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Manipulation of Mtdna Heteroplasmy in All Striated Muscles of Newborn Mice by AAV9-Mediated Delivery of a Mitochondria-Targeted Restriction Endonuclease

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Manipulation of Mtdna Heteroplasmy in All Striated Muscles of Newborn Mice by AAV9-Mediated Delivery of a Mitochondria-Targeted Restriction Endonuclease Gene Therapy (2012) 19, 1101 --1106 & 2012 Macmillan Publishers Limited All rights reserved 0969-7128/12 www.nature.com/gt ORIGINAL ARTICLE Manipulation of mtDNA heteroplasmy in all striated muscles of newborn mice by AAV9-mediated delivery of a mitochondria-targeted restriction endonuclease SR Bacman1, SL Williams1, D Duan2 and CT Moraes1,3 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. Gene Therapy (2012) 19, 1101--1106; doi:10.1038/gt.2011.196; published online 1 December 2011 Keywords: mtDNA; heteroplasmy; AAV9; myopathy; mouse model INTRODUCTION muscle and brain6 or to target specific organs, such as the liver (with adenovirus) or heart (with AAV6).9,10 However, manipulation Optimal expression of genes encoded by mitochondrial DNA of mtDNA heteroplasmy in all skeletal muscles in the body (mtDNA) is required for the biogenesis of the oxidative remained elusive. phosphorylation system (OXPHOS), impairment of which is central The prevalence of mitochondrial disease in the pediatric to the etiology of most mitochondrial disorders. Heteroplasmic population has been difficult to determine but is believed to be mtDNA mutations cause a variety of mitochondrial disorders, 11 1,2 similar to that seen in the adult population. We decided which can become clinically apparent during infancy and therefore to test whether we could accomplish endonuclease- generally include cardiovascular, skeletal muscle or neurological mediated heteroplasmy shift in all skeletal muscles at early ages manifestations. Among the genetic therapies being considered as using a well-characterized heteroplasmic mouse model containing strategies for the treatment of such disorders, manipulating two polymorphic mtDNA sequence variants, NZB and BALB/c.12 In mtDNA heteroplasmy to reduce mutation loads has been a goal of these mice, both mtDNA haplotypes behave as neutral variants our group and others for a number of years. except that with age there is an increase in the percentage of Cells contain hundred of copies of mtDNA for every copy of the NZB mtDNA in the liver and slight reduction in the spleen.10,13 --15 nuclear genome. High heteroplasmic mtDNA mutation loads, NZB mtDNA levels in cardiac and skeletal muscle do not change generally above 80%, are required to trigger OXPHOS defects in 3,4 with age. The BALB/c mtDNA variant harbors a unique ApaLI site specific tissues. Reducing mutation loads below these threshold that is not present in the NZB variant. Our goal was to deliver the levels, can restore OXPHOS function, and a complete clearance of recombinant RE to all muscles in the body after a single injection. mutant mtDNA is unnecessary. For this reason, endonuclease- To accomplish this, we produced recombinant AAV9 carrying the mediated heteroplasmy shift is an attractive strategy for genetic mito-RE, ApaLI, and delivered the virus via intraperitoneal (IP) or therapy. In situations where a heteroplasmic mtDNA mutation temporal vein (TV) injection in neonates. Our studies were based results in a unique restriction site, targeting the appropriate on previous reports that robust transduction in the skeletal muscle restriction enzyme (RE) to mitochondria causes a heteroplasmy could be achieved in mice after delivery of AAV9 vectors by shift by reducing the proportion of mutated mtDNA with systemic injection.16,17 the restriction site. We have demonstrated this mechanism in a number of model systems.5,6 SmaI, targeted to mitochondria of cells harboring the T8399G NARP/LHON mutation drastically reduced the mutated mtDNA, followed by repopulation by the RESULTS wild-type mtDNA and restoration of normal intracellular ATP levels Mito-ApaLI-HA expressed from AAV9 vectors is targeted to and mitochondrial membrane potential.7,8 In vivo work has shown mitochondria the efficacy of the system in mouse models when viral vectors Cultured mouse hepatocytes were infected with 1000 viral carrying the recombinant RE were administered focally to the genomes (vg) per cell of rAAV9[mito-ApaLI-HA] and analyzed 1Department of Neurology, University of Miami School of Medicine, Miami, FL, USA; 2University of Missouri School of Medicine, Department of Molecular Biology and Immunology, Columbia, MO, USA and 3Department of Cell Biology and Anatomy, University of Miami School of Medicine, Miami, FL, USA. Correspondence: Dr CT Moraes, Department of Neurology, University of Miami School of Medicine, 1420 NW 9th Avenue, Miami, FL 33136, USA. E-mail: [email protected] Received 6 July 2011; revised 17 October 2011; accepted 28 October 2011; published online 1 December 2011 Systemic manipulation of muscle mtDNA heteroplasmy SR Bacman et al 1102 10 days post-transduction by immunocytochemistry. Cells trans- AAV9[mito-ApaLI-HA] expression in muscle does not induce duced with rAAV9[mito-ApaLI-HA] showed HA expression that mtDNA depletion co-localized with the mitochondrial dye Mito Tracker Red CMXRos qPCR of DNA from gastrocnemius, quadriceps (Figure 5), heart and (Supplementary Figure S1). liver (data not shown) was used to determine whether transgene AAV9[mito-ApaLI-HA] efficiently transduces neonatal mouse muscle Temporal Injection Initially, we administered 5 Â 1011 vgs/mouse IP at P2-P3. The 6 weeks 6 weeks expression of rAAV9[mito-ApaLI-HA] and a control vector expres- AAV9-mitoApaLI AAV9-AP sing alkaline phosphatase, AAV9[AP], were analyzed at 6, 12 and 24 weeks post-injection. We also tested the efficacy of TV injection with 5 Â 1011 vg per mouse at P2-P3. Animals were also killed at the same time points. TV injection of AAV9[AP], resulted in increased AP activity in skeletal and cardiac muscle, as illustrated in tibialis anterior, heart and extensor digitorum longus after 6 weeks (Figure 1a) and after TA 12 and 24 weeks (data not shown). Only a few cells were stained in liver and no activity was observed in the spleen, lung, brain or kidney (data not shown). The same pattern of expression was observed after IP injection with AAV9[AP] at 6, 12 and 24 weeks post-injection (Figure 1b). Likewise, after IP-injection, high expression of AAV9[mito-ApaLI-HA] was observed by western blotting in all skeletal and cardiac muscles analyzed, including: gastrocnemius, quadriceps, soleus, heart, tibialis anterior (Figure 2), Heart and extensor digitorum longus (data not shown), with only a weak signal in liver and no detectable expression in the spleen, brain, lung or kidney, at 6 weeks (Figure 2), 12 and 24 weeks post- injection (Supplementary Figure S2a). Transgene expression of AAV9[mito-ApaLI-HA] in muscle was also confirmed after IP injection by immunocytochemistry staining (Figure 1c). TV injection also proved to be an efficient way to deliver the transgene to all muscles as HA-expression was also observed by western blotting in skeletal and cardiac muscle after 6 weeks EDL (Supplementary Figure S2b). Systemic delivery of AAV9[mito-ApaLI-HA] induces shifts in IP Injection mtDNA heteroplasmy 6 months 6 months Changes in the relative levels of NZB and BALB/c mtDNA were AAV-mito-ApaLI AAV-AP evaluated using ApaLI restriction fragment polymorphism in tissue samples taken at 6, 12 and 24 weeks post-injection (Figures 3 and 4). As shown graphically in Figure 3 (gel images in Supplementary Figure S3), a significant increase was observed in the percentage of NZB mtDNA in all skeletal and cardiac tissues analyzed in mice injected with AAV9[mito-ApaLI-HA], when compared with the percentage of NZB mtDNA present in tail samples from the same mice before injection. A significant increase in the levels of NZB mtDNA was also observed in liver. No change in the percentage of NZB mtDNA was seen in other organs. Likewise, no changes in heteroplasmy levels were observed in mice injected with AAV9[AP] at any time point (Figures 3 and 4). Long-term transduction at 24 weeks post-delivery of AAV9[mito-ApaLI-HA] showed lower changes in NZB levels compared with those recorded at 6 weeks or 12 weeks post-delivery of the transgene Quadriceps Gastro (Figures 3 and 4). HA-Immunostaining HA-Immunostaining 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]. (a) Temporal vein (TV) injections: tibialis anterior (TA), heart and extensor digitorum longus (EDL) at 6 weeks post-injection. (b) intraperitoneal (IP) injections: gastrocnemius (gastro) and quadriceps at 24 weeks. A similar pattern of expression was observed for Mito-ApaLI-HA in Quadriceps quadriceps at 24 weeks post-delivery of AAV9[ApaLI-HA] using anti-HA immunostaining of in 20 mm cryo-sections counterstained using DAPI (c). Bar ¼ 100 mm. Gene Therapy (2012) 1101 --1106 & 2012 Macmillan Publishers Limited Systemic manipulation of muscle mtDNA heteroplasmy SR Bacman et al 1103 AP Mito-ApaLI-HA 120 AAV9-ApaLI-HA 12 weeks 100 HA 80 actin 60 G QSHLiLi G Q S H 40 HA 20 0 actin 10 9 AAV9-AP 12 weeks Sp B Lu K Sp B Lu K TA 8 Figure 2. Expression of Mito-ApaLI-HA in targeted tissues.
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