Lentiviral transduction of face and limb flaps: Implications for immunomodulation of vascularized composite allografts

Los Angeles, Calif.; Boston, Mass.; and Palermo, Italy 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. Proofof-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 andlong-termconstitutivegeneexpression( 150days).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, constitutiveexpressionof immunomodulatoryproteins in faceandhandallografts.Futuregoals include replacement of the luciferase and eGFP reporter genes with key immunomodulatory proteins. (Plast. Reconstr. Surg. 129: 391, 2012.)