Chemically inducible systems represent valuable synthetic biology tools that enable the external control of biological processes. However, their translation to therapeutic applications has been limited due to unfavorable ligand characteristics or immunogenicity of non-human protein domains. Therefore, we developed novel dimerization systems that are based on human protein components and respond to clinically approved drugs or physiologically important ligands. First, we developed a strategy, where ligand-binding proteins of human origin are split into two fragments that reassemble in the presence of a ligand (INSPIRE). We show that the INSPIRE platform can be used for the dynamic, orthogonal, and multiplex control of gene expression in mammalian cells. Furthermore, we demonstrate the functionality of our INSPIRE platform in vivo and apply it for perturbing an endogenous regulatory network. INSPIRE presents a generalizable approach toward designing small molecule-responsive systems that can be implemented for the construction of new sensors, regulatory networks, and therapeutic applications. In addition to the INSPIRE platform, we have also developed dimerization systems utilizing separated antibody variable fragments (Fv) that bind to small molecules. This approach involves saperating Fv into two domains, which dimerize in the presence of the target ligand. To illustrate the broad utility of this approach, we present three examples. First, we demonstrate that saperated Fv can be employed to regulate gene expression and construction of logic gate functions. Second, we develop a fluorescein-responsive CID system and apply it for the recruitment of tumor-targeting scFv adaptor molecules to the universal CAR receptor expressed on T cells. Lastly, we showcase the fluorescein inducible regulation of a bispecific T cell engager (BiTE) complex capable of redirecting T cells to kill tumor cells. Additionally, we have developed two-input switches that are based on ligand-binding domains of nuclear receptors and their interacting peptides. These switches integrate two distinct competing signals, enabling precise control over cellular processes. We exemplify this controllability through the fine regulation of gene expression in mammalian cells. The designed dimerization systems offer an exciting opportunity to rapidly expand the current repertoire of small molecule-responsive systems for use in research and therapeutic applications. Together, these systems hold great potential for achieving chemogenetic control over gene, protein, and cell-based therapeutics, ultimately leading to enhanced safety and efficacy compared to existing treatments.
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