Synthetic mRNA is an attractive alternative to DNA for gene therapies due to the transient nature and improved safety profile of mRNA, but broadly applicable methods to control expression from delivered mRNA are lacking. Regulation of gene expression is a critical aspect of gene therapies as the imbalance of an expressed protein could lead to adverse clinical effects, yet most current therapeutics rely on constitutive expression. For DNA, there are well-established methods to control the strength and timing of transgene expression. However, similar control over synthetic mRNA expression has remained elusive.
Drawing on principles established by synthetic biologists for DNA genetic circuits, scientists from MIT developed an “ON” and “OFF” platform for regulation of proteins expressed from mRNA. Scientists sought to control regulation of protein expression by designing RNA to produce RNA-binding proteins (RBPs) that are receptive to a small molecule drug. The RBPs were delivered as self-replicating RNAs (replicons) and designed to be delivered along with the mRNA encoding the therapeutic protein. These mRNA regulatory devices successfully controlled expression of multiple proteins from synthetic mRNA directly transfected into mammalian cells.
They initially established turning OFF expression by fusing RBPs to destabilization domains, which are targeted for degradation by the proteasome unless the domain is stabilized by a cognate small molecule. The destabilizing domain from E. coli dihydrofolate reductase (DDd) was fused to L7Ae, an RBP capable of binding to kink-turn motifs in a 5’ UTR to repress expression. This engineered RNA was then co-transfected with an mVenus reporter with two kink-turns in its 5′ UTR. In the presence of FDA-approved small molecule drug trimethoprim, the DDd was stabilized, thereby allowing the fused protein, DDd-L7AE, to bind and repress mVenus, turning OFF expression.
For turning ON expression, the opposite strategy was deployed, and an external small molecule input was required only when the regulated protein is needed in the system. Scientists fused TetR RNA aptamers, which bind doxycycline (Dox), with mammalian dead box helicase6 protein, which enhances TetR/aptamer-mediated translational repression, in the 5′ UTR of an mKate fluorescent reporter. The introduction of the Dox binds TetR and prevents TetR from binding its cognate RNA aptamer, thereby turning ON expression.
By coupling these two ON and OFF mechanisms, two-output switches were also generated, with the timing and level of reporter protein expression externally regulated by the two small molecules. This study exemplified the potential of applying synthetic biology gene circuitry principles to mRNA designs, supporting the use of small molecule regulation in emerging RNA-based gene therapies.
The article titled “Small-molecule-based regulation of RNA-delivered circuits in mammalian cells” was published in Nature Chemical Biology.