Gene therapy for progeny of mito-mice carrying pathogenic mtDNA by nuclear transplantation

Abstract

Pathogenic mutations in mtDNAs have been shown to be responsible for expression of respiration defects and resultant expression of mitochondrial diseases. This study directly addressed the issue of gene therapy of mitochondrial diseases by using nuclear transplantation of zygotes of transmitochondria mice (mito-mice). Mito-mice expressed respiration defects and mitochondrial diseases due to accumulation of mtDNA carrying a large-scale deletion (DeltamtDNA). Second polar bodies were used as biopsy samples for diagnosis of mtDNA genotypes of mito-mouse zygotes. Nuclear transplantation was carried out from mito-mouse zygotes to enucleated normal zygotes and was shown to rescue all of the F(0) progeny from expression of respiration defects throughout their lives. This procedure should be applicable to patients with mitochondrial diseases for preventing their children from developing the diseases.

Fig. 1.

Determination of ΔmtDNA proportions in mito-mice by using second polar bodies and tails as biopsy samples. (A) Comparison of ΔmtDNA proportions in zygotes and their second polar bodies of mito-mice. The least-squares correlation coefficient is 0.95. The best fit is indicated by a line. (B) Effect of the ΔmtDNA proportions on respiratory function. Subclones of cultivated mouse cells possessing various proportions of ΔmtDNA were used for measurement of O₂ consumption rates. (C) Comparison of ΔmtDNA proportions between tails and kidneys of mito-mice. The least-squares correlation coefficient is 0.80. The best fit is indicated by a line.

Fig. 2.

Increase in ΔmtDNA proportions during development and aging of mito-mice. (A) Accumulation of ΔmtDNA proportions during gestation. Second polar bodies were used as biopsy samples to estimate ΔmtDNA proportions in zygotes. ΔmtDNA proportions were compared in second polar bodies and neonates at 0.5 and 19.5 days postcoitum. Open triangles represent ΔmtDNA proportions. (B) Accumulation of ΔmtDNA proportions during postnatal stages and aging. Open triangles represent ΔmtDNA proportions in tails. × and red triangles represent ΔmtDNA proportions in tails and kidneys, respectively, examined after the death of mito-mice.

Fig. 3.

Effects of nuclear transplantation of mito-mouse zygotes on the proportions of ΔmtDNA during embryogenesis, postnatal development, and aging. ΔmtDNA proportions in nuclear-transplanted mito-mice (A) and in nontransplanted mito-mice (B). Open triangles indicate ΔmtDNA proportions in second polar bodies or tails. Averages and SD of ΔmtDNA proportions are indicated by red circles and bars. × and red triangles indicate the proportions of ΔmtDNA in tails and kidneys, respectively, examined after the death of mito-mice. Red asterisks indicate the proportions of ΔmtDNA in tails from mito-mice killed for precise investigation of their clinical phenotypes shown in Fig. 4 D--G.

Fig. 4.

Effects of nuclear transplantation of mito-mouse zygotes on clinical phenotypes. (A) Measurement of body weights. Red and blue circles indicate mito-mice developed from nuclear-transplanted and nontransplanted zygotes, respectively. Open and filled circles indicate males and females, respectively. (B) Concentration of blood lactate after glucose loading. (C) Concentration of blood urea nitrogen examined 200 days after birth. (D) Kidneys. (E) Histopathology of kidneys. (F) Cytochrome c oxidase histochemistry of kidneys. (G) Cytochrome c oxidase histochemistry of hearts from normal control B6 mouse (Left), nuclear-transplanted (Center), and nontransplanted mito-mouse (Right) killed 210 days after birth. (Scale bar in D, 5 mm; scale bars in E-G, 50 μm.)