Session Information
Session Title: T cell Biology in Rheumatoid Arthritis and Other Arthritis
Session Type: Abstract Submissions (ACR)
Background/Purpose
RNA interfering (RNAi)-mediated gene silencing holds great promise for manipulating T cells to study basic T cell biology and for developing potential T cell targeted therapeutics. However, efficient delivery of small interfering RNA (siRNA) specifically into primary T cells represents a major hurdle to the widely use of RNAi technology. We explored the use of single-stranded oligonucleotide aptamers as vehicle to deliver small hairpin RNA (shRNA) to target Th17 cells.
Methods
An RNA aptamer specifically binds to CD4 was previously selected and sequenced. siRNA to retinoic acid related orphan receptor (ROR)-γt was previously designed and tested. A cDNA encoding the CD4 aptamer and RORγt shRNA was constructed and the CD4 aptamer-RORγt shRNA (CD4-AshR-RORγt) chimera 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+ human T lymphoma cells, Karpas 299 constitutively expressing RORγt were tested for CD4-AshR-RORγt internalization. Human CD4+ T cells were polarized to Th17 cells for analysis of CD4-AshR-RORγt delivery efficiency and suppression of RORγt expression and IL-17 production.
Results
The CD4 aptamer and RORγt shRNA was transcribed to form a stable single chimeric aptamer-shRNA molecule, CD4-AshR-RORγt. CD4-AshR-RORγt was labeled with flurochrome Cy3 via Cy3-CTP incorporation during RNA transcription. CD4-AshR-RORγt was specifically uptaken by CD4+ Karpas 299 cells and primary human CD4+ T cells as visualized by confocal microscopy and flow cytometry. The RORγt shRNA moiety of CD4-AshR-RORγt chimera was cleaved and released by endoribonuclease Dicer. CD4-AshR-RORγt suppressed RORγt gene expression in Karpas 299 cells and CD4+ T cells in a dose dependent manner, but did not affect Tbox21 gene expression. Furthermore, 50-70% IL-17A production by CD4+ T cells was inhibited by CD4-AshR-RORγt, but not by mock-CD4-AshR-RORγt or CD4-AshR-scrambled sequence.
Conclusion
The present data in our study revealed that CD4 aptamer can serve as a delivery vehicle for shRNA that targets a specific gene in CD4+ human T cells. CD4-AshR-RORγt specifically silenced the targeted RORγt gene expression and led to a marked decrease of Th17 differentiation and IL-17 production. CD4-AshR-RORγt can be evaluated for the development of a therapeutic agent in treatment of Th17 mediated inflammatory disorders.
Disclosure:
C. Q. Chu,
None;
P. Song,
None;
Y. K. Chou,
None;
X. Zhang,
None;
R. Meza-Romero,
None;
K. Yomogida,
None;
G. Benedek,
None.
« Back to 2014 ACR/ARHP Annual Meeting
ACR Meeting Abstracts - https://acrabstracts.org/abstract/cd4-aptamer-ror%ce%b3t-shrna-chimera-inhibits-il-17-synthesis-by-human-cd4-t-cells/