On July 15, 2019, Nature Biotechnology published a research article titled "Programmable RNA editing by recruiting endogenous ADAR using engineered RNAs" from Prof. Wei Wensheng's group, reporting a novel single-base RNA editing technology called LEAPER. Unlike conventional nucleic acid editing technology that requires simultaneous delivery of editing enzymes (such as Cas protein) and guide RNAs into cells, LEAPER only needs to express the guide RNA in the cell to achieve targeted target RNA editing by recruiting endogenous cellular deaminase. Using this novel technology, researchers have achieved efficient and precise editing in a series of disease-related gene transcripts and successfully repaired α-L-iduronidase-deficient cells from Hurler syndrome patients. The establishment of LEAPRER provides a new tool for scientific research and disease treatment.

In recent years, genome editing technologies using engineered nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), and Cas proteins of the CRISPR system, have been applied to manipulate the genome in a myriad of organisms. However, the current gene editing technologies encountered bottlenecks in therapeutic applications. One of the root causes is that the current gene editing technologies depend on exogenous editing enzymes (such as Cas9 nuclease, APOBEC, TadA deaminase). The ectopic expression of foreign editing enzymes in mammalian cells could cause a series of issues such as substantial global off-targeting edits on genomic DNA and RNA transcripts, immunogenicity, oncogenicity, and in vivo delivery barrier, which have severely hindered the applications of gene-editing technologies in disease treatment. Therefore, there is a need to develop novel gene-editing technologies, especially those independent of exogenous editing enzymes.

ADAR (Adenosine deaminase acting on RNA) is a type of adenosine deaminase that is widely expressed in various tissues in humans. This enzyme can catalyze the conversion of adenosine (A) to inosine (I) (guanosine, G) in double-strand RNAs. Compared with DNA editing, RNA editing does not cause permanent changes in the genome sequence. This reversible and tunable editing method may have advantages in therapeutic application. Although it has been reported that overexpression of Cas13-ADAR fusion protein and guide RNA could achieve precise editing of target RNA (Science, 2017), this tool still relies on the ectopic expression of foreign protein. To address this, Dr. Wei’s research team present an approach, called leveraging endogenous ADAR for programmable editing of RNA (LEAPER), that employs short engineered ADAR-recruiting RNAs (arRNAs) to recruit native ADAR1 or ADAR2 enzymes to change specific adenosine to inosine, and thus achieved precise editing does not require the introduction of any exogenous effector protein (Figure 1).

Leveraging endogenous ADAR1 protein for targeted RNA editing

Researchers demonstrated that LEAPER could achieve precise and efficient RNA targeted editing on a range of endogenous genes’ RNA transcripts, and also achieve targeted editing in a variety of human cell types, including primary T cells, and cells from mice and monkeys. In the therapeutic applications, LEAPER could restore the transcriptional regulatory function of p53 mutants by repairing the pathogenic mutations in the tumor suppressor gene TP53. Moreover, in primary cells derived from patients with Hurler syndrome, LEAPER could successfully repair pathogenic mutations and restore the catalytic activity of α-L-iduronidase in the cells (Figure 2).

 LEAPER could restore the transcriptional regulation function of TP53 and the catalytic function of IDUA in Hurler Syndrome patient cells

Recent studies have shown that DNA single-base editing technologies, including cytosine base editors and adenine base editors, have resulted in substantial off-targets at the RNA or DNA level (Science 2019; Nature 2019). Using RNA-Seq technology to evaluate LEAPER technology at the transcriptome level, no obvious off-target phenomenon was observed, showing the high specificity of the new technology. In addition, LEAPER does not affect the normal function of endogenous ADAR protein, nor does it induce the innate immune response in cells, indicating that it is safe.

Similar to RNAi, LEAPER makes full use of the mechanism that naturally exists in the cell: using only one RNA to achieve precise and efficient single-base editing of RNA, thus avoiding any potential problems caused by the ectopic expression of foreign effector proteins. This new technology points out the direction for the development of gene-editing technology based on cell endogenous mechanisms. Recently, Thorsten Stafforst's research group reported an RNA editing method named RESTORE (Nature Biotechnology 2019). Like LEAPER, RESTORE can also use endogenous ADAR for precise editing of targeted RNA; the difference is that the guide RNA in RESTORE is a chemically synthesized oligonucleotide, which requires substantial chemical modifications to maintain its stability. LEAPER can function in cells through stable expression, so it is suitable for loading into an adeno-associated virus (AAV), lentivirus, and other vectors, and continues to function after being delivered to the body.

Qu Liang (PTN), Yi Zongyi (CLS), Wang Chunhui (BIOPIC), Cao Zhongzheng (CLS), postdoctoral fellow Zhu Shiyou, associate investigator Dr. Zhou Zhuo, and Dr. Yuan Pengfei from Edigene are the co-first authors of this paper. Prof. Wei Wensheng is the corresponding author. This project was supported by funds from Beijing Municipal Science & Technology Commission, the National Science Foundation of China, Beijing Advanced Innovation Center for Genomics at Peking University and the Peking-Tsinghua Center for Life Science, the National Science Foundation of China, the National Major Science & Technology Project for Control and Prevention of Major Infectious Diseases in China, and the Beijing Nova Program.

Article link: https://www.nature.com/articles/s41587-019-0178-z