Discovery Of RNA Interference Research Articles

Biosafety aspects of RNAi-based pests control.

While the overuse of classical chemical pesticides has had a detrimental impact on the environment and human health, the discovery of RNA interference (RNAi) offered the opportunity to develop new and sustainable approaches for pest management. RNAi is a naturally occurring regulation and defense mechanism that can be exploited to effectively protect crops by silencing key genes affecting the growth, development, behavior or fecundity of pests. However, as with all technologies, there is a range of potential risks and challenges associated with the application of RNAi, such as dsRNA stability, the potential for off-target effects, the safety of non-target organisms, and other application challenges. A better understanding of the molecular mechanisms involved in RNAi and in-depth discussion and analysis of these associated safety risks, is required to limit or mitigate potential adverse effects. © 2024 Society of Chemical Industry.

Activity and Silencing of Transposable Elements in C. elegans

Since the discovery of transposable elements (TEs) in maize in the 1940s by Barbara McClintock transposable elements have been described as junk, as selfish elements with no benefit to the host, and more recently as major determinants of genome structure and genome evolution. TEs are DNA sequences that are capable of moving to new sites in the genome and making additional copies of themselves while doing so. To limit the propagation of TEs, host silencing mechanisms are directed at transposon-encoded genes that are required for mobilization. The mutagenic properties of TEs, the potential of TEs to form new genes and affect gene expression, together with the host silencing mechanisms, shape eukaryotic genomes and drive genome evolution. While TEs constitute more than half of the genome in many higher eukaryotes, transposable elements in the nematode C. elegans form a relatively small proportion of the genome (approximately 15%). Genetic studies of transposon silencing, and the discovery of RNA interference (RNAi) in C. elegans, propelled Caenorhabditis elegans (C. elegans) to the forefront of studies of RNA-based mechanisms that silence TEs. Here, I will review the transposable elements that are present and active in the C. elegans genome, and the host defense mechanisms that silence these elements.

Advances in RNA Interference for Plant Functional Genomics: Unveiling Traits, Mechanisms, and Future Directions.

RNA interference (RNAi) is a conserved molecular mechanism that plays a critical role in post-transcriptional gene silencing across diverse organisms. This review delves into the role of RNAi in plant functional genomics and its applications in crop improvement, highlighting its mechanistic insights and practical implications. The review begins with the foundational discovery of RNAi's mechanism, tracing its origins from petunias to its widespread presence in various organisms. Various classes of regulatory non-coding small RNAs, including siRNAs, miRNAs, and phasiRNAs, have been uncovered, expanding the scope of RNAi-mediated gene regulation beyond conventional understanding. These RNA classes participate in intricate post-transcriptional and epigenetic processes that influence gene expression. In the context of crop enhancement, RNAi has emerged as a powerful tool for understanding gene functions. It has proven effective in deciphering gene roles related to stress resistance, metabolic pathways, and more. Additionally, RNAi-based approaches hold promise for integrated pest management and sustainable agriculture, contributing to global efforts in food security. This review discusses RNAi's diverse applications, such as modifying plant architecture, extending shelf life, and enhancing nutritional content in crops. The challenges and future prospects of RNAi technology, including delivery methods and biosafety concerns, are also explored. The global landscape of RNAi research is highlighted, with significant contributions from regions such as China, Europe, and North America. In conclusion, RNAi remains a versatile and pivotal tool in modern plant research, offering novel avenues for understanding gene functions and improving crop traits. Its integration with other biotechnological approaches such as gene editing holds the potential to shape the future of agriculture and sustainable food production.

Using dsRNA Targeting unc-22 to Demonstrate Gene Knockdown Phenotypes in C.elegans in the Undergraduate Biology Lab Setting

The discovery of RNA interference (RNAi) and its role in gene silencing has revolutionized the study of genetics. In this study, we utilized Caenorhabditis elegans (C. elegans) to demonstrate the effects of RNAi in an undergraduate laboratory setting. Specifically, we targeted the unc-22 gene, responsible for coding the 'twitchin' protein which affects muscle coordination in C. elegans. By employing the L4440 vector, we inserted the unc22 gene into E.coli cells and then induced the production of double-stranded RNA (dsRNA) targeting unc-22 using IPTG. Feeding the C. elegans with these dsRNA-expressed E.coli resulted in a 'twitcher' phenotype, indicative of the unc-22 gene knockdown. This streamlined project offered students not only hands-on molecular experimentation but also experience in data analysis of worm movement. Moreover, through gel electrophoresis and bacterial transformation, students observed the nuances of RNAi machinery in action. The study reaffirms the importance of using C. elegans as a model organism for genetics and evolution studies and offers an experiential learning platform for students to understand the fundamental mechanisms of gene silencing.

RNA interference: from target validation to therapeutics

Generally, a new targeted medicine takes a long time, usually 5 to 15 years, from the discovery and validation of its target to its actual use in treating a disease. There is no doubt that steps such as target validation and target evaluation are essential in preclinical development of new drugs to ensure safety, bioavailability, and efficacy of the drug, and in the target validation phase, RNA interference (RNAi) is a very important method. RNAi drug has double-stranded RNA function, so the targeted mRNA of a specific gene sequence is degraded, its transcription process is abnormal, which leads to gene silencing, and related protein synthesis failure. RNAi technology could specifically shut off specific genes, and, it is a targeted drug with great potential. Therefore, RNAi technology can be used to treat some rare diseases, such as acute hepatic porphyria (AHP) in adults, by using drugs as carriers. In 1998, Andrew Fire and Craig C. Mello first defined the RNAi phenomenon and was honored with the Nobel Prize in Physiology or Medicine in 2006 for their discovery of RNAi and their outstanding contributions to the field of gene silencing. Since then, RNAi drugs have been studied by pharmaceutical scientists around the world, but many of the previous drugs have been stopped at the evaluation stage due to their chemical instability. Until October 2018, the worlds first RNAi drug (patisiran) was successfully marketed so that the RNAi drug development became popular again because of remarkable pesticide effect.

A perspective to weaponize microRNAs against lung cancer.

microRNAs are regulatory RNAs that silence specific mRNA by binding to it, inducing translational repression. Over the recent decades since the discovery of RNA interference, the field of microRNA therapeutics has expanded tremendously. The role of miRNAs in disease development has attracted researchers to investigate their potential in therapeutics. In lung cancer, multiple miRNAs are deregulated, and their involvement is observed in cell proliferation, immunomodulation, angiogenesis, and epithelial-mesenchymal transition. Thus, synthetic oligonucleotides are developed to downregulate the overexpressed miRNA or to upregulate the repressed miRNA. However, their clinical efficiency is limited due to the requirement for an effective delivery strategy. Advances in the current understanding of nanotechnology, biomaterial science, and disease molecular pathology have increased the chances of overcoming the limitations of miRNA-based therapy. This review enlists downregulated and upregulated miRNAs in lung cancer. This review also highlights the major contributions to miRNA-based therapeutics for lung cancer and strategies to overcome endosomal barriers. It also attempts to understand the nuances between current advancements in delivery methods, advantages, disadvantages, and practical issues for the large-scale development of miRNA-based therapeutics.

Opportunities and Challenges of RNA Interference Therapeutics in Oncology

随着核酸干扰机制的发现及一系列技术难题的逐步攻克，非肿瘤领域的多款核酸干扰药物在国外获批上市，其以特异性高、疗效持续时间长、研发成功率高等优势，成为令全球瞩目的新兴领域。在肿瘤领域尚未有此类新药上市，人们对这一领域的突破寄予厚望。本文阐述核酸干扰药物的特点、潜在抗肿瘤机制、递送系统的难点及在抗肿瘤领域的进展，分析其在非肿瘤领域的成功难以在肿瘤领域被简单复制的潜在原因，为研发及临床转化提供参考。

RNA functions in bacterial infections and its application to antimicrobial therapy

In the concept of central dogma (RNA is transcribed from DNA to produce proteins), RNA was thought to be merely an intermediary for genetic information to synthesize proteins from DNA. Since the discovery of RNA interference in 2000, research on RNA has progressed remarkably, especially in mammals. On the other hand, the role of RNA in bacterial infections was largely unknown. At that time, we started research on RNA and bacterial infection and revealed that miR-210, a small RNA in the gastric epithelial cells, is involved in gastric diseases caused by Helicobacter pylori in-fection. Furthermore, we have successfully developed sequence-specific antimicrobials by loading CRISPR-Cas13, an RNA-targeting CRISPR-Cas, on bacteriophage. The constructed antimicrobials were effective against at least Escherichia coli and Staphylococcus aureus. In this paper, we would like to introduce the importance of RNA in bacteriology.

Liposomal siRNA Delivery in Papillary Thyroid Carcinoma Cells.

The discovery of RNA interference (RNAi) has opened a new strategy in cancer therapy, especially by silencing target genes. Pharmacologically it can be achieved by introducing of small (19-21 base pairs) dsRNA molecules known as small interfering RNA (siRNA) targeting interested genes. siRNA mediated gene has been widely investigated for its utility in treating various diseases including cancer. However, the systemic delivery of interested siRNA via non-viral methods remains a major challenge with large numbers of polymeric and liposomal systems being tested. The most effective methods involving cationic liposomes delivery to cells. Nonetheless, systemic delivery of siRNA via cationic lipid particles is often poor due to rapid uptake by reticuloendothelial organs, resulting in decreased delivery of these particles to the site of interest. Polyethylene glycol (PEG) has been used in siRNA-liposomes formulation to minimize reticuloendothelial uptake. Also, PEGylation permits the accumulation of the liposomes-loaded siRNA at the tumor sites with defective vasculatures such as enhanced permeability and retention phenomena. Thus, a simple method to prepare stable PEGylated siRNA-loaded lipid particles could provide better systemic delivery system in treating various cancers, including papillary thyroid carcinoma. Here we illustrate a simple protocol for the formulation of siRNA-loaded lipid particles by hydration of freeze-dried matrix (HFDM) method for effective delivery of target specific siRNA to papillary thyroid carcinoma cells.

TNFα siRNA delivery by nanoparticles and photochemical internalization for psoriasis topical therapy

Psoriasis is a chronic inflammatory skin disease that presents increased expression of tumor necrosis factor α (TNFα), a proinflammatory cytokine. The discovery of RNA interference (RNAi), mediated by short interfering RNA (siRNA), made it possible for the expression of some genes to be eliminated. However, for its application, it is necessary to use carriers that can protect siRNA and release it in the target cells. Herein, we developed a delivery system for siRNA based on hybrid polymer-lipid nanoparticles (PLNs) and combined this system with photochemical internalization (PCI), photoactivating the photosensitizer TPPS2a, to optimize the endosomal escape of TNFα siRNA in the cytoplasm, aiming to use the system as a topical formulation to treat psoriasis. The PLNs composed of 2.0% of Compritol® 888 ATO (lipid), 1.5% of poloxamer 188 and 0.1% of the cationic polymer poly(allylamine hydrochloride) showed an average nanoparticle size of 142 nm, a zeta potential of +25 mV, and the ability to efficiently coencapsulate TPPS2a and complexed siRNA. In addition, these materials did not present cellular toxicity and showed high cellular uptake. In vitro delivery studies using porcine skin model revealed that the PLNs delivered siRNA and TPPS2a into the skin. The efficacy was verified using an in vivo psoriasis animal (hairless mouse) model induced by imiquimod (IMQ) cream. The results revealed that PLN-TPPS2a-TNFα siRNA combined with PCI resulted in a decrease in the levels of TNFα, showing the efficiency of the treatment to silence this cytokine in psoriatic lesions, which was accompanied by a reduction in the redness and scaling of the mouse skin. The results showed the potential of the developed PLNs in combined silencing gene therapy and PCI for topical treatment of psoriasis.

TME-Responsive Multistage Nanoplatform for siRNA Delivery and Effective Cancer Therapy.

Since the discovery of RNA interference (RNAi), RNAi technology has rapidly developed into an efficient tool for post-transcriptional gene silencing, which has been widely used for clinical or preclinical treatment of various diseases including cancer. Small interfering RNA (siRNA) is the effector molecule of RNAi technology. However, as polyanionic macromolecules, naked siRNAs have a short circulatory half-life (<15 min) and is rapidly cleared by renal filtration, which greatly hinders their clinical application. Furthermore, the anionic and macromolecular characteristics of naked siRNAs impede their readiness to cross the cell membrane and therefore delivery vehicles are required to facilitate the cellular uptake and cytosolic delivery of naked siRNAs. In the past decade, numerous nanoparticles (NPs) such as liposomes have been employed for in vivo siRNA delivery, which have achieved favorable therapeutic outcomes in clinical disease treatment. In particular, because tumor microenvironment (TME) or tumor cells show several distinguishing biological/endogenous factors (eg, pH, enzymes, redox, and hypoxia) compared to normal tissues or cells, much attention has recently paid to design and construct TME-responsive NPs for multistaged siRNA delivery, which can respond to biological stimuli to achieve efficient in vivo gene silencing and better anticancer effect. In this review, we summarize recent advances in TME-responsive siRNA delivery systems, especially multistage delivery NPs, and discuss their design principles, functions, effects, and prospects.

Topical Application of Double-Stranded RNA Targeting 2b and CP Genes of Cucumber mosaic virus Protects Plants against Local and Systemic Viral Infection.

Cucumber mosaic virus (CMV) is a destructive plant virus with worldwide distribution and the broadest host range of any known plant virus, as well as a model plant virus for understanding plant–virus interactions. Since the discovery of RNA interference (RNAi) as a major antiviral defense, RNAi-based technologies have been developed for plant protection against viral diseases. In plants and animals, a key trigger of RNAi is double-stranded RNA (dsRNA) processed by Dicer and Dicer-like (DCL) family proteins in small interfering RNAs (siRNAs). In the present study, dsRNAs for coat protein (CP) and 2b genes of CMV were produced in vitro and in vivo and applied onto tobacco plants representing a systemic solanaceous host as well as on a local host plant Chenopodium quinoa. Both dsRNA treatments protected plants from local and systemic infection with CMV, but not against infection with unrelated viruses, confirming sequence specificity of antiviral RNAi. Antiviral RNAi was effective when dsRNAs were applied simultaneously with or four days prior to CMV inoculation, but not four days post inoculation. In vivo-produced dsRNAs were more effective than the in vitro-produced; in treatments with in vivo dsRNAs, dsRNA-CP was more effective than dsRNA-2b, while the effects were opposite with in vitro dsRNAs. Illumina sequencing of small RNAs from in vivo dsRNA-CP treated and non-treated tobacco plants revealed that interference with CMV infection in systemic leaves coincides with strongly reduced accumulation of virus-derived 21- and 22-nucleotide (nt) siRNAs, likely generated by tobacco DCL4 and DCL2, respectively. While the 21-nt class of viral siRNAs was predominant in non-treated plants, 21-nt and 22-nt classes accumulated at almost equal (but low) levels in dsRNA treated plants, suggesting that dsRNA treatment may boost DCL2 activity. Taken together, our findings confirm the efficacy of topical application of dsRNA for plant protection against viruses and shed more light on the mechanism of antiviral RNAi.

Investigation of water-insoluble hydrophobic polyethylenimines as RNAi vehicles in chronic myeloid leukemia therapy.

The discovery of RNA interference (RNAi) more than two decades ago opened avenues for avant-garde cancer treatments that possess the ability to evade issues hampering current chemotherapeutic strategies, owing to its specific gene sequence-driven mechanism of action. A potent short interfering RNA (siRNA) delivery vehicle designed to overcome physiological barriers is imperative for successful RNAi therapy. For this purpose, this study explored the characteristics and therapeutic efficacy of low-molecular weight (MW) polyethylenimine (PEI) with high cholesterol substitution, yielding water-insoluble polymers, in chronic myeloid leukemia (CML) K562 cells. A strong impact of cholesterol grafting on the physicochemical attributes of the resultant polymers and their corresponding complexes with siRNA was observed, with the siRNA binding capacity of polymers increasing and complex dissociation sensitivity decreasing with increase in cholesterol content of the polymers. The modified polymer complexes were significantly smaller in size and possessed higher cationic charge compared to the parent polymer. The interaction with anionic heparan sulfate preoteoglycans present on the cell surface was significant in cellular uptake of the complexes. The therapeutic efficacy of siRNA/polymer complexes was reflected in their ability to effectively silence the reporter green fluorescent protein gene and endogenous CML oncogene BCR-ABL as well as significantly inhibit colony formation by K562 cells post BCR-ABL silencing. The results of this study demonstrated beneficial effects of high levels of hydrophobic substitution on low MW PEI on their functional performance bestowing them the potential to be potent RNAi agents for CML therapy.

Recent Advances in Engineered Nanoparticles for RNAi-Mediated Crop Protection Against Insect Pests

Since the discovery of RNA interference (RNAi) in the nematode wormCaenorhabditis elegansin 1998 by Fire and Mello et al., strides have been made in exploiting RNAi for therapeutic applications and more recently for highly selective insect pest control. Although triggering mRNA degradation in insects through RNAi offers significant opportunities in crop protection, the application of environmental naked dsRNA is often ineffective in eliciting a RNAi response that results in pest lethality. There are many possible reasons for the failed or weak induction of RNAi, with predominant causes being the degradation of dsRNA in the formulated pesticide, in the field or in the insect once ingested, poor cuticular and oral uptake of the nucleic acid and sometimes the lack of an innate strong systemic RNAi response. Therefore, in the last 10 years significant research effort has focused on developing methods for the protection and delivery of environmental dsRNA to enable RNAi-induced insect control. This review focuses on the design and synthesis of vectors (vehicles that are capable of carrying and protecting dsRNA) that successfully enhance mRNA degradation via the RNAi machinery. The majority of solutions exploit the ability of charged polymers, both synthetic and natural, to complex with dsRNA, but alternative nanocarriers such as clay nanosheets and liposomal vesicles have also been developed. The various challenges of dsRNA delivery and the obstacles in the development of well-designed nanoparticles that act to protect the nucleic acid are highlighted. In addition, future research directions for improving the efficacy of RNA-mediated crop protection are anticipated with inspiration taken from polymeric architectures constructed for RNA-based therapeutic applications.

Small RNA research and the scientific repertoire: a tale about biochemistry and genetics, crops and worms, development and disease.

The discovery of RNA interference in 1998 has made a lasting impact on biological research. Identifying the regulatory role of small RNAs changed the modes of molecular biological inquiry as well as biologists' understanding of genetic regulation. This article examines the early years of small RNA biology's success story. I query which factors had to come together so that small RNA research came into life in the blink of an eye. I primarily look at scientific repertoires as facilitators of rapid scientific change. I show that for a short period of time, between the years 1998 and 2002, different model organism communities, investigative strategies, technological innovations, and research interests could be successfully aligned to take small RNA research off the ground. I discuss how the keystone discoveries were situated in specific experimental traditions and what strategies were employed to establish these discoveries as more general phenomena. Providing thus a practice-based approach of rapid scientific change, I ask how to relate the change in propositional bits of scientific knowledge with changes in scientific practice.

Progress in the tumor environment (TME)-responsive nanocarriers for &amp;lt;italic&amp;gt;in vivo&amp;lt;/italic&amp;gt; siRNA delivery

Since the discovery of RNA interference (RNAi), RNAi technology has rapidly developed into an efficient tool for post-transcriptional gene silencing, which has been widely used for gene function analysis and clinical or preclinical treatment of various diseases (e.g., malignant tumor, Hepatitis B, obesity, andmultiple neuropathy). Small interfering RNA (siRNA) is the effector molecule of RNAi technology. As a biological macromolecule with negative charges, naked siRNA has a short half-life in vivo , and is easily attacked by nucleases and filtered by glomeruli. Therefore, the clinical application of naked siRNA is significantly limited. The key challenge for clinical translation of RNAi technology is to construct safe and efficient delivery carriers to improve the siRNA stability in vivo and gene silencing efficacy in target cells. Currently, numerous nanocarriers such as liposomes have been employed for in vivo siRNA delivery, which have achieved favorable therapeutic outcomes in clinical or preclinical disease treatment. It has been demonstrated that solid tumors show specific vascular structures and physiological microenvironment (e.g., weakly acidic environment, hypoxia, over-expression of specific chemicals and enzymes). Based on this fact, researches have recently paid great attention to designing and constructingtumor microenvironment (TME)-responsive nanocarriers to improve the accumulation of siRNA in tumor sites and enhance gene silencing in tumor cells. In this review, we summarize the design principles and functional characteristics of recently developed TME-responsive siRNA delivery nanocarriers, analyze their advantages and disadvantages, and explore the development trend of TME-responsive siRNA delivery nanocarriers in the future.

The 50th anniversary of reverse transcriptase-and its ironic legacy in the time of coronavirus.

The 50th anniversary of reverse transcriptase-and its ironic legacy in the time of coronavirus.

RNA interference of tyrosine hydroxylase caused rapid mortality by impairing cuticle formation in Nilaparvata lugens (Hemiptera: Delphacidae).

The application of RNA interference (RNAi) technique in controlling agricultural insect pests has been receiving much attention since the discovery of RNAi. The brown planthopper (BPH) Nilaparvata lugens, a notorious pest of rice, has evolved a high level of resistance to many kinds of insecticides. Tyrosine hydroxylase (Th) is an indispensable survival gene in holometabolous insects, playing key roles in cuticle tanning and immunity. In this study, we investigated whether Th could be used as a potential target in controlling N. lugens. Here, we demonstrated that NlTh had a periodical expression pattern during molting with the highest level observed in epidermis. Dysfunction of NlTH by dsNlTh microinjection or 3-IT feeding similarly caused rapid death of N. lugens. Compared with dsGFP control BPHs, dsNlTh injected BPHs (i) had cuticle pigmentation and sclerotizaton defects; (ii) had less endocuticle lamella in tergum integument; (iii) showed higher mortality during the molting process as a result of defective cuticle shedding; (iv) showed feeding disorders indicated by a low number of probe wound dots on rice; (v) had more vulnerable cuticle. This study demonstrated that TH orthologues play a conservative and crucial role for exocuticle tanning in both holometabolous and hemimetabolous insects, and NlTh could be targeted for RNAi-mediated BPH control. The rapid lethal phenotype of NlTH dysfunction BPHs partly induced by cuticle formation defects. © 2020 Society of Chemical Industry.

RNA interference therapeutics in organ transplantation: The dawn of a new era.

RNA interference (RNAi) is a natural process through which double-stranded RNA molecules can silence the gene carrying the same code as the particular RNA of interest. In 2006, the discovery of RNAi was awarded the Nobel Prize in Medicine and its success has accumulated since. Gene silencing through RNAi has been used successfully in a broad range of diseases, and, more recently, this technique has gained interest in the field of organ transplantation. Here, genes related to ischemia-reperfusion injury (IRI) or graft rejection may be silenced to improve organ quality after transplantation. Several strategies have been used to deliver siRNA, and pretransplant machine perfusion presents a unique opportunity to deliver siRNA to the target organ during ex situ preservation. In this review, the potential of RNAi in the field of organ transplantation will be discussed. A brief overview on the discovery of RNAi, its mechanism, and limitations are included. In addition, studies using RNAi to target genes related to IRI in liver, kidney, lung, and heart transplantation are discussed.

Management of Pest Insects and Plant Diseases by Non-Transformative RNAi.

Since the discovery of RNA interference (RNAi), scientists have made significant progress towards the development of this unique technology for crop protection. The RNAi mechanism works at the mRNA level by exploiting a sequence-dependent mode of action with high target specificity due to the design of complementary dsRNA molecules, allowing growers to target pests more precisely compared to conventional agrochemicals. The delivery of RNAi through transgenic plants is now a reality with some products currently in the market. Conversely, it is also expected that more RNA-based products reach the market as non-transformative alternatives. For instance, topically applied dsRNA/siRNA (SIGS – Spray Induced Gene Silencing) has attracted attention due to its feasibility and low cost compared to transgenic plants. Once on the leaf surface, dsRNAs can move directly to target pest cells (e.g., insects or pathogens) or can be taken up indirectly by plant cells to then be transferred into the pest cells. Water-soluble formulations containing pesticidal dsRNA provide alternatives, especially in some cases where plant transformation is not possible or takes years and cost millions to be developed (e.g., perennial crops). The ever-growing understanding of the RNAi mechanism and its limitations has allowed scientists to develop non-transgenic approaches such as trunk injection, soaking, and irrigation. While the technology has been considered promising for pest management, some issues such as RNAi efficiency, dsRNA degradation, environmental risk assessments, and resistance evolution still need to be addressed. Here, our main goal is to review some possible strategies for non-transgenic delivery systems, addressing important issues related to the use of this technology.