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CD4 aptamer-ROR?t shRNA chimera inhibits IL-17 synthesis by human CD4(+) T cells.

ABSTRACT: Cell type specific delivery of RNAi to T cells has remained to be a challenge. Here we describe an aptamer mediated delivery of shRNA to CD4(+) T cells targeting ROR?t to suppress Th17 cells. A cDNA encoding CD4 aptamer and ROR?t shRNA was constructed and the chimeric CD4 aptamer-ROR?t shRNA (CD4-AshR-ROR?t) was generated using in vitro T7 RNA transcription. 2'-F-dCTP and 2'-F-dUTP were incorporated into CD4-AshR-ROR?t for RNase resistance. CD4-AshR-ROR?t was specifically uptaken by CD4(+) Karpas 299 cells and primary human CD4(+) T cells. The ROR?t shRNA moiety of CD4-AshR-ROR?t chimera was cleaved and released by Dicer. Furthermore, CD4-AshR-ROR?t suppressed ROR?t gene expression in Karpas 299 cells and CD4(+) T cells and consequently inhibited Th17 cell differentiation and IL-17 production. These results demonstrate that aptamer-facilitated cell specific delivery of shRNA represents a novel approach for efficient RNAi delivery and is potentially to be developed for therapeutics targeting specific T cells subtypes.

SUBMITTER: Song P

PROVIDER: S-EPMC4216182 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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CD4 aptamer-RORγt shRNA chimera inhibits IL-17 synthesis by human CD4(+) T cells.

Song Pingfang P Chou Yuan K YK Zhang Xiaowei X Meza-Romero Roberto R Yomogida Kentaro K Benedek Gil G Chu Cong-Qiu CQ

Biochemical and biophysical research communications 20140918 4

Cell type specific delivery of RNAi to T cells has remained to be a challenge. Here we describe an aptamer mediated delivery of shRNA to CD4(+) T cells targeting RORγt to suppress Th17 cells. A cDNA encoding CD4 aptamer and RORγt shRNA was constructed and the chimeric CD4 aptamer-RORγt shRNA (CD4-AshR-RORγt) was generated using in vitro T7 RNA transcription. 2'-F-dCTP and 2'-F-dUTP were incorporated into CD4-AshR-RORγt for RNase resistance. CD4-AshR-RORγt was specifically uptaken by CD4(+) Karpa ...[more]

PMID: 25241192

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Project description:BackgroundRORγt is a transcription factor that enables elaboration of Th17-associated cytokines (including IL-17 and IL-22) and is proposed as a pharmacological target for severe asthma.MethodsIL-17 immunohistochemistry was performed in severe asthma bronchial biopsies (specificity confirmed with in situ hybridization). Primary human small airway epithelial cells in air liquid interface and primary bronchial smooth muscle cells were stimulated with recombinant human IL-17 and/or IL-22 and pro-inflammatory cytokines measured. Balb/c mice were challenged intratracheally with IL-17 and/or IL-22 and airway hyperreactivity, pro-inflammatory cytokines and airway neutrophilia measured. Balb/c mice were sensitized intraperitoneally and challenged intratracheally with house dust mite extract and the effect of either a RORγt inhibitor (BIX119) or an anti-IL-11 antibody assessed on airway hyperreactivity, pro-inflammatory cytokines and airway neutrophilia measured.ResultsWe confirmed in severe asthma bronchial biopsies both the presence of IL-17-positive lymphocytes and that an IL-17 transcriptome profile in a severe asthma patient sub-population. Both IL-17 and IL-22 stimulated the release of pro-inflammatory cytokine and chemokine release from primary human lung cells and in mice. Furthermore, IL-22 in combination with IL-17, but neither alone, elicits airway hyperresponsiveness (AHR) in naïve mice. A RORγt inhibitor specifically blocked both IL-17 and IL-22, AHR and neutrophilia in a mouse house dust mite model unlike other registered or advanced pipeline modes of action. Full efficacy versus these parameters was associated with 90% inhibition of IL-17 and 50% inhibition of IL-22. In contrast, anti-IL-17 also blocked IL-17, but not IL-22, AHR or neutrophilia. Moreover, the deregulated genes in the lungs from these mice correlated well with deregulated genes from severe asthma biopsies suggesting that this model recapitulates significant severe asthma-relevant biology. Furthermore, these genes were reversed upon RORγt inhibition in the HDM model. Cell deconvolution suggested that the responsible cells were corticosteroid insensitive γδ-T-cells.ConclusionThese data strongly suggest that both IL-17 and IL-22 are required for Th2-low endotype associated biology and that a RORγt inhibitor may provide improved clinical benefit in a severe asthma sub-population of patients by blocking both IL-17 and IL-22 biology compared with blocking IL-17 alone.

Dataset Information

CD4 aptamer-ROR?t shRNA chimera inhibits IL-17 synthesis by human CD4(+) T cells.

Publications

CD4 aptamer-RORγt shRNA chimera inhibits IL-17 synthesis by human CD4(+) T cells.

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