Exon replacement therapy for Rett syndrome using CRISPR/Cas9 editing technologies

Abstract

Rett syndrome (RTT) is a rare neurodevelopmental disorder resulting in regression of learnt behaviours, loss of purposeful hand movements and speech. Mutations in the Methyl CpG Binding Protein 2 (MECP2) gene is the underlying cause as MECP2 is important in regulating transcription and maturation of the CNS. MECP2 is a ‘goldilocks’ gene, requiring tight regulation, where both over- and under-expression are equally detrimental. 98% of mutations lie in exons 3 and 4 of MECP2. These exons are the target for the therapy outlined below.

Conventional gene replacement therapies used for other monogenic disorders are not applicable to RTT due to its tight expression. Alternatively, CRISPR/Cas9 gene editing was developed to restore wild type MECP2 expression under endogenous regulation. An exon replacement strategy, utilising CRISPR editing to excise and replace exons 3 and 4 at the native locus was designed. Guides directing Cas9 to cut at targeted sites in non-coding regions flanking exons 3 and 4 of MECP2 were assessed in vitro. The most efficient guide pair were selected for use in the repair construct. The repair construct was designed to include the guides, a fused wild-type exons 3 and 4 flanked by guide target sites, and a reporter gene. The therapy was delivered using dual AAV vectors.

Efficacy assessment was performed using human cellular models (patient derived fibroblasts containing a c.806delG mutation and cortical brain organoids derived from the same line) and a Mecp2T158A/y mouse model. Replacement was confirmed at low levels in fibroblast and cortical brain organoids. AAV.PHPeB was selected to deliver the therapy and a 2:1 ratio (Repair: Cas9) was shown to replace exons most effectively in mice. Rescue of the RTT phenotype was not determined as a severe toxic phenotype was observed in treated mice.

This study provides preliminary data into the potential for an editing therapy for RTT, addressing the underlying challenges faced in regulating MECP2 expression.