We present a general method for in-cellulo delivery of 2′-O-methyl (2′-OMe) RNA oligonucleotides (oligos) to mitochondria for antisense applications, with potential for implementation in other mitochondrial DNA (mtDNA)-targeted therapies. Exosomes, which are nanoscale, naturally occurring extracellular vesicles (EVs), have been employed for biotechnology applications in oligonucleotide delivery in recent years. We discovered that exosomes from fetal bovine serum (FBS) can be used as a simple and biologically compatible delivery agent of 2′-OMe RNA antisense oligonucleotides to cellular mitochondria, leading to target protein knockdown. While most RNA interference and antisense mechanisms occur in the cytoplasm or nucleus, the need for mitochondrial targeting has become increasingly apparent. Mitochondrial disease describes a variety of currently incurable syndromes that especially affect organs requiring significant energy including the muscles, heart, and brain. Many of these syndromes result from mutations in mtDNA, which codes for the 13 proteins of the oxidative phosphorylation system and are thus often implicated in inherited metabolic disorders.
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References
1.
WallaceDC, SinghG, LottMT, et al.Mitochondrial DNA mutation associated with Leber’s hereditary optic neuropathy. Science, 1988; 242:1427–1430.
2.
WeissigV, TorchilinVP. Cationic bolasomes with delocalized charge centers as mitochondria-specific DNA delivery systems. Adv Drug Deliv Rev, 2001; 49:127–149.
3.
WangZ, GuoW, KuangX, et al.Nanopreparations for mitochondria targeting drug delivery system: Current strategies and future prospective. Asian J Pharm Sci, 2017; 12:498–508.
4.
YoshinagaN, NumataK. Rational designs at the forefront of mitochondria-targeted gene delivery: Recent progress and future perspectives. ACS Biomater Sci Eng, 2022; 8:348–359.
5.
AnandS, TrounceIA, GangodaL. Role of extracellular vesicles in mitochondrial eye diseases. IUBMB Life, 2022; 74:1264–1272.
6.
JeenaMT, KimS, JinS, et al.Recent progress in mitochondria-targeted drug and drug-free agents for cancer therapy. Cancers (Basel), 2019; 12:4.
7.
ChernegaT, ChoiJ, SalmenaL, et al.Mitochondrion-targeted RNA therapies as a potential treatment strategy for mitochondrial diseases. Mol Ther Nucleic Acids, 2022; 30:359–377.
8.
SoldatovVO, KubekinaMV, SkorkinaMY, et al.Current advances in gene therapy of mitochondrial diseases. J Transl Med, 2022; 20:562.
9.
Di DonfrancescoA, MassaroG, Di MeoI, et al.Gene Therapy for mitochondrial diseases: Current status and future perspective. Pharmaceutics, 2022; 14:1287.
10.
LlopisJ, McCafferyJM, MiyawakiA, et al.Measurement of cytosolic, mitochondrial, and golgi pH in single living cells with green fluorescent proteins. Proc Natl Acad Sci U S A, 1998; 95:6803–6808.
11.
YamadaY, AkitaH, KamiyaH, et al.MITO-Porter: A liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion. Biochim Biophys Acta, 2008; 1778:423–432.
12.
YamadaY, FurukawaR, YasuzakiY, et al.Dual function MITO-Porter, a nano carrier integrating both efficient cytoplasmic delivery and mitochondrial macromolecule delivery. Mol Ther, 2011; 19:1449–1456.
13.
YamadaY, HarashimaH. Delivery of bioactive molecules to the mitochondrial genome using a membrane-fusing, liposome-based carrier, DF-MITO-Porter. Biomaterials, 2012; 33:1589–1595.
14.
YamadaY, KawamuraE, HarashimaH. Mitochondrial-targeted DNA delivery using a DF-MITO-Porter, an innovative nano carrier with cytoplasmic and mitochondrial fusogenic envelopes. J Nanoparticle Res, 2012; 14:1013.
15.
YamadaY, NakamuraK, FurukawaR, et al.Mitochondrial delivery of bongkrekic acid using a MITO-Porter prevents the induction of apoptosis in human HeLa cells. J Pharm Sci, 2013; 102:1008–1015.
16.
FurukawaR, YamadaY, KawamuraE, et al.Mitochondrial delivery of antisense RNA by MITO-Porter results in mitochondrial RNA knockdown, and has a functional impact on mitochondria. Biomaterials, 2015; 57:107–115.
17.
KawamuraE, HibinoM, HarashimaH, et al.Targeted mitochondrial delivery of antisense RNA-containing nanoparticles by a MITO-Porter for safe and efficient mitochondrial gene silencing. Mitochondrion, 2019; 49:178–188.
18.
YamadaY, NakamuraK, AbeJ, et al.Mitochondrial delivery of Coenzyme Q10 via systemic administration using a MITO-Porter prevents ischemia/reperfusion injury in the mouse liver. J Control Release, 2015; 213:86–95.
19.
YamadaY, BurgerL, KawamuraE, et al.Packaging of the Coenzyme Q10 into a liposome for mitochondrial delivery and the intracellular observation in patient derived mitochondrial disease cells. Biol Pharm Bull, 2017; 40:2183–2190.
20.
HibinoM, MaekiM, TokeshiM, et al.A system that delivers an antioxidant to mitochondria for the treatment of drug-induced liver injury. Sci Rep, 2023; 13:6961.
21.
ShiraishiM, SasakiD, HibinoM, et al.Human cardiosphere-derived cells with activated mitochondria for better myocardial regenerative therapy. J Control Release, 2024; 367:486–499.
22.
AbeJ, YamadaY, HarashimaH. Validation of a strategy for cancer therapy: Delivering aminoglycoside drugs to mitochondria in HeLa cells. J Pharm Sci, 2016; 105:734–740.
23.
YamadaY, MunechikaR, KawamuraE, et al.Mitochondrial delivery of doxorubicin using MITO-Porter kills drug-resistant renal cancer cells via mitochondrial toxicity. J Pharm Sci, 2017; 106:2428–2437.
24.
YamadaY, MunechikaR, KubotaF, et al.Mitochondrial delivery of an anticancer drug Via systemic administration using a mitochondrial delivery system that inhibits the growth of drug-resistant cancer engrafted on mice. J Pharm Sci, 2020; 109:2493–2500.
25.
AbeJ, YamadaY, TakedaA, 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; 269:177–188.
26.
YamadaY, DaikuharaS, TamuraA, et al.Enhanced autophagy induction via the mitochondrial delivery of methylated β-cyclodextrin-threaded polyrotaxanes using a MITO-Porter. Chem Commun (Camb), 2019; 55:7203–7206.
27.
YamadaY, SomiyaK, MiyauchiA, et al.Validation of a mitochondrial RNA therapeutic strategy using fibroblasts from a Leigh syndrome patient with a mutation in the mitochondrial ND3 gene. Sci Rep, 2020; 10:7511.
28.
KawamuraE, MaruyamaM, AbeJ, et al.Validation of gene therapy for mutant mitochondria by delivering mitochondrial RNA using a MITO-Porter. Mol Ther Nucleic Acids, 2020; 20:687–698.
29.
YamadaY, IshimaruT, IkedaK, et al.Validation of the mitochondrial delivery of vitamin B1 to enhance ATP production using SH-SY5Y cells, a model neuroblast. J Pharm Sci, 2022; 111:432–439.
30.
Pasquale CerratoC, KivijärviT, TozziR, et al.Intracellular delivery of therapeutic antisense oligonucleotides targeting mRNA coding mitochondrial proteins by cell-penetrating peptides. J Mater Chem B, 2020; 8:10825–10836.
31.
KulkarniCA, FinkBD, GibbsBE, et al.A novel triphenylphosphonium carrier to target mitochondria without uncoupling oxidative phosphorylation. J Med Chem, 2021; 64:662–676.
32.
TrayfordC, WilhalmA, HabibovicP, et al.One-pot, degradable, silica nanocarriers with encapsulated oligonucleotides for mitochondrial specific targeting. Discov Nano, 2023; 18:161.
LangW, TanW, ZhouB, et al.Mitochondria-Targeted gene silencing facilitated by Mito-CPDs. Chemistry, 2023; 29:e202204021.
35.
MahapatraS, GhoshS, BeraSK, et al.The D arm of tRNATyr is necessary and sufficient for import into Leishmania mitochondria in vitro. Nucleic Acids Res, 1998; 26:2037–2041.
36.
MukherjeeS, BhattacharyyaSN, AdhyaS. Stepwise transfer of tRNA through the double membrane of Leishmania mitochondria. J Biol Chem, 1999; 274:31249–31255.
37.
BhattacharyyaSN, MukherjeeS, AdhyaS. Mutations in a tRNA import signal define distinct receptors at the two membranes of Leishmania mitochondria. Mol Cell Biol, 2000; 20:7410–7417.
38.
BhattacharyyaSN, ChatterjeeS, AdhyaS. Mitochondrial RNA import in Leishmania tropica: Aptamers homologous to multiple tRNA domains that interact cooperatively or antagonistically at the inner membrane. Mol Cell Biol, 2002; 22:4372–4382.
39.
MukherjeeS, BasuS, HomeP, et al.Necessary and sufficient factors for the import of transfer RNA into the kinetoplast mitochondrion. EMBO Rep, 2007; 8:589–595.
40.
MukherjeeS, MahataB, MahatoB, et al.Targeted mRNA degradation by complex-mediated delivery of antisense RNAs to intracellular human mitochondria. Hum Mol Genet, 2008; 17:1292–1298.
41.
LiY, ParkJ-S, DengJ-H, et al.Cytochrome c oxidase subunit IV is essential for assembly and respiratory function of the enzyme complex. J Bioenerg Biomembr, 2006; 38:283–291.
42.
BorowskiLS, DziembowskiA, HejnowiczMS, et al.Human mitochondrial RNA decay mediated by PNPase–hSuv3 complex takes place in distinct foci. Nucleic Acids Res, 2013; 41:1223–1240.
43.
SantonocetoG, JurkiewiczA, SzczesnyRJ. RNA degradation in human mitochondria: The journey is not finished. Hum Mol Genet, 2024; 33:R26–R33.
AdrianoB, CottoNM, ChauhanN, et al.Milk exosomes: Nature’s abundant nanoplatform for theranostic applications. Bioact Mater, 2021; 6:2479–2490.
46.
ZhangY, LiuQ, ZhangX, et al.Recent advances in exosome-mediated nucleic acid delivery for cancer therapy. J Nanobiotechnology, 2022; 20:279.
47.
LeeY, JeongM, ParkJ, et al.Immunogenicity of lipid nanoparticles and its impact on the efficacy of mRNA vaccines and therapeutics. Exp Mol Med, 2023; 55:2085–2096.
48.
IzumiH, TsudaM, SatoY, et al.Bovine milk exosomes contain microRNA and mRNA and are taken up by human macrophages. J Dairy Sci, 2015; 98:2920–2933.
49.
TaoH, XuH, ZuoL, et al.Exosomes-coated bcl-2 siRNA inhibits the growth of digestive system tumors both in vitro and in vivo. Int J Biol Macromol, 2020; 161:470–480.
50.
Del Pozo-AceboL, Hazas M-Cl deL, Tomé-CarneiroJ, et al.Bovine milk-derived exosomes as a drug delivery vehicle for miRNA-based therapy. Int J Mol Sci, 2021; 22:1105.
51.
DidiotM-C, HallLM, ColesAH, et al.Exosome-mediated delivery of hydrophobically modified siRNA for huntingtin mRNA silencing. Mol Ther, 2016; 24:1836–1847.
52.
ZhaoL, GuC, GanY, et al.Exosome-mediated siRNA delivery to suppress postoperative breast cancer metastasis. J Control Release, 2020; 318:1–15.
53.
YangJ, LuoS, ZhangJ, et al.Exosome-mediated delivery of antisense oligonucleotides targeting α-synuclein ameliorates the pathology in a mouse model of Parkinson’s disease. Neurobiol Dis, 2021; 148:105218.
54.
MunagalaR, AqilF, JeyabalanJ, et al.Exosome-mediated delivery of RNA and DNA for gene therapy. Cancer Lett, 2021; 505:58–72.
55.
ShelkeGV, LässerC, GhoYS, et al.Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum. J Extracell Vesicles, 2014; 3.
56.
KornilovR, PuhkaM, MannerströmB, et al.Efficient ultrafiltration-based protocol to deplete extracellular vesicles from fetal bovine serum. J Extracell Vesicles, 2018; 7:1422674.
57.
LehrichBM, LiangY, KhosraviP, et al.Fetal bovine serum-derived extracellular vesicles persist within vesicle-depleted culture media. Int J Mol Sci, 2018; 19:3538.
58.
LehrichBM, LiangY, FiandacaMS. Foetal bovine serum influence on in vitro extracellular vesicle analyses. J Extracell Vesicles, 2021; 10:e12061.
59.
SteinCA, HansenJB, LaiJ, et al.Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents. Nucleic Acids Res, 2010; 38:e3.
60.
SchmajukG, SierakowskaH, KoleR. Antisense oligonucleotides with different backbones: Modification of splicing pathways and efficacy of uptake*. J Biol Chem, 1999; 274:21783–21789.
61.
SazaniP, KangS-H, MaierMA, et al.Nuclear antisense effects of neutral, anionic and cationic oligonucleotide analogs. Nucleic Acids Res, 2001; 29:3965–3974.
62.
WuC-D, ChouH-W, KuoY-S, et al.Nucleolin antisense oligodeoxynucleotides induce apoptosis and may be used as a potential drug for nasopharyngeal carcinoma therapy. Oncol Rep, 2012; 27:94–100.
63.
SchindelinJ, Arganda-CarrerasI, FriseE, et al.Fiji: An open-source platform for biological-image analysis. Nat Methods, 2012; 9:676–682.
64.
BolteS, CordelièresFP. A guided tour into subcellular colocalization analysis in light microscopy. J Microsc, 2006; 224:213–232.
65.
AdlerJ, ParmrydI. Quantifying colocalization by correlation: The Pearson correlation coefficient is superior to the Mander’s overlap coefficient. Cytometry A, 2010; 77:733–742.
66.
BarlowAL, MacLeodA, NoppenS, et al.Colocalization analysis in fluorescence micrographs: Verification of a more accurate calculation of Pearson’s correlation coefficient. Microsc Microanal, 2010; 16:710–724.
67.
DunnKW, KamockaMM, McDonaldJH. A practical guide to evaluating colocalization in biological microscopy. Am J Physiol Cell Physiol, 2011; 300:C723–C742.
68.
NabbiA, RiabowolK. Rapid isolation of nuclei from cells in vitro. Cold Spring Harb Protoc, 2015; 2015:769–772.
69.
SchagemanJ, ZeringerE, LiM, et al.The complete exosome workflow solution: From isolation to characterization of RNA cargo. Biomed Res Int, 2013; 2013:e253957.
70.
WitwerKW, BuzásEI, BemisLT, et al.Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles, 2013; 2:20360.
71.
MondalA, AshiqKA, PhulpagarP, et al.Effective visualization and easy tracking of extracellular vesicles in glioma cells. Biol Proced Online, 2019; 21:4.
72.
VaswaniK, MitchellMD, HollandOJ, et al.A method for the isolation of exosomes from human and bovine milk. J Nutr Metab, 2019; 2019:e5764740.
73.
GrossenP, PortmannM, KollerE, et al.Evaluation of bovine milk extracellular vesicles for the delivery of locked nucleic acid antisense oligonucleotides. Eur J Pharm Biopharm, 2021; 158:198–210.
74.
JangYJ, ChaBS, KimD, et al.Extracellular vesicles, as drug-delivery vehicles, improve the biological activities of astaxanthin. Antioxidants, 2023; 12:473.
75.
FengX, ChenX, ZhengX, et al.Latest trend of milk derived exosomes: Cargos, functions, and applications. Front Nutr, 2021; 8:747294.
76.
UrzìO, Olofsson BaggeR, CrescitelliR. The dark side of foetal bovine serum in extracellular vesicle studies. J Extracell Vesicles, 2022; 11:e12271.
77.
OrtegaA, Martinez-ArroyoO, FornerMJ, et al.Exosomes as drug delivery systems: Endogenous nanovehicles for treatment of systemic lupus erythematosus. Pharmaceutics, 2020; 13:3.
78.
LeeY, KimM, HaJ, et al.Brain-targeted exosome-mimetic cell membrane nanovesicles with therapeutic oligonucleotides elicit anti-tumor effects in glioblastoma animal models. Bioeng Transl Med, 2023; 8:e10426.
79.
GrayWD, MitchellAJ, SearlesCD. An accurate, precise method for general labeling of extracellular vesicles. MethodsX, 2015; 2:360–367.
80.
Calderón-PeláezM-A, CastellanosJE, Velandia-RomeroML. A protocol for loading Calcein-AM into extracellular vesicles from mammalian cells for clear visualization with a fluorescence microscope coupled to a deconvolution system. PLoS One, 2025; 20:e0317689.
81.
RavalN, JogiH, GondaliyaP, et al.Method and its composition for encapsulation, stabilization, and delivery of siRNA in Anionic polymeric nanoplex: An in vitro- in vivo Assessment. Sci Rep, 2019; 9:16047.
82.
AbdelmohsenK, GorospeM. RNA-binding protein nucleolin in disease. RNA Biol, 2012; 9:799–808.
83.
Anonymous. TransIT®-Oligo Transfection Reagent—0.4 mL | Mirus Bio. n.d.
84.
ReersM, SmithTW, ChenLB. J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry, 1991; 30:4480–4486.
AschrafiA, SchwechterAD, MamezaMG, et al.MicroRNA-338 regulates local cytochrome c oxidase IV mRNA levels and oxidative phosphorylation in the axons of sympathetic neurons. J Neurosci, 2008; 28:12581–12590.
87.
DasS, FerlitoM, KentOA, et al.Nuclear miRNA regulates the mitochondrial genome in the heart. Circ Res, 2012; 110:1596–1603.
88.
Bienertova-VaskuJ, SanaJ, SlabyO. The role of microRNAs in mitochondria in cancer. Cancer Lett, 2013; 336:1–7.
89.
DuarteFV, PalmeiraCM, RoloAP. The role of microRNAs in mitochondria: Small players acting wide. Genes (Basel), 2014; 5:865–886.
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