Many diseases are caused by a missing or defective copy of a single gene. For decades, scientists have been working on gene therapy treatments that could cure such diseases by delivering a new copy of the missing genes to the affected cells.Despite those efforts, very few gene therapy treatments have been approved by the FDA. One of the challenges to developing these treatments has been achieving control over how much the new gene is expressed in cells — too little and it won’t succeed, too much and it could cause serious side effects.To help achieve more precise control of gene therapy, MIT engineers have tuned and applied a control circuit that can keep expression levels within a target range. In human cells, they showed that they could use this method to deliver genes that could help treat diseases including Fragile X syndrome, a disorder that leads to intellectual disability and other 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 and the senior author of the new study.MIT graduate student Kasey Love is the lead author of the paper, which appears today in Cell Systems. Other authors of the paper include MIT graduate students Christopher Johnstone, Emma Peterman, and Stephanie Gaglione, and Michael Birnbaum, an associate professor of biological engineering at MIT.Delivering genesWhile gene therapy holds promise for treating a variety of diseases, including hemophilia and sickle cell anemia, only a handful of treatments have been approved so far, for an inherited retinal disease and certain blood cancers.Most gene therapy approaches use a virus to deliver a new copy of a gene, which is then integrated into the DNA of host cells. Some cells may take up many copies of the gene, while others don’t receive any.“Simple overexpression of that payload can result in a really wide range of expression levels in the target genes as they take up different numbers of copies of those genes or just have different expression levels,” Love says. “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.”To try to overcome this, scientists have experimented with different types of control circuits that constrain expression of the therapeutic gene. In this study, the MIT team decided to use a type of circuit called an incoherent feedforward loop (IFFL).In an IFFL circuit, activation of the target gene simultaneously activates production of a molecule that suppresses gene expression. One type of molecule that can be used to achieve that suppression is microRNA — a short RNA sequence that binds to messenger RNA, preventing it from being translated into protein.In this study, the MIT team designed an IFFL circuit, called “ComMAND” (Compact microRNA-mediated attenuator of noise and dosage), so that a microRNA strand that represses mRNA translation is encoded within the therapeutic gene. The microRNA is located within a short segment called an intron, which gets spliced out of the gene when it is transcribed into mRNA. This means that whenever the gene is turned on, both the mRNA and the microRNA that represses it are produced in roughly equal amounts.This approach allows the researchers to control the entire ComMAND circuit with just one promoter — the DNA site where gene transcription is turned on. By swapping in promoters of different strengths, the researchers can tailor how much of the therapeutic gene will be produced.In addition to offering tighter control, the circuit’s compact design allows it to be carried on a single delivery vehicle, such as a lentivirus or adeno-associated virus, which could improve the manufacturability of these therapies. Both of those viruses are frequently used to deliver therapeutic cargoes.“Other people have developed microRNA based incoherent feed forward 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,” Galloway says.Precise controlTo demonstrate this system, the researchers designed ComMAND circuits that could deliver the gene FXN, which is mutated in Friedreich’s ataxia — a disorder that affects the heart and nervous system. They also delivered the gene Fmr1, whose dysfunction causes Fragile X syndrome. In tests in human cells, they showed that they could tune gene expression levels to about eight times the levels normally seen in healthy cells.Without ComMAND, gene expression was more than 50 times the normal level, which could pose safety risks. Further tests in animal models would be needed to determine the optimal levels, the researchers say.The researchers also performed tests in rat neurons, mouse fibroblasts, and human T-cells. For those cells, they delivered a gene that encodes a fluorescent protein, so they could easily measure the gene expression levels. In those cells, too, the researchers found that they could control gene expression levels more precisely than without the circuit.The researchers now plan to study whether they could use this approach to deliver genes at a level that would restore normal function and reverse signs of disease, either in cultured cells or animal models.“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,” Love says.Other diseases that this approach could be applied to include Rett syndrome, muscular dystrophy and spinal muscular atrophy, the researchers say.“The challenge with a lot of those is they’re also rare diseases, so you don’t have large patient populations,” Galloway says. “We’re trying to build out these tools that are robust so people can figure out how to do the tuning, because the patient populations are so small and there isn’t a lot of funding for solving some of these disorders.”The research 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.
