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Thursday, January 22, 2004


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Gene Therapy in Salivary Glands Could Lead to Promising Applications in Oral Diseases

Although gene therapy has shown much promise over the past decade, one of its major challenges continues to be controlling the expression of a transplanted gene once it has been delivered into a cell. As many scientists already have reported, transplanted genes may switch off prematurely, or, in some cases, they might not turn off fast enough, causing an undesirable overproduction of its replacement protein.

One way around this problem is to control the expression of the transplanted gene with a system controlled by a small molecule, for example rapamycin, a well-characterized immunosuppressive drug. As scientists have envisioned the strategy, they stitch a chemical switch next to the gene that only rapamycin (or derivative) molecules can control. Upon administration of the drug, the gene will turn on leading to protein production. Already, researchers have demonstrated the effectiveness of this approach in the liver, muscle, and eye.

Now, a team of scientists report in an article published online today in the journal Gene Therapy they succeeded in getting the so-called "rapamycin gene-activation" system to work in the salivary glands. As the scientists noted, their finding could one day have important implications in treating a variety of oral and possibly systemic conditions with gene therapy. "Our data mark an important first step toward applying the technique in the salivary glands," said Dr. Bruce Baum, the senior author on the paper and a scientist at the NIH's National Institute of Dental and Craniofacial Research. "Our next goal will be to refine the technique further and apply it to treat disease."

For decades, most biologists have subscribed to the dogma that glands in the body either secrete proteins into the bloodstream, called endocrine secretion , or channel them outward through a duct, for example, to the mouth or intestine, called exocrine secretion . But, according to the dogma, they can't do both.

Salivary glands are an exception to the dogma. They not only secrete proteins into saliva then into the mouth in an exocrine manner, but also partly into the bloodstream in an endocrine fashion. This dual secretory feature has made the salivary glands an intriguing, though often overlooked, target for gene therapy experiments. "What's fascinating is, in theory, one could use gene therapy in the salivary glands to treat either oral and systemic conditions or single-gene disorders, such as diabetes and growth hormone deficiency," said Baum, who has studied the salivary glands for nearly three decades.

Baum's group and others already have confirmed this theory in animal studies. They found that, depending on the signal for the protein, the salivary glands can secrete in either direction. The group also established in a series of gene therapy experiments that the salivary glands will secrete the transferred gene's protein products in a normal, physiological way, showing that their potential as a site for gene therapy will extend well beyond the mouth.

In the current Gene Therapy paper, Wang et al. hypothesized that the rapamycin system also might work in the salivary glands. In a series of experiments, the scientists showed that the production of human growth hormone (hGH) and its secretion into the saliva of rats could be regulated with rapamycin for at least three times in 16 days. Rats that were not given rapamycin had no hGH in their saliva. hGH was used as a surrogate exocrine protein in this study. Normally, however, hGH is secreted via a "regulated pathway" in the anterior pituitary gland, which leads to its secretion in blood. However, in salivary glands, secretion from a regulated pathway secretion leads into the saliva. The scientists found only a small amount of hGH in the blood compared to saliva, showing the salivary gland released this protein in the same manner as the pituitary gland normally does.

To deliver the therapeutic gene and the rapamycin-based system, two adenoviruses were injected through the oral duct of the rat's salivary glands. "The system has several important components," Dr. Jianghua Wang, a scientist in Dr. Baum's lab and lead author on the paper, explained. "One virus contains the hGH gene and the second virus supplies genes encoding a DNA-binding domain protein and an activation domain protein." Wang added that the DNA-binding domain protein can attach to a site next to the hGH gene in the first virus. Both the activation and DNA-binding domain proteins are fused to a rapamycin binding site. Upon administration, rapamycin will link both domains leading to the activation of the hGH gene. Without rapamycin, no linkage and therefore no activation can occur.

Rapamycin is generally used to prevent rejection in patients receiving organ transplantation. It suppresses the immune system and currently, other scientists are investigating whether it also works to treat cancer. The application of rapamycin in a gene-regulating system could be complicated by its immunosuppressive actions. When used in patients, it could lead to infections the body is unable to respond to. Several scientists in the field have tried to find a way around this problem and examined analogs of rapamycin. Some of the analogs turned out to be as effective as rapamycin itself, but are without the immunosuppressive effects.

Wang noted that adenoviruses are not ideal vectors for gene transfer in people because they evoke a large immune response. However, they are helpful to study potential gene therapy in animals. Also, they may be useful for short term applications in humans. To help suppress the immune reaction in the rats initiated by the adenovirus, the scientists also injected dexamethasone, a drug commonly used to reduce immune reactions in clinical practice and animal studies.

"Given these results," said Wang, "it may be possible to transfer, and control, genes into the salivary glands to treat a variety of oral conditions." For example, oral ulcers or infections are common, but difficult to treat. With the rapamycin approach, the duration of the treatment could be controlled."

Fungal and bacterial infections, for instance, usually require treatment for about 10-14 days, a period the scientists showed could be achieved with their system. Oral ulcers, which can result for example from irradiation of the mouth or chemotherapy, could benefit from therapy with a single gene. Certain growth factors or cytokines, which are proteins involved in regulating immune responses, have been tested for ulcers. These protein drugs need to be given with frequent injections or require local application with creams. "The large advantage of gene therapy is, that only one gene delivery, instead of multiple protein injections, is needed, making it less expensive and easier to tolerate for patients," explained Wang. Additionally, therapeutic genes injected into the salivary gland could provide a higher local concentration of medication than is possible with general injections into blood or muscle.

"The next step will be the use of rapamycin in the delivery of genes for long-term expression with an adeno-associated virus (AAV)," Baum said. AAV has the advantage over adenovirus in that only a minimal immune response is seen after delivery, meaning this virus has the potential for broader use in humans. Also, the expression of the protein is much longer than can be achieved with adenovirus. "Our group has shown protein expression with an AAV up to one year in mice."

Collaborating with were Drs. Baum and Wang were Drs. Antonis Voutetakis and Changyu Zheng. The study is titled, "Rapamycin control of exocrine protein levels in saliva after adenoviral vector-mediated gene transfer" and it was published online in the journal Gene Therapy on Thursday, January 22, 2004.


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