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2008 36: 97Toxicol PatholAna P. Cotrim and Bruce J. Baum

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

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97

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 manipulategenetic material via recombinant DNA technology has brought this goal to reality. While originally conceived as a way to treat life-threateningdisorders (inborn errors, cancers) refractory to conventional treatment, gene therapy now is considered for many non–life-threatening conditions,including those adversely affecting a patient’s quality of life. The lack of suitable treatment has become a rational basis for extending the scope ofgene 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 prob-lems, gene therapy has made substantive progress, including tangible success, albeit much slower than was initially predicted. Although gene ther-apy is still at a fairly primitive stage, it is firmly science based. There is justifiable optimism that with increased pathobiological understanding andbiotechnological improvements, gene therapy will become a standard part of clinical practice within 20 years.

Keywords: Salivary gland; animal models; cell(ular) pathology.

Rosenberg and his colleagues used a retroviral vector to trans-fer the neomycin resistance marker gene into tumor-infiltratinglymphocytes obtained from 5 patients with metastatic melanoma.These lymphocytes then were expanded in vitro and later rein-fused into the respective patients. Since this first study showedthat retroviral gene transfer was safe and practical, it led tomany other studies. Indeed, since 1989, more than 900 clinicaltrials have been approved worldwide (Edelstein et al., 2004).What made gene therapy possible between 1963 and 1990 wasthe development of recombinant DNA technology.

VECTORS

There are two general approaches for introducing genes into acell: viral and nonviral (Table 1). Viral vectors have been used in~70% of the clinical trials to date (Edelstein et al., 2004). Viralvectors are extremely efficient at transferring genes but can createsome safety risks. Gene transfer mediated by viral vectors isreferred to as transduction. Nonviral vectors are considered to bemuch safer than viral vectors, but at present, they are fairly ineffi-cient at transferring genes. Gene transfer mediated by nonviralvectors is referred to as transfection.

As indicated, viral vectors have the advantage of achievinghighly efficient gene transfer in vivo. Although replication-deficient vectors are used, viral vectors still pose significantsafety concerns. The two most common viral vectors used in clin-ical 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 alsolead to stable gene transfer because they integrate randomlyinto chromosomes of the target cell. A major disadvantage of

INTRODUCTION

Gene therapy typically involves the insertion of a functioninggene into cells to correct a cellular dysfunction or to provide anew cellular function (Culver, 1994). For example, diseases suchas cystic fibrosis, combined immunodeficiency syndromes, mus-cular dystrophy, hemophilia, and many cancers result from thepresence of defective genes. Gene therapy can be used to corrector replace the defective genes responsible. Gene therapy hasbeen especially successful in the treatment of combined immun-odeficiency syndromes, showing lasting and remarkable thera-peutic benefit (Cavazzana-Calvo et al., 2000; Cavazzana-Calvoet al., 2001; Cavazzana-Calvo and Fischer, 2007).

However, it is important to remember that gene therapy isnot a new idea. In 1963, Joshua Lederberg wrote, “We mightanticipate the . . . interchange of chromosomes and segments.The ultimate application of molecular biology would be thedirect control of nucleotide sequences in human chromosomes,coupled with recognition, selection and integration of the desiredgenes. . . . It will only be a matter of time . . . before polynu-cleotide sequences can be grafted by chemical procedures ontoa virus DNA.” Less than 30 years later, the first clinical studyusing gene transfer was reported (Rosenberg et al., 1990).

Address correspondence to: Dr. Ana Cotrim, GTTB, NIDCR, NIH, 10 CenterDrive, Bethesda, MD 20892-1190; e-mail: acotrim@nidcr.nih.gov.

Abbreviations: Ad5, serotype 5 adenovirus; MoMLV, Moloney murineleukemia virus; SG, salivary gland; GI, gastrointestinal; AAV2, serotype 2adeno-associated virus; IR, irradiation; MnSOD-PL, manganese superoxidedismutase-plasmid liposome; AQP1, aquaporin-1; AdhAQP1, Ad5 vectorencoding human AQP1; SS, Sjogren’s syndrome; VIP, vasoactive intestinalpeptide; IL-10, interleukin-10; NOD, nonobese diabetic; h, human; Epo, ery-thropoietin; FDA, Food and Drug Administration; NIH, National Institutes ofHealth; SCID, severe combined immunodeficiency disorder.

Toxicologic Pathology, 36:97-103, 2008Copyright © 2008 by Society of Toxicologic PathologyISSN: 0192-6233 print / 1533-1601 onlineDOI: 10.1177/0192623307309925

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MoMLV vectors is the risk of insertional mutagenesis caused bythe integration of the retroviral genome into the host genome.Also, since retroviral vectors require dividing cells for success-ful transduction, they are not useful for targeting gene transfer towell-differentiated, quiescent cell types, such as in epithelialtissues. Ad5 vectors are able to transduce both dividing andnondividing cells and facilitate highly efficient gene transfer.Importantly, Ad5 vectors only very rarely integrate into a chro-mosome, that is, they exist in a target cell nucleus in an epi-chromosomal location. Thus, if the target cell divides, only onedaughter cell will receive the transferred gene, and with subse-quent cell-division cycles, the gene will be dramatically diluted.The main disadvantage of Ad5 vectors is that they induce apotent host-immune response. It is also important to recognizethat different viral vectors will vary in their ability to transducedifferent cell types. Often, this reflects the presence or absenceof cell membrane receptor proteins that mediate viral entry intothe target cell.

Of the developed nonviral gene-transfer approaches, twomethods have been used fairly often in clinical trials. Oneinvolves simply the direct injection of plasmids containing thetransgene (termed “naked DNA”) into a tissue. This has beenused in ~14% of approved clinical trials, most often in muscle.The second method uses cationic lipids (so-called liposomes) tosurround the plasmid DNA and is termed lipofection. Thismethod has been used in ~9% of approved trials. The cationiclipids facilitate plasmid entry into the cell. Nonviral gene transfertypically does not result in integration of the transgene.

THE TARGET TISSUE—SALIVARY GLANDS

Our studies have focused on gene transfer to salivary glands(SGs).These are encapsulated organs whose main function is tosecrete fluid and proteins into the oral cavity and upper gas-trointestinal (GI) tract via saliva. Importantly, although consid-ered classical exocrine glands, SGs can also secrete proteinsinto the bloodstream. SGs have a considerable ability to pro-duce proteins, facilitating their protective and digestive roles inthe mouth and upper GI tract (Amerongen and Veerman, 2002).Humans have six major SGs (the bilateral parotid, submandibu-lar, and sublingual) and numerous minor glands. A gene-therapytreatment for SGs involves transfer of a new gene via retroductalcannulation of the main excretory ducts of a major SG. Thiscould lead to the production of a cellular therapeutic protein(Baum et al., 2006; Kok et al., 2003) or to secretion either insaliva or in the bloodstream (Voutetakis et al., 2005; Wang et al.,2005). Cannulation of the main excretory ducts of major SGs isa fairly simple procedure that is used routinely in the clinic forcontrast radiographs (sialograms). This is a very effective deliv-ery method because virtually all of the epithelial cells in SGs arecontinuous with the duct system. Since SGs in humans areencapsulated organs, vectors delivered through the ductal systemare limited in reaching other organs or the bloodstream.

In preclinical studies, animals are anesthetized before the can-nulation procedure for restraint only; no anesthesia is needed clin-ically. In mice and rats, we typically target the submandibularglands for gene transfer, as their ducts are easier to cannulate thanthose of the parotid glands. However, in miniature pigs and non-human primates, we target the parotid glands for their ease andconvenience and because they are well encapsulated. The volumein which the vector is suspended for administration varies accord-ing to animal size (Table 2). For example, to deliver vectors to amouse submandibular gland, we use 50 µ1 of suspension buffer.Conversely, to deliver a vector to the parotid gland of a miniaturepig, we use a volume of 4,000 µ1.

For our applications of gene therapy studies to SGs(described below), we have used mainly Ad5 and serotype 2adeno-associated viral (AAV2) vectors (Table 3). Ad5 vectorscan transduce up to ~40% of virtually all cell types in SGs, andthey mediate a robust short-term transgene expression, withpeak expression at ~48–72 hours. Typically, because Ad5 vec-tors elicit a potent immune response, transgene expression is

98 COTRIM AND BAUM TOXICOLOGIC PATHOLOGY

TABLE 1.—General approaches used in gene therapy.a

Nonviral Viral-based

Naked/plasmid DNA Retrovirus (MoMLV)Lipofection Adenovirus (Ad5)Gene gun Adeno-associated virus (AAV2)

LentivirusHerpes Simplex virus

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

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

Animal Weight Gland Volume (µl) Vector type Transgenesb

Mouse ~20 g SMG 50 Ad5, AAV2 Epo, GH, IL-10Rat ~300 g SMG 200 Ad5, AAV2 AQP1; Epo; GH; α1-antitrypsinMiniature pig ~30 kg Parotid 4,000 Ad5, AAV2 histatin-3 AQP1; Epo; GHNonhuman primate ~5 kg Parotid 500 Ad5, AAV2 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, retro-viral, and nonviral vectors with this tissue. In addition, several reporter genes have been used in salivary gland gene-transfer studies, e.g., luciferase, β-galactosidase, and green fluores-cence protein. See text for additional details. Ad5: serotype 5 adenovirus; AAV2: serotype 2 adeno-associated virus; SMG: submandibular gland; Epo: erythropoietin; GH: growthhormone; IL-10: interleukin-10; AQP1: aquaporin-1.

b Many different transgenes have been tested, and representative ones are listed.

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dramatically reduced after 7 to 10 days and reduced to backgroundlevels by 14 days (Kagami et al., 1998). AAV2 vectors transducemainly ductal cells and require ~8–12 weeks to achieve maximallevels of transgene expression (Voutetakis et al., 2004). AAV2vectors elicit only a modest immune response, and transgeneexpression 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 neckcancers. Most patients receive between 50 and 70 Gray (Gy)of irradiation (IR), which typically is divided into doses of 2 to2.5 Gy/day, 5 days a week, for 5 to 7 weeks (Dobbs et al.,1999). Unfortunately, normal SG tissue in the IR field is dam-aged, and patients suffer considerable morbidity from the IR-induced salivary hypofunction. Therapeutic IR generatesdouble-strand DNA breaks in target cells and also results inoxidative stress via the generation of potentially damaging freeradicals. Cells that divide more rapidly (e.g., cancer cells) areusually considered more sensitive to IR. The relative radiosen-sitivity of a cell is cell-cycle dependent, with cells’ being mostradiosensitive in the G(2)-M phase (Pawlik et al., 2004). SGsare considered to be postmitotic, well-differentiated epithe-lial cells with a slow turnover rate. Therefore, it is expectedthat SGs would be relatively radio-resistant. However, SGs areextremely sensitive to IR, and the mechanism of this damage isstill not clear (Nagler et al., 2002; O’Connell, 2000). While IRappears primarily to affect the acinar cells, from which all fluidis secreted, the acinar cell damage could be secondary to local-ized vascular injury, interstitial edema, or inflammatory infil-tration (Vissink et al., 2003). For example, we have exploredthe possibility that microvascular endothelial cells within SGsare the primary target of IR damage, a concept first suggestedby Paris et al. (2001) in their studies on gastrointestinal radiation

damage. Our results generally are consistent with this concept(Cotrim et al., 2007). This suggests that efforts to protect micro-vascular cells in glands during IR may be useful to prevent acinarcell damage.

Normally, superoxide generated during IR is dismutated bythree forms of cellular superoxide dismutase to hydrogen per-oxide, which is then further metabolized by catalase and glu-tathione peroxidase to water and oxygen (Epperly et al., 2004;Oberley and Buettner, 1979). Interestingly, Greenberger andEpperly (2007) have shown that administration of manganesesuperoxide dismutase-plasmid liposomes (MnSOD-PL) canprovide mucosal IR protection in the lung, esophagus, oral cavity,urinary bladder, and intestine. Although the effects of MnSOD-PL on SG function have not been studied, this approach to pre-venting SG damage from IR appears promising.

Repair of SG Damage from IR

A major focus of our work has been to restore SG functionin patients who have already received IR. For this goal, ourstrategy has used transfer of the aquaporin-1 (AQP1) com-plementary DNA (cDNA). AQP1 was the first water-channelprotein discovered (Preston and Agre, 1991). SGs present inthe IR field show a dramatic loss of acinar cells; acinar cellsare considered water-permeable secretory epithelia. Ductal cellstypically survive the IR, but they are considered to be relativelywater-impermeable absorptive epithelia. We reasoned that ductcells in an IR-damaged SG would be capable of generating anosmotic gradient sufficient to allow the movement of water ina basal to apical direction, that is, into the lumen (Delporteet al., 1997). We speculated that the gradient would be based onforming potassium bicarbonate in the lumen: potassium enteringthe lumen in exchange for a proton via a potassium-protonexchanger present in the apical membranes of duct cells. Wefurther hypothesized that all that was lacking for the duct cellsto secrete fluid in an IR-damaged gland was a facilitated water-permeability pathway, a water channel protein.

We constructed an Ad5 vector encoding human AQP1(AdhAQP1) and showed that this vector leads to a dramaticincrease in fluid secretion when administered 90 or 120 daysafter IR in rats (Delporte et al., 1997) or miniature pigs (Shanet al., 2005; Table 4), to ~80% of control levels when measured3 days after transduction. A control Ad5 vector was withoutany significant effect on salivary flow. Additionally, afteradministration of AdhAQP1 to SGs, no significant toxicologi-cal effects were observed, that is, in multiple measured clinicalchemistry and hematology values (Zheng et al., 2006). Basedon these aggregate results, we developed a phase 1 (vectorsafety with some efficacy measures) clinical trial protocol totest AdhAQP1 in patients who received IR for head and neckcancer at least 5 years previously. Although Ad5 vectors onlylead to transient gene expression, because of the generatedimmune response, we chose this type of vector because verylittle is known about human ductal cell physiology. We assumethat human duct cells generally will be similar to those of rats

Vol 36, No. 1, 2008 GENE THERAPY 99

TABLE 3.—Some general characteristics of serotype 5 adenoviral and serotype 2 adeno-associated viral vectors.a

Characteristic Ad5 AAV2

Genome size 37 kb 4.7 kbDNA Double-stranded Single-strandedVirus particles Labile StableCellular targets Acinar, duct DuctTransgene expression High ModestStability of expression No YesImmune response Potent ModestPackaging (recombinant) Easy Laborious

aAd5 vectors are especially useful for experiments when high transient transgene expres-sion 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: serotype5 adenovirus; AAV2: serotype 2 adeno-associated virus; kb: kilobases.

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and miniature pigs, that is, also able to generate an osmotic gra-dient and fluid flow as described above. However, we do notknow that. In the event that human ductal cells are incapable ofthis response, the AdhAQP1 presence in the tissue will be rel-atively limited because of the immune response, which we con-sider an important safety consideration in the absence of anybenefit. However, if irradiated human SGs are able to secretefluid following AQP1 gene transfer, we have developed an AAV2vector capable of mediating long-term AQP1 expression, andpresumably, providing patients with the stable SG repairrequired. 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 candidia-sis remains a common opportunistic infection observed amongimmunosuppressed patients. In HIV-1–infected patients,this can lead to significant morbidity (Sroussi et al., 2007).Antifungal azole-type drugs are the principal management toolfor such candidal infections. However, the occurrence of azole-resistant Candida species necessitates the development ofalternative treatment strategies.

Histatins are a family of histidine-rich, cationic peptidescomposed of up to 38 amino acids. They are secreted by theSGs of humans and some primates and are a major componentof the innate host nonimmune defense system in the oral cav-ity against bacteria and fungal infections. The importance ofhistatins in azole-resistant candidiasis is twofold. Histatin lev-els in saliva are reduced in HIV-1–infected patients. Secondly,the mechanism of action of histatins in targeting candidalspecies is distinctly different from that of azole-type drugs.While azole drugs inhibit the synthesis of ergosterol, a majorplasma-membrane sterol (Amerongen et al., 2002), histatinsact by binding to the ergosterol present in the fungal mem-brane. We reasoned that transfer of the histatin-3 cDNA to SGswould result in an increased secretion of histatins in the oralcavity (O’Connell et al., 1996) and be useful in managing azole-resistant candidal species.

In animal model studies, we successfully expressed histatin-3 in rat SGs using an Ad5 vector (AdCMVH3). Theconcentration of histatin-3 in rat submandibular-gland salivacollected from treated rats 3 days after transduction with theAdCMVH3 was as high as 1 mg/ml, with a mean value of 302µg/ml. The fungicidal activity of the recombinant histatin-3mediated by the AdCMVH3 vector was tested in vitro in atimed-kill assay. At a concentration of 100 µg/ml, 90% of theazole-resistant Candida albicans were killed in 60 minutes(O’Connell et al., 1996).

Autoimmune Disorders

Sjogren’s syndrome (SS) is the second most commonautoimmune disease in the United States, affecting between 1million and 4 million persons, primarily female (~90%). Theetiology of SS is unclear, and current treatment is only pallia-tive. SS is characterized by the presence of a focal lymphoidcell infiltration in the salivary and lacrimal glands, althoughother organs may also be involved (Pillemer et al., 2001). Inthe absence of any suitable conventional treatments, we havesuggested that gene therapy may be beneficial for SS patients.We have hypothesized that transfer of immunomodulatorygenes into SGs may reduce the autoimmune sialadenitis andlead to increased salivation as well as symptomatic relief (Koket al., 2003). For example, the transfer of genes encoding anti-inflammatory cytokines such as interleukin-10 (IL-10) or vasoac-tive intestinal peptide (VIP; Lodde et al., 2006) could lead to adecrease in the expression of proinflammatory cytokines, andthus, protect SGs and preserve their secretory function.

To test this hypothesis, we used a common animal model ofSS, the female nonobese diabetic (NOD) mouse. We deliveredthe human (h) IL-10 and VIP cDNAs using AAV2 vectorsbecause they provide stable transgene expression with littleimmune reactivity. Both AAVhIL-10 and AAVhVIP, as well as a control vector, AAVLacZ encoding β-galactosidase, wereadministered locally via retrograde cannulation of the sub-mandibular glands. We compared salivary flow and sialadenitis~8–12 weeks later. Administration of AAVhIL-10 led to preser-vation of salivary flow rates as well as a reduction of thefocal autoimmune sialadenitis (Table 5; Kok et al., 2003).Administration of AAVhVIP also resulted in a preservation ofsalivary flow; however, no reduction of the focal sialadenitiswas observed with this transgene (Lodde et al., 2006).

These initial studies show that immunomodulatory genetransfer may be useful in managing the autoimmune sialadeni-tis and resultant salivary hypofunction that occur in SS patients.Nonetheless, since we do not understand SS pathogenesis, thisgene-transfer strategy is nonspecific and still requires consid-erable study.

Systemic Protein Deficiencies

As mentioned previously, SGs show several features that arecommon to many endocrine glands, particularly the ability toproduce high levels of protein for export and the ability to

100 COTRIM AND BAUM TOXICOLOGIC PATHOLOGY

TABLE 4.—Effect of AdhAQP1 on salivary secretion in irradiated rats and miniature pigs.a

Salivary flow Irradiation (% of control)

Species dose (Gy) Post-IR Post-AdhAQP1

Rat 21 ~35 ~84Miniature pig 20 ~20 ~81

aThis table provides a summary of data previously reported in Delporte et al. (1997) andShan et al. (2005). Before administration of AdhAQP1, IR resulted in a marked decreasein salivary flow to ~35% (rats) or ~20% (miniature pigs) of control values. Three daysafter 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 follow-ing 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 vectorencoding human aquaporin-1; IR: irradiation.

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secrete proteins into the bloodstream. We have suggested atherapeutic application to take advantage of these features: thetreatment of systemic single-protein deficiency disorders (Baumet al., 2004). Current treatment of these conditions involves theregular administration of a recombinant protein by bolus injec-tion (e.g., insulin for diabetes mellitus and erythropoietin [Epo]for anemias related to chronic renal failure). For example, manyof our studies have involved transferring the cDNA for Epo(Voutetakis et al., 2004; Voutetakis et al., 2007). Epo is producedin kidney epithelial cells and secreted by the constitutive secre-tory 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 vectorsencoding either human or rhesus Epo (AAV2hEpo; AAV2rhEpo)to transfer the Epo cDNA into the SGs of mice and rhesusmacaques. After male mice received 109 particles of AAV2hEpointo their submandibular glands, serum Epo reached maximumlevels by 8 to 12 weeks and remained relatively stable for 54weeks (the longest time studied). Hematocrit levelswere similarlyincreased. In male mice, Epo is secreted almost entirely into thebloodstream (160:1; Table 6; Voutetakis et al., 2004). After deliv-ery of AAV2rhEpo (3 × 1011 particles/gland) to parotid glands ofrhesus macaques, rhesus Epo expression increased significantlyafter 1 week, and levels remained relatively stable in serum andsaliva after 6 months (the longest time studied). When total rhe-sus Epo levels were measured, most rhesus Epo also was foundin serum, but at a much lower ratio than found in mice (~7:1serum/saliva ratio). While there is still a need to understand whatdirects transgenic secretory protein sorting into either serum orsaliva, it appears from our studies that use of SGs as a targetdepot organ for gene transfer to treat systemic single-proteindeficiency disorders has significant clinical potential.

PROBLEMS AND PROSPECTS

Since the first clinical gene-therapy trial was conducted(Rosenberg et al., 1990), much attention and considerablepromise has been given to the field. There has been substantial

public- and private-sector investment, as well as increasinglyhigher levels of research activity. Numerous preclinical animal-model studies have provided proofs of concept for multiplepotential clinical applications. Also, major advances have beenmade in understanding vector biology and improving vectordesign and production.

However, clinical progress has been slow. A major setbackfor the field occurred in September 1999, when a widely pub-licized death resulting from a gene-therapy trial was reported(Raper et al., 2003). Jesse Gelsinger, an 18-year-old man, diedin a clinical trial at the University of Pennsylvania, which useda modified Ad5 vector to deliver the gene for ornithine decar-boxylase, a deficient hepatic enzyme. According to an inves-tigation by the university, Gelsinger died from a massive immunereaction to the Ad5 vector. This widely publicized case ledto congressional and Food and Drug Administration (FDA)hearings on the conduct of clinical gene-therapy trials as wellas a transient hold, subsequently lifted, on all adenoviral-vectorclinical trials. An investigation by the FDA found numerouspossible violations in the way that this clinical trial had beenconducted and monitored. After 5 years of investigations, inFebruary 2005, the case was settled. As a result of the Gelsingercase, gene therapy experienced an intense phase of criticismand skepticism. Clearly, mistakes were made in that particularclinical trial, and as an appropriate outcome, all gene-therapytrials are now subject to much tighter regulation by the NationalInstitutes of Health (NIH) and FDA.

Fortunately for the gene therapy field, less than 1 year afterGelsinger died, the first report of a dramatically successful gene-therapy trial was published. In 2000, Cavazzana-Calvo and hercolleagues in Paris described results from a study involving twochildren suffering from a severe combined immunodeficiencydisorder (SCID-XI), which had restricted them to life in anisolated environment (Cavazzana-Calvo et al., 2000). These inves-tigators used a MoMLV vector to transfer a curative gene (γccytokine 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 normallives. Subsequently, several other patients were treated and appar-ently cured in these studies. However, there was a downside. Of~11 early patients treated with the MoMLV vector, 3 developedleukemia directly as a result of the gene-transfer procedure

Vol 36, No. 1, 2008 GENE THERAPY 101

TABLE 5.—Effect of IL-10 and VIP cDNA transfer on salivary flow and inflammatory focus score in NOD mice.a

Baseline Salivary Treatment salivary flow flow at Focusgroup (8 weeks) Treatment 16 weeks score

IL-10 186 µl rAAVhIL-10 168 µl 1.4125 µl rAAVLacZ 28 µl 3.0

VIP 3.8 µl/g rAAVhVIP 4.3 µl 1.93.8 µl/g rAAVLacZ 2.05 µl 1.85

aFor 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 glandularinflammatory infiltrates (focus score; Greenspan et al., 1974) compared to controls treatedwith rAAVLacZ. For all VIP (vasoactive intestinal peptide) experiments, saliva output waspresented as microliters of saliva per gram of animal weight (Lodde et al., 2006). VIP hadno effect on the presence of inflammatory infiltrates, but it was able to prolong prediseasesalivary secretion levels compared to controls treated with rAAVLacZ. See text and originalreferences for more details.

TABLE 6.—Total amount of erythropoietin secretion into serum and saliva after rAAV2Epo transduction of murine

and macaque salivary glands.a

Serum Salivary Ratio,Species Epo Epo serum:saliva

Mouse 80 0.4 160:1Rhesus macaque 1000 140 7:1

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

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(Fischer et al., 2004). In all of these patients, the MoMLV vectorhad integrated apparently in a nonrandom manner near the LM02(LIM domain only 2) gene. The LM02 gene was activated bythis integration, and leukemia was the result. The patients weretreated for the leukemia, and a large, collaborative scientific effortbegan to understand what mechanisms influence MoMLVintegration.

The SCID-XI trial likely reflects the path that gene therapy willfollow during the next 1 to 2 decades: success, but with some com-plications. Last year, a somewhat similar scenario occurred in aGerman clinical trial of a MoMLV vector to treat chronic granu-lomatous disease—that is, some clinical success, but subsequently,a patient died (Ott et al., 2006). Also, a clinical trial to correct aclotting disorder, Factor IX deficiency, by hepatic gene transferusing an AAV2 vector recently showed that transient correctionwas possible but quite limited in time because of subsequentimmune reactivity (Manno et al., 2006). These experiences addfurther credence to the general viewpoint offered by Leiden in a1995 editorial that gene therapy is a field in its infancy, and despitesome pitfalls, it is well grounded in fundamental scientific princi-ples with real clinical promise for the future (Leiden, 1995).

ACKNOWLEDGMENT

The authors’ research is supported by the intramural researchprogram of the National Institute of Dental and CraniofacialResearch.

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Baum, B. J., Zheng, C., Cotrim, A. P., Goldsmith, C. M., Atkinson, J. C.,Brahim, J. S., Chiorini, J. A., Voutetakis, A., Leakan, R. A., Van Waes, C.,Mitchell, J. B., Delporte, C., Wang, S., Kaminsky, S. M., and Illei, G. G.(2006). Transfer of the AQP1 cDNA for the correction of radiation-induced salivary hypofunction. Biochim Biophys Acta 1758, 1071-77.

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