Abstract
PURPOSE: Development of a human mitochondrial gene delivery vector is a critical step in the ability to treat diseases arising from mutations in mitochondrial DNA. Although we have previously cloned the mouse mitochondrial genome in its entirety and developed it as a mitochondrial gene therapy vector, the human mitochondrial genome has been dubbed unclonable in E. coli, due to regions of instability in the D-loop and tRNA(Thr) gene.
METHODS: We tested multi- and single-copy vector systems for cloning human mitochondrial DNA in E. coli and Saccharomyces cerevisiae, including transformation-associated recombination.
RESULTS: Human mitochondrial DNA is unclonable in E. coli and cannot be retained in multi- or single-copy vectors under any conditions. It was, however, possible to clone and stably maintain the entire human mitochondrial genome in yeast as long as a single-copy centromeric plasmid was used. D-loop and tRNA(Thr) were both stable and unmutated.
CONCLUSIONS: This is the first report of cloning the entire human mitochondrial genome and the first step in developing a gene delivery vehicle for human mitochondrial gene therapy.
Original language | English |
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Pages (from-to) | 2863-2870 |
Number of pages | 8 |
Journal | Pharmaceutical Research |
Volume | 28 |
Issue number | 11 |
DOIs | |
Publication status | Published - Nov 2011 |
Keywords
- base sequence
- clone cells
- DNA, mitochondrial
- drug compounding
- drug delivery systems
- Escherichia coli
- genetic therapy
- genetic vectors
- genome, human
- genome, mitochondrial
- humans
- mitochondria
- molecular sequence data
- molecular targeted therapy
- plasmids
- recombination, genetic
- Saccharomyces cerevisiae
- sequence analysis, DNA
- yeasts