Liposome-siRNA-Peptide Complexes as Therapy to Cure Prion Diseases in Mouse Model

描述（由申请人提供）：传染性海绵状脑病（TSE）包括绵羊瘙痒病；牛的牛海绵状脑病（BSE）；鹿和麋鹿的慢性消耗性疾病（CWD）；以及人类的库鲁病和克雅氏病。 PrPRES 是正常朊病毒蛋白的一种蛋白酶 K (PK) 抗性形式，PrPC 的积累似乎是这些疾病发病机制的核心。 TSE 总是致命的疾病，无法进行早期诊断或治疗。 RNA干扰（RNAi）是植物和动物中相对较新的发现，它可以调节可翻译成蛋白质的RNA量，已在实验中用于限制病毒、细菌和癌细胞的复制量。朊病毒疾病有效疗法的最新进展是利用RNAi来抑制神经元中正常宿主蛋白PrPC的表达以及随后的朊病毒神经病理学，利用疾病严重程度和进展与神经元中存在的PrPC数量相关的现象。然而，由于侵入性递送方法、有限的面积以及 PrPC 抑制的不可逆性和安全性问题，用于 RNAi 的编码 PrP 短发夹 RNA 的慢病毒递送在实验上仅表现出有限的有效性，并且在临床上的使用有限。我们最近开发了一种新型小干扰 RNA (siRNA) 递送系统，使用脂质体-siRNA-肽复合物 (LSPC) 来保护 siRNA 免于血清降解，将 siRNA 特异性递送至神经元，并充分敲低 PrPC 以治愈两种细胞培养模型中的朊病毒病。在这里，我们建议扩展这项工作，使用静脉注射 (iv) LSPC 将 PrP siRNA 穿过血脑屏障 (BBB) 传递到整个大脑的神经元，从而治愈小鼠中的两种不同的朊病毒疾病。我们的具体目标如下。具体目标 1：确定小鼠中 LSPC 递送和 PrPC 敲低的动力学，以优化 LSPC 治疗，以治疗朊病毒病 具体目标 2：确定含有 PrP siRNA 的 LSPC 是否可以治愈瘙痒症和慢性消耗性疾病 (CWD) 小鼠模型中已确定的朊病毒感染用小鼠适应的痒病和 CWD 朊病毒株感染小鼠 b．在临床疾病发作前监测小鼠的早期行为和认知缺陷 c．使用不同剂量和时间的方案用 PrP 治疗小鼠并对照 LSPC

DESCRIPTION (provided by applicant): Transmissible spongiform encephalopathies (TSEs) include scrapie in sheep; bovine spongiform encephalopathy (BSE) in cattle; chronic wasting disease (CWD) in deer and elk; and Kuru and Creutzfeldt-Jakob disease in humans. Accumulation of PrPRES, a proteinase K (PK) resistant form of the normal prion protein, PrPC appears central to the pathogenesis of these diseases. TSEs are invariably fatal diseases for which no early diagnosis or treatment exists. RNA interference (RNAi), a relatively new discovery in both plants and animals that regulates the amount of RNA available for translation into protein, has been used experimentally to limit the amount of viral, bacterial and cancer cell replication. Recent advances toward an effective therapy for prion diseases employ RNAi to suppress expression of a normal host protein, PrPC, in neurons and subsequent prion neuropathology, exploiting the phenomenon that disease severity and progression correlate with the amount of PrPC present in neurons. However, delivery of lentivirus encoding PrP short hairpin RNA for RNAi has demonstrated only modest effectiveness experimentally and limited use clinically due to the invasive delivery method, limited area and irreversibility of PrPC suppression and safety concerns. We have recently developed a novel small interfering RNA (siRNA) delivery system using liposome-siRNA-peptide complexes (LSPCs) to protect siRNAs from serum degradation, deliver siRNA specifically to neurons and knockdown PrPC sufficiently to cure prion disease in two cell culture models. Here we propose to extend this work to cure two distinct prion diseases in mice using LSPCs injected intravenously (iv) to deliver PrP siRNA across the blood-brain-barrier (BBB) to neurons throughout the brain. Our specific aims are as follows. SPECIFIC AIM 1: Determine kinetics of LSPC delivery and PrPC knockdown in mice to optimize LSPC therapy to treat prion disease SPECIFIC AIM 2: To determine whether PrP siRNA-containing LSPCs can cure established prion infection in mouse models of scrapie and chronic wasting disease (CWD) a. Infect mice with mouse-adapted strains of scrapie and CWD prions b. Monitor mice for early behavioral and cognitive defects prior to onset of clinical diseas c. Treat mice with PrP and control LSPCs using regiments varying in dosage and timing