Single chain Fv: a ligand in receptor-mediated gene delivery.
We have used an anti-human polymeric immunoglobulin receptor (pIgR) single chain Fv (scFv) to deliver reporter genes to epithelial cells in vitro. The scFv was constructed from a monoclonal antibody directed against pIgR and a cysteine residue was added at the carboxyl end to facilitate its conjugation to polylysine (polyK) via the heterobifunctional cross-linker SPDP. ScFv-cys was expressed in Drosophila S2 cells and purified to homogeneity using conventional column chromatography. ScFv-polyK, and polyK as control, were condensed with a DNA expression plasmid containing the luciferase reporter gene driven by the CMV promoter into unimolecular (with respect to DNA) complexes under high salt conditions. Target cells were MDCK cells transfected with human pIgR and repeatedly sorted for high-level receptor expression, with untransfected MDCK cells as control. Receptor-bearing MDCK cells were readily transfected by scFv-cys containing, pIgR directed complexes, and expression could be blocked by addition of excess human secretory component (SC), the extracellular portion of pIgR. In contrast, MDCK cells that did not express pIgR were not transfected. Nontargeted complexes were not effective in transfecting MDCK cells with or without pIgR. Targeted complexes also transfected human tracheal epithelial cells in primary culture, corroborating the pIgR-mediated gene delivery. These data indicate that a scFv directed against human pIgR can direct foreign genes specifically into receptor-bearing cells in vitro. We have expressed and purified a ligand that is efficient and specific in pIgR-mediated gene delivery.
Gupta
S
S
Department of Pediatrics, Case Western Reserve University School of Medicine, Biomedical Research Bldg, Rm 831, 2109, Adelbert Road, Cleveland, Ohio 44106-6006, USA.
Eastman
J
J
Silski
C
C
Ferkol
T
T
Davis
P B
PB
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D018929:Cell Culture Techniques; D004330:Drosophila / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000378:metabolism; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D008024:Ligands; D011961:Receptors, Fc / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D014132:Trachea / Q000378:metabolism; D014162:Transfection
D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D008024:Ligands; D011961:Receptors, Fc