The Mouth is a Gateway to the Body: Gene Therapy in 21st Century Dental Practice

By Bruce J. Baum, DMD, PhD;
Jane C. Atkinson, DDS;
Lorena Baccaglini, DDS, MS;
Mark E. Berkman;
Jaime S. Brahim, DDS, MS;
Clifford Davis, BS;
Henry E. Lancaster, DMD;
Yitzhak Marmary, DMD;
Anne C. O'Connell, BDS, MS;
Brian C. O'Connell, BDS, PhD;
Songlin Wang, DDS, PhD;
Yanying Xu, DDS, PhD;
Hisako Yamagishi, DDS, PhD;
Philip C. Fox, DDS

Gene therapy may become an integral tool in dental practice early in the 21st century. It, and other biological therapies, are expected to be applied to oral diseases and disorders during the midpractice lifetime of today's dental students. If the applications of oral gene transfer are expanded to systemic diseases, oral health care providers in the future could routinely be "gene therapists" with therapeutic targets well outside the oral cavity.

Article copyright 1998 Journal of the California Dental Association.
Photographs copyright of the authors.

Currently, the practice of dentistry reflects an educational system whose essential elements were structured following the publication of the Gies Report in 1926.1 Dental students today are taught basic biomedical sciences and excellent technical skills in the course of a four-year predoctoral curriculum in preparation for careers as the primary oral health providers in our system of health care delivery. Gies' report brought dental education fully into the university community, where it remains today. However, while many relevant components of this community have experienced significant pedagogical changes as a result of new knowledge and scholarship (e.g., in biology, medicine, materials science, and bioengineering), the impact has been limited within dental education.2,3 The basic formula for producing a dentist -- and, thus, by extension the definition of oral health care in America -- has changed little in the past 50 years with a few notable exceptions.4 Elsewhere in health care, the exponential growth in biology has dramatically altered approaches to clinical care.5 Arguably, the most dramatic example of such change is in the transfer of nucleic acids into cells for the purpose of altering a disease or disorder, so-called gene therapy.6 It is the authors' expectation that gene therapy will also become an integral tool in oral health care, i.e., dental practice, early in the 21st century.

Gene Transfer

Three years ago, two of the authors wrote an essay in the Journal of the American Dental Association7 titled, "The impact of gene therapy on dentistry." In it, they described the fundamental biological principles that underlie gene transfer, as well as methods in place at that time for its clinical application. They also reported several initial attempts, by their own and other laboratories, to apply gene transfer technology for orally relevant problems. Since that time, progress has been considerable, markedly exceeding the authors' most optimistic expectations. Although not yet ready for human use, many potential applications derived from test tube/cell culture experiments have entered the preclinical, animal model stage. The technology is still far from perfect and certainly has substantive problems, but clinical gene therapy is maturing and showing even more, and broader, potential then originally believed.6

One area of application is of particular importance for the oral health community to recognize: the use of genes as pharmaceutical agents. This is a use of clinical gene transfer barely appreciated initially by medicine.6,8 There are certainly many oral-specific, corrective applications of gene transfer possible, e.g., repair of irradiated salivary glands or treatments of oral cancer. However, it is conceivable that somewhat analogous to the conventional medications taken by mouth, a number of systemic gene therapeutics may also follow a route of oral delivery because it is convenient.

How might such future gene therapeutics work? Could dentists routinely perform gene transfer? Many natural gene products (proteins) intended for use in one body locale have their site of synthesis elsewhere. Because of the ease of access, it seems reasonable to consider oral tissues as a target for use in systemic gene therapeutics. And who is better trained to manipulate oral tissues than dentists? In the mouth, there are many sites to which a foreign gene might be delivered advantageously (and presumably be expressed) and from which the gene product might enter the upper gastrointestinal tract or the circulation. These sites include the salivary glands, mucosa, gingival crevice, and tongue. While other health care practitioners can be trained to perform oral procedures,9 this area is within the purview of dentistry. The following describes examples of such applications using salivary glands.

Methods of Gene Transfer

The delivery of genes or other nucleic acids into salivary glands is straightforward, employing the approach clinically used for sialography.10 While sialography is not a procedure commonly performed by most dentists, the manual skills required are within a dentist's repertoire. The procedure involves cannulating the main excretory ducts (Stensen's and Wharton's) of the major salivary glands (parotid and submandibular/sublingual, respectively). A suspension of the gene transfer vector is subsequently retrograde-infused slowly into the gland. Since almost all epithelial cells in a gland abut the duct lumen,11 the gene transfer vector potentially has most cells in the gland as targets.

Table 1 Vectors Used to Transfer Genes in Vivo. Viruses Retrovirus* ** Adenovirus* Adeno-associated virus* ** Lentivirus** Nonviral Methods Cationic liposomess* Macromolecular conjugates * In clinical use at present (for retroviruses, ex civo only).  Al l virus ventors used are replication deficient. ** Can lead to integrated (within the host chromosome), stable gene transfer.

How are genes transferred (Table 1)? A vector is the carrier of the gene to be transferred. There are essentially two ways to transfer genes at present, i.e., two types of vectors: viral and nonviral. Viruses have evolved highly efficient mechanisms to transfer genes. However, they may pose a safety risk, even though all forms currently used are replication-deficient (cannot multiply). For example, viruses can lead to a potent immune reaction that limits their activity and precludes their readministration. Nonviral gene transfer usually involves a formulation of condensed DNA within a lipid capsule. Nonviral methods have a low safety risk but thus far have proven to be markedly less efficient than viruses at gene transfer in vivo. Whatever the vector used, it is recognized in some way (a specific protein receptor or electrostatic charge) by the target cell, internalized, and transported to the nucleus with varying degrees of efficiency. Once in the nucleus, the gene is either integrated into the chromosome (e.g., retrovirus, adeno-associated virus, or lentivirus) or it exists in an epichromosomal (free) location (adenovirus and nonviral methods). There is no single idealized gene transfer vector for all clinical purposes at present, nor is there likely to be one in the near future. There have been major improvements in vector technology recently,6 but the methods used now leave considerable room for improvement.

Salivary glands seem to provide an excellent target site for gene transfer (Table 2), including gene transfer for the production of a secreted protein product. As noted above, these glands are easily accessed, and almost all of the parenchymal cells contact the lumen into which the gene transfer vector is infused. Further, most of the cells in the gland are acinar and thus designed to manufacture considerable protein for export, albeit typically into the mouth. The latter, normal secretory process could be augmented, for example, by transferring a gene into the gland, which would prevent or correct disorders of the upper gastrointestinal tract. Alternatively, since it is known that at least some exocrine proteins can reach the bloodstream,12 glands could be engineered to secrete a gene product for general systemic use. Recent in vivo animal studies have shown that both of these possibilities appear feasible in the near future.

Upper GI Tract Gene Therapeutics

One of saliva's physiological roles is to protect and nurture the tissues of the upper GI tract.13 Despite the fact that saliva contains many beneficial factors, including various antimicrobial, lubricatory, remineralizing and cell growth-promoting proteins, the tissues of the upper GI tract do suffer significant morbidities. Dentists know well the continuing problems of caries, periodontal diseases, aphthous ulcers, and mucosal candidiasis. Despite advances in conventional tools to manage several of these conditions, these oral disorders remain significant. The authors chose to address mucosal candidiasis as a prototypical problem. While it is less common for dentists to treat candidiasis than caries or periodontal diseases, this infection is potentially life-threatening in a medically compromised individual. At the time the authors began their efforts in gene transfer (late 1991), it was widely accepted that gene transfer technology primarily should be applied to clinical conditions with mortal risk, a view no longer widely held.14 The initial strategy was quite simple. Saliva contains naturally potent anticandidal proteins called histatins.15,16 These are believed to help control oral flora and are reported to be reduced in AIDS-immunosuppressed patients,17 thus rendering the individuals susceptible to mucosal candidiasis. This is particularly dangerous if Candida species develop that are resistant to common azole-type, oral antifungal drugs (e.g., fluconazole). The authors reasoned that if they could increase the production of histatins in such patients by gene transfer to the salivary glands, they could prevent or eliminate such infections. Although the specific mechanism of action by which histatins kill Candida is not yet clearly known, it appears to be different from that of azole drugs. Therefore, histatins may be effective against azole-resistant Candida species. Furthermore, the authors reasoned, it would not be necessary for the gene transfer to be permanent. Rather, it seemed likely that a "therapeutic" course of gene expression (10 to 14 days) would be adequate for this purpose. This meant that the authors could probably employ the current generation of adenoviruses as a gene transfer vector.10

They constructed a recombinant adenovirus encoding histatin 3,18 one of the histatin protein family members with demonstrated anticandidal action.16,17 They showed that this virus was able to direct the expression of authentic histatin 3 in a cell culture model and, more importantly, lead to the secretion of histatin 3 in rat saliva after infection of rat salivary glands with the vector. This is particularly noteworthy since rats do not normally make histatin 3. Further, the levels of histatin 3 found in rat saliva were as high as ~1.5 mg/ml, more than tenfold that seen normally in humans.18 Of greatest significance, recombinant histatin 3 was able to kill azole-resistant Candida in vitro.18 The authors are now engaged in a preclinical series of experiments, using immunosuppressed rabbits, to determine if this virus, and the researchers' approach, will be useful under more therapeutic conditions. If these studies are successful, the authors expect to be able to apply this gene transfer treatment to patients within a reasonable time.

The authors have also begun to test various strategies to augment saliva with proteins that could disrupt or limit dental plaque formation.19 Although this has not yet provided the positive results seen with the anti-Candidal strategy described above, they believe it shows considerable promise conceptually. The value of limiting dental plaque to prevent dental disease has been long recognized and is widely applied with conventional pharmacotherapeutics.20 Thus far, these conventional approaches have been only partially successful. A gene transfer approach likely would be entirely complementary to conventional methods and may substantially increase the extent to which dental plaque can be diminished. The authors expect that this approach, or another not yet conceived, will be able one day to reduce dental plaque formation substantially and eliminate caries and periodontal disease.

Systemic Gene Therapeutics

For many years, scientists have suggested that salivary glands are able to secrete in an endocrine (directly to the bloodstream) manner as well as use their common exocrine (external secretion) pathway.12,21 While the data supporting such a view are intriguing, they have not been fully convincing, and the notion of salivary glands as secondary endocrine organs never widely took hold. Gene transfer offered a way to test this possibility in a clear manner. The authors transferred the gene for human 1-antitrypsin (hAT), using an adenoviral vector, into the salivary glands of adult rats.22 HAT is a protein normally made in the liver and secreted into the bloodstream to function as an inhibitor of proteolytic enzymes. It is not normally made by salivary glands. Furthermore, the authors were able to measure hAT without any interference from the rat homologue. Hence, any hAT in the rat bloodstream would have had to come from the salivary glands subsequent to gene transfer. They were able to show this clearly, and their studies demonstrated unequivocally that a mammalian salivary gland was capable of endocrine secretion.

The human disease spectrum includes many conditions that result from a deficiency of a single protein. There are inborn errors of metabolism present from birth, including an endocrinopathy such as growth hormone deficiency or a hematologic (bleeding) disorder such as Factor VIII deficiency (hemophilia A), or conditions that develop later in life, such as diabetes. Although it is currently possible to treat such protein deficiencies via the injection of purified recombinant (genetically engineered) proteins, it is widely recognized that such injections do not represent an ideal therapeutic approach.23 Consequently, there is considerable effort in the biomedical science community to develop novel, more practical, convenient, and cost-effective ways to treat single, circulating-protein deficiency disorders. Gene transfer offers a viable approach to these issues.

The authors hypothesized that it might be possible to use salivary glands as a natural, endogenous slow-release device to secrete therapeutic proteins into the bloodstream after a single gene transfer. For a test case, they studied the expression of human growth hormone (hGH) by salivary glands. As above with hAT, they had excellent measurement tools available to assay protein production. Further, and most importantly, physiological responses to this increase in hGH could be followed preclinically in a rat model because, conveniently, rats are able to respond to the human hormone. They constructed an adenovirus encoding hGH and showed that it directed the production of hGH in, and secretion into the bloodstream by, rat salivary glands.24 The levels of hormone achieved were on average well-above that needed for therapy in humans. In adult rats, these hGH levels were also able to induce several serologic responses clearly indicative of the hormone's systemic activity (increased triglycerides and increased BUN/creatinine ratio).24 Thus, at least in rats for a short-term experiment, the authors' hypothesis was proved.

Another research group, at the University of California at San Francisco, has addressed this hypothesis in a slightly different manner.25 They transferred genes into adult rat salivary glands, however they used nonviral means rather than viruses. The genes transferred include hGH and insulin. Although the serum hormone levels achieved were substantially lower than those seen when viral-mediated gene transfer is employed, they were adequate to induce certain physiological responses. Thus, two separate studies support the use of salivary glands for the secretion of therapeutic proteins into the bloodstream. These results prove a principle. While the approach is not ready for application to patients, it lacks only refinement. The gene transfer field is experiencing explosive growth, and it can reasonably be anticipated that such refinement will come in the near future.

Dentists as Gene Therapists

The title of this section may at first sound strange, however it represents a real possibility in the next 20 or more years. To most people, and most dentists, clinical dentistry is primarily directed at the technical repair of the dentition and supporting structures. In its simplest form, gene transfer can also be viewed as a "technique," albeit one with a seemingly more obvious biological basis than treating caries or periodontitis. If it can be used to prevent or treat oral conditions more successfully than conventional techniques, why not use it?

A major advantage for all such studies is the ready accessibility of oral tissues. The mouth has long been said to serve as a convenient window to the body. The authors are confident that gene transfer, and other biological therapies, will be applied to oral diseases and disorders during the midpractice lifetime of today's dental students. If the applications of oral gene transfer are expanded to systemic diseases, such as those mentioned above, oral health care providers in the future could routinely be "gene therapists" with therapeutic targets well outside the oral cavity. However, for dentists to be able to use such techniques, and maintain their place as key oral health care providers, they must understand modern biology at a practical level. This means that today's schools of dentistry need to give students a biological foundation for their future.3 It also means that active practitioners, especially those who are relatively recent graduates and who have limited facility in biology and biomedicine, would be well-served by acquiring this same foundation.

Authors

Bruce J. Baum, DMD, PhD, is the chief of NIDR's Gene Therapy and Therapeutics Branch (GTTB).
Jane C. Atkinson, DDS, is a senior staff dentist (oral medicine) in the GTTB.
Lorena Baccaglini, DDS, MS, is an oral medicine fellow at NIDR.
Mark E. Berkman is a dental student at Ohio State University and for 1997-98 is a Howard Hughes Medical Institute-NIH research scholar working in the GTTB.
Jaime S. Brahim, DDS, MS, is a senior staff dentist (oral and maxillofacial surgery) at NIDR.
Clifford Davis, BS, is a dental student at UCLA and for 1997-98 is a NIH-Clinical Research Training Program fellow working in the GTTB.
Henry E. Lancaster, DMD, is an oral medicine fellow at NIDR.
Yitzhak Marmary, DMD, was a visiting scientist in the GTTB during 1996-97 and is head of oral and maxillofacial radiology at the Hebrew University-Hadassah School of Dental Medicine, Jerusalem, Israel.
Anne C. O'Connell, BDS, MS, is director of the NIDR Clinical Research Core Facility.
Brian C. O'Connell, BDS, PhD, is the head of the Gene Regulation and Expression Unit, GTTB.
Songlin Wang, DDS, PhD, was a visiting scientist at the GTTB during 1995-97 and is head of the Salivary Gland Disease Center at Capital University, Beijing.
Yanying Xu, DDS, PhD, was a visiting scientist at the GTTB during 1996-97 and is an associate professor in the Department of Oral Medicine, School of Stomatology, Beijing Medical University, Beijing.
Hisako Yamagishi, DDS, PhD, is a visiting fellow from Tokyo Dental College, Tokyo, working in the GTTB.
Philip C. Fox, DDS, is clinical director, NIDR, and chief, Clinical Investigations Section, GTTB.

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To request printed copies of this article, please contact/Bruce J. Baum, TKTK, GTTB/NIDR/NIH, 10 Center Drive, MSC 1190, Bldg. 10, Room 1N113, Bethesda, MD 20892.