Towards a CRISPR-mediated therapy for Rett Syndrome

Abstract

Rett syndrome (RTT) is a severe neurological disorder which is caused by mutations in the X-linked gene MECP2 (methyl-CpG binding protein 2). RTT-like symptoms can be reversed in Mecp2-null mice by restoring MeCP2 expression, which suggests that the disorder may be curable. Based on this, there has been a major focus on developing therapeutic strategies which can restore MeCP2 levels. However, MeCP2 overexpression also leads to neurological dysfunction, and so achieving safe but effective MeCP2 levels is a significant challenge for conventional gene therapy approaches. Most RTT-causing mutations affect two discrete domains which are necessary and sufficient for MeCP2 function. However, some RTT-causing mutations affect the region C-terminal to these domains. These include the missense mutation P322L and a group of C-terminal deletions which account for approximately 10% of RTT cases. These mutations cause RTT due to a dramatic reduction in MeCP2 protein levels. Since mouse models lacking the C-terminus of MeCP2 express normal levels of MeCP2 and do not have RTT-like symptoms, we hypothesised that removal of the mutant C-terminus would restore MeCP2 protein levels and alleviate RTT-like symptoms. This work investigates the potential of using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) to restore protein levels of P322L and the most prevalent C-terminal deletion. Guide RNAs can be designed which target Cas9 to the mutant allele, where it introduces a double-stranded break in the DNA. Since endogenous repair of CRISPR/Cas9-induced lesions often generates frameshift-causing mutations, we predicted that most repair products would generate stable C-terminally truncated MeCP2. The advantages of this approach are that MECP2 remains under the control of its regulatory elements, circumventing any issues with gene dosage, and that cutting by transient expression of CRISPR/Cas9 components should provide permanent correction. Using cell culture models I have demonstrated that CRISPR/Cas9 targeting of P322L and a C-terminal deletion predominantly generates repair products with increased protein levels. The stage is therefore set to determine whether CRISPR/Cas9 targeting in vivo also increases MeCP2 protein levels in mouse models of RTT, and whether this is sufficient to alleviate RTT-like symptoms. The promising results in cell culture suggest that there is potential to translate these findings into a therapy for RTT patients.