CD4 aptamer-RORγt 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 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(+) Karpas 299 cells and primary human CD4(+) T cells. The RORγt shRNA moiety of CD4-AshR-RORγt chimera was cleaved and released by Dicer. Furthermore, CD4-AshR-RORγt suppressed RORγt 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.

Keywords: Aptamer–shRNA; RORγt; Th17.

Figure 1. CD4-AshR-RORγt chimera

(A) Chimera in vitro transcribed by T7 RNA polymerase was analyzed by denatured PAGE and ethidium bromide staining. Lane 1, ssRNA ladder; Lane 2, CD4-AshR-RORγt chimera; Lane 3, mock-CD4-AshR-RORγt chimera. (B and C) Predicted secondary structure of CD4-AshR-RORγt chimera (B) and mock-CD4-AshR-RORγt chimera (C). The region of the CD4 aptamer (clone 9 [26]) responsible for binding to CD4 is outlined. The shRNA portion of the chimera consists of targeted RORγt siRNA with 2 overhang nucleotides at its 3′end and a 7 nucleotide loop. (D) Cleavage analysis of synthesized chimeras by Dicer. Lane 1, ssRNA ladder; Lane 2, antisense siRNA to RORγt; Lane 3, intact CD4-AshR-RORγt chimera; Lane 4–6, chimeras were digested with Dicer: Lane 4, Mock CD4-AshR-RORγt chimera; Lane 5, CD4-AshR-RORγt chimera; Lane 6, CD4-AshR-scrambled control chimera (representative of two experiments).

Figure 2. CD4-AshR-RORγt chimera efficiently entered CD4⁺ human T cells

(A) CD4- and mock-CD4-AshR-RORγt chimeras were labeled by incorporating Cy3-CTP during in vitro transcription. Cy3 scanning (right panel) showed strong Cy3-signaling bands that were at an appropriate size of transcripts shown in ethidium bromide imaging (left panel). Lane 1, ssRNA ladder; Lane 2, CD4-AshR-RORγt chimera; Lane 3, mock-CD4-AshR-RORγt chimera. (B and C) Uptake of Cy3-labeld CD4-AshR-RORγt chimera by CD4⁺ human T cell line Karpas 299 cells and CD4⁺ T cells in PBMCs. (D) Flow cytometric analysis showed that Cy3-labeled CD4-AshR-RORγt chimera was significantly internalized in CD4⁺ Karpas 299 cells and CD4⁺ T cells but not in CD8⁺ T cells. There is no uptake of Cy3-labeled mock-CD4-AshR-RORγt chimera by Karpas 299 cells or T-cell enriched PBMCs. Gray: PBS; Red line: Cy3-labeled CD4-AshR-RORγt chimera; Blue line: Cy3-labeled mock-CD4-AshR-RORγt chimera (representative of 2–5 experiments).

Figure 3. Specific silencing of RORγt in human CD4⁺ T cells by CD4-AshR-RORγt chimera

Karpas 299 cells and PBMCs were treated as described in methods. (A through C) Quantitative real-time PCR assay for RORγt gene expression. RORγt gene expression was significantly reduced by CD4-AshR-RORγt chimera in a concentration-dependent manner in Kapas 299 cells (A) and T-cell enriched PBMCs (B). Mock-CD4-AshR-RORγt chimera, CD4-AshR-scrambled control or CD4-AshR-CCR5 chimera had no effect on RORγt gene expression in T-cell enriched PBMCs (C). Additionally, all the chimeras lacked a significant inhibition on TBX21or GATA3 in T-cell enriched PBMCs (C). (Data are presented as mean ± SD of three experiments). 1, PBS; 2, CD4-AshR-RORγt chimera; 3, mock-CD4-AshR-RORγt chimera; 4, CD4-AshR-scrambled control chimera; 5, CD4-AshR-CCR5 chimera. (D and E) RORγt protein expression was analysed by flow cytometry. (D) Karpas 299 cells were stimulated with PMA 50 ng/ml for 24 h. Red line, CD4-AshR-RORγt chimera; Blue line, Mock-CD4-AshR-RORγt chimera (representative of three experiments). (E) PBMCs were stimulated with anti-CD3/CD28 and LPS for 48 h. RORγt expression was reduced by CD4-AshR-RORγt chimera (red line), but not by mock-CD4-AshR-RORγt chimeras (blue line) or CD4-AshR-scrambled control chimera (purple line). Black line, PBMCs without stimulation; green line, PBMCs with stimulation but without chimeras (representative of three experiments). (F) The percentage of RORγt⁺ cells in stimulated T-cell enriched PBMCs was reduced by CD4-AshR-RORγt chimeras, but not by mock-CD4-AshR-RORγt chimera or CD4-AshR-scrambled control chimera. 1, PBMCs without stimulation; 2, PBMCs with stimulation but without chimeras; 3, Stimulated PBMCs were treated with CD4-AshR-RORγt chimeras; 4, Stimulated PBMCs were treated with mock-CD4-AshR-RORγt chimera; 5, Stimulated PBMCs were treated with CD4-AshR-scrambled control chimera (Data are presented as mean ± SD of three experiments).

Figure 4. Reduction of IL-17A synthesis in CD4⁺ T cells by CD4-AshR-RORγt chimera

Karpas 299 cells were stimulated with PMA and T-cell enriched PBMCs were stimulated with anti-CD3/CD28 and cytokines with anti-cytokine antibodies or LPS for Th1, Th2 or Th17 polarization respectively. (A and B) IL-17A in the supernatant was measured by ELISA. IL-17A production was significantly decreased by CD4-AshR-RORγt chimera in a concentration-dependent fashion (Data are presented as mean ± SD of three experiments). (C and D) Reduction of IL-17A-producing CD4⁺ T cells by CD4-AshR-RORγt chimera, but not by mock- CD4-AshR-RORγt or CD4-AshR-scrambled control chimeras. (E, F, G and H) CD4-AshR-RORγt chimera had no significant impacts on IFN-γ- and IL-4-producing CD4⁺ T cells. 1, PBS; 2; CD4-AshR-RORγt chimera; 3, mock-CD4-AshR-RORγt chimera; 4, CD4-AshR-scrambled control chimera (Data are presented as mean ± SD of three experiments).