5 How to efficiently deliver small interfering RNA into living cells？

CreativeBiolabs is a contract research organization for mRNA studies. They produce podcasts on mRNA technology. Today's podcast discusses delivering small interfering RNA into living cells for cancer treatment. Immunotherapy has shown promise in curing cancer. RNA interference can regulate immune cells. Tumor vaccines use tumor antigens to activate the immune system. Cancer vaccines face challenges due to immunosuppressive mechanisms. Designing effective cancer vaccines involves stimulating dendritic cells and inhibiting immunosuppressive factors. Indolamine-2, 3-dioxygenase-positive dendritic cells can inhibit T-cell activation. RNA interference is a potential strategy to interfere with negative regulatory mechanisms. siRNA is a promising therapeutic tool that can activate innate immunity. Targeting interleukin-10 expression can enhance the effectiveness of cancer treatment. Dendritic cells process and present antigens by activating toll-like receptors. Stimulating dendritic cell matu Welcome to CreativeBiolab Science Channel. CreativeBiolabs is a specialized contract research organization supporting mRNA studies with all-round solutions covering mRNA synthesis, modification, and mRNA therapeutics development. With an unwavering pursuit of innovation and lifelong learning, we keep on producing podcast series related to mRNA technology based on our knowledge and practical experience gained through years of exploration in this area. Subscribe to our channel and keep updated with our podcasts. Good evening, dear friends. Thank you for tuning in to CreativeBiolabs podcast. Today we are going to talk about how to efficiently deliver small interfering RNA into living cells. Traditional cancer treatments, such as surgeries, radiotherapy, and drug treatment, all have certain limitations. Due to poor targeting, radiotherapy and drug treatment are easy to damage normal cells and produce adverse effects. Cancer has the biological characteristics of easy invasion and recurrence, so it needs a better targeted and less toxic treatment plan. In the past decade, immunotherapy has become an alternative form of cancer therapy with the potential to eradicate tumor metastasis. Today, we again invited our old friend David to our program. Thank you for joining us, David. To begin, can you introduce us to immunotherapy? Thank you for your invitation. I'm very excited to be here. Let's start from immunotherapy. In the past few decades, the emerging immunotherapy has convinced the medical community for the first time that it is possible to cure cancer, or, in the future, we can control cancer as a chronic disease like hypertension and diabetes. When we say immunotherapy, it is not just one type, but a wide range of cancer therapies, including cell-based therapies, vaccines, and immune system regulators. At present, the overall remission rate of chimeric antigen receptor T cell immunotherapy approved by FDA for specific blood tumor types can reach more than 70%. These treatments have been shown to induce significant therapeutic responses. Even patients with advanced stage of cancer whose survival time is only a few months can be expected for a complete remission, and in some cases the strong response lasts for months or even years. But isn't its therapeutic potential generally limited by immunosuppressive proteins that negatively regulate the function of dendritic cells and T cells? Is this something that any scientific and technological means can help? Yes. The recent discovery of RNA interference promotes the study of gene function in immune cells. Recent data show that the maturation, function and survival of dendritic cells can be regulated by small interfering RNA, targeting immunosuppression-related genes. That's exciting. And I know that with the development of tumor genomics, biological immunotherapy has become a powerful means of tumor treatment. Studies have shown that tumor cells have tumor antigens, and the immune system can distinguish tumor cells from normal cells by recognizing tumor antigens. Assisted by cytokines, camokines and other adjuvants, tumor vaccines can activate or enhance the body's own anti-tumor immunity by expressing specific and immunogenic tumor antigens, so the tumor vaccine can then be used to kill and remove tumor cells. Tumor vaccines are pretty popular as a research topic right now in the field of tumor therapy. What can you tell us about cancer vaccines? Right, cancer vaccines use tumor cell-related antigens to wake up the human immune system against cancer. The commonly used cells are antigen-presenting cells, mainly dendritic cells. However, there is a problem in the use of cancer vaccine, you know, there is active immunosuppression in the tumor microenvironment. Tumor cells have been suspected to evade immune detection by not expressing surface antigen. In fact, escape is not the whole ability of tumor cells. Now cancer cells are pretty cunning, they can also stimulate regulatory T-cells or higher suppressor cells from bone marrow to induce immunosuppression. In other words, one of the main reasons for the failure of cancer vaccines is that there are many immunosuppressive mechanisms that inhibit the activation of dendritic cells. As a result, things become more difficult, and we have to find a way to neutralize this immunosuppression so that the vaccine can really work. So I guess the cancer vaccines seem to be one of the essential ways in the future to deal with cancers. Any group thought of a good method to design effective cancer vaccines? Yeah. Some are actually developing drugs that stimulate the function of dendritic cells or inhibit the expression of immunosuppressive factors such as interleukin-10 and indolamine-2, 3-dioxygenase. Interleukin-10 is involved in the development of tolerogenic dendritic cells. Indolamine-2, 3-dioxygenase is an enzyme which is involved in inhibiting the activation of the low-reactive T-cells, and we know this can lead to tumor escape from the host immune system. If indolamine-2, 3-dioxygenase was used to help tumor cells escape immune system clearance, how can it improve the efficacy of cancer vaccine? Yeah, that's a good question. We see from recent research results that indolamine-2, 3-dioxygenase-positive dendritic cells are expected to naïve T-cells transformed into regulatory T-cells. This can induce tolerance rather than activate T-cells. A naïve T-cell is a T-cell that has matured and been released by the thymus but has not yet encountered its corresponding antigen. In other words, naïve T-cells are in the stage between maturity and activation. Each naïve T-cell has a unique T-cell receptor that recognizes a specific antigen. So you mean indolamine-2, 3-dioxygenase-positive dendritic cells can inhibit the activation of T-cells? But through what substance? Right. These cells produce a variety of tryptophan metabolites, such as N-formylcanuronine, which has a general immunosuppressive effect on T-cell activation. Therefore, siRNAs blocking the expression of indolamine-2, 3-dioxygenase may enhance the efficacy of tumor vaccine. It seems that in order to interfere with the negative regulatory mechanisms in dendritic cells, such as the expression of interleukin-10, RNA interference is a very potential strategy? You're absolutely right. RNA interference is a sequence-specific gene-silencing mechanism triggered by double-stranded RNA. It is no doubt a powerful tool for studying gene function in a wide range of organisms. So, can we say that as the effector of RNA interference, siRNA is also a promising therapeutic tool? Of course. siRNAs can be used alone or in combination with other traditional therapies. Some researchers have shown that when chemically synthesized siRNA is delivered to the endosome via cationic liposomes, it can activate innate immunity. I have also heard that siRNA has an effect on the expression of interferon. How does that happen? Both single-stranded and double-stranded siRNA can induce the expression of cytokines and interferon in a sequence-specific manner, and the response is mainly mediated by toll-like receptors. Many believe that siRNA's activated interferon pathway can hinder the treatmentability of siRNAs. But we can also use a combination strategy to block the expression of immunosuppressive factors and activate innate immunity. Can this lead to more effective cancer treatment? Great thought. Yes. In this case, some researchers have shown that targeting interleukin-10 expression by bifunctional siRNA can block interleukin-10 expression and activate toll-like receptors signaling in human monocytes and dendritic cells. They also showed that the cytoplasmic delivery of siRNA containing 5-triphosphate transcribed in vitro was more efficient. This is more likely to induce cytokine production by activating retinoic acid-induced gene I, which recognizes a variety of RNA viruses. Specifically, retinoic acid-inducible gene I senses RNA containing terminal 5-triphosphate. But I thought under physiological conditions, dendritic cells do not process autoantigens and present them to T-cells. So how are these antigens processed and presented? The treatment and presentation of non-self and autoantigen by dendritic cells, you know, need to activate the toll-like receptor. The development of drugs that stimulate the maturation of dendritic cells through endosome toll-like receptors inhibit the expression of immunosuppressive factors or regulate the function of T-cells may break self-tolerance and activate high-affinity self-reactive T-cells. Thank you for sharing your insight with us, David. As you mentioned, dendritic cells are essential for connecting innate and adaptive immune responses. In addition, they play a key role in tumor immunity. So we have again reached the end of today's program. Thanks everyone for listening. I'll see you next time.