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.

所有小鼠饲养，并保持在实验室动物资源，由协会进行评估和实验动物管理国际资格认证按照科罗拉多州立大学批准的机构动物护理和使用委员会认可的协议。 1. LSPCs的制备<ol><li>使用1:1 DOTAP（1,2-二油酰基-3-三甲基铵 - 丙烷）:胆固醇比率LSPCs。对于4纳摩尔脂质体制剂，混合的DOTAP的2纳摩尔和2纳摩尔胆固醇入10毫升1:1的氯仿:在烧瓶中的甲醇溶液。 </li><li>蒸发9毫升的氯仿:采用N 2气在通风橱中的甲醇溶液。蒸发最后1毫升溶剂在通风橱中过夜无气。观察在烧瓶的底部的薄的脂质膜。 </li><li>一个10％的蔗糖溶液至55℃的热10毫升</li><li>倒入加热10％蔗糖溶液到薄脂质膜缓慢， 即 1毫升/分钟，而戈ntly漩烧瓶中。保持10％的蔗糖和烧瓶在55℃的脂质，使得脂质分子保留在凝胶 - 液体相。补液结果多层囊泡（MLV）形成。 </li><li>允许脂质上浆脂质体之前在55℃下再水合为至少1小时。 </li><li>组装按制造商的说明挤压机用1.0微米的过滤器，或使用1.0微米的注射器过滤器的大小。 </li><li>将1ml加热脂质体悬浮液添加到挤压机中的注射器的一个，并通过两个注射器之间的悬浮液11倍。确保该悬浮液是在低于11道次后的起始注射器相反注射器。 </li><li>大小脂质体再通过0.45微米那么0.2微米的过滤器来产生大单层囊泡（的LUV）。保持更容易上浆加热脂质体悬浮液。 </li><li>孵育20微升4纳摩尔脂质体悬浮液（200μM）的用200μl20μM的（4纳摩尔总数）在室温下10分钟的库存siRNA溶液。 </li><li>孵育80微升500μM的（40纳摩尔总）的股票与在RT 10分钟的siRNA /脂质体溶液RVG-9R溶液。 LSPCs应尽快用作可能的，但可以在制备后可用于高达几个小时。在4°C储存LSPCs准备几经小时。 </li></ol> 2. PALETS的制备<ol><li>使用55:40:要么DSPE（1,2-二硬脂酰-sn-甘油基-3-磷酸乙醇胺）的5摩尔比:胆固醇:DSPE-PEG或DOTAP:胆固醇:DSPE-PEG为PALETS。对于4纳摩尔脂质体制剂，混合2.2纳摩尔或者DSPE或DOTAP的，胆固醇1.6纳摩尔，和0.2纳摩尔DSPE-PEG成10毫升2:1氯仿:在烧瓶中的甲醇溶液。 </li><li>如在步骤1.1和1.2以上描述的确切制备脂质体悬浮液。再水合DSPE脂质体在10ml 1×PBS中加热到75℃，加入1毫升/分钟。允许脂质在75℃在补充水分上浆前至少1个小时。 </li><li>大小的脂质体悬浮液完全按照步骤1.6-1.8中所述。 </li><li>吸管20微升每4纳摩尔（200μM）脂质体悬浮液的进DOTAP和DSPE脂质体后单次使用的等分试样一直尺寸，和冻干为使用台式冷冻干燥器（ 图2）30分钟。 </li><li>孵育200微升20μM的（4纳摩尔总）库存siRNA溶液与1.4微升在RT硫酸鱼精蛋白的26.6微米溶液10分钟，用于siRNA要被封装入DSPE PALETS脂质体的。 </li><li>再水化DOTAP PALETS脂质体用200μl的siRNA 4纳摩尔原液或用201.4微升的siRNA /鱼精蛋白溶液的再水化DSPE PALETS脂质体。孵育脂质体/ siRNA溶液在室温下10分钟。 </li><li>添加10微升60毫摩尔的1-乙基-3-（3-二甲基氨基丙基）碳二亚胺盐酸盐（EDC）溶液到两个DOTAP和DSPE PALETS脂质体/ siRNA溶液。 </li><li>加入10微升150毫摩尔N-羟基（硫代NHS）溶液到两个DOTAP和DSPE PALETS脂质体/ siRNA溶液。 </li><li>孵育EDC和磺基-NHS与该siRNA /脂质体悬浮液在RT 2小时。 </li><li>孵育80微升500微米（40毫微摩尔总数）股同的siRNA /脂质体交联在室温下10分钟的解决方案RVG-9R的解决方案。 注:EDC /磺基NHS允许RVG-9R以共价结合的PEG脂质。准备经过几次小时内使用PALETS。在4°C储存PALETS准备几经小时。 </li><li>要确定PALETS的siRNA的包封率: <ol><li>之前和在加入鱼精蛋白和使用分光光度计设定在260nm的脂质体后测量的siRNA的浓度。 </li><li>使用50 kDa的离心过滤器的脂质体/封装的siRNA悬架过滤未封装的siRNA。离心在1000 xg离心20分钟。 </li><li>测量的浓度在滤液未包封的siRNA和包封的siRNA在滞留物用分光光度计在260 nm处的浓度。 </li></ol></li></ol> 3.注射用LSPCs或PALETS小鼠<ol><li>暴露5-10分钟小鼠热灯扩张血管。 </li><li>麻醉鼠标采用了2-3％的异氟烷​​/氧流量注射前，和注射过程中5-10分钟。确认时，麻醉动物不再走动和呼吸都做一个脚趾捏放缓。放置在其背面或侧面的鼠标来访问尾静脉。使用鼠标的眼睛兽医药膏，以防止干燥时的麻醉下。 </li><li>擦拭/用70％乙醇喷尾，并用＆G胰岛素注射器注入300微升LSPCs或PALETS的到鼠标的尾静脉。开始向远侧注入尾静脉，如果静脉塌陷或破裂近侧移动。 </li><li>将鼠标在干净的笼子里，直到它保持胸骨斜卧。不要把莫与其他队友笼使用，直到它已完全恢复。小鼠应在5至15分钟，回收。它们可以被放置在一个加热垫或加热灯保持体温如果恢复时间较长下。小鼠应密切监测，以防止过热。 </li><li>监测动物几个小时注射后，以确保麻醉消退，并有治疗没有不良影响。允许LSPCs或PALETS循环至少24小时。 </li></ol> 4.通过流式细胞仪蛋白表达的分析<ol><li>安乐死用20％CO 2的流量为15分钟的小鼠。 </li><li>通过切开使用剪刀的小鼠的皮肤和颅骨解剖出脑的一半的半球。剥开一半大脑半球使用钳子或剪刀年底头骨之遥。 </li><li>从每只小鼠脑的通过使用2.5 FACS缓冲液中的溶液40微米网状细胞过滤（按半个半球1倍的PBS，1％胎牛血清，10mM EDTA的）在培养皿中，用注射器的柱塞。 </li><li>冲洗细胞过滤和培养皿一个额外的2.5毫升流式细胞仪缓冲。把单细胞悬浮液在冰上15毫升锥形管中。 </li><li>吸管100微升5 ml单细胞悬液至1.5 ml微量离心管中并离心，在350×g下5分钟。丢弃在1ml FACS缓冲液的上清，重悬细胞沉淀。旋在350×g离心5分钟。与再过1毫升流式细胞仪缓冲的重复。置于冰上的细胞悬液。 </li><li>孵育细胞在100μl的1:100稀释在FACS缓冲液0.5毫克/毫升的大鼠抗小鼠Fc块在冰上30-60分钟。沉淀和洗涤细胞如在步骤4.5。置于冰上的细胞悬液。 </li><li>孵育于100μl20μg/ ml的荧光抗小鼠朊病毒下在FACS缓冲液在冰上抗体在7％小鼠血清的细胞30-60分钟。沉淀和洗涤细胞如在步骤4.5。保持细胞悬浮液在冰上。 </li><li>沉淀和再悬浮细胞于1ml RBC裂解缓冲液（1×PBS中，155毫摩尔氯化铵 ，12毫碳酸氢钠 ，0.1毫摩尔EDTA）处理1分钟。粒料如在步骤4.5和重悬细胞于1ml FACS缓冲液中。置于冰上的细胞悬液。 </li><li>使用流式细胞仪如前面所述10 PrP的分析C的表达。门从住总细胞群的细胞。门的PrP C +活细胞群的细胞，以确定MFI（平均荧光强度）。 </li></ol>

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The authors declare that they have no competing financial interests.

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...

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&gt;。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。

<table><tbody><tr><td>DOTAP lipid</td><td>Avanti Lipids</td><td>890890</td><td></td></tr><tr><td>Cholesterol</td><td>Avanti Lipids</td><td>700000</td><td></td></tr><tr><td>DSPE</td><td>Avanti Lipids</td><td>850715</td><td></td></tr><tr><td>DSPE-PEG</td><td>Avanti Lipids</td><td>880125</td><td></td></tr><tr><td>Chloroform</td><td>Fisher Scientific</td><td>AC268320010</td><td></td></tr><tr><td>Methanol</td><td>EMD Millipore</td><td>113351</td><td></td></tr><tr><td>N&lt;sub&gt;2&lt;sub&gt; Gas</td><td>AirGas</td><td></td><td></td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>S5-500</td><td></td></tr><tr><td>Extruder</td><td>Avanti Lipids</td><td>610023</td><td></td></tr><tr><td>1.0, 0.4, and 0.2 &amp;#956;m filters</td><td>Avanti Lipids</td><td>610010, 610007, 610006</td><td></td></tr><tr><td>PBS</td><td>Life Technologies</td><td>70011-044</td><td></td></tr><tr><td>Protamine sulfate</td><td>Fisher Scientific</td><td>ICN10275205</td><td></td></tr><tr><td>EDC</td><td>Thermo Scientific</td><td>22980</td><td>Aliquoted for single use</td></tr><tr><td>Sulfo-NHS</td><td>Thermo Scientific</td><td>24510</td><td>Aliquoted for single use</td></tr><tr><td>40 &amp;#956;m Cell Strainer</td><td>Fisher Scientific</td><td>08-771-1</td><td></td></tr><tr><td>Rat anti-mouse CD16/CD32 Fc block</td><td>BD Pharmigen</td><td>553141</td><td></td></tr><tr><td>Anti-PrP antibody (PRC5)</td><td>Proprietary - PRC</td><td></td><td></td></tr></tbody></table>

delivery of therapeutic sirna to the cns using cationic and anionic liposomes

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.

以增加的siRNA包封的阴离子PALETS内的效率，所述siRNA与鱼精蛋白混合。以确定用于所述siRNA最好鱼精蛋白浓度，所述siRNA用不同浓度的鱼精蛋白混合，从1:1到2:1（ 图3A）。有阴离子脂质体60-65％的siRNA包封率，而无需使用鱼精蛋白。样品鱼精蛋白:siRNA的摩尔比从1:1到1.5:1（133-266纳米）有80〜90％的siRNA包封。摩尔比大于1.5:1导致的siRNA /鱼精蛋白复合物的沉淀。这些?...

Watch this Scientific Journal Video about Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes at JoVE.com

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

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.

治疗的siRNA递送至CNS使用阳离子和阴离子脂质体

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

delivery-therapeutic-sirna-to-cns-using-cationic-anionic

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

10.4K 次观看.  Colorado State University. 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. 

视频: Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Preparation of Exosomes for siRNA Delivery to Cancer Cells

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, and intrinsic capability in intercellular delivery of various biomolecules. This work establishes an isolation protocol to achieve high yield and high purity of exosomes for siRNA delivery. Human Embryonic Kidney cells (HEK-293 cells) are cultured in bioreactor flasks and the culture supernatant (hereon referred to as conditioned medium) is harvested on a weekly basis to allow for enrichment of HEK-293 exosomes. The conditioned medium (CM) is pre-cleared of dead cells and cellular debris by differential centrifugation and is subjected to ultracentrifugation onto a sucrose cushion followed by a washing step, to collect the exosomes. Isolated HEK-293 exosomes are characterized for yield, morphology and exosomal marker expression by nanoparticle tracking analysis, protein quantification, electron microscopy and flow cytometry, respectively. Small interfering RNA (siRNA), fluorescently labeled with Atto655, is loaded into exosomes by electroporation and excess siRNA is removed by gel filtration. Cell uptake in PANC-1 cancer cells, after 24 h incubation at 37 &#176;C, is confirmed by flow cytometry. HEK-293 exosomes are 107.0 &#177; 8.2 nm in diameter. The exosome yield and particle-to-protein ratio (P:P) ratio are 6.99 &#177; 0.22 &#215; 1012 particle/mL and 8.3 &#177; 1.7 &#215; 1010 particle/&#181;g, respectively. The encapsulation efficiency of siRNA in exosomes is ~ 10-20%. Forty percent of the cells show positive signals for Atto655 at 24 h post-incubation. In conclusion, exosome isolation by ultracentrifugation onto sucrose cushion offers a combination of good yield and purity. siRNA could be successfully loaded into exosomes by electroporation and subsequently delivered into cancer cells in vitro. This protocol offers a standard procedure for developing siRNA-loaded exosomes for efficient delivery to cancer cells.

An exosome is a new generation of drug delivery carriers. We established an exosome isolation protocol with high yield and purity for siRNA delivery. We also encapsulated fluorescently labelled non-specific siRNA into exosomes and investigated the cellular uptake of siRNA-loaded exosomes in cancer cells.

用于向癌细胞输送 sirna 的外质体的制备

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, ...

科研

癌症研究

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

多孔硅微粒进行交付的siRNA治疗的

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

生物工程

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

啮齿动物的中枢神经系统药物直接脑室交货

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

神经科学

Using Lipid Nanoparticles for the Delivery of Chemically Modified mRNA into Mammalian Cells

In recent years, chemically modified messenger RNA (mRNA) has emerged as a potent nucleic acid molecule for developing a wide range of therapeutic applications, including a novel class of vaccines, protein replacement therapies, and immune therapies. Among delivery vectors, lipid nanoparticles are found to be safer and more effective in delivering RNA molecules (e.g., siRNA, miRNA, mRNA) and a few products are already in clinical use. To demonstrate lipid nanoparticle-mediated mRNA delivery, we present an optimized protocol for the synthesis of functional me1&#936;-UTP modified eGFP mRNA, the preparation of cationic liposomes, the electrostatic complex formation of mRNA with cationic liposomes, and the evaluation of transfection efficiencies in mammalian cells. The results demonstrate that these modifications efficiently improved the stability of mRNA when delivered with cationic liposomes and increased the eGFP mRNA translation efficiency and stability in mammalian cells. This protocol can be used to synthesize the desired mRNA and transfect with cationic liposomes for target gene expression in mammalian cells.

The protocol presents in vitro transcription (IVT) of chemically modified mRNA, cationic liposome preparation, and functional analysis of liposome enabled mRNA transfections in mammalian cells.

In recent years, chemically modified messenger RNA (mRNA) has emerged as a potent nucleic acid molecule for developing a wide range of therapeutic ...

生物化学

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.

MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

一个简单的替代，以立体定向注射miRNA的脑特异性敲除

MicroRNAs (miRNAs) are key regulators of gene expression.  In the brain, vital processes like neurodevelopment and neuronal functions depend on the ...

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive properties of in vitro transcribed (IVT) mRNA. With the help of IVT mRNA, the de novo synthesis of a desired protein can be induced without changing the physiological state of the target cell. Moreover, protein biosynthesis can be precisely controlled due to the transient effect of IVT mRNA.
For the efficient transfection of cells, nanoliposomes (NLps) may represent a safe and efficient delivery vehicle for therapeutic mRNA. This study describes a protocol to generate safe and efficient cationic NLps consisting of DC-cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a delivery vector for IVT mRNA. NLps having a defined size, a homogeneous distribution, and a high complexation capacity, and can be produced using the dry-film method. Moreover, we present different test systems to analyze their complexation and transfection efficacies using synthetic enhanced green fluorescent protein (eGFP) mRNA, as well as their effect on cell viability. Overall, the presented protocol provides an effective and safe approach for mRNA complexation, which may advance and improve the administration of therapeutic mRNA.

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

Here we describe a protocol for the generation of cationic nanoliposomes, which is based on the dry-film method and can be used for the safe and efficient delivery of in vitro transcribed messenger RNA.

阳离子纳米脂质体的生成, 用于高效输送体外转录信使 rna

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive ...

Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical success for treating previously &#8216;undruggable&#8217; hepatic disease targets with siRNA has been achieved. However, efficient tumor siRNA delivery necessitates additional pharmacokinetic design considerations, including long circulation time, evasion of clearance organs (e.g., liver and kidneys), and tumor penetration and retention. Here, we describe the preparation and in vitro physicochemical/biological characterization of polymeric nanoparticles designed for efficient siRNA delivery, particularly to non-hepatic tissues such as tumors. The siRNA nanoparticles are prepared by electrostatic complexation of siRNA and the diblock copolymer poly(ethylene glycol-b-[2-(dimethylamino)ethyl methacrylate-co-butyl methacrylate]) (PEG-DB) to form polyion complexes (polyplexes) where siRNA is sequestered within the polyplex core and PEG forms a hydrophilic, neutrally-charged corona. Moreover, the DB block becomes membrane-lytic as vesicles of the endolysosomal pathway acidify (&#60; pH 6.8), triggering endosomal escape and cytosolic delivery of siRNA. Methods to characterize the physicochemical characteristics of siRNA nanoparticles such as size, surface charge, particle morphology, and siRNA loading are described. Bioactivity of siRNA nanoparticles is measured using luciferase as a model gene in a rapid and high-throughput gene silencing assay. Designs which pass these initial tests (such as PEG-DB-based polyplexes) are considered appropriate for translation to preclinical animal studies assessing the delivery of siRNA to tumors or other sites of pathology.

Methods to prepare and characterize the physicochemical properties and bioactivity of neutrally-charged, pH-responsive siRNA nanoparticles are presented. Criteria for successful siRNA nanomedicines such as size, morphology, surface charge, siRNA loading, and gene silencing are discussed.

用于细胞质 siRNA 传递的带电反应性聚合物纳米粒子的制备

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical ...

A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the central nervous system (CNS). Recent studies demonstrate the use of a peptide, RVG9R, incorporating the minimal receptor-binding domain of the Rabies virus glycoprotein, in eliciting the delivery of siRNA into neurons in the brain. In this protocol, the peptide-siRNA formulation is delivered intranasally with a pipette in the dominant hand, while the anesthetized mouse is restrained by the scruff with the nondominant hand in a "head down-and-forward position" to avoid drainage into the lung and stomach upon inhalation. This precise gripping of mice can be learned but is not easy and requires practice and skill to result in effective CNS uptake. Furthermore, the process is long-drawn, requiring about 45 min for the administration of a total volume of ~20-30 &#181;L of solution in 1-2 &#181;L droplet volumes per inhalation, with 3-4 min rest periods between each inhalation. The objective of this study is to disclose a mouse positioning device that enables the appropriate placement of mice for efficient IN administration of the peptide-siRNA formulation. Multiple features are incorporated into the design of the device, such as four or eight positioning chairs with adjustable height and tilt to restrain anesthetized mice in the head down-and-forward position, enabling easy visualization of the mice's nares and a built-in heating pad to maintain the mice's body temperatures during the procedure. Importantly, the ability to treat four or eight mice simultaneously with RVG9R-siRNA complexes in this manner enables studies on a much quicker time scale, for the testing of an IN therapeutic siRNA approach. In conclusion, this device allows for appropriate and controlled mouse head positioning for IN application of RVG9R-siRNA and other therapeutic molecules, such as nanoparticles or antibodies, for CNS delivery.

Here, we present a protocol using a mouse positioning device that enables the appropriate placement of mice for the intranasal administration of a brain-targeting peptide-siRNA formulation enabling effective gene silencing in the central nervous system.

在鼻内siRNA输送到中枢神经系统期间放置小鼠的定位装置

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the ...

免疫与感染

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Previous studies showed that knockdown of ITSN-1s (KDITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition1. Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce2. Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration3Here, efficient and specific KDITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN throughout the study4. Mouse tissues collected at several time points post-siRNAITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

Repeated retro-orbital injections of cationic liposomes/siRNA/ITSN-1s complexes in mice, every 72 hours for 24 days, efficiently deliver the siRNA duplex to the mouse lung microvasculature reducing ITSN-1s mRNA and protein expression by 75%. This technique is highly reproducible in an animal model, has no adverse effects and avoids fatalities.

长期沉默的1S Intersectin的在小鼠肺的特异性siRNA，通过阳离子脂质体重复交货。通过电子显微镜的击倒效果评价

Previous  studies showed that knockdown of ITSN-1s (KDITSN), an endocytic  protein involved in regulating lung vascular permeability and endothelial cells ...

生物学

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central nervous system (CNS). However, its existence also dramatically lowers the accessibility of systemically administered therapeutic agents to the CNS. One method to overcome this, is to inject those agents directly into the cerebrospinal fluid (CSF), thus bypassing the BBB. This can be done via implantation of a catheter for either continuous infusion using an osmotic pump, or for single bolus delivery. In this article, we describe a surgical protocol for delivery of CNS-targeting antisense oligonucleotides (ASOs) via a catheter implanted directly into the cauda equina space of the adult rat spine. As representative results, we show the efficacy of a single bolus ASO intrathecal (IT) injection via this catheterization system in knocking down the target RNA in different regions of the rat CNS. The procedure is safe, effective and does not require expensive equipment or surgical tools. The technique described here can be adapted to deliver drugs in other modalities as well.

Here, we describe a method for delivering drugs to the rat central nervous system by implanting a catheter into the lumbar intrathecal space of the spine. We focus on the delivery of antisense oligonucleotides, though this method is suitable for delivery of other therapeutic modalities as well.

大鼠中枢神经系统中反义寡核苷酸的细胞内传递

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central ...