Towards a CRISPR-mediated therapy for Rett Syndrome
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Date
07/12/2021Item status
Restricted AccessEmbargo end date
07/12/2022Author
FitzPatrick, Laura
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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.