Modelling and correction of retinal pathologies caused by mitochondrial DNA mutations via genetic engineering

Mitochondria, widely known for their energy production role, contain their own DNA (mtDNA), which is susceptible to mutations. These mutations underlie primary mitochondrial diseases, often affecting high energy-demand tissues such as the retina, brain, heart, and skeletal muscle. Consequently, they constitute a common cause of inherited retinopathies and optic neuropathies – conditions that currently lack curative treatments and lead to progressive visual loss. Although advances in genetic engineering have enabled the modelling and correction of nuclear DNA mutations in the retina, challenges remain in modifying mtDNA. The generation of accurate models that mimic retinal mtDNA diseases and development of curative gene correction approaches have been hindered largely due to the limited tools available for mtDNA editing. Thus far, most methods have relied on programmable nucleases. The recent emergence of mitochondrial base editors has broadened the scope of mtDNA genetic engineering approaches. However, the potential of programmable nucleases or base editors for modifying retinal mtDNA remains unexplored. This thesis first characterised the retinal pathophysiology in an existing mouse model carrying a mtDNA heteroplasmic mutation. The multiscale retinal phenotyping study identified pathologies consistent with those seen in patients carrying mtDNA mutations, validating this model for therapeutic assessment. Next, a panel of programmable nucleases and mitochondrial base editors were evaluated in this pre-clinical setting for their capacity to modify retinal mtDNA. These correction studies demonstrated the first proof-of-concept for the use of mtDNA engineering to eliminate mutant mtDNA molecules in the retina, paving the way for future translation applications. Lastly, leveraging novel mitochondrial base editors with expanded targeting scope, this work established the first mouse model bearing the most common mtDNA mutation causing LHON, creating a unique platform to test translational therapies aimed at correcting it. Altogether, this thesis demonstrated the potential of mitochondrial genetic engineering for correcting a retinal mtDNA mutation in a mouse model characterised here with retinal pathology and proved the use of mitochondrial base editors in modelling the most frequent optic neuropathy mtDNA mutation, establishing a comprehensive study of mtDNA modifying tools to advance therapy development for retinal mtDNA pathologies.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2025

Author dc:creator

• Luengo Gutierrez, Lucia

Advisors dc:contributor.advisor

• Minczuk, Michal
• Yu-Wai-Man, Patrick

• Gene therapy
• Mitochondrial DNA
• Retina

Licence

• https://www.repository.cam.ac.uk/bitstreams/10beee46-45b9-403e-b639-9c2af6685944/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.122995

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/392224