title: Vector serotype screening for use in ovine perinatal lung gene therapy.
abstract: OBJECTIVE
  Successful in utero or perinatal gene therapy for congenital lung diseases, such as cystic fibrosis and surfactant protein deficiency, requires identifying clinically relevant viral vectors that efficiently transduce airway epithelial cells. The purpose of the current preclinical large animal study was to evaluate lung epithelium transduction of adeno-associated viral (AAV) vector serotypes following intratracheal delivery.
  
  
  METHODS
  Six different AAV vector serotypes (AAV1, AAV5, AAV6, AAV8, AAV9, and AAVrh10) expressing the green fluorescent protein (GFP) as the transgene were injected into the right upper lobe of perinatal sheep via bronchoscopy. At 1 week, samples were harvested, analyzed by fluorescent stereomicroscopy and immunohistochemistry, and quantified using a radial grid and quantitative real-time polymerase chain reaction (qPCR).
  
  
  RESULTS
  Fluorescent stereomicroscopy demonstrated GFP expression in the right upper lobe following injection of all AAV serotypes assessed except AAV5. Immunohistochemistry analysis confirmed GFP expression in small- and medium-sized airways following intratracheal injection of AAV1, 6, 8, 9, and rh10. However, only AAV8 and AAVrh10 resulted in transgene expression in large airways. These results were confirmed by qPCR, yet, after 40 cycles, AAV1 did not show GFP gene amplification.
  
  
  CONCLUSIONS
  Adeno-associated viral vector serotypes 6, 8, 9, and rh10 demonstrated efficient GFP transgene expression at early time points, and AAV8 demonstrated efficient transduction of all airway sizes with high pulmonary GFP expression tested using qPCR.
authors.affiliation: The Center for Fetal Research, The Children's Hospital of Philadelphia, Abramson Research Building, Philadelphia, PA.
authors.forename: Lauren E
authors.initials: LE
authors.lastname: McClain
authors.affiliation: The Center for Fetal Research, The Children's Hospital of Philadelphia, Abramson Research Building, Philadelphia, PA.
authors.forename: Marcus G
authors.initials: MG
authors.lastname: Davey
authors.affiliation: The Center for Fetal Research, The Children's Hospital of Philadelphia, Abramson Research Building, Philadelphia, PA.
authors.forename: Phillip W
authors.initials: PW
authors.lastname: Zoltick
authors.affiliation: Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA.
authors.forename: Maria P
authors.initials: MP
authors.lastname: Limberis
authors.affiliation: The Center for Fetal Research, The Children's Hospital of Philadelphia, Abramson Research Building, Philadelphia, PA.
authors.forename: Alan W
authors.initials: AW
authors.lastname: Flake
authors.affiliation: The Center for Fetal Research, The Children's Hospital of Philadelphia, Abramson Research Building, Philadelphia, PA. Electronic address: peranteauw@email.chop.edu.
authors.forename: William H
authors.initials: WH
authors.lastname: Peranteau
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D000229:Dependovirus / Q000145:classification* / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000145:classification* / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008168:Lung / Q000378:metabolism / Q000821:virology; D008171:Lung Diseases / Q000151:congenital / Q000235:genetics / Q000628:therapy*; D060888:Real-Time Polymerase Chain Reaction; D065288:Serogroup; D012703:Serotyping; D012756:Sheep; D034561:Sheep, Domestic; D014161:Transduction, Genetic; D019076:Transgenes
chemical_list: D015415:Biomarkers; D049452:Green Fluorescent Proteins
keywords: AAV; Gene therapy; Lung; Screening; Sheep