title: Gene Therapy for Pyoderma Gangrenosum: Optimal Transfection Conditions and Effect of Drugs on Gene Delivery in the HaCaT Cell Line Using Cationic Liposomes.
abstract: OBJECTIVE
  Pyoderma gangrenosum (PG) is a rare ulcerative skin disease, currently treated empirically with immunosuppression. PG is a good target for gene therapy since the skin is easily accessible. This study used the FDA-approved vector Lipofectamine® 2000 to investigate in vitro transfection of skin keratinocytes. The aim was to determine an optimum transfection protocol, including the effect of drugs currently used to treat PG on the efficiency of gene transfer, since gene therapy is unlikely to be used as monotherapy.
  
  
  METHODS
  Cells of the HaCaT line were transfected with the lacZ reporter gene, and transgene expression was measured after a given time period. Conditions tested were: relative concentrations of DNA and Lipofectamine®, time from transfection to measurement of expression, pH, and exposure to clinically relevant drugs (hydrocortisone, methotrexate, infliximab).
  
  
  RESULTS
  The greatest levels of β-galactosidase expression were observed using a DNA:Lipofectamine® ratio of 1:5 (μg/μl) on day 3 after transfection, using culture medium at pH 7, and in the presence of hydrocortisone. Transfection efficiency was reduced by the presence of methotrexate and not significantly affected by infliximab.
  
  
  CONCLUSIONS
  Gene therapy is a potential future strategy for the management of PG; this study is a step towards the development of a topical gene-based agent.
authors.affiliation: Department of Surgery, School of Medicine, Cardiff University, Cardiff, UK.
authors.forename: Alexandra R
authors.initials: AR
authors.lastname: Teagle
authors.forename: James C
authors.initials: JC
authors.lastname: Birchall
authors.forename: Rachel
authors.initials: R
authors.lastname: Hargest
mesh_terms: D002412:Cations / Q000378:metabolism; D002460:Cell Line; D018014:Gene Transfer Techniques / Q000639:trends; D015316:Genetic Therapy / Q000379:methods* / Q000639:trends; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects; D008055:Lipids / Q000008:administration & dosage* / Q000235:genetics*; D008081:Liposomes; D017511:Pyoderma Gangrenosum / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism; D014162:Transfection / Q000379:methods*
chemical_list: D002412:Cations; D008055:Lipids; C086724:Lipofectamine; D008081:Liposomes