Many diseases stem from a missing or faulty copy of a single gene. For decades, researchers have pursued gene therapies that could treat these conditions by delivering a healthy replacement gene to the right cells.
Even with years of progress, only a limited number of gene therapies have been approved by the U.S. Food and Drug Administration. One major hurdle is controlling how strongly the introduced gene is expressed. If expression is too low, the treatment may not work; if it is too high, it can trigger serious side effects.
To address this problem, engineers at MIT have refined and tested a genetic control circuit designed to keep gene expression within a defined range. In human cells, they showed the strategy could help deliver genes relevant to conditions such as Fragile X syndrome, which causes intellectual disability and other developmental challenges.
“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,” said Katie Galloway, the W. M. Keck Career Development Professor in Biomedical Engineering and Chemical Engineering at MIT and the study’s senior author.
The study was led by MIT graduate student Kasey Love and published in Cell Systems. Co-authors include MIT graduate students Christopher Johnstone, Emma Peterman, and Stephanie Gaglione, as well as Michael Birnbaum, an associate professor of biological engineering at MIT.
Delivering genes
Gene therapy has long been viewed as a potential route to treating disorders such as hemophilia and sickle cell disease. Still, approvals remain relatively rare, with existing treatments largely focused on certain inherited eye diseases and specific blood cancers.
Many gene therapy approaches rely on viruses to deliver a functional gene into cells, where it can integrate into the host DNA. But delivery is uneven: some cells may receive multiple copies of the gene, while others receive none. This variability can cause gene expression levels to differ widely from cell to cell.
“Simple overexpression of that payload can result in a really wide range of expression levels,” Love said. “If it’s not expressing enough, that defeats the purpose of the therapy. But expressing at too high levels is also a problem, as that payload can be toxic.”
To tighten control, scientists have explored genetic “circuits” that limit or stabilize expression of a therapeutic gene. In the new work, the MIT team focused on a design known as an incoherent feedforward loop (IFFL), a circuit in which activation of a target gene also triggers a suppressor that dampens expression.
One way to create that suppression is through microRNAs—short RNA sequences that bind to messenger RNA (mRNA) and prevent it from being translated into protein. The team created an IFFL-based circuit they call ComMAND (Compact microRNA-mediated attenuator of noise and dosage). In their design, the microRNA that represses translation is encoded within the therapeutic gene itself.
Specifically, the microRNA is placed inside an intron, a short gene segment that is removed during RNA processing. As a result, when the therapeutic gene is turned on, both the mRNA and the microRNA that represses it are produced in roughly matched amounts, helping keep expression from rising too high.
This architecture also allows the entire ComMAND circuit to be controlled with a single promoter—the DNA element that initiates transcription. By choosing promoters with different strengths, researchers can tune how much of the therapeutic protein is ultimately produced.
Beyond tighter control, the compact design may make therapies easier to manufacture and deliver. Because the full circuit can fit into a single viral vector—such as lentivirus or adeno-associated virus—it can be packaged and delivered more efficiently. Both vectors are widely used in gene therapy research and development.
According to Galloway, similar microRNA-based IFFL concepts have been explored before, but this single-transcript approach offers a key advantage when delivery varies from cell to cell.
More precise control
To demonstrate the system, the researchers built ComMAND circuits to deliver FXN, a gene mutated in Friedreich’s ataxia, a condition that affects the nervous system and heart. They also delivered Fmr1, whose dysfunction causes Fragile X syndrome.
In experiments in human cells, the team reported they could tune expression to roughly eight times the levels typically seen in healthy cells. Without the ComMAND circuit, expression rose to more than 50 times normal—levels that could raise safety concerns. The researchers noted that further work in animal models would be needed to identify the most appropriate therapeutic ranges.
The team also tested the approach in rat neurons, mouse fibroblasts, and human T cells. In these experiments, they delivered a gene encoding a fluorescent protein to make expression levels easy to measure. Across these cell types, the circuit consistently improved the precision of expression compared with standard delivery.
Next, the researchers plan to explore whether this strategy can deliver genes at levels that restore normal function and reduce disease-related symptoms in cultured cells and animal models. They also believe the platform could be adapted to other genetic disorders, including Rett syndrome, muscular dystrophy, and spinal muscular atrophy.
Galloway noted that many monogenic disorders are rare, which can limit available funding and the size of patient populations for clinical research. The team’s goal is to develop robust, tunable tools that others can use to better calibrate gene therapies for these conditions.
The research was supported by the National Institute of General Medical Sciences, the National Science Foundation, the Institute for Collaborative Biotechnologies, and the Air Force Research Laboratory.