Web Date: November 22, 2016
A step toward mitochondrial gene therapy
Mutations in mitochondrial DNA underlie many diseases, including cancer, diabetes, heart disease, and age-related neurodegenerative disorders. But a safe, reliable method to deliver DNA to mitochondria and correct these mutations doesn’t currently exist.
Now, a team led by biochemistry professor Keiji Numata and postdoctoral researcher Jo-Ann Chuah of Japan’s RIKEN research institute have developed a peptide-based system that delivers a functional gene to human cells without toxic effects. The system could pave the way to gene therapy for mitochondrial diseases (Biomacromolecules 2016, DOI: 10.1021/acs.biomac.6b01056).
Unlike methods that deliver genes to chromosomes inside the cell nucleus, the new system delivers genes to the mitochondria, which house their own DNA and chromosomes. Earlier attempts to develop mitochondria-targeted gene delivery systems posed limitations. Many were toxic, and although researchers had shown that mitochondria took up peptides used in the various delivery systems, no one had shown whether mitochondria actually expressed the genes delivered.
Numata’s team built and tested several peptide-based systems and found that one had the highest uptake by cells: a dual-domain peptide made of a subunit of a yeast enzyme, cytochrome c oxidase (Cytcox), fused to another peptide containing only lysine and histidine residues, which are positively charged and can cross negatively charged cell membranes. The researchers packaged DNA with this modified Cytcox by mixing plasmid DNA encoding a green fluorescent protein (GFP) with the modified peptide and allowing the components to self-assemble into spherical particles.
Nuclear magnetic resonance showed that the lysine-histidine residues bound the DNA to form the inner core of the particle, with the α-helical Cytcox peptide strands radiating out toward the surface. Earlier studies had shown that maintaining an α-helical structure is important for the peptides to target and penetrate the mitochondria. In an α-helical structure, positively charged residues lie on one side of the helix, while uncharged hydrophobic residues lie on the other, a configuration recognized by receptor proteins on the surface of the mitochondria that trigger protein transport into the organelles.
To see if the system could deliver the DNA, the researchers added the peptide-DNA complexes to human embryonic cells whose mitochondria they stained with red fluorescent dye. Confocal microscopy revealed that the glow from the GFP and the red dye overlapped, indicating that 82% of the mitochondria had taken up and expressed the plasmid DNA. The researchers observed gene expression for about a week and saw that the cells remained viable throughout.
But the system requires further analysis, experts say. Michael A. Teitell of the University of California, Los Angeles, says successful delivery of a gene involved in mitochondrial disease would confirm the system’s clinical usefulness. Still, the researchers “learned a lot about self-assembly…and how to get things into the mitochondria,” he says. “The structural characterization of their particle looks robust.” Chuah says follow-up studies will test the system in an animal model.
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