Organ-specific shifts in mtDNA heteroplasmy following systemic delivery of a mitochondria-targeted restriction endonuclease

Abstract

Most pathogenic mtDNA mutations are heteroplasmic and there is a clear correlation between high levels of mutated mtDNA in a tissue and pathology. We have found that in vivo double-strand breaks (DSBs) in mtDNA lead to digestion of cleaved mtDNA and replication of residual mtDNA. Therefore, if DSB could be targeted to mutations in mtDNA, mutant genomes could be eliminated and the wild-type mtDNA would repopulate the cells. This can be achieved by using mitochondria-targeted restriction endonucleases as a means to degrade specific mtDNA haplotypes in heteroplasmic cells or tissues. In this work, we investigated the potential of systemic delivery of mitochondria-targeted restriction endonucleases to reduce the proportion of mutant mtDNA in specific tissues. Using the asymptomatic NZB/BALB mtDNA heteroplasmic mouse as a model, we found that a mitochondria-targeted ApaLI (that cleaves BALB mtDNA at a single site and does not cleave NZB mtDNA) increased the proportion of NZB mtDNA in target tissues. This was observed in heart, using a cardiotropic adeno-associated virus type-6 (AAV6) and in liver, using the hepatotropic adenovirus type-5 (Ad5). No mtDNA depletion or loss of cytochrome c oxidase activity was observed in any of these tissues. These results show the potential of systemic delivery of viral vectors to specific organs for the therapeutic application of mitochondria-targeted restriction enzymes in mtDNA disorders.

Figure 1. Expression of the rAAV6[mito-ApaLI-HA] in targeted tissues

(A) Alkaline phosphatase (AP) staining performed in heart showed expression of AP after 8 weeks of injection of rAAV6[AP] (1x10e11vg). The staining persisted in heart after 12 weeks (1x10e11vg (not shown) and 5x10e11vg). (B) HA expression post-delivery of the transgenes. We observed high expression in the heart after 8 and 12 weeks, but few cells expressing the transgene in liver, and no expression in the tissues targeted with control rAAV6[AP] . (C) The expression of the rAAV6[mito-ApaLI-HA] was also analyzed by western blotting using the same anti-HA antibody as in figures 1B. Positive expression was detected in heart (H) and liver (Li) after 2 weeks, 8 weeks (1x10e11vg) and 12 weeks (5x10e11vg) post-delivery of the rAAV6[mito-ApaLI-HA], with reduced expression to undetectable levels in other tissues analyzed such as quadriceps muscle (M), brain (B), lung (Lu), spleen (S). A negative control of a heart sample from a non-injected animal (H-) was also analyzed, with no detectable expression of HA. Samples were compared to tubulin used as control.

Figure 2. [Mito-ApaLI-HA] expression in liver or heart is not associated with COX deficiencies

(A) Expression of Mito-ApaLI-HA in liver after 7 days of the systemic delivery of the transgene rAd[Mito-ApaLI-HA]. Animals were sacrificed and liver samples frozen in liquid nitrogen-cooled isopentane as described in Methods. Twenty-μm liver sections were immunostained for HA (magnification 20x). eGFP liver staining was observed after 7 days systemic delivery of rAd5[eGFP] (magnification 20x). (B) COX staining in liver samples after 1 week post-delivery of the rAd5[mito-ApaLI-HA] samples did not show Cox deficiency when compared to rAd5[eGFP] transduced liver samples (magnification 20x). (C) Transduction of rAAV6[mito-ApaLI-HA] in heart showed no changes in the levels of subunit I of COX (MT-CO1)(green)/SDH (red) immunostaining after 12 weeks post-delivery of the transgene when compared to rAAV6[AP] control samples (magnification 40x).

Figure 3. Expression of mito-ApaLI-HA in heart and liver does not lead to mtDNA depletion

(A) MtDNA levels in heart were analyzed by Southern blot in samples obtained 2, 8 and 12 weeks after rAAV6[mito-ApaLI-HA] or rAAV6[AP] injections. No changes in the ratios of mtDNA/nuclear DNA were observed (B) rAd5[mito-ApaLI-HA] or controls rAd5[eGFP] transduced liver samples showed no changes in the ratios of mtDNA/nuclear DNA when comparing pre-injection liver samples to the post-injection samples after 1 or 2 weeks. Each double bar represents one animal sample before and after the delivery of the transgene. mtDNA/nDNA is expressed as ratios of arbitrary densitometric units.

Figure 4. [mito-ApaLI-HA] induces a significant shift in mtDNA heteroplasmy in targeted tissues after systemic injection of the transgenes

The percentage of NZB mtDNA genotype was quantified by the last-cycle hot PCR/RFLP analysis (as described in Methods). (A) DNA samples from injected animals with rAAV6[AP] or rAAV6[mito-ApaLI-HA] were evaluated for the increase of NZB mtDNA after 2 weeks (2w), 8 weeks (8w) and 12 weeks (12w) systemic delivery of the transgenes (one animal per group, 6 animals total). Significant increase of the NZB mtDNA was observed in heart after injection of rAAV6[mito-ApaLI-HA] over time, with no change when the control rAAV6[AP] virus was delivered (3 independent samples from the same mouse *p<0.05). (B) Because of the natural increase of NZB mtDNA in liver and kidney, and decrease in spleen (S1), the NZB mtDNA ratio from samples obtained after delivery of the rAAV6[mito-ApaLI-HA] were normalized to the rAAV6[AP] injected samples, and the ratios were compared to muscle samples before the injection. Only liver and heart showed significantly increased NZB mtDNA above 1, when compared to muscle-pre-injected tissue after 12 weeks (3 independent samples from the same mouse *p<0.05). (C) NZB mtDNA was increased in liver of injected mice 1 or 2 weeks after delivery of rAd5[mito-ApaLI-HA] (n=6) with no changes in the level of NZB mtDNA in the control animals after systemic delivery of the rAd5[eGFP] (n=3). Each line connects “Liver Pre” (as values obtained from liver DNA samples before injection) and “Liver 2w Post” as post-injection liver samples after 2 weeks of delivery of the transgenes. Each line represents one animal (**p<0.01). (D) Ratios of NZB percentages after/before treatment with rAd5[mito-ApaLI-HA]. No changes in NZB mtDNA percentages were observed in kidney and spleen. However, liver samples showed a marked increase in the percentages of NZB mtDNA after treatment. Each cluster of bars represents one animal (n=3 for rAd5[eGFP], n=2 for rAd5[mito-ApaLI-HA] 1 week and, n=2 for rAd5[mito-ApaLI-HA] 2 weeks).