Biessen E A, Vietsch H, Rump E T, Fluiter K, Kuiper J, Bijsterbosch M K, van Berkel T J
Division of Biopharmaceutics, LACDR, Leiden University, P.O. Box 9503, 2300 RA Leiden, The Netherlands.
Biochem J. 1999 Jun 15;340 ( Pt 3)(Pt 3):783-92.
Anti-sense oligodeoxynucleotides (ODNs) hold great promise for correcting the biosynthesis of clinically relevant proteins. The potential of ODNs for modulating liver-specific genes might be increased by preventing untimely elimination and by improving the local bioavailability of ODNs in the target tissue. In the present study we have assessed whether the local ODN concentration can be enhanced by the targeted delivery of ODNs through conjugation to a ligand for the parenchymal liver cell-specific asialoglycoprotein receptor. A capped ODN (miscellaneous 20-mer sequence) was derivatized with a ligand with high affinity for this receptor, N2-[N2-(N2,N6-bis{N-[p-(beta-d-galactopyranosyloxy) anilino] thiocarbamyl}-L-lysyl)-N6-(N-{p-[beta-D -galactopyranosyloxy] anilino} thiocarbamyl)-L-lysyl]-N6-[N- (p-{beta-D-galactopyranosyloxy}anilino)thiocarbamyl]-L-lysine (L3G4) (Kd 6.5+/-0.2 nM, mean+/-S.D.). Both the uptake studies in vitro and the confocal laser scan microscopy studies demonstrated that L3G4-ODN was far more efficiently bound to and taken up by parenchymal liver cells than underivatized ODN. Studies in vivo in rats showed that hepatic uptake could be greatly enhanced from 19+/-1% to 77+/-6% of the injected dose after glycoconjugation. Importantly, specific ODN accumulation of ODN into parenchymal liver cells was improved almost 60-fold after derivatization with L3G4, and could be attributed to the asialoglycoprotein receptor. In conclusion, the scavenger receptor-mediated elimination pathway for miscellaneous ODN sequences can be circumvented by direct conjugation to a synthetic tag for the asialoglycoprotein receptor. In this manner a crucial requisite is met towards the application of ODNs in vivo to modulate the biosynthesis of parenchymal liver cell-specific genes such as those for apolipoprotein (a), cholesterol ester transfer protein and viral proteins.
反义寡脱氧核苷酸（ODNs）在纠正临床相关蛋白质的生物合成方面具有巨大潜力。通过防止ODNs过早清除并提高其在靶组织中的局部生物利用度，可能会增强ODNs调节肝脏特异性基因的潜力。在本研究中，我们评估了通过与肝实质细胞特异性去唾液酸糖蛋白受体的配体结合，将ODNs靶向递送至肝脏，是否能够提高局部ODN浓度。一种加帽的ODN（杂合的20聚体序列）用对该受体具有高亲和力的配体进行衍生化，该配体为N2-[N2-(N2,N6-双{N-[对-(β-D-吡喃半乳糖氧基)苯胺基]硫代氨基甲酰}-L-赖氨酰)-N6-(N-{对-[β-D-吡喃半乳糖氧基]苯胺基}硫代氨基甲酰)-L-赖氨酰]-N6-[N-(对-{β-D-吡喃半乳糖氧基}苯胺基)硫代氨基甲酰]-L-赖氨酸（L3G4）（解离常数Kd为6.5±0.2 nM，均值±标准差）。体外摄取研究和共聚焦激光扫描显微镜研究均表明，与未衍生化的ODN相比，L3G4-ODN与肝实质细胞的结合及摄取效率要高得多。大鼠体内研究表明，糖缀合后肝脏摄取量可从注射剂量的19±1%大幅提高至77±6%。重要的是，用L3G4衍生化后，ODN在肝实质细胞中的特异性积累提高了近60倍，这可归因于去唾液酸糖蛋白受体。总之，通过直接与去唾液酸糖蛋白受体的合成标签结合，可以规避杂合ODN序列的清道夫受体介导的清除途径。通过这种方式，满足了将ODNs用于体内调节肝实质细胞特异性基因（如载脂蛋白(a)、胆固醇酯转运蛋白和病毒蛋白的基因）生物合成的一个关键要求。
