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

描述(申请人提供)：传染性海绵状脑病(TSE)包括绵羊的瘙痒病；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Kuru和CreutzFeldt-Jakob病。PrPRES是一种抵抗蛋白酶K(PK)的正常蛋白，PrPC的积聚似乎是这些疾病发病的核心。TSE总是致命的疾病，没有早期诊断或治疗方法。RNA干扰(RNAi)是在植物和动物中相对较新的发现，它调节可转化为蛋白质的RNA的量，已被实验用于限制病毒、细菌和癌细胞的复制量。最近在有效治疗普恩病毒疾病方面的进展是利用RNAi来抑制神经元中正常宿主蛋白PrPC的表达，以及随后的普恩神经病理，利用疾病的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，由于有侵入性的递送方法、PrPC抑制的有限区域和不可逆性以及安全性问题，RNAi的慢病毒编码PrP短发夹RNA的递送在实验上仅显示出适度的有效性，临床应用也有限。我们最近开发了一种新型的小干扰RNA(SiRNA)递送系统，使用脂质体-siRNA-肽复合体(LSPC)来保护siRNA免受血清降解，将siRNA特异性地递送到神经元，并在两种细胞培养模型中充分敲除PrPC以治愈普恩病毒疾病。在这里，我们建议将这项工作扩展到治疗小鼠的两种不同的普恩病毒疾病，方法是静脉注射LSPC(Iv)，将PrP siRNA穿过血脑屏障(BBB)传递到整个大脑的神经元。我们的具体目标如下。特异性目标1：确定小鼠体内LSPC的传递动力学和PrPC基因敲除，以优化LSPC疗法以治疗特定的PrP病。目的2：确定含有PrP siRNA的LSPC能否治愈在瘙痒病和慢性衰弱病(CWD)小鼠模型中已建立的Prion感染：a.用小鼠适应株的瘙痒病和CWD Prion感染小鼠；b.在临床疾病发生之前监测小鼠的早期行为和认知缺陷；c.用PrP治疗小鼠，并使用不同剂量和时间的方案控制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