Gene delivery to mitochondria by targeting modified adenoassociated virus suppresses Leber’s hereditary optic neuropathy in a mouse model

Yu, Hong; Koilkonda, Rajeshwari D.; Chou, Tsung Han; Porciatti, Vittorio; Ozdemir, Sacide S.; Chiodo, Vince A.; Boye, Sanford L.; Boye, Shannon E.; Hauswirth, William W.; Lewin, Alfred S.; Guy, John

doi:10.1073/pnas.1119577109

Cited by 169 publications

(182 citation statements)

References 46 publications

(50 reference statements)

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“…scHSP-mutND4FLAG+mCherry was constructed as previously described (17,18). In brief, human mutND4FLAG and mitochondrial-encoded mCherry (mCherry) were generated by site-directed mutagenesis (Quikchange II XL sitedirected mutagenesis kit; Stratagene) with the substitution of A for G at 11778 in ND4 and A for C at 559 in mCherry.…”

Section: Methodsmentioning

confidence: 99%

“…Taking advantage of this property, we redirected the adeno-associated virus (AAV) to target mitochondria efficiently, rather than its typical target, the nucleus, by adding the 23-aa cytochrome oxidase subunit 8 (COX8) presequence into the AAV2 capsid ORF (17,18). Here we use this vector to deliver the mutant human G11778A ND4 DNA into zygotes to generate transgenic LHON mice.…”

Section: Significancementioning

confidence: 99%

“…After packaging with the mitochondria-targeted capsid (cox8GFP) (Fig. 1B) (17,18), the recombinant AAV (rAAV) was injected into fertilized oocytes ( Fig. 1 C and D).…”

Section: Mutant Human Nd4 In Transgenic Micementioning

confidence: 99%

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Consequences of zygote injection and germline transfer of mutant human mitochondrial DNA in mice

Koilkonda

Chou

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Considerable evidence supports mutations in mitochondrial genes as the cause of maternally inherited diseases affecting tissues that rely primarily on oxidative energy metabolism, usually the nervous system, the heart, and skeletal muscles. Mitochondrial diseases are diverse, and animal models currently are limited. Here we introduced a mutant human mitochondrial gene responsible for Leber hereditary optic neuropathy (LHON) into the mouse germ line using fluorescence imaging for tissue-specific enrichment in the target retinal ganglion cells. A mitochondria-targeted adeno-associated virus (MTS-AAV) containing the mutant human NADH ubiquinone oxidoreductase subunit 4 (ND4) gene followed by mitochondrial-encoded mCherry was microinjected into zygotes. Female founders with mCherry fluorescence on ophthalmoscopy were backcrossed with normal males for eight generations. Mutant human ND4 DNA was 20% of mouse ND4 and did not integrate into the host genome. Translated human ND4 protein assembled into host respiratory complexes, decreasing respiratory chain function and increasing oxidative stress. Swelling of the optic nerve head was followed by progressive demise of ganglion cells and their axons, the hallmarks of human LHON. Early visual loss that began at 3 mo and progressed to blindness 8 mo after birth was reversed by intraocular injection of MTS-AAV expressing wild-type human ND4. The technology of introducing human mitochondrial genes into the mouse germ line has never been described, to our knowledge, and has implications not only for creating animal models recapitulating the counterpart human disorder but more importantly for reversing the adverse effects of the mutant gene using gene therapy to deliver the wild-type allele.gene therapy | adeno-associated virus | mitochondria | Leber hereditary optic neuropathy | blindness

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Section: Methodsmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

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Consequences of zygote injection and germline transfer of mutant human mitochondrial DNA in mice

Koilkonda

Chou

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…2,13,16,18,31 Specifically, preclinical studies have provided evidence that a number of gene therapies may provide beneficial effects for LHON. 2,3,32,33 Of note, results obtained from this study provide the first demonstration that therapeutic effects may be obtained using a stem cell-based approach for LHON. A number of RPC-secreted factors may be aiding recovery from rotenone-induced trauma and may be of assistance in treating a wide range of optic nerve pathologies.…”

Section: Molecular Markersmentioning

confidence: 71%

Cell therapy using retinal progenitor cells shows therapeutic effect in a chemically-induced rotenone mouse model of Leber hereditary optic neuropathy

et al. 2014

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Primary mitochondrial disorders occur at a prevalence of one in 10 000; B50% of these demonstrate ocular pathology. Leber hereditary optic neuropathy (LHON) is the most common primary mitochondrial disorder. LHON results from retinal ganglion cell pathology, which leads to optic nerve degeneration and blindness. Over 95% of cases result from one of the three common mutations in mitochondrial genes MTND1, MTND4 and MTND6, which encode elements of the complex I respiratory chain. Various therapies for LHON are in development, for example, intravitreal injection of adeno-associated virus carrying either the yeast NDI1 gene or a specific subunit of mammalian Complex I have shown visual improvement in animal models. Given the course of LHON, it is likely that in many cases prompt administration may be necessary before widespread cell death. An alternative approach for therapy may be the use of stem cells to protect visual function; this has been evaluated by us in a rotenone-induced model of LHON. Freshly dissected embryonic retinal cells do not integrate into the ganglion cell layer (GCL), unlike similarly obtained photoreceptor precursors. However, cultured retinal progenitor cells can integrate in close proximity to the GCL, and act to preserve retinal function as assessed by manganese-enhanced magnetic resonance imaging, optokinetic responses and ganglion cell counts. Cell therapies for LHON therefore represent a promising therapeutic approach, and may be of particular utility in treating more advanced disease. INTRODUCTIONPrimary mitochondrial disorders (those caused by mutations in the mitochondrial genome) occur at a prevalence of B1 in 10 000, with 1 in 200 individuals carrying at-risk mutations. 1 Approximately 50% demonstrate some form of ocular pathology. 2 Mitochondria provide energy in the form of ATP generated by oxidative phosphorylation; therefore, mitochondrial mutations primarily affect tissues with the greatest energy requirements, such as the retina, the inner ear, central and peripheral nervous systems and cardiac and skeletal muscle. 2,3 Leber hereditary optic neuropathy (LHON) is the most common primary mitochondrial disorder, with a prevalence of B1 in 31 000-50 000 in northern Europe. The prevalence of at-risk carriers is much higher, estimated at 1 in 8 500 in the UK; furthermore, primary LHON mutations were found in 1 out of 350 neonatal umbilical cord samples. 1,4 LHON is maternally inherited, often results from homoplasmy of the causative mitochondrial mutation, and is incompletely penetrant; visual loss occurs in 50% of males and 10% of females. Symptoms result from retinal ganglion cell (RGC) pathology, which leads to RGC death via apoptosis, resulting in optic nerve degeneration and subsequent blindness. Nonocular symptoms of mitochondrial dysfunction may also be present. 1,4 Although many mutations implicated in LHON have been described, 90-95% of the cases result from one of the three common mutations in mitochondrial genes including MTND1, MTND4 and MTND6, which encode elements of ...

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“…As with any gene therapy, the efficacy and longevity of the treatment will in part depend on the vector used for gene delivery. For example, DNA electroporation or AAV-mediated delivery into retinal ganglion cells of a wild type ND4 complex I subunit incorporating a MTS prevented RGC loss and optic nerve degeneration in mice expressing the ND4 R340H mutant [53,76], providing support for progression of ND4-based therapeutics for LHON towards clinical trial. In a recent study, inclusion of an MTS from the gene encoding mitochondrial cytochrome oxidase subunit 8 into the viral protein 2 (VP2) region of the AAV2 capsid directed delivery of the AAV cargo, the ND4 gene, to the mitochondrial compartment; however, the mode of action of this targeted delivery remains elusive [76].…”

Section: Trends In Geneticsmentioning

confidence: 99%

Mitochondrial disorders: aetiologies, models systems, and candidate therapies

et al. 2013

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Gene delivery to mitochondria by targeting modified adenoassociated virus suppresses Leber’s hereditary optic neuropathy in a mouse model

Cited by 169 publications

References 46 publications

Consequences of zygote injection and germline transfer of mutant human mitochondrial DNA in mice

Consequences of zygote injection and germline transfer of mutant human mitochondrial DNA in mice

Cell therapy using retinal progenitor cells shows therapeutic effect in a chemically-induced rotenone mouse model of Leber hereditary optic neuropathy

Mitochondrial disorders: aetiologies, models systems, and candidate therapies

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