A Novel, Safer Strategy For Regulating Gene Expression

The system, described in a proof-of-principle paper in the Sept. 23 edition of the journal Nature, is simpler than current methods of gene regulation, and the technology exists to make it work with virtually any drug, making it suitable for a broad range of therapeutic and research applications.

The technique involves inserting a special DNA sequence into a patient's own genes, or into a gene introduced by gene therapy. This sequence encodes a ribozyme, a sequence of RNA that has the unique ability to spontaneously cut itself in half. The ribozyme becomes part of the gene's messenger RNA (mRNA), the template that carries instructions for making the protein encoded by that gene. When the ribozyme cuts itself in half, the mRNA is cut in half, too – disabling it and, in effect, turning off the gene. Inhibiting ribozyme breakage, which can be done with various drugs, leaves the mRNA intact; this allows the gene to turn "on" and make the desired protein – such as a hormone or growth factor needed by the body.

"Perhaps the most exciting aspect of the new work is that, in conjunction with other technologies, we will likely be able to 'tailor' gene regulation systems to respond to any drug or chemical," says Dr. Richard Mulligan, director of gene therapy research at Children's Hospital Boston and director of the Harvard Gene Therapy Initiative. "Ultimately, the system should also enable the 'release' of a therapeutically useful protein in response to changing concentrations of chemicals in cells. For instance, it may be possible to develop a gene therapy whereby cells are engineered to secrete insulin in response to a rise in glucose. Such 'biological sensing' could have a wide range of applications."

Current methods of gene regulation usually involve a complicated three-part system that requires a "promoter" (a DNA sequence near the gene that allows it to transfer its information to RNA), a specialized activating protein that makes the promoter work, and a drug that, in turn, enables the activating protein. Together, these elements turn the gene on and off. However, there are concerns that the activating protein could trigger the immune system and cause unwanted side effect. In addition, the current systems work with only a handful of specific drugs. In contrast, the ribozyme-based system can, in principle, be designed to regulate genes using any drug, or any chemical change in the body. The system is also easier to turn on and off than existing systems, allowing a treatment to be stopped for safety reasons.

"With recent concerns about the development of leukemia in several children treated with gene therapy, this new method adds an important new safety feature to the gene therapy toolbox," says Mulligan, who is also a professor of genetics at Harvard Medical School.

Dr. Laising Yen and other members of Mulligan's laboratory began by evaluating hundreds of known ribozymes, and found two that function well in human cells. One ribozyme was especially prone to cutting itself in two, and the researchers tweaked it to make it even more efficient. Next, they identified two compounds that strongly inhibit ribozyme self-cutting, and showed that they could be used to turn on ribozyme-containing genes in mammalian cells, inducing the cells to make the desired protein. Finally, they proved that the technique works in live animals. They introduced the gene for a protein called luciferase, containing an embedded ribozyme, into the retinas of mice. When they treated the mice with a ribozyme-inhibiting drug, the gene turned on and the animals' retinas began producing luciferase. Without the drug, the gene remained "off," and no luciferase was produced.

Ribozymes occur naturally in plants, animals, and bacteria. Recent studies suggest that bacteria use ribozymes to regulate their own gene activity, by "sensing" and reacting to changing levels of natural compounds in their environment. Scientists have theorized that these ribozymes might have evolved before proteins did and functioned as an ancient gene-control system. Mulligan, Yen and colleagues have taken the first step to demonstrate that this natural system could be adapted and exploited to treat human disease.