ADARs and Site-Directed RNA Editing as a Potential Therapeutic

ADARs and Site-Directed RNA Editing as a Potential Therapeutic

by SeHee Park

In humans, Adenosine (A) to Inosine (I) conversion within RNA is mediated by deaminase enzymes known as ADARs (Adenosine deaminases acting on dsRNA). This change in RNA component by ADARs has numerous effects on biological pathways as discussed in my previous blog, "How Does RNA Editing Affect Human Health?".

Site-directed RNA editing is a strategy to correct single mutations within a specific target RNA through the conversion of RNA nucleobases, such as A-to-I changes catalyzed by ADARs. Unlike CRISPR-Cas9 genome editing system that were adapted from the bacteria immune system, ADARs naturally exist in humans. This makes ADAR enzymes a compelling target enzymes that can be engineered as a site-directed RNA editing tool to correct genetic diseases. When it comes to developing an RNA editing tool as a therapeutic, it is important to have high specificity to a target RNA to prevent unintended mutations within RNA, which is often referred to as RNA off-target editing. ADARs can be a precise editing tool because ADARs only edit adenosine within a duplex RNA. This makes it possible to use a guide RNA (gRNA) to control ADARs activity to specifically edit the target adenosine within a duplex RNA since the gRNA will be complementary to the target RNA and therefore lead to a duplex structure. Importantly, ADARs prefer to deaminate adenosine across from cytidine in a duplex structure. Together, these intrinsic preferences of ADARs provide further benefits to precisely edit a target RNA with minimal RNA off-target editing.

There are two major approaches to adapt ADARs for a site-directed RNA editing tool. One approach is to use gRNAs to recruit endogenous ADARs in humans. As mentioned above, ADARs are found in humans, which means that these existing ADARs can be harnessed for a site-specific conversion of A-to-I editing. Different systems have been developed to harness the endogenous ADARs including RESTORE and LEAPER. For example, RESTORE strategy uses a gRNA containing both a target binding sequence and a recruiting sequence that can be recognized and bound by RNA binding domains (dsRBDs) of ADARs as a means to recruit ADAR enzymes to the target editing site. Harnessing the endogenous ADARs through this approach has high potential as a therapeutic because enzymes no longer need to be delivered into the human body for therapeutic application, which can be challenging especially when therapeutic enzymes are larger as seen in CRISPR-Cas based genome editing tools.

The other approach is using a gRNA along with an engineered ADAR for site-directed RNA editing. This approach usually fuses ADAR enzymes with other protein components as a way to recruit an engineered ADARs to the target RNA mediated by gRNA. For example, one of such systems uses ADAR enzyme that is fused with a specific protein motif called λN peptide. λN peptide is a small RNA binding domain of λ bacteriophage anti-terminator protein N and this λN peptide selectively binds to boxB RNA. When gRNA containing boxB sequence is used, λN peptide that is fused with ADAR enzyme selectively binds to the boxB sequence within gRNA. This then brings ADAR enzyme close to the target editing site and promotes the deamination of adenosine 4 (Figure 1).

Site-directed RNA editing is a potential approach to revert diseases associated mutations within RNA. Rett syndrome is a genetic neurological disease that occurs primarily in female and results in several impairments including breathing problem and loss of speech. Rett syndrome is caused by mutations on the X chromosome on a gene called MECP2 (Methyl CpG Binding Protein 2). There are multiple mutations within the MECP2 gene that leads to Rett syndrome, some of which are caused by Guanosine (G) to Adenosine (A) mutations. These G-to-A point mutations in the MECP2 gene produces the mutated mRNA that leads to improper functioning of the MeCP2 protein. Since a product of A-to-I editing, inosine, is recognized as guanosine by cellular machinery, ADARs can be used to correct these G-to-A point mutations in the RNA level. To test if ADARs can be used to correct Rett Syndrome mutations, a recent study used an engineered ADAR enzyme with λN peptide and gRNA approach to correct Rett Syndrome causing mutations within MECP2 mRNA resulting in the restoration of proper functioning of the MeCP2 protein. This confirms that site-directed RNA editing is a promising strategy for the treatment of Rett syndrome and other neurological diseases 5 (Figure 2).

Although there are several genome editing tools to correct genetic diseases, site-directed RNA editing provides a safer option when it comes to therapeutics. For example, any unintended mutations in DNA caused by genome editing are permeant. In other words, these unwanted mutations cannot be restored to the original DNA sequence, which can be detrimental. However, mutations made within RNA is reversible. This means when RNA off-target mutations are produced, these off-target are not as detrimental as that of DNA due to the transient nature of RNA. Also, RNA-editing can be applied to any metazoan cells without limitations, which is not true for CRISPR-Cas based genome editing. Delivery of RNA editing tools are significantly less challenging compared to that of genome editing tools due to the endogenous nature of ADAR enzymes. In conclusion, site-directed RNA editing tools with ADAR enzymes have a great potential to correct diseases causing mutations within RNA as therapeutics. 

References

Aquino-Jarquin, G. Novel Engineered Programmable Systems for ADAR-Mediated RNA Editing. Mol. Ther. - Nucleic Acids 2020, 19, 1065–1072. https://doi.org/10.1016/j.omtn.2019.12.042.

Matthews, M. M.; Thomas, J. M.; Zheng, Y.; Tran, K.; Phelps, K. J.; Scott, A. I.; Havel, J.; Fisher, A. J.; Beal, P. A. Structures of Human ADAR2 Bound to DsRNA Reveal Base-Flipping Mechanism and Basis for Site Selectivity. Nat. Struct. Mol. Biol. 2016, 23 (5), 426–433. https://doi.org/10.1038/nsmb.3203.

Montiel-Gonzalez, M. F.; Diaz Quiroz, J. F.; Rosenthal, J. J. C. Current Strategies for Site-Directed RNA Editing Using ADARs. Methods 2019, 156, 16–24. https://doi.org/10.1016/j.ymeth.2018.11.016.

Nishikura, K. Functions and Regulation of RNA Editing by ADAR Deaminases. Annu. Rev. Biochem. 2010, 79 (1), 321–349. https://doi.org/10.1146/annurev-biochem-060208-105251.

Sinnamon, J. R.; Kim, S. Y.; Corson, G. M.; Song, Z.; Nakai, H.; Adelman, J. P.; Mandel, G. Site-Directed RNA Repair of Endogenous Mecp2 RNA in Neurons. Proc. Natl. Acad. Sci. 2017, 114 (44), E9395–E9402. https://doi.org/10.1073/pnas.1715320114.