In recent years, the research and transformation of genome editing technology represented by CRISPR/Cas9 in biomedicine and other fields have been in full swing. But for most scientists, the work of modifying the human "codebook of life" is still being carefully further refined.
RNA (ribonucleic acid) editing is a new gene editing technology that has emerged in recent years, and international pharmaceutical companies have laid out this technology. One of the technologies, named "LEAPER", is an RNA editing technology independently innovated by Professor Wei Wensheng of the School of Life Sciences of Peking University. Previously, in July 2019, Wei Wensheng's team reported on LEAP technology in the international academic journal Nature Biotechnology. In the early morning of February 11, Beijing time, the research team once again reported the upgraded LEAPER 2.0 technology in Nature Biotechnology.
"Unlike CRISPR-based DNA or RNA editing techniques, LEAPER only needs to express specially designed RNA (ADAR-recruiting RNA, arRNA) in cells to recruit endogenous deaminase ADAR in cells to achieve editing that targets adenosine A inosine I (guanosine G) in the target RNA." Wei Wensheng said in an interview with the surging news (www.thepaper.cn) reporter that because there is no need to introduce exogenous editing enzymes or effector proteins, the resulting off-target effects on the genome and transcriptome, delivery burden and related immunogenicity problems are avoided.
It is worth noting that LEAPER completely got rid of the dependence on the CRISPR system, and is the underlying core technology with independent intellectual property rights, which has important original innovation significance.
Wei Wensheng also mentioned that as an accurate editing tool for RNA, LEAPER does not cause changes in the genome sequence, and has advantages in terms of safety." In this latest study, the research team further optimized and upgraded THE EDITORIAL EFFICIENCY AND OFF-TARGET PROBLEMS OF LEAPER.
It is worth noting that Nature Biotechnology also published a study by Prashant Mali, an assistant professor at the University of California, San Diego (UCSD), and Mali et al. also found that the use of cyclic RNA that can recruit endogenous ADAR deaminase can improve the efficiency of RNA editing. ADAR1 deaminase is a class of adenosine deaminases that are widely expressed in tissues in the human body, which can catalyze the conversion of adenosine A inosine I (guanosine G) in the target RNA molecule.
"Although most of the world's research on RNA editing technology is currently in the laboratory stage, it is getting closer and closer to clinical application." Wei Wensheng said that at the technical level, the use of endogenous mechanisms to achieve safer RNA editing, "we are in the leading echelon internationally, and we have a competitive advantage in this field of research." ”
Solve the two limitations of LEAP
Genome editing technology represented by CRISPR/Cas9 still has a series of problems, and it also encounters bottlenecks in clinical treatment applications. In the view of Wei Wensheng et al., one of the root causes of the problem is that the current gene editing system relies on the expression of editing enzymes or effector proteins of bacterial or viral origin, such as Cas9 nuclease in bacteria.
This dependency can cause multiple problems. For example, protein molecular weight is too large to be loaded by viral vectors and delivered in vivo is very difficult, dna/RNA level off-target effect caused by protein overexpression, body immune response and damage caused by exogenous protein expression, pre-stored antibodies in the body make foreign editing enzymes or effector proteins neutralized by antibodies, resulting in gene editing failure.
The solution of Wei Wensheng et al. is to use the mechanisms that naturally exist in cells. For the first time in their previous study, they found that simply transferring to a specially designed ADAR-recruiting RNAs (arRNAs) could produce efficient and accurate editing of specific adenosine on the target gene transcript by recruiting endogenous ADAR1 deaminase without the need to introduce any exogenous effector proteins. This new RNA editing technique is called LEAP (Leveraging Endogenous ADAR for Programmable Editing on RNA).
The research team has previously demonstrated that LEAPER can accurately edit the vast majority of adenylate sites on RNA molecules. In human primary cells, including lung fibroblasts, bronchial epithelial cells, and T cells, LEAPER can be edited with up to 80% efficiency.
"Despite LEAP's considerable potential in scientific research and disease treatment, the technology has certain limitations." Wei Wensheng talked about two points, one is that LEAP uses endogenous editing enzymes, and its editing efficiency will be limited; 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 can significantly improve the editing efficiency of the LEAP system, "indicating that the abundance of arRNA in cells is critical to editing efficiency." ”
Another key point is that since linear arRNAs are easily degraded within cells, the team came up with the idea of using cyclization. Since there is no 5' or 3' end, cyclic RNA avoids cleavage of nucleic acid exonucleases, and has better stability and longer half-lifes in cells than linear RNA.
"We designed engineered cyclic arRNAs (circ-arRNAs)." Wei Wensheng said that the study found that circ-arRNA can maintain high levels of expression for a long time. In multiple endogenous transcription sites, the average editing efficiency of circ-arRNA is more than 3 times higher than that of linear versions, and it can also maintain effective editing for nearly half a month.
The study further found that genetically encoded circ-arRNAs can enable long-term RNA editing in human primary cells and organoids delivered by adeno-associated virus (AAV). In addition, in vitro synthesized circ-arRNAs can also enable efficient targeted editing and have similar characteristics to genetically encoded circ-arRNAs.
Wei concludes that because LEAP 2.0 still uses endogenous editing enzymes, there is no need for exogenous overexpression of editing enzymes, so delivery difficulties and overexpression of exogenous editing enzymes caused by off-target editing in the whole transcriptome range can be avoided.
Another important thing is that LEAPER 2.0 eliminates a special bystander off-target editing through special design, which greatly improves safety and accuracy.
Circ-arRNAs enable efficient, long-lasting RNA editing of endogenous transcripts.
RNA editing technology is being transformed at home and abroad
Similar to previous versions, the research team also evaluated the application potential of LEAP 2.0 in this latest study.
Their study showed that using LEAPER 2.0 technology, it is possible to successfully activate the Wnt signaling pathway (a class of signaling pathways that are highly conserved during species evolution) and repair pathogenic mutations in the TP53 gene (a tumor suppressor gene) to restore the p53 protein it expresses to normal transcriptional regulatory functions.
The research team also conducted preliminary technical validation on mouse models. Using an adeno-associated virus to deliver circ-arRNA to mice in a hurler syndrome disease model, they could successfully repair the disease-causing mutation on the IDU transcript and restore the normal catalytic function of the IDUA.
Hurler syndrome is a complex, progressive, multisystem-affected hereditary lysosomal disease that affects organs and tissues throughout the body. The accumulation of glycamine polysaccharides (GAGs) in lysosomes due to the loss of Edu glucuronidase a-L-Iduronidase (IDUA) causes multi-organ lesions. The disease is an autosomal recessive disorder.
Activation and restoration of protein function by circ-arRNAs in cell culture and in mice with Hurler syndrome.
"There is currently no cure for mucopolysaccharide storage disorder, and the main therapies include symptomatic therapy to improve quality of life, enzyme replacement therapy, and bone marrow transplantation/hematopoietic stem cell transplantation," Wei said. Existing therapies are too expensive, and patients and families need to spend a lot of time in the hospital, making it difficult to apply. ”
He believes that RNA editing has advantages for this type of disease, "and our newly developed LEAPER 2.0 combined with the AAV delivery system can achieve long-term effective editing, which is a good choice for treating many genetic diseases such as Hurler syndrome." In addition to Hurler syndrome, Wei Wensheng mentioned that LEAPER 2.0 has also observed very good results in the treatment of genetic diseases of the eye or brain.
Overall, Wei Wensheng believes that LEAPER technology has its unique advantages and potential in clinical application and disease treatment. "LEAPER is an RNA editing technology, its editing is reversible, adjustable, and the editing effect is dose-dependent, which is safer in principle. At the same time, LEAPER technology by delivering arRNA to the cell, recruiting endogenous ADAR protein to complete editing, the delivery burden is light, no need to introduce foreign editing enzymes, more suitable for in vivo gene editing therapy. ”
As mentioned above, at the technical level, the use of endogenous mechanisms to achieve safer RNA editing, domestic scientists have been in the leading echelon internationally, in this field of research has a great competitive advantage. In terms of industrial transformation, what stage is it at home and abroad?
Wei Wensheng said that there are already some biomedical companies in the world carrying out related technology transformation and development work, especially last year, RNA editing technology has received great attention.
For example, in August 2021, multinational pharmaceutical companies Roche and Shape Therapeutics entered into an R&D collaboration and licensing agreement to develop gene therapies for the treatment of Alzheimer's, Parkinson's and rare diseases using their RNA editing technology platform, RNAfix, and AAVid. Subsequently, in September 2021, Eli Lilly entered into a global licensing and research collaboration agreement with ProQR Therapeutics, which will leverage ProQR's proprietary Axiomer RNA editing technology platform to advance the clinical development and commercialization of new drug targets for liver and nervous system genetic diseases, collaborating on up to five RNA editing targets.
As for China, Wei Wensheng mentioned that Boya Jiyin is also carrying out the transformation of LEAP technology. Founded in 2015 and headquartered in Beijing, Boya Ji is a pioneer in the field of gene editing in China, with offices in Guangzhou, Shanghai and Cambridge, and Wei Wensheng is the founder of the Department of Gene Editing.
Wei Dong, CEO of Boya Jiyin, previously said in an interview with the surging news (www.thepaper.cn) reporter that for the special innovative technology of gene editing, "its primary task is to really see the products transformed by technology in different diseases, whether there are enough benefits, and safe and controllable." "Particularly serious diseases for which there are no effective treatments, such as genetic diseases and cancer, are the main "validation areas" for innovative therapies at present.
Wei Dong mentioned that the company's current layout of the entire pipeline includes in vitro therapy and in vivo therapy. Among them, in vivo therapy is based on LEAPer technology, which is being developed for ophthalmic, nervous system and other diseases. In 2020, the company also reported at the 2020 ASGCT annual meeting in the United States, through the accurate, sequence-specific adenosine A inosine I conversion of the 402nd codon in the IDUA messenger RNA sequence, the generation of messenger RNA and proteins of the wild-type IDUA gene, and the treatment of patients with W402X mutations in Hurler syndrome, the most severe subtype of mucopolysaccharide storage disease type I.
Wei Wensheng also mentioned in this interview that for LEAP 2.0 technology, Boya Jiyin is carrying out relevant transformation work. The company conducted preclinical studies through AAV delivery and obtained good data in multiple study models, enabling LEAPER 2.0 to translate into a proof-of-concept for in vivo editing therapies.
In November last year, Boya Jiyin also reached a research collaboration with the team of Professor Sui Ruifang of Peking Union Medical College Hospital to explore and promote in vivo gene editing therapies based on the genetic variant characteristics of the mainland hereditary retinal degeneration (IRD) population; in the same month, the company also reached a research collaboration with the David Gamm laboratory of the University of Wisconsin-Madison to evaluate the pharmacological properties of the company's RNA base editing candidate therapy based on LEAPer technology for specific genetic diseases.