title: Oral gene delivery: Strategies to improve stability of pDNA towards intestinal digestion.
abstract: OBJECTIVE
  Gastrointestinal (GI) nucleases are responsible for a rapid presystemic degradation of orally administered transgenes. Within the current study, the activity of these degrading enzymes as well as the effect of various nuclease inhibitors on the degradation process were evaluated in order to assess their potential as auxiliary agents in oral gene delivery.
  
  
  METHODS
  Digestion assays of pDNA with DNaseI and in GI juices were performed in absence and presence of inhibitors. Consequently, a chitosan conjugate with covalently bound ethylendiaminetetraacetic acid disodium salt dihydrat (EDTA) was synthesized and its nuclease inhibitory properties were evaluated.
  
  
  RESULTS
  Small intestinal juice was shown to possess a nuclease activity per millilitre corresponding to 0.02 Kunitz units of DNaseI. Inhibition studies revealed that inhibitory activity followed the ranking: EDTA > sodium dodecyl sulfate (SDS) > aurintricarboxylic acid (ATA) > poly (acrylic acid) > cysteine. The chitosan-EDTA conjugate offered good nuclease inhibiting properties.
  
  
  CONCLUSIONS
  This study determined the nuclease activity of native porcine small intestinal juice as well as enterocytes homogenate. Moreover, several promising strategies to overcome this enzymatic barrier were identified.
authors.affiliation: Institute of Pharmacy, Leopold-Franzens-University Innsbruck, Department of Pharmaceutical Technology, Innrain 52, 6020, Innsbruck, Austria. brigitta.loretz@uibk.ac.at
authors.forename: Brigitta
authors.initials: B
authors.lastname: Loretz
authors.forename: Florian
authors.initials: F
authors.lastname: Föger
authors.forename: Martin
authors.initials: M
authors.lastname: Werle
authors.forename: Andreas
authors.initials: A
authors.lastname: Bernkop-Schnürch
mesh_terms: D000284:Administration, Oral; D018938:Caco-2 Cells; D048271:Chitosan / Q000008:administration & dosage* / Q000138:chemical synthesis / Q000737:chemistry / Q000493:pharmacokinetics; D003850:Deoxyribonuclease I / Q000037:antagonists & inhibitors / Q000378:metabolism; D004492:Edetic Acid / Q000008:administration & dosage* / Q000138:chemical synthesis / Q000737:chemistry / Q000493:pharmacokinetics; D005750:Gastric Juice / Q000378:metabolism; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000201:enzymology; D010957:Plasmids / Q000008:administration & dosage* / Q000737:chemistry / Q000493:pharmacokinetics*
chemical_list: D048271:Chitosan; D004492:Edetic Acid; D003850:Deoxyribonuclease I