Nuclease-free precise genome editing corrects MECP2 mutations associated with Rett syndrome

Rett syndrome is an acquired progressive neurodevelopmental disorder caused by de novo mutations in the X-linked MECP2 gene which encodes a pleiotropic protein that functions as a global transcriptional regulator and a chromatin modifier. Rett syndrome predominantly affects heterozygous females while affected male hemizygotes rarely survive. Gene therapy of Rett syndrome has proven challenging due to a requirement for stringent regulation of expression with either over- or under-expression being toxic. Ectopic expression of MECP2 in conjunction with regulatory miRNA target sequences has achieved some success, but the durability of this approach remains unknown. Here we evaluated a nuclease-free homologous recombination (HR)-based genome editing strategy to correct mutations in the MECP2 gene. The stem cell-derived AAVHSCs have previously been shown to mediate seamless and precise HR-based genome editing. We tested the ability of HR-based genome editing to correct pathogenic mutations in Exons 3 and 4 of the MECP2 gene and restore the wild type sequence while preserving all native genomic regulatory elements associated with MECP2 expression, thus potentially addressing a significant issue in gene therapy for Rett syndrome. Moreover, since the mutations are edited directly at the level of the genome, the corrections are expected to be durable with progeny cells inheriting the edited gene. The AAVHSC MECP2 editing vector was designed to be fully homologous to the target MECP2 region and to insert a promoterless Venus reporter at the end of Exon 4. Evaluation of AAVHSC editing in a panel of Rett cell lines bearing mutations in Exons 3 and 4 demonstrated successful correction and rescue of expression of the edited MECP2 gene. Sequence analysis of edited Rett cells revealed successful and accurate correction of mutations in both Exons 3 and 4 and permitted mapping of HR crossover events. Successful correction was observed only when the mutations were flanked at both the 5′ and 3′ ends by crossover events, but not when both crossovers occurred either exclusively upstream or downstream of the mutation. Importantly, we concluded that pathogenic mutations were successfully corrected in every Rett line analyzed, demonstrating the therapeutic potential of HR-based genome editing.

(1.4 kb) obtained with the edited hemizygous male S134C Rett syndrome cells with 5' TI assay (M: Marker; UT: Untransduced S134C cells; T: Transduced S134C cells).B. Sanger Sequence analysis of the edited MECP2 gene in hemizygous male S134C Rett syndrome cells.Shown are the chromatograms of 5' genome-vector junction, SNP in Intron 2, Linker 1 sequence and Linker 2 sequence, representing the 2 editing outcomes (B1 and B2) observed in S134C cells with the 5' TI assay.The primers used for 5' TI analyses are depicted as thick red arrows.

Figure S1. 5 '
Figure S1.5' Sequence analysis of edited MECP2 gene in S134C cells.A. Specific PCR product (1.4 kb) obtained with the edited hemizygous male S134C Rett syndrome cells with 5' TI assay (M: Marker; UT: Untransduced S134C cells; T: Transduced S134C cells).B. Sanger Sequence analysis of the edited MECP2 gene in hemizygous male S134C Rett syndrome cells.Shown are the chromatograms of 5' genome-vector junction, SNP in Intron 2, Linker 1 sequence and Linker 2 sequence, representing the 2 editing outcomes (B1 and B2) observed in S134C cells with the 5' TI assay.The primers used for 5' TI analyses are depicted as thick red arrows.

Figure S2. 3 '
Figure S2.3' Sequence analysis of edited MECP2 gene in S134C cells.A. Specific PCR product (2.7 kb) obtained with the edited hemizygous male S134C Rett syndrome cells with 3' TI assay (M: Marker; UT: Untransduced S134C cells; T: Transduced S134C cells).B. Sequence analysis of the edited MECP2 gene in hemizygous male S134C Rett syndrome cells.Shown are the chromatograms of Linker 2 sequence, codon encoding WT S or mutant C at position 134, Exon 4-3' UTR junction sequence and 3' vector-genome junction sequence, representing the 2 editing outcomes (C1 and C2) observed in S134C cells with the 3' TI assay.The primers used for 3' TI analyses are depicted as thick red arrows.

Figure S3. 5 '
Figure S3.5' Sequence analysis of edited MECP2 gene in R106W cells A. Specific PCR product (1.5 kb) obtained with the edited heterozygous female R106W Rett syndrome cells with 5' TI assay (M: Marker; UT: Untransduced R106W cells; T: Transduced R106W cells).B. Sequence analysis of the edited MECP2 gene in R106W Rett syndrome cells.Shown are the chromatograms of 5' genome-vector junction, SNP in Intron 2, Linker 1 sequence, codon encoding WT R or mutant W at position 106, SNP in Intron 3 and Linker 2 sequence, representing the 4 editing outcomes (1, 2, 3 and 4) observed in R106W cells with the 5' TI assay.The primers used for 5' TI analyses are depicted as thick red arrows.

Figure S4. 5 '
Figure S4.5' Sequence analysis of edited MECP2 gene in R282X cells.A. Specific PCR product (1.4 kb) obtained with the edited heterozygous female R282X Rett syndrome cells with 5' TI assay (M: Marker; UT: Untransduced R282X cells; T: Transduced R282X cells).B. Sequence analysis of the edited MECP2 gene in R282X Rett syndrome cells.Shown are the chromatograms of 5' genome-vector junction, SNP in Intron 2, Linker 1 sequence, SNP in Intron 3 and Linker 2 sequence, representing the 4 editing outcomes (B1, B2, B3 and B4) observed in R282X cells with the 5' TI assay.The primers used for 5' TI analyses are depicted as thick red arrows.

Figure S5. 3 '
Figure S5.3' Sequence analysis of edited MECP2 gene in R282X cells.A. Specific PCR product (2.7 kb) obtained with the edited heterozygous female R282X Rett syndrome cells with 3' TI assay (M: Marker; UT: Untransduced R282X cells; T: Transduced R282X cells).B. Sequence analysis of the edited MECP2 gene in R282X Rett syndrome cells.Shown are the chromatograms of Linker 2 sequence, codon encoding WT R or mutant X at position 282, SNP encoding for S at position 423, Exon 4-3' UTR junction sequence and 3' vector-genome junction sequence, representing the 2 editing outcomes (C1 and C2) observed in R282X cells with the 3' TI assay.The primers used for 3' TI analyses are depicted as thick red arrows.
Consensus sequence of Edited Outcome 2 in R106W cells represented in Figure5.