COVALENT MODIFICATION OF THERAPEUTIC OLIGONUCLEOTIDES

该计划项目的目标是， 通过减少细胞表面表达的 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物 免疫排斥反应 所采取的方法是处理 移植前离体移植组织或器官，含寡核苷酸 其阻断MHC蛋白的表达。 作用于 通过包括反义双链体形成、反义三链体 蛋白质的形成、适体蛋白质结合或诱饵结合 转录所需的。 这个特定项目的目标是通过 寡核苷酸的共价修饰，以增加它们对 细胞内靶点。 大多数寡核苷酸被大多数 细胞被包含在内质小泡中，因此不能到达 其细胞质和/或细胞核靶向。 将采取两项战略 与增加寡核苷酸细胞质递送平行， 它们可以同时攻击细胞质和细胞核。 第一种策略将修饰一种多肽的骨架磷酸盐， 寡核苷酸，使其分配到膜脂质和扩散 通过膜直接进入细胞质，从而模拟 天然大分子运输系统。 这部小说的可行性 将使用H-磷酸盐化学和氨基磷酸酯测试策略 寡核苷酸 将首先筛选寡核苷酸的 与细胞结合并部分与外界隔绝的能力 通过KI猝灭荧光标记的 寡核苷酸 如果证明可行性， 将被合成，其中递送增强修饰基团被 通过细胞内酶去除以产生未修饰的寡核苷酸 在细胞内。 第二种策略是将一种小分子（芘、聚乙烯 二醇或多胺）与预期 通过内吞途径引起增加的内化。 这是一 传统的策略，显然依赖于寡核苷酸， 从内吞囊泡中泄漏到达细胞内靶点。 的 小分子还可以增加药物的表观效力， - 通过稳定寡核苷酸靶复合物来制备寡核苷酸，以及 通过增加寡核苷酸的核酸酶抗性。 将寡核苷酸递送到HeLa细胞的细胞质和细胞核中 将使用德克萨斯红标记的荧光寡核苷酸进行评估， 共聚焦显微镜 修饰的寡核苷酸更好地阻断MHC蛋白的能力 表达和减少宿主免疫应答的能力将在 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.