Abstract
Targeting specific T cell subtypes and intervening in their function are emerging a critical strategy for treatment of autoimmune diseases. Here we report that an RNA CD30 aptamer was utilized to deliver short hairpin RNA (shRNA) to CD30+ T cells to target retinoic acid receptor-related orphan receptor gamma t (RORγt), leading to impaired expression of RORγt and suppression of IL-17A and IL-17F. A DNA template consisting of CD30 aptamer and RORγt shRNA sequences was synthesized and was transcribed CD30 aptamer-RORγt shRNA chimera (CD30-AshR-RORγt). Insertion of 2′-F-dCTP and 2′-FdUTP was incorporated during CD30-AshR-RORγt transcription to increase its resistance to RNase. CD30-AshR-RORγt was specifically up-taken by CD30+ Karpas 299 cells, but not by Jurkat cells which lack CD30. It was also up-taken by activated, CD30 expressing human CD4+T cells, but not by resting CD4+ T cells. The RORγt shRNA moiety of CD30-AshR-RORγt chimera was cleaved and released by Dicers. Then, CD30-AshR-RORγt suppressed RORγt gene expression in Karpas 299 cells and activated human CD4+ T cells. Consistently, silence of Th17 cell differentiation and IL-17A and IL-17F synthesis with CD30-AshR-RORγt was demonstrated in activated human CD4+ T cells from healthy donors and RA patients. CD30-AshR-negative control chimera and prostate specific membrane antigen (PSMA)-AshR-RORγt had no significant impact on the expression of RORγt or IL-17A and IL-17F. These data present a novel strategy for shRNA delivery using CD30 RNA aptamers to down-regulate CD30+ Th17 cells and can be developed as a targeted therapy for treating Th17 cell mediated conditions.
Highlights
Away from inflammatory signaling[22,23,24]
It has been demonstrated that aptamer based delivery system can effectively delivery small interference RNA (siRNA) into CD4+ T cells to inhibit replication of human immunodeficiency virus[26,27], we have shown that CD4 specific RNA aptamer can serve as a delivery vehicle to deliver RORγt into primary human CD4+ T cells and suppress IL-17 production[25]
The ideal agent will need to and directly act on Th17 cells to down regulate Th17 cytokines and spare these cytokines produced by other immune cells[32]
Summary
Away from inflammatory signaling[22,23,24] These compounds are not RORγt specific, but rather affecting ROR family of transcription factors and whereby they will produce undesired adverse effects. RNAi based blockade of RORγt inhibition provides more specific suppression of Th17 cells[25] and is more desirable to clinical development for precision therapy. It has been demonstrated that aptamer based delivery system can effectively delivery siRNA into CD4+ T cells to inhibit replication of human immunodeficiency virus[26,27], we have shown that CD4 specific RNA aptamer can serve as a delivery vehicle to deliver RORγt into primary human CD4+ T cells and suppress IL-17 production[25]. CD4 expressed by monocytes will compete with CD4+ T cells in up-taking CD4 aptamers To overcome these issues, we produced CD30 aptamer-RORγt shRNA (CD30-AshR-RORγt) to suppress RORγt expression. CD30 is expressed by activated T cells including Th17 cells[28,29,30] and is target on activated T cells and may represent a desirable aptamer-shRNA chimera for development for Th17 targeted therapy
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