COVALENT MODIFICATION OF THERAPEUTIC OLIGONUCLEOTIDES

该计划项目的目标是 通过减少细胞表面蛋白的表达促进移植 主要组织相容性复合体(MHC)I类和II类蛋白。这些 蛋白质是触发宿主的主要抗原刺激。 免疫排斥反应。所采取的方法是治疗 体外移植组织或器官，移植前，含寡核苷酸 阻断MHC蛋白的表达。起作用的寡核苷酸 通过反义双链形成、反基因三链形成等机制 蛋白质的形成、适体蛋白结合或诱饵结合 使用了转录所需的代码。 这个特定项目的目标是通过以下方式改进移植 寡核苷酸的共价修饰以增加其获得 细胞内目标。大多数寡核苷酸被大多数人内化 细胞包含在内质泡中，因此不能到达 它的细胞质和/或核目标。将遵循两个战略 在增加寡核苷酸的细胞质递送的同时，从 他们可以接触到细胞质和核目标。 第一种策略将修改一种主链磷酸盐 寡核苷酸使其分配到膜脂中并扩散 通过膜直接进入细胞质，从而模仿 天然大分子运输系统。这部小说的可行性 策略将使用H-磷酸化学和磷酰胺进行测试 寡核苷酸。将对寡核苷酸进行初步筛选，以确定其 能够与细胞结合并与外部细胞部分隔离 荧光标记的KI猝灭测定介质 寡核苷酸。如果可行性被证明，前药构建 将合成其中促进传递的修饰基团是 被胞内酶去除以生成未修饰的寡核苷酸 在细胞内。 第二种策略是将一种小分子(芘、聚乙烯) 乙二醇或多胺)转化为寡聚核苷酸，预计 通过内吞途径引起更多的内化。这是一个 显然依赖于寡核苷酸的传统策略 从内吞小泡泄漏到细胞内的靶点。这个 小分子也可以增加一种表观效力 通过稳定寡核苷酸靶标复合体和 通过增加寡核苷酸的核酸酶抗性。 寡核苷酸进入HeLa细胞胞浆和胞核的研究 将使用德克萨斯州红色标记的荧光寡核苷酸和 共聚焦显微镜。 修饰寡核苷酸更好地阻断MHC蛋白的能力 表达和减少宿主免疫反应将在 模型系统。

The goal of the Program Project, of which this is a component, is to improve transplantation by decreasing the cell surface expression of major histocompatibility complex (MHC) class I and II proteins. These proteins are the primary antigenic stimuli that trigger the host immunologic rejection response. The approach taken is to treat the graft tissue or organ ex vivo, pretransplant, with oligonucleotides which block the expression of MHC proteins. Oligonucleotides that act by mechanisms including antisense duplex formation, antigene triplex formation, aptamer protein binding, or decoy binding of proteins required for transcription are used. The goal of this specific project is to improve transplantation through covalent modifications of oligonucleotides to increase their access to intracellular targets. Most of the oligonucleotide internalized by most cells is contained within endoplasmic vesicles, and thus cannot reach its cytoplasmic and/or nuclear target. Two strategies will be followed in parallel to increase cytoplasmic delivery of oligonucleotides, from which they can access both cytoplasmic and nuclear targets. The first strategy will modify the backbone phosphates of an oligonucleotide to cause it to partition into membrane lipid and diffuse through the membrane directly into the cytoplasm, and thereby mimic a native macromolecular transport system. The feasibility of this novel strategy will be tested using H-phosphate chemistry and phosphoramidate oligonucleotides. Oligonucleotides will be initially screened for their ability to bind to cells and be partially sequestered from the external media as determined by KI quenching of fluorescently labeled oligonucleotides. If feasibility is demonstrated, prodrug constructs will be synthesized in which the delivery-enhancing modifying groups are removed by intracellular enzymes to generate unmodified oligonucleotide within the cell. The second strategy will tether a small molecule (pyrene, polyethylene glycol, or a polyamine) to an oligonucleotide that is anticipated to cause increased internalization by an endocytotic route. This is a traditional strategy that apparently relies on the oligonucleotide that leaks from endocytotic vesicles to reach intracellular targets. The small molecules can also increase the apparent potency of an oligonucleotide by stabilizing the oligonucleotide target complex and by increasing the nuclease resistance of the oligonucleotide. Delivery of oligonucleotide into the cytoplasm and nucleus of HeLa cells will assessed using Texas Red-labeled fluorescent oligonucleotides and confocal microscopy. The ability of modified oligonucleotides to better block MHC protein expression and diminish the host immune response will be evaluated in model systems.