Synthetic mRNAs, which are produced by in vitro transcription, have been recently attracting attention because they can express any transgenes without the risk of insertional mutagenesis. Protein-based translational regulation systems enable the context-dependent production of therapeutic proteins and have the potential to further improve the efficacy and safety of synthetic mRNAs.
During gene expression, genes are first transcribed from DNA to messenger RNA (mRNA) and then translated from mRNA to protein. Thus, researchwers can make cells express exogenous genes by transfecting either DNA or mRNA. Although DNA transfection is the standard method for transgene expression, it can cause insertional mutagenesis of endogenous genes, which is a major drawback in medical applications. In contrast, synthetic mRNAs, which are produced by in vitro transcription, can be used for transgene expression without the risk of insertional mutagenesis. Thus, synthetic mRNAs have recently been attracting attention as tools for gene therapy, cellular reprogramming, and vaccine development .
Nevertheless, context-dependent regulation of transgene expression is more difficult in synthetic mRNA transfection than in DNA transfection. Upon DNA transfection, transgene expression can be tuned using transcriptional regulatory sequences, such as drug-inducible or tissue-specific promoters. On the other hand, such transcriptional regulation cannot apply to synthetic mRNA transfection as they must be transcribed in vitro prior to transfection. As some therapeutic genes may cause adverse effects when they are expressed in inappropriate contexts, context-dependent transgene regulation is helpful to cope with low adverse effects and high therapeutic efficacy. For example, cell-selective expression of pro-apoptotic genes [2,3,4,5,6] may enable cancer cell elimination without damaging healthy cells. Similarly, cardiomyocyte-selective expression of proliferation-promoting genes can be helpful to cope with myocardial regeneration without elevating fibrosis and immune response . Thus, the development of translational regulation systems will make synthetic mRNAs more valuable.
One promising approach is to develop protein-based systems for translational regulation of synthetic mRNAs. There are multiple advantages to protein-based translational regulation systems. First, when proteins are used as translational regulators, the regulators themselves can be translated from synthetic mRNAs, such that all components necessary for regulation can be delivered as synthetic mRNAs. Second, compared to ribozyme-embedded mRNAs  or caged mRNAs , mRNAs composing protein-based regulation systems have less concern regarding the alteration of mRNA properties during synthesis or storage, such as self-cleavage or decaging. Finally, in protein-based translational regulation systems, the first output protein can be used as the input for the second regulation, allowing layered gene circuits for sophisticated regulation ( Figure 1 ).