Antisense RNA oligonucleotides (ASOs) potentially allow gene silencing by mitochondrial delivery to target mtDNA-encoded mRNA. MITO-Porters are liposomal nanocarrier systems designed for mitochondrial delivery. Well-designed MITO-Porters may enable their use as therapeutics to regulate mitochondrial function. The efficient packaging of ASOs in a MITO-Porter via a nanoparticle packaging method has a 10-fold higher packaging efficiency than the conventional method. The delivery of the constructed carrier resulted in a decrease in the target mRNA levels and ATP production.
Mitochondrial diseases include multisystem disorders involving metabolic errors. The dysfunction of mitochondria appears to cause various diseases, including cancer, Alzheimer’s disease, Parkinson’s disease, diabetes mellitus, and others. Mitochondrial diseases most often affect the brain, retina, and skeletal muscles. However, multisystem damage can also involve the liver, gastrointestinal tract, pancreas, kidneys, etc.
Mitochondria are energy-generating cellular organelles with their own coding DNA, a circular mtDNA of approximately 16,000 base pairs. More than 1,000 genes from nuclear DNA (nDNA) and 37 genes from mitochondrial DNA (mtDNA) control the mitochondrial proteome. Mutations in more than 350 genes in both genomes appear to cause different mitochondrial diseases.
Figure 1: Mitochondrial electon transport chain (Adapted from Wiki commens. METC).
Gene therapy promises the correction of mitochondrial disorders. However, to be successful, the dynamics of mitochondrial genetics will need to be better understood. Current research focuses on increasing transfection efficiency while lowering cytotoxicity.
Historically, drug delivery into cells utilizes recombinant adeno-associated viruses as viral vectors. However, to selectively treat mitochondria dysfunctions, the targeted delivery of engineered genes or gene products to the nucleus of mitochondria is essential. Because large molecules, including plasmid DNA (pDNA), antisense oligonucleotides, and folded proteins, do not readily pass through the mitochondrial membrane, delivery into mitochondria is difficult. To address these limitations, Yamada et al. developed a liposome-based carrier for delivering cargo molecules called a “MITO-Porter.”
Two independent processes, “cytoplasmic delivery through the cell membrane” and “mitochondrial delivery through the mitochondrial membrane,” are required for efficient drug delivery to mitochondria.
MITO-Porters
Yamada et al., in 2008, described a liposome-based carrier for delivering macromolecular cargos to the mitochondrial interior via membrane fusion. The liposome particles utilized are called MITO-Porters. The nanoparticles carry octa-arginine (R8) surface modifications to enable their entry into cells as intact vesicles. The research team identified lipid compositions that promote the fusion of the nanoparticles with the mitochondrial membrane and the release of its cargo to the intra-mitochondrial compartment in living cells. This uptake process, called “macropinocytosis,” is a non-selective liquid-phase endocytic pathway to uptake extracellular substances.
High-density octa arginine-modified liposomes (R8-LPs) stimulate micropinocytosis by enabling intracellular trafficking. R8 is a synthetic peptide mimicking the trans-activating transcriptional activator derived from the human immunodeficiency virus. R8-LPs can escape from macropinosomes into the cytosol by keeping the encapsulated compounds intact. Low-density R8-LPs are taken up via clathrin-mediated endocytosis and degraded by lysosomal enzymes. MITO-Porter delivered to the cytosol binds to mitochondria via electrostatic interactions with R8. Encapsulated cargo is delivered to the intra-mitochondrial compartment via membrane fusion with the help of sphingomyelin or phosphatidic acid, the lipids that fuse with the mitochondrial membrane. Upon release from the macropinosomes, the MITO-Porter binds to the mitochondrial membrane via electrostatic interactions, inducing fusion between the MITO-Porter and mitochondrion.
The research team screened for liposomes fused with isolated rat liver mitochondria to find the best MITO-Porter liposome components. The variation of the lipid composition using a panel of liposomes and monitoring membrane fusion via fluorescence resonance energy transfer (FRET) analysis allowed the identification of two highly fusogenic lipid compositions, which form the basis of the MITO-Porter.
Green fluorescence protein (GFP) was used as a model macromolecule to validate the MITO-Porter. The use of confocal laser scanning microscopic analysis validated its delivery to mitochondria. Also, FRET analysis allowed the evaluation of membrane fusion between the MITO-Porter and mitochondria in living cells.
In 2019, Kawamura et el. reported that the MITO-Porter is a practical delivery vehicle for antisense oligonucleotides (ASOs) regulating mitochondrial function. The MITO-Porter showed a 10-fold higher packaging efficiency than conventional delivery methods. The use of ASO carriers resulted in a decrease in the targeted mRNA and ATP production.
In 2020, Gao et al. reported that transfected siRNAs could enter the mitochondrial matrix and allow targeted mitochondrial transcripts to be silenced. The study investigated whether siRNAs and small hairpin RNAs (shRNAs) can target mitochondria DNA (mtDNA) encoded transcripts.
DF-MITO-Porter
In 2011, Yamada et al. reported the development of a dual-function MITO-Porter called DF-MITO-Porter. The DF-MITO-Porter is a result of integrating R8-modified liposomes with the MITO-Porter. The research team showed that the DF-MITO-Porter delivers exogenous macro-biomolecules into the mitochondrial matrix. The DF-MITO-porter was adapted to contain an outer endosome-fusogenic envelope facilitating a more efficient escape from the endosome via membrane fusion.
As an example, the construction of a DF-MITO-Porter encapsulating DNase I requires the following three steps:
(i) the construction of nanoparticles containing DNase I;
(ii) coating the nanoparticles with a mitochondria-fusing envelope;
(iii) another coating endosome-fusogenic envelope step-wise, based on previous reports regarding gene packaging with two different types of lipid layers.
More recently, Chernega et al., in 2022, reviewed mitochondrion-targeted RNA therapies as a potential treatment strategy for mitochondrial diseases. The research team pointed out that a careful and thorough examination of possibly disease-associated mtDNA variants and heteroplasmic load is needed to identify genetic causes in patients with possible mitochondrial diseases.
The currently utilized RNA-based therapeutic agents include antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), and mRNA therapeutic agents. ASOs are single-stranded oligonucleotides that complementary bind to a target mRNA or premature mRNA, which induce degradation, altered splicing, or inhibited translation upon binding.
However, until this day, RNA-based therapeutic agents have yet to be approved for treating mitochondrial diseases. Targeting mitochondrial RNAs could be valuable therapeutic based on mitochondrial biology and conditions.
Also recently, Xu et al., in 2022, reviewed how to design mitochondria-targeted drugs for neurodegenerative diseases, the rescue mechanism of the drugs, and how to assess their therapeutic effect, including structures of small molecules and peptides targeting mitochondria.
References
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