The overall perspective presented in that 1995 report was that the impact of gene therapy on dentistry would be no different from its anticipated impact on all areas of science-based clinical practice—that is, pervasive and significant. The question was not whether, but rather when, the impact would be felt. Six years later, we now look back at what was written then and, with consideration of research progress over the intervening time, reassess the validity of the 1995 conclusion.

	GENERAL PROGRESS IN THE FIELD

TOP ABSTRACT GENERAL PROGRESS IN THE... GENE TRANSFER VECTORS AREAS OF IMPACT ON... CONCLUSION REFERENCES

 
Even using relatively simple measures, it seems quite reasonable to state that since 1995, the advances made in gene therapy have been extraordinary. For example, in 1995 there was one major research journal devoted to this discipline, and now there are at least four; Human Gene Therapy has been joined by Gene Therapy, Molecular Therapy and The Journal of Gene Medicine. In 1995 there were no national or international scholarly societies of scientists working in gene therapy, and now there are two major such societies, in the United States and Europe. Most importantly, in 1995 there was no single example of a clinical gene therapy treatment that was successful. In 2000, the first report of a fully successful gene therapy treatment—a French study involving a severe combined immunodeficiency in young children—was published.² Additionally, numerous studies have reported significant clinical benefits gained by augmenting more traditional therapies, and many investigators have shown "proof of concept"—that is, successful gene transfer in animal disease models. Most tissues and cell types have been targets for gene transfer experiments.

Nonetheless, there remain significant problems for this nascent field that are rate-limiting for clinical success.^3–5 Some recently have become very apparent with the first, and thus far only, death as a result of a clinical gene transfer procedure, which occurred in 1999.⁶ The major problems hindering gene transfer applications are biological, resulting from limitations in our knowledge of the essential components involved in the process. These include inadequate understanding of virus biology, recombinant vector interactions with different cell types and the targeted diseases.

Viral vectors are natural infectious agents for transferring genetic information and they are quite efficient.

For example, rapid progress in molecular biological technology has allowed researchers to make manipulations of genes easily. However, scientists may be unable to predict with precision the biological consequences of each manipulation. There is also much to be learned about host responses—immune and direct cell toxicities—to vectors, and an uneven appreciation of pathogenic mechanisms (that is, the molecular targets for gene transfer). For single-gene disorders, for which gene transfer was initially conceived, the pathophysiology may be reasonably well-understood. However, many diseases are of a much more complex etiology involving either multiple genes or interactions between genes and the environment (including both dental caries and periodontal diseases).⁷ Our understanding of these more complex conditions is less detailed. Not surprisingly, advances in applying gene transfer generally have been, and likely will continue to be, more rapid with tissues and diseases whose biology is better understood.

	GENE TRANSFER VECTORS

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As described in 1995,¹ there are two general ways to transfer genes (Table): viral and nonviral. Viral vectors are natural infectious agents for transferring genetic information. They are quite efficient, and at present they generally provide more pre-clinical and clinical utility than nonviral vectors, although that gap is diminishing. The principal viral vectors in clinical use today are based on modified adenoviruses, retroviruses and adeno-associated viruses. In addition, substantial progress has been made with lentiviruses, herpes viruses and hybrid viruses (which combine the positive features of more than one virus).

	AREAS OF IMPACT ON DENTISTRY

TOP ABSTRACT GENERAL PROGRESS IN THE... GENE TRANSFER VECTORS AREAS OF IMPACT ON... CONCLUSION REFERENCES

 
Bone repair. An area of real clinical importance to dentistry, with considerable research progress, uses gene therapy to repair bony lesions. Studies by researchers at the University of Michigan School of Dentistry have used ex vivo methods to transfer genes encoding bone morphogenetic proteins, or BMPs.^8,9 BMPs are agents well-established in induction of both orthotopic and ectopic bone formation. In ex vivo studies, researchers accomplish the actual gene transfer in a tissue culture environment and then place the transduced cells, carrying the foreign genes, back into the host. In animal models, the Michigan research group has shown that several different cell types—such as nonosteogenic fibroblasts (from human gingiva and dental pulp) and myoblasts, as well as osteoblasts—can express the BMP-7 gene after being infected with an adenoviral vector. These cells then are able to differentiate into bone-forming cells when placed in an osseous defect in vivo.

Other recent studies conducted by researchers at the Hebrew University-Hadassah Faculty of Dental Medicine in Jerusalem have used mesenchymal stem cell–mediated gene therapy for bone regeneration.^10,11 Genetically engineered mesenchymal stem cells expressing BMP-2 induced increased formation of new blood vessels as well as new bone. These studies also showed that the genetically engineered stem cells were able to engraft, differentiate and display regulatory behaviors. A recent investigation by Alden and colleagues¹² at the University of Virginia Medical School demonstrated that it is possible to directly deliver the BMP-2 gene in vivo to tissue via an adenoviral vector (vs. using ex vivo cellular re-engineering) and thus achieve healing of mandibular osseous defects (Figure 1).

View larger version (13K): [in this window] [in a new window]   Figure 1. Schematic depiction of the correction of a mandibular osseous defect using in vivo gene transfer. Transfer of the bone morphogenetic protein, or BMP,-2 gene is accomplished using a recombinant adenovirus, or Ad-BMP. The vector is administered directly to the osseous defect, and the expression of BMP-2 leads to repair of the osseous defect after three months. Based on the work of Alden and colleagues.>12

It is likely that other genes soon will be available to facilitate localized regeneration of bone for periodontal and oral surgical applications.

This general strategy tries to enhance a natural reparative response by supplementing the regenerative site with therapeutic proteins. It clearly is possible to manipulate a variety of cell types, by different methods, to express BMP genes and, thereafter, for these transduced cells to mediate bone regeneration. Additionally, it is likely that other genes soon will be available to facilitate localized regeneration of bone for periodontal and oral surgical applications. For example, a novel strategy, recently reported by another group at the University of Michigan,¹³ involved transfer of the platelet-derived growth factor gene to periodontal cells and resulted in DNA synthesis and cellular proliferation.

Gene transfer to salivary glands. Salivary glands are excellent target sites for gene transfer. They are capable of producing large amounts of proteins, and are a site where gene transfer can be readily accomplished in a minimally invasive manner (by means of intraductal cannulation). Human salivary glands also are encapsulated, a circumstance likely to minimize the undesirable access of administered vectors and transgenes to other tissues.

Our original goal in developing gene transfer with salivary glands was to provide novel and effective therapies for patients who suffer from irreversible salivary gland dysfunction resulting from either irradiation for head and neck cancers or the autoimmune damage occurring with Sjögren’s syndrome.¹ We later developed two additional clinical goals for salivary gland gene transfer, both involving the use of genes as pharmaceuticals (gene therapeutics). Salivary glands are by nature a secretory tissue and certainly are a logical site for local (oral, pharyngeal and esophageal) applications of gene therapeutics requiring the exocrine secretion of transgene products in saliva. Additionally, we suggested that salivary glands could be used for gene therapeutic applications with systemic single-protein deficiency disorders.¹⁴ Compared with salivary glands, other tissue sites have both advantages and disadvantages for systemic gene therapeutics. However, it is unlikely that any single tissue is ideal for all possible uses, and salivary glands may be useful for some specific purposes.

Since 1995,¹ there have been many studies reporting gene transfer to salivary glands. In addition to our own laboratory at NIDCR, groups at Genteric Inc. in California, Mount Sinai School of Medicine in New York and the Medical University of South Carolina, Charleston, as well as researchers participating in a collaborative effort between the University of Alabama School of Medicine, Birmingham, and the University of Regensburg in Germany, have published reports of successful gene transfer to salivary glands. A variety of genes have been transferred in these studies, including genes encoding hormones (growth hormone, insulin),^15,16 an antimicrobial agent (histatin 3, or H3),¹⁷ membrane proteins (aquaporin-1 and aquaporin-5),^18,19 a transcription factor (E2F-1),²⁰ protease inhibitors (1-antitrypsin and kallistatin),^14,21 a protein affecting apoptosis (Fas ligand)²² and several nonmammalian "reporter proteins" (ß-galactosidase, chloramphenicol transferase and luciferase).^23–25

The gene transfer application of immune modulation appears to have potential for treatment of autoimmune diseases such as Sjögren’s syndrome.

For repair of damaged salivary glands, our initial approach was to insert a gene encoding a water channel protein, aquaporin-1, or AQP1, into radiation-surviving (primarily ductal) salivary cells to convert these nonsecretory cells into a secretory phenotype.¹⁸ An adenovirus-encoding human AQP1, termed "AdhAQP1," was administered to hypofunctional rat submandibular glands that had been irradiated four months earlier with a dose of 21 gray. Three days after gene transfer, the treated glands were secreting saliva at flow rates indistinguishable from those of nonirradiated control glands.¹⁸ After these encouraging results, we tested the safety and efficacy of AdhAQP1 in rhesus monkeys.²⁶ At 20 weeks after irradiation, the animals received either AdhAQP1 or a control virus. The animals tolerated the single doses of AdhAQP1 well, but the salivary results were inconsistent. AdhAQP1 enhanced salivary secretion only modestly in some animals. We are not sure why the results were not as encouraging in the monkey model as in the rat model. One possible technical explanation is that in the monkey, the glands were underfilled by the vector/infusate volume used. A subsequent study in mice showed that maximal transgene expression occurred when glands were somewhat overfilled.²⁷ We are conducting additional animal studies to decide if it is useful to pursue the AQP1 gene transfer strategy clinically.

The second clinical goal was to use gene transfer to deliver a gene product locally to treat disorders of the mouth and upper gastrointestinal tract. The clinical condition we addressed initially was azole-resistant candidiasis in immunosuppressed patients. This leads to considerable morbidity and mortality, and there are no well-accepted alternative medications. We hypothesized that the overexpression of a naturally occurring salivary anticandidal polypeptide, histatin 3, or H3, could kill azole-resistant Candida species and manage the resulting mucosal candidiasis.¹⁷ We used a recombinant adenoviral vector encoding H3 to infect rat salivary glands. The glands began to produce copious amounts of H3 polypeptide, and the recombinant H3 was able to kill both azole-sensitive and azole-resistant Candida species with approximately equal efficiency.¹⁷

Our third clinical goal for gene transfer to salivary glands was to correct systemic single-protein disorders (Figure 2). Since 1995, we have demonstrated in rats that transgene products could be secreted from salivary glands into the bloodstream—in other words, endocrine secretion.¹⁴ When an adenovirus encoding human growth hormone, or hGH, was administered to adult rat salivary glands, serum hGH increased from background levels to ~16 nanograms per milliliter, well above the level considered therapeutic in humans, ~5 ng/mL.¹⁵ Importantly, these hGH levels induced serological responses indicative of systemic activity (increased insulinlike growth factor 1, triglycerides and blood urea nitrogen:creatinine ratio). Subsequently, we showed that to be efficient clinically, the direction (whether endocrine or exocrine) of transgene product secretion must be controllable.²⁸ Very recently, our research group at NIDCR ²⁹ reported that administration of the immunomodulatory drug hydroxychloroquine dramatically increases the efficiency of hGH endocrine secretion from rat submandibular glands.

View larger version (34K): [in this window] [in a new window]   Figure 2. Systemic gene therapeutics as an application of gene transfer to salivary glands. By means of an appropriate gene transfer vector—such as a recombinant adenovirus or an adeno-associated virus—a gene encoding a protein (a biopharmaceutical, or bio) is delivered to a healthy, functioning salivary gland via intraductal cannulation. The gene product, bio, is secreted across the basolateral membranes of the salivary epithelial cells into the bloodstream, rather than being secreted across the apical (lumenal) membrane into saliva. This is modeled after studies in the authors’ laboratories as described in the text and several references.14,15,28,29

SS is characterized by a focal mononuclear cell infiltrate in the salivary and lacrimal glands.³⁰ This chronic inflammation and the consequent secretion of proinflammatory cytokines are associated with dry mouth (xerostomia, often with a marked increase in dental caries) and dry eyes (keratoconjunctivitis sicca). SS presents in a primary and secondary form, existing without (primary) or with (secondary) another autoimmune disease, such as rheumatoid arthritis. The cellular infiltrates in SS consist mainly of CD4⁺ cells, which show divergence into T helper 1 and T helper 2, or Th1 and Th2, subsets. Th1 cells are associated with cell-mediated immunity, producing cytokines such as interleukin 2, or IL-2; interferon , or INF-; and tumor necrosis factor , or TNF-. Th2 cells produce IL-4, IL-6 and IL-10 and are associated with humoral immune responses. The Th1 cell subset induces inflammation, and Th1-related cytokines are likely to stimulate cytotoxic T cell processes within the gland. Th2-related cytokines tend to cause a decrease of inflammation.³² This situation gives rise to a general paradigm that has emerged for developing novel protein-based and, more recently, gene-based treatments for several autoimmune diseases, including SS. This strategy, which we use, is that biological factors that enhance Th2 functions and suppress Th1 cells likely will be efficacious for therapy (Figure 3).³³

View larger version (30K): [in this window] [in a new window]   Figure 3. General strategy adopted for gene therapy management of salivary gland pathology in Sjögren’s syndrome. Salivary glands in patients with Sjögren’s syndrome have a lymphocytic infiltrate with predominantly CD4+ cells, reflecting a predominance of the inflammation promoting T helper 1, or Th1, subset of cells. This leads to the local production of cytokines that can lead to tissue damage (tumor necrosis factor, or TNF, -; interleukin, or IL,-2; and interferon, or IFN, -), resulting in a derangement of the secretory acinar cells and their eventual loss. In this simplified and schematic representation, we depict our hypothesis that administration of a recombinant adeno-associated virus encoding human interleukin-10 (rAAVhIL10) to such tissue will restore an immunological balance in the salivary glands and result in a dissipation of the lymphocytic infiltrate. Consequently, we anticipate that the salivary glands will be able to undergo an endogenous repair process. This general strategy has proved useful for the correction of other autoimmune diseases in animal models, as described in the text and several references.22,31–33,39

One positive reason to consider immunomodulation of salivary glands in patients with SS using gene transfer is that the intervention uses a targeted, local delivery with selective tissue expression—that is, the encapsulated salivary gland.³⁴ However, a major concern about using gene transfer vectors with patients who have autoimmune disease is the possibility of an immunological reaction to the vector. In response to this concern, many investigators developing gene transfer–based treatments for autoimmune diseases, including SS, now are using recombinant adeno-associated virus, or AAV, serotype 2, as the vector of choice. AAV vectors are much less immunogenic than adenoviral vectors.

Our initial studies have focused on transferring the gene for human, or h, IL-10 using a recombinant AAV2 vector.³⁵ hIL-10 has a broad spectrum of biological effects. Among these is the inhibition of antigen-specific T-cell proliferation, of cytokine production by Th1-like cells and of macrophage-dependent antigen presentation. Conversely, it seems unlikely that overexpression of hIL-10 will cause severe disturbances in a host’s protective immune responses. hIL-10 immunomodulatory therapy has been tried and shown to be useful in preclinical models of other autoimmune diseases, including rheumatoid arthritis.³⁶ We recently began preclinical studies using a mouse with nonobese diabetes, or NOD, as a model of SS.³⁷ We hypothesized that hIL-10 gene transfer would lead to a shift in the Th1/Th2 lymphocyte subset distribution in salivary glands of the NOD mouse, resulting in an alteration in the cytokines expressed. Ultimately, we hope that this hypothesized change will reduce the glandular lymphocytic infiltrate and lead to an increase in saliva production (Figure 3).

Pain. Managing or eliminating pain is a major part of dental practice.³⁸ The use of gene transfer technology offers a potentially novel approach to manipulate specific, localized biochemical pathways involved in pain generation.³⁹ Gene transfer may be particularly useful for managing chronic and intractable pain.^40,41 Several studies in animal models, including studies from the NIDCR⁴⁰ and University of Pittsburgh School of Medicine,⁴² have shown that viral-mediated transfer of genes encoding opiate peptides to peripheral and central neurons can lead to antinociceptive effects. There also is a recent report from Okayama University Dental School in Japan showing the feasibility of direct gene delivery to the articular surface of the temporomandibular joint.⁴³ While considerably more research is needed before gene transfer can be tested clinically as a strategy for chronic pain management, the results of these recent studies suggest real promise.

DNA vaccinations. For many years, dental scientists have tried to use classical vaccination technology to eradicate dental caries or periodontal diseases, thus far achieving mixed success. In the last decade, gene transfer research has led to a novel way to achieve vaccination: directly delivering DNA in a plasmid vs. the traditional administration of a purified protein or an attenuated microbe.⁴⁴ The 1995 review¹ predicted that this approach might be useful in addressing dental diseases.

This prediction was demonstrated in an animal study published in 1999 by Kawabata and colleagues,⁴⁵ of Osaka University’s Faculty of Dentistry in Japan. They achieved a targeted salivary gland immunization using plasmid DNA encoding the Porphyromonas gingivalis fimbrial gene. This gene led to the production of fimbrial protein locally in the salivary gland tissue of mice, with the consequent production of specific salivary immunoglobulin A, or IgA, and immunoglobulin G, or IgG, antibodies and serum IgG antibodies.⁴⁵ Additionally, they observed the generation of antigen-specific cytotoxic T lymphocytes in immunized mouse spleen cells. Although it was not shown in their report, one might expect that the secretory IgA secreted in saliva could neutralize P. gingivalis and limit its ability to participate in plaque formation. Furthermore, any secreted fimbrial protein in saliva could bind to pellicle components and also inhibit the attachment of P. gingivalis to the developing plaque. Although applications of DNA vaccination are in the earliest stages of use with oropharyngeal tissues, it seems reasonable to suggest that these approaches will play a role in future strategies for preventing periodontal diseases and dental caries.

Gene transfer to keratinocytes. As noted in 1995,¹ there are several features that make epidermal and mucosal keratinocytes attractive for treating local tissue disorders and as systemic gene therapeutics.^46,47 First, monitoring is easy because the genetically modified tissue is accessible. Second, preclinical assessment is accurate since culture models are established. Third, expression of therapeutic genes can be achieved with use of topically applied agents. Fourth, procedures for transplanting keratinocyte sheets already are established because of their applications for burn patients. Finally, keratinocyte gene therapy is reversible because the genetically modified tissue can be excised readily.

Most preclinical studies of corrective gene transfer for epidermal pathology have focused on human skin tissue.⁴⁸ These efforts have relied on ex vivo gene transfer to keratinocytes via the use of retroviruses and resulted in a normalization of tissue architecture and epidermal function for conditions such as ichthyosis and epidermolysis bullosa.^49,50 These diseases require only that the corrective gene be expressed in the appropriate location in skin or mucosa.

Since 1995, keratinocyte gene therapy for systemic delivery of a missing protein has progressed substantially at both mucosal and cutaneous sites. For example, Mizuno and colleagues,⁵¹ working at the Department of Oral and Maxillofacial Surgery of Nagoya University Graduate School of Medicine in Japan, used a retroviral vector to express factor IX in human oral mucosal keratinocyte cultures. Thereafter, the transduced epithelial sheets were grafted into immunologically deficient nude mice. Human factor IX was detected in mouse plasma for more than three weeks in vivo and was biologically active. Similarly, several groups, including that of Taichman and colleagues⁴⁶ at the School of Dental Medicine at State University of New York at Stony Brook, have reported the use of human epidermal keratinocytes for the delivery of transgene products such as human apolipoprotein E, factor IX, growth hormone and IL-10 into the bloodstream of mice and rats. ⁴⁸

Head and neck cancer. Each year in the United States, approximately 40,000 head and neck squamous-cell carcinomas, or HNSCCs, occur, as well as 11,700 associated deaths.⁵² These numbers have not lessened for a long time, and any approach that improves their treatment obviously is welcome. In this context, the remarkable advances made using gene transfer technology during the last six years are especially important.

Although several gene transfer strategies to manage HNSCCs have been tested preclinically and clinically, we will highlight only the one that, to us, appears most promising. The general strategy is to express a gene product that will result in cancer cell death. In normal cells, the tumor suppressor protein p53 monitors the integrity of the genome and responds to any DNA damage by inducing cell cycle arrest to allow repair, or apoptosis if repair is impossible. In HNSCCs, the incidence of p53 tumor suppressor gene mutations is between 45 and 70 percent.⁵³ This observation has provided the impetus to develop a novel recombinant adenovirus that selectively replicates in, and kills, p53-deficient cells⁵⁴ (Figure 4).

View larger version (23K): [in this window] [in a new window]   Figure 4. Use of a recombinant adenoviral vector in the management of an oral squamous cell carcinoma. This depiction is based on the work of Heise and colleagues55 and Khuri and colleagues.56 The malignant tumor is a recurrent tumor and is refractory to chemotherapy. For clarity, the malignant tumor is shown as consisting entirely of cells with either a mutated/absent p53 tumor suppressor protein or a normal (wild type) p53 protein. The tumor tissue is injected directly with the recombinant adeno-virus ONYX-015. Cells in tumors with the mutated/absent p53 protein will be lysed (killed) directly, owing to the ability of ONYX-015 to replicate in these cells. Cells in tumors with the wild type p53 protein will be stimulated to divide by ONYX-015 and thus rendered susceptible to conventional chemotherapeutic agents.

Recently, after a large collaborative study involving investigators at The University of Texas M.D. Anderson Cancer Center in Houston, ONYX Pharmaceuticals and several academic centers in the United Kingdom, Khuri and colleagues⁵⁶ published clinical trial findings in which the ONYX-015 adenovirus was used with or without conventional chemotherapeutic drugs. As hypothesized, tumor cells with the mutant p53 protein underwent lysis at a higher rate than did tumors containing the wild-type p53 gene sequence (58 percent and 0 percent, respectively).⁵⁶

Additionally, using an adenoviral vector in tumor cells with a normally functioning p53 protein can be advantageous. The adenoviral E1A gene product will drive these tumor cells into S phase, making them more sensitive to conventional chemotherapy. Studies in vitro with cell cultures and in vivo with a nude mouse/human tumor xenograft model showed that the efficacy of ONYX-015 in combination with conventional cisplatin-based chemotherapy was additive or synergistic compared with that of either ONYX-015 or chemotherapy alone.⁵⁴ While intratumoral replication and tumor-selective tissue destruction were documented in clinical trials of ONYX-015 alone in patients who had recurrent, refractory HNSCC, durable responses and clinical benefit were seen in less than 15 percent of the patients treated.⁵⁴

Khuri and colleagues⁵⁶ tested combinations of ONYX-015, cisplatinum and 5-fluorouracil in patients with HNSCC that had recurred after treatment by surgery, radiotherapy, or both. Patients were injected at the largest or most symptomatic tumor mass with 10¹⁰ plaque-forming units of ONYX-015 per day for five consecutive days. Khuri and colleagues⁵⁶ described eight (27 percent) complete and 11 (36 percent) partial responses among the 30 patients included in the study. Two of four chemotherapy-refractory tumors responded to subsequent therapy with ONYX-015 plus the same chemotherapy regimen to which the tumors had been resistant. These results demonstrate tumor-selective augmentation of chemotherapy by ONYX-015; that is, ONYX-015 when combined with cisplatin and 5-fluorouracil was effective as a method of local tumor control in most patients. Whether this enhanced local control will be translated into a survival advantage remains to be confirmed by additional trials, but the median survival time of approximately 11 months described certainly is encouraging.⁵⁶

Most future gene therapy–based cancer treatment will be combined with other, more traditional regimens, such as chemotherapy, radiation therapy and surgery. Such augmentative approaches, rather than gene transfer alone, probably will be used to reduce tumor burden and help maintain quality of life in patients with head and neck cancer and other solid tumors.⁵⁶

	CONCLUSION

TOP ABSTRACT GENERAL PROGRESS IN THE... GENE TRANSFER VECTORS AREAS OF IMPACT ON... CONCLUSION REFERENCES

 
We believe, as outlined here, that the progress made during the last six years in gene transfer generally, as well as that specifically related to dentistry, have validated our 1995 conclusion.¹ Gene therapy is having a pervasive and significant impact on areas related to science-based dental practice. The 1995 review¹ addressed initial efforts in three areas of orally related gene transfer research. This new review addresses seven areas of relevant research, and for all seven, substantive proofs of concept have been shown in vivo in animal models.

Gene transfer studies related to the treatment of head and neck cancer have made the most significant progress since 1995, reaching well into the clinical testing stages. Cancer-related gene therapy appears efficacious as an adjunctive therapy for head and neck cancers. The progress made in our own studies has been much more rapid than we anticipated. Although we still consider current gene transfer methods to be fairly primitive, and associated with significant problems, gene therapy’s acceptance as part of the routine clinical armamentarium, at least for some applications (like head and neck cancer), seems very close.

Biological research is presenting dentists with more potential treatment options for the future. The 1995 prediction that biology would alter the nature of dental practice by 2015 still seems very much on track.