title: Optimization of Chemically Modified Oligonucleotides for Site-directed RNA Editing with Endogenous ADAR
Creators: name: Pfeiffer, Laura Sophia
Publisher: name: Universität Tübingen
Year: 2026
Subjects: subject: Biochemie , RNS , DNS , Desaminierung , Inosin , Adenosin , Nucleinsäuren , Gentherapie , Hypoxanthin
subject: 500
subject: 540
subject: 570
subject: 610
subject: RNA
subject: DNA
subject: Deamination
subject: ADAR
subject: Adenosine
subject: Inosine
subject: Hypoxanthine
subject: RNA Editing
subject: Editing
subject: Gene therapy
subject: Nucleic acid
subject: Nucleic acids
subject: Biochemistry

description: Current therapeutic developments are shifting their focus from the protein level to the informational oligonucleotide level. This includes the development of site-directed RNA editing (SDRE) approaches, which allow selective recoding of RNA nucleobases. Some SDRE-catalyzing enzymes are expressed endogenously in humans, including ADAR, which functionally recodes adenines to guanines. SDRE systems based on these enzymes are a potentially safer alternative to genome editing systems like CRISPR/Cas, which can induce permanent off-target effects. Several such SDRE approaches have already been developed, but these are often less effective, dependent on viral transduction, or require the expression of an exogenous fusion protein. Chemically modified oligonucleotides (ON) are an alternative that directly steer endogenous ADAR to the desired RNA site without additional components. However, there are no systematic studies on the requirements necessary for efficient and nuclease-resistant ONs. This thesis aims to uncover these requirements through stepwise development of the RESTORE 2.0 platform – an improvement of the previously published RESTORE system (recruiting endogenous ADAR to specific transcripts for oligonucleotide mediated RNA editing). Stabilized RESTORE 2.0 ON designs contained the commercially available 2’-fluoro, 2’-O-methyl, 2’-H (DNA) modifications and typically retained most of the editing yields from the corresponding unstable RNA-based designs. Fully modified RESTORE 2.0 ONs resisted serum and lysosomal nucleases for up to 31 days. Dense phosphorothioate modifications were essential to recruit endogenous ADAR. The modification principles were also applicable to the CLUSTER system, another guide RNA design platform. RESTORE 2.0 ONs could be shortened to just 32 nt and displayed twice the efficiency without requiring interferon-α supplementation compared to the 95nt long RESTORE 1.0 ONs. Efficient ON symmetries and favored placements of specific chemical modifications also aligned with known ADAR dsRNA binding patterns. Further, fully modified RESTORE 2.0 ONs were efficiently administered via clinically relevant delivery routes, such as GalNAc-mediated uptake into primary hepatocytes. Most importantly, optimized RESTORE 2.0 ON designs corrected multiple disease-relevant point mutations. This included a proof-of-concept treatment of an α-1-antitrypsin deficiency mouse model, where up to 42% correction on the RNA level was achieved, restoring therapeutically relevant levels of the corrected protein. Altogether, the RESTORE 2.0 ON platform provides simple guidelines to further develop chemically modified ONs for therapeutical purposes.
description: Die Dissertation ist gesperrt bis zum 02. Dezember 2026 !