title: Delivery and evaluation of recombinant adeno-associated viral vectors in the equine distal extremity for the treatment of laminitis.
abstract: BACKGROUND
  Our long-term aim is to develop a gene therapy approach for the prevention of laminitis in the contralateral foot of horses with major musculoskeletal injuries and non-weightbearing lameness.
  
  
  OBJECTIVE
  The goal of this study was to develop a practical method to efficiently deliver therapeutic proteins deep within the equine foot.
  
  
  METHODS
  Randomised in vivo experiment.
  
  
  METHODS
  We used recombinant adeno-associated viral vectors (rAAVs) to deliver marker genes using regional limb perfusion through the palmar digital artery of the horse.
  
  
  RESULTS
  Vector serotypes rAAV2/1, 2/8 and 2/9 all successfully transduced equine foot tissues and displayed similar levels and patterns of transduction. The regional distribution of transduction within the foot decreased with decreasing vector dose. The highest transduction values were seen in the sole and coronary regions and the lowest transduction values were detected in the dorsal hoof-wall region. The use of a surfactant-enriched vector diluent increased regional distribution of the vector and improved the transduction in the hoof-wall region. The hoof-wall region of the foot, which exhibited the lowest levels of transduction using saline as the vector diluent, displayed a dramatic increase in transduction when surfactant was included in the vector diluent (9- to 81-fold increase). In transduced tissues, no significant difference was observed between promoters (chicken β-actin vs. cytomegalovirus) for gene expression. All horses tested for vector-neutralising antibodies were positive for serotype-specific neutralising antibodies to rAAV2/5.
  
  
  CONCLUSIONS
  The current experiments demonstrate that transgenes can be successfully delivered to the equine distal extremity using rAAV vectors and that serotypes 2/8, 2/9 and 2/1 can successfully transduce tissues of the equine foot. When the vector was diluted with surfactant-containing saline, the level of transduction increased dramatically. The increased level of transduction due to the addition of surfactant also improved the distribution pattern of transduction.
authors.affiliation: Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, USA.
authors.forename: J B
authors.initials: JB
authors.lastname: Mason
authors.affiliation: Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, USA.
authors.forename: B L
authors.initials: BL
authors.lastname: Gurda
authors.affiliation: Utah Veterinary Diagnostic Laboratory, School of Veterinary Medicine, Utah State University, Logan, USA.
authors.forename: A
authors.initials: A
authors.lastname: Van Wettere
authors.affiliation: Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, USA.
authors.forename: J B
authors.initials: JB
authors.lastname: Engiles
authors.affiliation: Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, USA.
authors.forename: J M
authors.initials: JM
authors.lastname: Wilson
authors.affiliation: Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, USA.
authors.forename: D W
authors.initials: DW
authors.lastname: Richardson
mesh_terms: D000256:Adenoviridae / Q000502:physiology*; D000818:Animals; D005121:Extremities; D005534:Foot Diseases / Q000628:therapy / Q000662:veterinary*; D015971:Gene Expression Regulation, Enzymologic; D015967:Gene Expression Regulation, Viral; D015316:Genetic Therapy / Q000379:methods / Q000662:veterinary*; D005822:Genetic Vectors / Q000378:metabolism; D006724:Hoof and Claw / Q000473:pathology*; D006734:Horse Diseases / Q000628:therapy*; D006736:Horses; D007249:Inflammation / Q000628:therapy / Q000662:veterinary*; D019076:Transgenes; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D001616:beta-Galactosidase
keywords: gene therapy; horse; neutralising antibodies; recombinant adeno-associated viral vectors; regional limb perfusion; viral transduction