Elsevier

Journal of Controlled Release

Volume 274, 28 March 2018, Pages 109-117
Journal of Controlled Release

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

https://doi.org/10.1016/j.jconrel.2018.02.005Get rights and content

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.

Introduction

The vast majority of diseases of mitochondrial origin result from the incorporation of damaged or mutated proteins into complexes associated with the electron transport chain [[1], [2], [3]]. For example, mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) are caused by a mutation in protein synthesis in the encoding region of tRNA [4]. MELAS is one of the major mitochondria-related diseases that is responsible for mtDNA mutations, and the major point mutations found in MELAS are localized at bp 3243 (A to G; tRNALue) [4]. Since various diseases are caused by mutations in mtDNA, mitochondrial gene delivery would be expected to be a viable strategy for curing a mitochondrial disease. To achieve mitochondrial gene therapy, developing a mitochondrial transgene expression system to produce therapeutic proteins in mitochondria of diseased cells is essential.

We previously designed pCMV-mtLuc (CGG) that contains a promoter derived from Cytomegalovirus and a NanoLuc (Nluc) luciferase gene that records adjustments to the mitochondrial codon system (mtLuc (CGG) gene) [5]. As shown in Fig. S1A, the pCMV-mtLuc (CGG) contains the CMV promoter, the Nd 4 gene derived from the mouse mtDNA, mtLuc (CGG) gene and tRNAAsp with a 5′ untranslated region (UTR). The wild-type Nluc luciferase gene can be translated on cytoplasmic ribosomes, but not in mitochondria, because the AGG codon at amino acid 45 (bases 133–135) encodes for Arg in the nuclear genetic code, but in mitochondria, the AGG codon leads to the arrest of mitochondrial translation. While, in the case of the mtLuc (CGG) gene contained in pCMV-mtLuc (CGG), the AGG codon at amino acid 45 (bases 133–135) is changed into a CGG codon which encodes for Arg in both the nuclear genetic and the mitochondrial code. Moreover, the mtLuc (CGG) gene contains a TGA codon at amino acid 12 (bases 34–36), which encodes for mitochondrial Trp and a nuclear genetic stop codon. Therefore, it is not possible for mitochondrial Nluc luciferase to be produced outside mitochondria. We previously confirmed that luciferase activity was detected as the result of the transfection of pCMV-mtLuc (CGG) resulting from mitochondrial transgene expression [5]. The positions of point mutations in the wild-type Nluc luciferase gene in the design of mtLuc (CGG) are summarized in Table S1. The sequence information is shown in Supplementary vector sequences (Sequence S1).

Using MITO-Porter system, we also showed that the mitochondrial transfection of pCMV-mtLuc (CGG) resulted in the efficient production of the Nluc luciferase protein in human HeLa cells [5]. The MITO-Porter system is a liposome-based carrier for delivering cargoes to mitochondria via membrane fusion [6,7]. This mitochondrial transfection was performed using the KALA-MITO-Porter, which is modified with the KALA peptide, a cationic amphipathic cell-penetrating peptide [8]. As shown in Fig. 1, the MITO-Porter is internalized into cells, and the carriers then escape from the endosome into the cytosol. The carriers then bind to mitochondria via electrostatic interactions, and deliver pDNA into mitochondria via membrane fusion. Finally, the pDNA is transcribed to mRNA to produce the protein via translation inside the mitochondrion.

The purpose of this study was to confirm that mitochondrial transfection using the MITO-Porter system can achieve mitochondrial transgene expression in diseased cells. In this study, we used G625A fibroblasts obtained from a patient with a mitochondrial disease. The patient was suffering from MELAS-like symptoms including epilepsy, hearing loss and elevated lactate levels [9]. The G625A fibroblasts carry a heteroplasmic point mutation in the mtDNA localized at bp 625 (G to A) coding the tRNA for phenylalanine, leading to a decrease in mitochondrial complex III activity and membrane potential. The positively charged KALA-MITO-Porter binds to negatively charged mitochondria via electrostatic interactions. Our initial concern was whether the KALA-MITO-Porter could bind to mitochondria of diseased cells, which had a lower mitochondrial membrane potential than those in normal cells. Unfortunately, the mitochondrial transfection of pCMV-mtLuc (CGG) into G625A fibroblasts using the KALA-MITO-Porter or Lipofectamine 2000 (LFN 2000) resulted in no detectable luciferase activity (Fig. S1B).

To achieve mitochondrial transgene expression in mitochondrial diseased cells, we constructed a new type of mitochondrial DNA vector and optimized mitochondrial transfection using the KALA-MITO-Porter that was modified with a mitochondrial RNA aptamer. We first investigated the effect of promoters and the basic structure of pCMV-mtLuc (CGG) on gene expression efficiency by measuring luciferase activity in an attempt to produce a new mitochondrial DNA vector with a strong mitochondrial transgene expression. Moreover, we constructed an RP/KALA-MITO-Porter with an RNase P (RP) aptamer, a mitochondrial RNA aptamer that enhances cellular uptake and mitochondrial targeting [10]. To monitor the intracellular trafficking of the RP/KALA-MITO-Porter, cellular uptake analysis by flow cytometry and intracellular observation using confocal laser scanning microscopy (CLSM) were performed. Finally, the mitochondrial transfection of pDNA into G625A fibroblasts was performed using the optimized RP/KALA-MITO-Porter, and mitochondrial transgene expression was evaluated by means of a luciferase activity assay.

Section snippets

Materials

Cholesteryl hemisuccinate (CHEMS) was purchased from Sigma (St. Louis, MO, USA). 1,2-dioleoyl-sn-glycero-3-phosphatidyl ethanolamine (DOPE) and sphingomyelin (SM) were purchased from Avanti Polar lipids (Alabaster, AL, USA). The stearylated moiety that was covalently linked to the N-term of KALA sequence (WEKLAKALAKALAKHLAKALAKALKA-NH2) (STR-KALA) [11] was obtained from KURABO Industries Ltd. (Osaka, Japan). Cholesterol that was covalently linked to the 3′ end of 2′-O-Methyl RNAs containing the

Design of a mitochondrial DNA vector to express mitochondrial Nluc luciferase

We recently designed a pCMV-mtLuc (CGG) that contains a CMV promotor and an Nluc luciferase gene that records adjustments to the mitochondrial codon system (Fig. 2A), and showed that the mitochondrial transfection of the pDNA resulted in a strong mitochondrial gene expression in the liver and in cultured human HeLa cells [5]. However, the transfection of pCMV-mtLuc (CGG) into the G625A fibroblasts obtained from a mitochondrial disease patient resulted in no detectable luciferase activity (Fig.

Discussion

To develop practical applications of mitochondrial gene therapy via the mitochondrial transfection of a mitochondrial DNA vector, it is essential to demonstrate that the mitochondrial transfection methodology is capable of achieving mitochondrial transgene expression in patient-derived disease cells. We previously reported that mitochondrial transfection by the KALA-MITO-Porter results in achieving mitochondrial gene expression in human HeLa cells [5]. However, we were concerned as to whether

Acknowledgment

This work was supported, in part by, a Grant-in-Aid for Scientific Research (B) [grant 26282131 to Y.Y.] and a Grant-in-Aid for Challenging Exploratory Research [grant 25560219 to Y.Y.] from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government (MEXT), and the Platform Project for Supporting in Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science) from Japan Agency for Medical Research and

References (25)

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