Mitochondrial transgene expression via an artificial mitochondrial DNA vector in cells from a patient with a mitochondrial disease

Abstract

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.

Therapeutic Strategies for Regulating Mitochondrial Oxidative Stress

“…We also introduce cell therapy to reduce oxidative stress in mitochondria. Moreover, we outline the therapeutic strategy based on the use of a MITO-Porter that was developed in our laboratory to control mitochondrial oxidative stress [18][19][20][21][22].…”

Validation of a mitochondrial RNA therapeutic strategy using fibroblasts from a Leigh syndrome patient with a mutation in the mitochondrial ND3 gene

“…To circumvent this, we examined the use of a non-viral mitochondrial gene delivery system in an attempt to produce mitochondrial transgene expression by delivering a mitochondrial DNA vector to mitochondria. We used a MITO-Porter system, a liposome-based carrier that is capable of mitochondrial delivery via membrane fusion to achieve direct mitochondrial transfection 6,7 .…”

Lipid‐based gene delivery to macrophage mitochondria for atherosclerosis therapy

“…One of the latest nanomedical tendencies of targeted therapy of mitochondrial dysfunction is using nanocarriers for gene delivery directly to the mitochondrion. This strategy aims to correct the mtDNA damage 89 . Implementation of this strategy requires overcoming of several obstacles.…”

“…To date, various types of lipid carriers have been described that are characterized by different lipid composition and parameters of transfection efficacy. The next step in improving the nanocarrier design will be the use of specific mitochondria‐targeting molecules 89,109,110 …”