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Whole-brain in vivo base editing reverses behavioral changes in Mef2c-mutant mice

Nature Neuroscience volume 27, pages 116–128 (2024)Cite this article

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Abstract

Whole-brain genome editing to correct single-base mutations and reduce or reverse behavioral changes in animal models of autism spectrum disorder (ASD) has not yet been achieved. We developed an apolipoprotein B messenger RNA-editing enzyme, catalytic polypeptide-embedded cytosine base editor (AeCBE) system for converting C·G to T·A base pairs. We demonstrate its effectiveness by targeting AeCBE to an ASD-associated mutation of the MEF2C gene (c.104T>C, p.L35P) in vivo in mice. We first constructed Mef2cL35P heterozygous mice. Male heterozygous mice exhibited hyperactivity, repetitive behavior and social abnormalities. We then programmed AeCBE to edit the mutated C·G base pairs of Mef2c in the mouse brain through the intravenous injection of blood–brain barrier-crossing adeno-associated virus. This treatment successfully restored Mef2c protein levels in several brain regions and reversed the behavioral abnormalities in Mef2c-mutant mice. Our work presents an in vivo base-editing paradigm that could potentially correct single-base genetic mutations in the brain.

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Fig. 1: A de novo mutation in the MEF2C gene associated with ASD causes rapid degradation of the MEF2C protein.
Fig. 2: Reduced Mef2c protein levels and autistic-like behaviors in Mef2cL35P+/− mice.
Fig. 3: Design and validation of the AeCBE system.
Fig. 4: Restoration of Mef2c protein levels and abnormal inhibitory interneuron development in Mef2cL35P+/− mice after AeCBE editing.
Fig. 5: Restoration of spine density and impaired synaptic transmission in the medial PFC layer II and III neurons in Mef2cL35P+/− mice after AeCBE editing.
Fig. 6: Amelioration of autistic-like behaviors in Mef2cL35P+/− mice with AeCBE editing in vivo.

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Data availability

The datasets generated and analyzed during the present study are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

Code availability

The present study does not contain any customized code.

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Acknowledgements

We thank the ASD family for their participation in this study. We thank A. Gitler and members of the NPC for their critical comments on the manuscript. This work is supported by the Strategic Priority Research Program of the Chinese Academy of Sciences no. XDB32060202 (Z.-L.Q.), Fundamental Research Funds for the Central Universities (Z.-L.Q.), Natural Science Foundation of China grants to Z.-L.Q. (nos. 31625013, 82021001), T.-L.C. (nos. 32371144, 31600826), B.Y. (no. 32000726), and W.-J.H. (no. 82201632), Program of Shanghai Academic Research Leader no. 19XD1404300 (Z.-L.Q.), Shanghai Municipal Science and Technology Major Project (no. 2018SHZDZX05) (Z.-L.Q.), National Key R&D Program of China no. 2019YFA0111000 (T.-L.C.), Natural Science Foundation of Shanghai no. 20ZR1403100 (T.-L.C.), Shanghai Municipal Science and Technology no. 20JC1419500 (T.-L.C.), the Lingang Laboratory (no. LG-QS-202203-08) to T.-L.C, and Innovative Research Team of High-Level Local Universities in Shanghai (no. SHSMU-ZDCX20211100) (Z.-L.Q.). Z.-L.Q. is supported by the Guang Ci Professorship Program of Ruijin Hospital Shanghai Jiao Tong University School of Medicine.

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Author notes

  1. Wei-Ke Li

    Present address: Institute of Pediatrics, National Children’s Medical Center, Children’s Hospital, Institute for Translational Brain Research, State Key Laboratory of Medical Neurobiology, MOE Frontiers Center for Brain Science, Fudan University, Shanghai, China

  2. These authors contributed equally: Wei-Ke Li, Shu-Qian Zhang.

Authors and Affiliations

  1. Songjiang Research Institute, Songjiang Hospital & MOE-Shanghai Key Laboratory for Children’s Environmental Health, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Wei-Ke Li, Yi-Ting Yuan, Wen-Jian Han & Zi-Long Qiu

  2. Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, China

    Wei-Ke Li, Wan-Ling Peng, Yu-Han Shi, Bo Yuan, Jin-Cheng Wang, Zhi-Fang Chen, Shi-Fang Shan, Shu-Jia Zhu & Zi-Long Qiu

  3. Institute of Pediatrics, National Children’s Medical Center, Children’s Hospital, Institute for Translational Brain Research, State Key Laboratory of Medical Neurobiology, MOE Frontiers Center for Brain Science, Fudan University, Shanghai, China

    Shu-Qian Zhang, Jin-Long Chen, Cheng Zhang & Tian-Lin Cheng

  4. Department of Pediatrics, Qilu Hospital of Shandong University, Jinan, China

    Shu-Qian Zhang

  5. Department of Anesthesiology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Zhen-Yu Xue

  6. Institutes of Brain Science, State Key Laboratory of Medical Neurobiology, Ministry of Education Frontiers Center for Brain Science, Fudan University, Shanghai, China

    Bi-Qing Xue & Yi-Lin Tai

  7. Clinical Neuroscience Center, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Zi-Long Qiu

  8. National Clinical Research Center for Aging and Medicine, Huashan Hospital, Fudan University, Shanghai, China

    Zi-Long Qiu

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  1. Wei-Ke Li
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Contributions

T.-L.C. and Z.-L.Q. conceptualized the study. S.-Q.Z., C.Z. and J.-L.C. performed the base editing and plasmid construction. W.-K.L. and S.-J.Z. carried out the biochemical experiments. W.-K.L. and S.-Q.Z. carried out the cell experiments. W.-K.L., Y.-T.Y., Z.-Y.X., S.-F.S. and Z.-F.C. performed the mouse immunohistochemistry and behavioral experiments. W.-K.L., B.-Q.X., S.-Q.Z., C.Z. and Y.-L.T. carried out the laser dissections. Y.-H.S. and W.-K.L. performed the electrophysiology experiments. S.-Q.Z. and T.-L.C. carried out the GUIDE-seq. W.-K.L., S.-Q.Z. and W.-J.H. prepared the NGS samples. B.Y. carried out the NGS data analysis. W.-K.L. and J.-C.W. carried out the analysis of the animal data. W.-L.P. and W.-K.L. carried out the viral injections and analysis. W.-K.L., T.-L.C. and Z.-L.Q. wrote the manuscript.

Corresponding authors

Correspondence to Tian-Lin Cheng or Zi-Long Qiu.

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Extended data

Extended Data Fig. 1 Expression of MEF2C in the human brain and design of mouse Mef2c shRNA constructs.

(a) Annotation of the genomic and transcript information of L35P (based on the hg38 database). (b) Human brain transcriptomic data for MEF2C was obtained from the Human Brain Transcriptome (HBT), which ranges from embryonic development (periods 3-7) to postnatal development (periods 8–12) to adulthood (periods 13–15). Brain region abbreviations as follows, Hippocampus (HIP), neocortex (NCX), cerebellar cortex (CBC), mediodorsal nucleus of the thalamus (MD), striatum (STR), and amygdala (AMY). (c) Western blotting of MEF2C WT, L35F, L35P, L35I, or L35A expressed in HEK293 cells. (d) Quantifying relative MEF2C protein levels normalized to GAPDH (n = 7 per group). WT versus L35F: P < 0.0001; WT versus L35P: P < 0.0001; WT versus L35I: P = 0.0005; WT versus L35A: P = 0.0003, One-way ANOVA. (e) Schematic illustration of two Mef2c shRNA designed based on BLOCK-iT™ RNAi Designer (http://rnaidesigner.thermofisher.com/rnaiexpress/). (f) Quantifying Mef2c mRNA level of cultured primary cortical neurons infected with lentiviral vectors expressing scrambled or two Mef2c shRNA (P < 0.0001, n = 4 per group, One-way ANOVA). (g) Representative western blotting of cultured primary cortical neurons infected with lentiviral vectors expressing scrambled or Mef2c shRNA-1 for 72 h. Cells were harvested at DIV5. GAPDH was used as the internal control. (h) Quantitative analysis of relative Mef2c protein expression. Neurons infected with scrambled shRNA were normalized to 100% (P = 0.0327, n = 3, unpaired two-tailed Student’s t-test). n is biological repeat numbers of independent experiments. Statistical values represent the mean ± s.e.m. *P < 0.05, ***P < 0.001, ****P < 0.0001.

Source data

Extended Data Fig. 2 MEF2C-WT, but not MEF2C-L35P, could rescue impaired neuronal dendritic and axonal morphological development in Mef2c knockdown neurons.

(a) Representative image of immunofluorescent staining for GFP (green), MAP2 (red), and DAPI (blue) of cultured E14.5 mouse cortical neurons. Mouse cortical neurons were transfected with DsRed shRNA (Ctrl, n = 39), shMef2c (n = 36), shMef2c with human MEF2C-WT (n = 40) or MEF2C-L35P (n = 32) respectively at DIV1 via Lipofectamine 3000. Neuronal cells were immunofluorescent stained at DIV10. Scale bars, 60μm. Quantitative analysis of neuronal total dendritic length (b) and branch numbers (c) of neurons transfected with Ctrl, shMef2c, shMef2c + MEF2C-WT, and shMef2c + MEF2C-L35P. (b: all P < 0.0001; c: shMef2c+WT versus shMef2c + L35P: P = 0.0002) (d) Representative immunofluorescent staining images for GFP (green), MAP2 (red), and DAPI (blue) of cultured E14.5 mouse cortical neurons. Neuronal cells were transfected with DsRed shRNA (Ctrl, n = 34), shMef2c (n = 37), shMef2c with human MEF2C-WT (n = 33) or MEF2C-L35P (n = 34) respectively at DIV1. Neuronal cells were immunofluorescent stained at DIV4. Scale bars, 60μm. Quantitative analysis of neuronal axonal (e) and total neurite length (f) of neurons transfected with Ctrl, shMef2c, shMef2c + MEF2C-WT, and shMef2c + MEF2C-L35P. n is total neuron numbers collected from more than 3 independent experiments. (e, f: all P < 0.0001) Statistical values represent the mean ± s.e.m. ***P < 0.001, ****P < 0.0001, (b, c, e, f) One-way ANOVA.

Extended Data Fig. 3 Decreased protein expression and aberrant inhibitory interneuron development in Mef2c L35P+/- mice.

(a) Design of Mef2c L35P knock-in mice by CRISPR/Cas9 mediated homologous recombination. (b) Representative images of immunohistochemical staining for PV (red), Mef2c (green) and DAPI (blue) in the retrosplenial cortex (white dotted box indicates corresponding brain region) of Mef2c WT and L35P+/- mice. Scale bars, 200μm. Quantitative analysis of PV-positive neuronal cell density in the retrosplenial cortex (c), dentate gyrus (d), the somatosensory cortex (e), and visual cortex (f) of Mef2c WT (n = 11 brain slices from 4 mice) and L35P+/- mice (n = 12 brain slices from 4 mice). (c: P < 0.0001; d: P < 0.0001; e: P = 0.0004; f: P < 0.0001) (g) Representative images of immunohistochemical staining for SST (red), Mef2c (green), and DAPI (blue) in the retrosplenial cortex (White dotted box indicates corresponding brain region) of Mef2c WT and L35P+/- mice. Scale bars, 200μm. Quantification of SST-positive neuronal cell density in the retrosplenial cortex (h), hippocampus (i), and somatosensory cortex (j) of Mef2c WT (n = 8 brain slices from 3 mice) and L35P+/- mice (n = 7 brain slices from 3 mice). Statistical values represent the mean ± s.e.m. ***P < 0.001, **** P < 0.0001, (c, d, e, f, h, i, j) unpaired two-tailed Student’s t-test.

Extended Data Fig. 4 General development and cognitive ability of Mef2c L35P+/- mice.

(a) Body weights of Mef2c WT and L35P+/- mice of both sex from postnatal one week to nine weeks. (WT, n = 29 male mice, n = 17 female mice; L35P+/-, n = 17 male mice, n = 21 female mice). (b) Representative heat maps of Mef2c WT and L35P+/- mice in the novel object recognition task (the circle indicates a familiar object; the white box indicates a stranger object). Quantification of cumulative sniffing time of Mef2c WT (c) and L35P+/- mice (d) in the task (WT, n = 14; L35P+/-, n = 12; c: P = 0.0018; d: P = 0.0235). (e) Representative heat maps of Mef2c WT and L35P+/- mice in the elevated plus maze (open arm indicates top and bottom arms; closed arm indicates left and right arms). Quantification of cumulative duration exploring in the open arm (f) and closed arm (g) of Mef2c WT and L35P+/- mice (n = 14; f: P = 0.0003; g: P = 0.0006). (h) Representative locomotion track traces of Mef2c WT and L35P+/- mice in the light/dark exploration test. (i) Quantitative analysis of cumulative duration of Mef2c WT and L35P+/- mice in the light area (n = 8 per group). (j) Quantitative analysis of cumulative self-grooming and scratching time of Mef2c WT and L35P+/- mice (n = 14, j: P = 0.0004). (k) Quantitative analysis of primary latency spent locating the target hole of Mef2c WT and L35P+/- mice in the Barnes maze (n = 14 per group). Quantification of cumulative duration exploring in the target (l) and opposite zone (m) of Mef2c WT and L35P+/- mice in the apparatus (n = 14 per group). n represents mice number. Statistical values represent the mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001, (a, k) Two-way ANOVA test, (c,d) paired two-tailed Student’s t-test, (f, g, i, j, l, m) unpaired two-tailed Student’s t-test.

Extended Data Fig. 5 Female Mef2c L35P+/- mice displayed abnormal social novelty ability and object recognition.

(a) Representative heat maps for locomotion of female Mef2c WT and L35P+/- mice on the social approach and social novelty session in the three-chamber test. Quantification of cumulative duration of interacting with a mouse and empty cage of Mef2c WT (b) and L35P+/- mice (c) (n = 8, b: P < 0.0001; c: P = 0.0024). (d) The preference index of social approach test for female Mef2c WT and L35P+/- mice. Quantification of cumulative duration of interacting with novel stranger mouse and familiar mouse of female Mef2c WT (e) and L35P+/- (f) mice (n = 8, e: P = 0.0002). (g) The preference index of social novelty test for female Mef2c WT and L35P+/- mice. (P = 0.046) (h) Representative heat maps of female Mef2c WT and L35P+/- mice in the novel object recognition task (the circle indicates a familiar object; the white box indicates a stranger object). Quantification of cumulative sniffing time of Mef2c WT (i) and L35P+/- mice (j) in the task (n = 8, i: P = 0.0095; j: P = 0.163). n represents mice number. Statistical values represent the mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, (b, c, e, f, i, j) paired two-tailed Student’s t-test, (d, g) unpaired two-tailed Student’s t-test.

Extended Data Fig. 6 Female Mef2c L35P+/- mice exhibited hyperactive locomotion without anxiety behavior.

(a) Representative locomotion track traces of female Mef2c WT and L35P+/- mice in the open field test. Quantitative analysis of total distance (b), mean velocity (c), and duration in the center zone (d) of Mef2c WT and L35P + /- mice in the open field test (n = 8, b: P = 0.0054; c: P = 0.0054). (e) Representative locomotion track traces of female Mef2c WT and L35P+/- mice in the light/dark exploration test. (f) Quantitative analysis of cumulative duration of female Mef2c WT and L35P+/- mice in the light area (n = 8 per group). Representative heat maps of Mef2c WT and L35P+/- mice in the elevated plus maze (open arms are top and bottom arms; closed arms are left and right). (g) Quantification of cumulative duration exploring in the open arm (h) and closed arm (i) of female Mef2c WT and L35P+/- mice (n = 8, h: P = 0.0114; i: P = 0.038). (j) Representative locomotion track traces of female Mef2c WT and L35P+/- mice in the Barnes maze. (k) Quantitative analysis of primary latency spent locating the target hole of female Mef2c WT and L35P+/- mice in the Barnes maze (n = 8 per group). Quantification of cumulative duration exploring in the target (l) and opposite zone (m) of female Mef2c WT and L35P+/- mice in the apparatus (n = 8 per group). n represents mice number. Statistical values represent the mean ± s.e.m. * P < 0.05, ** P < 0.01, (b, c, d, f, h, i, l, m) unpaired two-tailed Student’s t-test, (k) Two-way ANOVA test.

Extended Data Fig. 7 Design of AeCBE.

(a) Illustration of various designs for classic CBE and APOBEC3A-embedding CBE with different embedding positions. (b) Screening for base editing efficiency of various APOBEC3A-embedding CBEs using sgRNA indicated in the HEK293 cells. (c) Representative FACS gating strategy for GFP, mCherry, and double positive samples in Fig. 3c. Quantitative analysis of base editing efficiency (C-to-T conversion) of bystander sites C103(d), C108(e), and C112(f) after different CBE applications in cultured mouse cortical neurons (n = 3 per group; n represents independent experiments, One-way ANOVA test). Statistical values represent the mean ± s.e.m.

Extended Data Fig. 8 Immunohistochemistry of SpCas9 and NeuN i in the Mef2c L35P+/- mice and evaluation of Off-targeting effects by AeCBE in vivo.

(a) Immunohistochemistry of SpCas9 and NeuN in the prefrontal cortex of AAV-AeCBE injected mice. (b) Quantification of SpCas9 and NeuN double-positive neurons in the total NeuN cells (n = 7 slices from 3 mice). (c) Immunostaining of SpCas9 and NeuN in the hippocampus of AAV-AeCBE injected mice. (d) Quantification of SpCas9 and NeuN double-positive neurons in the total NeuN cells (n = 12 slices from 4 mice). (e) Immunohistochemistry images of Mef2c and NeuN in the hippocampus of AAV-AeCBE injected mice. (f) Quantification of Mef2c and NeuN double-positive neurons in the total NeuN cells (n = 12 slices from 4 mice). (g) Off-target sites detected by GUIDE-seq, with sgRNA targeted to Mef2c L35P region. More reads indicated more likely to be targeted by SgC8 and SpG-Cas9. (h) Venn diagrams of off-target sites detected by the in-silico method and the GUIDE-seq method. (i) Off-target editing analysis at all 11 candidate sites by next-generation sequencing in vivo. (n = 3 mice, OT2-C7: P < 0.0001; OT4-C8: P = 0.0214; OT5-C8: P = 0.0243). Statistical values represent the mean ± s.e.m. * P < 0.05, *** P < 0.001, unpaired two-tailed Student’s t-test.

Extended Data Fig. 9 In vivo CBE gene editing did not restore decreased PV-positive inhibitory interneuron in the visual and somatosensory cortex of Mef2c L35P+/- mice.

(a) Representative images of immunohistochemical staining for PV (red) and DAPI (blue) in the Dentate Gyrus. (b) Quantification of PV-positive neurons in each group. WT-EGFP versus L35P-EGFP: P < 0.0001; L35P-EGFP: P = 0.0007 (c) Representative images of immunohistochemical staining for PV (red) and DAPI (blue) in the visual cortex and somatosensory cortex (White dotted box indicates corresponding brain region) of Mef2c WT mice injected with AAV-EGFP, Mef2c L35P+/- mice injected with either EGFP or AeCBE. Scale bars, 500μm. Quantitative analysis of PV-positive neuronal cell density in the visual (d) and somatosensory cortex (e) of three groups (n = 10 brain slices from 4 mice per group, d: P < 0.0001; e: P < 0.0001). Statistical values represent the mean ± s.e.m. *** P < 0.001, **** P < 0.0001, (b, d, e) unpaired two-tailed Student’s t-test.

Extended Data Fig. 10 No restoration of abnormal behaviors in Mef2c L35P+/- male mice injection with non-targeting sgRNA(sgNC) and AeCBE.

(a) Representative heat maps for locomotion of male Mef2c WT and L35P+/- mice injected with AAV-sgNC-AeCBE in the three-chamber test. Quantification of cumulative duration of Mef2c WT (b) and L35P+/- mice (c) (n = 11; b, c: P < 0.0001). (d) The preference index of social approach for Mef2c WT and L35P+/- mice (P = 0.462). Quantification of cumulative duration of Mef2c WT (e) and L35P+/- mice (f) (n = 11; e: P = 0.0003). (g) The preference index of social novelty for Mef2c WT and L35P+/- mice (P = 0.0134). Mean cumulative sniffing time (black curve) and individual mice (gray curve) in the social intruder test for Mef2c WT (h) and L35P+/- mice (i) with AAV-sgNC-AeCBE. (h: all P < 0.0001) (j) Cumulative sniffing time in the first trial of the social intruder test for Mef2c WT and L35P+/- mice (n = 11, P < 0.0001). (k) The social recognition index of the social intruder test for Mef2c WT and L35P+/- mice (n = 11, P < 0.0001). (l) Representative locomotion track traces of Mef2c WT and L35P+/- male mice in the light/dark exploration test. (m) Quantitative analysis of cumulative duration of Mef2c WT and L35P+/- mice in the light area (n = 9). (n) Quantitative analysis of cumulative time spent self-grooming and scratching of Mef2c WT and L35P+/- mice with AAV-sgNC-AeCBE. (n = 11, P = 0.0005). (o) Representative locomotion track traces of Mef2c WT and L35P+/- mice in the open field test. Quantitative analysis of duration in the center zone (p), mean velocity (q), and total distance (r) in the open field test (n = 11, q: P = 0.0112; r: P = 0.0099). n represents mouse number. Statistical values represent the mean ± s.e.m. (b, c, e, f) paired two-tailed Student’s t-test, (d, g, j, k, m, n, p, q, r) unpaired two-tailed Student’s t-test, (h, i) One-way ANOVA test.

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Li, WK., Zhang, SQ., Peng, WL. et al. Whole-brain in vivo base editing reverses behavioral changes in Mef2c-mutant mice. Nat Neurosci 27, 116–128 (2024). https://doi.org/10.1038/s41593-023-01499-x

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