Selective elimination of mutant mitochondrial genomes as therapeutic strategy for the treatment of NARP and MILS syndromes

Abstract

Mitochondrial diseases are not uncommon, and may result from mutations in both nuclear and mitochondrial DNA (mtDNA). At present, only palliative therapies are available for these disorders, and interest in the development of efficient treatment protocols is high. Here, we demonstrate that in cells heteroplasmic for the T8993G mutation, which is a cause for the NARP and MILS syndromes, infection with an adenovirus, which encodes the mitochondrially targeted R.XmaI restriction endonuclease, leads to selective destruction of mutant mtDNA. This destruction proceeds in a time- and dose-dependent manner and results in cells with significantly increased rates of oxygen consumption and ATP production. The delivery of R.XmaI to mitochondria is accompanied by improvement in the ability to utilize galactose as the sole carbon source, which is a surrogate indicator of the proficiency of oxidative phosphorylation. Concurrently, the rate of lactic acid production by these cells, which is a marker of mitochondrial dysfunction, decreases. We further demonstrate that levels of phosphorylated P53 and gammaH2ax proteins, markers of nuclear DNA damage, do not change in response to infection with recombinant adenovirus indicating the absence of nuclear DNA damage and the relative safety of the technique. Finally, some advantages and limitations of the proposed approach are discussed.

Figure 1

Ad.1891 infection results in the selective elimination of mutant mtDNA. (a) The structure of the Ad.1891 expression unit. P_CMV(CuO), cumate-inducible cytomegalovirus promoter; NLS, nuclear localization sequence; FLAG, FLAG-tag; M.SmaI, codon-optimized M.SmaI; IRES, internal ribosome entry site; cox, the mitochondrial targeting sequence of the human cytochrome c oxidase subunit VIII; Myc, myc-tag; R.XmaI, codon-optimised R. XmaI. (b) The dose–response of the M.SmaI and R.XmaI expression. MOI, multiplicity of infection; α-FLAG, expression of the FLAG-tagged M.SmaI; α-myc, expression of the myc-tagged R.XmaI; β-actin, loading control; arrow denotes the position of the R.XmaI-specific band. (c) The subcellular localization of R.XmaI. JCP239 cells were plated on coverslips and processed as described in the Materials and methods. α-myc, green staining for the myc-tagged R.XmaI; MT, MitoTracker red; overlay, the superimposition of the previous two images. Yellow, regions of colocalization of R.XmaI and MitoTracker red.

Figure 2

Biological activity of mitochondrially targeted R.XmaI. (a) Mitochondria were isolated from Ad1891-infected JCP239 cells as described in the Materials and methods and indicated amounts of total mitochondrial lysate were incubated with 2 μg of 915 bp PCR fragment either containing (mut) or not (WT) T8993G mutation. M, DNA size marker; SmaI, PCR fragment cut with commercial preparation of R.SmaI. Note that mitochondria possess nonspecific nuclease activity (smears). (b) Total cellular DNA from JCP213 (100% WT mtDNA) and JCP261 (100% mutant mtDNA) cells, which were either infected (+) or not (−) with Ad.1891 was subjected to Southern blotting as described in the Materials and methods. Positions of the signals from mtDNA and 18S rDNA are indicated by arrows. (c) Reduction in mutant mtDNA content in JCP239 cells in response to infection with Ad.1891 at various MOIs. M, DNA size markers. % Mutant mtDNA remaining and MOIs are indicated. (d) The time course for mutant mtDNA elimination during Ad.1891 infection. JCP239 cells were infected at MOI = 200, and mutant mtDNA content was evaluated at the indicated time points after infection. M, DNA size markers. (e) Effect of the repeated Ad.1891 applications on mutant DNA content. JCP239 cells were subjected to two rounds of Ad.1891 infection. Both MOIs and remaining mutant mtDNA content after the second round of infection are indicated.

Figure 3

Ad.1891 infection does not result in nuclear DNA damage. JCP239 cells were infected at various MOIs for 48 h or were treated with 20 μM etoposide and 60 μg of total protein were probed with antibodies against total P53, S15 phospho-P53; γ-H2ax, or β-actin (loading control).

Figure 4

Ad.1891 infection improves the ability of JCP239 cells to grow on galactose. The ability of JCP239 cells to utilize glucose (a) and galactose (b) as the sole carbon source before (JCP239) and after (2–50) two rounds of Ad.1891 infection at MOI = 50 was determined as described in the Materials and methods. The data are mean±s.e.m. (n = 3)

Figure 5

Ad.1891 infection improves biochemical parameters compromised by T8993G mutation. Total ATP content (a), the ATP synthesis rate (b), oxygen consumption (c) and lactate production (d) were determined in JCP239 cells before (JCP239) and after (2–50) two rounds of Ad.1891 infection at MOI = 50. The data are mean ± s.e.m. The statistical comparisons were performed using two-tailed t-test.