MIT Engineers Create a Tunable System to Control Transgene Expression in Cells

MIT engineers have developed a method to precisely adjust and maintain protein production from synthetic genes after they are introduced into cells. The system, called DIAL, offers a way to set and later edit protein expression levels, or “set points,” allowing cells to produce consistent amounts of a desired molecule. Published in Nature Biotechnology, the study was led by Katie Galloway.

Synthetic gene circuits typically include a gene and a nearby promoter region where regulators bind to control gene activity. However, differences in how many gene copies cells take up, combined with their natural variability, make it difficult to achieve uniform protein expression. The MIT team addressed this by altering the physical distance between the gene and its promoter. When a longer spacer sequence separated them, the gene was expressed at lower levels. Shortening the spacer increased expression by bringing transcription factors closer to the gene.

To enable post-delivery adjustments, the researchers inserted enzyme recognition sites within the spacer that could be cut by recombinases such as Cre. Each excision point moved the promoter closer to the gene, allowing the set point for gene expression to be tuned to “high,” “medium,” “low,” or “off.” Once the genetic construct was introduced into cells, recombinases could be added later to shift the expression level without replacing the circuit.

The researchers validated this design using fluorescent protein genes and functional transgenes in both mouse and human cells. They demonstrated that DIAL produced uniform and stable protein expression across cell populations. “We achieved uniform and stable control. This is very exciting for us because lack of uniform, stable control has been one of the things that's been limiting our ability to build reliable systems in synthetic biology,” Galloway said.

To test its application, the team used DIAL to control expression of the HRas^G12V gene in mouse embryonic fibroblasts, influencing their conversion into neurons. Higher expression levels of the gene increased the proportion of successfully reprogrammed cells. The researchers plan to apply this approach to study how transcription factor levels affect cell identity and efficiency in reprogramming. 

The group also demonstrated that DIAL can integrate with ComMAND, a previously developed system that limits overexpression of therapeutic genes. Together, these modular technologies may support future gene therapies capable of maintaining precise protein levels in specific cell types or patient populations.