Gene therapy for rare genetic disease Keratitis-Ichthyosis-Deafness Syndrome - Correction of Faults of the Disease-Associated Gene GJB2 by Prime Gene

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Keratitis-Ichthyosis-Deafness (KID) syndrome is a rare autosomal dominant caused in 86% of cases by a recurrent mutation: c. 148G>A in the gene GJB2, which encodes the protein connexin 26. It is an ectodermal disorder that affects the epidermis, corneal epithelium, and inner ear, which lead to the clinical symptoms of corneal inflammation, hearing loss and ichthyosis. Furthermore, the life expectancy of KID patients aged between 20-40 is reduced by 20% due to the development of squamous cell carcinoma.KID syndrome is currently untreatable; therefore, it is vital to develop a new therapy for this disease.There is a potential in correcting mutations in GJB2 via primer edit technology. Compared to alternative gene editing options such as nuclease-mediated homology-directed repair which has relatively low editing efficiency and base-editing technologies which requires protospacer-adjacent motif (PAM) to be around 10 base pairs from the target DNA sequence, the novel prime editing technology has the advantage that can install point mutations over 30 base pairs from the PAM which offer a greater degree of flexibility when correcting, inserting, or deleting >20 nucleotides in a precise and targeted manner.In this project, I will develop the prime gene editing platform for the hot mutation c. 148G>A in KID syndrome. I will design and clone pegRNAs for the mutation c. 148G>A into the vector pAAC-PE2-N. This vector alongside the vector pAAVPE2-C will be delivered into patient cells harboring the c.148G>A by AAV viral system. To determine the frequencies of gene editing and off-target of the gene editing, Sanger sequencing, Edit-R analysis and next generation sequencing will be applied. In addition, the expression and functional recovery of connexin 26 will be further examined. Finally, this therapy strategy will be tested in a KID disease mouse model which was newly developed by Dr Wei Li-Di's group. This will reveal the feasibility of the gene therapy for this condition.