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AAV-mediated FOXG1 gene editing in human Rett primary cells


Variations in the Forkhead Box G1 (FOXG1) gene cause FOXG1 syndrome spectrum, including the congenital variant of Rett syndrome, characterized by early onset of regression, Rett-like and jerky movements, and cortical visual impairment. Due to the largely unknown pathophysiological mechanisms downstream the impairment of this transcriptional regulator, a specific treatment is not yet available. Since both haploinsufficiency and hyper-expression of FOXG1 cause diseases in humans, we reasoned that adding a gene under nonnative regulatory sequences would be a risky strategy as opposed to a genome editing approach where the mutated gene is reversed into wild-type. Here, we demonstrate that an adeno-associated viruses (AAVs)-coupled CRISPR/Cas9 system is able to target and correct FOXG1 variants in patient-derived fibroblasts, induced Pluripotent Stem Cells (iPSCs) and iPSC-derived neurons. Variant-specific single-guide RNAs (sgRNAs) and donor DNAs have been selected and cloned together with a mCherry/EGFP reporter system. Specific sgRNA recognition sequences were inserted upstream and downstream Cas9 CDS to allow self-cleavage and inactivation. We demonstrated that AAV serotypes vary in transduction efficiency depending on the target cell type, the best being AAV9 in fibroblasts and iPSC-derived neurons, and AAV2 in iPSCs. Next-generation sequencing (NGS) of mCherry+/EGFP+ transfected cells demonstrated that the mutated alleles were repaired with high efficiency (20–35% reversion) and precision both in terms of allelic discrimination and off-target activity. The genome editing strategy tested in this study has proven to precisely repair FOXG1 and delivery through an AAV9-based system represents a step forward toward the development of a therapy for Rett syndrome.

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Fig. 1: Plasmid strategy and design.
Fig. 2: Transfection experiments.
Fig. 3: Sequencing analysis in edited patient-derived cells and PAX6 analysis.
Fig. 4: Infection in patient-derived cells.


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We thank FOXG1 patients and their families. The “Cell lines and DNA bank of Rett Syndrome, X-linked mental retardation, and other genetic diseases,” member of the Telethon Network of Genetic Biobanks (project nos. GTB12001 and GFB18001), funded by Telethon Italy, and of the EuroBioBank network provided us with specimens. We thank Anna Cereseto, Antonio Casini, and Giulia Maule (Laboratory of Molecular Virology Department of Cellular, Computational, and Integrative Biology—CIBIO-Trento) for performing GUIDE-seq analysis and for critically revising the paper. This work was supported by a grant from FOXG1 Research Foundation. This work is generated within the ERN ITHACA (European Reference Network for Intellectual Disability Telehealth and Congenital Anomalies). We thank SienaGenTest srl, a Spin-off of the University of Siena ( for gene editing efficiency analysis.


This work was supported by a grant from FOXG1 Research Foundation for the project “A glimpse of hope for FOXG1.”

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SC, MLC, KC, AR, CS, and IM have made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data and have been involved in drafting the paper. SD, FD, FTP, EF, FN, RT, AG, SF, and EB have made substantial contributions to acquisition and analysis of the data. DL, VL, and CL have made substantial contributions to interpretation of data and clinical evaluation. All authors have been involved in drafting the paper; they have given final approval of the version to be published and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

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Correspondence to Alessandra Renieri.

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The study was approved by Azienda Ospedaliera Universitaria Senese Ethics Committee, Prot Name CRI, Prot n 12362_2018.

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Informed consent was provided to the patients before blood drowning and skin biopsies.

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Croci, S., Carriero, M.L., Capitani, K. et al. AAV-mediated FOXG1 gene editing in human Rett primary cells. Eur J Hum Genet 28, 1446–1458 (2020).

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