Advancing the Development of a CRISPR/Cas9-Based Gene Editing Therapy for Epidermolysis Bullosa

Epidermolysis bullosa (EB) is a heterogenous group of genetic fragile skin disorders. It is characterized by pathogenic mutations in genes encoding structural proteins essential for skin integrity and adhesion. Gene editing facilitated by CRISPR/Cas9 represents a promising approach for precisely repairing EB mutations and providing a durable therapy, but this is currently limited by suboptimal editing efficacy. Our research aimed to advance the development of an ex vivo CRISPR-based gene editing therapy by addressing key hurdles hindering clinical translation. To overcome the low rates of precise mutation correction typically achieved in the field, we established a Cas9-based strategy to remove faulty collagen VII (COL7A1) exons which cause recessive dystrophic EB (RDEB). We excised three previously untargeted exons and achieved highly efficient exon deletion rates of up to 95%. To improve the efficiency and safety of precise mutation repair, we then developed an efficient strategy targeting a prevalent laminin (LAMB3) mutation which causes junctional EB (JEB). Here, we achieved up to 54% precise repair using the safer Cas9 nickase variant. The gene editing efficiencies achieved with both strategies represent the highest in the field to date. Lastly, we investigated alternative methods for delivering the CRISPR/Cas9 reagents into primary skin cells. Using cell penetrating peptides, we achieved up to 81% gene editing targeting EB-relevant loci, including the first demonstration of precise gene editing in primary keratinocytes using this delivery method. This approach improved cell viability compared to the gold standard electroporation method and can potentially be repurposed for in vivo gene editing applications which represents a desirable future goal of EB research. Taken together, this research significantly advances therapeutic gene editing for EB, providing a foundation for future efforts to develop durable, personalized gene therapies for this life-threatening group of disorders.

Name thesis:degree_name

PhD

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Biological Sciences

Grantor dc:publisher

ResearchSpace@Auckland

Year dc:date.issued

2025

Author dc:creator

• du Rand, Alex

Advisors dc:contributor.advisor

• Sheppard, Hilary
• Verdon, Daniel
• Feisst, Vaughan
• Ganley, Austen

• orts to develop durable
• personalized gene therapies for this life-threatening group

Statement dc:rights

• Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

Licence dc:rights.uri

• https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm

Handle dc:identifier.uri

https://hdl.handle.net/2292/72927

OAI record oai:identifier

https://researchspace.auckland.ac.nz/server/oai/request?verb=GetRecord&metadataPrefix=dim&identifier=oai%3Aresearchspace.auckland.ac.nz%3A2292%2F72927