“`html
A multitude of ailments results from an absent or malfunctioning version of a solitary gene. For many years, researchers have been exploring gene therapy solutions that may remedy these conditions by introducing a fresh copy of the absent genes into the impacted cells.
In spite of these endeavors, only a limited number of gene therapy solutions have received approval from the FDA. A significant obstacle in creating these treatments has been managing the extent of gene expression in cells — insufficient expression may lead to failure, whereas excessive expression could induce severe adverse effects.
To facilitate more accurate control over gene therapy, MIT engineers have optimized and employed a control circuit that regulates expression levels within a desired range. In human cells, they demonstrated that this technique could be utilized to deliver genes capable of addressing disorders like Fragile X syndrome, a condition responsible for intellectual disabilities and other developmental challenges.
“In principle, gene supplementation could address monogenic disorders that vary widely but have a comparatively straightforward gene therapy solution if the therapy can be managed sufficiently well,” remarks Katie Galloway, the W. M. Keck Career Development Professor in Biomedical and Chemical Engineering and the principal author of the recent study.
MIT graduate student Kasey Love serves as the primary author of the manuscript, which is published today in Cell Systems. Other contributors to the paper include MIT graduate students Christopher Johnstone, Emma Peterman, and Stephanie Gaglione, along with Michael Birnbaum, an associate professor of biological engineering at MIT.
Gene Delivery
While gene therapy shows promise for addressing a range of illnesses, such as hemophilia and sickle cell anemia, only a few treatments have been sanctioned thus far, for a hereditary retinal disease and specific blood cancers.
The majority of gene therapy strategies employ a virus to introduce a new copy of a gene, which is subsequently integrated into the DNA of host cells. Some cells may absorb multiple copies of the gene, while others may end up with none.
“Simple overexpression of that payload can yield a considerable variation in expression levels in the target genes, as they take in different quantities of those genes or simply exhibit diverse expression levels,” Love states. “If it’s not expressing adequately, that undermines the purpose of the therapy. Conversely, overexpression poses a risk as that payload may be toxic.”
To tackle this issue, researchers have tested various types of control circuits designed to regulate the expression of the therapeutic gene. In this investigation, the MIT team chose to implement a circuit known as an incoherent feedforward loop (IFFL).
In an IFFL circuit, the activation of the target gene simultaneously triggers the production of a molecule that inhibits gene expression. One type of molecule that can facilitate this suppression is microRNA — a brief RNA sequence that attaches to messenger RNA, preventing it from being translated into protein.
In this research, the MIT team constructed an IFFL circuit termed “ComMAND” (Compact microRNA-mediated attenuator of noise and dosage), so that a microRNA strand that represses mRNA translation is integrated within the therapeutic gene. The microRNA resides within a short segment known as an intron, which is removed from the gene during transcription into mRNA. This ensures that whenever the gene is activated, both the mRNA and the microRNA that represses it are produced in approximately equal proportions.
This method empowers the researchers to control the entire ComMAND circuit using just one promoter — the DNA site that initiates gene transcription. By substituting promoters of varying strengths, the researchers can adjust the quantity of the therapeutic gene being produced.
In addition to providing tighter regulation, the compact nature of the circuit allows it to be transported on a single delivery vehicle, such as a lentivirus or adeno-associated virus, which could enhance the manufacturability of these therapies. Both of these viruses are commonly utilized for delivering therapeutic substances.
“Other researchers have created microRNA-based incoherent feedforward loops, but what Kasey has achieved is consolidating it all onto a singular transcript, which she demonstrated provides the utmost control when there is variable delivery to cells,” Galloway notes.
Accurate Regulation
To validate this system, the researchers designed ComMAND circuits capable of delivering the gene FXN, which is altered in Friedreich’s ataxia — a condition impacting the heart and nervous system. They also delivered the gene Fmr1, whose malfunction leads to Fragile X syndrome. In experiments using human cells, they revealed that they could adjust gene expression levels to roughly eight times those typically observed in healthy cells.
Without ComMAND, gene expression exceeded 50 times the normal level, which could impose safety hazards. Further trials in animal models would be necessary to ascertain the optimal levels, the researchers indicate.
The researchers also conducted tests in rat neurons, mouse fibroblasts, and human T-cells. For those cells, they delivered a gene encoding a fluorescent protein, enabling them to easily quantify the gene expression levels. In those instances as well, the researchers found they could manage gene expression levels more precisely than without the circuit.
The researchers are now preparing to explore whether they can use this technique to introduce genes at a level that would reinstate normal functionality and reverse signs of disease, either in cultured cells or animal models.
Other conditions that this technique could be applied to include Rett syndrome, muscular dystrophy, and spinal muscular atrophy, the researchers assert.
“The difficulty with many of these conditions is that they are also infrequent diseases, which limits the available patient populations,” Galloway points out. “We are striving to establish these robust tools so that individuals can learn how to achieve the necessary adjustments, given the small sizes of patient populations and the limited funding for addressing some of these disorders.”
The research received support from the National Institute of General Medical Sciences, the National Science Foundation, the Institute for Collaborative Biotechnologies, and the Air Force Research Laboratory.
“`