Manipulation of mtDNA heteroplasmy in all striated muscles of newborn mice by AAV9-mediated delivery of a mitochondria-targeted restriction endonuclease

Abstract

Mitochondrial diseases are frequently caused by heteroplasmic mitochondrial DNA (mtDNA) mutations. As these mutations express themselves only at high relative ratios, any approach able to manipulate mtDNA heteroplasmy can potentially be curative. In this study, we developed a system to manipulate mtDNA heteroplasmy in all skeletal muscles from neonate mice. We selected muscle because it is one of the most clinically affected tissues in mitochondrial disorders. A mitochondria-targeted restriction endonuclease (mito-ApaLI) expressed from AAV9 particles was delivered either by intraperitoneal or intravenous injection in neonate mice harboring two mtDNA haplotypes, only one of which was susceptible to ApaLI digestion. A single injection was able to elicit a predictable and marked change in mtDNA heteroplasmy in all striated muscles analyzed, including heart. No health problems or reduction in mtDNA levels were observed in treated mice, suggesting that this approach could have clinical applications for mitochondrial myopathies.

Figure 1. Expression of AAV9[AP] in targeted tissues

Alkaline phosphatase activity staining reveals high expression of AP in skeletal muscle and heart after delivery of AAV9[AP]. 1A, temporal vein (TV) injections: Tibialis anterior (TA), heart and extensor digitorum longus (EDL) at 6 weeks post injection. 1B, intraperitoneal (IP) injections: gastrocnemius (gastro) and quadriceps at 24 weeks. A similar pattern of expression was observed for Mito-ApaLI-HA in quadriceps at 24 weeks post-delivery of AAV9[ApaLI-HA] using anti-HA immunostaining of in 20 µm cryo-sections counterstained using DAPI (1C). Bar = 100 µm.

Figure 2. Expression of Mito-ApaLI-HA in targeted tissues

The expression of ApaLI-HA was analyzed by anti-HA western blotting 6 weeks after IP injection in P2-P3 mice. Homogenates from skeletal muscles, gastrocnemius (G), quadriceps (Q), soleus (S), heart (H) and tibialis anterior (TA) showed high expression. No expression was observed in spleen (Sp), brain (B), lung (Lu) or kidney (K), with weak expression in liver (Li). Actin was used as a loading control.

Figure 3. AAV9[mito-ApaLI-HA] induces a shift in mtDNA heteroplasmy in muscle

NZB/BALB/c mtDNA heteroplasmy quantified using last-cycle hot PCR/RFLP with DNA samples from mice injected with AAV9[mito-ApaLI-HA] at 6 weeks post IP injection. Increases in the percentage of NZB mtDNA were observed in all skeletal muscle and heart tissues when compared to the samples obtained from tail before injection. 3A, representative phospho-image of radiolabeled RFLP gel, Gastrocnemius (G), quadriceps (Q), soleus (S), before injection (BI), uncut DNA (U); 3B, quantification of phosphor-imager data, following injection of AAV9[mito-ApaLI-HA] (upper panel) and AAV9[AP] (lower panel); tibialis anterior (TA), extensor digitorum longus (EDL). No change in the percentage of NZB mtDNA was observed in spleen, brain, and kidney; a shift was observed in liver when compared to the AAV9[AP] injected sample that represents the age related shift in heteroplasmy in this tissue. Each bar corresponds to a different tissue from a single mouse.

Figure 4. AAV9[mito-ApaLI-HA] induces a long-term shift in mtDNA heteroplasmy

NZB/BALB/c heteroplasmy quantified using last-cycle hot PCR/RFLP using muscle DNA from mice injected with AAV9[mito-ApaLI-HA] at 12 weeks (4A) and 24 weeks (4B) post IP injection. Tissue abbreviations as in Figure 3. Increased percentage of NZB mtDNA was observed in all the skeletal muscles and heart when compared to before injection samples from tails. Again no change in the percentage of NZB mtDNA was observed in spleen, kidney, lung or brain, with slight shift in liver. Each bar corresponds to a different tissue from a single mouse.

Figure 5. AAV9[mito-ApaLI-HA] expression in muscle does not induce mtDNA depletion

Quantitative PCR of gastrocnemius and quadriceps DNA showed no sign of mtDNA depletion at 6, 12 and 24 weeks post injection (three independent samples from the each tissue *P<0.05).