De wereldgezondheidsorganisatie constateerde dat in 2020 37.7 miljoen mensen leefden met het humaan immunodeficiëntie virus (HIV) virus. Dit duidt aan dat de impact van HIV op de volksgezondheid nog steeds significant is. Echter, de vooruitgang in de behandeling van HIV resulteerde in een toegenomen levensverwachting van geïnfecteerde patiënten. Momenteel worden HIV-patiënten behandeld met combinatie antiretrovirale therapie, die de virale lading vermindert maar de ziekte niet geneest. De persistentie van geïntegreerd viraal DNA in een transcriptioneel stille modus in cellulaire reservoirs is de belangrijkste barrière voor genezing van HIV infectie. Mijn onderzoeksgroep focust op een block-and-lock genezingsstrategie met als doel een cellulair reservoir te genereren resistent tegen reactivatie na onderbreking van de behandeling. Zo werden LEDGINs ontwikkeld, kleine moleculen die de interactie tussen het viraal integrase en lens epithelium-derived-growth-factor (LEDGF/p75) inhiberen en zo de virale integratie inhiberen en het residuele provirus omleiden naar regio's resistent tegen reactivatie. Helaas zijn LEDGINs onvoldoende om de transcriptie van HIV volledig te blokkeren. Daarom is diepgaand onderzoek nodig naar andere co-factoren die belangrijk zijn in de regulatie van HIV expressie, zoals bromodomain-containing-protein 4 (BRD4) en mixed-lineage-leukemia-1 (MLL1), om het potentieel voor een block-and-lock genezingsstrategie te verhogen. De branched-DNA (bDNA) techniek laat toe om het het viraal DNA en RNA niveau per cel te analyseren. Bijgevolg is deze techniek een elegante methode om het effect van een inhibitor op de transcriptionele toestand van het provirus te bestuderen, om zo nieuwe doelwitten te identificeren voor een block-and-lock genezingsstrategie.
JQ1 is een bekende BRD4 remmer, gebruikt in de shock-and-kill strategie, die gericht is op het uitroeien van het latente reservoir na reactivatie. Verschillende studies toonden aan dat JQ1 de HIV replicatie stimuleert. In deze studie toonde de bDNA techniek dat JQ1 de basale transcriptie en de tumor necrosis factor-α (TNF-α) geïnduceerde reactivatie van HIV bevorderde op ‘single cell’ niveau met een optimum bij 1 μM JQ1. Deze optimale concentratie van JQ1 accentueert een complexe rol van BRD4 in de transcriptionele regulatie van HIV.
Figuur 1. Het aantal vRNA spots per geïnfecteerde cel, na de bDNA analyse, zijn geplot voor telkens 72 cellen voor elke conditie van de niet gereactiveerde (oranje spots) en gereactiveerde (blauwe spots) cellen. Elke stip representeert het aantal vRNA spots per single cell en de balk representeert de mediaan. Statistische significantie werd berekend met een Krushkall-Wallis test (ns=non-significant, *p<0.05, **p<0.01, ****p<0.0001) tussen de niet gereactiveerde en de geractiveerde cellen en tussen de cellen behandeld met JQ1 in vergelijking met de controle (0 µM JQ1).
Niu et al. rapporteerde, naast het gebruik van BRD4-inhibitors in de shock-and-kill strategie, onlangs de eerste BRD4-inhibitor die HIV replicatie onderdrukt, ZL0580. Helaas belemmerde de hoge toxiciteit en het gebrek aan effect op HIV expressie in deze studie het potentieel van ZL0580 in een block-and-lock genezingsstrategie.
Figuur 2. Het aantal vRNA spots per geïnfecteerde cel, na de bDNA analyse, zijn geplot voor telkens 73 cellen voor elke conditie van de niet gereactiveerde (oranje spots) en gereactiveerde (blauwe spots) cellen. Elke stip representeert het aantal vRNA spots per single cell en de balk representeert de mediaan. Statistische significantie werd berekend met een Krushkall-Wallis test (ns=non-significant, *p<0.05) tussen de niet gereactiveerde en de geractiveerde cellen en tussen de cellen behandeld met ZL0580 in vergelijking met de controle (0 µM ZL0580).
LEDGINs inhiberen de HIV transcriptie maar residuele vRNA expressie blijft aanwezig. Het is het eerder aangetoond dat LEDGINs geen invloed hebben op integratie in de nabijheid van enhancer-regio’s, DNA elementen die transcriptie versterken. Omdat deze enhancer-regio’s wel afhankelijk zijn van BRD4, werd onderzocht of JQ1-gemedieerde inhibitie van BRD4 de residuele vRNA expressie na LEDGIN-gemedieerde retargeting zou inhiberen. In tegenstelling, JQ1 had geen inhiberend effect op residuele HIV replicatie. Interessant is dat deze studie wel aantoonde dat JQ1 de TNF-α geïnduceerde reactivatie van LEDGIN-geheroriënteerd provirus antagoniseert. Desondanks stimuleert JQ1 nog steeds de TNF-α geïnduceerde reactivatie van de cellen die niet behandeld zijn met LEDGINs of met een lage concentratie van LEDGINs met een optimum concentratie van JQ1. Het blijft onduidelijk waarom JQ1 de TNF-α geïnduceerde reactivatie van LEDGIN-geheroriënteerd provirus verhindert en verder onderzoek is nodig om dit te verklaren.
Figuur 3. De relative fold reactivation geïnduceerd door TNF-α (totaal aantal vRNA spots van TNF-α gereactiveerde cellen/ totaal aantal vRNA spots van niet gereactiveerde cellen) is gerepresenteerd voor cellen behandeld met verschillende concentraties LEDGIN en JQ1. De concentratie LEDGIN is aangeduid met verschillende kleuren, terwijl de concentratie JQ1 gedemonstreerd staat in de x-as.
Tenslotte rapporteerden Gao et al. een rol van MLL1 in reactivatie van HIV. Daarom werd het effect van MLL1 depletie op HIV replicatie verder onderzocht. Hiervoor werden infectie-experimenten opgezet waar de HIV replicatie vergeleken werd tussen cellen getransduceerd met lentivirale vectoren en cellen met een depletie voor MLL1 door middel van specifieke miRNA-gebaseerde lentivirale vectoren. De resultaten bewezen dat MLL1 depletie sterk de HIV-infectiviteit vermindert. Er blijven echter verschillende vragen over het moleculaire mechanisme achter deze bevindingen en verder onderzoek is nodig om MLL1 als een doelwit in een block-and-lock genezingsstrategie te identificeren.
Figuur 4. De relatieve luciferase counts, genormaliseerd voor de proteïne expressie (bepaald met de BCA assay) zijn geplot voor het wildtype en de cellen met een MLL1 depletie voor verschillende virus diluties. Elke kleur geeft een verschillende virus dilutie weer en de balken representeren de standaarddeviatie bepaald via technische duplicaten.
Dit onderzoek beklemtoont dat BRD4 en MLL1 cruciale determinanten zijn van de virale transcriptie van HIV. Een aanhoudende inspanning in academisch onderzoek naar de rol van deze co-factoren in de transcriptionele regulatie van HIV kan deuren openen naar een block-and-lock genezingsstrategie voor HIV.
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