Original Research ArticleTargeted mitochondrial delivery of antisense RNA-containing nanoparticles by a MITO-Porter for safe and efficient mitochondrial gene silencing
Introduction
Many useful drug delivery systems (DDS) in the field of gene therapy that target the nucleus or cytoplasm have been developed (Crowley and Rice, 2015; Miyata et al., 2012; Nakamura et al., 2012; Tammam et al., 2016), and delivering therapeutic nucleic acids for gene repair therapy and antisense therapy have been actively carried out, leading to clinical applications. Although this concept is applicable to mitochondria (Yamada and Harashima, 2008, Yamada and Harashima, 2017), mitochondrial gene therapy for clinical use has not been extensively examined. Thus, the development of mitochondrial DDS that are designed to regulate the mitochondrial genomic system are needed, if gene therapy for mitochondrial diseases caused by genetic defects in mitochondrial DNA is to be achieved.
In recent years, numerous reports have appeared showing that cytosolic RNA can be transported into the inside of mitochondria through the use of a mitochondrial membrane carrier (Endo et al., 2010; Schneider, 2011; Tarassov et al., 2007). A therapeutic strategy using this mechanism has been validated, and therapeutic wild-type tRNA has been transported to mitochondria containing mutant tRNA in disease cells via allotropic expression, a method for transfecting pDNA encoding an RNA into the nucleus and then delivering the nuclear-expressed RNA to mitochondria, resulting in the maintenance of a normal mitochondrial translation process (Karicheva et al., 2011; Kolesnikova et al., 2004; Kolesnikova et al., 2000). It has also been reported that the use of a mitochondrial import sequence resulted in the specific inhibition of the replication of mutant mitochondrial DNA, and that the wild-type mRNA was delivered to mitochondria to express a mitochondrial protein (Comte et al., 2013; Dovydenko et al., 2016; Tonin et al., 2014a; Tonin et al., 2014b; Wang et al., 2012).
Adhya and co-workers evaluated the antisense effect by using an antisense RNA oligonucleotide (ASO) that was complexed with an endogenous mitochondrial transport carrier, namely, the RNA import complex (RIC) protein (Goswami et al., 2003; Mahapatra et al., 1998; Mukherjee et al., 1999; Mukherjee et al., 2008). They used an ASO modified at the 5 ‘end with a Darm sequence, a sequence that binds to the RIC protein and has mitochondrial targeting activity, and assembled a complex containing a Darm ASO and the RIC protein. They confirmed the degradation of target mRNA and the suppression of protein expression by the transfection of this complex into cells, suggesting that ASO actually functions in mitochondria (Mukherjee et al., 2008).
In a previous study, we reported on the packaging of naked Darm ASO in a MITO-Porter, a liposomal nano carrier system for mitochondrial delivery that was developed in our laboratory (Abe et al., 2018; Takano et al., 2017; Yamada et al., 2008), using the ethanol dilution method, and verified that an antisense effect could be obtained (Furukawa et al., 2015; Yamada and Harashima, 2017). The sequence of ASO (5′- GGGACUGUAGCUCAAUUGGUAGAGCAUCUUGCGCUGCAUGUGCCAU -3′) for a cargo is the same one that was used by Adhya's group (Mukherjee et al., 2008), and the Darm ASO [COX2], where the Darm modified ASO that targeted mitochondrial mRNA encoded by mtDNA that codes for cytochrome c oxidase subunit II (COX2) was used in this study. COX2 is one of the mitochondrial proteins that make up complex IV of the electron transport system that functions in the inner membrane of mitochondria. When the ASO was delivered to mitochondria, it became bound to the target COX2 mRNA in mitochondria, followed by the degradation of the target mRNA or the inhibition of translation. Furthermore, the expression level of the target COX2 protein of the mitochondrial respiratory chain complex subunit would be expected to decrease mitochondrial membrane potential and decrease ATP production, eventually resulting in cell death (Fig. 1A).
As the result of qualitatively verifying the antisense effect, which included the quantitative reverse transcription PCR (RT-PCR) and immunostaining, it was shown that the expression of the target mRNA was suppressed, as was the production of the corresponding protein in mitochondria (Furukawa et al., 2015). These results suggest that delivering ASO to mitochondria using the MITO-Porter is an effective method for controlling mitochondrial gene expression. However, we found that the same dose of the empty MITO-Porter without ASO also caused a similar decrease in cell viability (Fig. 2A), suggesting that the empty carrier was strongly cytotoxic. One possible reason for this would be that the encapsulation efficiency of ASO in the MITO-Porter prepared by the ethanol dilution method was low, and the dose of lipid envelope was increased due to the high lipid to ASO ratio (Lipids/RNA molar ratio, 1680). In addition, because of this strong cytotoxicity, the experimental results varied considerably, and it was not possible to quantitatively show a decrease in protein expression or a decrease in mitochondrial function such as ATP production, thus making it impossible to verify an antisense effect.
Our findings described above stimulated us to attempt to construct carriers for delivering functional nucleic acids for gene therapy that are therapeutically effective but less toxic. In order to efficiently introduce nucleic acids into the cells with safety, a structure capable of efficiently packaging nucleic acids in the carrier is a very important issue. The image shown in Fig. 1B shows a multifunctional envelope-type nano device (MEND), a non-viral vector developed in our laboratory, in which various macromolecules can be packaged efficiently (Kogure et al., 2004; Nakamura et al., 2012; Yamada et al., 2012). In this study, we constructed a MITO-Porter encapsulating ASO with a reduced cytotoxicity based on the MEND concept, in which nanoparticles of ASO are complexed with a polycation, polyethyleneimine (PEI), a linear 10 kDa molecule, and are then encapsulated in the MITO-Porter, a process that increases the encapsulation efficiency. In an ideal situation (Fig. 1A), after reaching the MITO-Porter reaches the mitochondria, the nanoparticle of Darm modified ASO is delivered to the intermembrane space via membrane fusion with the outer membrane and is then imported into the mitochondrial matrix via the Darm import machinery. We also examined the mitochondrial antisense effect and mitochondrial membrane potential and ATP production of this preparation by delivering ASO targeting mitochondria using the MITO-Porter system.
Section snippets
Chemicals and materials
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and sphingomyelin (SM) were purchased from Avanti Polar lipids (Alabaster, AL). Cholesteryl hemisuccinate (CHEMS) and PEI (a 10 kDa, linear molecule) were purchased from Sigma (St Louis, MO). Stearylated octaarginine (STR-R8) (stearylated-RRRRRRRR-NH2) and Cholesteryl (Chol)-GALA (cholesteryl-WEAALAEALAEALAEHLAEALAEALEALAA-NH2) were obtained from KURABO Industries (Osaka, Japan). Darm modified antisense 2′ –O-Methyl (2′-OMe) RNA targeting COX
Construction of MITO-Porter encapsulating nano particle of ASO
In a previous study, we reported on the development of a MEND as an original gene vector and reported that nucleic acids could be efficiently packaged in the in the carrier by forming nanoparticles in which the nucleic acids were condensed with polycations, which mimics a virus envelope (Kogure et al., 2004; Nakamura et al., 2012; Yamada et al., 2012). Transmission electron microscopy analyses showed that the resulting MEND consisted of nanoparticles of nucleic acids covered with envelopes (
Discussion
We previously showed that the mitochondrial delivery of ASO using the MITO-Porter resulted in the suppression of the expression of target mRNA and protein in mitochondria (Furukawa et al., 2015). However, it was not possible to carry out a detailed investigation of the mitochondrial antisense effect, due to the strong cytotoxicity of the conventional MITO-Porter system (Fig. 2A). Because cytotoxicity is a major obstacle to the delivery of functional nucleic acids for gene therapy, we first
Acknowledgment
This work was supported, in part by, a Grant-in-Aid for Scientific Research (B) [Grant No. 26282131 and 17H02094 to Y.Y.] and Grants-in-Aid for Exploratory Research [Grant No. 26282131 and 15H12532 to Y.Y.] from the Ministry of Education, Culture, Sports, Science and Technology of Japanese Government (MEXT), a Grant-in-Aid for JSPS Research Fellow [Grant No. 14J02379 to E.K.] from Japan Society for the Promotion of Science (JSPS), the Uehara Memorial Foundation (to Y.Y.) and Akiyama Life
References (38)
- et al.
Validation of a strategy for cancer therapy: delivering aminoglycoside drugs to mitochondria in HeLa cells
J. Pharm. Sci.
(2016) - et al.
Cardiac progenitor cells activated by mitochondrial delivery of resveratrol enhance the survival of a doxorubicin-induced cardiomyopathy mouse model via the mitochondrial activation of a damaged myocardium
J. Control. Release
(2018) - et al.
Evolving nanoparticle gene delivery vectors for the liver: what has been learned in 30 years
J. Control. Release
(2015) - et al.
Method of carrier-free delivery of therapeutic RNA importable into human mitochondria: lipophilic conjugates with cleavable bonds
Biomaterials
(2016) - et al.
Mitochondrial matrix reloaded with RNA
Cell
(2010) - et al.
Mitochondrial delivery of antisense RNA by MITO-Porter results in mitochondrial RNA knockdown, and has a functional impact on mitochondria
Biomaterials
(2015) - et al.
Mitochondrial targeting functional peptides as potential devices for the mitochondrial delivery of a DF-MITO-Porter
Mitochondrion
(2013) - et al.
Development of a non-viral multifunctional envelope-type nano device by a novel lipid film hydration method
J. Control. Release
(2004) - et al.
Stepwise transfer of tRNA through the double membrane of Leishmania mitochondria
J. Biol. Chem.
(1999) - et al.
A pH-sensitive cationic lipid facilitates the delivery of liposomal siRNA and gene silencing activity in vitro and in vivo
J. Control. Release
(2012)
How successful is nuclear targeting by nanocarriers?
J. Control. Release
Characterization of chemically modified oligonucleotides targeting a pathogenic mutation in human mitochondrial DNA
Biochimie
Modeling of antigenomic therapy of mitochondrial diseases by mitochondrially addressed RNA targeting a pathogenic point mutation in mitochondrial DNA
J. Biol. Chem.
Mitochondrial drug delivery systems for macromolecule and their therapeutic application to mitochondrial diseases
Adv. Drug Deliv. Rev.
MITO-Porter: a liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion
Biochim. Biophys. Acta
Multifunctional envelope-type nano device (MEND) for organelle targeting via a stepwise membrane fusion process
Methods Enzymol.
A nanocarrier system for the delivery of nucleic acids targeted to a pancreatic beta cell line
Biomaterials
Mutations in a tRNA import signal define distinct receptors at the two membranes of Leishmania mitochondria
Mol. Cell. Biol.
Mitochondrial RNA import in Leishmania tropica: aptamers homologous to multiple tRNA domains that interact cooperatively or antagonistically at the inner membrane
Mol. Cell. Biol.
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These authors equally contribute this study.