title: Establishment of effective methods for transducing genes into iris pigment epithelial cells by using adeno-associated virus type 2.
abstract: OBJECTIVE
  To establish an efficient method of transferring the human brain-derived neurotrophic-factor (hBDNF) gene into human iris pigment epithelial (hIPE) cells by using recombinant adeno-associated virus type 2 (rAAV2).
  
  
  METHODS
  Cultured hIPE cells were treated with either hydroxyurea-sodium butyrate (HUSB; DNA synthesis inhibitor), or tyrphostin-1 (Tyr; epidermal growth factor receptor [EGFR] tyrosine kinase inhibitor), or a combination of HUSB and Tyr (HUSB-Tyr). After each treatment, cells were exposed to rAAV2 (rAAV-LacZ or rAAV-hBDNF). The levels of BDNF were measured by ELISA and also determined by Western blot analysis. Southern blot analysis was performed on each type of treated cell. The neuroprotective effect of BDNF on the retinal ganglion cells (RGCs) was quantitatively assessed by culturing rAAV-hBDNF-hIPE with RGCs.
  
  
  RESULTS
  The infection of hIPE cells was significantly lower than ARPE and HT1080 cells, which are highly permissive cells for rAAV2. The treatment of HUSB-Tyr enhanced the transgene expression more than that after treatment with one of these agents in rAAV-hIPE cells. Southern hybridization revealed that the amount of replicative form monomer (RFm) was less in Tyr than in HUSB or HUSB-Tyr treatment and there was no difference in conversion of virus genome to double stranded form after HUSB and HUSB-Tyr treatment. However, adding Tyr treatment stimulated the JNK1/2 and p38 pathways and modified the target transgene expression. BDNF had a significantly greater rescue effect of RGCs with the HUSB-Tyr-treated rAAV-hBDNF-hIPE cells (P < 0.01) than that with the HUSB-treated rAAV-hBDNF-hIPE cells (P > 0.05) compared with noninfected hIPE cells.
  
  
  CONCLUSIONS
  The combined treatment of HUSB-Try is an effective method of increasing transgene expression with the AAV-mediated gene transfer. The role of HUSB and Tyr in the increase of gene expression may be different and related to the conversion of virus into the host genome and the enhancement of the transcription, respectively.
authors.affiliation: Division of Biofunctional Science, Tohoku University Biomedical Engineering Research Organization, Tohoku University School of Medicine, Seiryomachi, Aobaku, Sendai 980-8574, Japan.
authors.forename: Eriko
authors.initials: E
authors.lastname: Sugano
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Tomita
authors.forename: Sei-ichi
authors.initials: S
authors.lastname: Ishiguro
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Abe
authors.forename: Makoto
authors.initials: M
authors.lastname: Tamai
mesh_terms: D015139:Blotting, Southern; D015153:Blotting, Western; D019208:Brain-Derived Neurotrophic Factor / Q000235:genetics* / Q000378:metabolism; D002087:Butyrates / Q000494:pharmacology; D002478:Cells, Cultured; D004261:DNA Replication / Q000187:drug effects; D000229:Dependovirus / Q000235:genetics*; D004338:Drug Combinations; D004797:Enzyme-Linked Immunosorbent Assay; D066246:ErbB Receptors / Q000037:antagonists & inhibitors; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression / Q000502:physiology*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D007498:Iris / Q000166:cytology*; D048055:Mitogen-Activated Protein Kinase 8 / Q000378:metabolism; D048056:Mitogen-Activated Protein Kinase 9 / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020032:Tyrphostins / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D019208:Brain-Derived Neurotrophic Factor; D002087:Butyrates; D004338:Drug Combinations; D012333:RNA, Messenger; D020032:Tyrphostins; D048056:Mitogen-Activated Protein Kinase 9; D066246:ErbB Receptors; D048055:Mitogen-Activated Protein Kinase 8; D048051:p38 Mitogen-Activated Protein Kinases; D006918:Hydroxyurea