safer strategy for regulating gene expression

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From Children's Hospital, Boston 22-Sep-2004

A novel, safer strategy for regulating gene expression

Technique provides 'on-off' control as easy as taking a pill

Researchers at Children's Hospital Boston and Harvard Medical School

have created a novel, elegant, and safer system for controlling gene

expression – turning genes on and off as needed – that involves an

intervention as simple as giving a drug. Potentially, with this

technique, a gene could even be activated by natural conditions in the

body – for example, in a diabetic patient, a rise in glucose

concentration would automatically turn on the gene responsible for

insulin production.

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.

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.