Design and Delivery of Hybrid Phosphorthioate/phosphodiester siRNA Duplexes

杂合硫代磷酸酯/磷酸二酯 siRNA 双链体的设计和交付

• 批准号: 7051920
• 负责人: Jeremy Heidel
• 金额: $ 10万
• 依托单位: CALANDO PHARMACEUTICALS, INC.
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7051920
• 关键词: SCID mouse; antisense nucleic acid; chemical conjugate; drug delivery systems; drug design /synthesis /production; laboratory mouse; luciferin monooxygenase; plasmids; small interfering RNA; thiophosphate

DESCRIPTION (provided by applicant): Gene silencing by RNA interference (RNAi) is rapidly becoming the method of choice for the elucidation of gene function and the identification of therapeutic targets. RNAi is envisioned to ultimately be used as a potent and selective human therapeutic. Short interfering RNAs (siRNA) can be chemically synthesized and do not require nuclear entry for efficacy. Delivery of siRNAs could potentially provide transient gene inhibition in humans for therapeutic use. However, effective systemic delivery of siRNA remains a challenge. Here, we investigate a new concept for the effective delivery of siRNA. siRNA duplexes containing phosphorothioate (PS) sense strands and phosphodiester (PO) antisense strands are formulated with proteins that are used to target cell surface receptors that provide endocytosis. Thus, the PS sense strand is employed to carry the antisense strand, impart stability to the duplex and to bind to proteins that can provide for cellular targeting and/or cellular uptake. No synthetic delivery systems are necessary for in vivo delivery. Hypothesis: A PS sense strand when combined with a PO antisense strand to form a siRNA duplex can elicit effective in vivo delivery of the duplex by binding to proteins that can provide cellular targeting and/or cellular uptake. Specific Aims: (1) Prepare siRNA with PS sense strands and PO antisense strands that give luciferase (LUC), FAS or EWS-FLI1 gene inhibition and investigate their in vitro behavior and their binding to proteins that can be ligands for cell surface receptors, e.g., asialofetuin (AF) for the asialoglycoprotein receptor on hepatocytes and transferrin (Tf) for transferrin receptor on cancer cells and the blood-brain barrier; (2) Measure the in vivo biocompatibility of the siRNA duplexes in mice by monitoring for toxicities and changes in blood chemistries; and (3) Measure the effectiveness of the siRNA for LUC, FAS and EWS-FLI1 gene inhibition by combining them with targeting proteins such as AF and Tf and using several dosing regimes in two mice models; namely, a transgenic model with liver luciferase expression and a metastatic tumor model that creates brain metastases to monitor the possibility of crossing of the blood-brain barrier. Phase II research will capitalize upon the findings in this Phase I program that is designed to define how to best target the PS(sense):PO(antisense) siRNA duplexes to liver and metastatic tumors and to define whether or not they cross the blood-brain barrier. Based on the results from the Phase I study, disease targets in the locations successfully reached will be explored. For example, we know from our preliminary results that hepatocytes can be targeted with this technology. Disease targets in the liver will be investigated in Phase II as well as possible tumor and brain targets. The expected outcome of the Phase II investigation will be a siRNA that can be taken into a human clinical study.

描述（由申请人提供）：通过RNA干扰（RNAi）进行基因沉默正在迅速成为阐明基因功能和识别治疗靶点的首选方法。 RNAi 预计最终将被用作一种有效的、选择性的人类治疗方法。短干扰 RNA (siRNA) 可以化学合成，不需要进入细胞核即可发挥作用。 siRNA 的递送可能会为人类提供短暂的基因抑制，用于治疗用途。然而，siRNA 的有效全身递送仍然是一个挑战。在这里，我们研究了有效递送 siRNA 的新概念。含有硫代磷酸酯 (PS) 有义链和磷酸二酯 (PO) 反义链的 siRNA 双链体与用于靶向提供内吞作用的细胞表面受体的蛋白质一起配制。因此，PS有义链用于携带反义链，赋予双链体稳定性并结合可提供细胞靶向和/或细胞摄取的蛋白质。体内递送不需要合成递送系统。假设：PS 有义链与 PO 反义链组合形成 siRNA 双链体时，可以通过与可提供细胞靶向和/或细胞摄取的蛋白质结合，引发双链体的有效体内递送。具体目标： (1) 制备具有 PS 有义链和 PO 反义链的 siRNA，以抑制荧光素酶 (LUC)、FAS 或 EWS-FLI1 基因，并研究它们的体外行为及其与可作为细胞表面受体配体的蛋白质的结合，例如，脱唾液酸胎球蛋白（AF）代表肝细胞上的脱唾液酸糖蛋白受体，转铁蛋白（Tf）代表癌细胞和血脑屏障上的转铁蛋白受体； (2) 通过监测毒性和血液化学变化来测量 siRNA 双链体在小鼠体内的生物相容性； (3) 通过将 siRNA 与 AF 和 Tf 等靶向蛋白结合，并在两种小鼠模型中使用多种给药方案，测量 siRNA 对 LUC、FAS 和 EWS-FLI1 基因抑制的有效性；即，具有肝脏荧光素酶表达的转基因模型和产生脑转移以监测穿过血脑屏障的可能性的转移性肿瘤模型。 II 期研究将利用这一 I 期项目的研究结果，该项目旨在确定如何最好地将 PS（有义）：PO（反义）siRNA 双链体靶向肝脏和转移性肿瘤，并确定它们是否穿过血液-脑屏障。根据第一阶段研究的结果，将探索成功达到的地点的疾病目标。例如，我们从初步结果中得知，这项技术可以靶向肝细胞。第二阶段将研究肝脏的疾病目标以及可能的肿瘤和大脑目标。 II 期研究的预期结果将是可用于人体临床研究的 siRNA。