Validation of Mitochondrial Gene Delivery in Liver and Skeletal Muscle

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Validation of Mitochondrial Gene Delivery in Liver and Skeletal Muscle via Hydrodynamic Injection Using an Artificial Mitochondrial Reporter DNA Vector Yukari Yasuzaki,† Yuma Yamada,† Takuya Ishikawa, and Hideyoshi Harashima* Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan S Supporting Information *

ABSTRACT: For successful mitochondrial transgene expression, two independent processes, i.e., developing a mitochondrial gene delivery system and construction of DNA vector to achieve mitochondrial gene expression, are required. To date, very few studies dealing with mitochondrial gene delivery have been reported and, in most cases, transgene expression was not validated, because the construction of a reporter DNA vector for mitochondrial gene expression is the bottleneck. In this study, mitochondrial transgene expression by the in vivo mitochondrial gene delivery of an artificial mitochondrial reporter DNA vector via hydrodynamic injection is demonstrated. In the procedure, a large volume of naked plasmid DNA (pDNA) is rapidly injected. We designed and constructed pHSP-mtLuc (CGG) as a mitochondrial reporter DNA vector that possesses a mitochondrial heavy strand promoter (HSP) and an artificial mitochondrial genome with the reporter NanoLuc (Nluc) luciferase gene that records adjustments to the mitochondrial codon system. We delivered the pDNA into mouse liver mitochondria by hydrodynamic injection, and detected exogenous mRNA in the liver using reverse transcription PCR analysis. The hydrodynamic injection of pHSP-mtLuc (CGG) resulted in the expression of the Nluc luciferase protein in liver and skeletal muscle. Our mitochondrial transgene expression reporter system would contribute to mitochondrial gene therapy and further studies directed at mitochondrial molecular biology. KEYWORDS: mitochondria, transgene expression, gene delivery, hydrodynamic injection, gene therapy

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INTRODUCTION

To date, several studies dealing with mitochondrial gene delivery have been reported, and most of these studies have been limited to in vitro experiments using cell lines and isolated mitochondria.7,8 We previously reported on the successful in vivo mitochondrial gene delivery in skeletal muscle of rats via hydrodynamic limb vein (HLV) injection.9 Liu et al.10 and Zhang et al.11 originally reported the hydrodynamic injection method, where a large volume of naked plasmid DNA (pDNA) is rapidly injected into the tail vein of mice, resulting in effective nuclear transgene expression in hepatocytes. Since those reports, HLV injection has been used for transgene expression

Mitochondria play a crucial role in homeostasis of our body. As a result, any dysfunction could trigger the development of many kinds of diseases such as cancer, diabetes, and neurodegenerative diseases.1−3 More recently, evolving evidence indicates that a variety of mitochondrial DNA (mtDNA) abnormalities are related to mitochondrial dysfunction and diseases.4−6 Thus, an understanding of mtDNA genotype− phenotype relationships and developing specific treatments for mtDNA-originated diseases would be expected to have great medical benefits including mitochondrial gene therapy. To achieve such an innovative therapy, successful mitochondrial transgene expression, two independent processes, i.e., the development of an in vivo mitochondrial gene delivery system and the construction of a DNA vector to achieve mitochondrial gene expression, are required. © 2015 American Chemical Society

Received: Revised: Accepted: Published: 4311

June 29, 2015 October 4, 2015 November 15, 2015 November 15, 2015 DOI: 10.1021/acs.molpharmaceut.5b00511 Mol. Pharmaceutics 2015, 12, 4311−4320

Article

Molecular Pharmaceutics

Figure 1. Gene designs to express exogenous protein in the mitochondria. (A) The gene design that contains the human HSP promoter, human ND4 gene, and FLAG previously reported by Yu et al.22 (B) The structure of the mitochondrial gene vector in this study. The structure contains the HSP promoter and the ND4 gene derived from mouse mtDNA, and artificial mitochondrial gene optimized for mitochondrial codon system. Moreover, additional sequence is linked to the 3′ terminus of the artificial mitochondrial gene for the improvement of mitochondrial protein translation.

in skeletal muscle.12−15 In our previous study, PCR analysis permitted us to confirm that the HLV injection achieved mitochondrial delivery of naked pDNA in the skeletal muscle,9 and furthermore showed that the mitochondrial delivery was enhanced by the condensation of pDNA.16 However, we were unable to validate transgene expression because the construction of a DNA vector for mitochondrial gene expression was a bottleneck. Even though this is a serious situation, there have been several reports regarding attempts to achieve mitochondrial transgene expression to date. It is known that the noncoding displacement (D) loop of mtDNA contains heavy strand mtDNA promoter (HSP) and light strand mtDNA promoter (LSP); especially the sequence “AAACCCC” in the HSP of human mtDNA is essential for successful mtDNA transcription.17 Jackson et al. attempted heterologous expression in isolated human mitochondria using a linear reporter construct containing the entire human D-loop with adjacent mitochondrial tRNA genes, heterologous rat mitochondrial tRNA genes, and the GFP sequence adapted to mitochondrial codon usage.18 In that study, the authors confirmed that the heterologous expression of the imported construct occurred at the RNA level using a circular RNA reverse transcription (RT)PCR assay and in organelle [α-32P]-UTP labeling. Weissig and co-workers developed DQAsomes, as mitochondriotropic nanocarriers.19 In a recent study, Lyrawati et al. validated mitochondrial transgene expression in mouse RAW264.7 cells by a combination of mitochondrial delivery using DQAsome and a pmtGFP, an artificial circular DNA that contains the mouse D-loop with adjacent mitochondrial tRNA genes, the mitochondrial OL region with mitochondrial tRNA genes, and the GFP sequence that was optimized for a mitochondrial codon system.20 The authors concluded that the mitochondrial delivery of pmtGFP resulted in the expression of mRNA and protein derived from GFP, albeit at a low efficiency. More recently, Cardoso et al. investigated the mitochondrial gene expression of pmtGFP mediated by gemini surfactant based formulations in human HeLa cells.21 The authors reported that gemini surfactant-based pmtGFP complexes, which are predicted to favorably interact with mitochondria exhibited a high ability to mediate mitochondrial gene expression. As described above, a useful mitochondrial delivery

system for pmtGFP has been reported. On the other hand, a further study, including the use of a highly sensitive reporter, would be expected to reveal additional details regarding exogenous mitochondrial protein expression. For mitochondrial transgene expression using a virus vector, Yu et al. constructed the adenoassociated virus (AAV) with mitochondrial targeting activity, which packages the human NADH ubiquinone oxidoreductase subunit 4 (ND4) gene.22 As shown in Figure 1A, the AAV possesses pTR-UF11 (AAV backbone) containing the wild type (WT) human ND4 gene linked with HSP and the FLAG epitope for immune detection (pTR-UF11-ND4FLAG). The authors showed that the expression of WT ND4 in cells with a point mutation in ND4 gene that was implicated with Leber’s hereditary optic neuropathy (LHON) restored defective ATP synthesis. Moreover, they demonstrated that the mitochondrial gene therapeutic strategy using an artificial construct of ND4 gene by MTS modified AAV suppressed LHON in a mouse model. The future study including a detailed investigation of the transcription/translation of an exogenous gene in mitochondrion would provide useful information regarding mitochondrial molecular biology. This report prompted us to consider whether mitochondrial gene expression using pDNA containing HSP and the ND4 gene could be achieved even without a virus vector, if the pDNA could be successfully delivered to mitochondria. In the present study, we validated in vivo mitochondrial gene delivery via hydrodynamic injection targeted to the mouse liver using an artificial mitochondrial reporter DNA vector. For this validation, we designed a mitochondrial reporter DNA vector possessing the HSP promoter and the ND4 gene, as shown in Figure 1B, based on the pTR-UF11-ND4FLAG.22 We chose the HSP promoter and the Nd4 gene derived from mouse mtDNA not human mtDNA, because the gene sequence derived from mouse mtDNA would effectively function on mouse mitochondria. Moreover, an additional sequence was linked to the 3′ terminus of the artificial mitochondrial gene to improve mitochondrial protein translation. Using PCR analysis, we first verified that the hydrodynamic injection of naked pDNA could achieve mitochondrial delivery in mouse liver. We also attempted to detect exogenous mRNA in the liver using RT-PCR analysis. In addition, we investigated the use of 4312

DOI: 10.1021/acs.molpharmaceut.5b00511 Mol. Pharmaceutics 2015, 12, 4311−4320

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Molecular Pharmaceutics

quantified using liver tissue from the same mouse (a total of 18 mice were sacrificed). In Figure 3, 2 mice were used at each time (7 points) and the PCR was done to detect Nd4flag (A) and Nd6 (B) after and before reverse transcription using liver tissue from the same mouse (a total of 14 mice were sacrificed). In Figure 5A, we used 1 mouse for PCR to detect Nluc luciferase after and before reverse transcription using liver tissue from the same mouse at each of the applied samples (saline, pHSP-ND4FLAG, pHSP-mtLuc (CGG), pHSP-mtLuc (TAG)) (a total of 4 mice were sacrificed). In Figure 5B, we used 5 mice to evaluate the transgene expression of pHSPmtLuc (CGG) and 3 mice to evaluate that of pHSP-ND4FLAG and pHSP-mtLuc (TAG), respectively (a total number of 11 mice were sacrificed). In this experiment, the 14 rats were sacrificed as described below. In Figure 6, we used 7 rats to evaluate the transgene expression of pHSP-mtLuc (CGG) at applied doses of 150 μg (4 rats) and 300 μg (3 rats), and 7 rats to evaluate the transgene expression of pHSP-mtLuc (TAG) at applied doses of 150 μg (3 rats) and 300 μg (4 rats). The animals were euthanized by cervical dislocation for this experiment. All animal protocols were approved by the institutional animal care and research advisory committee at the Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan (date: 22 March 2013, registration No. 130062). In Vivo Delivery of pDNA Using Hydrodynamic Injection. Hydrodynamic tail vein (HTV) injection was to deliver pDNA to mouse livers, as described in a previous report.10 The naked pDNA in a 5% (w/v) glucose solution (2 mL) was injected into the tail vein within a period of 5 s. In the case of pDNA3.1, 1−500 μg of the pDNA was injected. In the case of pHSP-ND4FLAG, pHSP-mtLuc (CGG), or pHSPmtLuc (TAG), 100 μg of the pDNA was injected. For the delivery of pDNA to skeletal muscle of rats, we performed HLV injection as previously described.9 Briefly, the naked pDNA in saline (3 mL) containing pHSP-mtLuc (CGG) or pHSP-mtLuc (TAG) was injected from a distal site of the dorsalis pedis vein at a dose of 150 or 300 μg of pDNA in 20 s. Quantification of pDNA in Mitochondria- and NucleiEnriched Fraction after HTV Injection. The pDNA3.1 was administered to mice by HTV injection. After 24 h injection, the livers were harvested and minced with scissors, and approximately 1 mL of ice-cold mitochondrial isolation buffer [250 mM sucrose, 2 mM Tris−HCl, 1 mM EDTA, pH 7.4] was then added. The suspension was homogenized using a PreCellys (Bertin Technologies, Montigny-le-Bretonneux, France), and the mitochondria- and nuclei-enriched fractions were isolated (see Supporting Information for details). We then extracted total DNA including pDNA, mtDNA, and nuclear DNA (nDNA) from the liver homogenate using SepaGene (Sanko Jun-yaku, Tokyo, Japan) to determine the copy numbers of mtDNA per total DNA in liver tissue (mtDNALiver [mtDNA-copy/g total DNA]) and the copy numbers of nDNA per total DNA in liver tissue (nDNALiver [nDNA-copy/g total DNA]). Copy numbers of mtDNA and nDNA were estimated by quantitative real-time PCR (q-PCR) (see Supporting Information for details), and concentrations of the total DNA were measured using a NanoDrop (ND-2000; Thermo Fisher Scientific, Inc. Waltham, MA, USA). We next extracted total DNA from the mitochondria-enriched fraction using SepaGene, and the copy numbers of pDNA and mtDNA were measured by q-PCR to determine the amount of pDNA per mtDNA in mitochondria-enriched fraction (pDNAmt [ng pDNA/mtDNA-

hydrodynamic injection of a mitochondrial reporter DNA vector with the reporter NanoLuc (Nluc) luciferase gene that records adjustments to the mitochondrial codon system that could lead to achieving transgene expression to produce a reporter Nluc luciferase protein in liver and skeletal muscle.

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EXPERIMENTAL SECTION Materials. The pcDNA3.1 (+)-luc plasmid (pDNA3.1) was used to validate mitochondrial DNA delivery using hydrodynamic injection, as detailed in a previous report. 9 Oligonucleotides were purchased from Sigma Genosys Japan (Ishikari, Japan) in purified form. pDNA used in this experiment was purified using an Endfree Plasmid Giga Kit (Qiagen GmbH, Hilden, Germany). All other chemicals used were commercially available reagent-grade products. Design and Construction of pDNA Containing Artificially Designed DNA. The gene fragment for pHSPND4FLAG, pCMV-ND4FLAG, pND4FLAG, and pHSPmtLuc (CGG) was synthesized by Biomatik Corporation (Cambridge, Canada), and the synthesized genes were inserted into the pBluescript II SK(−) (Stratagene, La Jolla, CA, USA) pretreated with the restriction enzymes (EcoRI and SmaI sites). For the construction of pHSP-mtLuc (TAG), the DNA fragment derived from pHSP-mtLuc (CGG) was amplified by PCR using the following pair of primers: 5′-aacctggaccaagtccttga-3′ (forward) and 5′-caccgctcaggacaatctattg-3′ (reverse). Sequences indicated by underline indicate sites for restriction enzymes (Tth111I and Bpu10I), and sequences in bold italics initiate mutation points in pHSP-mtLuc (CGG). The amplified mutated DNA fragment (Tth111I−Bpu10I fragment) was inserted into pHSP-mtLuc (CGG) pretreated with the same restriction enzymes to construct pHSP-mtLuc (TAG). The sequence information on these plasmids is summarized in the Supporting Information (sequences S1−S5). Experimental Animals. Female C57BL/6 mice (6 weeks old) and female Wistar Hannover rats (7−9 weeks old) were purchased from Sankyo Labo Service (Sapporo, Japan). In this experiment, the 57 mice were sacrificed as described below. In Figure 2, 3 mice were used at each of the applied doses (6 points) and the DNA delivery values for mitochondria- (closed circles) and nuclei-enriched fraction (open circles) were

Figure 2. Evaluation of exogenous pDNA levels in mitochondria- and nuclei-enriched fraction of liver tissue following HTV injection. Various doses of pDNA3.1 were delivered to mouse liver by HTV injection. At 24 h postinjection, the DNA delivery values for mitochondria-enriched fraction (closed circles) and nuclei-enriched fraction (open circles) were quantified. Circles indicate means ± SD (n = 3). 4313

DOI: 10.1021/acs.molpharmaceut.5b00511 Mol. Pharmaceutics 2015, 12, 4311−4320

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Figure 3. Gel images of exogenous mRNA in mouse liver following HTV injection of pHSP-ND4FLAG. 100 μg of pHSP-ND4FLAG was administered to mice via HTV injection, and the livers were harvested and total RNA was extracted at 0.5, 2, 6, 12, 24, 48, 72 h post injection. PCR was done with specific primers to detect exogenous mRNA coding Nd4flag derived from pHSP-ND4FLAG (A) and endogenous mRNA coding Nd6 derived from mouse mtDNA (B) after reverse transcription (RT+, upper panels) and before reverse transcription (RT−, lower panels). The PCR products were subjected to electrophoresis. M indicates DNA ladder marker (lane 1). The experiment was repeated twice for each time: 0.5 h (lanes 2, 3), 2 h (lanes 4, 5), 6 h (lanes 6, 7), 12 h (lanes 8, 9), 24 h (lanes 10, 11), 48 h (lanes 12, 13), 72 h (lanes 14, 15) after administration.

Figure 4. A nucleotide sequence and its transcription of pHSP-mtLuc (CGG). The pHSP-mtLuc (CGG) contains the HSP promoter, mtLuc (CGG) gene that codes mitochondrial Nluc luciferase and tRNAAsp with the 5′UTR. If the Nluc luciferase were to be translated in the cytosol, translational arrest was expected at codon “tgA” (nt34−36) that is a stop codon as nuclear genetic codon, while full length versions of mitochondrial Nluc luciferase were produced in mitochondrion, because “tgA” codon encodes for Typ in mitochondria. Positions of point mutation in Nluc luciferase gene to construct mtLuc (CGG) gene are indicated by uppercase.

Figure 5. Evaluation of transgene expression of mitochondrial Nluc luciferase in liver tissue following HTV injection. 100 μg of pHSP-mtLuc (CGG) that codes mitochondrial Nluc luciferase was administered to tail vein of mice via HTV injection, and the livers were harvested at 6 h postinjection. In this experiment, we also administrated pHSP-ND4FLAG without Nluc luciferase gene and pHSP-mtLuc (TAG) that contains the mitochondrial/ universal stop codon (TAG) in Nluc luciferase gene. For gel images of exogenous mRNA (A), total RNA was extracted from the harvested livers, and PCR was done with specific primers to detect Nluc luciferase gene (405 bp) after reverse transcription (RT+, upper panel) and before reverse transcription (RT−, lower panel). The PCR products were subjected to electrophoresis. Lane 1, DNA ladder marker; lane 2, saline administration; lane 3, pHSP-ND4FLAG administration; lane 4, pHSP-mtLuc (CGG) administration; lane 5, pHSP-mtLuc (TAG) administration. To evaluate transgene expression of mitochondrial Nluc luciferase (B), the luciferase activities of the harvested livers were measured. Bars represent the means ± SD (n = 3−5). *Significant differences between pHSP-mtLuc(CGG) and others were calculated by one-way ANOVA, followed by the Bonferroni test (p < 0.05).

described above. DNA delivery values (ng pDNA/g total DNA) in mitochondria- and nuclei-enriched fractions were calculated as follows:

copy]). We also extracted total DNA from the nuclei-enriched fraction, and determined the amount of pDNA per nDNA in nuclei-enriched fraction (pDNAnu [ng pDNA/nDNA-copy]) as 4314

DOI: 10.1021/acs.molpharmaceut.5b00511 Mol. Pharmaceutics 2015, 12, 4311−4320

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injection, the livers were harvested and the total RNA was reverse transcribed as described above. PCR was performed on cDNA to detect exogenous mRNA coding Nluc luciferase, using primers Nluc (+) and Nluc (−) (Table S1), and the PCR products were visualized as described above. Nluc Luciferase Assay of Liver and Muscle Tissue after Hydrodynamic Injection of pHSP-mtLuc (CGG). To evaluate the extent of the transgene expression of mitochondrial Nluc luciferase in liver tissue, 100 μg of pHSP-mtLuc (CGG) that codes mitochondrial Nluc luciferase was administered to the tail vein of mice via HTV injection. In this experiment, we also evaluated the transgene expression of pHSP-ND4FLAG without mitochondrial NLuc luciferase gene and pHSP-mtLuc (TAG) that contains a mitochondrial/universal stop codon (TAG) in the mitochondrial Nluc luciferase gene. At 6 h post injection, the livers were harvested and minced with scissors. Approximately 200 mg of the minced liver tissue was then homogenized using a PreCellys in 1 mL of lysis buffer (100 mM Tris−HCl, 2 mM EDTA, 0.1% Triton X-100, pH 7.8). After centrifugation at 15000g for 10 min at 4 °C, the supernatant was used for an Nluc luciferase assay using the Nano-Glo Luciferase Assay System (Promega). Protein concentrations were determined using a BCA protein assay kit (Pierce, Rockford, IL). Nluc luciferase activities are expressed as relative light units (RLU) per mg of protein. To evaluate the transgene expression of mitochondrial Nluc luciferase in skeletal muscle, 3 mL of saline containing pHSPmtLuc (CGG) or pHSP-mtLuc (TAG) was injected from a distal site of the dorsalis pedis vein of rats at a dose of 150 or 300 μg of pDNA via HLV injection. At 6 h post injection, the skeletal muscles were harvested and minced with scissors, approximately 250 mg of the minced tissue was then homogenized, and the resulting homogenate was used to estimate the Nluc luciferase activity, as described above. Statistical Analysis. For multiple comparisons in Figure 5B, one-way ANOVA followed by Bonferroni test was performed. In Figure 6, we performed two-way ANOVA analysis to compare the effect of two factors that are “pDNA type” and “applied dose”.

Figure 6. Evaluation of transgene expression in skeletal muscle following HLV injection of pHSP-mtLuc (CGG). 3 mL of saline containing pHSP-mtLuc (CGG) that codes mitochondrial Nluc luciferase was administered at a dose of 150 or 300 μg of pDNA via HLV injection. In this experiment, we also used pHSP-mtLuc (TAG) that contains the mitochondrial/universal stop codon (TAG) in the mitochondrial Nluc luciferase gene as a negative control. Bars represent the means ± SD (n = 3−4). We performed two-way ANOVA analysis to compare the effect of two factors that are “pDNA type” and “applied dose”. There was no significant interaction between the two factors (p = 0.40), and there were no significant differences among different “applied dose” groups (p = 0.66). **Significant differences between pHSP-mtLuc (CGG) group and pHSP-mtLuc (TAG) group were detected (p < 0.01 by two-way ANOVA analysis, followed by Bonferroni correction). DNA delivery value for mitochondria [ng pDNA/g total DNA] = mtDNALiver [mtDNA‐copy/g total DNA] × pDNA mt [ng pDNA/mtDNA‐copy]

(1)

DNA delivery value for nuclei [ng pDNA/g total DNA] = nDNALiver [nDNA‐copy/g total DNA] × pDNAnu nu [ng pDNA/nDNA‐copy]

(2)

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Detection of Exogenous mRNA in Mouse Liver Using RT-PCR. For the experiment shown in Figure 3, 100 μg of pHSP-ND4FLAG coding Nd4flag (exogenous mRNA) was administered via the tail vein of mice via HTV injection. At various time points after the HTV injection, the livers were harvested and minced with scissors. Total RNA was then purified with RNeasy (Qiagen, Hilden, Germany), combined with DNase I digestion for the degradation of DNA in the total RNA samples using RNase-Free DNase Set (Qiagen). The resulting RNA suspension was reverse transcribed using a High Capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA). A PCR was performed on cDNA using GoTaqGreen Master Mix kit (Promega, Madison, WI, USA) to detect exogenous mRNA coding Nd4f lag derived from pHSPND4FLAG and endogenous mRNA coding Nd6 derived from mouse mtDNA, using primers Nd4flag (+) and Nd4flag (−); primers M Nd6 (+) and M Nd6 (−) (Table S1). Each PCR product (5 μL) was subjected to electrophoresis in a 1.5% agarose gel in TAE (40 mM TrisHCl, 40 mM acetic acid, 1 mM EDTA, pH 8.0) at 100 V for 30 min. The DNA bands were visualized by UV after ethidium bromide staining. For the experiment shown in Figure 5A, 100 μg of pHSPmtLuc (CGG), pHSP-ND4FLAG, or pHSP-mtLuc (TAG) was administered to mice via HTV injection. At 6 h after the

RESULTS Gene Delivery to the Liver by HTV Injection and Evaluation of Exogenous pDNA-Levels in the Mitochondria- and Nuclei-Enriched Fraction Using Quantitative PCR. To validate in vivo mitochondrial gene delivery via hydrodynamic injection using an artificial mitochondrial reporter DNA vector, we chose liver as a target tissue for this validation. The liver is rich in mitochondria, and procedures for the isolation of the mitochondria from the tissue are well established. Because of this, we concluded that the use of liver tissue would be appropriate to evaluate small amounts of exogenous mitochondrial gene products. The hydrodynamic injection method has been reported to result in effective nuclear transgene expression in certain tissues such as livers and skeletal muscle,10−15 and this method without carriers, including viral vectors and cationic polymers, has been used to investigate various reporter vectors by many researchers. Previously, we demonstrated that naked pDNA was delivered into mitochondria in skeletal muscle of rats by hydrodynamic injection.9,16 In that experiment, we established a method to quantify the mitochondrial localization of exogenous pDNA following hydrodynamic injection. In this procedure, we used pDNA3.1 as applied pDNA and specific primer sets for the 4315

DOI: 10.1021/acs.molpharmaceut.5b00511 Mol. Pharmaceutics 2015, 12, 4311−4320

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transcriptional efficiency of pHSP-ND4FLAG and pND4FLAG was almost the same, suggesting that it is difficult to transcribe the downstream gene under HSP control in in vitro Transcription System. These results indicate that the pHSPND4FLAG could be only slightly transcribed in nuclei at the same levels as the negative control without a promoter (pND4FLAG). Design of pHSP-mtLuc (CGG) To Express Nluc Luciferase in Mitochondria. To evaluate mitochondrial protein expression, we designed a new candidate mitochondrial reporter DNA vector (pHSP-mtLuc (CGG)) which can express a functional reporter protein, namely, Nluc luciferase. The Nluc luciferase is a type of luminescent reporter protein and has a shorter amino acid length than that of the firefly luciferase.23 More preferably, the Nluc luciferase luminescent activity potential is reported to be ∼150-fold higher than that of firefly luciferase, thus making it a highly sensitive reporter protein. We designed pHSP-mtLuc (CGG) in consideration of the following points: the mitochondrial codon system and mitochondrial RNA processing (Figure 4). The conventional Nluc luciferase (Nluc) gene can be translated on cytoplasmic ribosomes not in the mitochondria, because the AGG codon at amino acid 45 (bases 133−135) encodes for Arg in the nuclear genetic code, while the AGG codon is leading to the arrest of mitochondrial translation (Table 1). Therefore, we designed the mitochondrial Nluc

quantification by PCR analysis as described in the Experimental Section. We herein performed the HTV injection using various doses of naked pDNA3.1, and harvested the livers at 24 h postinjection, followed by quantification of the DNA delivery values for mitochondria-enriched fraction and the nucleienriched fraction (Figure 2). The results showed that the amount of delivered pDNA was almost the same between the two organelles, and the pDNA was increased with increasing applied dose, suggesting that pDNA molecules might nonspecifically localize in liver mitochondria. This nonspecific distribution of pDNA in the target tissue via hydrodynamic injection was also observed in the case of an HLV injection targeted to skeletal muscle tissue as previously reported.9 Detection of Exogenous mRNA in Mouse Liver after HTV Injection of pDNA Vector Containing HSP. For successful transgene expression on mitochondria, the gene is required to be transcribed in mitochondria. We validated this transcription in liver in the case of our designed DNA vector, pHSP-ND4FLAG that possesses the HSP promoter and ND4 derived from mouse mtDNA and FLAG-tag (Figure S1A(a)), educated from pTR-UF11-ND4FLAG (Figure 1A) reported by Yu et al.22 We administered pHSP-ND4FLAG to the tail vein of mice via HTV injection, and evaluated the transcription efficiency of the gene by amplifying the Nd4flag sequences using RT-PCR. Briefly, the livers were harvested at several time points after the injection, followed by the extraction of total RNA. After reverse transcription of the RNA, PCR was carried out with specific primers to detect exogenous mRNA coding Nd4flag derived from pHSP-ND4FLAG (upper panels in Figure 3A) and endogenous mRNA coding Nd6 derived from mouse mtDNA (upper panels in Figure 3B). As a result, the transcription products of pHSP-ND4FLAG were detected in the gel image which reached a peak at ∼24 h. We confirmed the absence of contaminating DNA when this RT-PCR assay was peformed. Gel images of the RT-PCR detection showed that the target DNA bands appeared in the case of reverse transcription (RT+) (upper panels in Figures 3A and 3B) and were not observed in the absence of reverse transcription (RT−) (lower panels in Figures 3A and 3B). These results indicate that the DNase I treatment procedure during the RT-PCR assay was properly performed. The findings suggest that pHSP-ND4FLAG was transcribed inside cells. However, there was a concern as to whether the pHSPND4FLAG would function in nuclei, although HSP is wellknown as an endogenous mitochondrial promoter. We then evaluated the transcription efficiency of pHSPND4FLAG in nuclear conditions using a commercially available HeLaScribe Nuclear Extract in vitro Transcription System. We also evaluated the transcription efficiencies of the pCMVND4FLAG containing the cytomegalovirus (CMV) promoter as a positive control and pND4FLAG without a promoter as a negative control. It is well-known that the CMV promoter is frequently used to achieve effective nuclear transgene expression in mammalian cells. The sequence information on these plasmids is summarized in Figure S1A (see the Supporting Information for the details). pHSP-ND4FLAG, pCMV-ND4FLAG, and pND4FLAG were added to the nuclear extract, followed by in vitro transcription, and the amounts of the transcripts were quantified by real-time RT-PCR (Figure S1B). The results showed that the amount of the pCMVNDFLAG transcript was ∼50-fold higher than that of pHSPND4FLAG and pND4FLAG. Additionally, the level of

Table 1. Positions of Point Mutations in the Nluc Luciferase Gene To Construct mtLuc (CGG) and mtLuc (TAG) Genes gene Nluc

codon (position) tgg (34−36) agg (133−135)

mtLuc (CGG)

tgA (34−36) Cgg (133−135)

mtLuc (TAG)

tgA (34−36) TAg (133−135)

amino acid encoded by the codon Trp for universal and mitochondrial codon Arg for universal codon/Stop codon for mitochondrial codon Stop codon for universal codon/Trp for mitochondrial codon Arg for universal and mitochondrial codon Stop codon for universal codon/Trp for mitochondrial codon Stop codon for universal and mitochondrial codon

luciferase (mtLuc (CGG)) gene that is adjusted to the mitochondrial codon system so as to permit protein expression, as shown in Figure 4. In the mtLuc (CGG) gene, AGG codon at amino acid 45 (bases 133−135) was changed into CGG codon which encodes for Arg in both the nuclear genetic and the mitochondrial code. Moreover, the mtLuc (CGG) gene contains the TGA codon at amino acid 12 (bases 34−36), which encodes for mitochondrial Trp and nuclear genetic stop codon, therefore mitochondrial Nluc luciferase cannot be produced outside mitochondria. We also designed a pHSP-mtLuc (TAG) containing TAG codon at amino acid 45 (bases 133−135), mitochondrial/universal stop codon, as a dual stop codon variant, which would lead to nuclear and mitochondrial translational arrest. The positions of point mutations in the conventional Nluc gene in the design of new constructs are summarized in Table 1. The positions are indicated by uppercase letters. Regarding mitochondrial RNA processing, we noted that the mitochondrial RNA is polycistronically transcribed from mtDNA followed by mitochondrial RNA processing which is a different process from the cytosolic RNA transcription 4316

DOI: 10.1021/acs.molpharmaceut.5b00511 Mol. Pharmaceutics 2015, 12, 4311−4320

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Molecular Pharmaceutics

factors (p = 0.40) and no significant differences among the different “applied dose” groups (p = 0.66). Moreover, we detected significant differences between pHSP-mtLuc (CGG) group and pHSP-mtLuc (TAG) group. Based on these results, we concluded that the HLV injection of pHSP-mtLuc (CGG) resulted in successful mitochondrial gene delivery in skeletal muscle via HLV injection.

process. The transcribed mitochondrial RNA is processed by RNaseP and RNaseZ, which recognize the 5′ and 3′ side secondary structure of the mitochondrial tRNA respectively, and then splice the polycistronic RNAs to functional units such as mRNA, tRNA, and rRNA, followed by the translational process.24,25 Based on this background information, the gene sequence coding for mitochondrial tRNA was linked to the 3′ terminus of the mtLuc (CGG) gene in pHSP-mtLuc (CGG) so as to improve mitochondrial protein translation. As shown in Figure 4, the pHSP-mtLuc (CGG) was constructed by inserting the mtLuc (CGG) gene linked with gene sequence coding tRNAAsp with the 5′UTR into pHSP-ND4FLAG which was validated for mitochondrial transcription (please see the Supporting Information for detailed sequence information). Evaluation of in Vivo Transgene Expression of Mitochondrial Nluc Luciferase Following Hydrodynamic Injection. We evaluated Nluc luciferase activity on mouse liver mitochondria, after the administration of 100 μg of pHSPmtLuc (CGG) encoding mitochondrial Nluc luciferase protein to the tail vein of mice via HTV injection. We also evaluated the transgene expression of pHSP-ND4FLAG without mitochondrial Nluc luciferase and pHSP-mtLuc (TAG), which contains the mitochondrial/universal stop codon (TAG) as controls. Before measuring Nluc luciferase activity, we investigated the mRNA levels of Nluc luciferase following HTV injection (Figure 5A). PCR was done with specific primers to detect cDNA derived from exogenous mRNA coding Nluc luciferase after reverse transcription. As a result, the mRNAs coding Nluc luciferase derived from pHSP-mtLuc (CGG) and pHSP-mtLuc (TAG) were detected. Figure 5B shows that the value of Nluc luciferase activity in liver tissue administered pHSP-mtLuc (CGG) was significantly higher than that with pHSP-ND4FLAG and pHSP-mtLuc (TAG). We also confirmed that pHSP-mtLuc (CGG) produced no Nluc luciferase in the nucleo-cytoplasmic translation system. For this validation, mRNAs coding mtLuc (CGG) and mtLuc (TAG), which contain a universal stop codon “TGA”, and mRNA coding wild type Nluc luciferase (positive control) were used. These mRNAs were added to a Rabbit Reticulocyte Lysate for nucleo-cytoplasmic translation, and the Nluc luciferase activities were measured. Nluc luciferase activity was not detected when mRNAs coding mtLuc (CGG) and mtLuc (TAG) were used, while strong Nluc luciferase activity was observed in the case of mRNA coding for wild type Nluc luciferase (Y. Yamada et al., unpublished results). The result suggests that “TGA” codon substitution prevented the correct nucleo-cytoplasmic translation of the mitochondrial Nluc luciferase construct. These results suggest that HTV injection achieves liver mitochondrial gene delivery and pHSPmtLuc (CGG) functions to express mitochondrial Nluc luciferase protein. We subsequently attempted the mitochondrial delivery of pHSP-mtLuc (CGG) to skeletal muscle, where mitochondrial genomic dysfunctions are largely associated with various diseases.4−6,26 In this experiment, a 3 mL sample of saline containing pHSP-mtLuc (CGG) or pHSP-mtLuc (TAG) was administered at a dose of 150 or 300 μg of pDNA via HLV injection. As a result, skeletal muscle that had been administered pHSP-mtLuc (CGG) showed a higher Nluc luciferase activity than that with pHSP-mtLuc (TAG) (Figure 6). We also compared the effect of two factors, namely, the “pDNA type” and the “applied dose”, by a two-way ANOVA analysis. There were no significant interactions between the two

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DISCUSSION Hydrodynamic injection that Liu et al.10 and Zhang et al.11 originally reported is now frequently used as an efficient, simple and convenient transfection method for laboratory animals. Excellent review articles have been published with emphasis on various aspects of this method for in vivo nuclear gene delivery without the aid of viral and nonviral vectors.27,28 Taking these advantages into consideration, many researchers have used hydrodynamic injection as an in vivo nuclear gene transfer method for delivering naked pDNA in wide variety of basic and translational studies (please see excellent reviews for details27−29). As described above, we succeeded in in vivo mitochondrial gene delivery via hydrodynamic injection.9 Based on previous reports and our findings, we conclude that hydrodynamic injection can be a useful mitochondrial transfection method for evaluating the mitochondrial transgene expression of artificial mitochondrial reporter DNA vector (pHSP-mtLuc (CGG)), and we were able to detect mitochondrial transgene expression. It is noteworthy that the transfection of pHSP-mtLuc (CGG) into culture cells by lipofection showed no luciferase activity (data not shown). Thus, we validated mitochondrial the transgene expression of pHSP-mtLuc (CGG) in animal experiments using hydrodynamics injection in this study. In this study, we designed original artificial mitochondrial reporter DNA vectors based on the pTR-UF11-ND4FLAG previously reported by Yu et al.22 The pTR-UF11-ND4FLAG possesses HSP and ND4 gene sequences derived from human mtDNA, and transgene expression was validated using mice. We also constructed DNA vectors based on the sequence of mouse mtDNA and validated transgene expression using mice and rats. Unlike human HSP where the sequence “AAACCCC” located on the upstream of transcription start site of the human mitochondrial tRNAPhe is essential for mtDNA transcription (Figure 1A), the sequence information on the mouse HSP was not clearly determined. In this study, we chose the sequence containing “AAACCCC” which is located on the upstream of transcription start site of the mouse mitochondrial tRNAPhe as mouse HSP promoter (Figure 1B). The results confirmed that our artificial mitochondrial reporter DNA vector (pHSP-ND4FLAG) produced exogenous mRNA in the mouse liver (Figure 3). Moreover, we showed that pHSP-mtLuc (CGG) expressed Nluc luciferase protein (exogenous protein) in the mitochondria of rat skeletal muscle as well as the mouse liver. The results showing that the pHSPmtLuc (CGG) could achieve mitochondrial transgene expression regardless of the species might be due to the fact that sequence homology of the D-loop including the HSP region between mouse mtDNA and rat mtDNA was about 75%. On the other hand, although the sequence homology of the D-loop in mtDNA between the mouse and human was about 64%, Yu et al. succeeded in producing exogenous mRNA in mouse mitochondria using pTR-UF11-ND4FLAG designed based on human mtDNA. 4317

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volume, high injection rate), toxicity concerns remain. Regarding HTV injection, Liu’s group reported that there was no serious liver damage after hydrodynamic injection.10 It was also reported that HLV injection caused only minimal and transient muscle damage.41,42 Moreover, we investigated mitochondrial toxicity following HLV injection, and the results indicated no significant mitochondrial toxicity on the basis of COX activity and mitochondrial membrane potential measurements.9 We also observed that hydrodynamic injection caused no fatalities in the mice and rats used in our experiments. Recently, several attempts at establishing a hydrodynamic procedure for clinical use have been reported. Liu’s group reported on the image-guided, lobe-specific hydrodynamic gene delivery to liver using a computer-controlled injection device.43 This procedure resulted in the safe and efficient gene delivery targeted to liver in small and large animals.43,44 Wolff’s group reported that the modified HLV procedure provided a safe gene delivery technique targeted to muscle tissue in small and large research animals in an excellent review.14 The two groups, and others, are continuously conducting research on hydrodynamic gene delivery for human gene therapy. We believe that a clinically applicable hydrodynamic procedure will be established in the near future. In this study, we succeeded in constructing artificial mitochondrial reporter DNA vectors with large scale preparation possible. We also confirmed that hydrodynamic injection achieved mitochondrial delivery of naked pDNA in mouse liver, resulting in the production of exogenous mRNA in the liver. Moreover, we showed that the hydrodynamic injection of pHSP-mtLuc (CGG) could achieve mitochondrial Nluc luciferase protein expression in liver and skeletal muscle. The findings are the first report of the successful gene expression of an exogenous protein with a function as an enzyme in in vivo mitochondria. We expect that our findings would contribute to research regarding mitochondrial molecular biology as well as mitochondrial gene therapy.

To validate the mitochondrial transcription of our designed DNA vector in mouse livers, we administered pHSPND4FLAG via hydrodynamic injection, and evaluated the transcription efficiency of the gene by amplifying Nd4f lag sequences using RT-PCR (Figure 3). This result showed that the hydrodynamic injection of pHSP-ND4FLAG produced exogenous mRNA in the mouse liver, while the possibility that the gene did not function in mitochondria but in nuclei cannot be completely excluded. It would be expected that direct verification using in vitro mitochondrial transcription could be used to validate the mitochondrial transcription of pHSPND4FLAG. However, an in vitro transcription assay using mammalian mitochondria is not a generally widely used method, although there are reports of its use by Fisher et al.30 and Falkenberg et al.31 It is also necessary to prepare several special proteins including POLRMT, TFAM, TFB1M, and TFB2M. Therefore, we attempted to indirectly validate mitochondrial transcription of the pHSP-ND4FLAG based on the results from the transcription in the nuclei and transcription in whole cells, since it was difficult to verify the in vitro mitochondrial transcription directly. As shown in Figure S1, pHSP-ND4FLAG could be only slightly transcribed in nuclear extracts at the same levels as the negative control without a promoter, although pHSP-ND4FLAG was transcribed inside the liver after hydrodynamic injection (Figure 3). These results indicate the possibility that the pHSP-ND4FLAG might be transcribed in mouse liver mitochondria. This issue is the subject of a future investigation. To validate protein expression after HTV injection of pHSPND4FLAG, we performed Western blotting to detect ND4FLAG. No band corresponding to ND4-FLAG was detected (data not shown), although the transcription products of pHSP-ND4FLAG were detected in the liver (Figure 3). Based on these results, we concluded that the efficiency of mitochondrial transfection via hydrodynamic injection was too low to permit the exogenous protein to be detected using Western blotting. Therefore, we used pHSP-mtLuc (CGG), which produces a highly sensitive reporter protein, to validate mitochondrial gene delivery by hydrodynamic injection in this study. We are continuously making efforts directed toward improving mitochondrial transgene expression efficiency, to develop a new artificial DNA construct. It has been reported that the 5 terminus of the endogenous mitochondrial mRNA forms a higher order structure with difficulty.32−34 This characteristic of the 5 terminus of the endogenous mitochondrial mRNA would be necessary to be accessible to the tight mRNA entrance site of mitochondrial ribosomes.35 Moreover, recently reported findings suggest that mitochondrial ribosomes localize on the inner membrane of the cristae side36 and mitochondrial translation may progress while the protein is being inserted into the inner membrane.37−40 These reports prompted us to choose the ND4 (endogenous mitochondrial protein) as a leader sequence of Nluc luciferase at the 5 terminus rather than designing a new 5′UTR region of Nluc luciferase. It was expected that the mRNA transcribed from pHSP-mtLuc (CGG) containing ND4 as a leader sequence would easily gain access to the mitochondrial ribosome and be translated via the mitochondrial endogenous transcription/translation mechanism. In this study, we validated mitochondrial gene delivery in liver and skeletal muscle via hydrodynamic injection of pHSPmtLuc (CGG). Since the hydrodynamic injection requires somewhat severe conditions on the animals (large injection

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ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge at The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.5b00511. Materials and methods, table of primers, transcription assay figure, and vector sequences (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Kita 12, Nishi 6, Kita-ku, Sapporo 060-0812, Japan. Tel: +8111-706-3919. Fax: +81-11-706-4879. E-mail: harasima@pharm. hokudai.ac.jp. Author Contributions †

Y. Yasuzaki and Y. Yamada contributed equally as first author.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported, in part by, a Grant-in-Aid for Young Scientists (A) [Grant 23680053 to Y. Yamada], a Grant-in-Aid for Challenging Exploratory Research [Grant 25560219 to Y. Yamada], and a Grant-in-Aid for Scientific Research (B) [Grant 26282131 to Y. Yamada] from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese 4318

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Government (MEXT). We are grateful to Eriko Kawamura, Jiro Abe, Mai Tabata, and Yutaka Fukuda for their dedicated cooperation on purification of pDNA used in this experiment. We also thank Dr. Milton Feather for his helpful advice in writing the manuscript.

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