Name: evaluation of the efficacy and toxicity of rnas targeting hiv-1 production for use in gene or drug therapy
Uploaded: 2016-09-05T14:00:00.000+00:00
Duration: 12 min 3 s
Description: Watch this Scientific Journal Video about Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy at JoVE.com

这里介绍的工作是由卫生研究院（CIHR）加拿大研究院的支持（授予DCB-120266，PPP-133377和HBF-348967到AG）。

1.细胞转染与<ol><li>培养的HEK 293T细胞在Dulbecco改进的Eagle培养基（DMEM）中补充有10％胎牛血清（FBS）和1％青霉素/链霉素。制备在细胞培养基中的2×10 5个细胞/ ml悬浮液。加500，100和1000微升的细胞悬液以每孔24孔，96孔和12孔板，用于病毒生产，细胞生存力和免疫激活测定法，分别为（ 图2A）。 </li><li>轻轻摇动平板，用5％的CO 2孵育他们O / N在37℃。生长细胞50-70％汇合。 </li><li>根据计划，转染，在1.5ml微管（例如， 图2B）准备测试的RNA及其控制的稀释液。 <ol><li>对于病毒生成测定，准备HIV-1表达质粒的10纳克/微升稀释并加入10微升至每管。下一个准备测试RNA的5微米稀释和阴性对照RNA.添加2.5或10微升各试验RNA和阴性对照稀释到相应管25或100nM的最终浓度。 </li><li>对于细胞存活力测定法，制备试验RNA的5微米稀释和10毫克/毫升稀释的阳性对照RNA，低分子量聚I的:C。添加2微升试验RNA或阳性对照RNA稀释至用于分别100nM或200微克/毫升的最终浓度，相应的管中。 </li><li>对于免疫激活法，加入20微升试验RNA或步骤1.3.2准备阳性对照RNA稀释。为分别100nM或200微克/毫升的最终浓度，相应的管中。 注意:对于测试RNA表达质粒，在地方试验RNA的5μM稀释准备10毫微克/微升稀释液，给25和100最终金额纳克步1.3.1。和100纳克的步骤1.3.2。 1.3.3和。 </li></ol></li><li>添加50，25或75微升的DMEM为病毒督促各转管，减税，细胞活力和免疫激活测定，分别。用于病毒生产测定，带来细胞和制备转染管到生物安全水平3（BSL3）实验室下一步骤之前。 </li><li>添加2微升转染试剂依次向转染管和孵育15至20分钟以使配合物形成。见材料的表的具体的转染试剂中使用。 </li><li>从每个微管逐滴向在细胞培养板中的相应位置添加整个转染混合物。轻轻摇动并在37℃用5％CO 2孵育48小时将板。 </li><li>测量在细胞培养板（ 图2C）的HIV-1的生产（第2部分），细胞存活率（第3节）和免疫激活（第4节）。 </li></ol> 2.病毒生成测定<ol><li>除去从培养箱24孔细胞培养板，并轻轻摇动BSL3细胞培养内板引擎盖。转移150微升上清液从每孔在96孔平底板，这将被用于量化的HIV-1的生产相应的井。 注意:普通的病毒定量测定包括测定HIV-1的衣壳蛋白（p24的）通过酶联免疫吸附测定（ELISA）20，量化通过逆转录聚合酶链反应（RT-PCR）21病毒RNA的表达，并测量活性的HIV-1逆转录酶。步骤2.2至2.5解释的方法来量化HIV-1 RT活性13,15,16。 </li><li>转印5微升上清液在一个96孔板的相应孔，含有25微升的病毒破坏鸡尾酒15（ 表1）。在室温下孵育5分钟该混合物，将板转移到放射性工作站。 注:步骤2.2。只有必要的，如果一个放射性工作站不在BSL3实验室提供。如果板不需要从除去BSL3实验室，继续执行步骤2.3。和到位的25微升放射性鸡尾酒的加入50微升放射性/病毒破坏的鸡尾酒（ 表1）。 </li><li>制备放射性鸡尾酒15（ 表1），并添加25微升病毒上清和破坏鸡尾酒的每个孔中。孵育所述板在37℃下2小时。 </li><li>点5微升中的玻璃纤维二乙基氨基乙基（DEAE）滤纸纸，将反应混合物到相应的平方和允许斑点干燥10分钟。现货每隔广场反应混合物中，以便没有两个样品直接房客彼此。这有助于在步骤2.6确定每分钟计数（CPM），以避免样品之间交叉。 </li><li>洗纸5倍5分钟用2×盐水柠檬酸钠（SSC）缓冲液（ 表1），随后是两个1分钟洗涤用95％的乙醇。允许论文干，封住他们的样品袋。 </li><li>剪辑取样袋合作ntaining的纸滤纸成磁带，将磁带插入微板闪烁计数器。设置计数器读取的cpm为32磷与设置在实验中使用的批次[32 P] dTTP各基准日期。选择使用板地图读取和启动计数器进行采样。 </li><li>为任何病毒定量方法中，通过相邻的阴性对照划分为每个测试的RNA得到的值再乘以100这个值来得到的HIV-1生产的抑制百分率的测试RNA的每个重复。比较不同浓度不同的测试的RNA的结果的一个例子在图3中提供。 </li></ol> 3.细胞活力检测<ol><li>从培养箱中取出96孔板，并添加20微升5mg / ml的MTT（3- [4.5二甲基-2-噻唑基] -2,5-二苯基-2H-四唑鎓溴化物）的Dulbecco氏磷酸盐缓冲盐水稀释（ DPBS）到每个孔中。孵育板˚F或在37℃下3小时。 </li><li>添加150微升酸化异丙醇与去污剂（1％NP-40，4毫HCl的异丙醇）至各孔，孵育所述板在室温2小时。 </li><li>确定在微孔板分光光度计570nm处的吸光度。 </li><li>除以由相邻的转染控制每个采样得到的值计算每个阳性对照，检测RNA相对MTT代谢。比较不同测试RNA和阳性对照结果的一个例子在图4中提供。 </li></ol> 4.免疫活化分析<ol><li>从培养箱中取出12孔板并吸出培养基。轻轻用DPBS洗涤细胞两次，并添加70微升的冷裂解缓冲液22（包括蛋白酶和磷酸酶抑制剂， 表1）向每个孔中。孵育在冰上10分钟的板。 </li><li>转移细胞裂解液微管浸入快速冻结他们管在液氮中。允许样品解冻，并重复2更冻融循环，共3。 </li><li>离心15分钟的裂解物在4℃，15700 XG，以沉淀细胞碎片。将上清转移到新的微管中，并确定使用考马斯蓝（布拉德福德）方法23,24蛋白质浓度。 </li><li>解决75微克蛋白的从每个样品在10％变性聚丙烯酰胺凝胶并转移到硝酸纤维素膜上如前所述25,26。 </li><li>以下电泳并转移到膜上，通过丽春红S（ 表1）为1分钟，随后在双蒸馏水洗涤孵育膜揭示蛋白质带。使用带和蛋白质梯作为指导，在80和55 kDa的切割膜。 注意:在步骤4.4的样品可以在16×18 CM短运行2凝胶至34 kDa的，所以，它很容易在规定的位置上切割膜，并通过不切感兴趣的乐队。可替代地，多个凝胶可以用相同的样品来运行，以避免切割膜。 </li><li>洗出用含有0.05％吐温20（TBST， 表1）的Tris缓冲盐水的Ponceau S染色。添加TBST用5％无脂牛奶以完全覆盖膜。孵育在RT膜搅拌1小时。 </li><li>孵育膜O / N在TBST用3％牛血清白蛋白（BSA）和抗体在1000稀释到1对ADAR1（110和150 kDa的），磷酸PKR（62 kDa的），和磷酸化IRF3（47 kDa的），用于分别的顶部，中部和底部片膜。 </li><li>洗涤该膜5倍5分钟用TBST和在TBST用5％无脂奶和过氧化物酶标记的山羊抗兔次级抗体（在5000稀释到1）1小时孵育。 </li><li>洗涤该膜5×5分钟用TBST和应用电化学发光（ECL）溶液可视化上，根据制造商的说明电影的频带。 </li><li>可视化上薄膜的蛋白质条带后，洗膜的中部和底部件10分钟与抗体反萃液。孵育针对总PKR膜O / N在TBST用3％BSA和抗体（在500 1）和IRF3（在1000 1），用于分别的中部和底部件。重复步骤4.8。和4.9。使用过氧化物酶标记的山羊抗小鼠代替用于PKR膜（中）的抗 - 兔二级抗体。 </li><li>洗底部片膜5倍的5分钟用TBST和用抗肌动蛋白的抗体（在5000稀释到1）孵育，用3％BSA的TBST将膜1小时。重复步骤4.8。和4.9。使用过氧化物酶标记的山羊抗小鼠代替抗兔二级抗体。比较不同测试RNA和阳性对照结果的一个例子在图5中提供。参见材料的表中使用的特异性抗体。 </li></ol>

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F., Gatignol, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+translation+and+its+regulation+by+cellular+factors+PKR+and+PACT.">HIV-1 translation and its regulation by cellular factors PKR and PACT.</a> Virus Res. 193, 65-77 (2014).</li><li>ter Brake, O., Konstantinova, P., Ceylan, M., Berkhout, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+of+HIV-1+with+RNA+interference:+a+multiple+shRNA+approach.">Silencing of HIV-1 with RNA interference: a multiple shRNA approach.</a> Mol Ther. 14, 883-892 (2006).</li><li>Naito, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+design+and+validation+of+antiviral+siRNA+for+targeting+HIV-1.">Optimal design and validation of antiviral siRNA for targeting HIV-1.</a> Retrovirology. 4, 80 (2007).</li><li>Mandal, D., Feng, Z., Stoltzfus, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excessive+RNA+splicing+and+inhibition+of+HIV-1+replication+induced+by+modified+U1+small+nuclear+RNAs.">Excessive RNA splicing and inhibition of HIV-1 replication induced by modified U1 small nuclear RNAs.</a> J Virol. 84, 12790-12800 (2010).</li><li>Chang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+expression+and+HIV-1+inhibition+of+chimeric+tRNA(Lys3)-ribozymes+under+dual+U6+snRNA+and+tRNA+promoters.">Enhanced expression and HIV-1 inhibition of chimeric tRNA(Lys3)-ribozymes under dual U6 snRNA and tRNA promoters.</a> Mol Ther. 6, 481-489 (2002).</li><li>Chang, L. J., Liu, X., He, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+siRNAs+targeting+multiple+highly+conserved+RNA+sequences+of+human+immunodeficiency+virus+type+1.">Lentiviral siRNAs targeting multiple highly conserved RNA sequences of human immunodeficiency virus type 1.</a> Gene Ther. 12, 1133-1144 (2005).</li><li>Daniels, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+RRE+RNA+acts+as+an+RNA+silencing+suppressor+by+competing+with+TRBP-bound+siRNAs.">HIV-1 RRE RNA acts as an RNA silencing suppressor by competing with TRBP-bound siRNAs.</a> RNA Biol. 12, 123-135 (2015).</li><li>Castanotto, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+delivery+of+small+interfering+RNAs+reduces+RNAi+efficacy+by+selective+incorporation+into+RISC.">Combinatorial delivery of small interfering RNAs reduces RNAi efficacy by selective incorporation into RISC.</a> Nucleic Acids Res. 35, 5154-5164 (2007).</li><li>Vickers, T. A., Sabripour, M., Crooke, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=U1+adaptors+result+in+reduction+of+multiple+pre-mRNA+species+principally+by+sequestering+U1snRNP.">U1 adaptors result in reduction of multiple pre-mRNA species principally by sequestering U1snRNP.</a> Nucleic Acids Res. 39, e71 (2011).</li><li>Tai, C. J., Li, C. L., Tai, C. J., Wang, C. K., Lin, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Viral+Entry+Assays+for+the+Identification+and+Evaluation+of+Antiviral+Compounds.">Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds.</a> J Vis Exp. , (2015).</li><li>Riss, T. L., et al., Sittampalam, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Assay Guidance Manual. , (2004).</li><li>Reynolds, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+the+interferon+response+by+siRNA+is+cell+type-+and+duplex+length-dependent.">Induction of the interferon response by siRNA is cell type- and duplex length-dependent.</a> Rna. 12, 988-993 (2006).</li><li>Whitehead, K. A., Dahlman, J. E., Langer, R. S., Anderson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+or+stimulation?+siRNA+delivery+and+the+immune+system.">Silencing or stimulation? siRNA delivery and the immune system.</a> Annu Rev Chem Biomol Eng. 2, 77-96 (2011).</li></ol>

The authors have nothing to disclose.

用HEK293T细胞（ 图2）进行描述的HIV-1的生产法和类似用于筛选HIV-1RNA有效核酶13，shRNA的10,29，siRNA的30，和U1i的RNA 11,31靶位点测定。使用不同的方法来量化的HIV-1的生产，大多数研究已经测量的病毒生产48小时与候选的RNA的HIV-1的表达质粒共转染之后。继生产HIV-1的，不成熟的病毒粒子经历的多聚蛋白的蛋白裂解由HIV-1蛋白酶变得成熟的病毒粒子，能够感染...

的当前的HIV-1治疗的局限性在于，它们必须长期施用，以防止疾病的进展。 HIV-1的抗T淋巴细胞，或造血干细胞的移植，有可能提供HIV-1复制的长期控制在没有药物治疗1,2的电位，也可以是实现一种HIV-1治疗的有效方法3。渲染到HIV-1复制抗性细胞的一种方法是自体移植4中插入一个或多个基因编码抗-HIV-1的RNA或肽进入受感染个体的细胞。几个候选抗HIV-1基因已被设计有一些进入临床试验在两个5或3个6的组合，以防止HIV-1的抗性的发展的任何单一基因。 抗HIV-1 RNA的顶端候选组合的基因治疗中，由于它们的低电势以引起免疫反应和，因为它们从很短的基因序列转录。一些抗HIV-1的RNA已被设计为靶向病毒进入和整合。然而，大多数的抗HIV-1的RNA靶向病毒生命周期积分后步骤（ 图1）。后的整合酶抑制剂包括诱饵的RNA，针对HIV-1调控蛋白达或REV 1，和基于反义RNA的，在HIV-1 RNA针对不同的网站，如核酶7，8的shRNA的U1i和RNA的9。已用于比较的抗HIV-1 RNA的功效的方法包括监测病毒复制与编码基因候选RNA和测定用表达候选RNA的质粒瞬时转染细胞的病毒的生产和HIV-1的表达质粒转染细胞10 -13。我们先前使用过的HIV-1的生产试验，筛选新的核酶靶位点13-15 HIV-1 RNA。这些方法，至今已细化到优化的RNA的格式干扰分子从质粒DNA表达为一个shRNA的或作为合成的siRNA 16传递。该测定测量生产的人胚胎肾（HEK）293T细胞成熟的病毒，并且可以用于比较针对在HIV-1复制周期积分后步骤（ 图1）抑制剂的影响。对于面向预集成的步骤抑制剂，需要替代的试验，如TZM-BL细胞感染性测定17来评价抗病毒疗效。 在临床的抗HIV-1 RNA的递送主要的安全问题包括对人类RNA或蛋白质，以及天然免疫传感器的激活潜在的脱靶效应。为了评估抗HIV-1 siRNA的毒性，我们使用在不同的细胞系16的细胞活力测定。我们还测量了双链RNA的免疫传感器的激活，RNA活化蛋白激酶R（PKR）和Toll样受体3（TLR3），以及移干扰素的PRESSION刺激基因，ADAR1 P150。这些测定可用于证实抗HIV-1 RNA的效力不因对细胞活力或免疫传感器活化间接影响。他们也是不包括候选人的RNA与进一步发展的潜在的毒性非常有用。 在下面的协议，过程，以确定新的治疗RNA和优化现有的格式被描述。该方法可用于HIV-1复制的筛选基于RNA后整合抑制剂和可适于筛选其他集成后抑制剂如小分子靶向启介导的病毒RNA 18或CRISPR的出口/设计CAS大全MSDS系统到目标集成HIV-1 DNA 19。

<table><tbody><tr><td>DMEM HyClone</td><td>GE Healthcare</td><td>SH30243.01</td><td></td></tr><tr><td>FBS HyClone</td><td>GE Healthcare</td><td>SH30396.03</td><td></td></tr><tr><td>Penicillin/Streptomycin Gibco</td><td>Thermo Fisher</td><td>15140-122</td><td></td></tr><tr><td>Cell culture plates, 96 well, 24 well, 6 well.</td><td>Corning</td><td>353075, 353047, 353043</td><td></td></tr><tr><td>Micro tubes Axygen</td><td>Corning</td><td>311-08-051</td><td></td></tr><tr><td>Low molecular weight Poly I:C</td><td>InvivoGen</td><td>3182-29-6</td><td></td></tr><tr><td>DharmaFECT-1</td><td>Dharmacon</td><td>T-2001-01</td><td>transfection reagent for synthetic RNAs</td></tr><tr><td>TransIT-LT1</td><td>Mirus</td><td>MIR 2300</td><td>transfection reagent for RNA expression plasmids</td></tr><tr><td>Nonidet P40 (NP-40)</td><td>USB</td><td>19628</td><td></td></tr><tr><td>[&lt;sup&gt;32&lt;/sup&gt;P]dTTP</td><td>Perkin Elmer</td><td>BLU505H</td><td></td></tr><tr><td>poly(A) RNA template&amp;nbsp;</td><td>Sigma-Aldrich</td><td>10108626001</td><td></td></tr><tr><td>oligo(dT)&lt;sub&gt;12-18&lt;/sub&gt; DNA primer</td><td>Thermo Fisher</td><td>18418-012</td><td></td></tr><tr><td>DEAE filtermat paper&amp;nbsp;</td><td>Perkin Elmer</td><td>1450-522</td><td></td></tr><tr><td>Microplate scintillation counter</td><td>Perkin Elmer</td><td>1450-024</td><td></td></tr><tr><td>MTT</td><td>Sigma-Aldrich</td><td>M-2128</td><td></td></tr><tr><td>DPBS HyClone</td><td>GE Healthcare</td><td>SH30028.02</td><td></td></tr><tr><td>Microplate spectrophotometer</td><td>Bio-rad</td><td>1706930</td><td></td></tr><tr><td>Lysis buffer tablets</td><td>Roche</td><td>4693159001, 4906837001</td><td>protease and phosphatase inhibitors</td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>5415R</td><td></td></tr><tr><td>Bradford reagent</td><td>Bio-rad</td><td>500-0006</td><td></td></tr><tr><td>Gel running chamber</td><td>Hoefer</td><td>SE600</td><td></td></tr><tr><td>Semi-dry transfer cell</td><td>Bio-rad</td><td>1703940</td><td></td></tr><tr><td>Protein ladder EZ-Run</td><td>Thermo Fisher</td><td>BP3603-500</td><td></td></tr><tr><td>Nitrocellulose membrane</td><td>Bio-rad</td><td>162-0094</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A9647-1006</td><td></td></tr><tr><td>Antibody stripping solution</td><td>Millipore</td><td>2504</td><td></td></tr><tr><td>ECL - Pierce</td><td>Thermo Fisher&amp;nbsp;</td><td>PI32106</td><td></td></tr><tr><td>ADAR1 antibody</td><td></td><td></td><td>from Dr. B.L. Bass</td></tr><tr><td>phospho-T446-PKR antibody</td><td>Abcam</td><td>ab32036</td><td></td></tr><tr><td>phospho-S396-IRF3 antibody</td><td>Cell Signaling</td><td>4947</td><td></td></tr><tr><td>PKR antibody</td><td></td><td></td><td>from Dr. A. Hovanessian</td></tr><tr><td>IRF3 antibody</td><td>Cell Signaling</td><td>11904</td><td></td></tr><tr><td>Actin antibody</td><td>Millipore</td><td>MAB1501</td><td></td></tr><tr><td>Peroxidase-labeled goat anti-rabbit</td><td>KPL</td><td>474-1506</td><td></td></tr><tr><td>Peroxidase-labeled goat anti-mouse</td><td>KPL</td><td>474-1806</td><td></td></tr><tr><td>Ponceau S&amp;nbsp;</td><td>Sigma-Aldrich</td><td>6226-79-5</td><td></td></tr></tbody></table>

evaluation of the efficacy and toxicity of rnas targeting hiv-1 production for use in gene or drug therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

的程序的一般示意性示出在图2中为三个测试RNA和在图2B中提供了一个对照RNA的一例的转染方案。用于病毒生产和细胞生存力测定法，所读出的每个测试构建体是标准化为阴性对照。重复转染在集，使得每个测试RNA被归一化到其相邻的阴性对照。这样做是为了避免与配位和转染之间的时间，这可能会导致如果，例如，所有的阴性对照的是第一转染不...

Watch this Scientific Journal Video about Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy at JoVE.com

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

evaluation-efficacy-toxicity-rnas-targeting-hiv-1-production-for-use

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

7.8K 次观看.  Lady Davis Institute for Medical Research. Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones. 

视频: Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Here we describe a protocol for employing high-throughput RNAi screening to uncover host targets that can be manipulated to enhance oncolytic virus therapy, specifically rhabodvirus and vaccinia virus therapy, but it can be readily adapted to other oncolytic virus platforms or for discovering host genes that modulate virus replication generally.

全基因组的 rna 干扰筛查以确定调节溶瘤病毒治疗的宿主因素

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus ...

科研

癌症研究

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

发展的细胞类型特异性的抗艾滋病毒gp120基因的siRNA传递适配

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

免疫与感染

DNA Vector-based RNA Interference to Study Gene Function in Cancer

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing1, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible2,3. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression. 
We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. 
Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer4,5, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution.

RNA interference (RNAi) possesses many advantages over gene knockout and has been broadly used as a tool in gene functional studies. The invention of DNA vector-based RNAi technology has made long term and inducible gene knockdown possible, and also increased the feasibility of gene silencing in vivo.

基于DNA载体的RNA干扰在癌症研究基因功能

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA ...

医学

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

高通量筛选RNA病毒的广谱化学抑制剂

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

HIV逆转录酶和整合酶抑制剂的快速筛选

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter &beta;1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

细菌提供的RNAi效应：Transkingdom RNA干扰

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

生物学

MicroRNA-based Regulation of Picornavirus Tropism

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and understanding the biological properties of viruses. There are several mechanisms for regulating viral tropism, however they tend to be virus class specific and many result in virus attenuation. Additionally, many viruses, including picornaviruses, exhibit size constraints that do not allow for incorporation of large amounts of foreign genetic material required for some targeting methods. MicroRNAs are short, non-coding RNAs that regulate gene expression in eukaryotic cells by binding complementary target sequences in messenger RNAs, preventing their translation or accelerating their degradation. Different cells exhibit distinct microRNA signatures and many microRNAs serve as biomarkers. These differential expression patterns can be exploited for restricting gene expression in cells that express specific microRNAs while maintaining expression in cells that do not. In regards to regulating viral tropism, sequences complementary to specific microRNAs are incorporated into the viral genome, generally in the 3' non-coding regions, targeting them for destruction in the presence of the cognate microRNAs thus preventing viral gene expression and/or replication. MicroRNA-targeting is a technique that theoretically can be applied to all viral vectors without altering the potency of the virus in the absence of the corresponding microRNAs. Here we describe experimental methods associated with generating a microRNA-targeted picornavirus and evaluating the efficacy and specificity of that targeting in vitro. This protocol is designed for a rapidly replicating virus with a lytic replication cycle, however, modification of the time points analyzed and the specific virus titration readouts used will aid in the adaptation of this protocol to many different viruses.

We describe here a method for regulating picornavirus tropism by incorporating sequences complementary to specific microRNAs into the viral genome. This protocol can be adapted to all different classes of viruses with modifications based upon the length and nature of their life cycle.

小核糖核酸病毒取向的微小RNA基于调控

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and ...

High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune system and is not targeted by the current antiretroviral therapy (cART). Transcription and splicing have been shown to reinforce HIV-1 latency in resting CD4+ T cells. Reversal of latency by the use of latency reversal agents (LRAs) in the &#34;shock and kill&#34; approach has been studied extensively in an attempt to purge this reservoir but has thus far not shown any success in clinical trials due to the lack of development of adequate small molecules that can efficiently perturb this reservoir. The protocol presented here provides a method for reliably and efficiently assessing latency reversal agents (LRAs) on HIV transcription and splicing. This approach is based on the use of an LTR-driven dual color reporter that can simultaneously measure the effect of an LRA on transcription and splicing by flow cytometry. The protocol described here is adequate for adherent cells as well as the cells in suspension. It is useful for testing a large number of drugs in a high throughput system. The method is technically simple to implement and cost-effective. In addition, the use of flow cytometry allows the assessment of cell viability and thus drug toxicity at the same time.

A high throughput protocol for functional assessment of HIV efficient reactivation and clearance of latent proviruses is described and applied by testing the impact of interventions on HIV transcription and splicing. Representative results of the effect of latency reversing agents on LTR-driven transcription and splicing are provided.

hiv 转录和拼接中延迟逆转剂的高通量体外评价

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

荧光检测病毒RNA<em&gt;原位</em&gt;杂交（FISH）

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Oral Combinational Antiretroviral Treatment in HIV-1 Infected Humanized Mice

The human immunodeficiency virus (HIV-1) pandemic continues to spread unabated worldwide, and currently, there is no vaccine available against HIV. Although combinational antiretroviral therapy (cART) has been successful in suppressing viral replication, it cannot completely eradicate the reservoir from HIV-infected individuals. A safe and effective cure strategy for HIV infection will require multipronged methods, and therefore the advancements of animal models for HIV-1 infection are pivotal for the development of HIV cure research. Humanized mice recapitulate key features of HIV-1 infection. The humanized mouse model can be infected by HIV-1 and viral replication can be controlled with cART regimens. Moreover, cART interruption results in a prompt viral rebound in humanized mice. However, administration of cART to the animal can be ineffective, difficult, or toxic, and many clinically relevant cART regimens are unable to be optimally utilized. Along with being potentially unsafe for researchers, administration of cART by a commonly used intensive daily injection procedure induces stress by physical restraint of the animal. The novel oral cART method to treat HIV-1 infected humanized mice described in this article resulted in suppression of viremia below the detection level, increased rate of CD4+ restoration, and improved overall health in HIV-1 infected humanized mice.

This protocol describes a novel method to deliver oral combinational antiretroviral drugs that successfully suppress HIV-1 RNA replication in humanized mice.

口服联合抗逆转录病毒治疗HIV-1感染人源化小鼠

The human immunodeficiency virus (HIV-1) pandemic continues to spread unabated worldwide, and currently, there is no vaccine available against HIV. ...