Silencing RORγt in Human CD4+ T cells with CD30 aptamer-RORγt shRNA Chimera

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.

www.nature.com/scientificreports www.nature.com/scientificreports/ away from inflammatory signaling [22][23][24] . However, these compounds are not RORγt specific, but rather affecting ROR family of transcription factors and whereby they will produce undesired adverse effects. In contrast, RNAi based blockade of RORγt inhibition provides more specific suppression of Th17 cells 25 and is more desirable to clinical development for precision therapy. But critical challenge associated with therapy using RNAi technology is specifically and effectively transfer of small interference RNA (siRNA) or short hairpin RNA (shRNA) across the Th17 cellular membrane. 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 , moreover, 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 drawback of using CD4 aptamers as delivery vehicle is that they bind CD4 + resting T cells and probably poses an unwanted effect. 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 hence is more specifically target on activated T cells and may represent a desirable aptamer-shRNA chimera for development for Th17 targeted therapy.

Materials and Methods
CD30-AshR-RORγt chimera synthesis by in vitro transcription. CD30-AshR-RORγt chimera was synthesized as previously described 25 . In brief, commercial DNA oligos (Table 1) were purchased (Integrated DNA Technologies). cDNA templates holding T7 promoter used for synthesis of RNA chimeras were generated by PCR with Pfu DNA polymerase (Thermo Fisher Scientific) and purified with QIAquick Gel purification kit (Qiagen). The sequences of cDNA were verified by Sanger sequencing. Then, the RNA CD30-AshR-RORγt chimera was transcribed in vitro by T7 polymerase with DuraScribe kit (Illumina). 2′-FdCTP and F'-dUTP were incorporated to strengthen its resistance to RNases. Cy3-CTP (GE) was incorporated (Cy3-CTP/2′-FdCTP ratio = 1/9) for imaging and resolved on 10% dPAGE Gel for Cy3 scanning and then ethidium bromide staining prior to purification with P-30 micro Bio-Spin chromatography columns (Bio-Rad) and phenol extraction and sodium acetate/ethanol precipitation. The sequences of chimeras of CD30-AshR-RORγt and scrambled shRNA are shown in Table 2. In order to verify if the CD30-AshR-RORγt transcribed in vitro is a substrate for endoribonuclearase Dicer that catalyzes cleavage of longer endogenous RNA precursors into short RNA as an intracellular step of RNAi pathway, chimera cleavage was assayed in vitro with recombinant human Dicer Kit (Genlantis) in accordance to the guideline. Prostate specific membrane antigen (PSMA)-AshR-RORγt chimera was synthesized as previously described 26,31 .
www.nature.com/scientificreports www.nature.com/scientificreports/ (eBioscience). CD4 in PBMCs was staining with APC/FITC-anti-human CD4 and analyzed with fluorescent microscopy and flow cytometry. Binding of the Cy3-labeled chimeras on Karpas 299 and Jurkat cells was determined by flow cytometry. Uptake of Cy3-labeled chimeras by Karpas 299, Jurkat cells and PBMCs was analyzed with fluorescent microscopy. Intracellular cellular staining for RORγt, IL-17A and IL-17F in Karpas 299 cells and PBMCs was determined with flow cytometric analysis after cells were fixed and permeable with fixation/permeabilization buffer (eBioscience) and then incubated with PE-anti-mouse/human RORγt and PE-anti-human IL-17A and PE-Cy7-anti-IL-17F (eBioscience).

Real-time PCR.
Real-time PCR was carried out as previously described 25 . Total RNA was prepared with RNeasy mini Kit (Qiagen). The probes and primers for RORγt and GUSB were purchased from Thermo Fisher Scientific. mRNA levels of RORγt were normalized by GUSB.
Statistics. Data presented here were mean ± SD. Data of real-time PCR, and flow cytometry were analyzed by one-way ANOVA followed by Dunnett comparison test. If P value was less than 0.05, the differences were considered to be significant. www.nature.com/scientificreports www.nature.com/scientificreports/

Suppression of IL-17A and IL-17F synthesis in human CD4 + T cells from healthy donors and RA patients by CD30-AshR-RORγt chimera.
RORγt is a master transcription factor that controls Th17 subtype differentiation and synthesis of Th17 cytokines. Thus, it is expected that silencing RORγt by CD30-AshR-RORγt chimera will inhibit synthesis of Il-17A and IL-17F as well. As shown in Fig. 4A,B, CD30-AshR-RORγt significantly decreased intracellular staining of IL-17A and IL-17F by 60% in activated human CD4 + T cells from healthy donors, but CD30-AshR-negative control chimera and PSMA-AshR-RORγt chimera had no effects. It was similarly observed in activated human CD4 + T cells from RA patients that CD30-AshR-RORγt significantly suppressed IL-17A and IL-17F as demonstrated by decreased intracellular staining of these cytokines, while CD30-AshR-negative control chimera and PSMA-AshR-RORγt chimera had no effects on IL-17A or IL-17F synthesis (Fig. 4C,D). In order to rule out the possibility that reduction of IL-17A and IL-17F was due to cell death, the total number of cells in the culture was numerated and the number of CD4 + cells was derived from percentage of CD4 + cells in flow cytometry analysis. There were no significant difference in total CD4 + T cells observed between treatment groups. These results suggest that reduction of IL-17A and IL-17F producing cells was the result of suppression of RORγt expression rather than cell death.

Discussion
Pathogenic effector Th17 cells are highly desirable target for treating Th17 cell mediated diseases. However, the precise targeting is challenging. Current available therapeutics are targeting individual Th17 cytokines, but not the pathogenic Th17 cells. The ideal agent will need to specifically and directly act on Th17 cells to down regulate Th17 cytokines and spare these cytokines produced by other immune cells 32 . Recently, several small molecules have been shown to be able to suppress or deviate the signaling of the Th17 master transcription factor, RORγt, but these small molecules are not Th17 cell specific nor RORγt specific 10 .
RNAi based technology provides gene specific target but suffers from insufficient therapeutic level of delivery to the target cell types. Aptamers have been explored to serve as delivery vehicles to transfer siRNA or shRNA into target cells. A target cell specific aptamer is linked with an siRNA or shRNA to form a molecule complex which is then internalized by the target cell. Several methods are reported to link an aptamer with an siRNA or shRNA for the purpose of delivery 33,34 . McNamra et al. 31 pioneered in making an aptamer-siRNA chimeric RNA, where an RNA aptamer specific for PSMA was directly connected with siRNA targeting survival genes for cancer therapy. This approach was adopted by Wheeler et al. 26 and made a CD4 aptamer-CCR5 siRNA and/or CD4 aptamer-HIV siRNA for inhibition of HIV replication in an in vivo model. We have made a CD4 aptamer-shRNA for RORγt to inhibit Th17 cells 25 . The aptamer-shRNA chimera has significant technical advantage versus aptamer-siRNA. In aptamer-siRNA chimera, the single chained aptamer-anti-sense of the siRNA is transcribed and then the sense strand of the siRNA is annealed. This can be technically challenging since the annealing rate varies. Whereas, aptamer-shRNA is produced by one stage transcription and the production is readily high yield. The major barrier for in vivo use of CD4-AshR-RORγt was the uptake by monocytes which expressing low levels of CD4.
Since there is no cell surface molecule that has been identified to be specific for pathogenic Th17 cells, we took an alternative approach by using CD30 as a portal to deliver shRNA to RORγt. CD30 RNA aptamer that was developed by Mori et al. recognizes a common structure conserved in the TNF receptor family proteins, rather than the ligand-binding site and has a very high affinity of binding CD30 with dissociation constant 0.11 nM 35 . CD30 aptamer-based probes were synthesized and used for imaging of tumors expressing biomarker CD30, further confirming the binding specificity of CD30 RNA aptamers with high affinity 36 . Here we show that addition of RORγt shRNA did not affect the CD30 aptamer forming secondary structure. Indeed, the CD30-AshR-RORγt chimera shows specific binding to CD30 expressing tumor cells and activated primary human T cells expressing CD30 and successfully delivered shRNA to suppress RORγt. The silencing effects of CD30-AshR-RORγt chimera on Rorc gene expression are consistent with specific siRNA transfected by lipid transfection agents 37 . Importantly, CD30-AshR-RORγt chimera is specific for Rorc gene since Tbox 21 and Gata3 genes in CD4 + T cells were not affected, suggesting that CD30-AshR-RORγt chimera does not have off-target effects.
In summary, the data of this study revealed that CD30 can serve as a delivery vehicle for shRNA that targets specific gene, RORγt in CD30 expressing activated human T cells. The internalized RORγt shRNA via CD30 aptamer can be cleavage by Dicers and then specifically silence RORγt gene expression and led to marked decrease of Th17 cell differentiation and IL-17A and IL-17F synthesis. Therefore, CD30-AshR-RORγt chimera may potentially be developed into a therapeutic approach to treatment of autoimmune inflammatory diseases such as RA.