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Ana P. Cotrim and Bruce J. Baum Toxicol Pathol 2008 36: 97 DOI: 10.1177/0192623307309925 The online version of this article can be found at: http://tpx.sagepub.com/content/36/1/97

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Toxicologic Pathology, 36:97-103, 2008 Copyright 2008 by Society of Toxicologic Pathology ISSN: 0192-6233 print / 1533-1601 online DOI: 10.1177/0192623307309925

Gene Therapy: Some History, Applications, Problems, and Prospects

ANA P. COTRIM AND BRUCE J. BAUM
From the Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research, NIH, DHHS, Bethesda, Maryland, USA.
ABSTRACT The concept of transferring genes to tissues for clinical applications has been discussed for nearly half a century, but our ability to manipulate genetic material via recombinant DNA technology has brought this goal to reality. While originally conceived as a way to treat life-threatening disorders (inborn errors, cancers) refractory to conventional treatment, gene therapy now is considered for many nonlife-threatening conditions, including those adversely affecting a patients quality of life. The lack of suitable treatment has become a rational basis for extending the scope of gene therapy. This manuscript reviews the general methods by which genes are transferred as well as diverse examples of clinical applications (acquired tissue damage, upper gastrointestinal tract infection, autoimmune disease, systemic protein deficiency). Despite some well-publicized problems, gene therapy has made substantive progress, including tangible success, albeit much slower than was initially predicted. Although gene therapy is still at a fairly primitive stage, it is firmly science based. There is justifiable optimism that with increased pathobiological understanding and biotechnological improvements, gene therapy will become a standard part of clinical practice within 20 years. Keywords: Salivary gland; animal models; cell(ular) pathology.

INTRODUCTION Gene therapy typically involves the insertion of a functioning gene into cells to correct a cellular dysfunction or to provide a new cellular function (Culver, 1994). For example, diseases such as cystic fibrosis, combined immunodeficiency syndromes, muscular dystrophy, hemophilia, and many cancers result from the presence of defective genes. Gene therapy can be used to correct or replace the defective genes responsible. Gene therapy has been especially successful in the treatment of combined immunodeficiency syndromes, showing lasting and remarkable therapeutic benefit (Cavazzana-Calvo et al., 2000; Cavazzana-Calvo et al., 2001; Cavazzana-Calvo and Fischer, 2007). However, it is important to remember that gene therapy is not a new idea. In 1963, Joshua Lederberg wrote, We might anticipate the . . . interchange of chromosomes and segments. The ultimate application of molecular biology would be the direct control of nucleotide sequences in human chromosomes, coupled with recognition, selection and integration of the desired genes. . . . It will only be a matter of time . . . before polynucleotide sequences can be grafted by chemical procedures onto a virus DNA. Less than 30 years later, the first clinical study using gene transfer was reported (Rosenberg et al., 1990).
Address correspondence to: Dr. Ana Cotrim, GTTB, NIDCR, NIH, 10 Center Drive, Bethesda, MD 20892-1190; e-mail: acotrim@nidcr.nih.gov. Abbreviations: Ad5, serotype 5 adenovirus; MoMLV, Moloney murine leukemia virus; SG, salivary gland; GI, gastrointestinal; AAV2, serotype 2 adeno-associated virus; IR, irradiation; MnSOD-PL, manganese superoxide dismutase-plasmid liposome; AQP1, aquaporin-1; AdhAQP1, Ad5 vector encoding human AQP1; SS, Sjogrens syndrome; VIP, vasoactive intestinal peptide; IL-10, interleukin-10; NOD, nonobese diabetic; h, human; Epo, erythropoietin; FDA, Food and Drug Administration; NIH, National Institutes of Health; SCID, severe combined immunodeficiency disorder. 97

Rosenberg and his colleagues used a retroviral vector to transfer the neomycin resistance marker gene into tumor-infiltrating lymphocytes obtained from 5 patients with metastatic melanoma. These lymphocytes then were expanded in vitro and later reinfused into the respective patients. Since this first study showed that retroviral gene transfer was safe and practical, it led to many other studies. Indeed, since 1989, more than 900 clinical trials have been approved worldwide (Edelstein et al., 2004). What made gene therapy possible between 1963 and 1990 was the development of recombinant DNA technology. VECTORS There are two general approaches for introducing genes into a cell: viral and nonviral (Table 1). Viral vectors have been used in ~70% of the clinical trials to date (Edelstein et al., 2004). Viral vectors are extremely efficient at transferring genes but can create some safety risks. Gene transfer mediated by viral vectors is referred to as transduction. Nonviral vectors are considered to be much safer than viral vectors, but at present, they are fairly inefficient at transferring genes. Gene transfer mediated by nonviral vectors is referred to as transfection. As indicated, viral vectors have the advantage of achieving highly efficient gene transfer in vivo. Although replicationdeficient vectors are used, viral vectors still pose significant safety concerns. The two most common viral vectors used in clinical trials have been those derived from a serotype 5 adenovirus (Ad5; ~26%) and Moloney murine leukemia virus (MoMLV; ~28%), a retrovirus. MoMLV vectors target dividing cells with a reasonably high degree of efficiency. Importantly, they also lead to stable gene transfer because they integrate randomly into chromosomes of the target cell. A major disadvantage of

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TABLE 1.General approaches used in gene therapy.a

Nonviral Naked/plasmid DNA Lipofection Gene gun Viral-based Retrovirus (MoMLV) Adenovirus (Ad5) Adeno-associated virus (AAV2) Lentivirus Herpes Simplex virus

THE TARGET TISSUESALIVARY GLANDS Our studies have focused on gene transfer to salivary glands (SGs).These are encapsulated organs whose main function is to secrete fluid and proteins into the oral cavity and upper gastrointestinal (GI) tract via saliva. Importantly, although considered classical exocrine glands, SGs can also secrete proteins into the bloodstream. SGs have a considerable ability to produce proteins, facilitating their protective and digestive roles in the mouth and upper GI tract (Amerongen and Veerman, 2002). Humans have six major SGs (the bilateral parotid, submandibular, and sublingual) and numerous minor glands. A gene-therapy treatment for SGs involves transfer of a new gene via retroductal cannulation of the main excretory ducts of a major SG. This could lead to the production of a cellular therapeutic protein (Baum et al., 2006; Kok et al., 2003) or to secretion either in saliva or in the bloodstream (Voutetakis et al., 2005; Wang et al., 2005). Cannulation of the main excretory ducts of major SGs is a fairly simple procedure that is used routinely in the clinic for contrast radiographs (sialograms). This is a very effective delivery method because virtually all of the epithelial cells in SGs are continuous with the duct system. Since SGs in humans are encapsulated organs, vectors delivered through the ductal system are limited in reaching other organs or the bloodstream. In preclinical studies, animals are anesthetized before the cannulation procedure for restraint only; no anesthesia is needed clinically. In mice and rats, we typically target the submandibular glands for gene transfer, as their ducts are easier to cannulate than those of the parotid glands. However, in miniature pigs and nonhuman primates, we target the parotid glands for their ease and convenience and because they are well encapsulated. The volume in which the vector is suspended for administration varies according to animal size (Table 2). For example, to deliver vectors to a mouse submandibular gland, we use 50 1 of suspension buffer. Conversely, to deliver a vector to the parotid gland of a miniature pig, we use a volume of 4,000 1. For our applications of gene therapy studies to SGs (described below), we have used mainly Ad5 and serotype 2 adeno-associated viral (AAV2) vectors (Table 3). Ad5 vectors can transduce up to ~40% of virtually all cell types in SGs, and they mediate a robust short-term transgene expression, with peak expression at ~4872 hours. Typically, because Ad5 vectors elicit a potent immune response, transgene expression is

a Viral vectors lead to relatively efficient gene transfer and therefore are used in most clinical trials. However, viral vectors can pose significant safety risks. Nonviral vectors, although safer, are relatively inefficient for gene transfer to most tissues. See text for details. MoMLV: Moloney murine leukemia virus; Ad5: serotype 5 adenovirus; AAV2: serotype 2 adeno-associated virus.

MoMLV vectors is the risk of insertional mutagenesis caused by the integration of the retroviral genome into the host genome. Also, since retroviral vectors require dividing cells for successful transduction, they are not useful for targeting gene transfer to well-differentiated, quiescent cell types, such as in epithelial tissues. Ad5 vectors are able to transduce both dividing and nondividing cells and facilitate highly efficient gene transfer. Importantly, Ad5 vectors only very rarely integrate into a chromosome, that is, they exist in a target cell nucleus in an epichromosomal location. Thus, if the target cell divides, only one daughter cell will receive the transferred gene, and with subsequent cell-division cycles, the gene will be dramatically diluted. The main disadvantage of Ad5 vectors is that they induce a potent host-immune response. It is also important to recognize that different viral vectors will vary in their ability to transduce different cell types. Often, this reflects the presence or absence of cell membrane receptor proteins that mediate viral entry into the target cell. Of the developed nonviral gene-transfer approaches, two methods have been used fairly often in clinical trials. One involves simply the direct injection of plasmids containing the transgene (termed naked DNA) into a tissue. This has been used in ~14% of approved clinical trials, most often in muscle. The second method uses cationic lipids (so-called liposomes) to surround the plasmid DNA and is termed lipofection. This method has been used in ~9% of approved trials. The cationic lipids facilitate plasmid entry into the cell. Nonviral gene transfer typically does not result in integration of the transgene.

TABLE 2.Gene transfer to salivary glands in different species.a

Animal Mouse Rat Miniature pig Nonhuman primate Weight ~20 g ~300 g ~30 kg ~5 kg Gland SMG SMG Parotid Parotid Volume (l) 50 200 4,000 500 Vector type Ad5, AAV2 Ad5, AAV2 Ad5, AAV2 Ad5, AAV2 Transgenesb Epo, GH, IL-10 AQP1; Epo; GH; 1-antitrypsin histatin-3 AQP1; Epo; GH AQP1, Epo; GH; histatin-3

a Salivary glands have been used as gene-transfer targets in several species. Typically, Ad5 and AAV2 vectors have been used, although there are published reports using lentiviral, retroviral, and nonviral vectors with this tissue. In addition, several reporter genes have been used in salivary gland gene-transfer studies, e.g., luciferase, -galactosidase, and green fluorescence protein. See text for additional details. Ad5: serotype 5 adenovirus; AAV2: serotype 2 adeno-associated virus; SMG: submandibular gland; Epo: erythropoietin; GH: growth hormone; IL-10: interleukin-10; AQP1: aquaporin-1. b Many different transgenes have been tested, and representative ones are listed.

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TABLE 3.Some general characteristics of serotype 5 adenoviral and serotype 2 adeno-associated viral vectors.a
Characteristic Genome size DNA Virus particles Cellular targets Transgene expression Stability of expression Immune response Packaging (recombinant) Ad5 37 kb Double-stranded Labile Acinar, duct High No Potent Easy AAV2 4.7 kb Single-stranded Stable Duct Modest Yes Modest Laborious

a Ad5 vectors are especially useful for experiments when high transient transgene expression is sufficient. AAV2 vectors provide more stable and long-lasting transgene expression, albeit at much lower levels than seen for peak Ad5 vector-mediated expression. Ad5: serotype 5 adenovirus; AAV2: serotype 2 adeno-associated virus; kb: kilobases.

damage. Our results generally are consistent with this concept (Cotrim et al., 2007). This suggests that efforts to protect microvascular cells in glands during IR may be useful to prevent acinar cell damage. Normally, superoxide generated during IR is dismutated by three forms of cellular superoxide dismutase to hydrogen peroxide, which is then further metabolized by catalase and glutathione peroxidase to water and oxygen (Epperly et al., 2004; Oberley and Buettner, 1979). Interestingly, Greenberger and Epperly (2007) have shown that administration of manganese superoxide dismutase-plasmid liposomes (MnSOD-PL) can provide mucosal IR protection in the lung, esophagus, oral cavity, urinary bladder, and intestine. Although the effects of MnSODPL on SG function have not been studied, this approach to preventing SG damage from IR appears promising. Repair of SG Damage from IR

dramatically reduced after 7 to 10 days and reduced to background levels by 14 days (Kagami et al., 1998). AAV2 vectors transduce mainly ductal cells and require ~812 weeks to achieve maximal levels of transgene expression (Voutetakis et al., 2004). AAV2 vectors elicit only a modest immune response, and transgene expression in mice is quite stable (Voutetakis et al., 2004; Voutetakis et al., 2005). APPLICATIONS Acquired Tissue Damage Prevention of Irradiation Damage to SGs Radiotherapy is used to treat the majority of head and neck cancers. Most patients receive between 50 and 70 Gray (Gy) of irradiation (IR), which typically is divided into doses of 2 to 2.5 Gy/day, 5 days a week, for 5 to 7 weeks (Dobbs et al., 1999). Unfortunately, normal SG tissue in the IR field is damaged, and patients suffer considerable morbidity from the IRinduced salivary hypofunction. Therapeutic IR generates double-strand DNA breaks in target cells and also results in oxidative stress via the generation of potentially damaging free radicals. Cells that divide more rapidly (e.g., cancer cells) are usually considered more sensitive to IR. The relative radiosensitivity of a cell is cell-cycle dependent, with cells being most radiosensitive in the G(2)-M phase (Pawlik et al., 2004). SGs are considered to be postmitotic, well-differentiated epithelial cells with a slow turnover rate. Therefore, it is expected that SGs would be relatively radio-resistant. However, SGs are extremely sensitive to IR, and the mechanism of this damage is still not clear (Nagler et al., 2002; OConnell, 2000). While IR appears primarily to affect the acinar cells, from which all fluid is secreted, the acinar cell damage could be secondary to localized vascular injury, interstitial edema, or inflammatory infiltration (Vissink et al., 2003). For example, we have explored the possibility that microvascular endothelial cells within SGs are the primary target of IR damage, a concept first suggested by Paris et al. (2001) in their studies on gastrointestinal radiation

A major focus of our work has been to restore SG function in patients who have already received IR. For this goal, our strategy has used transfer of the aquaporin-1 (AQP1) complementary DNA (cDNA). AQP1 was the first water-channel protein discovered (Preston and Agre, 1991). SGs present in the IR field show a dramatic loss of acinar cells; acinar cells are considered water-permeable secretory epithelia. Ductal cells typically survive the IR, but they are considered to be relatively water-impermeable absorptive epithelia. We reasoned that duct cells in an IR-damaged SG would be capable of generating an osmotic gradient sufficient to allow the movement of water in a basal to apical direction, that is, into the lumen (Delporte et al., 1997). We speculated that the gradient would be based on forming potassium bicarbonate in the lumen: potassium entering the lumen in exchange for a proton via a potassium-proton exchanger present in the apical membranes of duct cells. We further hypothesized that all that was lacking for the duct cells to secrete fluid in an IR-damaged gland was a facilitated waterpermeability pathway, a water channel protein. We constructed an Ad5 vector encoding human AQP1 (AdhAQP1) and showed that this vector leads to a dramatic increase in fluid secretion when administered 90 or 120 days after IR in rats (Delporte et al., 1997) or miniature pigs (Shan et al., 2005; Table 4), to ~80% of control levels when measured 3 days after transduction. A control Ad5 vector was without any significant effect on salivary flow. Additionally, after administration of AdhAQP1 to SGs, no significant toxicological effects were observed, that is, in multiple measured clinical chemistry and hematology values (Zheng et al., 2006). Based on these aggregate results, we developed a phase 1 (vector safety with some efficacy measures) clinical trial protocol to test AdhAQP1 in patients who received IR for head and neck cancer at least 5 years previously. Although Ad5 vectors only lead to transient gene expression, because of the generated immune response, we chose this type of vector because very little is known about human ductal cell physiology. We assume that human duct cells generally will be similar to those of rats

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TABLE 4.Effect of AdhAQP1 on salivary secretion in irradiated rats and miniature pigs.a
Irradiation dose (Gy) 21 20 Salivary flow (% of control) Post-IR ~35 ~20

Species Rat Miniature pig

Post-AdhAQP1 ~84 ~81

a This table provides a summary of data previously reported in Delporte et al. (1997) and Shan et al. (2005). Before administration of AdhAQP1, IR resulted in a marked decrease in salivary flow to ~35% (rats) or ~20% (miniature pigs) of control values. Three days after AdhAQP1 delivery, salivary flow was markedly increased to ~80% of control values. The data shown are the average percentages of control salivary flow results seen following AdhAQP1 delivery. 100% would be equivalent to control (i.e., normal) salivary flow. See text and original references for more details. AdhAQP1: serotype 5 adenovirus vector encoding human aquaporin-1; IR: irradiation.

In animal model studies, we successfully expressed histatin-3 in rat SGs using an Ad5 vector (AdCMVH3). The concentration of histatin-3 in rat submandibular-gland saliva collected from treated rats 3 days after transduction with the AdCMVH3 was as high as 1 mg/ml, with a mean value of 302 g/ml. The fungicidal activity of the recombinant histatin-3 mediated by the AdCMVH3 vector was tested in vitro in a timed-kill assay. At a concentration of 100 g/ml, 90% of the azole-resistant Candida albicans were killed in 60 minutes (OConnell et al., 1996). Autoimmune Disorders Sjogrens syndrome (SS) is the second most common autoimmune disease in the United States, affecting between 1 million and 4 million persons, primarily female (~90%). The etiology of SS is unclear, and current treatment is only palliative. SS is characterized by the presence of a focal lymphoid cell infiltration in the salivary and lacrimal glands, although other organs may also be involved (Pillemer et al., 2001). In the absence of any suitable conventional treatments, we have suggested that gene therapy may be beneficial for SS patients. We have hypothesized that transfer of immunomodulatory genes into SGs may reduce the autoimmune sialadenitis and lead to increased salivation as well as symptomatic relief (Kok et al., 2003). For example, the transfer of genes encoding antiinflammatory cytokines such as interleukin-10 (IL-10) or vasoactive intestinal peptide (VIP; Lodde et al., 2006) could lead to a decrease in the expression of proinflammatory cytokines, and thus, protect SGs and preserve their secretory function. To test this hypothesis, we used a common animal model of SS, the female nonobese diabetic (NOD) mouse. We delivered the human (h) IL-10 and VIP cDNAs using AAV2 vectors because they provide stable transgene expression with little immune reactivity. Both AAVhIL-10 and AAVhVIP, as well as a control vector, AAVLacZ encoding -galactosidase, were administered locally via retrograde cannulation of the submandibular glands. We compared salivary flow and sialadenitis ~812 weeks later. Administration of AAVhIL-10 led to preservation of salivary flow rates as well as a reduction of the focal autoimmune sialadenitis (Table 5; Kok et al., 2003). Administration of AAVhVIP also resulted in a preservation of salivary flow; however, no reduction of the focal sialadenitis was observed with this transgene (Lodde et al., 2006). These initial studies show that immunomodulatory gene transfer may be useful in managing the autoimmune sialadenitis and resultant salivary hypofunction that occur in SS patients. Nonetheless, since we do not understand SS pathogenesis, this gene-transfer strategy is nonspecific and still requires considerable study. Systemic Protein Deficiencies As mentioned previously, SGs show several features that are common to many endocrine glands, particularly the ability to produce high levels of protein for export and the ability to

and miniature pigs, that is, also able to generate an osmotic gradient and fluid flow as described above. However, we do not know that. In the event that human ductal cells are incapable of this response, the AdhAQP1 presence in the tissue will be relatively limited because of the immune response, which we consider an important safety consideration in the absence of any benefit. However, if irradiated human SGs are able to secrete fluid following AQP1 gene transfer, we have developed an AAV2 vector capable of mediating long-term AQP1 expression, and presumably, providing patients with the stable SG repair required. This vector includes the same promoter, AQP1 cDNA, and polyadenylation signal as AdhAQP1 (Braddon et al., 1998). Infections of the Upper GI Tract Although rapid advances have been made in the detection, management, and biology of HIV-1, even today, oral candidiasis remains a common opportunistic infection observed among immunosuppressed patients. In HIV-1infected patients, this can lead to significant morbidity (Sroussi et al., 2007). Antifungal azole-type drugs are the principal management tool for such candidal infections. However, the occurrence of azoleresistant Candida species necessitates the development of alternative treatment strategies. Histatins are a family of histidine-rich, cationic peptides composed of up to 38 amino acids. They are secreted by the SGs of humans and some primates and are a major component of the innate host nonimmune defense system in the oral cavity against bacteria and fungal infections. The importance of histatins in azole-resistant candidiasis is twofold. Histatin levels in saliva are reduced in HIV-1infected patients. Secondly, the mechanism of action of histatins in targeting candidal species is distinctly different from that of azole-type drugs. While azole drugs inhibit the synthesis of ergosterol, a major plasma-membrane sterol (Amerongen et al., 2002), histatins act by binding to the ergosterol present in the fungal membrane. We reasoned that transfer of the histatin-3 cDNA to SGs would result in an increased secretion of histatins in the oral cavity (OConnell et al., 1996) and be useful in managing azoleresistant candidal species.

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TABLE 5.Effect of IL-10 and VIP cDNA transfer on salivary flow and inflammatory focus score in NOD mice.a
Treatment group IL-10 VIP Baseline salivary flow (8 weeks) 186 l 125 l 3.8 l/g 3.8 l/g Salivary flow at 16 weeks 168 l 28 l 4.3 l 2.05 l Focus score 1.4 3.0 1.9 1.85

TABLE 6.Total amount of erythropoietin secretion into serum and saliva after rAAV2Epo transduction of murine and macaque salivary glands.a
Species Mouse Rhesus macaque Serum Epo 80 1000 Salivary Epo 0.4 140 Ratio, serum:saliva 160:1 7:1

Treatment rAAVhIL-10 rAAVLacZ rAAVhVIP rAAVLacZ

a For all salivary flow measurements, saliva was collected for 20 minutes. For the IL-10 (interleukin-10) experiments, the total amount of saliva was presented (Kok et al., 2003). IL-10 prolonged normal salivary secretion and diminished the presence of focal glandular inflammatory infiltrates (focus score; Greenspan et al., 1974) compared to controls treated with rAAVLacZ. For all VIP (vasoactive intestinal peptide) experiments, saliva output was presented as microliters of saliva per gram of animal weight (Lodde et al., 2006). VIP had no effect on the presence of inflammatory infiltrates, but it was able to prolong predisease salivary secretion levels compared to controls treated with rAAVLacZ. See text and original references for more details.

a Total serum erythropoietin (Epo), expressed as mU, was calculated as serum Epo concentration volume (mice, 2 ml; macaques, 60 ml/kg). Total salivary Epo was calculated as salivary Epo concentration volume of collected saliva. See text and original reference (Voutetakis et al., 2007) for more details.

secrete proteins into the bloodstream. We have suggested a therapeutic application to take advantage of these features: the treatment of systemic single-protein deficiency disorders (Baum et al., 2004). Current treatment of these conditions involves the regular administration of a recombinant protein by bolus injection (e.g., insulin for diabetes mellitus and erythropoietin [Epo] for anemias related to chronic renal failure). For example, many of our studies have involved transferring the cDNA for Epo (Voutetakis et al., 2004; Voutetakis et al., 2007). Epo is produced in kidney epithelial cells and secreted by the constitutive secretory pathway into the bloodstream. In SGs, after gene transfer, much Epo is also secreted into the bloodstream (Table 6). For most of our experiments, we have used AAV2 vectors encoding either human or rhesus Epo (AAV2hEpo; AAV2rhEpo) to transfer the Epo cDNA into the SGs of mice and rhesus macaques. After male mice received 109 particles of AAV2hEpo into their submandibular glands, serum Epo reached maximum levels by 8 to 12 weeks and remained relatively stable for 54 weeks (the longest time studied). Hematocrit levels were similarly increased. In male mice, Epo is secreted almost entirely into the bloodstream (160:1; Table 6; Voutetakis et al., 2004). After delivery of AAV2rhEpo (3 1011 particles/gland) to parotid glands of rhesus macaques, rhesus Epo expression increased significantly after 1 week, and levels remained relatively stable in serum and saliva after 6 months (the longest time studied). When total rhesus Epo levels were measured, most rhesus Epo also was found in serum, but at a much lower ratio than found in mice (~7:1 serum/saliva ratio). While there is still a need to understand what directs transgenic secretory protein sorting into either serum or saliva, it appears from our studies that use of SGs as a target depot organ for gene transfer to treat systemic single-protein deficiency disorders has significant clinical potential. PROBLEMS AND PROSPECTS Since the first clinical gene-therapy trial was conducted (Rosenberg et al., 1990), much attention and considerable promise has been given to the field. There has been substantial

public- and private-sector investment, as well as increasingly higher levels of research activity. Numerous preclinical animalmodel studies have provided proofs of concept for multiple potential clinical applications. Also, major advances have been made in understanding vector biology and improving vector design and production. However, clinical progress has been slow. A major setback for the field occurred in September 1999, when a widely publicized death resulting from a gene-therapy trial was reported (Raper et al., 2003). Jesse Gelsinger, an 18-year-old man, died in a clinical trial at the University of Pennsylvania, which used a modified Ad5 vector to deliver the gene for ornithine decarboxylase, a deficient hepatic enzyme. According to an investigation by the university, Gelsinger died from a massive immune reaction to the Ad5 vector. This widely publicized case led to congressional and Food and Drug Administration (FDA) hearings on the conduct of clinical gene-therapy trials as well as a transient hold, subsequently lifted, on all adenoviral-vector clinical trials. An investigation by the FDA found numerous possible violations in the way that this clinical trial had been conducted and monitored. After 5 years of investigations, in February 2005, the case was settled. As a result of the Gelsinger case, gene therapy experienced an intense phase of criticism and skepticism. Clearly, mistakes were made in that particular clinical trial, and as an appropriate outcome, all gene-therapy trials are now subject to much tighter regulation by the National Institutes of Health (NIH) and FDA. Fortunately for the gene therapy field, less than 1 year after Gelsinger died, the first report of a dramatically successful genetherapy trial was published. In 2000, Cavazzana-Calvo and her colleagues in Paris described results from a study involving two children suffering from a severe combined immunodeficiency disorder (SCID-XI), which had restricted them to life in an isolated environment (Cavazzana-Calvo et al., 2000). These investigators used a MoMLV vector to transfer a curative gene (c cytokine receptor subunit) into the patients lymphocytes ex vivo, and after amplification of the cells, returned them to the patients. Both patients were able to leave the hospital and resume normal lives. Subsequently, several other patients were treated and apparently cured in these studies. However, there was a downside. Of ~11 early patients treated with the MoMLV vector, 3 developed leukemia directly as a result of the gene-transfer procedure

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(Fischer et al., 2004). In all of these patients, the MoMLV vector had integrated apparently in a nonrandom manner near the LM02 (LIM domain only 2) gene. The LM02 gene was activated by this integration, and leukemia was the result. The patients were treated for the leukemia, and a large, collaborative scientific effort began to understand what mechanisms influence MoMLV integration. The SCID-XI trial likely reflects the path that gene therapy will follow during the next 1 to 2 decades: success, but with some complications. Last year, a somewhat similar scenario occurred in a German clinical trial of a MoMLV vector to treat chronic granulomatous diseasethat is, some clinical success, but subsequently, a patient died (Ott et al., 2006). Also, a clinical trial to correct a clotting disorder, Factor IX deficiency, by hepatic gene transfer using an AAV2 vector recently showed that transient correction was possible but quite limited in time because of subsequent immune reactivity (Manno et al., 2006). These experiences add further credence to the general viewpoint offered by Leiden in a 1995 editorial that gene therapy is a field in its infancy, and despite some pitfalls, it is well grounded in fundamental scientific principles with real clinical promise for the future (Leiden, 1995). ACKNOWLEDGMENT The authors research is supported by the intramural research program of the National Institute of Dental and Craniofacial Research. REFERENCES
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