A Humanized Transcriptional Regulatory Program for Orthogonal Genetic Control

The ability to precisely control expression of genes in synthetic circuits without undesirably impacting native cellular processes is critical for emergent therapeutic applications. State-of-the-art gene therapy platforms currently lack strategies to limit the duration and spatial extent of therapeutic production post-delivery. While several technologies using synthetic regulatory proteins have been adopted for spatiotemporal genetic control in proof-of-concept in vitro circuits, fundamental limitations such as large payload and immunogenicity of heterologous parts prevent their translation into in vivo contexts. Here, we seek to develop synthetic biological controllers poised for genetic control in in vivo therapeutic contexts. We engineer genome-orthogonal genetic regulation schemes utilizing synthetic transcription factors and corresponding responsive promoters. Our humanized regulators are based on compact, mammalian-derived zinc finger scaffolds with specificities for genome-orthogonal DNA sites, fused to well-established regulatory domains. These engineered controllers are capable of efficiently regulating gene expression at their cognate target loci, while minimally altering expression of native genomic transcripts. We envision that our flexible genetic regulation platform will translate to a broad array of gene therapy applications and enable controllable expression of human therapeutics.