Circular RNAs (circRNAs), a type of non-coding RNA, are generated by non-canonical back-splicing events and are highly expressed in the central nervous system (CNS). Numerous studies have shown that they are involved in many pathological and physiological processes. In April 2024, the research team led by Professor Yao Honghong from Southeast University published an article titled "Engagement of N6-methyladenisine methylation of Gng4 mRNA in astrocyte dysfunction regulated by CircHECW2" in the journal Acta Pharmaceutica Sinica B (IF = 14.5). In this article, the authors found that the level of circular RNA HECW2 (circHECW2) was significantly increased in the plasma of patients with major depressive disorder (MDD) and in the mouse model of chronic unpredictable stress (CUS). Notably, the downregulation of circHECW2 could alleviate astrocyte dysfunction and CUS-induced depression-like behaviors. In addition, it was further demonstrated that the downregulation of circHECW2 increased the expression of the methyltransferase WTAP, leading to an increase in the expression of Gng4 through m6A modification, which provided functional insights into the correlation between circHECW2 and m6A methylation. This indicates that circHECW2 may represent a potential target for the treatment of MDD. circHECW2 is upregulated in CUS mice and MDD patients It is noteworthy that LPS- and CUS-induced models of depression are well-documented in studies relevant to depression. The author's previous study first demonstrated that circHECW2 levels were increased in the hippocampus of LPS treated mice. Thus, to investigate the potential involvement of circHECW2 in depression (Fig. 1C and D), the author isolated the hippocampus and collected plasma from CUS mice. Next, the author examined the levels of circHECW2 in the plasma of MDD patients and healthy control individuals (HCs), found that circHECW2 levels were markedly increased in MDD patients (Fig. 1E). Notably, our analysis revealed a positive correlation between circHECW2 levels and the scores of the Hamilton Rating Scale for depression 24 items (HAMD-24) (Fig. 1F), the scores of the Montgomery-Asberg Depression Rating Scale (MADRS) (Fig. 1G) and the scores of the Hamilton Anxiety Scale (HAMA) (Fig. 1H). Additionally, through linear regression analysis, the author uncovered that MDD patients with elevated circHECW2 expression levels and high scores on the childhood trauma questionnaire (CTQ) displayed more severe depression symptoms (Fig. 1J). To assess the predictive capacity of circHECW2 levels for MDD outcomes, the author examined the changes in circHECW2 level two weeks after treatment in MDD patient plasma and found that the level of circHECW2 was decreased 2 weeks after treatment in MDD patient plasma (Fig. 1K). The downregulation of circHECW2 ameliorates the behaviors induced by CUS After microinjection for 2 weeks, the author examined the efficacy of the lentiviral transduction and found that the expression of circHECW2 was decreased in shRNA-circHECW2-injected mice (Fig. 2C). Behavioural tests including sucrose preference test (SPT), forced swim test (FST), and tail suspension test (TST) were employed to evaluate the effect of circHECW2. The sucrose preference of the CUS-treated mice was decreased, indicative of anhedonia. Encouragingly, this deficit was significantly alleviated by the downregulation of circHECW2 expression (Fig. 2D). In both the FST and TST, the time of immobility was notably prolonged in the CUS mice, and these effects were markedly ameliorated in shRNA-circHECW2-injected mice (Fig. 2E and F). Role of circHECW2 on astrocyte dysfunction in CUS mice hippocampus Subsequently, the author investigated the cellular mechanism through which circHECW2 affects functional recovery after CUS. To further assess the cell types in which circHECW2 expression is upregulated, the author detected the expression of circHECW2 in astrocytes, microglia, neurons, and endothelial cells from the CUS mice brain (Fig. 3A). The results revealed a significant upregulation of astrocyte-derived circHECW2 in CUS compared to microglia-, neuronal-, or endothelial cell-derived circHECW2 (Fig. 3B). Additionally, the fluorescence in situ hybridization staining indicated that circHECW2 was abundant in astrocytes (Fig. 3C). Furthermore, shRNA-circHECW2 treatment significantly mitigated the decrease in GFAP expression observed in CUS mice (Fig. 3D and E). Then, the author detected the function of shRNA-circHECW2 on the astrocyte’s morphology using GFAP and 3D reconstruction (Fig. 3F). Sholl analysis indicated that astrocyte dysfunction was induced by CUS, as evidenced by a reduction in branch numbers, length, and volume of astrocytes. Importantly, these deficits were markedly improved by shRNA-circHECW2 treatment (Fig. 3GeI). Taken together, these results suggest that the abnormal upregulation of circHECW2 in astrocytes may represent a critical molecular event in the progression of depression. circHECW2 inhibits m6A methylation by downregulating WTAP Given the potential role of m6A methylation in MDD and the mutual regulation between circRNAs and m6A methylation, we embarked on an investigation to determine whether circRNA’s regulatory role in the pathological processes of depression, particularly in astrocyte-mediated mechanisms, involves m6A modifications. CUS led to a decrease in m6A levels in the hippocampus, an effect that was significantly mitigated by shRNAcircHECW2, indicating a regulation of circHECW2 on m6A methylation (Fig. 4A). We next measured the expression of m6Amodifying enzymes above in CUS mouse models both in mRNA and protein levels, found that only the protein level of WTAP was reduced in the hippocampus of CUS mice (Fig. 4B). Subsequently, we used mouse primary astrocytes transduced with shRNA-circHECW2 lentivirus or circHECW2-overexpressed plasmid for further investigations(Fig. 4C and D). The expression of WTAP was significantly increased in shRNA-circHECW2-treated cells (Fig. 4E), whereas the Western blot analysis indicated that circHECW2 did not alter the levels of METTL3, METTL14, FTO, and ALKBH5, suggesting a specific association between circHECW2 and WTAP. To confirm these findings, primary astrocytes were transduced with the circHECW2 circHECW2-overexpressed plasmid and WTAP was significantly decreased in astrocytes (Fig. 4F). Next, pulldown assay was used to explore the interaction between circHECW2 and WTAP, and circHECW2 showed a stronger affinity to WTAP (Fig. 4G). Furthermore, in vivo experiments demonstrated that shRNA-circHECW2 significantly improved the decrease in WTAP expression induced by the CUS model (Fig. 4H). To explore why WTAP decreased after CUS, we employed immunoprecipitation to detect ubiquitination. The lysine 48-linked ubiquitination (Ub-K48) level of WTAP was significantly decreased by circHECW2 knockdown in the CUS model (Fig. 4I). Next, we investigated the effect of circHECW2 and WTAP on the survival of astrocytes. Corticosterone was used to mimic the depression in vitro. Astrocytes transduced with shRNAcircHECW2 showed an amelioration of the decreased viability induced by corticosterone (Fig. 4J). In contrast, knockdown the expression of WTAP significantly aggravated the decreased viability of astrocytes treated with corticosterone (Fig. 4K). Furthermore, WTAP siRNA decreased viability of astrocytes was ameliorated by shRNA-circHECW2, further indicating a close relationship between circHECW2 and WTAP (Fig. 4L). Additionally, we constructed a brain astrocyte-specific AAVGFAP-WATP knockdown (KD) virus. Three weeks after the microinjection of AAV-GFAP-WATP KD and shRNAcircHECW2 lentivirus in the hippocampus, the mice were subjected to CUS or control. Behavioral experiments including SPT, FST, and TST were examined after 4 weeks of CUS exposure. WTAP regulates m6A modification of Gng4 mRNA in depression To seek the potential downstream molecule that participated in the CUS mouse, the author posted a transcriptome-wide detection of m6A modification in the hippocampus of the CUS mouse in previous study. The gene ontology biological processes (GO-BP) analysis showed that the downregulated genes were enriched in gene terms associated with the cellular process. The region of mRNA with altered m6A modification (downregulated genes) (Fig. 5B). Next, RNA-seq analysis was performed in the hippocampus of the CUS mouse model. We identified 288 genes that were differentially expressed in RNA-seq analysis (Cuffdiff adjusted P-value<0.05) (Fig. 5C). Of particular interest, there were 19 overlapping transcripts between the two comparisons (Fig. 5D), suggesting that these genes may be the target genes involved in astrocyte dysfunction after CUS. Notably, these 19 overlapping transcripts were verified at the mRNA level. In the 4 upregulated transcripts, no gene was validated. Additionally, GNG4 was significantly decreased in the plasma of MDD patients. Additionally, there was a negative correlation between GNG4 and the scores of the HAMD-Cognitive impairment. Based on these findings, GNG4 emerged as a central focus in our study of MDD. Furthermore, we analyzed the pathway involving GNG4 and its association with decreased m6A modification by Kyoto Encyclopedia of Genes and Genomes (KEGG) (Fig. 5E). Next, further results indicated that m6A modification was decreased in the peak region (chr13: 13825437e13825707) in the 30-UTR of Gng4 mRNA (Fig. 5F and G). The author employed luciferase assays to compare the wild-type (WT) and mutant m6A sites with and without WTAP siRNA treatment. These assays demonstrated that the mutation prevented methylation and increased the stability of Gng4 mRNA (Fig. 5H). As expected, overexpression of Gng4 markedly ameliorated the declined viability of astrocytes treated with corticosterone (Fig. 5I). CircHECW2 regulates m6A methylation of Gng4 mRNA via WTAP Furthermore, the author examined the expression and function of GNG4 in the CUS mouse model. The mRNA and protein levels of Gng4 were significantly decreased in the hippocampus of CUS mice, which was consistent with the decreased m6A modification of Gng4 (Fig. 6A and B). In addition, to understand the relationship between Gng4 levels and the WTAP, WTAP siRNA was transfected in mouse primary astrocytes. The expression of GNG4 and Gng4 mRNA are all decreased (Fig. 6C). Moreover, the upregulation of circHECW2 led to reduced mRNA and protein levels of Gng4 (Fig. 6D). Conversely, the downregulation of circHECW2 in astrocytes increased the mRNA and protein levels of Gng4 (Fig. 6E). Co-transfection of WTAP siRNA and shRNAcircHECW2 indicated that WTAP siRNA decreased the GNG4 level, which was markedly ameliorated by shRNA-circHECW2 (Fig. 6F). To validate these in vitro findings, Western blot analysis was performed to assess the levels of GNG4 in shRNA-circHECW2-treated CUS mice. The results showed that shRNA-circHECW2 treatment significantly mitigated the decrease in GNG4 expression observed in CUS mice (Fig. 6G and H). We also constructed a brain astrocyte-specific AAV-GFAP-Gng4 KD virus. Three weeks after the microinjection of AAV-GFAP-Gng4 KD and shRNA-circHECW2 lentivirus into the hippocampus, the mice were exposed to CUS or kept in a control condition. The results of SPT, FSF, and TST revealed that astrocytic Gng4 is involved in the regulation of circHECW2 in depression (Fig. 6IeK). Hippocampus functional connectivity to the prefrontal cortex was positively correlated with GNG4 in MDD patients Finally, the seed-to-voxel analysis (hippocampal functional connectivity) was performed and showed hippocampus functional connectivity (FC) with prefrontal cortex (PFC) in MDD patients. As shown in Fig. 7A-C, the FC between the hippocampus and the prefrontal cortex was significantly reduced in MDD patients when compared to HCs. We next identified a noteworthy decrease in the resting-state functional connectivity (rsFC) between the hippocampus and the dorsolateral prefrontal cortex (Fig. 7D), whereas other brain regions, such as the visual cortex and inferior temporal lobe, exhibited no significant difference. Moreover, we found a positive correlation between GNG4 levels and rsFC, suggesting that GNG4 may play a role in the cognitive brain function of individuals with MDD (Fig. 7E). Summary Our study demonstrated that upregulation of circHECW2 led to the decrease in Gng4 mRNA via WTAP-mediated m6A modification, and caused subsequent astrocyte dysfunction. Specifically, circHECW2 promoted the ubiquitin-mediated degradation of WTAP in the CUS mouse model, and the effect of GNG4 on maintaining normal astrocyte functions is abolished when WTAP is downregulated in astrocytes. Therefore, the circHECW2/WTAP/GNG4 axis regulates astrocyte dysfunction by decreasing GNG4 stability via WTAP-mediated m6A modification. In conclusion, our findings indicate that circHECW2 holds promise as a therapeutic target for the treatment of depression. In addition, our study sheds light on the functional link between circHECW2 and m6A methylation, offering novel insights for the development of preventive strategies and effective treatments for MDD.

Extracellular vesicles (EVs) are various membrane-structured vesicular structures (40-100 nm) released by cells. Due to their low immunogenicity, biodegradability, low toxicity, and the ability to cross the blood-brain barrier, EVs have become prospective drug carriers in the fields of immunotherapy, regenerative medicine, etc., and have emerged as a vehicle for novel drug delivery systems. Circular RNA DYM (circDYM) is derived from exons 4, 5, and 6 of the DYM gene. It can act as a "sponge" for miRNA 9 to adsorb and inhibit miRNA, thus playing a regulatory role. In 2018, the research team led by Yao Honghong from the Department of Pharmacology, School of Medicine, Southeast University discovered that the overexpression of circDYM in the hippocampal region could inhibit the activity of miRNA 9, ultimately reducing microglial cell activation and alleviating depression-like behaviors. On January 13, 2022, based on the above research achievements, the research team led by Yao Honghong constructed extracellular vesicles that target the central nervous system and encapsulate circDYM (RVG-circDYM-EVs), which effectively alleviated the depression-like behavioral disorders caused by chronic stress. They successfully developed circular RNA into a nucleic acid drug. In 2011, a new method for targeted delivery of siRNA using exosomes was developed. The surface membrane protein lamp2b of exosomes was modified and fused with the neuron-specific rabies virus glycoprotein (RVG) to form exosomes with the RVG-lamp2b fusion protein. Through the method of electroporation, exogenous short-chain RNA interference was made to enter the exosomes. The RVG on the surface of the exosomes binds to the acetylcholine receptor to specifically release the short-chain RNA interference into neurons, significantly down-regulating the expression of BACE1, a protein related to Alzheimer's disease. Through a similar method, the researchers constructed the RVG - circDYM - EVs system. Using exosomes as carriers, they achieved the over - expression of circDYM with nicotinic acetylcholine receptor targeting (Figure 1). Two hours after the tail - vein injection of 200 μg of RVG - circDYM - EVs, this exosome system began to enrich in the liver, kidneys, heart, spleen, brain and other parts of the mice (Figure 2). Among them, the liver had the highest enrichment and expression. Over time, the content of exosomes gradually decreased. Furthermore, RVG - circDYM - EVs can be specifically expressed on microglia, neurons and astrocytes in the hippocampal region. This indicates that the exosomes with nicotinic acetylcholine receptor - targeted over - expression of circDYM can penetrate the blood - brain barrier and enter the mouse brain, and are evenly distributed. In vitro cell experiments revealed that RVG - circDYM - EVs could reduce the activation of microglia induced by lipopolysaccharide: the expressions of iNOS, IL - 6, IL - 1β and MCP - 1 were decreased. Subsequently, after tail - vein injection of 100, 200, 300 and 400 μg of RVG - circDYM - EVs to mice with depression - like behavior caused by chronic stress, the 200, 300 and 400 μg dose groups could significantly improve the depression - like behavior of the mice (Figure 3). To further explore the molecular mechanism by which RVG - circDYM - EVs regulate the activity of microglia, the researchers found through single - cell sequencing that the transcription factor TATA - box binding protein - associated factor 1 (TAF1) protein could regulate more than 10 differentially expressed genes and interact with circDYM, co - localizing in the cytoplasm of microglia. In vitro experiments showed that over - expression of TAF1 promoted the expressions of Trpm6 and Cyp39a1, and over - expression of circDYM could inhibit this promoting effect. Lipopolysaccharide could promote the binding of TAF1 to the promoters of Trpm6 and Cyp39a1, and knockdown of either Trpm6 or Cyp39a1 could block the increase in iNOS levels caused by lipopolysaccharide. These molecular experiments indicated that circDYM could directly regulate the activation of microglia through TAF1. Chronic stress could cause a decrease in the expression of tight - junction proteins in the hippocampal region, an increase in the permeability of the blood - brain barrier, and ultimately lead to the infiltration of peripheral immune cells such as CD4 - positive T cells, CD8 - positive T cells and B220 - positive B cells into the brain parenchyma. Treatment with RVG - circDYM - EVs could partially repair the blood - brain barrier and reduce the infiltration of peripheral immune cells. It is reported that the RVG - circDYM - EVs developed in this paper was applied for a patent on February 13, 2020. The application name is: Exosomes with nicotinic acetylcholine receptor - targeted over - expression of circDYM and their preparation methods and applications.

"The 7th circRNA Research and Industry Forum" was successfully held in Guangzhou on November 11-12, 2023! We aim to provide an exchange platform to share the latest circRNA research results, discuss cutting-edge technical methods, and promote the development and application of circRNA in the biomedical industry. Over the past few years, there has been remarkable progress in circRNA research, revealing its important role in gene expression and regulation. Today, with the advancement of technology and in-depth research, we have begun to use circRNA as a biomarker for disease diagnosis, as well as a new target for drug development. At the same time, by combining circRNA research with advanced technologies such as artificial intelligence, we are starting a new wave of biomedical innovation. In this context, we specially planned a roundtable discussion to bring together top experts and industry leaders across disciplines to discuss circRNA's future development strategies, technology challenges and transformation opportunities. We are convinced that such dialogue and collision will not only promote the basic research of circRNA, but also accelerate its practical application in medicine, health, biotechnology and other fields. Let's take a step into this roundtable discussion, listen to the insights and insights of experts, and explore the infinite possibilities of circRNA. The high-quality development of circRNA from basic research to industrial transformation The high-quality development of circRNA from basic research to industrial transformation On the 11th, Chief Professor, Southeast University Prof. Yao Honghong, Prof. Yang Baihua, Professor of Experimental Medicine and Pathology, University of Toronto, Canada; Prof. Li Xiangdong, Professor of College of Biological Sciences and Medicine, University of Science and Technology of China; Prof. Peng Yong, Professor of State Key Laboratory of Biological Therapy, West China Hospital, Sichuan University; The topic of "High-quality development of circRNA from basic research to industrial transformation" was discussed.   The meeting mainly discussed the problems of circular RNA in basic research, application and industrial transformation. In terms of basic research, the key issues to be solved include the specificity of protein expression and the dynamic regulation of RNA structure. In terms of applications, circular RNA can be used as a diagnostic marker and is expected to be a tool for minimally invasive or non-invasive diagnosis. In terms of industrial transformation, problems such as delivery and scale production need to be solved, and the emergence of circular RNA drugs may be predicted in the future. Opportunities and challenges of circular RNA therapy On the 12th, Mr. Zhang Maolei, CTO of Geisai Biology, Mr. Yang Yun, CTO of Ring Code Biology, Mr. Gao Lu, CEO of Yuanyin Biology, Mr. Dai Dongsheng, CEO of Youhuan Biology, and Mr. Xu Congcong, young Distinguished Professor of Suzhou University, had a lively and multi-dimensional discussion on the topic of "Opportunities and Challenges of circular RNA therapy".   The meeting mainly discussed key issues such as the industrialization prospect of circular RNA, industrial chain construction, and delivery, as well as specific practices such as how to find blockbuster drug pipelines and promote clinical progress. The teachers believe that the industrialization of circular RNA has broad development prospects, but it needs to solve the problems at the process level and the bottleneck of industrial chain construction. They suggested strengthening industry-university-research-medical cooperation, promoting innovative drug research and development, and solving circulation problems to promote the development and application of circular RNA therapy.  

RNA-based vaccines are the heroes of the COVID-19 pandemic, setting the record for the fastest vaccine development time in history, taking just one year from development to FDA approval. More recently, mRNA technology has received Nobel Prize recognition, with Katalin Kariko and Drew Weissman winning the 2023 Nobel Prize in Physiology or Medicine for their discovery of nucleoside base modifications that led to the development of effective mRNA vaccines against COVID-19. It has long been recognized that RNA technology possesses a critical limitation: RNA typically exists in a linear configuration, which results in a relatively short lifespan for this form of mRNA. Within a few hours, intracellular nucleases degrade these molecules. While the transient nature of RNA is not problematic for vaccines, as it only requires a brief period to encode proteins that elicit an immune response, it poses challenges for most therapeutic applications where prolonged RNA stability is desirable. Circular RNAs (circRNAs), with their covalently closed ring structure, offer a significant advantage by protecting themselves from nuclease degradation, thereby enhancing stability and extending their lifespan. Theoretically, even at low dose levels, circular RNA could enhance therapeutic efficacy.   Discovery and development of circular RNA Circrnas were first discovered in 1976, but were thought to be by-products of mRNA splicing errors in cells. In 2013, two research papers on circular RNA were published in the journal Nature at the same time, pointing out that circular RNA is a kind of non-coding RNA with regulatory effect, and regulates the expression of other genes by acting as a sponge of miRNA. This makes circular RNA, which has been silent for more than 30 years, striking and becoming a new generation of star molecules, and has been found to play an important regulatory role in cell differentiation, tissue homeostasis, disease development, and immune metabolism.   In July 2018, a key turning point in the history of circular RNA development, Daniel Anderson and others from the Massachusetts Institute of Technology (MIT) published a paper in the journal Nature Communications, demonstrating for the first time that engineered circular RNA can stably and efficiently express proteins in eukaryotic cells. The novel application of foreign circular RNA for protein expression in eukaryotic cells has also proved that circular RNA is an effective substitute for linear mRNA.     Circular RNA entrepreneurship boom Based on this research, Daniel Anderson founded Orna Therapeutics, the world's first company to develop new therapies using circular RNA. The company's core technology lies in its development of RNA cyclization technology, where they achieved an ultra-long circular RNA construct to cyclify the 12,000 nucleotides long mRNA encoding Dystrophin, which is deficient in Duchenne muscular dystrophy (DMD). But Orna isn't the only startup working on cyclization, there are other companies taking a different approach to building circular Rnas. Torque Bio, for example, puts the instructions for making circular RNA into viruses and splices them inside their cells to make it. Chimera Therapeutics uses genetically engineered bacteria to produce circular RNA. In addition, there are two startups from China, Cyclocode Biology and Cremate, which developed new RNA cyclization technologies in separate papers published in preprints last year.   Advantages and progress of circular RNA Talking about the advantages of circular RNA, Howard Chang of Stanford University argues that you can get a durable enough protein with just one injection. In July 2022, Zhang Yuanhao's team published a paper in the journal Nature Biotechnology, which showed that through multiple optimization designs, the protein yield of successful circular RNA translation was increased by hundreds of times, which can achieve effective and durable protein production in vivo. Zhang, along with mRNA pioneer Drew Weissman, winner of the 2023 Nobel Prize in Physiology or Medicine, and others, founded A company called Orbital Therapeutics, which closed a $270 million Series A round in the first half of this year. Proponents of circular RNA technology expect it to become the RNA platform of choice for the pharmaceutical industry, potentially unlocking the next generation of vaccines, therapies for rare diseases, and anti-cancer drugs. When developing circular RNA as a therapy, we need to remove the extra sequence in the ring formation process to avoid causing an unnecessary immune response. Lingling Chen, a researcher at the Center of Excellence in Molecular and Cellular Sciences at the Chinese Academy of Sciences, said it really depends on the specific way the circular RNA is built. Her research, published in the journal Molecular Cell in November 2021, shows that when building circular RNA, the sequence left behind by the self-splicing motif distorts the RNA fold, resulting in an irregularly structured ring structure that triggers an immune response. However, in some cases, an immune response is desirable. In March 2022, a team led by Professor Wei Wensheng from Peking University published a paper in the journal Cell. The study demonstrated in mice and monkeys that compared with linear mRNA vaccines, circular RNA vaccines induced more neutralizing antibodies and more effective T-cell responses. Additionally, circular RNA is more stable at ambient temperatures than linear mRNA, meaning that vaccines based on circular RNA can be stored and transported without the need for a cold chain. Professor Wei Wensheng founded Cyranos Bio, and has begun human trials of a circular RNA COVID-19 vaccine, which is the first time a synthetic circular RNA drug has been tested in humans. In 2023, some other circular RNA-based drugs may enter clinical trials, including a cancer therapy from CureVac that uses circular RNA to encode the immune-stimulating molecule interleukin-12 (IL-12).Orna is preparing to start a clinical trial of a circular RNA therapy in 2024. This circular RNA therapy can reprogram immune cells to attack blood cancer. At the American Society of Gene and Cell Therapy (ASGCT) conference in May this year, Orna's scientists presented preclinical research results showing that injecting a low dose of LNP-delivered circular RNA into mice could reprogram T cells in situ and eliminate tumors in a mouse model of leukemia without the need for any complex cell engineering or high-intensity conditioning drug regimens.Synthetic circular RNAs can not only encode therapeutic proteins. When they fold into specific shapes, these circular RNAs can also directly bind to targets like antibodies, serving as a type of drug known as aptamers. They can capture and isolate different types of regulatory molecules, effectively removing them from the cellular environment. They can also act as "antisense factors" to bind to gene transcripts (mRNAs), blocking or altering their expression. Moreover, they can serve as guiding molecules for RNA editing applications, directing specific enzymes to the mutant gene transcripts that need correction. Various startups are actively exploring these applications.   Artificial intelligence boosts circular RNA research  In May 2023, Huang Liang and Zhang Liang (currently a professor at China Pharmaceutical University), both from Baidu Research USA, in collaboration with Sinovac, published a paper in the top international academic journal Nature. They used artificial intelligence (AI) tools to optimize the mRNA vaccine sequence, thereby helping to create more effective and stable mRNA. This research not only provides a timely and promising tool for mRNA vaccines, but also offers great potential for mRNA therapies to revolutionize healthcare. LinearDesign, a linear design tool developed in the study, optimizes mRNA encoding all therapeutic proteins, including monoclonal antibodies and anti-cancer drugs.   In July 2023, the team published a paper on the preprint bioRxiv, further developing the circDesign algorithm platform for circular RNA structure prediction and sequence design. The research team applied circDesign algorithm to the sequence optimization design of circRNa-based rabies vaccine and herpes zoster vaccine, which enhanced the sequence stability, protein translation efficiency and immunogenicity of circRNA in mouse models, and successfully verified the effectiveness of circDesign platform in the optimization of circRNA sequence design.   It is reported that this is the first case in the world to optimize the design of circular RNA through artificial intelligence algorithms, which is expected to simplify the sequence optimization design of circular RNA and improve the efficiency, stability and protein translation level.   Professor Zhang Liang of China Pharmaceutical University, co-creator of circDesign algorithm and LinearDesign, said that compared with linear RNA, sequence design for circular RNA needs to consider more factors, and the team is currently actively exploring design algorithms for different RNA platforms. The hope is that AI technology will accelerate the development of RNA vaccines and drugs. Problems with circular RNA New progress has been made in the field of circular RNA, but with the development of the field, some problems have been exposed.     In June, Laronde, the most funded company in the circular RNA field, whose core research project used circular RNA to express GLP-1 for weight loss, was exposed as having falsified data. The event also raised doubts about the potential of circular Rnas. Strand Therapeutics, a company that develops synthetic biology-based mRNA therapies, has also developed circra-based therapies, but the company's co-founder and CEO, Dr. Jake Becraft, said that cirrnas are junk! The number of challenges involved in developing a circular RNa-based drug or therapy is mind-boggling and often overlooked.   The first human trial of a circular RNa-based drug was launched in August. But circular Rnas are a long way from starting a revolution in drug development, or fulfilling Laronde's promise to develop 100 new drug programs based on circular Rnas by the end of the century. Whether the advantages of circular RNA will make it superior to other durable treatments, such as traditional gene therapy, as well as emerging gene-editing therapies, remains an area of ongoing investigation and scientific inquiry.   But Alexander Wesselhoeft, first author of the 2018 paper that pioneered new applications of foreign circular Rnas for proteins expressed in eukaryotic cells and co-founder of Orna Therapeutics, is still bullish on circular Rnas. He is now director of RNA Therapy at the Brigham Institute for Gene and Cell Therapy at Massachusetts General Hospital. He believes that despite the great success of linear mRNA vaccines, circular Rnas are the way of the future and will be the first choice for RNA therapeutic technologies.