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Yi Liu. Department of Chemical and Biomolecular Engineering, Faculty of Engineering, National University of Singapore, Singapore, Singapore.
Zhen Hua Chia. Department of Chemical and Biomolecular Engineering, Faculty of Engineering, National University of Singapore, Singapore, Singapore.
Johannes Nathaniel Min Hui Liew. Department of Chemical and Biomolecular Engineering, Faculty of Engineering, National University of Singapore, Singapore, Singapore.
Shi Min Or. Department of Chemical and Biomolecular Engineering, Faculty of Engineering, National University of Singapore, Singapore, Singapore.
Kyle K.L. Phua. Department of Chemical and Biomolecular Engineering, Faculty of Engineering, National University of Singapore, Singapore, Singapore.
Address correspondence to: Kyle K.L. Phua, PhD, Faculty of Engineering, Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore 117580, Singapore, E-mail: [email protected]
Introduction
In vitro transcribed messenger RNA (IVT mRNA) has demonstrated significant therapeutic potential in many preclinical studies and clinical trials [1-4]. It is well known that IVT mRNA triggers the innate immune system by being recognized by pattern recognition receptors such as toll-like receptors [5] (TLR3, TLR7, and TLR8), retinoic acid-inducible gene (RIG-I)-like receptors [6], and protein kinase RNA activated (PKR) [7], causing the activation of downstream effectors, including type I interferon (IFN) and other cytokines [8]. This feature of mRNA is favorable for vaccine applications because the induced cytokines are pro-inflammatory and will strengthen ensuing adaptive immune responses, leading to the development of many tumor targeted delivery systems [9-11]. However, for nonvaccine applications, the consequences of innate immune responses such as mRNA degradation, mRNA translation inhibition, and mRNA-induced toxicity are undesired. To address these concerns, effective strategies have been developed to tune down the immunogenicity of IVT mRNA. The most widely adopted approach is to use base modified nucleotides such as pseudouridine triphosphate to replace uridine triphosphate [12] and 5-methylcytidine triphosphate to replace cytidine triphosphate [13]. Transcribed with base modified nucleotides, IVT mRNA gains the ability to passively avoid RNA sensors and immune triggers. However, laboratory synthesized modified mRNAs still contain double-stranded RNA (dsRNA) fragments due to the inefficient nature of RNA polymerases [14]. As a result, even modified mRNA triggers innate immune responses although at a significantly lower level [15]. Subsequently, additional effort to remove dsRNA through HPLC purification of the in vitro transcripts with modified nucleotides is reported and shown, as a proof of concept,...