title: Ex vivo gene therapy prevents chronic graft vascular disease in cardiac allografts.
abstract: OBJECTIVE
  We hypothesized that ex vivo hyperbaric transfection of antisense oligodeoxynucleotides for blockade of intercellular adhesion molecule-1, an important mediator of cell adhesion and T-cell co-stimulation, would reduce chronic graft vascular disease in cardiac allografts.
  
  
  METHODS
  PVG hearts underwent ex vivo transfection with antisense, reverse antisense intercellular adhesion molecule-1 oligodeoxynucleotide (80 micromol/L), or saline solution at 3 atm pressure for 45 minutes at 4 degrees C and were transplanted heterotopically into ACI recipients with or without treatment with intercellular adhesion molecule-1 (1A29) or leukocyte function associated antigen-1 (WT.1) monoclonal antibodies. Transfection efficiency was confirmed with fluorescein isothiocyanate-labeled oligodeoxynucleotides and fluorescent microscopy. Efficacy of intracellular adhesion molecule-1 blockade was assessed with the use of immunohistochemistry. Graft reperfusion injury was evaluated at 6 to 24 hours by neutrophil infiltration (myeloperoxidase [MPO]), cardiac edema (%wt/wt), and histologic injury (percent contraction band necrosis). Grafts from recipients treated with cyclosporine A (5 mg/kg per day, days 0 to 9) were scored for chronic graft vascular disease on postoperative day 90 ranging from 0 (no involvement) to 4 (>50% vascular occlusion).
  
  
  RESULTS
  Transfection was highly efficient (fluorescein isothiocyanate-labeled oligodeoxynucleotides in 48%+/-5% of total myocardial nuclei) and effective at blocking intracellular adhesion molecule-1 expression (positive area in allografts taken on postoperative day 3 was reduced from 100%+/-0% to 52%+/-14%, n=4). Blockade with antisense oligodeoxynucleotides versus monoclonal antibodies was less effective at preventing reperfusion injury while more effective at reducing chronic graft vascular disease (score 0.98+/-0.48, p < 0.05). Reverse antisense oligodeoxynucleotides and vector control (antisense oligodeoxynucleotide infusion without pressure) groups failed to demonstrate this beneficial effect.
  
  
  CONCLUSIONS
  Hyperbaric transfection of antisense oligodeoxynucleotides proved highly efficient, effective at blockade of intracellular adhesion molecule-1, and demonstrated a sequence-specific reduction in chronic graft vascular disease. This highly targeted alteration of donor organ immunogenicity may have an important future role in clinical immunosuppressive strategies.
authors.affiliation: Department of Cardiothoracic Surgery, Stanford University School of Medicine, Calif 94305, USA.
authors.forename: R S
authors.initials: RS
authors.lastname: Poston
authors.forename: M
authors.initials: M
authors.lastname: Ennen
authors.forename: J
authors.initials: J
authors.lastname: Pollard
authors.forename: E G
authors.initials: EG
authors.lastname: Hoyt
authors.forename: M E
authors.initials: ME
authors.lastname: Billingham
authors.forename: R C
authors.initials: RC
authors.lastname: Robbins
mesh_terms: D000818:Animals; D003327:Coronary Disease / Q000209:etiology / Q000276:immunology / Q000517:prevention & control*; D015316:Genetic Therapy / Q000379:methods*; D006084:Graft Rejection / Q000276:immunology / Q000517:prevention & control; D016027:Heart Transplantation / Q000009:adverse effects; D007165:Immunosuppression Therapy / Q000379:methods; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics* / Q000502:physiology; D008297:Male; D015428:Myocardial Reperfusion Injury / Q000209:etiology / Q000276:immunology / Q000517:prevention & control*; D016376:Oligonucleotides, Antisense / Q000627:therapeutic use; D011183:Postoperative Complications / Q000209:etiology / Q000276:immunology / Q000517:prevention & control*; D011312:Pressure; D051381:Rats; D011912:Rats, Inbred ACI; D011919:Rats, Inbred Strains; D014162:Transfection; D016042:Transplantation, Heterotopic
chemical_list: D016376:Oligonucleotides, Antisense; D018799:Intercellular Adhesion Molecule-1