title: LHON gene therapy vector prevents visual loss and optic neuropathy induced by G11778A mutant mitochondrial DNA: biodistribution and toxicology profile.
abstract: OBJECTIVE
  To demonstrate safety and efficacy of allotopic human ND4 for treatment of a Leber's hereditary optic neuropathy (LHON) mouse model harboring the G11778A mitochondrial mutation.
  
  
  METHODS
  We induced LHON in mice by intravitreal injection of mutant (G11778A) human ND4 DNA, responsible for most cases of LHON, that was directed to mitochondria using an AAV2 vector to which we appended a mitochondrial targeting sequence to the VP2 capsid. We then attempted rescue of visual loss using our test article (ScAAV2-P1ND4v2) containing a synthetic nuclear encoded G11778G ND4 gene that was allotopically expressed. Control mice either were uninjected or received AAV2-GFP or AAV2-mCherry. We performed RT-PCR and confocal microscopy at 2 weeks post injection. Pattern electroretinograms (PERGs), spectral-domain optical coherence tomography (SD-OCT), histology, and transmission electron microscopy (TEM) were performed. For toxicology and biodistribution studies, the test article was administered intravitreally to rats and rhesus macaques at different doses.
  
  
  RESULTS
  Mutant and wild-type ND4 were efficiently expressed in the mitochondria of retinal ganglion cells (RGCs). Visual function assessed by serial PERGs and retinal structure by serial SD-OCT showed a significant rescue by the test article. Histology and ultrastructural analysis confirmed that loss of RGCs and demise of axons was prevented by ScAAV2-P1ND4v2. Rat and nonhuman primate biodistribution studies showed that vector spread outside the injected eye into spleen and lymph nodes was minimal. Histopathology of tissues and organs including the eyes was comparable to that of uninfected and saline-injected eyes.
  
  
  CONCLUSIONS
  Allotopically expressed wild-type ND4 prevents the phenotype induced by G11778A mitochondrial DNA with a toxicology profile acceptable for testing in a phase I clinical trial.
authors.affiliation: Bascom Palmer Eye Institute, University of Miami, Miller School of Medicine, Miami, Florida, United States.
authors.forename: Rajeshwari
authors.initials: R
authors.lastname: Koilkonda
authors.affiliation: Bascom Palmer Eye Institute, University of Miami, Miller School of Medicine, Miami, Florida, United States.
authors.forename: Hong
authors.initials: H
authors.lastname: Yu
authors.affiliation: Bascom Palmer Eye Institute, University of Miami, Miller School of Medicine, Miami, Florida, United States.
authors.forename: Venu
authors.initials: V
authors.lastname: Talla
authors.affiliation: Bascom Palmer Eye Institute, University of Miami, Miller School of Medicine, Miami, Florida, United States.
authors.forename: Vittorio
authors.initials: V
authors.lastname: Porciatti
authors.affiliation: Bascom Palmer Eye Institute, University of Miami, Miller School of Medicine, Miami, Florida, United States.
authors.forename: William J
authors.initials: WJ
authors.lastname: Feuer
authors.affiliation: Department of Ophthalmology, University of Florida, College of Medicine, Gainesville, Florida, United States.
authors.forename: William W
authors.initials: WW
authors.lastname: Hauswirth
authors.affiliation: Department of Ophthalmology, University of Florida, College of Medicine, Gainesville, Florida, United States.
authors.forename: Vince
authors.initials: V
authors.lastname: Chiodo
authors.affiliation: Department of Pediatrics, University of Florida, College of Medicine, Gainesville, Florida, United States.
authors.forename: Kirsten E
authors.initials: KE
authors.lastname: Erger
authors.affiliation: Department of Ophthalmology, University of Florida, College of Medicine, Gainesville, Florida, United States.
authors.forename: Sanford L
authors.initials: SL
authors.lastname: Boye
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Florida, College of Medicine, Gainesville, Florida, United States.
authors.forename: Alfred S
authors.initials: AS
authors.lastname: Lewin
authors.affiliation: Department of Pediatrics, University of Florida, College of Medicine, Gainesville, Florida, United States.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Conlon
authors.affiliation: Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, Oregon, United States.
authors.forename: Lauren
authors.initials: L
authors.lastname: Renner
authors.affiliation: Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, Oregon, United States.
authors.forename: Martha
authors.initials: M
authors.lastname: Neuringer
authors.affiliation: Charles River Pathology Associates-Illinois, Chicago, Illinois, United States.
authors.forename: Carol
authors.initials: C
authors.lastname: Detrisac
authors.affiliation: Bascom Palmer Eye Institute, University of Miami, Miller School of Medicine, Miami, Florida, United States.
authors.forename: John
authors.initials: J
authors.lastname: Guy
mesh_terms: D000818:Animals; D001369:Axons / Q000648:ultrastructure; D001766:Blindness / Q000235:genetics / Q000628:therapy*; D004272:DNA, Mitochondrial / Q000235:genetics*; D004195:Disease Models, Animal; D004596:Electroretinography; D015316:Genetic Therapy / Q000009:adverse effects / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D008253:Macaca mulatta; D051379:Mice; D046529:Microscopy, Electron, Transmission; D008928:Mitochondria / Q000235:genetics* / Q000378:metabolism; D009245:NADH Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D029242:Optic Atrophy, Hereditary, Leber / Q000235:genetics / Q000503:physiopathology / Q000628:therapy*; D051381:Rats; D012165:Retinal Ganglion Cells / Q000378:metabolism / Q000648:ultrastructure; D041623:Tomography, Optical Coherence
chemical_list: D004272:DNA, Mitochondrial; C098483:NADH dehydrogenase subunit 4; D009245:NADH Dehydrogenase
keywords: LHON; gene therapy; mitochondria