Animal Model Selection and Deep Phenotyping in Mitochondrial Genetic Diseases: Connecting Human Variants, Multisystem Pathology, and Preclinical Evidence

Abstract

Mitochondrial genetic diseases arise from variants in mitochondrial DNA (mtDNA) or nuclear genes required for mitochondrial function. Although many involve several organs, the predominant clinical manifestations may be confined to particular organs. Experimental models are used to investigate variant pathogenicity, organ vulnerability, disease progression, therapeutic response, and safety. We discuss how to select animal models and other experimental systems for specific research questions. We focus on genetically engineered mice (constitutive knockout, knock-in/humanized, conditional/tissue-specific, inducible, and heteroplasmic mtDNA models) and consider when yeast, worms, flies, zebrafish, rats, large animals, companion animals, and human in vitro models can complement or replace particular animal experiments. We define deep phenotyping by five minimum characteristics: longitudinal assessment across defined disease stages; prespecified relationships among molecular, mitochondrial, organ, and whole-animal measurements; integration of repeated and terminal endpoints; alignment with patient-relevant disease stages; and separation of primary from exploratory outcomes. Model selection depends on the experimental objective and the evidence needed to address it. We also propose a multidomain framework that distinguishes target engagement, molecular correction, downstream pharmacodynamic responses, organ function, and whole-animal or patient-relevant benefit without assuming that effective treatments must improve every domain sequentially. We also discuss reporting practices, the 3Rs (Replacement, Reduction, and Refinement), and the implications of mtDNA editing, iPSC-derived systems, organoids, and automated phenotyping for study design.