Merwin J R, Noell G S, Thomas W L, Chiou H C, DeRome M E, McKee T D, Spitalny G L, Findeis M A
TargeTech, Inc., Meriden, Connecticut 06450.
Bioconjug Chem. 1994 Nov-Dec;5(6):612-20. doi: 10.1021/bc00030a017.
In vivo gene therapy shows promise as a treatment for both genetic and acquired disorders. The hepatic asialoglycoprotein receptor (ASGPr) binds asialoorosomucoid-polylysine-DNA (ASOR-PL-DNA) complexes and allows targeted delivery to hepatocytes. The tris(N-acetylgalactosamine aminohexyl glycoside) amide of tyrosyl(glutamyl) glutamate [YEE(GalNAcAH)3] has been previously reported to have subnanomolar affinity for the ASGPr. We have used an iodinated derivative of YEE(GalNAcAH)3 linked to polylysine and complexed to the luciferase gene (pCMV-Luc) in receptor-binding experiments to establish the feasibility of substituting ASOR with the synthetic glycopeptide for gene therapy. Scatchard analyses revealed similar Kd values for both ASOR and the glycopeptide. Binding and internalization of 125I-Suc-YEE(GalNAcAH)3 were competitively inhibited with either unlabeled ASOR or glycopeptide. The reverse was also true; 125I-ASOR binding was competed with unlabeled YEE(GalNAcAH)3 suggesting specific binding to the ASGPr by both compounds. Examination of in vivo delivery revealed that the 125I-labeled glycopeptide complex mimicked previous results observed with 125I-ASOR-PL-DNA. CPM in the liver accounted for 96% of the radioactivity recovered from the five major organs (liver, spleen, kidney, heart, and lungs). Cryoautoradiography displayed iodinated glycopeptide complex bound preferentially to hepatocytes rather than nonparenchymal cells. In vitro, as well as in vivo, transfections using the glycopeptide-polylysine-pCMV-luciferase gene complex (YG3-PL-Luc) resulted in expression of the gene product. These data demonstrate that the YEE(GalNAcAH)3 synthetic glycopeptide can be used as a ligand in targeted delivery of DNA to the liver-specific ASGPr.
体内基因治疗作为治疗遗传性疾病和后天性疾病的一种方法显示出了前景。肝去唾液酸糖蛋白受体（ASGPr）能结合去唾液酸血清类黏蛋白-聚赖氨酸-DNA（ASOR-PL-DNA）复合物，并实现向肝细胞的靶向递送。先前报道酪氨酰（谷氨酰）谷氨酸的三（N-乙酰半乳糖胺氨基己糖苷）酰胺[YEE(GalNAcAH)3]对ASGPr具有亚纳摩尔亲和力。在受体结合实验中，我们使用了与聚赖氨酸相连的YEE(GalNAcAH)3的碘化衍生物，并使其与荧光素酶基因（pCMV-Luc）复合，以确定用合成糖肽替代ASOR用于基因治疗的可行性。斯卡查德分析显示ASOR和糖肽的解离常数（Kd）值相似。125I-Suc-YEE(GalNAcAH)3的结合和内化受到未标记的ASOR或糖肽的竞争性抑制。反之亦然；125I-ASOR的结合受到未标记的YEE(GalNAcAH)3的竞争，这表明两种化合物都能与ASGPr特异性结合。对体内递送的研究表明，125I标记的糖肽复合物模拟了先前用125I-ASOR-PL-DNA观察到的结果。肝脏中的每分钟计数（CPM）占从五个主要器官（肝脏、脾脏、肾脏、心脏和肺）回收放射性的96%。冷冻放射自显影显示碘化糖肽复合物优先与肝细胞而非非实质细胞结合。在体外以及体内，使用糖肽-聚赖氨酸-pCMV-荧光素酶基因复合物（YG3-PL-Luc）进行转染均导致了基因产物的表达。这些数据表明，YEE(GalNAcAH)3合成糖肽可作为一种配体，用于将DNA靶向递送至肝脏特异性ASGPr。
