Martina Bigatti, André Moser, Bas Dierssen, Shtjefen Frrokaj, Elena Covato, Christophe Pfleger, Joerg Lill, Yael Leiser, Joël Zuber, Andreas Staempfli, Filippo Sladojevich, Stefan G. Koenig
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引用次数: 0
Abstract
In this manuscript, we present a robust chemo-enzymatic approach for the production of single guide RNAs (sgRNAs), essential reagents for CRISPR-Cas9-based cell and gene therapy applications currently under development. Our method leverages ligase-mediated assembly of two RNA fragments, each synthesized using standard solid-phase chemistry. This versatile process has been applied, without modification, to produce a variety of GMP-grade sgRNAs, supporting our clinical ex vivo cell therapy pipeline. We demonstrate that our approach consistently achieves higher purity (10–15% improvement in LC-UV area%) and significantly greater yield (3–4 times higher) compared to traditional linear solid-phase synthesis, which is commonly used for sgRNA production. Importantly, the process utilizes T4 RNA ligase 2, a natural, nonengineered enzyme, which can be easily sourced from several vendors. We believe that openly sharing this method will drive significant progress in the development of cell and gene therapies, enabling the production of higher-quality sgRNAs at lower cost, ultimately improving accessibility and treatment outcomes for patients.
期刊介绍:
The journal Organic Process Research & Development serves as a communication tool between industrial chemists and chemists working in universities and research institutes. As such, it reports original work from the broad field of industrial process chemistry but also presents academic results that are relevant, or potentially relevant, to industrial applications. Process chemistry is the science that enables the safe, environmentally benign and ultimately economical manufacturing of organic compounds that are required in larger amounts to help address the needs of society. Consequently, the Journal encompasses every aspect of organic chemistry, including all aspects of catalysis, synthetic methodology development and synthetic strategy exploration, but also includes aspects from analytical and solid-state chemistry and chemical engineering, such as work-up tools,process safety, or flow-chemistry. The goal of development and optimization of chemical reactions and processes is their transfer to a larger scale; original work describing such studies and the actual implementation on scale is highly relevant to the journal. However, studies on new developments from either industry, research institutes or academia that have not yet been demonstrated on scale, but where an industrial utility can be expected and where the study has addressed important prerequisites for a scale-up and has given confidence into the reliability and practicality of the chemistry, also serve the mission of OPR&D as a communication tool between the different contributors to the field.