El trabajo presentado aquí fue apoyada por los Institutos Canadienses de Investigación en Salud (CIHR) (otorga DCB-120266, PPP-133377 y 348967 a HBF-AG).

1. Las células y transfecciones <ol><li> Cultura células HEK 293T en medio Eagle modificado de Dulbecco (DMEM) suplementado con 10% de suero fetal bovino (FBS) y 1% de penicilina / estreptomicina. Preparar una suspensión 2 x 10 5 células / ml en el medio de cultivo celular. Añadir 500, 100 y 1.000 l de la suspensión celular a cada pocillo de 24 pocillos, placas de 96 pocillos y 12 pocillos, para la producción viral, la viabilidad celular y ensayos de activación inmune, respectivamente (Figura 2A). </li><li> Hacer girar suavemente las placas y se incuban O / N a 37 ° C con 5% de CO2. Se cultivan las células a 50-70% de confluencia. </li><li> De acuerdo con un plan de la transfección, se preparan diluciones de ARN de prueba y sus controles en tubos de 1,5 ml (micro ejemplo, la Figura 2B). <ol><li> Para el ensayo de la producción viral, preparar una 10 ng / l de dilución de un plásmido de expresión del VIH-1 y añadir 10 l a cada tubo. Siguiente preparar 5 M diluciones de ARN de ensayo y un control negativo RNA. Añadir 2,5 o 10 l de cada dilución de ARN de ensayo y de control negativo a los tubos correspondientes para 25 ó 100 nM concentraciones finales. </li><li> Para el ensayo de viabilidad celular, preparar 5 M diluciones de ARN de prueba y un 10 mg / ml de dilución de la RNA control positivo, de bajo peso molecular de poli I: C. Añadir 2 l de ARN de ensayo o diluciones de ARN de control positivo a los tubos correspondientes para 100 nM o concentraciones finales ml 200 g /, respectivamente. </li><li> Para el ensayo de la activación inmune, añadir 20 l de RNA de ensayo o las diluciones de ARN de control positivo preparados en la etapa 1.3.2. a los tubos correspondientes para 100 nM o 200 mg / ml concentración final, respectivamente. Nota: Para las pruebas de ARN plásmidos de expresión, preparar 10 ng diluciones / l en lugar de las 5 micras diluciones de ARN de ensayo, dando cantidades finales de 25 y 100 ng para el paso 1.3.1. y 100 ng para los pasos 1.3.2. y 1.3.3. </li></ol></li><li> Añadir 50, 25 o 75 l de DMEM a cada tubo para la transfección viral producción, la viabilidad celular y ensayos de activación inmune, respectivamente. Para los ensayos de producción viral, llevar las células y los tubos de transfección preparados a un nivel 3 de laboratorio bio-seguridad (BSL3) antes del siguiente paso. </li><li> Añadir 2 l de la secuencialmente reactivo de transfección a los tubos de transfección y se incuba de 15 a 20 min para permitir que los complejos a la forma. Ver la Tabla de materiales para el reactivo de transfección específico a utilizar. </li><li> Añadir toda la mezcla de transfección de cada tubo micro gota a gota a las posiciones correspondientes en las placas de cultivo celular. Hacer girar suavemente y se incuban las placas durante 48 horas a 37 ° C con 5% de CO2. </li><li> Medir el VIH-1 de producción (sección 2), la viabilidad celular (sección 3) y la activación inmune (sección 4) en las placas de cultivo celular (Figura 2C). </li></ol> 2. Ensayo de la producción viral <ol><li> Eliminar las placas de cultivo celular de 24 pocillos de la incubadora y agitar suavemente las placas en el interior del cultivo celular BSL3capucha. Transferir 150 l de sobrenadante de cada pocillo a un pocillo correspondiente en una placa de 96 pocillos de fondo plano, que se utiliza para cuantificar la producción de VIH-1. Nota: ensayos de cuantificación virales comunes incluyen la medición de la expresión de la proteína de la cápside del VIH-1 (p24) por ensayo de inmunoabsorción ligado a enzimas (ELISA) 20, la cuantificación de ARN viral mediante transcripción inversa reacción en cadena de la polimerasa (RT-PCR) 21 y la medición de la actividad de la enzima de VIH-1 RT. Los pasos 2.2 a 2.5 explicar los métodos para cuantificar VIH-1 RT actividad 13,15,16. </li><li> Transferencia de 5 l de sobrenadante a los pocillos correspondientes en una placa de 96 pocillos, que contiene 25 l de un cóctel viral interrupción 15 (Tabla 1). Incubar la mezcla durante 5 min a TA y la transferencia de la placa a una estación de trabajo radiactividad. Nota: El paso 2.2. sólo es necesario si una estación de trabajo radiactividad no está disponible en el laboratorio NBS3. Si las placas no tienen que ser retirados de laNBS3 laboratorio, continúe en el paso 2.3. y añadir 50 l de cóctel de interrupción radiactivo / viral (Tabla 1) en lugar de los 25 l de cóctel radiactivo. </li><li> Preparar un cóctel radiactiva 15 (Tabla 1), y añadir 25 l a cada pocillo de sobrenadante viral y cóctel de interrupción. Incubar las placas a 37 ° C durante 2 horas. </li><li> Punto 5 l de la mezcla de reacción en las casillas correspondientes en una fibra de vidrio Di Etil amino etil (DEAE) Filtermat de papel y permitir que las gotas se sequen durante 10 min. Detectar la mezcla de reacción en cada otra cuadrado, de manera que no hay dos muestras Boarder directamente entre sí. Esto ayuda a evitar el cruce entre las muestras para determinar las cuentas por minuto (cpm) en el paso 2.6. </li><li> Lavar los papeles 5X para 5 min con tampón 2x citrato de sodio salino (SSC) (Tabla 1), seguido de dos lavados de 1 min con 95% de etanol. Permitir que los papeles se sequen y sellan en bolsas de muestra. </li><li> Clip bolsas de muestras containing el papel Filtermat en un casete e inserte el cassette en un contador de centelleo de microplacas. Poner el contador a leer cpm de 32P con la fecha de referencia que establece el lote de [32P] dTTP utilizado en el experimento. Seleccione el que las muestras de leer Cómo utilizar el mapa plato y activar el contador. </li><li> Para cualquier método de cuantificación viral, dividir los valores obtenidos para cada ARN de ensayo por el control negativo adyacente y multiplicar este valor por 100 para obtener el porcentaje de inhibición de producción de VIH-1 para cada réplica de los ARN de prueba. Un ejemplo de los resultados que comparan diferentes ARN de ensayo a diferentes concentraciones se proporciona en la Figura 3. </li></ol> 3. ensayo de viabilidad de la célula <ol><li> Retire las placas de 96 pocillos de la incubadora y se añaden 20 l de 5 mg / ml de MTT (3- [4,5-dimetil-2-tiazolil] -2,5-difenil-2H-tetrazolio) diluido en solución salina tamponada con fosfato de Dulbecco ( DPBS) a cada pocillo. Se incuban las placas fo 3 horas a 37 ° C. </li><li> Añadir 150 ml de isopropanol acidificado con detergente (1% NP-40, HCl 4 mM en isopropanol) a cada pocillo e incubar las placas durante 2 horas a RT. </li><li> Determinar la absorbancia a 570 nm en un espectrofotómetro de microplacas. </li><li> Calcular el metabolismo de MTT relativa para cada ARN de control positivo de la prueba y dividiendo el valor obtenido para cada muestra por su control de transfección adyacente. Un ejemplo de los resultados que comparan diferentes ARN de ensayo y un control positivo se proporciona en la Figura 4. </li></ol> 4. Ensayo de Activación Inmune <ol><li> Retire las placas de 12 pocillos de la incubadora y aspirar el medio de cultivo. Lavar suavemente las células dos veces con DPBS y añadir 70 l de tampón de lisis frío 22 (incluyendo inhibidores de la proteasa y de fosfatasa, la Tabla 1) a cada pocillo. Se incuban las placas durante 10 minutos en hielo. </li><li> Transferir lisados ​​celulares para microtubos y rápido congelarlos por inmersión de latubos en nitrógeno líquido. Dejar que las muestras se descongelen y se repiten durante 2 ciclos de congelación y descongelación más para un total de 3. </li><li> Centrifugar los lisados ​​durante 15 min a 4 ° C, 15.700 xg, para sedimentar los residuos celulares. Transferir el sobrenadante a tubos nuevos micro y determinar la concentración de proteína usando el método de azul de Coomassie (Bradford) 23,24. </li><li> Resolver 75 g de proteína de cada muestra en un gel de poliacrilamida desnaturalizante al 10% y la transferencia a una membrana de nitrocelulosa como se ha descrito previamente 25,26. </li><li> Después de la electroforesis y la transferencia a una membrana, revelar bandas de proteínas mediante la incubación de la membrana en Ponceau S (Tabla 1) durante 1 min seguido de lavado en agua doblemente destilada. Usar las bandas y la escalera de proteína como una guía para cortar la membrana en 80 y 55 kDa. Nota: En el paso 4.4 muestras se pueden ejecutar en 16 x 18 cm 2 geles abajo a 34 kDa, de manera que es fácil de cortar la membrana en las posiciones especificadas y no cortar a través de labandas de interés. Por otra parte, varios geles se pueden ejecutar con las mismas muestras para evitar el corte de la membrana. </li><li> Lavar la tinción Ponceau S con solución salina tamponada con Tris que contenía 0,05% de Tween 20 (TBST, Tabla 1). Añadir TBST con leche no grasa al 5% para cubrir completamente las membranas. Se incuban las membranas a temperatura ambiente con agitación durante 1 hr. </li><li> Se incuban las membranas de O / N en TBST con 3% de albúmina de suero bovino (BSA) y los anticuerpos se diluyó a 1 en 1000 contra ADAR1 (110 y 150 kDa), fosfo-PKR (62 kDa), y fosfo-IRF3 (47 kDa), para las piezas superior, media e inferior de la membrana, respectivamente. </li><li> Lavar las membranas 5X para 5 min con TBST y se incuban en TBST con leche 5% sin grasa y de cabra anti-conejo anticuerpos secundarios marcados con peroxidasa (diluido a 1 en 5000) durante 1 hora. </li><li> Lavar las membranas 5x durante 5 min con TBST y aplicar la solución de electroquimioluminiscencia (ECL) para visualizar las bandas en las películas según las instrucciones del fabricante. </li><li> Después de visualizar las bandas de proteína en las películas, lavar las piezas intermedias de la membrana durante 10 min con una solución de anticuerpo de extracción. Se incuban las membranas O / N en TBST con BSA al 3% y anticuerpos contra PKR total de (a 1 en 500) y IRF3 (a 1 de cada 1.000), por el medio y piezas inferiores, respectivamente. Repita los pasos 4.8. y 4.9. utilizando marcado con peroxidasa de cabra anti-ratón en lugar del anticuerpo secundario anti-conejo para la membrana de PKR (medio). </li><li> Lavar la pieza inferior de la membrana 5x durante 5 min con TBST y se incuba la membrana en TBST con BSA al 3% durante 1 hora con un anticuerpo contra actina (diluido a 1 en 5000). Repita los pasos 4.8. y 4.9. utilizando marcado con peroxidasa de cabra anti-ratón en lugar del anticuerpo secundario anti-conejo. Un ejemplo de los resultados que comparan diferentes ARN de ensayo y un control positivo se proporciona en la Figura 5. Ver la Tabla de materiales para los anticuerpos específicos para ser utilizados. </li></ol>

<ol>
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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.

El ensayo de la producción de VIH-1 descrito se realizó utilizando células HEK293T (Figura 2) y es similar a los ensayos utilizados para la detección de VIH-1 RNA de ribozima eficaz 13, shRNA 10,29, siRNA 30, y ARN U1i sitios 11,31 objetivo. El uso de diferentes métodos para cuantificar la producción de VIH-1, la mayoría de estudios han medido la producción viral 48 horas después de la co-transfección de un plásmido de expresión de VIH-1 con los AR...

Una limitación de los actuales tratamientos para el VIH-1 es que deben ser administrados crónicamente para prevenir la progresión de la enfermedad. Trasplante de VIH-1 resistentes a los linfocitos T, o células madre hematopoyéticas, tiene el potencial de proporcionar el control a largo plazo del VIH-1 de replicación en ausencia de la terapia con medicamentos 1,2 y también puede ser un enfoque eficaz para alcanzar una cura de VIH-1 3. Una forma de hacer que las células resistentes al VIH-1 de replicación es insertar uno o más genes que codifican anti-VIH-1 ARN o péptidos en células de un individuo infectado durante un trasplante autólogo 4. Varios genes candidatos anti-VIH-1 han sido diseñados con algunos ensayos clínicos que entran en combinaciones de dos o tres 5 6, para prevenir el desarrollo de resistencia del VIH-1 a un solo gen. Anti-VIH-1 ARN se encuentran entre los mejores candidatos para la terapia génica de combinación debido a su bajo potencial para provocar respuestas inmunes y porquese transcriben a partir de secuencias de genes muy cortos. Algunos ARN anti-VIH-1 se han diseñado para actuar sobre la entrada y la integración viral. Sin embargo, la mayoría de los anti-VIH-1 ARN diana pasos posteriores a la integración en el ciclo de vida viral (Figura 1). Inhibidores de la post-integración incluyen ARN señuelo, apuntando a los VIH-1 proteínas reguladoras Tat o Rev 1, y ARN antisentido basados ​​en, dirigidos a diferentes sitios en el VIH-1 ARN, como las ribozimas 7, 8 y shRNAs U1i ARNs 9. Los métodos que se han utilizado para comparar la eficacia de los anti VIH-1 ARN incluyen la supervisión de la replicación viral en las células transducidas con genes que codifican para los ARN candidatos y medición de la producción viral en células transfectadas transitoriamente con plásmidos que expresan ARN candidatos y un plásmido de expresión del VIH-1 10 -13. Hemos utilizado anteriormente un ensayo de producción de VIH-1 para detectar el VIH-1 RNA para el nuevo ribozima sitios diana 13-15. Estos métodos ya se han refinado para optimizar el formato de un ARNmolécula de interferencia expresa a partir de ADN plásmido como un shRNA o entregada como un ARNsi sintético 16. El ensayo mide la producción de virus maduros a partir de células 293T de riñón embrionario (HEK) humanos, y se puede utilizar para comparar los efectos de los inhibidores que se dirigen a pasos posteriores a la integración en el ciclo de replicación del VIH-1 (Figura 1). Para inhibidores que se dirigen a los pasos de pre-integración, se necesitan ensayos alternativos, tales como un ensayo de infectividad celular TZM-bl 17 para evaluar la eficacia antiviral. Las principales preocupaciones de seguridad para la entrega de los anti-VIH-1 ARN en la clínica incluyen los posibles efectos fuera de la meta de ARN o proteínas humanas, y la activación de sensores inmune innata. Para evaluar la toxicidad de anti-HIV-1 siRNAs, hemos utilizado un ensayo de viabilidad celular en diferentes líneas celulares 16. También se midió la activación de los sensores inmunes de ARN bicatenario, ARN proteína quinasa activada por R (PKR) y Toll like receptor 3 (TLR3), así como exsión del gen de interferón estimulado, p150 ADAR1. Estos ensayos se pueden usar para confirmar que la eficacia de anti-HIV-1 ARN no se debe a efectos indirectos sobre la viabilidad celular o la activación inmune sensor. También son útiles en la exclusión de los ARN candidatos con los potenciales efectos tóxicos de un mayor desarrollo. En los siguientes protocolos, procedimientos para identificar nuevos ARN terapéuticas y optimizar el formato de los existentes se describen. Los métodos son útiles para los inhibidores de la post-integración basadas ARN de detección de VIH-1 de replicación y pueden ser adaptados a la pantalla de otros inhibidores de la post-integración, tales como pequeñas moléculas de direccionamiento Rev exportación mediada por ARN viral 18 o CRISPR / sistemas de Cas diseñados para dirigir integrada VIH-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.

Un esquema general de los procedimientos se muestra en la Figura 2 con un plan de transfección ejemplo para tres ARN de prueba y un ARN de control proporcionada en la Figura 2B. Para los ensayos de producción y la viabilidad celular virales, La lectura para cada construcción de prueba es normalizada a un control negativo. Las réplicas se transfectan en conjuntos, de modo que cada ARN de ensayo se normalizó a su control negativo adyacente. Esto se ha...

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.

Evaluación de la eficacia y la toxicidad de los ARN del VIH-1 Orientación de la producción para uso en terapia génica o de drogas

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 vistas.  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. 

Video: 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.

Detección del genoma-ancha de RNAi para identificar los Host factores que modulan la terapia de Virus oncolíticos

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

Investigación

Investigación sobre el cáncer

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.

Desarrollo de programas específicos de células de tipo anti-gp120 del VIH aptámeros para la entrega de 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 ...

Inmunología e Infección

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.

ADN vectorial basado en el ARN de interferencia para estudiar la función génica en el cáncer

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.

Screening de alto rendimiento para los de amplio espectro químico Inhibidores de Virus ARN

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.

Detección rápida de la transcriptasa inversa del VIH y inhibidores de la integrasa

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 efectores: Transkingdom RNAi

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

Biología

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.

Reglamento basadas en microARN 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 rendimiento en la evaluación de Vitro de latencia inversión a en VIH transcripción y 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 ...

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.

La detección de ARN viral por fluorescencia<em&gt; In situ</em&gt; La hibridación (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.

Tratamiento antirretroviral combinado oral en ratones humanizados infectados con VIH-1

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