Periodic Reporting for period 1 - SNAP-ADAR Kit (Simplifying the SNAP-ADAR Tool for Broad Usage in Life Science Research and Drug Discovery)
Okres sprawozdawczy: 2022-06-01 do 2023-11-30
Site-directed RNA base editing has high potential to become both: a novel platform for therapy and an important tool for basic research (Rees et al., Nat. Rev. Gen. 2018, 770). While the clinical translation has a clear commercial path, which is already taken by several companies, the application in basic sciences would benefit from a commercialization of the technology, e.g. in form of an easy-to-handle kit. Currently, the available technologies, e.g. the Cas13-ADAR (Cox et al., Science 2017, 1019) or lambda-ADAR, suffer from the difficulty to predict guideRNAs that enable efficient and bystander off-target-free editing. Furthermore, viruses are needed to co-overexpress guideRNA and editing enzyme, which require viral delivery for cells beyond 293T and HeLa, like primary cells for example. The generation of viruses for each guideRNA make it cumbersome and impractical for many applications. Our SNAP-ADAR approach (Fig. 1a) solves these issues by us-ing chemically modified, short (20 nt) guideRNAs which are easily transfected into various (prima-ry) cells. The chemical design of the guideRNA enables highly precise and bystander-free editing by choosing from a small set of chemical modification patterns. Even though the guideRNA design is highly rational, the making of the guideRNA requires the choosing of the right chemical modifica-tion pattern, and furthermore, requires some organic chemistry skills to add a self-labeling moiety to the guideRNA followed by a urea-PAGE clean-up. The complex protocol is currently impeding the broad application of the SNAP-ADAR tool in basic research. Another practical restriction of the current SNAP-ADAR protocol is the limited duration of the editing reaction. For a one-week exper-iment in cell culture, we currently need to transfect the guideRNA twice. This is due to the limited stability of the current guideRNA design. With this PoC action, we want to explore the potential to simplify the SNAP-ADAR technology to make it more widely applicable for the broad life science community.
In the course of the action, we have successfully developed a synthetic protocol to make gram amounts of activated esters of the self-labeling moiety by using only recrystalization and similarly simple, industrially scalable methods. This enabled us to notably simplify the guide RNA modification protocol. Before that, the guide RNA was incubated with in situ activated self-labeling moiety and due to side-reactions, cumbersome PAGE purification was required. The new protocol allows for simple incubation with access of the active ester for few hours and very simple purification by standard ethanol precipitation. This now makes the generation of modified guide RNA "kit"-able. Finally, the protocol was also extended to the self-lableing moiety of Halo-tagged deaminases making the protocol even more useful. A manuscript describing the protocol and a side-by-side comparison of the products (modified guide RNAs) obtained form the new versus the old, cumbersome protocol has been written and will be submitted soon after the action. First, we need to ensure if Intellectual porperty can be generated about this protocol.
Regarding the application, we could broadly demonstrate application of RNA base editing to modulate signaling cues by a novel approach be termed PTM interference. A respective manuscript is currently under revision in a major journal. This data includes also NGS-based analyses of the RNA-base editing-driven PTM interference on the important JAK/STAT signaling pathway. We currently work on some control experiments ask by the reviewers, but we are confident that this work will soon be published very visibly, which is an important step for the SNAP-ADAR system and will open broader utility.
Regarding the stabilization and shortening of the guideRNA, we achieved to stabilize the guide RNA for >1week in 100% FBS and tritosomes. We stabilized both the RNA and the self-labeling moiety and linker. This, enabled editing with yields up to 40% in standard cell lines (e.g. 293) by means of naked uptake. However, it did not (yet) enable very longlasting editing, likely due to the high proliferative rate or high turnover of the SNAP-ADAR protein component in theses cell lines. In the future, we will focus more on appyling the tool in primary hepatocytes where a duration of a few days upon GalNAc mediated uptake would be sufficient for a variety of appealing applications.
Regarding the application, we could broadly demonstrate application of RNA base editing to modulate signaling cues by a novel approach be termed PTM interference. A respective manuscript is currently under revision in a major journal. This data includes also NGS-based analyses of the RNA-base editing-driven PTM interference on the important JAK/STAT signaling pathway. We currently work on some control experiments ask by the reviewers, but we are confident that this work will soon be published very visibly, which is an important step for the SNAP-ADAR system and will open broader utility.
Regarding the stabilization and shortening of the guideRNA, we achieved to stabilize the guide RNA for >1week in 100% FBS and tritosomes. We stabilized both the RNA and the self-labeling moiety and linker. This, enabled editing with yields up to 40% in standard cell lines (e.g. 293) by means of naked uptake. However, it did not (yet) enable very longlasting editing, likely due to the high proliferative rate or high turnover of the SNAP-ADAR protein component in theses cell lines. In the future, we will focus more on appyling the tool in primary hepatocytes where a duration of a few days upon GalNAc mediated uptake would be sufficient for a variety of appealing applications.
RNA base editing is a novel yet underdeveloped technology to manipulate the cell´s genetic information in particularly efficient and safe manner. With the help of this action, we could improve the general concept and the specific SNAP-ADAR tool.
Regarding the general concept, we can now demonstrate (manuscript invited for minor revision) applciation of RNA base editing for the editing of wild type allels in order to manipulate wildtype protein function. Specifically, we introduced the concept of PTM interference where native protein function is modulated by removal of typical sites of posttranslational modification, like phosphorylation, methylation, acetylation and ubiquitination. This is very important for the field as it moves the view away from the concept to "only" repair rare G-to-A point mutations but to rather use the technology more broadly for creating valuable phenotypes by acting on wildtyp allels.
Regarding the SNAP-ADAR technology, we achieved a major simplification in the protocol to generate these guide RNAs (manuscript in preparation). Furthermore, we tested the general applicability of the SNAP-ADAR tool broadly on over 70 different PTM sites. This breaks the ground for a rational application of the SNAP-ADAR tool for target screening and PTM interference in the future, by our and other labs. So, this was important to increase the potential market size of both, the screening tool but also for the pipeline development anyone interested in the drug program.
We started discussions around commercialization of the SNAP-ADAR tool. However, there remain questions about the potential marked size for a SNAP-ADAR kit and there is still more knowledge to be gained to fully understand and predict chemical modifications patterns of highyl efficient guide RNAs to make this tool reliable enough for direct commercialization.
Regarding the general concept, we can now demonstrate (manuscript invited for minor revision) applciation of RNA base editing for the editing of wild type allels in order to manipulate wildtype protein function. Specifically, we introduced the concept of PTM interference where native protein function is modulated by removal of typical sites of posttranslational modification, like phosphorylation, methylation, acetylation and ubiquitination. This is very important for the field as it moves the view away from the concept to "only" repair rare G-to-A point mutations but to rather use the technology more broadly for creating valuable phenotypes by acting on wildtyp allels.
Regarding the SNAP-ADAR technology, we achieved a major simplification in the protocol to generate these guide RNAs (manuscript in preparation). Furthermore, we tested the general applicability of the SNAP-ADAR tool broadly on over 70 different PTM sites. This breaks the ground for a rational application of the SNAP-ADAR tool for target screening and PTM interference in the future, by our and other labs. So, this was important to increase the potential market size of both, the screening tool but also for the pipeline development anyone interested in the drug program.
We started discussions around commercialization of the SNAP-ADAR tool. However, there remain questions about the potential marked size for a SNAP-ADAR kit and there is still more knowledge to be gained to fully understand and predict chemical modifications patterns of highyl efficient guide RNAs to make this tool reliable enough for direct commercialization.