MIT Engineers Develop Gene Circuit for
Precise Control of Gene Therapy

Many diseases are caused by a missing or defective gene, and for decades, scientists have worked on gene therapy as a potential cure by delivering a new copy of the gene to affected cells. However, achieving precise control over gene expression remains a significant challenge. Too little expression leads to failure, while too much could cause harmful side effects.

To tackle this, MIT engineers have developed a new gene circuit that precisely regulates gene expression. Their approach, demonstrated in human cells, shows potential for treating diseases such as fragile X syndrome, a disorder that causes intellectual disabilities and developmental problems.

“In theory, gene supplementation can solve monogenic disorders that are very diverse but have a relatively straightforward gene therapy fix if you could control the therapy well enough,” says Katie Galloway, the W. M. Keck Career Development Professor in Biomedical Engineering and Chemical Engineering at MIT and senior author of the study.

MIT graduate student Kasey Love, the lead author of the paper, helped design a control circuit called an incoherent feedforward loop (IFFL). The circuit uses microRNA, a molecule that represses gene expression, to keep expression levels within a target range.

“If it’s not expressing enough, that defeats the purpose of the therapy. But on the other hand, expressing at too high levels is also a problem, as that payload can be toxic,” Love says.

The IFFL circuit, which they called “ComMAND,” is designed to work with a single promoter, allowing the team to control the therapeutic gene’s expression more precisely. The microRNA is encoded within the therapeutic gene itself and helps suppress its translation, allowing for tight regulation of gene expression. The compact design also makes the circuit easy to deliver using common viral vectors, like lentivirus or adeno-associated virus, improving the manufacturability of these therapies.

“Other people have developed microRNA-based incoherent feedforward loops, but what Kasey has done is put it all on a single transcript, and she showed that this gives the best possible control when you have variable delivery to cells,” says Galloway.

In their experiments, the researchers tested the system with the FXN gene, mutated in Friedreich’s ataxia, and the Fmr1 gene, which causes fragile X syndrome when defective. The results showed they could precisely control gene expression levels, achieving around eight times the normal levels seen in healthy cells, which is significantly more controlled compared to gene expression without the ComMAND circuit.

“Without ComMAND, gene expression was more than 50 times the normal level, which could pose safety risks,” says Love. “There’s probably some tuning that would need to be done to the expression levels, but we understand some of those design principles, so if we needed to tune the levels up or down, I think we’d know potentially how to go about that.”

The researchers also tested the circuit in rat neurons, mouse fibroblasts, and human T-cells, demonstrating the ability to control gene expression in various cell types. The team now plans to explore whether this method can be used to restore normal gene function and reverse disease symptoms in animal models.

“This approach could be applied to other diseases, like Rett syndrome, muscular dystrophy, and spinal muscular atrophy,” says Galloway, noting the challenges of developing therapies for rare diseases with small patient populations.

The study, published in Cell Systems, was funded by the National Institute of General Medical Sciences, the National Science Foundation, the Institute for Collaborative Biotechnologies, and the Air Force Research Laboratory.

Sources – Cell Systems, MIT News