TARGETED GENE DELIVERY TO ALVEOLAR MACROPHAGES VIA FC RECEPTOR-MEDIATED ENDOCYTOSIS

Alveolar macrophage (AM) plays important roles in lung homeostasis and pathogenesis of diseases. The study of macrophage gene function and r egulation as well as its potential therapeutic intervention will requi re the development of vectors capable of safe and efficient transfer o f DNA to the AM. In the present study, we report a new transfection sy stem that utilizes Fc receptor-mediated endocytosis as a means to targ et DNA to the AM. This system employs molecular conjugates consisting of a cognate moiety, in this case IgG which recognizes the AM Fc recep tor, covalently-linked to a DNA-binding moiety, such as a cationic pol yamine. A Complex was formed between immunoglobulin G-polylysine conju gate (IgG-pL) and plasmid DNA carrying the LacZ reporter gene (pSV bet a). The conjugate-DNA complex was added directly to the AMs in culture and incubated for 24 h, after which LacZ gene expression was analyzed for beta-galactosidase activity by microfluorometry using a fluorogen ic beta-galactosidase substrate, 5-dodecanoylaminofluorescein di-beta- D-galactopyranoside (C(12)FDG). The AMs treated with the IgG pL/DNA co mplex exhibited galactosidase activity significantly augmented over ba ckground levels. Effective gene transfer was shown to require both the DNA-binding moiety and cognate moiety for the cell surface receptor. Specific internalization of the complex by the Fc receptor pathway was verified by competitive inhibition using excess IgG. Under this condi tion, LacZ gene expression was inhibited, suggesting complex internali zation through the Fc mediated endocytosis pathway. The requirement of Pc receptors for complex internalization was further demonstrated usi ng cells that lack Fc receptors, e.g., alveolar epithelial cells. When exposed to the IgG-pL/pSV beta complex, these epithelial cells showed no susceptibility to gene transfer. Thus, the immune conjugate system may be used to accomplish targeted gene delivery to the AMs via the e ndocytosis pathway. Finally, the conjugate system was found to be nont oxic at concentrations effectively enhancing gene transfer, thereby, s uggesting its potential safety in vivo.