Modeling mtDNA Disease in vivo

Mitochondrial disease has a minimum prevalence of at least 1 in 5000 adults, with few or no effective treatments. Mitochondrial disease patients demonstrate enormous biological variation and diverse disorders. These include neurologic, cardiac, endocrine, kidney, visual, hearing, blood, and skeletal muscle systems.

Imaging and basic science of mitochondria showcase how this highly dynamic organelle responds differentially to extrinsic, intrinsic and unknown biological signals from roles in metabolism, organ homeostasis, apoptosis and aging. One unique feature of mitochondria is their dual genome nature where both nuclear and

mitochondrial genomes contribute to its form and function. Sequence variations in either genome each contribute to human mitochondrial genetic disease. The mitochondrial genome is highly conserved in all vertebrates, for example the zebrafish mitochondrial genome is nearly identical in size (16kb+) and encodes

the same complement of genes that are organized in the same order as the human mitochondrial genome. A key bottleneck in the field has been the historical lack of mitochondrial DNA (mtDNA) gene manipulation tools that has greatly restricted the options for studying the differential roles of genetic variation in biology and

disease. However, the advent of mtDNA base editors has enabled a series of new cellular and animal models. With advanced methods and effective delivery, near-complete editing efficiency capable of introducing over 80% programmed editing efficiency in the pioneering animal the zebrafish (Danio rerio) is now possible,

enabling the establishment of the first designer in vivo models of mtDNA disease. This research resource project will be accomplished in three aims: 1) Generating animal models of mtDNA disease using the established mitoFUSXTBE cytosine base editor. Designer models with single nucleotide variants will be generated in both protein-coding and tRNA mitochondrial genes. 2) To enhance the

kind of alleles that can be modeled and to develop new, tissue-specific mtDNA animal models, mtDNA modeling work will be expanded using new mitoFUSXTBE adenine base editor. 3) Community engagement for allele selection and to enhance access to these new models through education, outreach and sharing plans.

The outcomes of this work will include a series of validated zebrafish lines harboring designer mtDNA variants suitable for hypothesis testing as well as discovery science. The molecular toolbox will also be optimized for utility in helping generate other animal models from work by mitochondrial scientists in the field.

Together, these gene editors and in vivo avatars will enable new approaches for diagnoses and therapies for these terrible diseases.