title: Lentiviral transduction of face and limb flaps:  implications for immunomodulation of vascularized composite allografts.
abstract: BACKGROUND
  Ex vivo introduction of an immunomodulatory transgene into a face or hand allograft may improve the risk-to-benefit ratio of vascularized composite allografts. Abrogation of the immunogenicity of the skin component of a face or hand allograft may decrease alloreactivity and permit the induction of immunologic tolerance. Proof-of-principle demonstrations of transduction of composite tissue have been established using adenoviral vectors, producing transient gene expression. The authors hypothesized that transduction, integration, and long-term expression of transgenes in a vascularized composite allograft could be achieved using lentiviral vectors.
  
  
  METHODS
  Ex vivo transduction of heterogeneous primary rat cell lines representative of a composite tissue flap's cellular architecture was performed using a luc-enhanced green fluorescent protein (eGFP) human immunodeficiency virus-1-based lentiviral vector. Ex vivo injections of rat superficial inferior epigastric artery flaps with the viral vector were performed intraarterially, intramuscularly, and intradermally.
  
  
  RESULTS
  Quantifiable reporter expression by flow cytometry (fluorescence-activated cell sorting) analysis and in vitro bioluminescence was observed. The luc-eGFP vector exhibited broad tropism and allowed transgene expression in relevant cell lines and throughout the flaps. Ex vivo intradermal transfection resulted in genomic integration and long-term constitutive gene expression (>150 days). Similarly, efficient intradermal transfection of face and hand flaps in a rat model corroborated this approach. Ex vivo intravascular perfusion of the vector proved inferior to intradermal injection.
  
  
  CONCLUSIONS
  Intradermal delivery of the transgenes proved superior to intravascular perfusion. Optimization of this gene-delivery approach may allow long-term, constitutive expression of immunomodulatory proteins in face and hand allografts. Future goals include replacement of the luciferase and eGFP reporter genes with key immunomodulatory proteins.
authors.affiliation: Los Angeles, Calif.; Boston, Mass.; and Palermo, Italy From the Laboratory for Stem Cell-Based Microsurgical Tissue Engineering, Division of Plastic and Reconstructive Surgery, the Tissue Engineering and Regeneration Research Laboratory, Department of Pathology, and the Molecular Imaging Center, Department of Radiology, Keck School of Medicine, University of Southern California; the Division of Plastic and Reconstructive Surgery, Massachusetts General Hospital, Harvard Medical School; and the Dipartimento di Discipline Chirurgiche ed Oncologiche, Sezione di Chirurgia Plastica e Ricostruttiva, Universita' degli Studi di Palermo.
authors.forename: Angelo A
authors.initials: AA
authors.lastname: Leto Barone
authors.forename: Zhao Y
authors.initials: ZY
authors.lastname: Zhou
authors.forename: Michael W
authors.initials: MW
authors.lastname: Hughes
authors.forename: Ryan
authors.initials: R
authors.lastname: Park
authors.forename: Ruth M
authors.initials: RM
authors.lastname: Schulman
authors.forename: Steven
authors.initials: S
authors.lastname: Lee
authors.forename: Evan N
authors.initials: EN
authors.lastname: Vidar
authors.forename: Travis L
authors.initials: TL
authors.lastname: Shiba
authors.forename: Erin L
authors.initials: EL
authors.lastname: Weber
authors.forename: Curtis L
authors.initials: CL
authors.lastname: Cetrulo
mesh_terms: D000818:Animals; D005121:Extremities / Q000601:surgery; D005145:Face / Q000601:surgery; D018014:Gene Transfer Techniques; D006801:Humans; D056747:Immunomodulation; D016086:Lentivirus / Q000235:genetics*; D051381:Rats; D013524:Surgical Flaps / Q000098:blood supply* / Q000276:immunology*; D014161:Transduction, Genetic