3-5 Advances in RNA Sensing by the Immune System Separation of siRNA Unwanted Effects from RNA Inter

Creative Biolabs is a contract research organization based in New York that specializes in antibody discovery, engineering, and biomanufacturing solutions. In a recent program, they discussed the inhibition of 2-feed modified RNA and its inability to trigger immune activation. They also discussed the molecular basis of RNA sensing and the off-target effects of small interfering RNA (siRNA) therapy. To ensure the specificity of siRNA targeting, scientists use the basic local comparison search tool (BLAST) and analyze global gene expression. Chemical modifications, such as 2-pheduridine modification, can help overcome off-target effects. Dendritic cells play a role in the immune response, and their maturation is crucial for immunity and tolerance. Toll-like receptor signaling pathway is effective in activating dendritic cell maturation. Immunostimulatory siRNA can stimulate dendritic cells and inhibit the expression of negative regulators, leading to the development of effective tumor va Welcome to Creative Biolabs, 100% of the effort, 100% of the service. As a dynamic contract research organization, we are based in New York and serve the whole world. Our seasoned scientists are skilled in antibody discovery, antibody engineering, and biomanufacturing solutions. Good evening, dear friends. Thanks for joining us. In the last program, we talked about the inhibition of 2-feed modified RNA. We understand that unmodified RNA, rather than 2-feed modified RNA, is an effective trigger for innate immunity. We then discussed the function of 2-feed modification, and why 2-feed modified RNA could not trigger immune activation. Beyond that, the molecular basis of RNA sensing by RNA helicases retinoic acid inducible gene 1 is also very important, as concluded by many studies. Today, David will bring us more knowledge about the effect of chemical modifications on small interfering RNA off-target effects, and potential beneficial effects of immunostimulatory small interfering RNAs. Let's welcome David. Hello David, thanks for being with us again. Thank you for your invitation. I'm very excited to be here. And yes, let's continue with small interfering RNAs. They can specifically silence target genes, which have been widely used to elucidate gene function, identify drug targets, and develop more effective therapeutic drugs. However, their off-target effects complicate the interpretation of phenotypic effects in gene silencing experiments, which leads to toxic and side effects of small interfering RNA therapy. Before we start discussing the off-target effects of small interfering RNA, can you talk about the targeting effects of microRNA? Sure. In mammals, microRNAs target transcripts by incomplete base pairing with multiple sites in three untranslated regions. Watson-Crick base pairing with the 5' end of microRNAs, especially the so-called seed sequence, composed of 2-7 nucleotides, is crucial for targeting. And recent data suggest that imperfect microRNA-messenger RNA hybrids with 9-12 nucleotide central protrusions can cause translation inhibition or exonuclease-messenger RNA degradation. In addition, microRNA can also guide DNA methylation. What is the difference between microRNAs and small interfering RNA targeting? The diversity of the mechanisms by which microRNAs are used to silence gene expression highlights the need to re-examine the specificity of small interfering RNAs. Scientists have used microarray to show that small interfering RNA may have extra target effects compared to microRNAs that must be thoroughly studied before use. But how do we ensure the specificity of small interfering RNA targeting? The most common strategy is to use the basic local comparison search tool, BLAST. However, the BLAST program may not be able to detect short sequence extensions. And the recognition of this sequence does not necessarily indicate the occurrence of off-target effects. Similarly, the absence of short homology does not exclude off-target effects. The best way to solve this problem is to analyze global gene expression, especially when small interfering RNAs will be used in functional genomics or therapy. Can a chemical modification of small interfering RNA overcome the off-target effects? Some researchers have found that 2-pheduridine modification can evade the activation of TLR7 or TLR8 and block most TLR7 or TLR8 independent effects, including off-target effects. Although the mechanism of escape is not clear, the interaction between small interfering RNA sequence and unexpected cell messenger RNA is expected to be affected by chemical modification. RNA modification may specifically disrupt the binding of small interfering RNA to mismatched sequences. So is the 2-phed modification the current strategy to overcome the off-target effects of small interfering RNA? Some researchers found that chemical modification of small interfering RNA, such as 2-phedoxymethyl at the second position of the guide chain, inhibited the silencing effect of the off-target gene, but did not affect the small interfering RNA silencing of the target gene. In general, these data provide a simple strategy to reduce small interfering RNA off-target effects. Can you give us more detail on immune activation? Dendritic cells play an important role in the innate immune receptors that connect innate and acquired immunity. They are the only cell type that can initiate the acquired immune response by activating immature T cells. These cells are derived from myeloid or lymphoid bone marrow progenitor cells, which are home to potential antigen entry sites, where they are localized into immature dendritic cells. I'm aware that some recent studies showed controlled immunity and tolerance by dendritic cells. What does that mean to you? Yes, I saw the publication as well. Immature dendritic cells mediate peripheral immune tolerance, without danger signals, and that the lack of appropriate co-stimulation of CD80 or CD86 leads to T cell incompetence and deletion. After antigen capture in the presence of maturation signal, dendritic cells undergo a complex maturation process, which results in the upregulation of major histocompatibility complex class I and II molecules, co-stimulatory molecules such as CD40, CD80 and CD86, and the production of interleukin-12. So we say that the mature state of dendritic cells is closely related to immunity and tolerance. Speaking of the mature state of dendritic cells, under what conditions do they become mature cells? Under physiological conditions, dendritic cells can capture antigens derived from self and non-self compounds with the same efficiency. However, only antigens captured in the presence of danger signals, such as microbes and inflammatory cytokines, can induce dendritic cell maturation, indicated by the expression of CD80 or CD86 molecules. The interaction between these molecules expressed on dendritic cells and CD28 expressed on T cells is crucial for the activation of naive T cells. Which signaling pathways are the most effective in activating dendritic cell maturation? Generally speaking, Toll-like receptor signaling pathway is the most effective. Unlike infectious pathogens, tumors do not induce an effective inflammatory response, leading to dendritic cell activation. What about pathogen-mediated dendritic cell maturation? Well this is mainly mediated by Toll-like receptors expressed on immature dendritic cells. So the optimal dendritic cell maturation may require the combination of cytokines and Toll-like receptor ligands. Can you talk about the potential benefits of immunostimulatory small interfering RNA? Since we are on the topic of dendritic cell maturation here, I think one benefit here would be to stimulate dendritic cells for these cells to mature in secret cytokines, including interleukin-6 and interleukin-12. While interleukin-12 is required for Th1 type response, interleukin-6 may render CD4 effector T-cells refractory to regulatory T-cells-mediated suppression. To extend these findings, researchers designed a bifunctional small interfering RNA and found that they can induce Toll-like receptor signal and block the expression of immunosuppressive factors. Note here, inappropriate expression of immunosuppressive cytokines and other negative regulators is expected to hinder immunity to tumor and virus-infected cells. Therefore, the development of drugs that stimulate dendritic cells and subsequently inhibit the expression of negative regulators will contribute to the development of effective tumor vaccines. Sounds exciting. I heard that in a recent study, researchers evaluated the possibility of combining gene silencing and immune stimulation in a single small interfering RNA molecule. Yes. According to the difference of design and chemical modification, they also found that they can design single function small interfering RNA without immune stimulation function, or double function small interfering RNA with gene silencing and immune stimulation activity. Thank you for sharing with us David. Okay everyone, that's the end of today's program. Thank you for listening. I'll see you next time.