title: Differential rAAV2 transduction efficiencies and insulin secretion profiles in pure and co-culture models of human enteroendocrine L-cells and enterocytes.
abstract: BACKGROUND
  Cell-based therapies for treating insulin-dependent diabetes (IDD) can provide a more physiologic regulation of blood glucose levels in a less invasive fashion than insulin injections. Previously, we developed an engineered human enteroendocrine L-cell model for regulated insulin release via recombinant adeno-associated virus serotype 2, or rAAV2, transduction. The aim of this study was to evaluate the efficiency and selectivity of rAAV2-mediated insulin gene delivery to enteroendocrine L-cells in co-culture with a prevailing number of enterocytes, which are the predominant cell type in intestinal epithelium.
  
  
  METHODS
  We tested rAAV2 transduction in pure and co-culture models of human cell lines of enterocytes (Caco-2 and T84 cell lines) and enteroendocrine L-cells (NCI-H716 cell line). Non-viral, chemical-mediated transfection was used as a control. Transduced and transfected co-cultures were subjected to insulin secretion studies.
  
  
  RESULTS
  In pure cultures, rAAV2 exhibited a low transduction efficiency towards both Caco-2 and T84 enterocytes, as opposed to a strong reporter expression in permissive NCI-H716 L-cells. In co-cultures of NCI-H716 L-cells and Caco-2 or T84 enterocytes, rAAV2 exhibited differential transduction efficiency with a strong preference towards NCI-H716 L-cells. The rAAV2-transduced co-culture achieved regulated insulin release against stimulation, whereas the chemically transfected co-culture failed to respond.
  
  
  CONCLUSIONS
  This study demonstrated that rAAV2-mediated insulin gene transfer can differentiate human intestinal cell types in vitro, in particular enterocyte and enteroendocrine L-cell lines. We consider the AAV2 vector a useful tool in developing enteroendocrine L-cell-specific insulin gene delivery for IDD treatment, in terms of AAV2 avoiding enterocytes and targeting selectively L-cells.
authors.affiliation: School of Chemical and Biomolecular Engineering, Georgia Tech-Emory Center for the Engineering of Living Tissues, Georgia Institute of Technology, Atlanta, GA 30332, USA.
authors.forename: Shiue-Cheng
authors.initials: SC
authors.lastname: Tang
authors.forename: Athanassios
authors.initials: A
authors.lastname: Sambanis
mesh_terms: D002460:Cell Line; D018920:Coculture Techniques; D000229:Dependovirus / Q000235:genetics* / Q000502:physiology; D003922:Diabetes Mellitus, Type 1 / Q000628:therapy*; D020895:Enterocytes / Q000378:metabolism / Q000821:virology; D019858:Enteroendocrine Cells / Q000378:metabolism* / Q000821:virology; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D015316:Genetic Therapy; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007328:Insulin / Q000096:biosynthesis / Q000235:genetics*; D008055:Lipids; D014161:Transduction, Genetic
chemical_list: C411955:FuGene; D007328:Insulin; D008055:Lipids; D049452:Green Fluorescent Proteins