siRNA stabilization prolongs gene knockdown in primary T lymphocytes

Abstract

RNA interference (RNAi)-mediated knockdown of target gene expression represents a powerful approach for functional genomics and therapeutic applications. However, for T lymphocytes, central regulators of immunity and immunopathologies, the application of RNAi has been limited due to the lack of efficient small interfering RNA (siRNA) delivery protocols, and an inherent inefficiency of the RNAi machinery itself. Here, we use nucleofection, an optimized electroporation approach, to deliver siRNA into primary T lymphocytes with high efficiency and negligible impairment of cell function. We identify siRNA stability within the cells as the critical parameter for efficient RNAi in primary T cells. While generally short-lived and immediately lost upon T-cell activation when conventional siRNA is used, target gene knockdown becomes insensitive to cell activation and can persist for up to 2 wk in non-dividing cells with siRNA stabilized by chemical modifications. Targeting CD4 and the transcription factor GATA-3, we show that the use of stabilized siRNA is imperative for functional gene analysis during T lymphocyte activation and differentiation in vitro as well as in vivo.Key words: Gene expression . Immune regulation . T cells Introduction RNA interference (RNAi) is an evolutionarily conserved process by which double-stranded small interfering RNA (siRNA) induces sequence-specific, post-transcriptional gene silencing [1][2][3]. Given its ease of application, its high efficiency and remarkable specificity, RNAi holds great promise for broad in vitro and in vivo application in all biomedical areas. Most importantly, RNAi is directly applicable to essentially all somatic cell types including human primary cells, which makes it an invaluable tool to study human gene function and may enable new therapeutic approaches [4,5].Within the immune system T lymphocytes are one major target for siRNA-based gene silencing, since they are key regulators of immune responses and involved in many immune related disorders, like autoimmunity, chronic inflammation or lymphoma.Despite this high potential, the lack of protocols for efficient and sustained siRNA delivery into primary mammalian cells is currently the major obstacle to the use of RNAi. In particular, primary lymphocytes are highly resistant to non-viral transfection using cationic lipids and polymer reagents [6][7][8]. Although lymphocytes can be transfected by electroporation in vitro, this method so far has been rather inefficient, limited to activated cells and was complicated by a severe impairment of cell function and cell viability [9,10]. Recently, a promising approach for the 2616in vivo targeting of lymphocytes has been reported using antibody-protamine fusion proteins to deliver siRNA [11]. However, recent data from small-hairpin RNA transgenic mice indicate that in T lymphocytes the RNAi machinery itself works inefficiently as compared with other cell types [12]. In fact, due to the aforementioned problems with siRNA delivery, the parameters determining RNAi efficien...

Docking of Lytic Granules at the Immunological Synapse in Human CTL Requires Vti1b-Dependent Pairing with CD3 Endosomes

“…A recently reported modification of siRNA can efficiently downregulate protein expression in primary T cells (44). Introducing this modification into Vti1b-siRNA, we were able to efficiently downregulate Vti1b in activated primary human CD8 + T cells as confirmed by qRT-PCR (Fig.…”

“…The following siRNAs were used: vti1b siRNA [VTi1B_8_4479, Qiagen; modified by Qiagen as described by Mantei et al (44)], stx11 siRNA (1642453; Qiagen), and stx6 siRNA (L-017164-00; Dharmacon). A modified nonsilencing siRNA (Qiagen) was used as control.…”

Prolonged gene silencing by combining siRNA nanogels and photochemical internalization

“…One of the limitations of non-viral carriers for synthetic siRNA delivery, especially when long-term treatment is advised, is the transient nature of the gene silencing effect as a result of cell division and intracellular siRNA degradation [13][14][15][16]. On the other hand, major concerns are raised with regard to adverse effects of siRNA therapeutics, such as unwanted immune activation [17], off-target silencing [18] and saturation of the RNAi machinery [19].…”

IRF4 is essential for IL-21-mediated induction, amplification, and stabilization of the Th17 phenotype

“…WT CD4 ϩ T cells were nucleofected with IRF4-specific si1, a mixture of three different IRF4-specific siRNA constructs, or scrambled siRNA. The siRNAs were designed as described (34) and prepared by IBA. Nucleofection was performed as described (33), then the cells were cultured under the conditions indicated in the experiments.…”