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

O trabalho aqui apresentado foi apoiado pelos Institutos Canadenses de Pesquisa em Saúde (CIHR) (concede DCB-120.266, PPP-133377 e HBF-348967 para AG).

1. Células e transfecções <ol><li> Culturas de células HEK 293T em meio de Eagle modificado por Dulbecco (DMEM) suplementado com 10% de soro fetal de bovino (FBS) e 1% de penicilina / estreptomicina. Prepara-se uma suspensão de 2 x 10 5 células / mL no meio de cultura celular. Adicionar 500, 100 e 1.000 mL da suspensão de células a cada poço de 24 poços, 96 poços e 12 poços placas, para a produção virai, a viabilidade celular e ensaios de activação imunitária, respectivamente (Figura 2A). </li><li> Agite suavemente as placas e incubar O / N a 37 ° C com 5% de CO 2. Cultivar células a 50-70% de confluência. </li><li> De acordo com um plano de transfecção, preparar diluições de ARN de teste e os controlos em tubos de 1,5 ml (micro exemplo, a Figura 2B). <ol><li> Para o ensaio de produção viral, preparar uma diluição de 10 ng / ul de um plasmídeo de expressão de HIV-1 e adicionar 10 uL a cada tubo. Próximo preparar 5 uM diluições de ARN de teste e um controlo negativo RNA. Adicionar 2,5 ou 10 ul de cada diluição de ARN de teste e de controlo negativo aos tubos correspondentes para 25 ou 100 nM de concentração final. </li><li> Para o ensaio de viabilidade de células, preparar 5 uM diluições de ARN e um teste de 10 mg / ml de diluição do ARN de controlo positivo, de baixo peso molecular, poli I: C. Adicionar 2 mL de ARN de teste, ou de diluições de ARN controlo positivo para os correspondentes tubos de 100 nM ou 200 ug / ml de concentração final, respectivamente. </li><li> Para o ensaio de activação imunitária, adicionar 20 ul de ARN de teste, ou de diluições de ARN de controlo positivo, preparado no passo 1.3.2. aos tubos correspondentes para 100 nm ou 200 ug / ml de concentração final, respectivamente. Nota: Para os plasmídeos de expressão de ARN de teste, preparar diluições de 10 ng / ul em vez dos 5 uM diluições de ARN de teste, dando quantidades finais de 25 e 100 ng para o passo 1.3.1. e 100 ng para etapas 1.3.2. e 1.3.3. </li></ol></li><li> Adicionam-se 50, 25 ou 75 ul de DMEM a cada tubo de transfecção para prod viralução, a viabilidade celular e ensaios de activação imunitária, respectivamente. Para os ensaios de produção virais, trazem células e tubos de transfecção preparados para a 3 (BSL3) laboratório nível bio-segurança antes da próxima etapa. </li><li> Adicionar 2 ml da sequencialmente reagente de transfecção para os tubos de transfecção e incubar 15 min a 20 para permitir que os complexos de modo a formar. Ver a Tabela de Materiais para o reagente de transfecção específico a ser utilizado. </li><li> Adicionar toda a mistura de transfecção de cada gota a gota, para as posições correspondentes nas placas de cultura de células micro tubo. Agite suavemente e incuba-se as placas durante 48 h a 37 ° C com 5% de CO 2. </li><li> Meça HIV-1 de produção (seção 2), a viabilidade celular (seção 3) e ativação imune (secção 4) nas placas de cultura de células (Figura 2C). </li></ol> 2. Ensaio de produção virai <ol><li> Remover as placas de cultura celular de 24 poços da incubadora e agitar suavemente as placas no interior da cultura de células BSL3capuz. Transferir 150 ul de sobrenadante de cada poço para um poço correspondente de uma placa de 96 poços de fundo plano, que vai ser utilizado para quantificar a produção de HIV-1. Nota: Os ensaios de quantificação virais mais comuns incluem a medição da expressão da proteína da cãpside de HIV-1 (p24) pelo ensaio imunossorvente ligado a enzima (ELISA) 20, a quantificação de ARN viral por transcrição reversa de reacção em cadeia da polimerase (RT-PCR) 21 e medindo a actividade da enzima RT HIV-1. Passos 2.2 a 2.5 explicar métodos para quantificar HIV-1 actividade RT 13,15,16. </li><li> Transferência de 5 ul de sobrenadante para poços correspondentes de uma placa de 96 poços, contendo 25 ul de uma perturbação viral cocktail 15 (Tabela 1). Incubar a mistura durante 5 minutos à temperatura ambiente e transferir a placa para uma estação de trabalho radioactividade. Nota: Passo 2.2. só é necessário se uma estação de trabalho de radioactividade que não está disponível no laboratório BSL3. Se as placas não precisam de ser removidos dolaboratório BSL3, vá para o passo 2.3. e adicionar 50 ul de radioactivos / viral cocktail ruptura (Tabela 1) em lugar dos 25 ul de coquetel radioactivos. </li><li> Prepara-se uma radioactivos cocktail 15 (Tabela 1), e adicionar 25 ul a cada poço de sobrenadante viral e cocktail de interrupção. Incubar as placas a 37 ° C durante 2 h. </li><li> Ponto 5 uL da mistura de reacção sobre quadrados correspondentes de uma fibra de vidro Di Etil amino-etil (DEAE) de papel filtermat e deixar que as gotas para secar durante 10 min. Manchar a mistura de reacção em todos os outros quadrados de modo que não há duas amostras Boarder diretamente um ao outro. Isto ajuda a evitar o cruzamento entre amostras para determinar as contagens por minuto (cpm) no passo 2.6. </li><li> Lavar os papéis 5x durante 5 min com tampão de 2x citrato de sódio salino (SSC) (Tabela 1), seguido por duas lavagens com um mínimo de 95% de etanol. Permitir que os papéis para secar e selá-los em sacos de amostras. </li><li> Clip sacos de amostras containing do papel filtermat em uma fita e insira a cassete em um contador de cintilação de microplacas. Definir o contador para ler cpm de 32 P com a data de referência prevista para o lote de [32 P] dTTP utilizado na experiência. Selecionar quais amostras de ler usando o mapa da placa e iniciar o contador. </li><li> Para qualquer método de quantificação viral, dividir os valores obtidos para cada teste de ARN pelo controlo negativo adjacente e multiplicar este valor por 100 para obter a percentagem de inibição da produção do VIH-1 para cada repetição dos ARN de teste. Um exemplo dos resultados que comparam diferentes RNAs de teste em diferentes concentrações é fornecido na Figura 3. </li></ol> 3. ensaio de viabilidade celular <ol><li> Remover as placas de 96 poços a partir da incubadora e adicionar 20 uL de 5 mg / ml de MTT (brometo de 3- [4,5-dimetil-2-tiazolil] -2,5-difenil-2H-tetrazólio), diluído em solução salina tamponada com fosfato de Dulbecco ( DPBS) a cada poço. Incubar as placas de fou 3 horas a 37 ° C. </li><li> Adicionar 150 uL de isopropanol, acidificado com detergente (1% de NP-40, de HCl 4 mM em isopropanol) a cada poço e incuba-se as placas durante 2 horas à temperatura ambiente. </li><li> Determinar a absorvância a 570 nm num espectrofotómetro de microplacas. </li><li> Calcular o metabolismo de MTT relativo para cada ARN de controlo e de teste positivo, dividindo o valor obtido para cada amostra pelo seu controlo de transfecção adjacente. Um exemplo dos resultados que comparam diferentes RNAs de teste e um controlo positivo é fornecida na Figura 4. </li></ol> 4. Activação Ensaio Imunológico <ol><li> Remover as placas de 12 poços a partir da incubadora e aspirar o meio de cultura. Lavar suavemente as células duas vezes com DPBS e adicionar 70 ul de tampão de lise frio de 22 (incluindo os inibidores de protease e fosfatase, quadro 1) a cada poço. Incubar as placas durante 10 min em gelo. </li><li> Transferir os lisados ​​celulares para micro tubos e rápido congelá-los por imersão dotubos em azoto líquido. Permitir que as amostras para descongelar e repetir por mais 2 ciclos de congelação descongelação para um total de 3. </li><li> Centrifugar os lisados ​​durante 15 minutos a 4 ° C, 15700 x g, para sedimentar os detritos celulares. Transferir o sobrenadante para novos tubos de micro e determinar a concentração de proteína usando o método de azul de Coomassie (Bradford) 23,24. </li><li> Resolver 75 ug de proteína de cada amostra num gel de poliacrilamida desnaturante a 10% e transferir para uma membrana de nitrocelulose como anteriormente descrito 25,26. </li><li> Após electroforese e transferência para uma membrana, revelam bandas de proteína por incubação da membrana em Ponceau S (Tabela 1) durante 1 min seguido de lavagem em água destilada duas vezes. Use as bandas e escada proteína como um guia para cortar a membrana em 80 e 55 kDa. Nota: No passo 4.4 amostras pode ser executado em 16 x 18 cm 2 géis para baixo a 34 kDa, de modo que é fácil de cortar a membrana nas posições especificadas e não cortar através dabandas de interesse. Alternativamente, vários géis podem ser executados com as mesmas amostras para evitar o corte da membrana. </li><li> Lavar a coloração de Ponceau S com solução salina tamponada com Tris contendo 0,05% de Tween 20 (TBST, Tabela 1). Adicionar TBST com leite não gordo a 5% para cobrir completamente as membranas. Incubam-se as membranas à temperatura ambiente com agitação durante 1 h. </li><li> Incubam-se as membranas de O / N em TBST, com 3% de albumina de soro bovino (BSA) e anticorpos diluídos a 1: 1000 contra ADAR1 (110 e 150 kDa), fosfo-PKR (62 kDa), e fosfo-IRF3 (47 kDa), para as peças de topo, meio e inferior da membrana, respectivamente. </li><li> Lavam-se as membranas 5x durante 5 min com TBST e incubar em TBST com 5% de leite sem gordura e os anticorpos secundários anti-coelho de cabra marcado com peroxidase (diluído a 1: 5000) durante 1 h. </li><li> Lava-se a membranas de 5x durante 5 min com TBST e aplicar a solução de electroquimioluminescência (ECL) para visualizar as bandas em películas de acordo as instruções do fabricante. </li><li> Depois de visualizar as bandas de proteína sobre os filmes, lavar as peças do meio e inferior da membrana durante 10 minutos com uma solução de anticorpo de extracção. Incubam-se as membranas de o / n em TBST, com 3% de BSA e anticorpos contra PKR total de (a 1 em 500) e IRF3 (a 1 em 1000), para o meio e pedaços de fundo, respectivamente. Repita os passos 4.8. e 4,9. usando marcado com peroxidase de cabra anti-ratinho em vez do anticorpo secundário anti-coelho para a membrana PKR (meio). </li><li> Lava-se a peça de fundo da membrana 5x durante 5 min com TBST e incubar a membrana em TBST, com 3% de BSA durante 1 hora com um anticorpo contra actina (diluído a 1: 5000). Repita os passos 4.8. e 4,9. usando marcado com peroxidase de cabra anti-ratinho em vez do anticorpo secundário anti-coelho. Um exemplo de resultados comparando diferentes RNAs de teste e um controlo positivo é fornecido na Figura 5. Veja a Tabela de Materiais para os anticorpos específicos a serem utilizados. </li></ol>

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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.

O ensaio de produção de HIV-1 descrita foi realizada utilizando células HEK293T (Figura 2) e é semelhante para os ensaios utilizados para rastrear ARN de HIV-1 para ribozima eficaz 13, shRNA 10,29, ARNsi 30, e U1i locais de ARN alvo 11,31. Usando diferentes métodos para quantificar a produção de HIV-1, a maioria dos estudos mediram a produção virai de 48 h após a co-transfecção de um plasmídeo de expressão do VIH-1 com RNAs candidatos. Após a pr...

Uma limitação dos actuais tratamentos do HIV-1 é que eles devem ser cronicamente administrado para prevenir a progressão da doença. Transplantação de VIH-1 resistente a linfócitos T, ou células-tronco hematopoiéticas, tem o potencial para proporcionar um controlo de longa duração de replicação do HIV-1 na ausência do tratamento medicamentoso 1,2 e pode também ser uma abordagem eficaz para atingir uma cura de HIV-1 3. Uma maneira de tornar as células resistentes ao HIV-1 é a replicação para inserir um ou mais genes que codificam para anti-HIV-1 RNA ou péptidos em células de um indivíduo infectado durante um transplante autólogo 4. Vários genes candidatos anti-VIH-1 foram concebidos com alguns estudos clínicos que entram em combinações de dois ou três 5 6, para prevenir o desenvolvimento de resistência do HIV-1 para um único gene. Anti-HIV-1 RNA estão entre os principais candidatos para a terapia de combinação de genes devido ao seu baixo potencial para provocar respostas imunes e porquesão transcritas a partir de sequências de genes muito curtos. Alguns-HIV-1 anti RNAs foram projetados para direcionar entrada e de integração viral. No entanto, a maioria dos ARNs anti-HIV-1 como alvo passos pós-integração no ciclo de vida viral (Figura 1). Inibidores pós-integração incluem RNAs de engodo, tendo como alvo os HIV-1 proteínas reguladoras Tat ou Rev 1, e RNAs à base de anti-senso, tendo como alvo locais diferentes em HIV-1 RNA, como as ribozimas 7, 8 e shRNAs U1i RNAs 9. Os métodos que foram utilizados para comparar a eficácia dos anti-HIV-1-RNA incluem a monitorização de replicação virai nas células transduzidas com genes que codificam para os ARN candidatas e medindo a produção virai em células transitoriamente transfectadas com plasmídeos que expressam os ARN candidatas e um plasmídeo de expressão de HIV-1 10 -13. Nós já usou um ensaio de produção HIV-1 para a tela HIV-1 RNA para sites de destino nova ribozimas 13-15. Estes métodos já foram refinados para optimizar o formato de um ARNmolécula de interferência expressa a partir de DNA de plasmídeo como um shRNA ou entregue como um siARN sintética 16. O ensaio mede a produção de vírus maduras a partir de células 293T de rim embrionário humano (HEK), e pode ser usado para comparar os efeitos dos inibidores que têm como alvo as etapas de pós-integração no ciclo de replicação do HIV-1 (Figura 1). Para os inibidores que têm como alvo as etapas de pré-integração, ensaios alternativos, tais como um ensaio de infecciosidade de células TZM BL-17 são necessários para avaliar a eficácia antiviral. As principais preocupações de segurança para a entrega de anti-HIV-1 RNA na clínica incluem potenciais efeitos fora do alvo em RNAs humanos ou proteínas, e ativação de sensores imune inata. Para avaliar a toxicidade do anti-HIV-1 siRNAs, utilizou-se um ensaio de viabilidade celular em diferentes linhas celulares 16. Nós também medido activação dos sensores imunes ARN de cadeia dupla, ARN activada proteína quinase R (PKR) e Toll like receptor 3 (TLR3), bem como expressão do gene de interferão estimulada, p150 ADAR1. Estes ensaios podem ser utilizados para confirmar que a eficácia do anti-HIV-1 RNA não é devido aos efeitos indirectos sobre a viabilidade celular ou a activação do sensor imune. Eles são também úteis na exclusão com RNAs candidatos potenciais toxicidades de desenvolvimento posterior. Nos seguintes protocolos, os procedimentos para identificar novos RNAs terapêuticos e optimizar o formato das existentes são descritos. Os métodos são úteis para inibidores pós-integração baseadas em ARN rastreio de HIV-1 da replicação e pode ser adaptado para o rastreio de outros inibidores pós-integração, tais como pequenas moléculas de direccionamento Rev exportação mediada por ARN viral 18 ou CRISPR / sistemas Cas concebidos para direccionar integrada 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.

Um esquema geral dos processos é mostrado na Figura 2, com um plano de transfecção exemplo para três RNAs de teste e um ARN de controlo fornecida na Figura 2B. Para os ensaios de viabilidade celular de produção e virais, a leitura para cada construto de teste é normalizada para um controlo negativo. Os duplicados estão transfectadas em conjuntos, de modo que cada ARN teste é normalizado para o seu controlo negativo adjacente. Isto é feito para ...

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.

Avaliação da eficácia e toxicidade de RNAs Segmentação HIV-1 Produção para uso em Gene ou Quimioterapia

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

Research

JoVE Journal

Immunology and Infection

7.8K visualizações.  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. 

Vídeo: 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.

Rastreio de RNAi todo o genoma para identificar fatores do hospedeiro que modulam o vírus Oncolíticos terapia

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

Pesquisa do câncer

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.

Desenvolvimento de células de tipo específico anti-HIV gp120 aptâmeros para entrega siRNA

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

Imunologia e Infecção

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.

DNA Vector baseado em RNA de interferência para estudar a função Gênica em Câncer

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

Medicina

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.

Triagem de alta capacidade para amplo espectro químico Inibidores de Vírus RNA

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.

Triagem rápida de HIV transcriptase reversa e integrase Inibidores

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.

Entrega bacteriana de 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 ...

Biologia

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.

Regulamento baseada MicroRNA de Picornavirus tropismo

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.

Alto Throughput em Vitro avaliação de latência invertendo agentes na transcrição de HIV e de emenda

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.

Detecção de RNA viral por fluorescência<em&gt; In situ</em&gt; Hybridization (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.

Tratamento Antirretroviral Combinacional Oral em Camundongos Humanizados Infectados pelo HIV-1

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