Evaluering af effekt og toksicitet af RNA Målretning HIV-1 Produktion til brug i Gene eller Drug Therapy
Summary
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.
Abstract
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.
Introduction
En begrænsning ved nuværende HIV-1 behandlinger er, at de skal være kronisk administreret for at forhindre progression af sygdommen. Transplantation af HIV-1 resistent T-lymfocyt, eller hæmatopoietiske stamceller, har potentialet til at give langsigtet kontrol af HIV-1-replikation i fravær af lægemiddelbehandling 1,2 og kan også være en effektiv tilgang til at opnå en HIV-1 kur 3. En måde at gøre celler modstandsdygtige mod HIV-1-replikation er at indsætte et eller flere gener der koder for anti-HIV-1 RNA'er eller peptider i en inficeret individs celler under en autolog transplantation 4. Adskillige kandidat anti-HIV-1-gener er blevet designet med nogle ind kliniske forsøg i kombinationer af to 5 eller tre 6, for at forhindre udviklingen af HIV-1 resistens over for en enkelt gen.
Anti-HIV-1 RNA er blandt de bedste kandidater til kombination genterapi på grund af deres ringe potentiale til at fremkalde immunresponser og fordi detranskriberes fra meget korte gensekvenser. Nogle anti-HIV-1 RNA er designet til at målrette viral indtrængen og integration. Men de fleste anti-HIV-1 RNA målrette post-integration trin i den virale livscyklus (figur 1). Post-integration hæmmere omfatter lokkefugle RNA, rettet mod de HIV-1 regulerende proteiner Tat eller Rev 1, og antisense-baserede RNA, rettet mod forskellige steder i HIV-1 RNA, såsom ribozymer 7, shRNAs 8 og U1i RNA 9. Metoder, der er anvendt til at sammenligne virkningen af anti-HIV-1 RNA inkluderer monitorering viral replikation i celler transduceret med gener, der koder for kandidat RNA'er og måle viral produktion i celler transient transficeret med plasmider, der udtrykker kandidat RNA'er og en HIV-1 ekspressionsplasmid 10 -13. Vi har tidligere anvendt et HIV-1-produktion assay til screening HIV-1 RNA til nye ribozym målsteder 13-15. Disse fremgangsmåder er siden blevet raffineret for at optimere udformningen af et RNAinterferens molekyle udtrykt fra plasmid-DNA som en shRNA eller leveres som et syntetisk siRNA 16. Assayet måler produktionen af modne virus fra humane embryoniske nyre (HEK) 293T-celler, og kan anvendes til at sammenligne virkningerne af inhibitorer, der er målrettet efter integration trin i HIV-1-replikation cyklus (figur 1). For inhibitorer, der er målrettet præ-integration trin, er alternative assays, såsom et TZM-bl celle infektivitet assay 17 er nødvendige til vurdering antiviral virkningsfuldhed.
Større sikkerhedsproblemer for levering af anti-HIV-1 RNA i klinikken omfatter potentielle off-target effekter på humane RNA eller proteiner og aktivering af medfødte immun sensorer. For at evaluere toksiciteten af anti-HIV-1 siRNAs, har vi anvendt en celleviabilitetstest i forskellige cellelinier 16. Vi målte også aktivering af det dobbeltstrengede RNA immune sensorer, RNA aktiveret protein kinase R (PKR) og Toll like receptor 3 (TLR3), samt expression af interferon stimulerede gen, ADAR1 P150. Disse assays kan anvendes til at bekræfte, at effektiviteten af anti-HIV-1 RNA'er ikke skyldes indirekte virkninger på cellernes levedygtighed eller immun sensor aktivering. De er også nyttige i at udelukke kandidat RNA'er med potentielle toksiciteter fra yderligere udvikling.
I de følgende protokoller, procedurer identificere nye terapeutiske RNA og optimere format eksisterende beskrives. Fremgangsmåderne er nyttige til screening RNA baserede post-integration inhibitorer af HIV-1 replikation og kan tilpasses til at screene andre post-integration inhibitorer, såsom små molekyler rettet Rev medieret eksport af viralt RNA 18 eller CRISPR / Cas systemer designet til at målrette integreret HIV-1-DNA 19.
Protocol
Representative Results
Discussion
HIV-1-produktion assayet beskrevet blev udført under anvendelse HEK293T celler (figur 2) og svarer til assays anvendes til at screene HIV-1 RNA til effektiv ribozym 13, shRNA 10,29, siRNA 30, og U1i RNA 11,31 målsteder. Hjælp af forskellige metoder til at kvantificere HIV-1-produktion, har de fleste undersøgelser målte virusproduktion 48 timer efter co-transfektion af en HIV-1-ekspressionsplasmid med kandidat- RNA'er. Efter produktionen af HIV-1…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Arbejdet præsenteres her blev støttet af den canadiske Institutes of Health Research (CIHR) (giver DCB-120.266, PPP-133.377 og HBF-348.967 til AG).
Materials
| DMEM HyClone | GE Healthcare | SH30243.01 | |
| FBS HyClone | GE Healthcare | SH30396.03 | |
| Penicillin/Streptomycin Gibco | Thermo Fisher | 15140-122 | |
| Cell culture plates, 96 well, 24 well, 6 well. | Corning | 353075, 353047, 353043 | |
| Micro tubes Axygen | Corning | 311-08-051 | |
| Low molecular weight Poly I:C | InvivoGen | 3182-29-6 | |
| DharmaFECT-1 | Dharmacon | T-2001-01 | transfection reagent for synthetic RNAs |
| TransIT-LT1 | Mirus | MIR 2300 | transfection reagent for RNA expression plasmids |
| Nonidet P40 (NP-40) | USB | 19628 | |
| [32P]dTTP | Perkin Elmer | BLU505H | |
| poly(A) RNA template | Sigma-Aldrich | 10108626001 | |
| oligo(dT)12-18 DNA primer | Thermo Fisher | 18418-012 | |
| DEAE filtermat paper | Perkin Elmer | 1450-522 | |
| Microplate scintillation counter | Perkin Elmer | 1450-024 | |
| MTT | Sigma-Aldrich | M-2128 | |
| DPBS HyClone | GE Healthcare | SH30028.02 | |
| Microplate spectrophotometer | Bio-rad | 1706930 | |
| Lysis buffer tablets | Roche | 4693159001, 4906837001 | protease and phosphatase inhibitors |
| Microcentrifuge | Eppendorf | 5415R | |
| Bradford reagent | Bio-rad | 500-0006 | |
| Gel running chamber | Hoefer | SE600 | |
| Semi-dry transfer cell | Bio-rad | 1703940 | |
| Protein ladder EZ-Run | Thermo Fisher | BP3603-500 | |
| Nitrocellulose membrane | Bio-rad | 162-0094 | |
| BSA | Sigma-Aldrich | A9647-1006 | |
| Antibody stripping solution | Millipore | 2504 | |
| ECL – Pierce | Thermo Fisher | PI32106 | |
| ADAR1 antibody | from Dr. B.L. Bass | ||
| phospho-T446-PKR antibody | Abcam | ab32036 | |
| phospho-S396-IRF3 antibody | Cell Signaling | 4947 | |
| PKR antibody | from Dr. A. Hovanessian | ||
| IRF3 antibody | Cell Signaling | 11904 | |
| Actin antibody | Millipore | MAB1501 | |
| Peroxidase-labeled goat anti-rabbit | KPL | 474-1506 | |
| Peroxidase-labeled goat anti-mouse | KPL | 474-1806 | |
| Ponceau S | Sigma-Aldrich | 6226-79-5 |
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