Merwin J R, Carmichael E P, Noell G S, DeRome M E, Thomas W L, Robert N, Spitalny G, Chiou H C
J Immunol Methods. 1995 Oct 26;186(2):257-66. doi: 10.1016/0022-1759(95)00150-9.
Specific DNA delivery has been achieved via interactions between an asialoorosomucoid-polylysine conjugate and the asialoglycoprotein receptor. We have now extended this technology to another cell type. In order to achieve DNA delivery uniquely to T cells, we have employed an antibody-polylysine conjugate which binds and is internalized via CD5. Binding analyses of the T101 monoclonal antibody to Jurkat cells and freshly isolated human peripheral T lymphocytes were performed and Scatchard plots revealed Kd values of 1.4 and 1.2 pM, respectively. To introduce DNA into the T cell, a complex of T101-polylysine and the luciferase plasmid was formed (T101-PL-DNA). 125I-labeled antibody alone or T101-PL-DNA complexes were both shown to internalize. Subcellular fractionation indicated that the complex remained in the endosomal compartment of the cell for up to 90 min. However, with the addition of adenovirus particles, there was a decrease of labeled complex in the endosomal fraction over time suggesting it was no longer 'tethered' to the endosome vesicle. In vitro transfections confirmed this result showing the addition of adenovirus particles during incubation resulted in increased expression of the luciferase protein. Without adenovirus, there was limited expression of the transduced gene. These data revealed that T101 can deliver DNA via an antibody-PL conjugate. The addition of adenovirus allowed the DNA to escape the endosome enabling expression of the reporter gene.
通过去唾液酸糖蛋白 - 聚赖氨酸偶联物与去唾液酸糖蛋白受体之间的相互作用实现了特异性DNA递送。我们现在已将这项技术扩展到另一种细胞类型。为了仅将DNA递送至T细胞，我们采用了一种抗体 - 聚赖氨酸偶联物，它通过CD5结合并内化。对Jurkat细胞和新鲜分离的人外周血T淋巴细胞进行了T101单克隆抗体的结合分析，Scatchard图显示Kd值分别为1.4和1.2 pM。为了将DNA引入T细胞，形成了T101 - 聚赖氨酸与荧光素酶质粒的复合物（T101 - PL - DNA）。单独的125I标记抗体或T101 - PL - DNA复合物均显示能内化。亚细胞分级分离表明该复合物在细胞的内体区室中保留长达90分钟。然而，加入腺病毒颗粒后，随着时间的推移，内体部分中标记复合物的量减少，这表明它不再与内体囊泡“拴系”在一起。体外转染证实了这一结果，显示在孵育期间加入腺病毒颗粒导致荧光素酶蛋白的表达增加。没有腺病毒时，转导基因的表达有限。这些数据表明T101可以通过抗体 - PL偶联物递送DNA。腺病毒的加入使DNA能够逃离内体，从而使报告基因得以表达。
