Mohammed Amarzguioui

Functional polarity is introduced by Dicer processing of short substrate RNAs

Synthetic RNA duplexes that are substrates for Dicer are potent triggers of RNA interference (RNAi). Blunt 27mer duplexes can be up to 100-fold more potent than traditional 21mer duplexes (1). Not all 27mer duplexes show increased potency. Evaluation of the products of in vitro dicing reactions using electrospray ionization mass spectrometry reveals that a variety of products can be produced by Dicer cleavage. Use of asymmetric duplexes having a single 2-base 3′-overhang restricts the heterogeneity that results from dicing. Inclusion of DNA residues at the ends of blunt duplexes also limits heterogeneity. Combination of asymmetric 2-base 3′-overhang with 3′-DNA residues on the blunt end result in a duplex form which directs dicing to predictably yield a single primary cleavage product. It is therefore possible to design a 27mer duplex which is processed by Dicer to yield a specific, desired 21mer species. Using this strategy, two different 27mers can be designed that result in the same 21mer after dicing, one where the 3′-overhang resides on the antisense (AS) strand and dicing proceeds to the ‘right’ (‘R’) and one where the 3′-overhang resides on the sense (S) strand and dicing proceeds to the ‘left’ (‘L’). Interestingly, the ‘R’ version of the asymmetric 27mer is generally more potent in reducing target gene levels than the ‘L’ version 27mer. Strand targeting experiments show asymmetric strand utilization between the two different 27mer forms, with the ‘R’ form favoring S strand and the ‘L’ form favoring AS strand silencing. Thus, Dicer processing confers functional polarity within the RNAi pathway.

Rational design and in vitro and in vivo delivery of Dicer substrate siRNA.

RNA interference is a powerful tool for target-specific knockdown of gene expression. The triggers for this process are duplex small interfering RNAs (siRNAs) of 21–25 nt with 2-bp 3′ overhangs produced in cells by the RNase III family member Dicer. We have observed that short RNAs that are long enough to serve as Dicer substrates (D-siRNA) can often evoke more potent RNA interference than the corresponding 21-nt siRNAs; this is probably a consequence of the physical handoff of the Dicer-produced siRNAs to the RNA-induced silencing complex. Here we describe the design parameters for D-siRNAs and a protocol for in vitro and in vivo intraperitoneal delivery of D-siRNAs and siRNAs to macrophages. siRNA delivery and transfection and analysis of macrophages in vivo can be accomplished within 36 h.

Ex vivo and In vivo Delivery of Anti-Tissue Factor Short Interfering RNA Inhibits Mouse Pulmonary Metastasis of B16 Melanoma Cells

Purpose: The coagulation trigger tissue factor has been implicated in tumor growth, angiogenesis, and metastasis. In this study, we explore the effects of ex vivo and in vivo delivery of short interfering RNA (siRNA) targeting tissue factor on B16 melanoma colonization of the lung in a murine model for metastasis. The purposes of this work are to establish a noncytotoxic in vivo model for investigation of tissue factor function and provide preclinical assessment of the therapeutic potential of tissue factor siRNA for prevention of metastasis. Experimental Design and Results: C57BL/6 mice were evaluated for pulmonary metastases following tail vein injection of B16 cells transfected with either active or inactive siRNA. Mice receiving cells transfected with active siRNA had significantly lower numbers of pulmonary tumors compared with mice injected with control cells (transfected with inactive siRNA). The average time point at which the mice started to exhibit tumor-associated stress was also increased significantly from 22 days for the control group to 27 days for the experimental group (P = 0.01). In a therapeutically more relevant model, where the siRNA was delivered i.p. and the cells (untransfected) by tail vein injection, an inhibitory effect on metastasis was observed when the siRNA treatment was initiated either before or at the time of cell injection. Conclusions: The results suggest that tissue factor has a crucial function in promoting lung tumor metastasis of blood-borne tumor cells in the early stages of the tumor take process and further suggest that treatment with tissue factor siRNA may become a viable clinical strategy for prevention of tumor metastasis.

Knockdown of C‐terminal Src kinase by siRNA‐mediated RNA interference augments T cell receptor signaling in mature T cells

C‐terminal Src kinase (Csk) controls the Src family kinase Lck, which is essential for T cell antigen receptor (TCR)‐mediated signaling. For the first time, we here report the effects of acuteelimination of Csk in Jurkat T cells and primary T cells using short interfering (si) RNA. In both cell types, 70–85% knockdown of Csk was achieved within 48 h. No alterations in surface expressionof CD3, CD4 or CD8, or in Lck protein level were observed. Phosphorylation of Y505 in Lck was markedly reduced and a concomitant 4–5‐fold increase in Lck Y394 phosphorylation was observed both in normal and Jurkat T cells. Kinase assays revealed 2–3‐fold higher Lck activity. In Jurkat cells, basal levels of ζ chain phosphorylation were elevated, and spontaneous NFAT‐AP‐1 activation occurred, indicating aberrant Lck kinase activity. After TCR triggering, Csk knockdown cells revealed faster and stronger, but not sustained, phosphorylation of Lck Y394 and ζ chains compared to control. TCR‐induced activation of NFAT‐AP‐1 and TCR/CD28‐stimulated IL‐2 secretion occurred at weaker stimuli and with augmented responses in Csk knockdown Jurkat and primary T cells, respectively. Altogether, these data suggest that acute elimination of Csk in T cells without evolution of compensatory mechanisms results in aberrant Lck activity and augmented TCR‐stimulated responses.

Principles of Dicer Substrate (D-siRNA) Design and Function

An efficient RNAi largely depends on optimal design of the siRNA. In recent studies, Dicer substrates were found to be more potent than classical synthetic 21-mer siRNAs, suggesting a coupling of the Dicer-mediated processing step to the efficient assembly of the silencing complex, RISC. We describe the fundamental principles and experimental results leading to optimal Dicer substrates.

Downregulation of tissue factor by RNA interference in human melanoma LOX-L cells reduces pulmonary metastasis in nude mice.

Tissue factor (TF) is the membrane receptor of the serine protease coagulation factor VIIa (FVIIa). Formation of the TF/FVIIa complex initiates the coagulation cascade. We used short hairpin RNA (shRNA)‐mediated RNA interference to knock down TF expression in the human metastatic melanoma cell line LOX‐L. After transfection with the shRNA construct, 3 stable clones with significantly downregulated TF expression were established. They exhibited decreased proliferation in vitro as determined by 14C thymidine incorporation and soft agar assay. The in vivo metastatic potential was assessed in an experimental pulmonary metastasis model in which cells from different clones were injected into the tail vein of nude mice. The incidence of pulmonary tumors was significantly lower in mice receiving shRNA‐expressing cells (33% ± 15%) than in control mice injected with wild‐type cells or cells stably transfected with empty expression vector (90% ± 10%). The mice injected with TF‐downregulated cells had markedly longer survival time (69 ± 17 days) compared to the control mice (35.6 ± 5 days; p = 0.03). Thus, reduction of TF levels in LOX‐L cells significantly delayed and reduced lung tumor formation. As a first step in elucidating the molecular basis for this effect, we compared the global gene expression profile in TF‐downregulated cells and control cells by using cDNA microarray analysis. Forty‐four known human genes were found to be significantly upregulated (> 2‐fold; p < 0.05) and 228 genes significantly downregulated (≥ 3‐fold; p < 0.05) in TF‐downregulated cells compared to control cells. The differentially expressed genes encode proteins functioning in transcription, translation, cell communication and cell growth/death. The results provide a basis for investigating molecular mechanisms underlying the effects of TF on the metastatic capacity of LOX‐L melanoma cells.

Short-Interfering RNA-Mediated Lck Knockdown Results in Augmented Downstream T Cell Responses

The Src family kinase Lck is essential for T cell Ag receptor-mediated signaling. In this study, we report the effects of acute elimination of Lck in Jurkat TAg and primary T cells using RNA interference mediated by short-interfering RNAs. In cells with Lck knockdown (kd), proximal TCR signaling was strongly suppressed as indicated by reduced ζ-chain phosphorylation and intracellular calcium mobilization. However, we observed sustained and elevated phosphorylation of ERK1/2 in Lck kd cells 30 min to 2 h after stimulation. Downstream effects on immune function as determined by activation of a NFAT-AP-1 reporter, and TCR/CD28-stimulated IL-2 secretion were strongly augmented in Jurkat and primary T cells, respectively. As expected, overexpression of SHP-1 in Jurkat cells inhibited TCR-induced NFAT-AP-1 activation, but this effect could be overcome by simultaneous kd of Lck. Furthermore, acute elimination of Lck also suppressed TCR-mediated activation of SHP-1, suggesting the possible role of SHP-1 in a negative feedback loop originating from Lck. This report underscores Lck as an important mediator of proximal TCR signaling, but also indicates a suppressive role on downstream immune function.