AAV-mediated FOXG1 gene editing in human Rett primary cells

Abstract

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.

Modeling Rett Syndrome with Human Pluripotent Stem Cells: Mechanistic Outcomes and Future Clinical Perspectives

“…Two major concerns regarding this potential therapeutic approach still persist, particularly viral toxicity and heterogenous distribution of MeCP2 gene expression throughout the brain, since local overexpression causes severe symptoms, as observed in MeCP2 duplication syndrome [ 111 ]. Recently, another publication showed that AAV9 coupled with the use of CRISPR/Cas9 was able to target and correct FOXG1 in both RTT hiPSCs and hiPSC-derived neurons, with an efficiency of 20–35% [ 112 ]. Very close to clinical trials is also an AAV-mediated MeCP2 gene expression cassette [ 113 ] with endogenous regulatory elements, already tested in pre-clinical mouse models [ 114 ].…”

Effects of homocysteine-induced endoplasmic reticulum protein on endoplasmic reticulum stress, autophagy, and neuronal apoptosis following intracerebral hemorrhage

“…After incubating for 4 h at 37 °C with 5% carbon dioxide, the medium was replaced with NEUROBASALTM medium supplemented with 2% B27. The neurons were used for experiments after 7‐d culture ( Croci et al, 2020 ; Yan et al, 2020 ).…”

Current and Future Prospects for Gene Therapy for Rare Genetic Diseases Affecting the Brain and Spinal Cord

“…Using MECP2-knockout mice, encouraging pre-clinical results with increased survival and body weight have been seen after intracisternal delivery of AAV vectors encoding MECP2 (Gadalla et al, 2017;Sinnett et al, 2017;Sandweiss et al, 2020). An alternative approach is the use of CRISPR/Cas9 genome editing that has been shown to be efficient at correcting FOXG1 variants in human RTT patient-derived fibroblasts and induced pluripotent stem-derived neurons using AAV9 vectors (Croci et al, 2020). Human clinical trials remain to be initiated.…”