Innovative DIAL System for Precise Control of Synthetic Gene Expression

For years, synthetic biologists have been advancing gene circuits that can be integrated into cells for various applications, including reprogramming stem cells into neurons and producing proteins that may aid in treating conditions like fragile X syndrome. Typically, these gene circuits are introduced into cells using carriers such as nonpathogenic viruses. However, ensuring that these cells generate the appropriate amount of the protein encoded by the synthetic gene has proven challenging. To address this issue, engineers at MIT have created an innovative control mechanism that enables the establishment of a desired protein level, or ‘set point,’ for any gene circuit. This new approach also allows for post-delivery adjustments to the set point. As stated by Katie Galloway, an assistant professor in Chemical Engineering at MIT and senior author of the study, “This tool is stable and multifunctional. Its modular design means a wide array of transgenes can be controlled using this system.” Utilizing this strategy, researchers demonstrated their ability to induce cells to produce consistent levels of target proteins. In one specific application, they successfully converted mouse embryonic fibroblasts into motor neurons by delivering elevated levels of a gene that facilitates this conversion. The lead author of the study, MIT graduate student Sneha Kabaria, alongside a team of researchers, published their findings in Nature Biotechnology. **Enhancing Gene Expression Control** Synthetic gene circuits are designed to not only include the gene of interest but also a promoter region where transcription factors and other regulators can bind to initiate gene expression. Achieving uniform expression of the desired gene across a population of cells can be difficult; some cells may take up just one copy of the circuit while others receive multiple copies. Furthermore, inherent biological variation among cells adds another layer of complexity to gene reprogramming efforts. In their recent paper, the team developed a method to control gene expression levels by manipulating the distance between the synthetic gene and its promoter. They discovered that increasing the DNA “spacer” between these components led to lower gene expression levels. This distance reduction decreases the likelihood that transcription factors attached to the promoter will activate gene transcription effectively. To create adjustable set points, the researchers introduced excision sites within the spacer that can be targeted by an enzyme known as Cre recombinase. As segments of the spacer are removed, transcription factors are brought closer to the gene of interest, thereby enhancing gene expression. The team successfully created spacers featuring multiple excision points, each designed for different recombinases. This innovative system, referred to as DIAL, enables them to establish set points classified as ‘high,’ ‘medium,’ ‘low,’ and ‘off’ for gene expression. Once the DNA segment containing the gene and its promoter is delivered into cells, recombinases can be introduced, allowing for real-time edits to the set points. The researchers validated their system in both mouse and human cells by delivering genes encoding various fluorescent proteins and functional genes, achieving uniform expression across cell populations at designated target levels. Galloway expressed enthusiasm about their results: “We achieved uniform and stable control. This is exciting because the lack of consistent control has been a significant barrier in developing reliable systems in synthetic biology. Too many variables can complicate matters, especially with natural biological variations.” **Applications in Cell Reprogramming** To illustrate potential applications of the DIAL system, researchers used it to deliver varying levels of the HRasG12V gene to mouse embryonic fibroblasts. Previous studies indicated that this HRas variant accelerates fibroblast-to-neuron conversion. The MIT team found that cells receiving higher doses of the gene displayed a greater success rate in transforming into neurons. Moving forward, researchers aim to conduct systematic studies on various transcription factors that could drive cells toward different identities. Such investigations could uncover how varying factor levels influence transformation success rates and whether adjustments in transcription factor levels might alter the resultant cell type. In ongoing work, researchers demonstrated that DIAL could be combined with a previously developed system called ComMAND, which employs a feedforward loop to regulate therapeutic gene expression and prevent overexpression. By integrating these systems, it may become feasible to customize gene therapies to produce precise and consistent protein levels tailored to individual patients’ target cells. Galloway remarked on the exciting prospects: “Both DIAL and ComMAND are modular systems, allowing us to create not only well-controlled gene therapies suitable for broader populations but also potentially personalized treatments for specific individuals or cell types.” This research received partial funding from the National Institute of General Medical Sciences, the National Science Foundation, and the Institute for Collaborative Biotechnologies.