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
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

Arbetet presenteras här stöddes av den kanadensiska Institutes of Health Research (CIHR) (ger DCB-120.266, PPP-133.377 och HBF-348.967 till AG).

1. Celler och transfektioner <ol><li> Kultur HEK 293T-celler i Dulbeccos modifierade Eagle-medium (DMEM) kompletterat med 10% fetalt bovint serum (FBS) och 1% penicillin / streptomycin. Bered en 2 x 10 5 celler / ml suspension i cellodlingsmediet. Tillsätt 500, 100 och 1000 | j, l av cellsuspensionen till varje brunn i 24-brunnars, 96-brunnars och 12-brunnars plattor, för viral produktion, cellviabiliteten och immunaktiveringsanalyser, respektive (Figur 2A). </li><li> Snurra försiktigt p...

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HIV-1-produktion analys som beskrivits utfördes med användning av HEK293T-celler (figur 2) och liknar analyser som användes för att screena HIV-1-RNA för effektiv ribozym 13, shRNA 10,29, siRNA 30, och U1i RNA 11,31 målställen. Med hjälp av olika metoder för att kvantifiera HIV-1-produktion, har de flesta studierna mättes viral produktion 48 timmar efter samtransfektion av en HIV-1-expressionsplasmid med kandidat RNA. Följer på produktionen av HIV-...

En begränsning av nuvarande HIV-1-behandlingar är att de måste vara administreras kroniskt för att förhindra sjukdomsprogression. Transplantation av HIV-1 resistent T-lymfocyter, eller hematopoietiska stamceller, har potential att skapa långsiktig kontroll av HIV-1-replikation i frånvaro av läkemedelsbehandling 1,2 och kan också vara en effektiv metod för att uppnå en HIV-1 botemedel 3. Ett sätt att göra celler resistenta mot HIV-1-replikation är att sätta in en eller flera gener som kodar för anti-HIV-1 RNA eller peptider i en infekterad individs celler under en autolog transplantation 4. Flera kandidat anti-HIV-1-gener har utformats med några in kliniska prövningar i kombinationer av två 5 eller tre 6, för att förhindra utvecklingen av HIV-1-resistens mot någon enskild gen. Anti-HIV-1 RNA är bland de bästa kandidaterna för kombinations genterapi på grund av deras låga potential att framkalla immunsvar och eftersom detranskriberas från mycket korta gensekvenser. Vissa anti-HIV-1 RNA har utformats för att rikta viralt inträde och integration. De flesta anti-HIV-1 RNA riktar efterintegrations steg i virusets livscykel (Figur 1). Post-integration inhibitorer innefattar lock RNA, med inriktning på HIV-1 reglerande proteiner Tat eller Rev 1, och antisense-baserade RNA, med inriktning på olika platser i HIV-1 RNA, såsom ribozymer 7, shRNAs 8 och U1i RNA 9. Metoder som har använts för att jämföra effekten av anti-HIV-1 RNA omfattar övervakning virusreplikation i celler transducerade med gener som kodar för kandidat RNA och mäta viral produktion i celler transient transfekterade med plasmider som uttrycker kandidat RNA och en HIV-1-expressionsplasmid 10 -13. Vi har tidigare använt en HIV-1-produktion analys för att screena HIV-1-RNA för ny ribozym målställen 13-15. Dessa metoder har sedan förfinats för att optimera utformningen av en RNAstörningar molekyl som uttrycks från plasmid-DNA som en shRNA eller levereras som en syntetisk siRNA 16. Analysen mäter produktionen av mogna virus från humana embryonala njur (HEK) 293T-celler, och kan användas för att jämföra effekterna av hämmare som riktar efterintegrations steg i HIV-1-replikationscykeln (Figur 1). För hämmare som riktar steg före integration, är alternativa analyser såsom TZM-bl cellinfektionsanalys 17 som behövs för att utvärdera antiviral effekt. Stora säkerhetsrisker för leverans av anti-HIV-1 RNA i kliniken är de potentiella off-target effekter på människors RNA eller proteiner och aktivering av medfödda immun sensorer. För att utvärdera toxiciteten av anti-HIV-1 siRNA, har vi använt en cellviabilitet analys i olika cellinjer 16. Vi mätte också aktivering av de dubbelsträngade RNA-immunsensorer, RNA aktiverat proteinkinas R (PKR) och Toll like receptor 3 (TLR3), såväl som expres av interferon stimulerade genen ADAR1 P150. Dessa analyser kan användas för att bekräfta att effekten av anti-HIV-1 RNA är inte på grund av indirekta effekter på cellviabilitet eller immunsensoraktivering. De är också användbara i exklusive kandidat RNA med potentiella toxicitet från vidareutveckling. I följande protokoll, förfaranden identifiera nya terapeutiska RNA och optimera formatet av befintliga beskrivs. Metoderna är användbara för screening RNA baserade efter integration hämmare av HIV-1-replikation och kan anpassas för att screena andra hämmare efter integration, såsom små molekyler som riktar Rev medierad export av viralt RNA 18 eller crispr / Cas-system utformade för att rikta integrerad HIV-1-DNA 19.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>[32P]dTTP</td>
			<td>Perkin Elmer</td>
			<td>BLU505H</td>
			<td></td>
		</tr>
		<tr>
			<td>poly(A) RNA template&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>10108626001</td>
			<td></td>
		</tr>
		<tr>
			<td>oligo(dT)12-18 DNA primer</td>
			<td>Thermo Fisher</td>
			<td>18418-012</td>
			<td></td>
		</tr>
		<tr>
			<td>DEAE filtermat paper&#160;</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&#160;</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&#160;</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.

Ett allmänt schema över de förfaranden som visas i figur 2 med ett exempel transfektion plan för tre test RNA och en styr RNA tillhandahålls i figur 2B. För virala produktion och cellernas viabilitet analyser är utläsningen för varje test konstrukt normaliserat till en negativ kontroll. Replikat transfekteras i satser, så att varje test RNA normaliserades till dess intilliggande negativ kontroll. Detta görs för att undvika felaktiga uppgifter...

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.

Utvärdering av effekt och toxicitet av RNA Targeting HIV-1-produktion för användning i Gene eller 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 ...

Research

JoVE Journal

Immunology and Infection

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Genotypisk Inferens av HIV-1 Tropism Använda Befolkning-sekvensering av V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Beredning och användning av HIV-1 infekterade Primär CD4 + T-celler som målceller i Natural Cytotoxiska Killer Cell analyser

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Generering av Multivirus-specifika T-celler för att förhindra / behandla virusinfektioner efter allogen hematopoetisk stamcellstransplantation

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Förstärka och kvantifiera HIV-1 RNA i hiv-infekterade individer med virusnivåer under gränsen för upptäckt med vanliga kliniska analyser

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

In vitro Uncoating of HIV-1 Cores

In vitro Uncoating of HIV-1 Cores

In vitro Uncoating av HIV-1 kärnor

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Avbildning av HIV-1 kuvert-inducerad Virologiskt Synapse och signalering av syntetiska lipiddubbelskikt

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

Conformational Utvärdering av HIV-1 trimera Envelope Glycoproteins användning av en cellbaserad ELISA-analys

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Hög-throughput kvantitativ realtids-RT-PCR-analys för bestämning av uttryck Profiler av typ I och III Interferon Subtyper

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Isolering av exosomes från plasma HIV-1 positiva individer

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

mätning av<em&gt; In Vitro</em&gt; Integration Aktivitet av HIV-1 Preintegration Anläggningar

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...