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

Die vorgestellten Arbeiten wurden von der kanadischen Institutes of Health Research (CIHR) unterstützt (gewährt DCB-120266, PPP-133377 und HBF-348967 zu AG).

1. Zellen und Transfektionen <ol><li> Kultur HEK 293T Zellen in Dulbecco-modifiziertem Eagle-Medium (DMEM) mit 10% fötalem Rinderserum (FBS) und 1% Penicillin / Streptomycin ergänzt. Vorbereiten eines 2 x 10 5 Zellen / ml - Suspension in dem Zellkulturmedium. In 500, 100 und 1000 &amp; mgr; l der Zellsuspension in jede Vertiefung von 24-Well, 96-Loch - und 12-Well - Platten, die für die virale Produktion, die Lebensfähigkeit der Zellen und Immunaktivierung Assays, bzw. (2A). </li><li> Vorsichtig schwenken die Platten und inkubieren O / N bei 37 ° C mit 5% CO 2. Wachsen Zellen zu 50-70% Konfluenz. </li><li> Nach einer Transfektion Plan, bereiten Verdünnungen von Test RNAs und ihre Kontrollen in 1,5 ml Mikroröhrchen (Beispiel 2B). <ol><li> Für die virale Produktion Assay, bereiten eine 10 ng / &amp; mgr; l Verdünnung einer HIV-1-Expressionsplasmid und 10 &amp; mgr; l zu jedem Röhrchen hinzufügen. Vorbereitung Die nächsten 5 uM Verdünnungen von Test RNAs und eine negative Kontrolle RNA. In 2,5 oder 10 &amp; mgr; l jeder Test RNA und negative Kontrolle Verdünnung auf die entsprechenden Rohre für 25 oder 100 nM Endkonzentrationen. </li><li> Für die Zelllebensfähigkeitstest vorbereiten 5 uM Verdünnungen von Test RNAs und eine 10 mg / ml Verdünnung der positiven Kontroll-RNA, niedermolekulare Poly I: C. Fügen Sie 2 ul Test RNA oder positive Kontroll-RNA-Verdünnungen zu den entsprechenden Röhrchen 100 nM oder 200 &amp; mgr; g / ml Endkonzentration, respectively. </li><li> Für die Immunaktivierung Test werden 20 ul Test RNA oder positive Kontroll-RNA Verdünnungen, hergestellt in Schritt 1.3.2. zu den entsprechenden Röhrchen 100 nM oder 200 &amp; mgr; g / ml Endkonzentration, respectively. Hinweis: Für den Test-RNA-Expressionsplasmide Herstellung von 10 ng / ul-Verdünnungen anstelle der 5 &amp; mgr; M Verdünnungen von Test RNAs, so dass endgültige Mengen von 25 und 100 ng für Schritt 1.3.1. und 100 ng für die Schritte 1.3.2. und 1.3.3. </li></ol></li><li> In 50, 25 oder 75 &amp; mgr; l DMEM zu jeder Transfektion Rohr für die virale production, die Lebensfähigkeit der Zellen und Immunaktivierung Assays sind. Für die virale Produktion Assays bringen Zellen und bereit Transfektion Rohre zu einem Bio-Sicherheitsstufe 3 (BSL3) Labor vor dem nächsten Schritt. </li><li> Fügen Sie 2 ul des Transfektionsreagenz sequentiell an die Transfektion Röhrchen und Inkubation 15 bis 20 min-Komplexen zu erlauben, zu bilden. Siehe Tabelle der Materialien für die spezifischen Transfektionsreagenz verwendet werden. </li><li> Fügen Sie die gesamte Transfektionsgemischs aus jedes Mikroröhrchen tropfenweise auf die entsprechenden Positionen in den Zellkulturplatten. Leicht geschwenkt und die Platten für 48 Stunden bei 37 ° C mit 5% CO 2 inkubieren. </li><li> Messen Sie HIV-1 - Produktion (Abschnitt 2), die Lebensfähigkeit der Zellen (Abschnitt 3) und Immunaktivierung (Abschnitt 4) in den Zellkulturplatten (2C). </li></ol> 2. Viral Produktion Assay <ol><li> Entfernen Sie die 24-Well-Zellkulturplatten aus dem Inkubator und vorsichtig schwenken die Platten im Inneren der BSL3 ZellkulturHaube. Übertragung von 150 &amp; mgr; l Überstand aus jeder Vertiefung auf eine gut entspricht, in einer 96-Well-Flachbodenplatte, die verwendet werden, HIV-1-Produktion zu quantifizieren. Hinweis: Common viral Quantifizierungsassays umfassen die Expression des HIV-1 - Kapsidprotein Messung (p24) durch enzyme-linked immunosorbent assay (ELISA) 20, viraler RNA durch reverse Transkriptase - Polymerase - Kettenreaktion quantifiziert (RT-PCR) 21 und Messen der Aktivität der HIV-1 RT-Enzym. Die Schritte 2.2 bis 2.5 erklären Methoden HIV-1 - RT - Aktivität 13,15,16 zu quantifizieren. </li><li> Transfer 5 &amp; mgr; l Überstand zu entsprechenden Vertiefungen in einer 96-Well - Platte, enthaltend 25 &amp; mgr; l einer viralen Störung Cocktail 15 (Tabelle 1). Inkubieren der Mischung für 5 min bei RT und übertragen die Platte auf eine Radioaktivität Workstation. Hinweis: Schritt 2.2. Wenn ein Radioaktivität Workstation ist nur notwendig, nicht im BSL3 Labor ist. Wenn die Platten müssen nicht aus der entfernt werdenBSL3 Labor, gehen 2.3 zu treten. und fügen Sie 50 ul radioaktivem / viralen Störung Cocktail (Tabelle 1) anstelle der 25 ul radioaktiver Cocktail. </li><li> Bereiten Sie eine radioaktive Cocktail 15 (Tabelle 1), und fügen Sie 25 ul in jede Vertiefung der viralen Überstand und Störungen Cocktail. Inkubiere die Platten bei 37 ° C für 2 Stunden. </li><li> Spot 5 ul der Reaktionsmischung auf entsprechenden Plätzen in einer Glasfaser Di Ethyl Aminoethyl (DEAE) Filtermat Papier und ermöglichen die Flecken für 10 min trocknen. Spot der Reaktionsmischung auf jedem anderen Platz, so dass keine zwei Proben unmittelbar aneinander Grenze. Dies hilft, cross-over zwischen den Proben zu vermeiden, wenn in Counts pro Minute (cpm) Schritt 2.6 bestimmt wird. </li><li> Waschen Sie die Papiere für 5 min 5x mit 2x Salz Natriumcitrat (SSC) Puffer (Tabelle 1), gefolgt von zwei 1 minütiges Waschen mit 95% Ethanol. Lassen Sie die Papiere, um zu trocknen und zu versiegeln sie in einen Probenbeutel verpackt. </li><li> Clip Probenbeutel containing die Filtermatte Papier in eine Kassette und legen Sie die Kassette in einem Mikrotiterplatten-Szintillationszähler. Stellen Sie den Zähler cpm für 32 P mit dem Referenzdatum für die Partie von [32 P] dTTP in dem Experiment verwendet zur Verfügung gestellt zu lesen. Wählen Sie die Proben zu lesen, die Platte Karte unterhalten und den Zähler zu starten. </li><li> Für jede viral Quantifizierungsmethode, teilen die Werte für jede Test RNA durch die benachbarte negative Kontrolle erhalten und multiplizieren diesen Wert mit 100 für jede Wiederholung der Test RNAs die prozentuale Hemmung der HIV-1-Produktion zu erhalten. Ein Beispiel der Ergebnisse verschiedener Test RNAs in unterschiedlichen Konzentrationen zu vergleichen ist in 3 bereitgestellt. </li></ol> 3. Zell Viability Assay <ol><li> Entfernen Sie die Platten mit 96 Vertiefungen aus dem Inkubator und füge 20 &amp; mgr; l von 5 mg / ml MTT (3- [4,5-Dimethyl-2-thiazolyl] -2,5-diphenyl-2H-tetrazolium-bromid), verdünnt in Dulbeccos phosphatgepufferter Salzlösung ( DPBS) in jede Vertiefung. Inkubieren Sie die Platten foder 3 Stunden bei 37 ° C. </li><li> Zugabe von 150 ul angesäuertes Isopropanol mit einem Detergens (1% NP-40, 4 mM HCl in Isopropanol) in jede Vertiefung und Inkubieren der Platten für 2 h bei RT. </li><li> Bestimmen Sie die Absorption bei 570 nm in einem Mikroplatten-Spektralphotometer. </li><li> Berechnen der relativen MTT-Metabolismus für jede positive Kontrolle und Test-RNA durch den Wert für jede Probe durch seinen benachbarten Transfektionskontrolle erhalten dividiert wird. Ein Beispiel der Ergebnisse verschiedener Test RNAs und eine positive Kontrolle Vergleich ist in Figur 4 vorgesehen. </li></ol> 4. Immunaktivierung Assay <ol><li> Entfernen Sie die 12-Well-Platten aus dem Inkubator und absaugen Kulturmedien. Die Zellen vorsichtig waschen zweimal mit DPBS und 70 ul Puffer kalt Lyse in jede Vertiefung 22 (einschließlich Protease und Phosphatase - Inhibitoren, Tabelle 1) hinzuzufügen. Inkubieren Sie die Platten für 10 Minuten auf Eis. </li><li> Übertragen Sie Zelllysate auf Mikroröhrchen und sie schnell frieren durch das EintauchenRöhrchen in flüssigem Stickstoff. Lassen Sie die Proben für 2 weitere Gefrier-Auftau-Zyklen für insgesamt 3 aufzutauen und zu wiederholen. </li><li> Zentrifugation der Lysate 15 min bei 4 ° C, 15.700 xg, um Zelltrümmer zu pelletieren. Den Überstand auf neue Mikroröhrchen und bestimmen die Proteinkonzentration unter Verwendung des Coomassie - Blau (Bradford) Methode 23,24. </li><li> Auflösen 75 ug Protein aus jeder Probe in einem 10% igen denaturierenden Polyacrylamid - Gel und Transfer auf eine Nitrocellulosemembran , wie zuvor beschrieben 25,26. </li><li> (Tabelle 1) für 1 min , gefolgt von Waschen in doppelt destilliertem Wasser nach der Elektrophorese und Transfer auf eine Membran, offenbaren Proteinbanden durch die Membran in Ponceau S inkubiert. Verwenden Sie die Bänder und Proteinleiter als Führung der Membran bei 80 und 55 kDa zu schneiden. Hinweis: In Schritt 4.4 kann mit Hilfe von 16 x 18 cm 2 Gelen ausgeführt werden , bis zu 34 kDa, so dass es einfach ist , die Membran an den angegebenen Positionen zu schneiden und nicht durch den ausgeschnittenenBands von Interesse. Alternativ können mehrere Gele mit den gleichen Proben durchgeführt werden, um zu vermeiden, dass die Membran zu schneiden. </li><li> Auswaschen der Ponceau S Färbung mit Tris-gepufferter Kochsalzlösung , enthaltend 0,05% Tween 20 (TBST, Tabelle 1). In TBST mit 5% fettfreie Milch, um vollständig die Membranen abdecken. Inkubieren der Membranen bei RT unter Rühren für 1 Stunde. </li><li> Inkubieren der Membranen O / N in TBST mit 3% Rinderserumalbumin (BSA) und 1 in 1000 gegen ADAR1 verdünnten Antikörper (110 und 150 kDa), phospho-PKR (62 kDa) und phospho-IRF3 (47 kDa), für die oberen, mittleren und unteren Teile der Membran sind. </li><li> Waschen Sie die Membranen für 5 min mit TBST 5x und in TBST mit 5% fettfreie Milch und Peroxidase-markierten Ziege-Anti-Kaninchen-Sekundärantikörper (verdünnt auf 1 in 5000) für 1 Stunde inkubiert. </li><li> Waschen Sie die Membranen 5x 5 min mit TBST und gelten Elektrochemilumineszenz (ECL) Lösung, die Bänder auf Filme zu visualisieren den Anweisungen des Herstellers entsprechend. </li><li> Nach Visualisierung auf Filme, die die Proteinbanden, wasche die mittleren und unteren Stücke der Membran für 10 min mit einem Antikörper Ausstreiflösung. Inkubieren der Membranen O / N in TBST mit 3% BSA und Antikörper gegen Gesamt PKR (bei 1 in 500) und IRF3 (bei 1 in 1000), für die mittleren und unteren Teile, respectively. Wiederholen Sie die Schritte 4.8. und 4.9. unter Verwendung von Peroxidase-markiertem Ziege-anti-Maus anstelle des anti-Kaninchen-Sekundärantikörpers für die PKR-Membran (mittel). </li><li> Waschen Sie das Unterteil der Membran 5x für 5 min mit TBST und die Membran in TBST mit 3% BSA für 1 h inkubiert mit einem Antikörper gegen Actin (verdünnt auf 1 in 5000). Wiederholen Sie die Schritte 4.8. und 4.9. unter Verwendung von Peroxidase-markiertem Ziege-anti-Maus anstelle des anti-Kaninchen-Sekundärantikörpers. Ein Beispiel der Ergebnisse verschiedener Test RNAs und eine positive Kontrolle zu vergleichen ist in 5 vorgesehen. Siehe Tabelle der Materialien für die spezifischen Antikörpern verwendet werden. </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.

Die HIV-1 - Produktion beschriebenen Assay wurde unter Verwendung von HEK293T - Zellen (Figur 2) durchgeführt und ist ähnlich Assays verwendet HIV-1 - RNA für eine effektive Ribozym 13, shRNA 10,29, siRNA 30 und U1i RNA 11,31 Zielstellen zu screenen. Mit Hilfe verschiedener Methoden HIV-1-Produktion zu quantifizieren, wurden die meisten Studien virale Produktion 48 Stunden nach der Co-Transfektion eines HIV-1-Expressionsplasmid mit Kandidaten RNAs gemessen....

Eine Einschränkung der derzeitigen HIV-1-Behandlungen ist, dass sie chronisch Fortschreiten der Krankheit zu verhindern, verabreicht werden müssen. Transplantation von HIV-1 resistent T - Lymphozyten oder hämatopoetischen Stammzellen, hat das Potenzial , Langzeitkontrolle der HIV-1 - Replikation in Abwesenheit von Arzneimitteltherapie 1,2 und kann auch ein wirksamer Ansatz sein , um eine HIV-1 Heilung zu erreichen 3. Ein Weg , um Zellen resistent gegen HIV-1 - Replikation rendern ist ein oder mehrere Gene einzuführen , kodierend für anti-HIV-1 RNAs oder Peptide in einem infizierten Individuum Zellen während einer autologe Transplantation 4. Mehrere Kandidaten anti-HIV-1 - Gene wurden mit einigen klinischen Studien Eingabe in Kombinationen von zwei oder drei 5 6, die die Entwicklung von HIV-1 Resistenz gegenüber einem einzelnen Gen zu verhindern. Anti-HIV-1 RNAs sind unter den ersten Kandidaten für die Kombination Gentherapie aufgrund ihrer niedrigen Potential Immunantworten hervorzurufen, und weil siesind von sehr kurzen Gensequenzen transkribiert. Einige anti-HIV-1 RNAs wurden entwickelt Viruseintritt und Integration zu zielen. Allerdings sind die meisten anti-HIV-1 - RNA das Ziel nach der Integrationsschritte im viralen Lebenszyklus (Abbildung 1). Nachintegration Inhibitoren umfassen Decoy RNAs, die Targeting - HIV-1 - regulatorischen Proteine ​​Tat oder Rev 1 und Antisense-RNAs beruhen, verschiedene Standorte in HIV-1 - RNA - Targeting, wie Ribozyme 7, 8 und shRNAs U1i RNAs 9. Verfahren, die die Wirksamkeit von Anti-HIV-1 RNAs gehören die Überwachung der viralen Replikation in Zellen , transduziert mit Genen , kodierend für Kandidaten RNAs und Mess virale Produktion in Zellen , transient transfiziert mit Plasmiden exprimieren Kandidaten RNAs und eine HIV-1 - Expressionsplasmid 10 zum Vergleich verwendet wurden -13. Wir haben zuvor eine HIV-1 - Produktion zu screenen Assay HIV-1 RNA für neue Ribozym - Zielstellen 13-15 verwendet. Diese Verfahren sind seit verfeinert das Format einer RNA zu optimieren,Interferenz - Molekül von dem Plasmid DNA als shRNA oder geliefert als synthetisches siRNA 16 ausgedrückt. Der Assay misst die Produktion von reifen Viren aus menschlichen embryonalen Nieren (HEK) , 293T - Zellen, und kann verwendet werden , die Auswirkungen von Inhibitoren zu vergleichen , die nach der Integrationsschritte in der HIV-1 - Replikationszyklus Ziel (Abbildung 1). Für Inhibitoren , die Pre-Integrationsschritte Ziel, alternative Assays wie eine TZM-bl Zelle Infektiosität Assay 17 nötig sind , um antivirale Wirksamkeit zu bewerten. Wichtige Sicherheitsbedenken für die Lieferung von Anti-HIV-1-RNA in der Klinik sind potenzielle Nebeneffekte auf die menschliche RNAs oder Proteine, und die Aktivierung der angeborenen Immunsensoren. Um die Toxizität von anti-HIV-1 siRNAs bewerten, haben wir einen Zelllebensfähigkeitstest in verschiedenen Zelllinien 16 verwendet. Wir maßen auch die Aktivierung der doppelsträngigen RNA-Immunsensoren, RNA aktivierte Proteinkinase R (PKR) und Toll-like-Rezeptor 3 (TLR3), sowie expression des Interferon-stimulierte Gen, ADAR1 p150. Diese Assays können verwendet werden, um zu bestätigen, dass die Wirksamkeit von Anti-HIV-1-RNA ist nicht auf indirekte Auswirkungen auf die Lebensfähigkeit der Zellen oder Immunsensoraktivierung. Sie sind auch nützlich in Ausnahme von Kandidaten RNAs mit potentiellen Toxizitäten von der weiteren Entwicklung. In den folgenden Protokolle, Verfahren neue therapeutische RNAs zu identifizieren und zu optimieren das Format der bestehenden beschrieben werden. Die Verfahren sind nützlich für das Screening RNA basieren Nachintegration Inhibitoren der HIV-1 - Replikation und angepasst werden könnten andere post-Integration - Inhibitoren, wie kleine Moleküle Targeting Rev vermittelte Export von viraler RNA 18 oder CRISPR / CAS - Systeme entwickelt , um screenen integrierten Ziel 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.

Eine allgemeine schematische Darstellung der Verfahren ist in Figur 2 mit einem Beispiel der Transfektion Plan für drei Test RNAs und einer Steuer RNA bereitgestellt in 2B gezeigt. Für die virale Produktion und Zelllebensfähigkeitstests, die Auslese für jedes Testkonstrukt wird auf eine negative Kontrolle normalisiert. Replikate werden in Sätzen transfiziert, so daß jeder Test RNA an seinen benachbarten negativen Kontrolle normiert. Dies geschieht,...

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.

Bewertung der Wirksamkeit und Toxizität von RNAs Targeting HIV-1-Produktion für den Einsatz in Gene oder Arzneimitteltherapie

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

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

7.8K Aufrufe.  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. 

Serie: 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.

Genomweiten RNAi-Screening, Host Faktoren zu identifizieren, die onkolytische Viren Therapie zu modulieren

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

Forschung

Krebsforschung

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.

Entwicklung von Zell-spezifischen Anti-HIV-gp120-Aptamere für 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 ...

Immunologie und Infektion

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-Vektor-basierte RNA-Interferenz, um Genfunktionen in Cancer Study

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

Medizin

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.

Hochdurchsatz-Screening für Breitspektrum-Chemische Inhibitoren der RNA-Viren

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.

Rapid-Screening von HIV-Reverse-Transkriptase und Integrase-Inhibitoren

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.

Bakterielle Lieferung von RNAi Effektoren: Transkingdom RNAi

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

Biologie

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.

MicroRNA-basierten Regulation der Picornavirus Tropism

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.

Hoher Durchsatz In Vitro Bewertung der Latenz Umkehrung Agenten auf HIV Transkription und Spleißen

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.

Nachweis viraler RNA durch Fluoreszenz<em&gt; In-situ-</em&gt; Hybridisierung (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.

Orale kombinierte antiretrovirale Behandlung bei HIV-1-infizierten humanisierten Mäusen

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