SYNTHETIC RNA CIRCUITS WITH SMALL MOLECULE INDUCTION FROM REPLICATING RNA VACCINES

BACKGROUND: Synthetic biology has the potential to create more improved nucleic acid therapies by adding increased control in the design space. Synthetic engineered vectors have been shown to provide cell type specificity, enhanced expression dynamics, and variable protein expression dynamics. Using these tools, better vaccines and therapies can be built on top of existing therapeutic modalities by harnessing the power of various synthetic biology toolboxes. METHODS: Here, we have further developed the RNA alphavirus and modified RNA platforms into vectors capable of carrying synthetic circuitry payloads that can provide a variety of desirable dynamics. Utilizing natural and synthetic RNA-binding proteins (RBPs), we have constructed a toolbox capable of regulating expression via non-degrative, reversible binding to transcripts expressing therapeutic proteins of interest. Using FDA-approved small molecules, we can modify protein stability. RESULTS: Using this approach, we ultimately created advanced circuitry that can be contained on single replicons or a multitude of modified RNA transcripts, expressing up to five proteins at once and exhibiting cascade topology. We have used this technology to create therapeutic circuits for muscular dystrophy. Particularly, we have created a two-state inducible system that is capable of switching between expressing the muscle differentiation promoting protein kinase B (Akt), and then switching to muscle growth promoting factor follistatin. Normally, Akt has been shown to be very toxic after long expression in muscle. We created a circuit topology capable of expressing Akt for a defined period of time, and then sequentially switching to follistatin in order to rescue the muscle health. This circuit has shown an increase in muscle differentiation compared to constitutive expression from traditional expression vectors. CONCLUSIONS: Our platform enables precise control of RNA-only expression platforms, including the use of self-replicating RNA to express therapeutic proteins. We leverage this technology as a potential future controllable vaccine, allowing external control for antigen or antibody expression, hopefully increasing the potential to vaccinate against increasingly complex targets.