Kim Tae-Gyun, Kang Seog-Youn, Kang Ju-Hye, Cho Mi-Young, Kim Joo-Il, Kim Seung-Hee, Kim Jin-Seok
Department of Pharmacology, National Institute of Toxicological Research, Korea Food and Drug Administration, Seoul 122-704, Republic of Korea.
Bioconjug Chem. 2004 Mar-Apr;15(2):326-32. doi: 10.1021/bc0340262.
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.
受体相关蛋白（RAP）是低密度脂蛋白（LDL）受体家族所有成员的配体。RAP通过受体介导的内吞运输被内化到细胞中，这使其成为一种高效基因递送的有吸引力的机制。在本研究中，我们开发了一种以RAP作为靶向配体的基因递送系统。通过聚合酶链反应（PCR）从人肝脏cDNA文库中扩增出缺失C端肝素结合域的RAP cDNA，并使用在其羧基端含有半胱氨酸密码子的引物进行再扩增，以促进其与聚赖氨酸（polyK）偶联。使用细菌表达系统纯化RAP，并通过异双功能交联剂SPDP将其与平均分子量为50 kDa的聚-D-赖氨酸（PDL）或聚-L-赖氨酸（PLL）偶联。使用荧光标记的RAP配体，与PLL偶联的RAP相比，转染复合物对HepG2细胞的细胞摄取显示出高效性且对PDL偶联的RAP更具特异性。含有荧光素酶报告基因的质粒DNA与RAP-PDL或RAP-PLL凝聚。在10%胎牛血清存在的情况下，用RAP-PDL偶联物对HepG2细胞进行体外转染，与未偶联的PDL、RAP-PLL或LipofecAMINE/DNA复合物相比，荧光素酶表达水平显著更高。添加过量的RAP可抑制荧光素酶表达。用洛伐他汀处理细胞，洛伐他汀可抑制HMG-Co还原酶并增加LDL受体的表达，刺激荧光素酶表达，表明基因递送是由LDL受体特异性介导的。因此，RAP-PDL偶联物有潜力用作一种新型非病毒基因递送载体。
