治疗的siRNA递送至CNS使用阳离子和阴离子脂质体
Summary
The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.
Abstract
Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects.
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.
Introduction
朊病毒是影响中枢神经系统严重的神经退行性疾病。朊病毒病从正常细胞朊病毒蛋白，朊蛋白C的错误折叠通过所谓的PrP res的传染性异构体造成的。这些疾病影响的各种物种包括牛的牛海绵状脑病，羊瘙痒病绵羊，在cervids慢性消耗性疾病，和Creutzfeldt-Jakob病中的人类1-3。朊病毒引起神经退行性变与突触丧失开始，发展到空泡化，胶质细胞增生，神经元丢失和斑块沉积。最终，导致动物/个体4的死亡。几十年来，研究人员已经调查了旨在减缓或阻止朊病毒疾病的进展的化合物。然而，研究人员还没有发现任何一个成功的治疗或有效的全身递送载体。
内源性的PrP C表达式所需的朊病 毒疾病5的开发</s了>。因此，减少或消除的PrP C的表达可能导致延迟或疾病的改善。几个研究小组创建了转基因小鼠直接表达shRNA的进鼠脑组织调查朊蛋白C的表达水平的朊病 毒疾病中的作用朊蛋白的C水平降低或注射lentivectors。发现减少的神经元的PrP C量这些研究导致停止朊病毒病的渐进的神经病理学和延长了动物6-9的寿命。我们已经报道了在小鼠神经母细胞瘤10朊蛋白的Res间隙的朊蛋白C小干扰治疗效果。这些研究表明，使用的疗法以降低朊病毒C表达式水平，如小干扰RNA（siRNA），其裂解的mRNA，可以充分地延迟朊病毒病的进展。然而，研究了朊病毒病大多数治疗的方式，是不实际交付在临床环境中。因此，siRNA的治疗需要全身递送系统，该系统通过静脉内递送并靶向中枢神经系统。
研究者已经研究了使用脂质体作为递送媒介物的基因治疗产品。阳离子和阴离子脂质都在脂质体的形成中使用。阳离子脂质比阴离子脂质更广泛地使用，因为阳离子脂质和DNA / RNA之间的电荷差允许有效的包装。阳离子脂质的另一个优点是，它们更容易穿过细胞膜比其它脂质11-14。然而，阳离子脂质是比阴离子脂质13,14更具免疫原性。因此，研究人员已经开始使用阳离子和阴离子脂质体脂肪移位。基因治疗产品可以有效地包装成使用带正电荷的肽鱼精蛋白硫酸盐，其中凝结的DNA / RNA分子15-19阴离子脂质体。由于阴离子梨皮DS比阳离子脂质它们可能增加循环时间，并且可以在动物模型13,14被更耐受的免疫原性更低。脂质体定位到使用定位附加到脂质体肽的特异性的组织。使RVG-9R神经肽，其结合烟碱乙酰胆碱受体，已被用于靶向siRNA和脂质体至CNS 17-20。
这个报告概述的协议以产生三个siRNA递送车辆，打包和递送的siRNA神经元细胞（ 图1）。脂质体的siRNA肽复合物（LSPCs）由用siRNA脂质体和RVG-9R靶向肽静电连接于脂质体的外表面。肽处理脂质体包封的治疗性的siRNA（PALETS）由siRNA和鱼精蛋白在脂质体中包封的，具有RVG-9R共价结合到脂质的PEG基团。使用以下方法来生成LSPCs和PALETS，朊蛋白C小干扰降低朊蛋白C的表达高达神经细胞90％，其中蕴含着巨大的承诺治愈或基本延缓朊病毒疾病病理的发作。
Protocol
Representative Results
Discussion
这份报告描述了一个协议，创建高效的siRNA输送至CNS 2靶向递送系统。递送的siRNA至CNS的以前的方法包括直接注射的siRNA / shRNA的载体入脑，靶向siRNA的静脉注射，或非靶向脂质体的siRNA复合物的静脉注射。的siRNA / shRNA的载体进入中枢神经系统的注射却引起靶蛋白表达水平下降。然而，所述siRNA / shRNA的不能自由通过中枢神经系统扩散。另外，这些注射导致对邻近的神经组织6-9的损伤。靶向siRN…
Declarações
The authors have nothing to disclose.
Acknowledgements
We would like to acknowledge the following funding sources: the CSU Infectious Disease Translational Research Training Program (ID:TRTP) and the NIH research grant program (R01 NS075214-01A1). We would like to thank the Telling lab for the use of their monoclonal antibody PRC5. We would also like to thank the Dow lab for DOTAP liposomes, and for sharing their expertise in generating liposomes.
Materials
| DOTAP lipid | Avanti Lipids | 890890 | |
| Cholesterol | Avanti Lipids | 700000 | |
| DSPE | Avanti Lipids | 850715 | |
| DSPE-PEG | Avanti Lipids | 880125 | |
| Chloroform | Fisher Scientific | AC268320010 | |
| Methanol | EMD Millipore | 113351 | |
| N2 Gas | AirGas | ||
| Sucrose | Fisher Scientific | S5-500 | |
| Extruder | Avanti Lipids | 610023 | |
| 1.0, 0.4, and 0.2um filters | Avanti Lipids | 610010, 610007, 610006 | |
| PBS | Life Technologies | 70011-044 | |
| Protamine sulfate | Fisher Scientific | ICN10275205 | |
| EDC | Thermo Scientific | 22980 | Aliquoted for single use |
| Sulfo-NHS | Thermo Scientific | 24510 | Aliquoted for single use |
| 40um Cell Strainer | Fisher Scientific | 08-771-1 | |
| Rat anti-mouse CD16/CD32 Fc block | BD Pharmigen | 553141 | |
| Anti-PrP antibody (PRC5) | Proprietary – PRC |
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