MitoTALEN reduces mutant mtDNA load and restores tRNAAla levels in a mouse model of heteroplasmic mtDNA mutation

Abstract

Mutations in the mitochondrial DNA (mtDNA) are responsible for several metabolic disorders, commonly involving muscle and the central nervous system¹. Because of the critical role of mtDNA in oxidative phosphorylation, the majority of pathogenic mtDNA mutations are heteroplasmic, co-existing with wild-type molecules¹. Using a mouse model with a heteroplasmic mtDNA mutation², we tested whether mitochondrial-targeted TALENs (mitoTALENs)^3,4 could reduce the mutant mtDNA load in muscle and heart. AAV9-mitoTALEN was administered via intramuscular, intravenous, and intraperitoneal injections. Muscle and heart were efficiently transduced and showed a robust reduction in mutant mtDNA, which was stable over time. The molecular defect, namely a decrease in transfer RNA^Ala levels, was restored by the treatment. These results showed that mitoTALENs, when expressed in affected tissues, could revert disease-related phenotypes in mice.

Fig. 1 |. Development of a mitoTALEN for the mouse mutant m.5024C>T mtDNA.

a,b, Structure of mitoTALEN monomers, which include: (i) a basic TAL-binding domain; (ii) a mitochondrial localization signal in the N terminus (Cox8-Sub9 or SOD2); (iii) a unique tag (HA or FLAG) for immunological detection; (iv) a GFP or mCherry separated from the mitoTALEN by a picornaviral 2A-like sequence (T2A) for sorting of transfected cells; (v) a 3′UTR untranslated region from a nuclear gene (ATP5B or SOD2 mRNA); (vi) FokI that works as a heterodimer (NEL only dimerizes with CKK). Panel a also shows the binding site of the monomers to the mtDNA. The mutation is at position T0 (arrow). c,d, Tag detection in HeLa cells transfected with mitoTALEN monomers. Mitochondrial localization was determined with Mitotracker Red (left panels) and protein size by western blots (right panels). Scale bar, 5μm. Monomer A, 9.5 RVDs, 84.2 kDa; monomer B, 15.5 RVDs, 112,5 kDa. Experiments were performed twice with similar results. MW, molecular weight markers; Unt, untransfected cells; Transf, transfected cells; TUB, tubulin. e, MtDNA analyses of transfected heteroplasmic MEFs after sorting. Cells were sorted for the fluorescent markers 24 h after transfection and analyzed by PCR/RFLP to differentiate the mutant from the WT mtDNA. WT refers to DNA from a WT C57BL/6J mouse. Experiments were repeated four times with similar results. Cl1, clone 1; Bl, sorted “black” population; Yell, sorted “yellow” population. f,g, Quantification of heteroplasmy, using mitoTALEN A + B (mean ± s.d.; n = 4 independent experiments) or the mitoTALEN A + C (mean ± s.d.; n = 4 independent experiments). h, MtDNA depletion (ND1 mtDNA gene/18S nuclear gene) was not observed after sorting (24h) (mean ± s.d.; n = 4 independent experiments). Statistics done by two-tail Student’s t-test.

Fig. 2 |. AAV9-MitoTALEN is expressed in skeletal muscle and shifts mtDNA heteroplasmy in a predicted manner.

a,b, Western blots showing expression of mitoTALENs in the right (R) and GFP in the left(L) tibialis anterior 4 weeks after recombinant AAV9 intramuscular injections. HA (a), FLAG (b), and GFP (a,b). (+): positive controls are homogenates of HeLa cells transiently transfected with each monomer. NI represents non-injected tibialis anterior muscle. These experiments were repeated for every injected tibialis anterior. c, Immunocytochemistry analysis showed positive GFP expression in the right tibialis anterior (24 weeks post-injection). These experiments were performed twice with similar results. Scale bar, 100μm. d, Representative PCR/RFLP showing a decrease in the mutant mtDNA 4–24 weeks after injection. e, Percentage of mutant mtDNA in left and right tibialis anterior in each mouse. f, Averages of “time groups” from panel e (4 week, n = 3; 6 weeks, n = 6; 10 weeks, n = 3; 12 weeks, n = 6; 24 weeks, n = 3). Statistics done by two-tail Student’s t-test. g–j, High-resolution northern blots showing the tRNA^Ala and RNA^Trp levels in the left and right tibialis anterior of two mice after 24 weeks post-injection (g,i) and six additional mice after 4, 10, 12, and 24 weeks post-injection (h,j). These experiments were repeated twice with similar results. k,l, The percentage of mutant mtDNA change from the right tibialis anterior (injected with AAV9-mitoTALEN) to the left (injected with AAV9-GFP) is also shown for the same mice analyzed for tRNAs levels (data extracted from panel e).

Fig. 3 |. AAV9-mitoTALENs induce a significant and persistent shift in mtDNA heteroplasmy in heart and skeletal muscle after systemic injection.

a, Expression of mitoTALEN (western blot for FLAG, HA and GFP) observed in heart and skeletal muscle after 6 weeks. Tissues analyzed: heart (H), gastrocnemius (G), quadriceps (Q), tibialis anterior (TA), liver (Li), kidney (K), and brain (Br). (+): positive controls are homogenates of HeLa cells transiently transfected with each monomer. Load equalis total proteins in membrane. These experiments were performed three times with similar results. b,c, Quantification of the mutant/wild-type (MUT/WT) mtDNA load in heart (b) and quadriceps (c) normalized to kidney, a tissue not targeted by AAV9, after 6 and 12 weeks post systemic delivery of the AAV9-mitoTALENs and AAV9-GFP (mean ± s.d.; 6 week, n = 3; 12 weeks, n = 3; combined, n = 6).