Engineering a regulatory framework for synthetic self-amplifying RNA circuits
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Self-amplifying RNA replicons are an attractive alternative to traditional nucleic acid therapeutics, providing high, sustained expression from a low dose, without the risk of genomic integration. Replicons are derived from a viral genome, but utilize viral self-amplification to produce heterologous genes instead of the structural proteins necessary to form virions. Despite a variety of therapeutic applications, ranging from vaccination to genetic reprogramming, regulating expression from replicons remains relatively unexplored, as the field continues to rely solely on constitutive expression. However, we will demonstrate that dose cannot be used to control constitutive expression levels from a replicon. Without any means of regulation, the inability to adjust expression is a major safety concern that must be addressed before this platform can be used for more clinically-driven specifications. In this dissertation, we have employed synthetic biology to expand the potential of replicon-based platforms to include sequence-level and small molecule mediated control of expression levels. Synthetic biology aims to create and characterize libraries of highly predictable and modular genetic parts that can be combined to produce genetic circuits. To this end, we generated a collection of parts that can modulate replicon subgenomic transcription, explored existing and novel replicon-based expression platforms, and designed small molecule responsive replicon circuits. We established sequence elements that can be used to predictably control constitutive expression levels of up to three genes driven from a single self-amplifying RNA strand. We verified that this regulatory framework was functional for multiple replicon-based platforms, including multi-SGP replicons, DNA-launched replicons, and a novel self-cleaving, amplifying RNA platform. Finally, we coupled these genetic parts with small molecule responsive elements to form RNA-only circuits delivered on a single replicon that could control expression of multiple proteins based on external inputs. By introducing regulatory genetic circuits to self-amplifying RNA, we demonstrate control over the strength, timing, and location of expression, enhancing the utility of RNA for gene delivery and establishing a framework for the next generation of RNA-based therapeutics.