2026-07-10 | Functional Editing of the OTC locus by Targeted Integration with Phenotype Correction and Restoration of Physiological Patterns of Expression.

Here we report highly efficient functional repair of the ornithine transcarbamylase (OTC) locus in mutant mouse and human hepatocytes in vivo using a dual AAV system delivering CRISPR-Cas9 editing reagents and a promoterless donor for targeted integration. The approach was mutation agnostic and targeted intronic sequences to prevent inadvertent inactivation of hypomorphic alleles. Notably, in a murine model, we corrected the metabolic defect and simultaneously achieved liver-wide restoration of physiological metabolic zonation of Otc expression by capturing native cis-acting regulatory elements. The effectiveness of this approach was confirmed using a universally configured therapeutic cassette in patient-derived primary human hepatocytes in vivo. These data provide a powerful template to guide further optimization of this approach and, given the high editing efficacy required for phenotypic effect in OTC deficiency, have broader relevance to other liver disease phenotypes.

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2026-04-23 | Profiling and Targeting of Regulatory RNAs to Upregulate Gene Expression

Abstract Transcription of long noncoding RNAs (lncRNAs), including enhancer RNAs (eRNAs) and promoter-associated RNAs (paRNAs), collectively termed regulatory RNAs (regRNAs), is a hallmark of active gene expression, yet it remains unknown whether regRNAs can be targeted to selectively enhance transcription in cis . We developed regRNA Capture-seq, a high-throughput method to profile regRNAs, and applied it to primary human hepatocytes, annotating thousands of regRNAs at ∼2,000 enhancers and promoters. Using this approach, we interrogated a genetically validated enhancer of the ornithine transcarbamylase ( OTC ) gene, mutations of which cause OTC deficiency (OTCD), the most common urea cycle disorder. Antisense oligonucleotides (ASOs) targeting enhancer-derived regRNAs led to dose-dependent upregulation of OTC in hepatocytes. Mechanistically, ASOs altered regRNA structure, elevated regRNA levels, displaced transcriptional repressors, and increased H3K27 acetylation at the targeted enhancer. This work establishes a potential therapeutic strategy for addressing haploinsufficiency and highlights regRNAs as actionable targets for ASO-mediated upregulation of gene expression.

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2026-04-03 | Enzyme Structure and Substrate Binding: From Atomic Mechanisms to Therapeutic Drug Development

This comprehensive technical whitepaper explores the intricate relationship between enzyme structure, substrate binding mechanisms, and catalytic efficiency, highlighting their profound implications for drug discovery and industrial biotechnology. The article begins by detailing the four hierarchical levels of enzyme architecture, explaining how covalent and non-covalent forces stabilize these structures to create highly specific active sites. It critically evaluates molecular recognition models, transitioning from the classical lock-and-key hypothesis to the more dynamic induced-fit and conformational selection models, which better account for the structural flexibility required during the catalytic cycle. A significant portion of the text is dedicated to advanced methodologies used to interrogate enzyme function. Experimental techniques such as X-ray crystallography, cryo-electron microscopy, and nuclear magnetic resonance spectroscopy are discussed alongside cutting-edge computational approaches, including molecular docking, molecular dynamics simulations, and machine learning frameworks. These tools enable researchers to visualize transition states, map complex energy landscapes, and predict kinetic parameters like the Michaelis constant and turnover number. The article also emphasizes the critical role of non-protein cofactors and the surprising impact of distal mutations, residues located far from the active site that significantly enhance catalytic efficiency through allosteric networks and structural dynamics. Practical applications are heavily featured, particularly rational drug design strategies aimed at overcoming drug resistance through multi-target-directed ligands, allosteric inhibitors, and structure-based optimization. Case studies on isovaleric acidemia and ornithine transcarbamylase deficiency illustrate how disease-associated mutations disrupt enzyme stability and function, underscoring the necessity of integrating multi-omics data with structural kinetics. Ultimately, the integration of AI-driven predictive models and high-throughput experimental validation is presented as a transformative paradigm for engineering robust biocatalysts and developing targeted therapeutics. Source: https://www.enzymestudy.com/posts/enzyme-structure-and-substrate-binding-from-atomic-mechanisms-to-therapeutic-drug-development

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