LAUSR

&NA; To achieve mitochondrial gene therapy, developing a mitochondrial transgene expression system that produces therapeutic proteins in mitochondria of disease cells is essential. We previously reported on the design of pCMV‐mtLuc (CGG) containing a CMV promotor and a NanoLuc (Nluc) luciferase gene that records adjustments to the mitochondrial codon system, and showed that the mitochondrial transfection of pCMV‐mtLuc (CGG) resulted in the efficient production of the Nluc luciferase protein in human HeLa cells. This mitochondrial transfection was achieved using a MITO‐Porter, a liposome‐based carrier for delivering a cargo to mitochondria via membrane fusion. We report herein that mitochondrial transfection using the MITO‐Porter results in mitochondrial transgene expression in G625A fibroblasts obtained from a patient with a mitochondrial disease. We investigated the effect of promoters and the basic structure of pCMV‐mtLuc (CGG) on gene expression efficiency, and were able to construct a high performance mitochondrial DNA vector, pCMV‐mtLuc (CGG) [hND4] that contains a human mitochondrial endogenous gene. We also constructed an RP/KALA‐MITO‐Porter composed of the KALA peptide (cell‐penetrating peptide) with a mitochondrial RNA aptamer to enhance cellular uptake and mitochondrial targeting. Finally, the mitochondrial transfection of pCMV‐mtLuc (CGG) [hND4] in G625A fibroblasts using the RP/KALA‐MITO‐Porter resulted in strong mitochondrial transgene expression. Graphical abstract Figure. No caption available.