Abstract
Alveolar macrophage (AM) plays important roles in lung homeostasis and pathogenesis of diseases. The study of macrophage gene function and regulation as well as its potential therapeutic intervention will require the development of vectors capable of safe and efficient transfer of DNA to the AM. In the present study, we report a new transfection system that utilizes Fc receptor-mediated endocytosis as a means to target DNA to the AM. This system employs molecular conjugates consisting of a cognate moiety, in this case IgG which recognizes the AM Fc receptor, covalently-linked to a DNA-binding moiety, such as a cationic polyamine. A Complex was formed between immunoglobulin G-polylysine conjugate (IgG-pL) and plasmid DNA carrying the LacZ reporter gene (pSVβ). 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 β-galactosidase activity by microfluorometry using a fluorogenic β-galactosidase substrate, 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C12FDG). The AMs treated with the IgG-pL/DNA complex exhibited galactosidase activity significantly augmented over background 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 condition, LacZ gene expression was inhibited, suggesting complex internalization through the Fc mediated endocytosis pathway. The requirement of Fc receptors for complex internalization was further demonstrated using cells that lack Fc receptors, e.g., alveolar epithelial cells. When exposed to the IgG-pL/pSVβ 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 endocytosis pathway. Finally, the conjugate system was found to be nontoxic at concentrations effectively enhancing gene transfer, thereby, suggesting its potential safety in vivo.
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REFERENCES
P. L. Felgner and G. Rhodes. Gene therapeutics. Nature 349:351–352 (1991).
W. F. Anderson. Prospects for human gene therapy. Science 226:401–409 (1984).
F. L. Graham and A. Van de Eb. A new technique for the assay of infectivity of human adenovirus IV DNA. Virology 52:456 (1973).
M. P. Carlos, J. S. Lebkowski, and M. R. Botchan. High mutation frequency in DNA transfected into mammalian cells. Proc. Natl. Acad. Sci. USA 80:3015 (1983).
W. A. Keown, C. R. Campbell, and R. S. Kucherlapti. Methods for introducing DNA into mammalian cells. In D. V. Goeddel (ed.), Methods in Enzymology, Vol. 185, Academic Press, New York, 1990. pp. 527–537.
P. L. Felgner, T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J. P. Northrop, G. M. Ringold, and M. Danielsen. Lipofectin: a highly efficient lipid-mediated DNA transfection procedure. Proc. Natl. Acad. Sci. USA 84:7413–7417 (1987).
A. Pettenazzo, A. Jobe, M. Ikegami, R. Abra, E. Hogue, and P. Mihalko. Clearance of phosphatidylcholine and cholesterol from liposomes, liposome loaded with metaproterenol, and rabbit surfactant from adult rabbit lungs. Am. Rev. Respir. Dis. 139:752–758 (1989).
D. Meisner, J. Pringle, and M. Mezei. Liposomal pulmonary drug delivery. J. Microencap. 6:379–387 (1989).
M. A. Rosenfeld, W. Siegfried, K. Yoshimura. Adenovirus-mediated transfer of a recombinant ∞1-antitrypsin gene to the lung epithelium in vivo. Science 252:431–434 (1991).
A. Berken and B. Benacerraf. Properties of antibodies cytophilic for macrophages. J. Exp. Med. 123:119–144 (1966).
Y. Z. Zhang, J. J. Naleway, K. D. Larison, Z. Huang, and R. P. Haugland. Detecting LacZ gene expression in living cells with new lipophilic, fluorogenic, β-galactosidase substrates. FASEB J 5:3108–3113 (1991).
J. K. Kang, D. J. Lewis, V. Castranova, Y. Rojanasakul, D. E. Banks, and J. K. H. Ma. Inhibition of macrophage production of IL-1 and IL-1-mediated thymocyte proliferation by tetrandrine. Exp. Lung Res. 18:715–729 (1992).
L. Y. Wang, D. Toledo-Vasquez, D. Schwegler-Berry, J. K. H. Ma, and Y. Rojanaskul. Transport and hydrolysis of enkephalins in cultured alveolar epithelial monolayers. Pharm. Res. 10:1662–1667 (1993).
J. Carlsson, H. Drevin, and R. Axen. Protein thiolation and reversible protein-protein conjugation. Biochem. J. 173:723–737 (1978).
W. C. Shen and H. J. P. Ryser. Conjugation of poly-L-lysine to albumin and horseradish peroxidase: a novel method of enhancing the cellular uptake of proteins. Proc. Natl. Acad. Sci. USA 75:1872–1876 (1978).
H. J. P. Ryser, I. Drummond, and W. C. Shen. The cellular uptake of horseradish peroxidase and its poly(lysine) conjugate by cultured fibroblasts are qualitatively similar despite a 900-fold difference in rate. J. Cell. Physiol. 113:167–178 (1982).
E. Wagner, M. Zenke, M. Cotten, H. Beug, and M. L. Birnstiel. Transferrin-polycation conjugated as carriers for DNA uptake into cells. Proc. Natl. Acad. Sci. USA 87:3410–3414 (1990).
M. Cotten, F. Langle-Rouault, H. Kirlappos. Transferrin-polycation-mediated introduction of DNA into human leukemic cells: stimulation by agents that affect the survival of transfected DNA or modulate transferrin receptor levels. Proc. Natl. Acad. Sci. USA 87:4033–4037 (1990).
D. T. Curiel, S. Agarwal, M. U. Romer, E. Wagner, M. Cotten, M. L. Birnstiel, and R. C. Boucher. Gene transfer to respiratory epithelial cells via receptor-mediated endocytosis pathway. Am. J. Respir. Cell Mol. Biol. 6:247–252 (1992).
R. P. Haugland. Handbook of Fluorescence Probes and Research Chemicals. Molecular Probes Inc., Eugene, OR, 1992, pp. 172–180.
E. Crouch. Pathobiology of pulmonary fibrosis. Am. J. Physiol. 259:L159–L184 (1990).
R. Stribling, E. N. Brunette, and R. J. Debs. The mouse as a model for cationic liposome-based, aerosolized gene delivery. J. Biopharm. Sci. 3(1/2):255–263 (1992).
T. A. Hazinski, P. A. Ladd, and C. A. DeMatteo. Localization and induced expression of fusion genes in the rat lung. Am. J. Respir. Cell Mol. Biol. 4:206–209 (1991).
K. L. Brigham, B. Meyrick, B. Christman, M. Magnuson, G. King, and L. C. Berry, Jr. Rapid communication: in vivo transfection of murine lungs with a functioning prokayotic gene using a liposome vehicle. Am. J. Med. Sci. 298:278–281 (1989).
R. Debs, M. Plan, K. Gaensler, J. Clements, D. S. Friend, and L. Dobbs. Prolonged transgene expression in rodent lung cells. Am. J. Respir. Cell. Mol. Biol. 7:406–413 (1992).
L. G. Johnson, J. C. Olsen, M. Wong-Sun, J. Yankaskas, and R. C. Boucher. Optimization of retroviral-mediated gene transfer in transformed and primary human airway epithelial cells. Am. Rev. Respir. Dis. 143(suppl.):A139 (1991).
G. Y. Wu and C. H. Wu. Receptor-mediated gene delivery and expression in vivo. J. Biol. Chem. 263:14621–14624 (1988).
G. Y. Wu, J. M. Wilson, F. Shalaby, M. Grossman, D. A. Shafritz, and C. H. Wu. Receptor-mediated gene delivery in vivo. Partial correction of genetic analbuminemia in nagase rats. J. Biol. Chem. 266:14338–14342 (1991).
J. Heyder, J. Gebhart, and E. Stahlhofen. Inhalation of aerosols: particle deposition and retention. In K. Willeke (ed.), Generation of Aerosols. Ann Arbor Science Publishers, Ann Arbor, 1980.
O. G. Rabbe, M. A. Al-Bayati, S. V. Teague. Regional deposition of inhaled monodisperse coarse and fine particles in small laboratory animals. Ann. Occup. Hyg. 32:53–63 (1988).
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Rojanasakul, Y., Wang, L.Y., Malanga, C.J. et al. Targeted Gene Delivery to Alveolar Macrophages via Fc Receptor-Mediated Endocytosis. Pharm Res 11, 1731–1736 (1994). https://doi.org/10.1023/A:1018959231951
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DOI: https://doi.org/10.1023/A:1018959231951