title: Gene transfer into human hepatoma cells by receptor-associated protein/polylysine conjugates.
abstract: Receptor-associated protein (RAP) is a ligand for all members of low-density lipoprotein (LDL) receptor families. RAP is internalized into cells via receptor-mediated endocytic trafficking, making it an attractive mechanism for efficient gene delivery. In this study, we have developed a gene delivery system using RAP as a targeting ligand. A RAP cDNA lacking a C-terminal heparin-binding domain was amplified by polymerase chain reaction (PCR) from a human liver cDNA library and was reamplified by using a primer containing a cysteine codon at its carboxyl end to facilitate its conjugation to polylysine (polyK). RAP was purified using a bacterial expression system and coupled to poly-D-lysine (PDL) or poly-L-lysine (PLL) of average MW 50 kDa via the heterobifunctional cross-linker SPDP. Using fluorescence-labeled RAP ligand, cellular uptake of the transfection complexes into HepG2 cells was shown to be highly efficient and more specific to PDL-conjugated RAP compared with PLL-conjugated one. Plasmid DNA containing a luciferase reporter gene was condensed with either RAP-PDL or RAP-PLL. In vitro transfection into HepG2 cells with RAP-PDL conjugate resulted in significantly higher luciferase expression levels in comparison to either nonconjugated PDL, or RAP-PLL, or LipofecAMINE/DNA complexes in the presence of 10% fetal bovine serum. Luciferase expression was inhibited by the addition of excess RAP. Treatment of the cells with Lovastatin, which inhibits HMG-Co reductase and increases expression of LDL receptor, stimulates luciferase expression, suggesting that the gene delivery is specifically mediated by LDL receptor. Thus, RAP-PDL conjugates have the potential to be used as a new nonviral gene delivery vector.
authors.affiliation: Department of Pharmacology, National Institute of Toxicological Research, Korea Food and Drug Administration, Seoul 122-704, Republic of Korea.
authors.forename: Tae-Gyun
authors.initials: TG
authors.lastname: Kim
authors.forename: Seog-Youn
authors.initials: SY
authors.lastname: Kang
authors.forename: Ju-Hye
authors.initials: JH
authors.lastname: Kang
authors.forename: Mi-Young
authors.initials: MY
authors.lastname: Cho
authors.forename: Joo-Il
authors.initials: JI
authors.lastname: Kim
authors.forename: Seung-Hee
authors.initials: SH
authors.lastname: Kim
authors.forename: Jin-Seok
authors.initials: JS
authors.lastname: Kim
mesh_terms: D001667:Binding, Competitive; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D018014:Gene Transfer Techniques; D006801:Humans; D026563:LDL-Receptor Related Protein-Associated Protein / Q000235:genetics* / Q000378:metabolism; D008148:Lovastatin / Q000494:pharmacology; D011107:Polylysine / Q000235:genetics* / Q000378:metabolism; D014162:Transfection / Q000379:methods
chemical_list: D026563:LDL-Receptor Related Protein-Associated Protein; D011107:Polylysine; D008148:Lovastatin