vitro\u003c/em\u003e efficiency verification. K.Y., Y-X.G., Y-T.Y. and W-K.L. performed mouse virus injection and \u003cem\u003ein vivo\u003c/em\u003e editing efficiency verification. K.Y., T-Y.Z., Z-H.L., W-X.W. and J-W.W. performed animal behavioral experiments. Z-Y.Y., L.G., W.Z., S-H.W., W-X.Y., J-S.W., K.Y. and Y-X.G. performed intrathecal injection and expression efficiency verification in monkeys. F.L., Z-L.Q., K.Y., T-L.C. and W-K.L. provided the experimental funds. K.Y., Y-X.G. and Z-L.Q. wrote the manuscript. Z-L.Q., T-L.C., F.L. and J-S.L. revised the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eSnijders Blok, L.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e CHD3 helicase domain mutations cause a neurodevelopmental syndrome with macrocephaly and impaired speech and language. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 4619 (2018). https://doi.org:10.1038/s41467-018-06014-6\u003c/li\u003e\n \u003cli\u003eTie, X.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Insights From a Novel Splicing Variant and Recurrent Arginine Variants in the CHD3 Gene Causing Snijders Blok-Campeau Syndrome. \u003cem\u003eAm J Med Genet A\u003c/em\u003e, e63930 (2024). https://doi.org:10.1002/ajmg.a.63930\u003c/li\u003e\n \u003cli\u003eCoursimault, J.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Hypersociability associated with developmental delay, macrocephaly and facial dysmorphism points to CHD3 mutations. \u003cem\u003eEur J Med Genet\u003c/em\u003e \u003cstrong\u003e64\u003c/strong\u003e, 104166 (2021). https://doi.org:10.1016/j.ejmg.2021.104166\u003c/li\u003e\n \u003cli\u003eMizukami, M.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e A de novo CHD3 variant in a child with intellectual disability, autism, joint laxity, and dysmorphisms. \u003cem\u003eBrain Dev\u003c/em\u003e \u003cstrong\u003e43\u003c/strong\u003e, 563-565 (2021). https://doi.org:10.1016/j.braindev.2020.12.004\u003c/li\u003e\n \u003cli\u003eDrivas, T. 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Here, we report a \u003cem\u003ede novo\u003c/em\u003e single-nucleotide variant (c.C3073T, p.R1025W) in \u003cem\u003eCHD3\u003c/em\u003e identified in a child with SNIBCPS, which leads to accelerated degradation of the CHD3 protein. Using a \u003cem\u003eChd3\u003c/em\u003e\u003csup\u003e\u003cem\u003eR1025W/+\u003c/em\u003e\u003c/sup\u003e knock-in mouse model, we observed impaired vocalization, cognition, and autism-like behaviors. To address these deficits, we developed an improved TadA-embedded adenine base editor (TeABE) and delivered into the mouse brain via Adeno-associated virus (AAV). Base editing \u003cem\u003ein vivo\u003c/em\u003e significantly restored CHD3 protein levels in the mouse brain and ameliorated various behavioral abnormalities. Furthermore, we validated the AAV-mediated delivery efficacy of TeABE in nonhuman primate, highlighting its translational potential. These findings establish \u003cem\u003ein vivo\u003c/em\u003e base editing as a promising therapeutic strategy for SNIBCPS and pave the way for clinical applications targeting brain disorders.\u003c/p\u003e","manuscriptTitle":"Behavioral Improvement Through In Vivo Base Editing in a Mouse Model of Snijders Blok-Campeau Syndrome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-28 10:29:48","doi":"10.21203/rs.3.rs-5690634/v1","editorialEvents":[],"status":"published","journal":{"display":false,"email":"[email protected]","identity":"nature","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nature","sideBox":"Learn more about [Nature](http://www.nature.com/nature/)","snPcode":"","submissionUrl":"","title":"Nature","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7dc45350-49c6-47f5-b4fc-0914c8ae128d","owner":[],"postedDate":"January 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":43377834,"name":"Health sciences/Health care/Therapeutics/Gene therapy/Targeted gene repair"},{"id":43377835,"name":"Health sciences/Diseases/Neurological disorders/Neurodevelopmental disorders/Autism spectrum disorders"}],"tags":[],"updatedAt":"2026-02-19T08:14:46+00:00","versionOfRecord":{"articleIdentity":"rs-5690634","link":"https://doi.org/10.1038/s41586-026-10113-6","journal":{"identity":"nature","isVorOnly":false,"title":"Nature"},"publishedOn":"2026-02-18 05:00:00","publishedOnDateReadable":"February 18th, 2026"},"versionCreatedAt":"2025-01-28 10:29:48","video":"","vorDoi":"10.1038/s41586-026-10113-6","vorDoiUrl":"https://doi.org/10.1038/s41586-026-10113-6","workflowStages":[]},"version":"v1","identity":"rs-5690634","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5690634","identity":"rs-5690634","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}