Department of Biochemistry and Molecular Biology and Institute for Genetic Medicine, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90033, USA.
Bioconjug Chem. 2009 Mar 18;20(3):427-31. doi: 10.1021/bc800540j.
PEGylated-carbon nanotubes (PNTs) were evaluated as nanocarriers of antisense oligonucleotides into T-cells using protein tyrosine phosphatase N22 (PTPN22) as a model target gene. PTPN22 is an important predisposing gene and drug target in type 1 diabetes and several other human autoimmune diseases. Here, we generated the first anti-PTPN22 20-mer antisense oligonucleotides (ASOs) and tethered them to PNTs through a cleavable disulfide bond. Spectroscopic and atomic force microscopy analyses were used to determine the loading of ASO onto PNTs, whereas the cleavable nature of the disulfide bond connecting the oligonucleotide to the nanocarrier was confirmed by incubation with dithiothreitol followed by agarose gel electrophoresis. PNT-conjugated ASOs achieved efficient (>50%) knockdown of PTPN22 expression in T-lymphocytes in culture at the mRNA and protein level, as measured by quantitative real-time PCR and Western blotting, respectively. Considering the high biocompatibility and low in vivo toxicity of PNTs, we expect that our approach will be easily translated to achieve in vivo knockdown of PTPN22 and other T lymphocyte targets, thus enabling novel ASO-mediated immunotherapies for type 1 diabetes and other autoimmune diseases.
聚乙二醇化碳纳米管（PNTs）被评估为将反义寡核苷酸递送到 T 细胞的纳米载体，使用蛋白酪氨酸磷酸酶 N22（PTPN22）作为模型靶基因。PTPN22 是 1 型糖尿病和其他几种人类自身免疫性疾病的重要易感基因和药物靶标。在这里，我们生成了第一个针对 PTPN22 的 20 个碱基对反义寡核苷酸（ASO），并通过可裂解的二硫键将其连接到 PNTs 上。光谱和原子力显微镜分析用于确定 ASO 加载到 PNTs 上的情况，而通过与二硫苏糖醇孵育并进行琼脂糖凝胶电泳来确认连接寡核苷酸和纳米载体的二硫键的可裂解性质。PNT 缀合的 ASO 在培养的 T 淋巴细胞中实现了 PTPN22 表达的高效（>50%）下调，分别通过定量实时 PCR 和 Western 印迹进行测量。考虑到 PNTs 的高生物相容性和低体内毒性，我们预计我们的方法将很容易转化为实现体内 PTPN22 和其他 T 淋巴细胞靶标的下调，从而为 1 型糖尿病和其他自身免疫性疾病提供新的 ASO 介导的免疫疗法。
