Pku team upgraded RNA editing technology: the safety and efficiency were greatly improved, and it was closer to clinical practice

2022-06-17 0 By

In recent years, genome editing technology represented by CRISPR/Cas9 has been in full swing in biomedical research and transformation attempts.But for most scientists, the work of tinkering with the human “life code” is still being carefully refined.RNA (ribonucleic acid) editing is a new gene editing technology emerging in recent years, and international pharmaceutical companies have been in the layout of this technology.One of them, named “LEAPER”, is an RNA editing technology independently developed by Professor Wei Wensheng’s team at Peking University’s School of Life Sciences.Earlier in July 2019, Wei’s team reported on LEAPER technology in the international academic journal Nature Biotechnology.The team reported on an updated version of LEAPER 2.0 technology in Nature Biotechnology on Tuesday morning.”Unlike CRISPR-based DNA or RNA editing techniques, LEAPER only needs to express specifically designed RNA (ADAR-RECRUITING RNA) in cells to recruit the endogenous deaminase ADAR.Editing adenosine A→ inosine I (guanosine G) in targeted RNA.”In an interview with (, Wei Wensheng said that because there is no need to introduce exogenous editing enzymes or effectors, the problems of off-target effect, delivery burden and related immunogenicity on the genome and transcriptome are avoided.It is worth noting that LEAPER completely gets rid of the dependence on CRISPR system and is the underlying core technology with independent intellectual property rights, which has important original innovation significance.As an ACCURATE RNA editing tool, “LEAPER does not cause genome sequence changes and has an advantage in terms of safety,” Wei said.In this latest study, the team further refined LEAPER’s editing efficiency and off-target issues.Of interest, nature Biotechnology also published a study by the team of Prashant Mali, assistant professor at the University of California, San Diego (UCSD). Mali et al. also found that rnas that recruit intracellular ADAR deaminase can improve RNA editing efficiency.ADAR1 deaminase is A kind of adenosine deaminase widely expressed in various tissues in human body, which can catalyze the conversion of adenosine A to inosine I (guanosine G) in target RNA molecule.”While most RNA editing research around the world is currently in the laboratory, clinical applications are getting closer.”On the technical level, wei said, using endogenous mechanisms to achieve safer RNA editing, “we are in the leading echelon internationally and have a competitive advantage in this area of research.”At present, genome editing technology represented by CRISPR/Cas9 still has a series of problems, and its clinical application has also encountered bottlenecks.According to Wei and others, one of the root causes of the problem is that current gene-editing systems rely on the expression of editing enzymes or effector proteins of bacterial or viral origin, such as Cas9 nuclease in bacteria.This dependency leads to multiple problems.Such as too much protein molecular weight made by virus vector for loading and delivery is very difficult, in the human body caused by the protein expression through the level of DNA/RNA off-target effects, caused by exogenous protein expression of the body’s immune response and damage, the body within the pre-existing antibodies that exogenous enzyme or effect editor protein by neutralizing antibodies to cause the failure of gene editing, etc.Wei wensheng et al. ‘s solution is to use mechanisms that exist naturally in cells.In this study, we first discovered that a specially designed ADAR-recruiting RNAs (arRNAs) can efficiently and precisely edit specific adenosine on the targeted gene transcript by recruiting intracellular ADAR1 deaminase without introducing any exogenous effector protein.This new RNA Editing technology was named LEAPER (Leveraging Endogenous ADAR for Programmable Editing on RNA).The team has previously shown that LEAPER can accurately edit most of the adenylate sites on the RNA molecule.In human primary cells, including lung fibroblasts, bronchial epithelial cells and T cells, LEAPER editing efficiency was up to 80%.”While LEAPER has considerable potential for research and disease treatment, there are limitations to the technology.”Wei Wensheng mentioned two points. First, LEAPER uses endogenous editing enzymes, which will limit its editing efficiency.In addition, arRNA with a certain length may cause off-target editing of bases adjacent to the target editing site.In this latest study, they first found that enhancing arRNA expression by optimizing the promoter in the expression vector significantly improved the editing efficiency of the LEAPER system, “suggesting that the abundance of arRNA in cells is critical for editing efficiency.”Another key point is that because linear arRNA is easily degraded in cells, the team thought of using cyclization.Annular Rnas, which do not have 5 ‘or 3’ ends and can avoid exonuclease cleavage, have better stability and a longer half-life in cells than linear Rnas.”We engineered cirC-ARRNA.”Circ-arrna was found to be able to maintain high levels of expression for a long time, Wei said.At multiple endogenous transcription sites, the average editing efficiency of CIRC-ARRNA was more than 3 times higher than that of the linear version, while maintaining effective editing for up to half a month.Furthermore, cirC-ARRNA can be delivered by adeno-associated virus (AAV) to achieve long-term RNA editing in human primary cells and organoids.In addition, cirC-ARRNA synthesized in vitro can also achieve efficient targeted editing and has similar characteristics to genetically encoded CIRC-ArRNA.Wensheng Wei concluded that because LEAPER 2.0 still uses intracellular editing enzymes and does not require exogenous overexpression of editing enzymes, it can avoid delivery difficulties and off-target editing within the whole transcriptome caused by overexpression of exogenous editing enzymes.More importantly, LEAPER 2.0 was designed to eliminate a particular type of bystander off-target editing, resulting in significant improvements in security and precision.Circ-arrnas are capable of efficient and durable RNA editing of endogenous transcripts.Similar to previous versions of the RNA editing technology being developed in China and abroad, the team evaluated the potential of LEAPER 2.0 in this latest study.We demonstrated that LEAPER 2.0 successfully activated the Wnt signaling pathway, a highly conserved signaling pathway during evolution, and repaired pathogenic mutations in the TP53 gene, a tumor suppressor gene, to restore the expression of p53 protein to its normal transcriptional regulatory function.The team also performed preliminary validation of the technique in a mouse model.They delivered CIRC-ARRNA into Hurler syndrome disease model mice using adeno-associated virus, which successfully repaired pathogenic mutations in IDUA transcripts and restored the normal catalytic function of IDUA.Hurler syndrome is a complex, progressive, multi-system involved genetic lysosomal disease that affects organs and tissues throughout the body.The loss of Iduronidase A-L-IDuronidase (IDUA) in patients leads to the accumulation of glycoamine polysaccharide (GAGs) in lysosomes, resulting in multi-organ lesions.This disease is an autosomal recessive hereditary disease.Activation and restoration of protein function in cell culture and Hurler syndrome mice by CIRC-ARRNAS.”There is currently no cure for mucopolysaccharide storage disease,” wei said. “The main therapies include symptomatic treatment to improve quality of life, enzyme replacement therapy and bone marrow transplantation/hematopoietic stem cell transplantation.Existing treatments are too expensive and difficult to use because they require patients and their families to spend a lot of time in hospital.”He believes that RNA editing has advantages for these diseases, “and our new LEAPER 2.0, combined with AAV delivery system, can achieve effective editing over a long period of time, which is a good option for treating many genetic diseases such as Hurler’s syndrome.”In addition to Hurler syndrome, Wei noted that LEAPER 2.0 has also been shown to be effective in the treatment of genetic diseases of the eye or brain.In general, Wei Believes that LEAPER technology has its unique advantages and potential in clinical application and disease treatment.”LEAPER is an RNA editing technology that is reversible, controllable and dose-dependent, making it safer in principle.Meanwhile, LEAPER technology can deliver arRNA into cells and recruit endogenous ADAR protein to complete editing. The delivery burden is light and there is no need to introduce exogenous editing enzymes, so it is more suitable for gene editing therapy in vivo.”As mentioned above, at the technical level, domestic scientists have taken the lead internationally in achieving safer RNA editing using endogenous mechanisms, and have a great competitive advantage in this research field.In terms of industrial transformation, what is the stage at home and abroad?Wei wensheng said that there are already some international biomedical companies in the transformation and development of related technologies, especially last year, RNA editing technology has attracted great attention.For example, in August 2021, roche and Shape Therapeutics entered into a research and development collaboration and licensing agreement to develop gene therapies for Alzheimer’s disease, Parkinson’s disease and rare diseases using their RNA editing technology platform RNAfix and their AAVid technology platform.In September 2021, Lilly entered into a global licensing and research collaboration agreement with ProQR Therapeutics that will leverage ProQR’s proprietary Axiomer RNA editing platform to advance the clinical development and commercialization of novel drug targets for liver and neurological genetic diseases.The collaboration includes up to five RNA editing targets.As for China, Wei wensheng mentioned that Boya Jiyin is also carrying out the transformation of LEAPER technology.As a pioneer in the field of gene editing in China, Boya Jiyin was founded in 2015 with its headquarters in Beijing and offices in Guangzhou, Shanghai and Cambridge, USA. Wei Wensheng is the founder of the company.Wei Dong, CEO of Boya Jiyin, said in an interview with the Paper ( that for such innovative technology as gene editing, “the primary task is to be able to see whether the products transformed from the technology in different diseases have enough benefits and are safe and controllable.”Particularly serious diseases for which there are no effective treatments, such as genetic diseases and cancer, are currently the main “validation zones” for innovative therapies.Wei noted that the company’s current pipeline includes both in vitro and in vivo therapies.Among them, in vivo therapies based on LEAPER technology are being developed for ophthalmic and neurological diseases.In 2020, the company also reported at the 2020 ASGCT Annual Meeting in the United States that the mrna and protein of wild-type IDUA gene were generated by the precise adenosine A→ inosine I transformation of the 402nd codon in the IDUA MRNA sequence.Data from an early study of patients with W402X mutation in Hurler syndrome, the most severe subtype of mucopolysaccharide storage syndrome Type I.In this interview, Wei Wensheng also mentioned that For LEAPER 2.0 technology, Boya Jiyin is carrying out relevant transformation work.The company has conducted preclinical studies with AAV delivery and has achieved good data in multiple research models, achieving a proof of concept for LEAPER 2.0 to in vivo editing therapy.In November last year, Boya Jiyin also entered into a research collaboration with Professor Suiruifang’s team at Peking Union Medical College Hospital to explore and promote gene editing therapy in vivo based on the characteristics of genetic variation in The population of Hereditary retinal degeneration (IRD) in China.That same month, the company also entered into a research collaboration with David Gamm’s lab at the University of Wisconsin-Madison to evaluate the pharmacological properties of the company’s LEAPER technology based RNA base editing candidate therapy for specific genetic diseases.Source: