MECHANISM OF ANTISENSE OLIGONUCLEOTIDE UPTAKE IN KIDNEY

最有前途的癌症分子治疗方法之一， 病毒感染是使用反义DNA。 反义寡核苷酸 是体外基因表达的有效抑制剂，并且已被证明 阻断一些与肿瘤生长或病毒相关的细胞基因 感染培养的细胞。 双链的化学修饰 寡核苷酸，以便它们可以全身施用， 为体内分子治疗提供了手段。 可惜 在特定组织中负责反义摄取的机制仍然存在 不清楚，虽然这是一个深入调查的领域。 给药时 系统地，反义寡核苷酸主要被 肝脏和肾脏。 因此，这两个器官是理想的治疗 目标，但它们也可能是毒性的主要部位。 到 在未来，直接治疗肾脏和远离肾脏， 必须确定反义摄取的分子基础。 这项建议 旨在通过定义分子基础来解决这一关键问题， 肾脏中的反义摄取。 我们已经提纯了 结合蛋白，我们已经确定， 这种蛋白质结合寡核苷酸的电泳迁移率变化 我们已经证明，这种蛋白质允许电导 电流通过人工脂质双层的存在下， 寡核苷酸 已测定了部分氨基酸序列， 揭示了该蛋白质是新的，与已知的具有部分同源性， 转录因子、DNA结合蛋白和跨膜穿梭 proteins. 因此，这项建议的具体目标是， 简单明了：1.为了克隆主要基因的全长cDNA， 使用简并引物和猜测体的寡核苷酸通道蛋白 从已知氨基酸序列推导的肾上皮cDNA文库 将为此进行筛选。 2.为了确定函数 细胞培养和人工培养中DNA通道活性的特征 脂质双层，并通过位点- 定向诱变和3.利用获得的信息设计 使用动物体内抑制基因表达的有效策略 单基因诱导型肾衰竭模型。 这些研究 应该为提出研究提供所需的实验基础 人类肾脏疾病系统性反义治疗

One of the most promising approaches to molecular therapy for cancer and viral infection is the use of antisense DNA. Antisense oligonucleotides are potent inhibitors of gene expression in vitro, and have been shown to block a number of cellular genes associated with tumor growth or viral infection in cultured cells. The chemical modification of double stranded oligonucleotides so that they can be administered systemically has now provided the means for molecular therapy in vivo. Unfortunately, the mechanisms responsible for antisense uptake in specific tissues remains unclear, although an area of intense investigation. When administered systemically, antisense oligonucleotides are taken up predominantly by liver and kidney. As a result, these two organs are ideal therapeutic targets but they will likely be the major sites of toxicity as well. To direct therapy both to and away from the kidney in the future, the molecular basis of antisense uptake must be defined. This proposal intends to address this critical issue by defining the molecular basis of antisense uptake in the kidney. We have already purified the major DNA binding protein in renal brush border membrane, we have established that this protein binds oligonucleotides in electrophoretic mobility shift assays, and we have demonstrated that this protein allows the conductance of current through an artificial lipid bilayer in the presence of oligonucleotides. Partial amino acid sequence has been determined and reveals the protein to be novel with partial homology to known transcription factors, DNA binding proteins, and transmembrane shuttle proteins. The specific aims of this proposal are, therefore, straightforward: 1. To clone the full-length cDNA for the major oligonucleotide channel protein by using degenerate primers and guessmers deduced from known amino acid sequence; a renal epithelium cDNA library will be screened for this purpose. 2. To determine the functional characteristics of DNA channel activity in cell culture and in artificial lipid bilayers and to map the functional peptide domains through site- directed mutagenesis. and 3. To use the information obtained to design effective strategies to inhibit gene expression in vivo using an animal model of a single gene-induced form of renal failure. These studies should provide the needed experimental basis for proposing studies of systemic antisense treatment for renal diseases in man.