USE OF SYNTHETIC OLIGONUCLEOTIDES IN BONE MARROW PURGING

合成寡核苷酸在骨髓净化中的用途

• 批准号: 2464559
• 负责人: R BERGAN
• 金额: --
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2464559
• 关键词: antineoplastics; antisense nucleic acid; bone marrow purging; electroporation; human tissue; laboratory mouse; lymphoma; neoplasm /cancer chemotherapy; neoplasm /cancer genetics; neoplasm /cancer pharmacology; nonhuman therapy evaluation; nucleic acid sequence; oligonucleotides; protooncogene; synthetic nucleic acid; transfection

This project sought to improve the delivery of oligodeoxynucleotides (ODNs) in a clinical model. A series of investigations designed to increase the efficiency of cell uptake led to the use of electroporation (EP). We characterized EP mediated ODN uptake, and demonstrated that: transfection rates of 100% were typically attained, overall cell uptake was increased by up to l0 fold, uptake was immediate, and ODN localized primarily to the nucleus and cytoplasm (sites of ODN pharmacologic action). Increased efficacy was demonstrated by showing that suppression of c-myc protein by c-myc antisense ODNs was seen in a variety of cell lines within 90 minutes. Sequence-dependent decreases in cell viability within 24 hours were also demonstrated. Using the U937 human lymphoma cell line in a murine xenograft model, prolonged animal survival was demonstrated for mice receiving cells preloaded with c-myc antisense ODN. A thorough analysis of EP upon normal hematopoiesis demonstrated little to no effect upon normal hematopoietic activity. These tests included: CFU-GM and CFU-S colony forming assays, and competitive bone marrow repopulation assays in murine systems, and CFU-GM assays with human bone marrow cells. By performing a mixing assay in which normal human bone marrow was contaminated with U937 cells, we demonstrated that c-myc antisense, introduced into cells by EP, had no significant effect upon normal CFU-GM activity, while colony forming activity of U937 cells was decreased by 87%.

该项目旨在改善寡聚脱氧核苷酸的交付 (ODN)在临床模型中。一系列旨在 细胞摄取效率的提高导致了电穿孔的使用 (EP)。我们对EP介导的ODN摄取进行了表征，并证明： 通常达到100%的转染率，细胞总摄取率 增加高达10倍，摄取即刻，ODN本地化 主要分布于胞核和胞浆(ODN药理部位 行动)。药效的提高是通过显示抑制 C-myc反义寡核苷酸在多种细胞中表达c-myc蛋白 90分钟内排队。 24小时内细胞活力的顺序依赖性下降也是 演示了。U937人淋巴瘤细胞系在小鼠体内的应用 在异种移植模型中，小鼠的动物存活时间延长 接受预先负载c-myc反义ODN的细胞。 对正常造血的EP的彻底分析显示 对正常的造血活动没有影响。这些测试包括： CFU-GM和CFU-S集落形成试验及竞争骨髓 小鼠系统的再繁殖检测和人骨的CFU-GM检测 骨髓细胞。通过对正常人骨进行混合测试， 骨髓被U937细胞污染，我们证明c-myc 反义基因被EP导入细胞后，对细胞生长无明显影响 CFU-GM活性正常，而U937细胞集落形成活性 下降了87%。