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, 6 (4), 458-66

Mitochondrially Targeted ZFNs for Selective Degradation of Pathogenic Mitochondrial Genomes Bearing Large-Scale Deletions or Point Mutations

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Mitochondrially Targeted ZFNs for Selective Degradation of Pathogenic Mitochondrial Genomes Bearing Large-Scale Deletions or Point Mutations

Payam A Gammage et al. EMBO Mol Med.

Abstract

We designed and engineered mitochondrially targeted obligate heterodimeric zinc finger nucleases (mtZFNs) for site-specific elimination of pathogenic human mitochondrial DNA (mtDNA). We used mtZFNs to target and cleave mtDNA harbouring the m.8993T>G point mutation associated with neuropathy, ataxia, retinitis pigmentosa (NARP) and the "common deletion" (CD), a 4977-bp repeat-flanked deletion associated with adult-onset chronic progressive external ophthalmoplegia and, less frequently, Kearns-Sayre and Pearson's marrow pancreas syndromes. Expression of mtZFNs led to a reduction in mutant mtDNA haplotype load, and subsequent repopulation of wild-type mtDNA restored mitochondrial respiratory function in a CD cybrid cell model. This study constitutes proof-of-principle that, through heteroplasmy manipulation, delivery of site-specific nuclease activity to mitochondria can alleviate a severe biochemical phenotype in primary mitochondrial disease arising from deleted mtDNA species.

Figures

Figure 1
Figure 1
Improved design and localization of mtZFNs.

Schematic structure of previous mtZFN architecture. Mitochondrial targeting is facilitated by a 49-amino acid-long MTS from subunit F1β of human mitochondrial ATP synthase. To ensure that the MTS cleavage site is upstream of the epitope tag, its length was adjusted using proteomic data (Carroll et al, 2009) that identified the N-terminus of the mature subunit at Ala48. NES, nuclear export signal; ZFP, zinc finger peptide; M, Myc tag; HA, haemagglutinin tag.

Schematic of the novel obligatory heterodimeric mtZFN monomers. “+” and “−”, ELD and KKR modified FokI domain, respectively (Doyon et al, 2011); F, FLAG tag; AA, two-alanine linker; GS, glycine-serine linker (Kim et al, 1996).

The intracellular localization of mtZFNs by immunofluorescence. mtZFN(+) or mtZFN(−) was transiently expressed in HOS 143B cells. Cell nuclei were stained with DAPI (blue, 1 and 5). mtZFN(+) and mtZFN(−) were detected by anti-HA and anti-FLAG antibodies, respectively, and visualized by Alexa Fluor 594-conjugated secondary antibodies (red, 2 and 6). Mitochondria were detected with anti-TOM20/Alexa Fluor 488 antibodies (green, 3 and 7). Co-localization appears yellow on digitally merged images (4 and 8). Scale bars: 10 μm. R8-4 and R13-2 mtZFNs were used in this example (Supplementary Table S2).

Location of mtZFNs in subcellular fractions. HOS 143B cells stably transfected with mtZFNs were fractionated into cell debris/nuclei (D, lane 2), cytosol (C, lane 3) and mitochondria (lanes 4–5). The mitochondrial fraction was treated with 25 μg/ml proteinase K (lane 5). “T, total cell lysate. The fractions were analysed by western blotting with anti-HA [mtZFN(+)] and anti-FLAG [mtZFN(−)]. The following marker proteins were used: mtSSB1 (mitochondrial matrix), TOM22 (mitochondrial outer membrane), β-actin (cytosol) and histone H4 (nucleus). “p” indicates mtZFN precursor (still containing MTS), and “m” indicates the mature form. “fl” indicates TOM22 of full length; “tr” indicates proteinase K-truncated TOM22. R8-4 and R13-2 were used in this example.

Source data are available for this figure.
Figure 2
Figure 2
Targeting m.8993T>G with mtZFNs.

Schematic of targeting mtDNA point mutations by mtZFNs. Red box, the binding site for the m.8993T > G-specific mtZFN monomer (NARPd); Blue box, binding site for the companion mtZFN on the opposite strand (COMPa)

Western blotting of ˜93% m.8993T > G cybrid cells transfected with NARPd and/or COMPa and vector controls. β-Actin, loading control.

RFLP analysis of last cycle hot PCR products (mtDNA nt positions 8339–9334) amplified from total DNA samples of ˜93% m.8993T > G cybrid cells transfected with vectors, NARPd and/or COMPa. Wild-type cells and 99% m.8993T > G cybrids were used as controls.

Results of RFLP analysis as per (C) from three independent transfections. P value according to two-tailed Student's t-test.

Source data are available for this figure.
Figure 3
Figure 3
Targeting mtDNA “common deletion” (CD) with mtZFNs.

Wild-type mtDNA is not cleaved as mtZFN R8-n(+) and R13-n(−) monomers (where n is a specific construct number) bind several kilobases apart preventing dimerization of the FokI domain. Red and blue boxes, the binding sites for mtZFN monomers on either side of the CD (Supplementary Table 2); Black boxes, the 13-bp direct repeats.

mtDNA harbouring CD is cleaved as adjacent binding of the mtZFN R8-n(+) and R13-n(−) monomers results in FokI dimerization.

Western blotting of bulk populations of H39 CD cybrid cells transfected with the R8-4(+) and/or R13-1(−) constructs and vector controls. β-Actin, loading control.

Southern blot analysis of total DNA from wild-type (WT), mock-or mtZFN-transfected cells digested with BamHI and probed with a radioactive mtDNA-specific probe (region: 14986–15607), 18S rDNA probe is used as a loading control.

Analysis of mtDNA heteroplasmy in clones expressing CD-specific mtZFNs as indicated. % wild-type mtDNA was measured by Southern blotting. Grey dots indicate individual clones; coloured bars indicate the average value for each mtZFN pair. P values calculated in two-tailed Student's t-test.

Source data are available online for this figure.
Figure 4
Figure 4
Recovery of OXPHOS function upon elimination of CD mtDNA by mtZFNs.

Oxygen consumption rate (OCR) measured in quadruplicate for wild-type (WT, white), Rho0 (black) and clonal cells expressing CD-specific mtZFNs from the same transfection/selection experiment.

Southern blot analysis of total DNA from wild-type (WT), Rho0 and mock-or mtZFN-transfected clonal cells used in OCR analysis.

Western blot analysis of steady-state levels of OXPHOS subunits in clonal cell lines as per (A) and (B). Mitochondrial preparations of WT and CD cells (Mitoprep) were also analysed in this experiment. Coomassie gel staining was used as a control for protein loading.

Source data are available for this figure.

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