DNA delivery to the mitochondria sites using leader peptide conjugated polyethylenimine

Abstract

Background: Some genetic diseases are associated with defects of the mitochondrial genome. Mitochondria gene therapy by direct DNA delivery to mitochondria has been suggested as a therapeutic option for the diseases. However, a few studies about DNA delivery to mitochondria has been reported and no definite delivery method is available. Although numerous viral and non-viral gene carriers have been developed, these carriers were designed for the delivery of DNA into the nucleus. Thus, mitochondrial gene therapy has been thought as theoretical and speculative. In this research, we hypothesized that mitochondrial leader peptide (LP) conjugated polyethylenimine (PEI) could deliver DNA to the mitochondrial sites. Methods: PEI-LP was synthesized by conjugation LP to PEI. The complex formation of PEI-LP with DNA was confirmed by a gel retardation assay. In vitro delivery tests into isolated mitochondria or living cells were performed with rhodamin-labeled DNA and PEI-LP. Mitochondria in living cells were counter-stained with Mitotrack green. The toxicity was measured by MTT assay. Results: DNA was completely retarded at a 0.4:1 PEI-LP:DNA weight ratio in the gel retardation assay, confirming the complex formation of PEI-LP and DNA. In vitro cell-free delivery assay with isolated mitochondria showed that PEI-LP/DNA complexes were localized at mitochondria sites. However, naked DNA or PEI/DNA complex did not show this effect. Furthermore, the PEL-LP/DNA complexes were localized at the mitochondrial sites in living cells. However, a control carrier, PEI, did not show this effect. In addition, MTT assay showed that PEI-LP showed lower cytotoxicity than PEI.