The Impact of Gene Therapy on Dentistry

The Impact of Gene Therapy on Dentistry G ene therapy is a term that has appeared with increasing frequency in both the popular and the scientific literature. It is commonly used in reference to any clinical application of the transfer of a foreign gene. As a field of biomedicine, its impact has been nothing less than explosive.' In the last four years, the number of gene therapy clinical protocols, scientific papers, meetBRUCE J. BAUM,

D.M.D., PH.D.;

BRIAN C. O'CONNELL, D.D.S.,

PH.D.

ings and associated com-

therapy trials.5) Because of this focus, it could seem to the casual observer that gene therapy will have little impact on dentistry. Such a conclusion, however, would be shortsighted. This report aims to provide the practicing dentist with a general understanding of what gene therapy entails as well as to provide several examples of how this science is being applied to dental and oral problems today. THE DEVELOPMENT OF A SCIENCE

We are able to accomplish gene transfers today because of the incredibly rapid progress made in molecular biology, the biochemistry of the genome (which is the cell's genetic information). Figure 1 shows a time line depicting some of the seminal advances made in the past 50 years that have led to this ability.6 Each of these events was a key step, but practical progress has been particularly rapid following the discovery of enzymes like reverse transcriptase and various

mercial ventures has increased exponentially. There is even a journal simply titled Human Gene Therapy. Initially, gene therapy was associated with either the correction of inherited genetic disorders or the treatment of lifethreatening conditions.24 Indeed, approved gene therapy protocols for humans include experimental studies with severe combined immune deficiency (SCID) due to a deficiency of adenosine deaminase, cystic fibrosis, AIDS, malignant melanoma, various carcinomas, hypercholesterolemia and brain tumors. (Table 1 lists several currently approved human gene

JADA, Vol. 126, February 1995 179

CORE[ STOBY

N STational Cancero mstithwte University of Mchgan! University of Pennsylvania

University of Michigan

restriction endonucleases. These (and other) enzymes are the tools of today's molecular biologist, permitting the ready manipulation of genetic material. A scientist can, for example, copy a known gene from one organism almost at will and then place the copy into the DNA of a recipient organism, much as a cinematographer would cut and splice a film. This process is what has come to be known as cloning genes. If the gene is placed in a virus, the resulting recombinant virus can then be used to transfer the gene into a mammalian target cell in vitro (in a test tube) or in vivo (in a living organism).2'7 This article will not review 180 JADA, Vol. 126, February 1995

how these and other necessary molecular biological processes occur. The reader interested in investigating these processes further may wish to consult two good introductory texts that can provide a full mechanistic understanding.89 What is essential to recognize is that there are tools, whose use can be readily learned, that enable gene manipulation to occur in the laboratory and, ultimately, in the clinic. GENERAL PRINCIPLES OF GENE TRANSFER

While gene cloning can be quite routine in the test tube, it is a significant leap to transfer a foreign gene correctly into a

laboratory animal, let alone a human. Accordingly, there are some important general principles necessary to discuss here before proceeding further. Figure 2 depicts a typical mammalian gene in a schematic, linear array. A gene actually consists of many modular elements with names like promoters, enhancers, and coding regions. These elements are of two general types. The coding regions are the modules that encode the protein of interest (the gene product). The promoters and enhancers are regulatory elements that play a key role in modulating the extent to which the coding regions will be expressed-in

;E7tCrLeD

COVER STIOY studies have pointed to the value of using tissue-specific promoters-promoters that direct gene expression only in a specific tissue-for tissuetargeted, more stable gene expression."1 METHODS OF GENE TRANSFER

As Table 2 shows, there are two general methods for transferring genes into cells: viral and non-viral (or physical) methods.3'4'2

Figure 1. Time line of major scientific advances leading to gone therapy. Reprinted with permission from Baum BJ. Has modem biology entered the mouth? The clinical Impact of biological research. J Dent Educ 1'991;55:299-303.

other words, how much messenger RNA (mRNA), and thus protein, will be made by a cell. Using the enzymatic tools mentioned above, a researcher can arrange these modules in a desired manner. However, successful gene function requires coding regions and regulatory elements to be present and in the correct alignment. A major technological challenge of gene therapy is designing the correct genetic architecture. Particu-

larly, the promoter chosen for use seems to be critical for obtaining stable, high-level expression

of

a foreign gene.

Most early experiments in gene transfer employed viral promoters that act promiscuously and drive the expression of many mammalian genes.210 Not all promoters are equal, and often different promoters must be tested to determine which of them will yield the most efficient gene expression. Recent

Viral methods. While viruses entail more of a safety risk, they have developed as nature's way of efficiently transferring genes; that is their role in life. In practice, viral methods are more often employed for gene therapy because they are generally more efficient. Many viruses could be used for gene transfer, yet only a few-retroviruses, adenoviruses, adeno-associated viruses and herpesviruses-have been widely employed. Each of these viral gene carriers, or vectors, has advantages and disadvantages. This review will not include a consideration of herpesviruses, as there have been no significant oral applications for them as yet. Some of the main concerns that should be considered in selecting a vector to use for gene transfer are the tissue target; the desired stability of gene expression;

the size of the gene that is to be transferred.

Enhancer f.

:;

;;

SiA

...

00 f:X _; 09

Figure 2. Schematic depiction of a mammalian gene. The must be In the proper alignment for optimal function.

gene consists

of several modular elements that

JADA, Vol. 126, February 1995 181

~COVER STORY-

Aid.-D01't

For example, retroviruses infect only dividing cells, while both adenoviruses and adenoassociated viruses infect both dividing and non-dividing cells.'4'" Retroviruses integrate the foreign DNA into the host cell chromosome and, thus, lead to stable expression. However, the gene insertion is not controlled, and it can occur in such a way as to cause a mutation of the cell.4"' Wild-type, natural adeno-associated viruses also can integrate the new gene into the host cell genome, but the insertion site appears to be consistent and not located in a chromosome region of risk to the cell.'4

Adenoviruses do not integrate the foreign DNA into the

host cell's genome; rather, the foreign DNA exists independently in the nucleus (a so-called episome).4"'2 Every time the cell divides, the number of cells containing the foreign gene decreases and, thus, adenovirus-mediated gene expression TABLE 2

4j

Af4

Accommodates gene of "reasonable" size

[

is quite transient. However, adenoviruses can be grown in high concentrations sufficient to directly infect a target tissue in ViVo.0,1215 Retroviruses have primarily been used with cells temporarily removed from the body (for example, blood cells), infected, and then returned to the host individual (see the

thus produce large numbers of viruses and potential damage to the host. The viruses in use today have been rendered replication-deficient by deleting one or more elements in their genome (as above) that are

discussion of ex vivo gene

"wild" outside world. Non-viral or physical methods. The non-viral or physical methods have greatest appeal as gene delivery systems

transfer below). Finally, viruses cannot accommodate a foreign gene of unlimited size and still produce functional, infective viruses. Adeno-associated viruses, the smallest of the three vectors listed, can accommodate only about half as much foreign DNA as the others.'4

Another key to using viruses is the need to render them replication-deficient, an important safety consideration."2'3

The optimum virus would be a recombinant virus that will infect target cells and transfer a foreign gene but will not be able to grow in the cell and

necessary for replication.16'17 These viruses will grow only in a laboratory setting, not in the

because of their safety.3'4"2'18

Generally, however, they are considered to be much less efficient mechanisms for gene transfer. We mention here the two most promising methods of physical gene transfer: liposomes (essentially bags of lipids containing the DNA) and macromolecular conjugates (the negatively charged DNA mixed with a large positively charged molecule that is linked to a specific cell ligand). These methods are capable of transfer-

I

o

182 JADA, Vol. 126, February 1995

YXsJ

y

Y

Yes

COVER STORY E6

E7

HPV 10/W18

1LZZ-

-dZ IJ4 0ZZ r

I0:-fyS -

p

E7 mRN 1:

AdS

;tC

Gene therapy

;

000 %%Ot

d

E7 - Specific ribozymes

,d f.: A::ft, e:d

U If V, f ~ Transformation to cancer cell type Figure 3. Schematic depiction of adenovirus-transferred ribozymes disrupting HPV-16/18 E7 function In an oral cancer cell. The function of ES could be similarly targeted.

ring fairly large genes, but expression is quite transient.'9 However, there is a much lower potential for an adverse inflammatory or immune reaction against them, compared with viruses (especially adenoviruses4"12). Also, there is no risk of host cell mutation. Hence, repeated administration of genes is feasible with these physical methods. With both physical approaches, the DNA-carrier complex associates with the target cell membrane. For liposomes, it is believed that the lipid bags are taken into the cell and release their packaged DNA once inside.20 The macro-

molecular conjugate method has the unique possibility to target a cell type via a specific protein ligand or antibody that is covalently linked to the positively charged molecule component.'8 These complexes can then be internalized by the specifically targeted cell using a normal intracellular routing pathway. USES OF OCENE TRANSFER

Gene transfer can be used clinically for two purposes: therapy (the term with which it is most associated), the correction of an inherited or acquired defect through the transfer of a foreign gene; and therapeutics,

the use of gene transfer to produce biomolecules with pharmacological functions. Gene therapeutics could be employed for either treatment or prophylactic purposes. Clinical use of gene transfer can be accomplished in either of two ways: - completely in vivo, when the foreign gene is administered to the patient by viral or physical methods, or - ex vivo, when the foreign gene is applied to certain of the patient's cells (for example, lymphocytes or fibroblasts) that are temporarily maintained outside of the body in a sterile environment and, after a suitable period, returned to the JADA, Vol. 126, February 1995 183

COVER STORY body.2'12 Both approaches are actively employed (see Table 1). It is important to recognize that while virtually all currently approved clinical trials of gene transfer involve lifethreatening conditions (such as severe genetic diseases or terminal cancer), the technology is certainly not so restricted. Initial applications to the federal government for the performance of gene transfer to patients were evaluated in an environment of uncertainty and caution appropriate for such a novel endeavor. All applications, for example, were required to have a special

public examination by the National Institutes of Health as well as a private review by the Food and Drug Administration. The public review was conducted by the NIH Recombinant DNA Advisory Committee. Given such an atmosphere of scrutiny, appropriate as it might be, most investigators reasoned that any clinical problem approved for gene transfer research had better be quite severe and without any effective alternative therapy. Because of this view, and because of the initial feeling that gene therapy and therapeutics was a heroic measure,

many acquired and non-lifethreatening conditions were overlooked as potential candidates for management by gene transfer. The mood has changed and, as reported recently in Science,21 there no longer is a federal requirement for the public review-indicating that "gene therapy has come of age."21 It is now widely recognized that gene transfer offers the possibility for ingenious treatments for a host of clinical disorders. Many clinical disciplines that are not normally involved in managing life-threatening conditions are recognizing areas in which gene transfer can be applied. Three groups in dentistry are actively using this science, all for quite different purposes and with different approaches. While the development of gene transfer tools is still in its infancy, this variety of applications provides an impressive spectrum of the possible applications of modern biology to dentistry. Accordingly, we will review each group's work in some detail. APPLYING GENE THERAPY TO ORAL CANCER

Figure 4. Gene transfer to keratinocytes using retroviruses ex vivo. In step A, a biopsy Is performed to obtain keratinocytes that are then grown in vitro (step B). These cells are then Infected with a recombinant retrovirus (step C) that transfers the foreign gene. The keratinocytes are grown In tissue culture further (step D) to form a sheet of cells, which Is returned to the patient as an autologous graft (stop E). Reprinted with permission from Talchman LB. Keratinocyte gene therapy: using genetically altered keratinocytes to deliver new gene products. In: Burgdorf WHC, Katz ST, eds. Dermatology: progress and perspectives. Pearl River, N.Y.: Parthenon; 1993:53-6.

184 JADA, Vol. 126, February 1995

Studies at the University of Texas Dental Branch in Houston, led by Dr. E.J. Shillitoe, use gene therapy for the treatment of oral cancer and precancerous lesions.2223 Oral cancers are fairly prevalent in the United States; each year, about 35,000 new cases are reported, and each year it results in about 13,000 deaths.23 These prevalence rates have not improved in decades, and no new treatment approaches have been developed recently. Shillitoe and colleagues reasoned

5f$i,20_Fd69o/rg41!fi-.;SLX:
0=_.StC0;zfg.1D42Srj5R0ai:;E1t.7dLy0|DXu_;

COVER STORY

GEKNE THERAPY ~~~Tenr of gen_*o a to correc.t en d or acq__m de

.

SS

a}0 .-.S

..

"

'S'0 ''-' SiD 'iV

'X'55

.' " D

lyd

saliva MO s7c

t;

X,' fiC:

ld;

i:

_ _ _ id;;f :00000

id DUCst 0 0

'_Z00f;

der. e bys, bw|t>.ad.bS0..on y

C,...

contaln inVg u

n,

1't

74:.

rX tS

'' .:; ''-!S-:0''"g-'S:X-'-'':- 'SE-7_ ::d fuSt

0;00;$

00

R

Vf;

|

tS

7;

|

"t't:0 i: t0'-$:-

-00 -S0 0S:|- -# - ;: ;F+# t#d 0'.

l0'0f-'D00iSE

T;

1:0: :0 0 0'tAV: 0:10 0- 'f't-'tif 1;; 0':'x'd;:f70001Sa;

$ff,1S,,00 d0 0

\z,,,,X,,, w,40

t'SSStXSst'7.s. XT ff: ?_ .y

'.f: :RX?

X,--y,,:00 0f,;

,,000.00 -S; 0100

W-

*09sbI rWv"

7gl:'s

',

0: Wl:ft::','t

,

''S;

;V00 :: :fE, D:

t'Ve_ ;0

0000 Ws0 6000 i:

reRD;

--

|

ff20 f DS?00

', 0-; 0S0 0 ;-000 .000000f;00 '}

tss _ ':j:

w

7;; 0; t; ; _

:f ffa. f:ff33 S;

:0 0

0 D-0 ^ 0

f::: sa: t:::

C_0

-t

Ls

fwnnue

Figure 5. Schematic depiction of a gene therapy approach to salivary glands damaged by irradiation.

that therapy for cancer is likely to be most effective when directed at targets expressed in cancer cells but lacking in normal healthy cells.22'23 They chose as a target human papillomaviruses, which are present in many oral neoplasms.22'3 HPVs are DNA viruses with an affinity for epithelium. Among the many types of HPVs isolated, types 16 and 18 (often found in oral tumors) are classified as mucosal high-risk types.22'23 HPV-16 and -18 can convert or transform normal keratinocytes in vitro into an immortal, malignantlike phenotype. This process has

repairing

been shown to require the high level of expression of two HPV genes, termed E6 and E7.2223 It seems that the E6/E7 gene products alone are necessary, but not sufficient, to cause tumor development; an additional factor such as trauma or an environmental irritant is needed.2223 Hence, interference in the expression of the HPV genes can prevent cells from causing a tumor-and so it appears that HPV-16 and -18 genes, such as E6 and E7, might be reasonable targets for a gene therapy approach to oral cancer.

Shillitoe and others have

used molecules called ribozymes to disrupt the function of HPV16 and HPV-18 E6/E7 (Figure 3).24 Ribozymes are a specialized class of RNA molecules that can act as enzymes-in other words, catalyze biochemical reactions. Specifically, ribozymes cleave other RNA molecules at very well-defined sites. Ribozymes have been designed to cut the mRNA transcripts of HPV-18 at three distinct sites.2 By damaging the mRNA transcripts of E6/E7 in such a way, it would be impossible for these genes to produce the proteins that interfere with cellular growth control. Indeed, in in vitro studies, these investigators have shown that such ribozymes dramatically cleave the target E6/E7 mRNA.24 Recently, Shillitoe and colleagues have put the DNA encoding for these ribozymes into a replication-deficient adenovirus vector.24 This will enable the ribozyme gene to be delivered in the laboratory to target cancer cells. Further, this also will permit study of the ribozymes' effectiveness in cleaving E6/E7 mRNA within cells-a significant step beyond the test tube experiments. If this novel approach continues to prove successful, one can envision its use not only for primary tumors, but also perhaps for targeting residual disease after conventional treatment and for removing dysplastic cells that are not yet fully transformed into malignant cells.224 GENE TRANSFER TO ORALIMUCOSAL KERATINOCYTES

A second area of highly productive study has examined the

possibility of using genetically JADA, Vol. 126, February 1995 185

OVER STIOY-

Figure 6. Schematic depiction of the use of gone therapeutics with salivary glands to deliver blopharmaceuticals to the oral cavity and the upper gastrointestinal tract.

altered keratinocytes to deliver various new gene products. These studies, led by Dr. L.B. Taichman of the Department of Oral Biology and Pathology at the State University of New York at Stony Brook, have focused on the technology of reengineering keratinocytes ex vivo using retroviruses.2 As noted earlier, this type of virus will introduce a gene into the host cell genome permanently. Taichman and colleagues have coupled this gene transfer tool with a useful property of keratinocytes: they can be grown in vitro in sheets of cells 186 JADA, Vol. 126, February 1995

that then can be returned to the donor (patient) as a stable autograft, a technique developed for treating burn patients. The process used by these investigators is shown in Figure 4. This strategy has been used to transfer foreign genes into both epidermal2 and oral26

keratinocytes. While no specific oral disease has yet been targeted by Taichman and others, their initial results show considerable promise for conditions in the oral cavity and for systemic conditions. Their general approach is one of gene

therapeutics, using the foreign gene products as pharmaceutical agents. Keratinocytes have high rates of protein synthesis and do in fact secrete an array of proteins outside of the cell.27 Importantly, the studies by these investigators have moved into a whole-animal laboratory model.28 Taichman and colleagues have, for example, engineered human skin keratinocytes to produce a secretory protein, apolipoprotein E, which is important in regulating cholesterol and lipoprotein metabolism. Epithelial sheets derived from these cells have been grafted to athymic mice, immunocompromised animals whose systems are incapable of rejecting the graft. These cells were able to secrete considerable levels of apolipoprotein E into the mice's circulation. This same approach can be used for many other purposes, such as to replace absent or inadequate levels of a systemic hormone or to deliver a locally active agent in the oral cavity for the treatment of mucosal and other disorders. Additional possible uses of keratinocyte gene transfer include correcting genetic disorders of the epidermis, like xeroderma pigmentosum, and acting as metabolic sinks for trapping high levels of toxic products associated with inborn metabolic errors or even derived from bacterial infections.29 This strategy appears to be quite flexible and, with time, undoubtedly will have considerable clinical impact. GENE TRANSFER TO SALIVARY GLANDS

Our own laboratory at the National Institute of Dental Research in Bethesda, Md., has

COVER STORY been actively studying the application of gene transfer technology to salivary glands. These tissues present an inviting target for in vivo gene transfer because of their anatomic location and ease of access.'5

Our initial studies examined the feasibility of using replication-deficient recombinant adenovirus vectors to transfer foreign genes into rat salivary glands in vivo. Preliminary studies had shown that such vectors could infect salivary epithelial cells in vitro. We administered the vectors to cannulated glands through the duct orifice via retrograde injection.15 We found that all major rat salivary glands (parotid, submandibular and sublingual) could be infected by adenoviruses.'5 Histological examinations showed that both acinar and ductal epithelial cells could act as recipients for gene transfer. In addition, we were able to demonstrate that these same vectors were suitable for transferring exogenous genes to human labial glands ex vivo. Gene therapy. After these early studies, we began two lines of clinical application: one directed at gene therapy, the other at gene therapeutics. The former studies are aimed at the repair of salivary glands whose acinar cells have been irreversibly damaged (Figure 5). Salivary glands consist of two general parts: acinar cells that secrete salt and are waterpermeable and ductal cells that absorb salt and are waterimpermeable.30 Two relatively common situations that result in acinar cell damage, and thus the inability of glands to secrete fluid, are therapeutic irradiation (for head and neck tumors

with salivary glands in the radiation field) and Sjogren's syndrome (an autoimmune exocrinopathy).3' Our project aims to convert surviving ductal cells into acinarlike cells that secrete salt and fluid. This is an example of what is termed

for human ac-antitrypsin, a secretory protein normally made by the liver and secreted into the circulation.'5 In fact, we observed high levels of this foreign protein secreted in rat saliva after adenovirusmediated gene transfer.'5

Our first experimental proj-

Our fist experimental project has explored the possibility of treating, or even preventing, the severe mucosal candidiasis that accompanies mmunosuppression due to infection or to therapeutic treatment organ engineering, changing

the basic function of a cell type. These studies are still at the test-tube level; our present efforts are directed at creating a recombinant adenovirus that contains a water channel gene. Gene therapeutics. The second line of our experimentation, gene therapeutics, has made considerably more progress. The approach here is to use normally functioning salivary glands to deliver, in their secretions, biopharmaceuticals encoded by transferred foreign genes (Figure 6). We have demonstrated the feasibility of such an approach by transferring into rat submandibular glands in vivo the gene

ect has explored the possibility

of treating, or even preventing, the severe mucosal (oralpharyngeal-esophageal) candidiasis that accompanies immunosuppression due to infection (as in AIDS) or to therapeutic treatment (for example, as a result of transplants). We hypothesized that overexpression of a naturally occurring salivary anticandidal peptide (histatin) might be useful for such an approach.32 In collaboration with Drs. F. Oppenheim and T. Xu of Boston University, we have created a recombinant adenovirus containing a histatin gene, and we have transferred that gene into human and rat salivary cells in vitro.32 Further, we also have successfully transferred the gene into rat salivary glands in vivo. We are now beginning studies to test the functional ability of the histatin protein produced after gene transfer to kill candida and prevent fungal infections in animal models. A second example of this experimental tack is just beginning in collaboration with Drs. R. Genco and A. Sharma of the SUNY-Buffalo Periodontal Disease Research Center. These investigators have isolated the gene for fimbrillin, a surface protein of the important periodontopathic bacterium Porphyromonas gingivalis. We are constructing a recombinant adenovirus containing this gene and will transfer it into salivary JADA, Vol. 126, February 1995 187

COVER STOBYglands. We anticipate that the soluble protein product of this gene will be secreted locally around the gland as well as in saliva. We expect the locally secreted fimbrillin to elicit an immune response leading to the production of a specific secretory IgA. This secretory IgA would be secreted in saliva and neutralize P. gingivalis, inhibiting its ability to participate in plaque formation. Similarly, secreted fimbrillin in saliva could bind to pellicle components, blocking the attachment of P. gingivalis. This strategy, or a similar one, although in its infancy, could prove to be a very useful new tool against periodontal diseases, especially in populations at high risk. THE FUTURE OF GENE TRANSFER AND ITS IMPACT ON DENTISTRY

Using gene transfer for therapy and therapeutics is now being accepted as feasible by the general biomedical community. It is no longer considered to be an esoteric exercise without real practical application. Because its potential is so vast, growth in this field has been incredibly rapid. Today, it is not surprising to find articles on

Dr. Baum Is clinical director of the National Institute of Dental Research and chiof of the Clinical Investigations and Patient Care Branch of NIDR, National Institutes of Health, Bethesda, Md.

20892-1190. Address reprint requests to Dr. Baum.

Dr. 0 Connell Isa senior stafffallow In the Clinical

Investigations and Patient Care Branch of the National institute of Dental Research, National Institutes of HealthX Bethesda, Md.

188 JADA, Vol. 126, February 1995

gene transfer in mainstream general medical journals, such as The New England Journal of Medicine, a journal read widely by practicing physicians. Indeed, NEJM just launched a series of articles33 to educate practitioners about the tools and language of molecular biology so that the clinical impact of this field will be recognized

Using oene transferfor therapy and therapeutics is no longer considered to be an esoteric exercise without real practical application. and exploited. However, gene transfer as it stands today is not a panacea for all of society's clinical ills. It is a field in a primitive stage of its development, somewhat analogous to the status of antibiotics after World War II and perhaps even less advanced than that. Science and medicine-all medicine, including dental medicine-are just beginning to sense the possibilities that gene transfer can provide by way of novel, specific and highly corrective treatments. The tools we have available to us today are very crude compared to what is on the horizon. For example, the vectors available for in vivo gene transfer-especially the viral vectors-have real (but likely correctable) problems.34"2 These include the transient and inflammatory nature of adeno-

virus use and the low titers and mutagenic potential (safety concerns) of retroviruses. A burgeoning part of the biotechnology industry has recognized these shortcomings and, not surprisingly, is working hard to develop better, more efficientindeed, user-friendly-viral and other types of vectors.4 Why? Because of the field's tremendous commercial potential. Such treatment may seem heroic to many, but the mechanics of gene transfer, while technically demanding, are rather mundane. The challenge to the research investigator is in the idea, not in the technical steps. Scientific progress will only hasten the process. CONCLUSION

We have briefly presented the work on gene transfer of three research groups within dentistry. These initial applications, like the crude state of our vectors, undoubtedly will be viewed as simple or naive in 10 years. We must realize that they are merely building blocks, a start, for the future. However, and importantly, these early efforts show that dentistry will be broadly affected by the impact of molecular biology, so the issue is not whether but when. Initially, it is likely that gene transfer approaches will not be used for any routine care, but rather for patients whose conditions are refractory to more conventional treatment, such as people at especially high risk for caries or periodontal diseases. Thereafter, we suspect gene transfer will become more common. It may well be possible to use these methods to disrupt plaque formation efficiently through some transferred gene, as suggested earlier. If the

COVER STORY transfer was stable, it obviously would have very far-reaching effects on dental health and dental practice. Conditions such as oral ulcers, periodontal bone loss and delayed tooth eruption all can be placed in scenarios in which gene transfer could dramatically change the current standards of care. What is important for dentists to recognize, and what we hope this review will convey, is the notion that biology is changing rapidly and giving the clinical scientist-and, before long, the clinician-options for care that 10, 15 or 20 years ago were in the realm of science fiction. Today's dentists certainly must be able to provide care for their patients that meets the current standards of practice. But because science is acquiring the power to change those standards in unconventional and dramatic ways, our profession must keep a close eye on biology if we are to continue as an essential part of mainstream health care. Regardless of whether dentistry embraces the new molecular medicine exemplified by gene transfer procedures, this field certainly will change the nature of the practice of dentistry within the next 20 years. . The authors especially wish to thank Drs. E.J. Shillitoe and L.B. Taichman for their help in preparing portions of this article that refer to their studies and for sharing with us

various findings prior to publication. We also appreciate the excellent assistance of Kathi Barnes in preparing Figures 2, 3, 5 and 6. Finally, we appreciate the encouragement, support and constructive criticism we received about our work from our many colleagues and collaborators. 1. Morgan RA, Anderson WF. Human gene therapy. Annu Rev Biochem 1993;62:191-217. 2. Culver KW, Anderson WF, Blaese RM. Lymphocyte gene therapy. Hum Gene Ther 1991;2:107-9. 3. Roemer K, Friedmann T. Concepts and strategies for human gene therapy. Eur J Biochem 1992;208:211-25. 4. Meager A, Griffiths E. Human somatic gene therapy. Trends Biotechnol 1994;12:10813. 5. Currently approved human gene transfer studies. Hum Gene Ther 1994;5:1067-74.

6. Baum BJ. Has modern biology entered the mouth? The clinical impact of biological research. J Dent Educ 1991;55:299-303. 7. Zabner J, Couture LA, Gregory RJ, Graham SM, Smith AE, Welsh MJ. Adenovirus-mediated gene transfer transiently corrects the chloride transport defect in nasal epithelia of patients with cystic fibrosis. Cell

1993;75:207-16.

8. Watson JD, Gilman M, Witkowski J, Zoller M. Recombinant DNA. 2nd ed. New York: Scientific American Books;1992. 9. Lewin B. Genes V. Oxford: Oxford

University Press;1993. 10. Rosenfeld MA, Yoshimura K, Trapnell BC, et al. In vivo transfer of the human cystic

fibrosis transmembrane conductance regulator gene to the airway epithelium. Cell 1992:68:143-55. 11. Dai Y, Roman M, Naviaux RK, Verma IM. Gene therapy via primary myoblasts: long-term expression of factor IX protein following transplantation in vivo. Proc Natl Acad Sci USA 1992;89:10892-5. 12. Mulligan RC. The basic science of gene therapy. Science 1993;260:926-32. 13. Cormetta K, Morgan RA, Anderson WF. Safety issues related to retroviral-mediated gene transfer in humans. Hum Gene Ther

1991;2:5-14. 14. Kotin RM. Prospects for the use of adeno-associated virus as a vector for human gene therapy. Hum Gene Ther 1994;5:793801. 15. Mastrangeli A, O'Connell B, Aladib W, Fox PC, Baum BJ, Crystal RG. Direct in vivo adenovirus-mediated gene transfer to salivary glands. Am J Physiol 1994;266:1146-55. 16. Berkner KL. Expression of heterologous sequences in adenoviral vectors. Curr Top Microbiol Immunol 1992;158:39-66.

17. Taichman LB. Keratinocyte gene therapy: using genetically altered keratinocytes to deliver new gene products. In: Burgdorf WHC, Katz SI, eds. Dermatology: progress and perspectives. Pearl River, N.Y.: Parthenon;1993:53-6. 18. Wu GY, Wu CH. Delivery systems for gene therapy. Biotherapy 1991;3:87-95. 19. Zhu N, Liggitt D, Liu Y, Debs R. Systemic gene expression after intravenous DNA delivery into adult mice. Science 1993;261:209-11. 20. Wolff JA, ed. Gene therapeutics: methods and applications of direct gene transfer. Boston: Birkhauser;1994. 21. Marshall E. One less hoop for gene therapy. Science 1994;265:599. 22. Shillitoe EJ, Lapeyre J-N, Adler-Storthz K Gene therapy: its potential in the management of oral cancer. Oral Oncol Eur J Cancer 1994;30B:143-54. 23. Shillitoe EJ, Kamath P, Chen Z. Papilloma viruses as targets for cancer gene therapy. Cancer Gene Therapy 1994;in press. 24. Shillitoe EJ, Chen Z, Kamath P, Zhang S. Anti-papilloma virus ribozymes for gene therapy of oral cancer. Head Neck 1994;16:506. 25. Garlick JA, Katz AB, Fenjvec ES, Taichman LB. Retrovirus mediated transduction of cultured epidermal keratinocytes. J Invest Dermatol 1991;97:824-9. 26. Garlick JA, Taichman LB. The fate of genetically marked human oral keratinocytes in vitro. Arch Oral Biol 1993;38:903-10. 27. Katz AB, Taichman LB. The epidermis as a secretory tissue: an in vitro tissue model to study keratinocyte secretion. J Invest Dermatol 1994;102:55-60. 28. Fenjves ES, Smith J, Zaradic S, Taichman LB. Systemic delivery of secreted protein by grafts of epidermal keratinocytes: prospects for keratinocyte gene therapy. Hum Gene Ther 1994;5:1243-50. 29. Carroll JM, Fenjves ES, Garlick JA, Taichman LB. Keratinocytes as a target for gene therapy. In: Darmon M, Blumenberg M, eds. Molecular biology of the skin: the keratinocyte. San Diego: Academic Press;1993:269-84. 30. Baum BJ. Principles of saliva secretion. Ann NY Acad Sci 1993;694:17-21. 31. Baum BJ. Age-related vulnerability. Otolaryngol Head Neck Surg 1992;106:730-2. 32. O'Connell BC, Mastrangeli A, Oppenheim FG, Crystal RG, Baum BJ. Adenovirus mediated gene transfer of histatin 3 to rat salivary glands in vivo. J Dent Res 1993;73:310. 33. Rosenthal N. Molecular medicine. Tools of the trade-recombinant DNA. N Engl J

Med 1994;331:315-7.

JADA, Vol. 126, February 1995 189