title: Proof-of-concept: neonatal intravenous injection of adeno-associated virus vectors results in successful transduction of myenteric and submucosal neurons in the mouse small and large intestine.
abstract: BACKGROUND
  Despite the success of viral vector technology in the transduction of the central nervous system in both preclinical research and gene therapy, its potential in neurogastroenterological research remains largely unexploited. This study asked whether and to what extent myenteric and submucosal neurons in the ileum and distal colon of the mouse were transduced after neonatal systemic delivery of recombinant adeno-associated viral vectors (AAVs).
  
  
  METHODS
  Mice were intravenously injected at postnatal day one with AAV pseudotypes AAV8 or AAV9 carrying a cassette encoding enhanced green fluorescent protein (eGFP) as a reporter under the control of a cytomegalovirus promoter. At postnatal day 35, transduction of the myenteric and submucosal plexuses of the ileum and distal colon was evaluated in whole-mount preparations, using immunohistochemistry to neurochemically identify transduced enteric neurons.
  
  
  RESULTS
  The pseudotypes AAV8 and AAV9 showed equal potential in transducing the enteric nervous system (ENS), with 25-30% of the neurons expressing eGFP. However, the percentage of eGFP-expressing colonic submucosal neurons was significantly lower. Neurochemical analysis showed that all enteric neuron subtypes, but not glia, expressed the reporter protein. Intrinsic sensory neurons were most efficiently transduced as nearly 80% of calcitonin gene-related peptide-positive neurons expressed the transgene.
  
  
  CONCLUSIONS
  The pseudotypes AAV8 and AAV9 can be employed for gene delivery to both the myenteric and the submucosal plexus, although the transduction efficiency in the latter is region-dependent. These findings open perspectives for novel preclinical applications aimed at manipulating and imaging the ENS in the short term, and in gene therapy in the longer term.
authors.affiliation: Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Antwerp, Belgium.
authors.forename: R
authors.identifier: http://orcid.org/0000-0001-9976-2889
authors.initials: R
authors.lastname: Buckinx
authors.affiliation: Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Antwerp, Belgium.
authors.forename: S
authors.initials: S
authors.lastname: Van Remoortel
authors.affiliation: Laboratory for Viral Vector Technology & Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium.|Leuven Viral Vector Core, KU Leuven, Leuven, Belgium.
authors.forename: R
authors.initials: R
authors.lastname: Gijsbers
authors.affiliation: Gene Transfer Technology Group, Institute for Women's Health, University College London, London, UK.|Wits/SAMRC Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa.
authors.forename: S N
authors.initials: SN
authors.lastname: Waddington
authors.affiliation: Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Antwerp, Belgium.
authors.forename: J-P
authors.initials: JP
authors.lastname: Timmermans
mesh_terms: D000818:Animals; D003106:Colon; D000229:Dependovirus; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry; D007275:Injections, Intravenous; D007421:Intestine, Small; D051379:Mice; D023421:Models, Animal; D009197:Myenteric Plexus / Q000821:virology*; D009474:Neurons / Q000821:virology*; D013368:Submucous Plexus / Q000821:virology*; D014161:Transduction, Genetic
chemical_list: D049452:Green Fluorescent Proteins
keywords: AAV; enteric nervous system; myenteric plexus; submucosal plexus; viral transfection