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

Materials and Methods

Measurement of O₂ Consumption Rates in Mouse Cell Lines with ΔmtDNA. Mouse hybrid subclones with various proportions of ΔmtDNA (7) were grown in normal RPMI medium 1640 (Nissui Seiyaku, Tokyo) containing 10% FCS, 50 μg/ml uridine, and 0.1 mg/ml pyruvate. The rate of oxygen consumption was measured by trypsinizing cells, incubating the suspension in PBS, and recording oxygen consumption in a 1.0-ml polarographic cell at 37°C with a Clark-type oxygen electrode (Yellow Springs Instruments).

Estimation of ΔmtDNA Proportions by Southern Blot Analysis. For estimation of ΔmtDNA proportions in neonates and adult tissues, Southern blot analysis was carried out. Briefly, XhoI-digested total DNA was separated in 0.6% agarose gel and transferred to a nylon membrane. Hybridization was carried out with a mtDNA probe (nucleotide positions 1859-2762). Signal calculation was carried out with nih image program. Average and standard deviation of ΔmtDNA ratios were obtained from three independent measurements of each sample.

Estimation of ΔmtDNA Proportions by Real-Time Monitoring PCR. For estimation of ΔmtDNA proportions in zygotes and polar bodies, real-time monitoring PCR was carried out by using a TaqMan PCR reagent kit and a PRISM 7900HT Sequence Detection System (Applied Biosystems). The primer set specific for ΔmtDNA was nucleotide positions 7697-7725 and 12528-12508. The reporter dye 6-carboxyfluorescein (FAM)-labeled TaqMan MGB probe (Applied Biosystems) specific for ΔmtDNA was nucleotide positions 7750-7758 and 12455-12464. The primer set specific for wild-type mtDNA was nucleotide positions 11933-11952 and 12076-12047. The reporter dye FAM and the quencher dye 6-carboxy-tetramethyl rhodamine (TAMRA)-labeled probe was nucleotide positions 12012-12037. For protein digestion, isolated zygotes and polar bodies were incubated at 55°C overnight in 20 μl of PCR buffer/nonionic detergent and proteinase K solution (50 mM KCl/10 mM Tris·HCl, pH 8.3/1.5 mM MgCl₂/0.1% gelatin/0.45% Nonidet P-40/0.45% Tween-20/100 μg/ml proteinase K), and proteinase K was inactivated at 95°C for 15 min. Samples of 2 μl were used directly as real-time monitoring PCR templates. Independent reactions were repeated three times for both ΔmtDNA and wild-type mtDNA copy number estimation. Proportions of ΔmtDNA were estimated from the obtained copy numbers of ΔmtDNA and wild-type mtDNA.

Collection of Oocytes and the Second Polar Bodies. Four- to 8-week-old mito-mice and C57BL/6J strain (B6) mice were induced to superovulate by consecutive injections of pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) with an interval of 48 h between injections. Unfertilized oocytes were collected from the oviducts 15 h after hCG injection. In vitro fertilization was carried out by using B6 sperm in human tubal fluid (HTF) medium. The second polar bodies were collected with a piezo-driven micromanipulator.

Nuclear Transplantation. Enucleation of zygotes from mito-mice and B6 mice were carried out in M2 medium with 5 μg/ml cytochalasine B and 0.3 μg/ml nocodazole. Karyoplasts from mito-mice zygotes were introduced into enucleated B6 zygotes with a micromanipulator. Manipulated zygotes were placed between electrodes positioned 100 μm apart in a fusion chamber (Nepa Gene, Ichikawa, Japan) filled with 30 ml of mannitol solution (0.3 M mannitol/0.05 mM CaCl₂/0.1 mM MgSO₄/0.001% BSA). Membrane fusion was induced by applying a single electric pulse (750 V/cm for 50 μsec) with a prepulse AC current (1 MHz, 150 V/cm for 15 sec). Fused zygotes were transferred to the oviducts of pseudopregnant females.

Biochemical Analyses. For determination of blood lactate concentration, tail vein blood was prepared from mito-mice and B6 mice. Mito-mice and B6 mice were starved overnight, and glucose (1.5 g/kg of body weight) was orally administered at 30 min before the measurement. Blood lactate concentration was measured with the automatic blood lactate test meter Lactate Pro (ARKRAY, Kyoto). For determination of blood urea nitrogen concentration, tail vein blood was prepared from nonfasting mito-mice and B6 mice. Blood urea nitrogen was measured with a Urea N B test (Wako Pure Chemical, Osaka).

Results

Diagnosis of mtDNA Genotypes by Using Polar Bodies and Tails. An important problem in applying gene therapy to mito-mouse zygotes was how we could diagnose their proportion of ΔmtDNA without killing them. Recently, polar bodies and blastomeres were shown to be effective for deducing the status of heteroplasmy in embryos of mice carrying NZB and BALB wild-type mtDNAs (13). For examination of this possibility, we compared the proportions of ΔmtDNA in mito-mouse zygotes and their second polar bodies.

After in vitro fertilization, 37 zygotes (pronuclear stage embryos) and their second polar bodies were separated with a micromanipulator, and the proportions of ΔmtDNA were estimated by using real-time monitoring PCR (Fig. 1A). The results showed that all of the 37 zygotes we examined possessed 8-78% ΔmtDNA (Fig. 1 A). The least-squares correlation coefficient for the proportions of ΔmtDNA in second polar bodies and zygotes was 0.95 (Fig. 1 A). This observation indicated that second polar bodies could be used as biopsy samples of zygotes.

The maximum proportion of ΔmtDNA in mito-mouse zygotes was 78% (Fig. 1 A), suggesting that oogonia carrying >78% ΔmtDNA did not differentiate into oocytes because of respiration defects induced by the accumulated ΔmtDNA. This idea was supported by the observation that cultivated cells carrying >78% ΔmtDNA showed <50% respiratory function (Fig. 1B).

We also examined whether tails could be used as biopsy samples of mito-mice for monitoring the proportions of ΔmtDNA after postnatal stages. Because renal failure caused by ΔmtDNA-induced respiration defects was always responsible for the death of mito-mice (7), we compared the proportions of ΔmtDNA in the kidneys and tails. The least-squares correlation coefficient for the proportions of ΔmtDNA in the kidneys and tails was 0.80 (Fig. 1B). Therefore, tails could be used for monitoring the proportions of ΔmtDNA in the kidneys, although tails had slightly lower proportions.

The maximum proportion of ΔmtDNA in the kidneys of mito-mice was 90% (Fig. 1C), which could provide only 10% respiratory function in cultivated cells (Fig. 1B) and was slightly higher than the maximum proportion of ΔmtDNA in zygotes (78%; Fig. 1 A). These observations suggested that the kidneys of mito-mice were more resistant to respiration defects than oocytes and zygotes, and that mito-mice with the kidneys having >90% ΔmtDNA died because of renal failure.

Change in the Proportions of ΔmtDNA During in Utero Development. Nuclear transplantation would introduce small amounts of ΔmtDNA from mito-mice into normal zygotes. Because of the smaller size of ΔmtDNA by 4696-bp deletion, it possesses replication advantages over wild-type mtDNA, leading to its accumulation during aging of mito-mice. Thus, for success of gene therapy of mito-mouse zygotes, we had to determine the limiting amount of ΔmtDNA in mito-mouse zygotes required to avoid expression of respiration defects throughout the life of adults. For this determination, we monitored the accumulation rates of ΔmtDNA in mito-mice during embryogenesis, postnatal stages, and aging.

First we estimated the accumulation rate of ΔmtDNA during embryogenesis (in utero development) by comparison of the proportions of ΔmtDNA in the second polar bodies of zygotes and whole bodies of neonates developed from the same zygotes. The second polar bodies were collected from 72 zygotes (pronuclear stage embryos) to estimate the proportion of ΔmtDNA, and the 72 zygotes were transferred to the oviducts of 72 pseudopregnant female mice. We obtained 13 neonates from 72 zygotes transferred into the oviducts of 72 pseudopregnant female mice, and we compared the proportions of ΔmtDNA in the second polar bodies and corresponding neonates (Fig. 2A).

The zygotes used in this study possessed 2-78% ΔmtDNA in their second polar bodies, and the resultant neonates possessed 0-75% ΔmtDNA. No neonates developed from zygotes carrying >60% ΔmtDNA (Fig. 2 A). Considering that the average increase in the proportion of ΔmtDNA during gestation (for 19 days) was 17 ± 10% (mean ± SD) (Fig. 2 A), neonates developed from such zygotes possessing >60% ΔmtDNA would carry >77% ΔmtDNA during gestation and would be lethal in the uterus. All of these observations suggested that zygotes and neonates carrying >77-78% ΔmtDNA could not survive because of respiration defects induced by accumulated ΔmtDNA (Fig. 1B).

Change in the Proportions of ΔmtDNA During Postnatal Development and Aging. Next, we estimated the accumulation rates of ΔmtDNA in mito-mice after birth. We used tails of 12 neonates obtained from eight pregnant female mito-mice carrying 23-48% ΔmtDNA in their tails (Fig. 2B).

Comparison of the proportions of ΔmtDNA in neonates and weaned mice showed an increase of 8 ± 6% in the 30 days after birth. Then we monitored the proportions of ΔmtDNA in the tail every 100 days and found an increase rate of 8 ± 6% in the first 100 days and then of 6 ± 3% in every 100 days later (Fig. 2B). Therefore, the rate of increase was 17% during gestation (19 days after fertilization; Fig. 2 A), 8% before weaning after birth (for 30 days after birth), 8% in the first 100 days after weaning, and 6% in every subsequent 100-day period (Fig. 2B). The significant decrease in the rate of ΔmtDNA accumulation after birth would correspond to decrease of the mtDNA replication frequency because of decrease in frequency of cell division.

Of 12 neonates, 10 survived for 200-430 days and 1 survived for 765 days after birth (Fig. 2B). Deaths were due to renal failure of mice in which the kidneys always possessed >82% ΔmtDNA (Fig. 2B). On the other hand, the one remaining mouse that survived for 917 days after birth did not show renal failure and possessed 39% ΔmtDNA in its kidney, suggesting that its death was probably due to aging (Fig. 2B).

These observations suggested that mito-mice carrying 5% ΔmtDNA at their zygote stage should possess 22% at birth, 30% before weaning, and 38% ΔmtDNA in the next 100 days. Moreover, they should possess 78% ΔmtDNA for 800 days after birth, assuming that the accumulation rate of ΔmtDNA became 6% in every subsequent 100-day period. Therefore, gene therapy of zygotes has to be carried out so that mito-mouse zygotes include <5% ΔmtDNA to avoid expression of disease phenotypes caused by ΔmtDNA-induced respiration defects within their lifetimes.

Nuclear Transplantation of Mito-Mouse Zygotes for Their Gene Therapy. Mouse zygotes possess 5 × 10⁵ mtDNA copies, but the maximum mtDNA copies that could be introduced into mouse zygotes by cytoplasmic transplantation is limited to 10⁴. These amounts are insufficient for mito-mouse zygotes to be rescued from expressing respiration defects after their development.

On the other hand, nuclear transplantation would be more effective than cytoplasmic transplantation to exclude ΔmtDNA from mito-mouse zygotes. Nuclear transplantation is accomplished by the fusion of enucleated zygotes derived from normal mice with karyoplasts obtained by enucleation of mito-mouse zygotes. Because karyoplasts possess 3 × 10⁴ mtDNA copies, only 6% exogenous mtDNA would be cointroduced along with nucleus from mito-mice zygotes into normal recipient zygotes. Thus, nuclear transplantation would result in production of zygotes with all nuclear genome and <5% ΔmtDNA from mito-mice, even when the mito-mouse zygotes possessed the maximum proportion of ΔmtDNA (78%, cf. Fig. 2 A).

Therefore, we carried out nuclear transplantation of 39 mito-mouse zygotes for testing whether it was effective in gene therapy of F₀ progeny of mito-mice. First, we estimated their ΔmtDNA proportions by using second polar bodies as biopsy samples. The results showed that 17-53% ΔmtDNA (average 35%) was present in 39 zygotes (Fig. 3A). For nuclear transplantation, we prepared karyoplasts from 39 zygotes and fused them with enucleated normal zygotes. In this experiment, the amounts of ΔmtDNA in nuclear-transplanted zygotes would be 2% on average (Fig. 2 A), because karyoplasts carried 35% ΔmtDNA on average and because only 6% mtDNA in nuclear-transplanted zygotes was derived from karyoplasts. The resultant 39 nuclear-transplanted zygotes were transferred together into 2 pseudopregnant females, and 11 newborn mice were obtained. Then, we used their tails for monitoring the proportions of ΔmtDNA after birth and found that 11 mice at the weaned stage (20-40 days after birth) possessed 6-21% ΔmtDNA (average 11%), and that the average increase was 12% in the subsequent 300 days to possess 5-44% ΔmtDNA (average 23%) in their tails (Fig. 3A).

As nontransplanted controls, we used 34 mito-mouse zygotes possessing 11-47% ΔmtDNA (32% on average) for transfer into 2 pseudopregnant females, and obtained 9 newborn mito-mice (Fig. 3B). Their tails possessed 51-73% ΔmtDNA (66% on average) at the weaned stage. Thus, the increase of ΔmtDNA was ≈34% during the 50-day period from fertilization to the weaned stage. We used tails of all 9 weaned-stage mice for further monitoring the proportions of ΔmtDNA during aging and showed that they increased 14% during the further 170 days after weaning (Fig. 3B).

Effects of Nuclear Transplantation on Expression of Disease Phenotypes. Nontransplanted mito-mice grew much slower than did nuclear-transplanted mito-mice, and they subsequently showed decrease of body weight on day 150 after birth (Fig. 4A). Therefore, we estimated the amounts of lactate and urea nitrogen in the blood in both groups on day 200 after birth. The results showed significant increase of lactate and urea nitrogen levels in the bloods of nontransplanted mito-mice but steady levels in all nuclear-transplanted mito-mice (Fig. 4 B and C).

On the day 210, we killed one nontransplanted mito-mouse carrying 88% ΔmtDNA in its tail and one nuclear-transplanted mito-mouse carrying 37% ΔmtDNA in its tail for precise investigation of renal failure, which we had shown to be a common abnormality of mito-mice (7, 8). A nontransplanted mito-mouse showed enlarged kidneys with a granulated surface (Fig. 4D) as well as dilatation of the cortical proximal and distal tubules (Fig. 4E). The kidney had 85% ΔmtDNA, and most cells in the kidney and heart showed significant reduction of cytochrome c oxidase activity (Fig. 4 F and G). On the other hand, a nuclear-transplanted mito-mouse showed no abnormalities (Fig. 4).

All remaining nontransplanted mito-mice died at 218-277 days. Although some of their tails possessed <70% ΔmtDNA at their deaths, their kidneys possessed >88% mtDNA (Fig. 3A, red triangles) and had a granulated surface (data not shown). Thus, the death of all nontransplanted mito-mice should be attributable to renal failure caused by significant ΔmtDNA-induced respiration defects.

On the other hand, all remaining nuclear-transplanted mito-mice survived for >300 days after birth (Fig. 3A). They possessed 6-21% ΔmtDNA (11% on average) at weaning, and the average increase of ΔmtDNA was 12% in the subsequent 300 days (Fig. 3A). Thus, the proportion of ΔmtDNA on day 800 after birth would be 43%, suggesting that most nuclear-transplanted mice would not die because of respiration defects caused by accumulated ΔmtDNA, and that application of gene therapy to mito-mouse zygotes by nuclear transplantation is very effective for their rescue from mitochondrial diseases throughout their lives.

Discussion

Our recent studies provided convincing evidence for genetic complementation between exogenous mitochondria with ΔmtDNA of Mus musculus domesticus and endogenous mitochondria with wild-type mtDNA of Mus spretus by generating mito-mice (14, 15). If there were no genetic complementation between exogenous and endogenous mitochondria, ΔmtDNA with replication advantages would expand clonally and accumulate focally in some tissues to express significant respiration defects. Thus, protection of nuclear-transplanted F₀ progeny of mito-mice from expressing disease phenotypes found in this study would be explained by assuming genetic complementation between endogenous mitochondria in recipient zygotes and exogenous mitochondria with ΔmtDNA, which was cointroduced into zygotes along with nucleus from mito-mouse zygotes.

However, nuclear transplantation is not suitable for CPEO patients carrying ΔmtDNA for the following reasons. One reason is the much longer life span of human subjects than mice. It is true that nuclear transplantation is effective to protect progeny of mito-mice from sufficient accumulation of ΔmtDNA required to express respiration defects for 800 days after birth. However, it would not rescue progeny of the patients, because they eventually express disease phenotypes before finishing their natural life span.

Another reason why nuclear transplantation is not appropriate to CPEO patients is the absence of maternal transmission of ΔmtDNA in human cases (1, 2). Our previous study explained the discrepancy of maternal transmission of ΔmtDNA in mouse but not in human cases by assuming frequent creation of partially duplicated forms in mito-mice by recombination between wild-type and ΔmtDNA (7). However, our recent study provided convincing evidence for rare creation of mtDNA recombinants in mammalian species (15), and partially duplicated forms found in mito-mice (7) corresponded to partial digestion products of wild-type mtDNA (data not shown). Moreover, Marchington et al. (16) reported that 10-50% ΔmtDNA was present in oocytes from patients with Kern-Sayre syndrome (KSS), suggesting maternal transmission of human ΔmtDNA at least to F₀ embryos. Probably, the 13 times longer prenatal period in humans than in mice resulted in accumulation of a sufficient proportion of ΔmtDNA in embryos to be lethal before birth. Assuming that the proportion of ΔmtDNA increases 18% during 3-week embryogenesis as in mice (Fig. 2), human embryos would accumulate 90% ΔmtDNA to be lethal within a 15-week period of embryogenesis, even when human oocytes possessed only 1% ΔmtDNA.

Therefore, application of nuclear transplantation would be restricted to patients with mitochondrial diseases only when pathogenic mtDNAs in patients were inherited maternally and did not possess significant replication advantages over wild-type mtDNA. For example, zygotes from patients with MELAS and MERRF caused by 3,243 and 8,344 point mutations in mtDNAs, respectively, would be appropriate for applying nuclear transplantation to rescue their F₀ progeny. In these cases, a small proportion of wild-type mtDNAs was sufficient to suppress disease phenotypes (17, 18). Recently, we generated respiration-deficient mito-mice due to homoplasmy of pathogenic mtDNA with a point mutation in the COI gene by using female-type ES cells (our unpublished data). Because the pathogenic mtDNA does not possess replication advantages, investigating them as typical models for applying gene therapy to zygotes from patients with mitochondrial diseases will be useful.

In human cases, however, there are two additional problems to be resolved before applying gene therapy to zygotes. One problem is that human oocytes, but not zygotes, should be used for nuclear transplantation. When oocytes are used instead of zygotes, first polar bodies have to be used as biopsy samples for diagnosis of mtDNA genotypes of oocytes, as suggested by Thorburn and Dahl (19). Our preliminary experiments showed that the proportions of ΔmtDNA in first polar bodies were almost comparable to those in oocytes of mito-mice (data not shown). When first polar bodies of all oocytes possessed predominant amounts of pathogenic mtDNAs, their nuclear transplantation to enucleated normal oocytes and subsequent in vitro fertilization have to be carried out for obtaining zygotes carrying lower proportions of pathogenic mtDNA. On the other hand, we do not have to apply gene therapy to oocytes, particularly when first polar bodies showed heteroplasmy of wild-type and pathogenic mtDNAs and oocytes carrying predominant amounts of wild-type mtDNAs were available.

Another problem raised by nuclear transplantation is artificial formation of heteroplasmic mtDNAs and possible creation of new recombinant mtDNA haplotypes in F₀ progeny. Exclusive transmission of mtDNAs from mothers to following generations in mammalian species (10-12) represented asexual inheritance of mammalian mtDNAs. Therefore, the mixture of mtDNAs derived from different individuals by nuclear transplantation seems to be an unusual event and may induce respiration defects by unknown mechanisms. However, no respiration defects were observed in human somatic hybrids carrying heteroplasmic mtDNAs derived from different human subjects (20) or in mice carrying heteroplasmic mtDNAs derived from different individuals (21). Moreover, our recent study showed an extremely low frequency of recombinant formation in human somatic hybrids and in mice carrying heteroplasmic mtDNAs (15). Even when mtDNA recombinants were created (22) and became predominant for some unknown reasons, there was no evidence and no rational explanation that the heteroplasmic state itself or resultant creation of mtDNA recombinants induced respiration defects.

An additional biological problem in nuclear transplantation is that resultant zygotes possess nuclear genomes from both parents but possess mitochondrial genomes from normal donors. However, all genes encoded by mtDNA were exclusively involved in respiratory function (1, 2) and thus would not affect individuality. The most important issue of nuclear transplantation is that it unambiguously rescues progeny of the patients from expressing mitochondrial diseases (Fig. 3).