Mutations in mitochondrial DNA can be inherited or occur de novo leading to several debilitating myopathies with no curative option and few or no effective treatments. Allotopic expression of recoded mitochondrial genes from the nucleus has potential as a gene therapy strategy for such conditions, however progress in this field has been hampered by technical challenges. Here we employed codon optimization as a tool to re-engineer the protein-coding genes of the human mitochondrial genome for robust, efficient expression from the nucleus. All 13 codon-optimized constructs exhibited substantially higher protein expression than minimally-recoded genes when expressed transiently, and steady-state mRNA levels for optimized gene constructs were 5–180 fold enriched over recoded versions in stably-selected wildtype cells. Eight of thirteen mitochondria-encoded oxidative phosphorylation (OxPhos) proteins maintained protein expression following stable selection, with mitochondrial localization of expression products. We also assessed the utility of this strategy in rescuing mitochondrial disease cell models and found the rescue capacity of allotopic expression constructs to be gene specific. Allotopic expression of codon optimized ATP8 in disease models could restore protein levels and respiratory function, however, rescue of the pathogenic phenotype for another gene, ND1 was only partially successful. These results imply that though codon-optimization alone is not sufficient for functional allotopic expression of most mitochondrial genes, it is an essential consideration in their design.

线粒体 DNA 突变可以遗传或从头发生，导致几种衰弱性肌病，没有治疗选择，也很少或根本没有有效的治疗方法。从细胞核中重新编码的线粒体基因的同位素表达具有作为此类疾病的基因治疗策略的潜力，但是该领域的进展受到技术挑战的阻碍。在这里，我们采用密码子优化作为工具来重新设计人类线粒体基因组的蛋白质编码基因，以实现从细胞核中稳健、高效的表达。当瞬时表达时，所有 13 个密码子优化的构建体都表现出比最低限度重新编码的基因高得多的蛋白质表达，并且优化基因构建体的稳态 mRNA 水平比稳定选择的野生型细胞中的重新编码版本富集 5-180 倍。13 种线粒体编码的氧化磷酸化 (OxPhos) 蛋白中的 8 种在稳定选择后保持蛋白表达，且表达产物定位于线粒体。我们还评估了该策略在拯救线粒体疾病细胞模型中的效用，并发现同位素表达构建体的拯救能力具有基因特异性。在疾病模型中密码子优化的 ATP8 的同位素表达可以恢复蛋白质水平和呼吸功能，然而，拯救另一个基因 ND1 的致病表型仅部分成功。这些结果意味着，虽然单独的密码子优化不足以实现大多数线粒体基因的功能性同位素表达，但它是其设计中的一个重要考虑因素。