Gene transfer into human hepatoma cells by receptor-associated protein/polylysine conjugates
- Authors
- Kim, TG; Kang, SY; Kang, JH; Cho, MY; Kim, JI; Kim, SH; Kim, JS
- Issue Date
- Mar-2004
- Publisher
- AMER CHEMICAL SOC
- Citation
- BIOCONJUGATE CHEMISTRY, v.15, no.2, pp 326 - 332
- Pages
- 7
- Journal Title
- BIOCONJUGATE CHEMISTRY
- Volume
- 15
- Number
- 2
- Start Page
- 326
- End Page
- 332
- URI
- https://scholarworks.sookmyung.ac.kr/handle/2020.sw.sookmyung/15903
- DOI
- 10.1021/bc0340262
- ISSN
- 1043-1802
1520-4812
- 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.
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