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Review Interpretation: Delivery Strategies Targeting Mitochondrial DNA.
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Review Interpretation: Delivery Strategies Targeting Mitochondrial DNA.
Mitochondria are energy factories in cells and play an important role in cellular metabolism. Mitochondrial DNA (mtDNA) encodes a variety of proteins, and mutations in mtDNA can lead to disturbances in mitochondrial function and ultimately lead to the occurrence of various congenital diseases.
Currently, mitochondria-targeted gene delivery systems have been developed to ameliorate mtDNA mutations. However, the application of these strategies in mitochondrial gene therapy is still being explored and optimized.
This review, by Naoto Yoshinaga and Keiji Numata, highlights recent mitochondrial-targeted strategies for gene therapy and discusses future directions for mitochondrial-targeted gene delivery.
Summary Figure: Overview of mitochondrial targeting strategies
Drug delivery technology can target a variety of drugs (small molecule drugs, nucleic acid drugs, and proteins) to specific organelles, tissues or cells.
Targeting organelles can improve therapeutic effects and reduce side effects. Mitochondria have become promising targets in subcellular organelles due to their ability to generate energy (ATP) , control the levels of intracellular reactive oxygen species and calcium ions, and regulate autophagy.
In addition, human mtDNA is multi-copy, circular and double-stranded, containing 16,568 base pairs, which can encode 37 genes, 22 tRNAs, 13 important proteins related to ATP synthesis, and 2 rRNAs.
Unlike nuclear DNA, mtDNA is not packaged and protected by histones and is chronically exposed to ROS, thus increasing the risk of mutation over time. Mitochondrial dysfunction caused by mtDNA mutations contributes to a variety of genetic diseases , such as neurodegenerative diseases, diabetes, and cancer.
In addition to gene therapy targeting human mitochondria, plant mitochondria are also important target organelles in agriculture, so the development of delivery strategies targeting mtDNA has always been a research hotspot.
Figure 1 Structure of mtDNA
Mitochondrial gene delivery
As mentioned above, human mtDNA mutations are associated with a variety of mitochondrial diseases and severely disrupt the balance of cellular metabolism. In particular, mutations in proteins associated with oxidative phosphorylation inhibit ATP production.
This mitochondrial dysfunction occurs when the ratio of mutated mtDNA to healthy wild-type mtDNA in mitochondria, known as mitochondrial heterogeneity, exceeds a certain threshold .
For mitochondrial disease therapy, improving mitochondrial heterogeneity by augmenting wild-type mtDNA or using genome editing to repair mutated mtDNA and eliminate mutated mtDNA is an effective strategy (Figure 2).
In the early stages of mitochondrial gene therapy, the cytoplasm of healthy cells can be fused with mitochondria-deficient cells in an in vitro model to provide intact mitochondria through which mitochondrial function is restored. To date, multiple strategies for mitochondrial gene therapy have been developed (Table 1) .
This article will highlight recent achievements in mitochondrial gene therapy based on several nucleic acid delivery, categorized according to mitochondrial targeting approaches.
Image Figure 2 Schematic diagram of mitochondrial heterogeneity leading to mitochondrial diseases
Table 1 Overview of mitochondria-targeted nucleic acid delivery vehicles
Mitochondrial Targeting Signal Peptide (MTS)
Introduction of plasmid DNA (pDNA) into mitochondria is the most straightforward method to provide essential mitochondrial proteins using gene therapy methods.
However, since the mitochondrial outer membrane is only permeable to low or medium molecular weight substances, an efficient delivery system is required to introduce plasmids into mitochondria .
MTS is one of the commonly used tools for mitochondrial-targeted delivery.
Most mitochondrial proteins are encoded in the nuclear genome and then transported to mitochondria. Mitochondrial proteins typically have an MTS at the N-terminus and exhibit an amphipathic α-helical structure flanked by cationic and hydrophobic residues, respectively.
MTS sequences are recognized by negatively charged translocases in the outer mitochondrial membrane (TOM) complex and transferred to the mitochondrial intermembrane space by the barrel TOM40 translocase.
Inner membrane translocase (TIM) , also a negatively charged complex, recognizes MTS and ultimately allows mitochondrial proteins to enter the mitochondrial matrix.
This mechanism has been extended to mitochondria-targeted delivery.
Bennett designed a gene delivery system based on MTS of a functional protein , called protofection. It consists of three domains: (i) a protein transduction domain (PTD) , which is similar in sequence to viral proteins and used to enhance cellular uptake (ii) an MTS domain for mitochondrial targeting (iii) a mitochondrial transcription factor A (TFAM) , essential for mtDNA replication.
Wild-type mtDNA containing PTD-MTS-TFAM was successfully transported into mitochondria, resulting in increased respiration rate and mtDNA copy number in diseased cell lines and decreased mutant mtDNA .
Notably, intravenous injection of the PTD-MTS-TFAM/mtDNA complex into the tail vein of miceIncreases NADH ubiquinone oxidoreductase-driven respiration rate in brain and skeletal muscle mitochondria .
They also reported mitochondrial-targeting micelles prepared from pDNA and MTS-conjugated peptides (Fig. 3) . The formation of nanoparticles (NPs) and pDNA is facilitated by electrostatic interactions between MTS containing part of the cytochrome c oxidase (cytocox) subunit IV sequence and lysine-histidine (KH) repeats.
Figure 3 Schematic diagram of peptide/pDNA complex
MTS-based mitochondrial targeting strategies extend to viral gene delivery . In 2012, John Guy et al. developed an MTS-bound adeno-associated virus (AAV) loaded with pDNA encoding NADH ubiquinone oxidoreductase subunit 4 (pND4) .
MTS delivery of the AAV vector enhanced pND4 in mitochondria. accumulation in optic nerve atrophy in mice with vision loss.
Mitochondrial targeting based on lipophilic cations
Lipophilic cations, such as TPP, as ligand molecules are reliable methods for mitochondria-targeted gene delivery as well as small drug delivery , because lipophilic cations can enhance electrostatic interactions between delivery vehicles and mitochondria.
Faria and colleagues conjugated TPP to the high molecular weight reagent poly(ethylene glycol)-poly(ethyleneimine) (PEG-PEI) via amide coupling , which enhanced pDNA internalization to mitochondria and cellular uptake . Finally, PEG-PEI-TPPNPs significantly enhanced the expression of reporter gene and ND4 protein (Fig. 4) .
Figure 4 Schematic diagram of PEG-PEI-TPP
A research group at BeiraInterior University has developed a rhodamine 123 (Rho123) -modified gene delivery vector for mitochondrial targeting. To construct pDNA-loaded Rho123-labeled NPs, the authors used a co-precipitation method induced by the interaction between Ca and pDNA in the presence of carbonate .
Interestingly, the size of the nanoparticles was controlled by adding cellulose or gelatin as stabilizers, and the fluorescence intensity in mitochondria was observed by confocal laser microscopy, and it could be found that NPs mainly accumulated in mitochondria and effectively promoted the reporter gene.
Figure 5 Schematic diagram of Rho123-modified gene delivery vector
NPs based on Dequalinium (DQA: 1,1′-(1,10-decamethylene-bis-[aminoquinaldinium])-chloride) complexed with pDNA, termed DQAsomes , are also considered promising mitochondria-targeting vehicles .
DQA is an FDA-approved cationic lipid that can form liposome-like structures with pDNA through electrostatic interactions. DQAsomes loaded with pDNA encoding GFP induce mitochondrial gene expression in cells.
However, the transfection efficiency of DQAsomes in mitochondria is limited to 5%. To address this issue, Choi and colleagues combined DQA with other liposomes—1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3 – Phosphoethanolamine (DOPE) was mixed to further improve the function of DQAsomes.
The combined use of DQA and DOTAP/DOPE enhanced cellular uptake and endosomal escape, further enhancing mitochondrial gene expression .
Figure 6 Schematic diagram of DQA-DOTAP/DOPE mitochondrial targeting carrier
Lipid-based mitochondrial fusion delivery
Another lipid-based delivery vehicle is MITO-Porter , which consists of DOPE, sphingomyelin (SM) and cell penetrating peptide (CPP) , eight arginines (R8) .
MITO-Porter can deliver loaded drugs to mitochondria through DOPE-induced membrane fusion mechanism, and R8 can increase the rate of cellular uptake.
This membrane fusion system is capable of delivering various drugs to mitochondria. To improve the efficiency of transfection of exogenous pDNA into mitochondria using MITO-Porter, Yamada et al. used another CPP, the KALA peptide with a lysine-leucine-alanine repeat, to deliver drugs into cells .
The transfection efficiency of the KALA- modified MITO-Porter construct (KALA-MITO-Porter) is about 10-fold higher than that of the R8-modified MITO-Porter construct; however, the replacement of R8 with KALA unfortunately increases cytotoxicity, which is likely a Mitochondrial membrane destabilization induced by KALA peptide .
The authors further enhanced the function of KALA-MITO-Porter by incorporating additional mitochondrial RNA aptamer (RP) ligands to enhance cellular uptake and mitochondrial targeting activity.
RP modification significantly increased the transfection efficiency of pDNA-loaded KALA-MITO-Porter constructs into mitochondria.
Furthermore, the administration of RP/KALA-MITO-Porter did not alter the biomarker levels in mouse serum, indicating its good biocompatibility for in vivo applications (Figure 7) .
Figure 7 Working diagram of two MTIO-Porters (R8 and KALA)
Mitochondrial genome editing
Another direction of mitochondrial gene therapy is genome editing of mtDNA , which can further improve our understanding of the underlying mechanisms of intramitochondrial biosynthesis and mitochondrial disease.
Numerous studies on nuclear genome editing such as restriction endonucleases (RE) , zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) contribute to the development of mtDNA editing technology.
Since these editing systems are based on the interaction between the edited protein and DNA, binding MTS to them can accelerate introduction into mitochondria.
In addition, in 2020, Mok et al. can convert C·G in mtDNA to T·A with high specificity and accuracy by using MTS and TALE-conjugated double-stranded DNA deaminase toxin A (DddA) , which can be used for Construction of disease models with specific sequences of mtDNA mutations .
In addition, the technology has been extended to mouse and plant mitochondrial and chloroplast genome editing .
Mitochondria play a vital role in the normal operation of life activities in cells , such as fatty acid oxidation, TCA cycle, autophagy, etc.
Therefore, DNA disorders can lead to the occurrence and development of many diseases . There are currently three common methods for repairing mutated mtDNA in vivo: mitochondrial targeting based on lipophilic cations; lipid-based mitochondrial fusion delivery; mitochondrial targeting signal peptide (MTS)-based targeting technology (Figure 8) .
Furthermore, genome editing approaches have been shown to be effective in vivo, but may cause cytotoxicity in host cells.
Therefore, it should be safer to deliver genes directly to mitochondria. To achieve safe in vivo delivery , vectors must possess other basic capabilities in addition to targeting mitochondria, including high colloidal stability to protect the loaded genes from nuclease degradation.
An additional consideration is that since mitochondrial dysfunction due to mitochondrial disease can affect various organs, the delivery vehicle must be able to deliver the loaded gene to multiple organs and not be rapidly cleared by the liver or spleen.
For better biodistribution, MTS-based NPs may not be suitable because MTSs composed of cationic and hydrophobic residues may interact with biological macromolecules and form aggregates in physiological environments. Therefore, more complex and diverse NP assembly designs need to be designed to optimize biodistribution.
In conclusion, future development should mainly focus on vector design to overcome the barriers in mitochondrial delivery in vivo, and when these limitations are finally overcome, the mechanisms of mitochondrial biosynthesis will be deeply understood, and it will be possible to develop targeted therapies for inherited mitochondrial diseases. treatment method.
Figure 8 Mitochondria are closely related to multiple pathways; common targeted mitochondrial delivery systems
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Review Interpretation: Delivery Strategies Targeting Mitochondrial DNA.
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