Short Double-Stranded RNA with Immunostimulatory Activity: Sequence Dependence

Abstract

Small interfering RNAs (siRNA) are able to activate the mammalian innate immune system depending on their structure, sequence, and method of delivery. The immunostimulatory activity of double-stranded RNA can be applied to antiviral and antitumor therapy. Here we identified a set of 19-bp RNA duplexes with 3-nucleotid overhangs in the 3' ends that display immunostimulating activity (here and after immunostimulating RNA, or isRNA) and studied their sequence/activity relationships. It was found that the introduction of substitutions in the middle part of the isRNA sequence (10-16 positions counting from the 5' end of strand 1) does not alter the antiproliferative activity, while substitutions in the 3' end region of isRNA substantially reduce it. isRNAs efficiently inhibit the proliferation of human oral epidermoid carcinoma cells [half-maximal inhibitory concentration (IC(50)) values varied from 10 to 100 nM]. Our research demonstrated that antiproliferative effects of isRNAs are related to cell growth arrest, rather than the induction of apoptosis. These isRNAs strongly stimulate the synthesis of interferon-α (IFN-α), and to a lesser extent the synthesis of tumor necrosis factor (TNF-α) and interleukin-6 (IL-6), in adherent peripheral blood mononuclear cells. An intravenous injection of isRNA/Lipofectamine complexes into C57BL mice increases IFN-α and IL-6 levels in the blood serum up to 15-fold and 3-fold, respectively, compared to the control mice. The results obtained clearly demonstrate the pronounced immunostimulatory and antiproliferative properties of the isRNAs under study. Hence, these short double-stranded RNAs can be considered as potential agents for the therapy of oncological and viral diseases.

Immunotherapy of hepatocellular carcinoma with small double-stranded RNA

“…Although the control type 1 interferon inducer poly(I:C) [27] activates the synthesis of IFN-α more effectively than isRNA, it induces as high as an 17-fold increase of IL-6 level (Figure 1B). The data obtained are in agreement with our previous results [25] demonstrated that isRNA after intravenous administration mainly induces the synthesis of IFNα and to a lesser extent the synthesis of IL-6. It should be noted, that after i.p.…”

“…In the present study we used the most effective among previously tested [25] immunostimulatory isRNA molecules of 19-bp long with 3-nt overhangs at the 3′-ends. As the positive control, poly(I:C), which is known to induce an interferon response, was used [27].…”

“…Our results show that 19-bp-long isRNAs with 3-nt overhangs at the 3′-ends induce IFN-α (Figure 1), but not proinflammatory cytokines IL-6 and TNF-α secretion in CBA/LacSto mice [25]. It is very important for HCC treatment to use type I IFN inducers, because in many cases HCC develops on a background of chronic inflammatory liver disease (hepatitis B and C, cirrhosis) and secretion of pro-inflammatory cytokines could additionally complicate the course of disease.…”

“…Recently, we designed a series of short double-stranded RNAs that possess pronounced antiproliferative activity in cancer cells [24,25]. The study of their sequence/activity relationships showed [25] that the introduction of substitutions in the middle part of isRNA sequence does not alter the antiproliferative activity.…”

“…The study of their sequence/activity relationships showed [25] that the introduction of substitutions in the middle part of isRNA sequence does not alter the antiproliferative activity. Disruption of GUGU motif at the 5′-end of strand 1, which is similar to the reduced version of immunostimulatory sequence according to [26], caused a minor decrease of the antiproliferative activity compared with the parent isRNA.…”