Localization of exogenous DNA to mitochondria in skeletal muscle following hydrodynamic limb vein injection
Graphical abstract
Introduction
Mitochondrial dysfunction has been implicated in a variety of human diseases [1], [2], [3]. It is noteworthy that such disorders are mainly associated with tissues that have high energy requirements, such as the brain, heart, muscle and liver. It is now well accepted that mutations and defects in the mitochondrial genome form the basis of these diseases [4], [5], [6], [7]. Therefore, mitochondrial gene therapy and diagnosis would be expected to have substantial medical benefits. However, their utility has not yet been realized because mitochondrial gene delivery technology is the bottleneck. Even through in vitro experiments have been reported, actual studies of mitochondrial gene delivery are very few [8], [9], [10], [11]. The use of a variety of applications for nuclear gene delivery in vitro/in vivo have been reported [11], [12], [13], [14], [15], [16], in attempts to accelerate progress in the field of nuclear gene therapy. Thus, successful mitochondrial gene transfer would largely contribute to mitochondrial gene therapy.
Skeletal muscle represents an attractive target tissue for mitochondrial gene therapy as well as nuclear gene therapy, because mitochondrial diseases, including mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) [5] and myoclonic epilepsy and ragged-red fiber disease (MERRF) [7] are largely associated with mitochondrial genomic dysfunction in skeletal muscle. Vascular delivery procedures have recently been used to deliver plasmid DNA (pDNA) to the skeletal muscle of rodents and nonhuman primates by hydrodynamic limb vein (HLV) injection [17], [18], [19], [20]. The hydrodynamic injection method originally was reported by Liu et al. [21] and Zhang et al. [22], and was used to achieve effective nuclear transgene expression in hepatocytes in mice by the rapid injection of large volumes of naked pDNA into the tail vein. Recently, Yan et al. reported on a facile hydrodynamic injection method via the retro-orbital sinus [23].
In the HLV injection procedure, a tourniquet is used to limit the delivery area to one limb per injection and naked pDNA is rapidly injected into the vein in the anterograde direction [20]. A sufficient volume of saline is used to facilitate extravasation of the pDNA from the vasculature and into the muscle tissue through multiple physical barriers. It has been suggested that hydrodynamic force could induce the transient opening of cellular membrane to permit pDNA to be internalized into cells, the subsequent localization of pDNA into the nucleus may be achieved via some mechanisms such as active nuclear import, cell division or transient opening of nuclear membrane. More recently, it has been reported that the HLV injection of condensed pDNA was even more effective than naked pDNA in achieving nuclear transgene expression in skeletal muscle [24], [25], [26]. Based on these reports, we hypothesized that the HLV injection technique might be useful for delivering pDNA into, not only the nucleus, but also the mitochondria.
The purpose of this study was to validate the possibility of mitochondrial DNA delivery in skeletal muscle of rats by HLV injection (Fig. 1a). We first verified, using PCR analysis, that the HLV injection technique could be used to deliver naked pDNA into myofibrillar mitochondria. We also investigated the effects of injection volume and dose of pDNA on mitochondrial delivery by HLV injection. A combination of immunostaining for mitochondrial proteins and fluorescent in situ hybridization to detect pDNA (Immuno-FISH) permitted us to visualize the pDNA that was delivered to skeletal muscle tissue. Finally, we assessed the mitochondrial toxicity in skeletal muscle following HLV injection in terms of cytochrome c oxidase (COX) activity and mitochondrial membrane potentials.
Section snippets
Materials
The pcDNA3.1 (+)-luc plasmid was constructed by inserting the firefly luciferase gene (Hind III-Xba I fragment) of the pGL3-Control plasmid (Promega, Madison, WI, USA) into the pcDNA3.1 (+) plasmid (Invitrogen, Carlsbad, CA, USA) pretreated with the same restriction enzymes. The luciferase gene in the pDNA is expressed under the control of the cytomegalovirus promoter. The pDNA was purified using an Endfree Plasmid Giga Kit (Qiagen GmbH, Hilden, Germany). Oligonucleotides were purchased from
Gene delivery to skeletal muscle by HLV injection and evaluation of nuclear transgene expression
We first evaluated nuclear transgene expression in hind limb muscle groups following HLV injection, to determine the optimal part of the limb muscle for the evaluation of mitochondrial gene delivery. In this experiment, pDNA encoding the luciferase gene was intravenously injected into the distal hind limb of rats, as shown in Fig. 1a. Prior to each pDNA injection, a tourniquet was placed on the upper hind limb to restrict blood flow into and out of the hind limb. Basically, suspensions of naked
Discussion
To date, several groups have reported successful examples of mitochondrial RNA delivery [29], [30], [31], since the fact that the mitochondrion possesses endogenous RNA imported via an RNA signal tag system in these studies [32], [33]. Moreover, it has been reported that allotropic expression in the nucleus, followed by the transfer of a signal tag fused protein to the mitochondria appears promising [11]. However, these signal tags may not function in mitochondrial diseased cells. For example,
Conclusion
In this study, we attempted the mitochondrial delivery of exogenous pDNA in skeletal muscle using HLV injection. Our results indicated that an HLV injection resulted in the mitochondrial delivery of naked pDNA. Moreover, the evaluation of COX activity and mitochondrial membrane potentials showed that HLV injection was not toxic to mitochondria. The findings are the first report regarding in vivo mitochondrial gene delivery using the HLV injection procedure. We expected that the findings
Acknowledgment
This work was supported, in part by, the Advanced Research for Medical Products Mining Programme of the National Institute of Biomedical Innovation (NIBIO), a Grant-in-Aid for Young Scientists (A) and a Grant-in-Aid for Scientific Research (S) from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government (MEXT). We thank Sapporo General Pathology Laboratory Co., Ltd., for the technical assistance with the histopathology (Sapporo, Japan). We are grateful to Dr.
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2017, BiomaterialsCitation Excerpt :To achieve such an innovative strategy, several promising studies focusing on the combination of using a mitochondrial codon and a mitochondrial endogenous promotor have been reported to date [6–9]. Based on previous interesting reports, we are in the process of continuously developing more efficient methods of mitochondrial gene delivery via hydrodynamic injection [14]. The hydrodynamic injection originally reported by Liu et al. [21] and Zhang et al. [34] is frequently as an efficient in vivo nuclear gene transfer method of naked pDNA [11–13].
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These authors contributed equally as first author.