Turner, Thomas; (2024) Correcting Dominant Negative Neurological Disease-Causing Mutations with in vivo CRISPR Genome Editing. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Episodic ataxia type 1 (EA1) is a rare, autosomal dominantly inherited neurological movement disorder caused by mutations in the KCNA1 gene, which encodes the voltage-gated potassium channel Kv1.1. The phenotypes of patients and their responses to drugs are inconsistent, sometimes even for carriers of the same mutation. Many EA1 mutations have dominant negative (DN) effects that antagonise the activity of the wild-type (WT) protein and other Kv1 family members that coassemble into heteromultimeric channels, which makes conventional gene therapy approaches challenging. This thesis investigates the therapeutic potential of using CRISPR genome editing to insert the healthy KCNA1 coding sequence (CDS) and a stop codon upstream of the endogenous genomic copy to co-opt genomic promoter activity to express healthy Kv1.1. This could restore physiological Kv1.1 expression and bioprocessing while bypassing all EA1 mutations, including DN ones. First, I characterised the phenotype of a new transgenic mouse model of a DN EA1 mutation that truncates Kv1.1 - R417X. I found that posttranslational glycosylation of Kv1.1 was impaired in Kcna1R417X/+ mice, while in vitro in primary cortical Kcna1R417X/+ neurons there was a reduction in potassium currents. I also showed that Kcna1R417X/+ mice exhibit hyperexcitability, with a reduced threshold for kainic acid-induced seizures, reflecting the overrepresentation of epilepsy in EA1 patients. I then developed a CDS replacement approach for the murine Kcna1 using CRISPR. I confiirmed that knock-in occurs in the correct orientation in Kcna1 in vitro and in vivo in neurons and was able to show that it is transcribed and spliced normaly in vivo in the brain. I have yet to show functional expression of the knock-in or a consequent therapeutic effect in Kcna1R417X/+ mice, despite developing several novel assays. Overall, this thesis provides promising groundwork for development of CDS replacement therapy for EA1 and expands the EA1 phenotypes modelled in mice.

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