Stereo-random Oligonucleotides Enable Efficient Recruitment of ADAR in vitro and in vivo
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ABSTRACT:
PROVIDER: PRJNA1295427 | ENA |
REPOSITORIES: ENA
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Project description:Liver-originated diseases remain a significant global health burden, with limited treatment options available. RNA therapeutics, particularly adenosine deaminase acting on RNA (ADAR)-mediated editing using editing oligonucleotides (EONs) offer a novel approach to address these unmet medical needs. This study evaluates in vitro and in vivo liver models to predict the therapeutic potential of chemically modified EONs in human liver tissue, using ACTB as target. Multiple liver models, including primary human hepatocytes (PHH), cancer-derived cell lines, and iPSC-derived hepatocytes in both 2D and 3D formats, were characterized for ADAR expression, liver functionality, and RNA editing efficiency. The results show consistent ADAR expression across most models, with 3D models, particularly PHH spheroids and liver microtissues, closely mimicking human liver tissue in gene expression and editing capacity. In vivo, non-human primate liver biopsies showed high levels of ACTB editing, consistent with findings from PHH models. These findings highlight the critical role of preclinical models in EON selection, where multiple factors must be carefully considered. A multimodel assessment utilizing composite scoring to comprehensively assess the key parameters seems to be the approach of choice to maximize translatability across preclinical models and increase the likelihood of success in clinical applications
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Project description:RNA editing can be a promising therapeutic approach. However, ectopic expression of RNA editing enzymes was found to trigger off-target editing, and the recruitment of endogenous adenosine deaminase acting on RNA (ADAR) suffers from low efficiency and fluctuating ADAR expression. Here, we identified ADAR inhibitors (ADIs) that suppressed the activity of the fused ADAR2 deaminase domain (ADAR2DD). Using ADI, we developed an RNA transformer adenine base editor (RtABE) with both high specificity and high efficiency. With the fusion of ADI to ADAR2DD, RtABE remains inactive until it binds its target site. After binding to the target site, ADI is cleaved from ADAR2DD and RtABE becomes active. RtABE induced efficient on-target editing in various cells with different ADAR expression levels. Delivering RtABE via an adeno-associated virus enabled up to a 45% RNA correction rate in Hurler syndrome mice with no significant off-target editing, and -L-iduronidase activity was restored. RtABE is a highly specific and efficient RNA editing system with broad applicability.
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