Elsevier

Biomaterials

Volume 136, August 2017, Pages 56-66
Biomaterials

Validation of the use of an artificial mitochondrial reporter DNA vector containing a Cytomegalovirus promoter for mitochondrial transgene expression

https://doi.org/10.1016/j.biomaterials.2017.05.016Get rights and content

Abstract

Mitochondria have their own gene expression system that is independent of the nuclear system, and control cellular functions in cooperation with the nucleus. While a number of useful technologies for achieving nuclear transgene expression have been reported, only a few have focused on mitochondria. In this study, we validated the utility of an artificial mitochondrial DNA vector with a virus promoter on mitochondrial transgene expression. We designed and constructed pCMV-mtLuc (CGG) that contains a CMV promotor derived from Cytomegalovirus and an artificial mitochondrial genome with a NanoLuc (Nluc) luciferase gene that records adjustments to the mitochondrial codon system. Nluc luciferase activity measurements showed that the pCMV-mtLuc (CGG) efficiently produced the Nluc luciferase protein in human HeLa cells. Moreover, we optimized the mitochondrial transfection of pCMV-mtLuc (CGG) using a MITO-Porter system, a liposome-based carrier for mitochondrial delivery via membrane fusion. As a result, we found that transfection of pCMV-mtLuc (CGG) by MITO-Porter modified with the KALA peptide (cationic amphipathic cell-penetrating peptide) showed a high mitochondrial transgene expression. The developed mitochondrial transgene expression system represents a potentially useful tool for the fields of nanoscience and nanotechnology for controlling the intracellular microenvironment via the regulation of mitochondrial function and promises to open additional innovative research fields of study.

Introduction

Controlling the function of an intracellular microenvironment such as an organelle could potentially create innovative research opportunities and discoveries in nanoscience and nanotechnology. Mitochondria, a prime target organelle, carry out various essential cellular functions including ATP production, regulating apoptosis and mitochondrial biosynthesis. Mitochondria possess their own genome, mitochondrial DNA (mtDNA), with a gene expression system that is independent of the nuclear system, and regulate cellular functions in cooperation with the nucleus [1]. To date, many useful technologies regarding nuclear transgene expression have been reported [2], [3], [4], [5], and a wide variety of plasmid DNA (pDNA) vectors have been developed and are now used as convenient and useful tools for transgene expression by many researchers and clinicians world-wide [5]. While, there are a few reports regarding the development of pDNA vectors for mitochondrial transgene expression, and a convenient and established method for achieving mitochondrial transgene would be highly desirable. The acceleration of mitochondrial DNA vector development would open innovative fields including the life sciences, drug discovery and gene therapy.

It is well known that mitochondria possess their own transcription/translation system and a unique codon usage that is different from universal codon usage. Because of this, a mitochondrial DNA vector needs to be designed to meet essential components for mitochondrial transgene expression, including an optimal promoter for mitochondrial transcription and mitochondrial codon usage. Several studies regarding artificial mitochondrial DNA vectors have been reported, most of which involved the design of DNA vectors containing a gene optimized for the mitochondrial codon system and a mitochondrial endogenous promotor such as a heavy strand mtDNA promoter (HSP) [6], [7], [8], [9]. Among the previous excellent reports, we focused on the use of a virus vector for achieving mitochondrial transgene expression that was originally reported by Yu et al. [9]. In this study, the authors constructed the Adenoassociated virus (AAV) with mitochondrial targeting activity, in which the human NADH ubiquinone oxidoreductase subunit 4 (ND4) gene linked with HSP (pTR-UF11-ND4FLAG) was packaged, and showed that the expression of WT ND4 in cells with a point mutation in the ND4 gene, which is implicated in Leber's hereditary optic neuropathy (LHON) restored a defect in ATP synthesis [9]. This report prompted us to hypothesize that a pDNA containing HSP and the endogenous gene (ND4) could achieve mitochondrial transgene expression even without the need for a virus vector, if the successful mitochondrial delivery of pDNA could be achieved.

We recently constructed a pHSP-mtLuc (CGG) that possesses the HSP, Nd 4 gene derived from mouse mtDNA and an artificial mitochondrial genome with the reporter NanoLuc (Nluc) luciferase gene that records adjustments to the mitochondrial codon system [10]. The basic structure of pHSP-mtLuc (CGG) was designed based on pTR-UF11-ND4FLAG [9], and an additional sequence was further linked to the 3′ terminus of the artificial mitochondrial gene to improve mitochondrial protein translation. In this study, we examined the in vivo mitochondrial delivery of pHSP-mtLuc (CGG) to the liver and skeletal muscle of mice via hydrodynamic injection, and confirmed that mitochondrial transgene expression had occurred. Hydrodynamic injection, in which a large volume of naked pDNA is rapidly injected, is an efficient method for the in vivo nuclear delivery of naked pDNA and has been used in a wide variety of basic and translational studies [11], [12], [13]. When naked pDNA was delivered to the livers of mice by hydrodynamic tail vein (HTV) injection, 2 mL of saline containing naked pDNA was injected into the tail vein within a period of 5 s [14]. A sufficient volume of saline was used to facilitate the extravasation of the pDNA from the vasculature and into the liver tissue through multiple physical barriers. It has been suggested that hydrodynamic force could induce the transient opening of the cellular membrane, permitting pDNA to be internalized into cells. This may account for the subsequent localization of pDNA in the nucleus. We also succeeded in in vivo mitochondrial gene delivery via hydrodynamic injection [14]. Moreover, we showed that the hydrodynamic injection of pHSP-mtLuc (CGG) resulted in the expression of the mitochondrial Nluc luciferase protein in liver and skeletal muscle [10].

We next investigated the mitochondrial transgene expression of pHSP-mtLuc (CGG) using cultured cells, because it can be assumed that a mitochondrial transgene expression system for living cells would accelerate mitochondrial gene therapy and further studies directed at mitochondrial molecular biology. However, the transfection of pHSP-mtLuc (CGG) into cultured HeLa cells by lipofection using Lipofectamine 2000 resulted in no luciferase activity [10] (Fig. S1). The efficiency of the transfection of pHSP-mtLuc (CGG) using Lipofectamine 2000 therefore appears to be lower than that for hydrodynamic injection, and, as a result, lipofection failed to result in successful mitochondrial transgene expression. Based on these results, our research has focused on improving mitochondrial transgene expression efficiency, to develop a new artificial DNA construct, which can achieve mitochondrial transgene expression in living cells, even when a non-viral vector is used.

In this study, we focused on the Cytomegalovirus (CMV) promoter to improve mitochondrial transgene expression efficiency. The CMV promoter has been used frequently to achieve effective nuclear transgene expression in mammalian cells [15], [16]. However, a virus promotor that is capable of functioning in mitochondrial transgene expression has not been reported. In this study, we validated the utility of an artificial mitochondrial DNA vector with a virus promoter on mitochondrial transgene expression. For this validation, we designed pCMV-mtLuc (CGG) that contains a CMV promotor and an Nluc luciferase gene optimized for a mitochondrial codon system based on pHSP-mtLuc (CGG) [10] (Fig. 1A). We first investigated whether pCMV-mtLuc (CGG) could lead to transgene expression to produce a reporter Nluc luciferase protein in in vivo and in vitro experiments based on Nluc luciferase activity measurements. Hydrodynamic injection and lipofection were used for in vivo and in vitro transfection, respectively. Moreover, in vitro transcription and translation assays in a nucleo-cytoplasmic translation system were performed to validate the mitochondrial transgene expression of pCMV-mtLuc (CGG) in detail. Finally, we attempted to optimize the mitochondrial transfection of pCMV-mtLuc (CGG) using MITO-Porter system, which liposome-based carrier for mitochondrial delivery via membrane fusion [17], [18].

Section snippets

Materials

Cholesteryl hemisuccinate (CHEMS) were 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). Stearylated octaarginine (STR-R8) [19] and stearylated KALA (STR-KALA) [20] was obtained from KURABO Industries Ltd (Osaka, Japan). Protamine was purchased from CALBIO CHEM (Darmstadt, Germany). pNL1.1 CMV [Nluc/CMV] Vector was purchased from Promega (Madison, WI, USA).

Design of pCMV-mtLuc (CGG) with a CMV promotor to express mitochondrial Nluc luciferase

We recently constructed pHSP-mtLuc (CGG) that contained HSP as a promoter, and showed that the hydrodynamic injection of pHSP-mtLuc (CGG) resulted in the expression of the mitochondrial Nluc luciferase protein in the liver and skeletal muscle [10]. However, the transfection of pHSP-mtLuc (CGG) into cultured HeLa cells resulted in no luciferase activity [10] (Fig. S1). Thus, we focused on the use of a CMV promoter to improve mitochondrial transgene expression efficiency, and designed a new

Discussion

To achieve nuclear transgene expression, a number of useful technologies, including pDNA vectors have been reported to date [2], [3], [4], [5]. Based on such a current trend, many researchers have focused on the mitochondrial targeting of exogenous protein via allotropic expression, where an engineered gene coding a protein fused with a mitochondrial targeting signal peptide (MTS) is transcribed/translated via nuclear transcription/cytosolic translation, and the gene product is then delivered

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), the Uehara Memorial Foundation [to Y.Y.], and the Platform Project for Supporting in Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science) [grant

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