Researchers at the Massachusetts Institute of Technology have engineered a novel control mechanism that allows for precise, adjustable regulation of synthetic genes introduced into cells. This new tool provides a stable and modular way to establish and even change the level of protein expression after a gene circuit has been delivered, a significant step forward for gene therapy and cell reprogramming. The system, named DIAL, addresses a long-standing challenge in synthetic biology: ensuring that engineered cells produce a consistent and correct amount of a therapeutic protein.
The ability to finely tune gene expression is critical for both research and therapeutic applications. For decades, synthetic biologists have worked to develop gene circuits for tasks like turning stem cells into neurons or producing proteins to treat diseases. However, achieving uniform expression levels across a population of cells has been a persistent obstacle. The new DIAL system overcomes this by allowing scientists to create set points for gene activity—such as “high,” “medium,” “low,” or “off”—and then use specific enzymes to edit those set points as needed. This level of control promises more effective and systematic studies of how gene expression levels influence cell fate and function.
Overcoming Expression Variability
Synthetic gene circuits are essential tools in modern biotechnology. These circuits, which include the gene of interest and a promoter region to control it, are often delivered into cells using viral vectors. A major difficulty has been that not all cells in a population will express the inserted gene at the same level. This variability can hinder the effectiveness of therapies that rely on a specific dosage of a protein. For example, in gene therapies, producing too little of a protein may render the treatment ineffective, while overproduction could be toxic to the cells.
Previous methods to control gene expression often involved synthetic transcription factors, such as zinc fingers, which bind to promoter regions to activate a gene. However, these transcription factors frequently need to be redesigned for each specific gene they target, a process that is both time-consuming and complex. Another approach developed at MIT in 2013 utilized the CRISPR system to create transcription factors that could more easily control naturally occurring genes. While a significant advance, the challenge of precisely regulating externally introduced transgenes with a predictable, full-spectrum system remained.
The DIAL System Mechanism
To address the need for more precise control, MIT engineers designed the DIAL system to be highly modular and editable. The core of the system is a DNA segment carrying the synthetic gene and its promoter, which is delivered into the target cells. This promoter is engineered with specific sites that can be acted upon by enzymes called recombinases.
By adding different recombinases to the cells, researchers can edit the promoter’s structure, effectively dialing the gene’s expression level up or down. This process allows them to establish clear and stable set points for protein production. The team successfully created settings for “high,” “medium,” “low,” and “off,” providing a range of predictable expression levels. A key advantage of this design is that the set point is not permanent; it can be adjusted at any time after the initial gene circuit is in place simply by introducing the appropriate recombinase.
CRISPR-Based Predecessors
The development of the DIAL system builds on earlier work in the field, including a separate but related MIT technology based on CRISPR proteins. In that system, a deactivated Cas9 protein (dCas9) is attached to a transcription activation domain. This complex is guided by a specific guide RNA to a synthetic promoter site, where it activates gene expression. The researchers created a library of these synthetic promoters and guide RNAs to achieve a spectrum of expression levels, from very low to very high. This CRISPR-based approach was shown to be highly consistent across different mammalian cell types, including the Chinese hamster ovary (CHO) cells commonly used in industrial manufacturing of therapeutic proteins like monoclonal antibodies.
Demonstrated Therapeutic Potential
The researchers rigorously tested the DIAL system in both mouse and human cells, demonstrating its effectiveness and stability. In one key experiment, they delivered genes for fluorescent proteins and showed they could achieve uniform expression across a cell population at the desired target level. This confirmed the system’s ability to produce consistent results, a critical requirement for any potential therapeutic.
In a more advanced application, the team used the system to reprogram cells. They successfully converted mouse embryonic fibroblasts into motor neurons by delivering a high level of a gene known to promote this specific cellular transition. This highlights the system’s potential for use in regenerative medicine and for creating specific cell types for research and therapeutic use. According to senior author Katie Galloway, an assistant professor at MIT, the tool is “very modular, so there are a lot of transgenes you could control with this system.”
Future Directions and Integration
The DIAL system opens the door for more systematic studies of how transcription factors—the proteins that regulate gene activity—drive cell differentiation. Researchers can now investigate how different levels of these factors affect the success rate and outcome of cell conversion processes. Such studies could reveal whether altering the expression levels at different times might change the final cell type that is generated.
Looking ahead, the research team is working to combine the DIAL system with other technologies they have developed. One such tool is known as ComMAND, a system that uses a feedforward loop to prevent cells from overexpressing a therapeutic gene. By integrating these systems, they hope to create even more robust and safer gene circuits for a wide range of biomedical applications, from producing protein-based drugs to engineering advanced cell therapies.