Suppression of the Immune Response Against Exogenous Desmoglein 3 in Desmoglein 3 Knockout Mice: An Implication for Gene Therapy

ORIGINAL ARTICLE See Commentary on pages vii and xi

Suppression of the Immune Response Against Exogenous Desmoglein 3 in Desmoglein 3 Knockout Mice: An Implication for Gene Therapy Manabu Ohyama,nz Takayuki Ota,n Miyo Aoki,n Kazuyuki Tsunoda,n Reiko Harada,z Shigeo Koyasu,w Takeji Nishikawa,n and Masayuki Amagain Departments of n Dermatology and wMicrobiology and Immunology, Keio University School of Medicine, Tokyo, Japan; zDivision of Dermatology, Tokyo Electric Power Company Hospital, Tokyo, Japan

Gene therapies for recessive genetic diseases may provoke unwanted immune responses against the introduced gene product because patients, especially those with null mutation of a certain protein, have no tolerance for the protein of interest. This study used desmoglein 3 knockout (Dsg3^/^) mice as a disease model for a genetic defect in DSG3, to investigate whether nonviral gene therapy induces an immune response against Dsg3 and whether the reaction against Dsg3 can be prevented. When mouse Dsg3 cDNA was injected in the skin of Dsg3^/^ mice, 50% of treated Dsg3^/^ mice developed anti-Dsg3 IgG, which can bind native Dsg3 in vivo. To prevent this response, we used an anti-CD40L monoclonal antibody, MR1, which blocks the costimulatory interaction between CD40 and CD40L. To evaluate the e¡ect of MR1, we grafted Dsg3 þ/ þ skin on Dsg3^/^ mice, to mimic stable gene

transfer of Dsg3. After skin grafting, all the recipient Dsg3^/^ mice were treated with either MR1 (n ¼ 8) or control hamster IgG (n ¼ 8). All of the control IgG-treated mice developed circulating anti-Dsg3 IgG about 2 wk after grafting, and IgG deposition was observed on the surfaces of keratinocytes in the grafted Dsg3 þ/ þ skin. Such anti-Dsg3 IgG production was signi¢cantly prevented, however, when the recipient mice were treated with MR1. These ¢ndings suggested that gene therapies for recessive diseases may provoke an immune response against the transgene product, and that the CD40^CD40L interaction might be a reasonable target for e¡ective prevention of such undesirable immune responses, leading, in turn, to a successful gene therapy. Key words: CD154/CD40 ligand/immunosuppression/recessive genodermatosis/skin graft. J Invest Dermatol 120:610 ^615, 2003

S

contrast, little attention has been paid to the immune reaction that might be provoked against the introduced gene products. As skin is a primary immunologic barrier against foreign subjects and contains many antigen-presenting cells (dendritic cells) (Tuting et al, 1998; Larregina and Falo, 2000), the immune response against a transgene product, and the prevention of this response, is a priority issue in gene therapy that is targeted on skin. This problem becomes particularly important when gene therapy is applied to correct a recessive genetic disease caused by null mutation. In these patients, the immune system has never encountered the missing protein. Accordingly, there is no tolerance of what would be a novel protein in such patients. Consequently, any gene product introduced to correct a genetic defect might be recognized as a foreign antigen, which would inevitably result in an unwanted immune response against the introduced gene product (Khavari, 1998; 2000). In contrast, patients with missense mutations may still express endogenous gene product that serves as self-protein to establish self-tolerance although its function is altered. Despite the signi¢cance of this phenomenon, such immune responses in the skin, or in other organs, have not been satisfactorily studied. Several investigators have reported immune reactions against transgene products. As gene delivery was performed using viral vectors, however, it transpires that most of these documented reactions were against the transgene vehicles (Dai et al, 1995; Tripathy et al, 1996; Yang et al, 1996; Ilan et al, 1997; Morral et al, 1997; Stein et al, 1999; 2000). This means that

kin is an attractive target for gene therapy, because it is readily accessible for gene transfer, gene expression is easily observed, and the skin can be removed when an unexpected side-e¡ect occurs (Vogel, 1993; Greenhalgh et al, 1994; Hengge et al, 1995; 1996). As in other organs, the genetic correction of recessive genodermatoses is of great potential value, as only palliative therapies are at present available (Khavari, 1998; 2000; Uitto and Pulkkinen, 2000). Therefore, attempts to develop gene therapies for forms of epidermolysis bullosa (Dellambra et al, 1998; Vailly et al, 1998; Seitz et al, 1999; Chen et al, 2000), X-linked ichthyosis (Jensen et al, 1993; Freiberg et al, 1997), lamellar ichthyosis (Choate et al, 1996), and xeroderma pigmentosum (Zeng et al, 1998) have been reported. In these studies, expression of the intended genes was successful and the impaired protein functions were corrected. Several hurdles remain to be cleared before practical gene therapy can be applied, however. One such problem is how to achieve long-term expression of the introduced gene at therapeutic levels. This matter has attracted great interest and has been investigated intensively. By Manuscript received July 17, 2002; revised October 13, 2002; accepted for publication November 5, 2002 Reprint requests to: Masayuki Amagai, M.D., Ph.D., Department of Dermatology, Keio University School of Medicine, 35 Shinanomachi, Shinjyuku-ku, Tokyo 160 -8582, Japan; Email: [email protected] Abbreviation: Dsg, desmoglein.

0022-202X/03/$15.00  Copyright r 2003 by The Society for Investigative Dermatology, Inc. 610

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the immune response elicited against a transgene product has not yet been evaluated. In addition, only a few studies have dealt with the issue of immune provocation against a corrected selfantigen in a gene therapy model of recessive disease (Ilan et al, 1997; Stein et al, 2000). Therefore, an adequate animal model and immune assay are required in order to further evaluate and prevent such undesirable immune responses. Desmoglein 3 (Dsg3) is an epithelial type transmembrane desmosomal component that belongs to the cadherin gene superfamily; it has been identi¢ed as the autoimmune target of pemphigus vulgaris (Amagai et al, 1991; 1994). In 1997, the Dsg3 knockout (Dsg3^/^) mouse was generated by genetic disruption of DSG3 (Koch et al, 1997). The adult mouse develops crusted erosions on traumatized skin, telogen-speci¢c hair loss, and weight loss due to decreased food intake as a result of painful erosions on the mucous membranes that are manifestations of Dsg3 dysfunction. Recently, we intensively investigated the gross and histopathologic phenotype of Dsg3^/^ mice (Ohyama et al, 2002) in order to characterize their pathophysiology. In addition, we have developed an enzyme-linked immunosorbent assay (ELISA) using recombinant mouse Dsg3, which should prove to be a valuable tool for detecting circulating anti-Dsg3 IgG in mice (Amagai et al, 2000). In this study, using the Dsg3^/^ mouse as a disease model for a genetic defect in DSG3, we investigated whether an undesirable immune response against the transgene product of Dsg3 was actually provoked after naked DNA injection of Dsg3 cDNA in Dsg3^/^ mice. Then, we showed that blockading the CD40^CD40L interaction prevented this undesirable immune response. MATERIALS AND METHODS Mice Dsg3^/^ mice were obtained by mating a male Dsg3^/^ mouse and female Dsg3 þ/^ mice (Koch et al, 1997; Amagai et al, 2000). These mice have a mixed genetic background of 129/SV (H-2b) and C57BL/6 J (H-2b). The C57BL/6 J mice used as skin graft donors were purchased from Japan Clea (Tokyo, Japan). All mice studies were approved by the animal ethics review board of Keio University, Japan. DNA constructs The plasmid pcDNA-mouse Dsg3 (mDsg3) was constructed by subcloning mDsg3 cDNA between the EcoR1 and Sph1 sites of pcDNA1 containing the CMV promoter (Invitrogen, Carlsbad, CA). Dr. Jouni Uitto (Je¡erson Medical College, Philadelphia, PA) generously provided the cDNA. Naked DNA injection Materials for naked DNA injection were prepared and the injection was performed essentially as previously described (Hengge et al, 1995; 1996). The puri¢ed plasmid DNA was diluted to the indicated concentrations with phosphate-bu¡ered saline. Injections into the super¢cial dermis of the footpad or shaved trunk skin were performed using syringes with ultra-¢ne 29G needles (U-100, Japan Becton Dickinson, Tokyo, Japan). The injected volume was 20^50 ml, depending on the site. Ten Dsg3^/^ mice were divided into four groups to undergo gene therapy with pcDNA-mDsg3 naked DNA injection, at doses of 50^200 mg per mouse per injection, and at injection frequencies that ranged from once every 2 wk to twice per week. The indicated quantity of DNA was dissolved in 100 ml of phosphate-bu¡ered saline per injection for each individual mouse. Two C57BL/6 J (Dsg3 þ/ þ ) mice were treated with 200 mg per mouse every 2 wk as controls. Serum was collected at weekly intervals for further analysis. To determine whether anti-Dsg3 IgG binds to the transgene product, pcDNA^mDsg3 (1 mg per ml) was injected into the footpads of a Dsg3^/^ mouse that had produced anti-Dsg3 IgG after naked DNA injection of pcDNA^mDsg3. Biopsy specimens from the footpads were taken 12 h after injection and frozen sections were prepared. These were incubated with 100-fold diluted £uorescein isothiocyanate anti-mouse IgG antibody for 1 h, washed, and embedded for microscopic investigation. Dsg3 expression assay To evaluate the potential of the constructed DNA to express Dsg3 in epidermis, plasmid solution of either pcDNA^ mDsg3 or pcDNA1 (5 or 10 mg per ml for pcDNA^mDsg3, 10 mg per ml for pcDNA1) was injected into one or two sites per footpad (six to eight sites per mouse) in Dsg3^/^ mice (n ¼ 2 for each concentration of DNA). Skin biopsies were performed 18 h after injection. Samples were embedded

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in OCT compound (Sakura Finetechnical, Tokyo, Japan) and cryostat sections of these samples were prepared for direct immuno£uorescence study. Sections were ¢rst incubated for 1 h with anti-mouse Dsg3 monoclonal antibody, AK9 (Tsunoda et al, manuscript in preparation). Sections were then incubated for 1 h with 100-fold diluted £uorescein isothiocyanate rabbit anti-mouse IgG antibody (Zymed Laboratories, San Francisco, CA). All these sections were examined using £uorescence microscopy (Eclipse E800, Nikon, Tokyo, Japan). ELISA The production of anti-Dsg3 and anti-Dsg1 IgG was evaluated by ELISA, using recombinant mouse Dsg3 and Dsg1, as previously reported (Ohyama et al, 2002). Brie£y, diluted sera (500-fold for sera of mice treated with naked DNA injection of Dgs3, 1000-fold for sera of Dsg3^/^ mice with Dsg3 þ/ þ skin graft) were incubated for 1 h on ELISA plates coated with 2.5 mg per ml mouse rDsg3, or rDsg1, expressing the entire extracellular domains using a baculovirus expression system (Amagai et al, 2000). After washing, the plates were incubated with 5000-fold diluted peroxidase-labeled goat anti-mouse IgG (Zymed Laboratories) for 1 h. After the second wash, color was developed using a solution containing equal amounts of tetramethylbenzidine and hydrogen peroxide (Medical Biological Laboratories, Nagano, Japan) for 30 min, and stopped by adding 100 ml of 4 N H2SO4. The OD was measured at 450 nm using a microplate reader (Bio-Rad Laboratories, Hercules, CA). We calculated the mean and standard deviation for this ELISA using OD values of control sera from 12 normal mice. Based on the mean þ 3SD, we set the cut-o¡ values for de¢nite positive reactivity as 0.090 for Dsg3 IgG and 0.067 for Dsg1 IgG. Sera were tested at least twice for Dsg3 ELISA and once for Dsg1 ELISA. Grafting Dsg3 þ/ þ skin onto Dsg3^/^ mice For the skin graft procedure, 8  8 mm pieces of dorsal skin were removed from sacri¢ced 6 - to 12-wk-old wild-type C57BL/6 J mice and placed in sterile saline. Seven 8- to 12-wk-old Dsg3^/^ mice, which have a mixed genetic background of 129/SV and C57BL/6 J, were carefully anesthetized with dimethyl ether and the dorsal skin was replaced with the skin graft. The graft was attached with nylon sutures. After topical application of ointment containing gentamicin sulfate (Schering-Plough, Osaka, Japan), the surgical site was dressed with gauze and an elastic bandage for 7^10 d. Typically, successful adaptation was obtained and grafts were maintained at the surgical site for about 3 wk. Then, grafts began to necrotize and were spontaneously removed within 4 wk probably due to allo-reaction. Then a second piece of Dsg3 þ/ þ skin was grafted onto the Dsg3^/^ mice to evaluate the in vivo reaction of anti-Dsg3 IgG. Serum samples from these mice with grafts were collected once a week. Sera diluted 1 : 1000 were analyzed for anti-Dsg1 and anti-Dsg3 IgG with ELISA using recombinant mouse Dsg1 and Dsg3. MR1/hamster IgG treatment Hamster anti-mouse CD40L monoclonal antibody MR1 was prepared according to the protocol described in the original report (Noelle et al, 1992). Hybridomas producing MR1 were injected intraperitoneally into hamsters and the collected ascites was puri¢ed by ion-exchange high performance liquid chromatography to obtain MR1. The Dsg3^/^ mice with skin grafts were injected intraperitoneally with either MR1 (n ¼ 8) or control hamster IgG (n ¼ 8) (Cappel Product, Aurora, OH). The dose of MR1 was determined based on previous reports (Durie et al, 1993; Yang et al, 1996; Stein et al, 1999; 2000) as well as our pilot study. In our pilot study 125 mg to 1 mg per mouse were tested at various schedules, but 125^250 mg per mouse showed only partial suppression. Therefore, expecting substantial suppression, we used 1 mg per mouse at day 0 and 0.5 mg per mouse at days 2, 4, 7, 14, 21, and 28. Serum samples from these mice were collected once a week and analyzed with ELISA using mouse rDsg3 and mouse rDsg1, as described above. To evaluate whether intercellular deposition of IgG in the epidermis of the skin graft occurred, E2  6 mm biopsy samples were taken from the second skin grafts, about 1 wk after grafting, and examined for IgG deposition by direct immuno£uorescence. Seven grafts from eight MR1-treated mice and ¢ve grafts from eight control-IgG-treated mice were investigated in this study.

RESULTS Development of anti-Dsg3 IgG in Dsg3^/^ mice after naked DNA injection of Dsg3 cDNA We used Dsg3^/^ mice as a model for recessive genodermatosis. To correct the defect in Dsg3 expression, we introduced Dsg3 cDNA by naked DNA injection. The proper expression of Dsg3 in the epidermis was con¢rmed by staining skin sections from the injection site for

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Figure 1. Dsg3 expressed on keratinocyte cell surfaces following naked DNA injection. Positive staining for Dsg3 was observed on the surfaces of keratinocytes at the site of pcDNAmDsg3 injection into a footpad of a Dsg3^/^ mouse (a), but not at the site of control DNA injection (b). Scale bar: 50 mm.

Dsg3. Distinct positive staining for Dsg3 was observed on the surfaces of keratinocytes at the sites injected with 5^10 mg per ml pcDNAmDsg3 (Fig 1a), whereas no such cell surface staining for Dsg3 was observed at the sites injected with pcDNA1 (Fig 1b). We then determined whether Dsg3^/^ mice develop anti-Dsg3 IgG after naked DNA injection of pcDNAmDsg3. Dsg3^/^ mice were injected with various amounts of pcDNAmDsg3 in various schedules (Table I), and serial serum samples were evaluated by mouse Dsg3 as well as mouse Dsg1 ELISA. Each mouse in the three groups of Dsg3^/^ mice injected with 50 mg pcDNAmDsg3 per mouse twice per week (n ¼ 2), or 100 mg per mouse once (n ¼ 4) or twice (n ¼ 2) developed anti-Dsg3 IgG at day 32. Although the anti-Dsg3 IgG level detected in these groups was relatively low, more distinct anti-Dsg3 IgG production was observed in two out of two Dsg3^/^ mice injected with 200 mg per mouse every 2 wk (Fig 2a). Therefore, anti-Dsg3 IgG production was observed altogether in ¢ve of the 10 mice examined when 500 mg per mouse or more total naked DNA was injected. The sera of these mice with anti-Dsg3 IgG showed no apparent reactivity against mouse Dsg1 at a mean OD value of 0.04670.011 (n ¼ 5). In contrast to Dsg3^/^ mice, no anti-Dsg3 IgG production was observed in two wild-type mice (C57BL/6 J) injected with the same amount of DNA (Fig 2a). Next, we determined whether the anti-Dsg3 IgG produced after the naked DNA injection binds to the exogenous Dsg3 expressed by naked DNA injection in Dsg3^/^ mice. Biopsies were taken at the injection sites (footpad) of Dsg3^/^ mice producing anti-Dsg3 IgG after naked DNA injection, and in vivo IgG deposition was examined by direct immuno£uorescence (Fig 2b). Obvious in vivo IgG deposition was noticed on keratinocyte cell surfaces in six out of 36 biopsy sections. This indicated that the anti-Dsg3 IgG produced after the naked DNA injection of Dsg3 cDNA could bind to gene therapy transgene products in vivo.

Figure 2. Anti-Dsg3 IgG production in Dsg3^/^ mice after naked DNA injection. (a) Anti-Dsg3 IgG production was measured by ELISA over time after naked DNA injection. Dsg3^/^ mice injected with pcDNAmDsg3 (200 mg per mouse, every 2 wk) developed anti-Dsg3 IgG. No such IgG elevation was observed in Dsg3 þ/ þ control mice, however. (b) In vivo deposition of IgG in the epidermis was observed (arrow) at the site of pcDNAmDsg3 injection in a Dsg3^/^ mouse that was producing anti-Dsg3 IgG. Scale bar: 50 mm.

Blockade of CD40L prevented anti-Dsg3 IgG production in Dsg3^/^ mice with Dsg3 þ/ þ skin graft Achieving stable gene expression of the transgene is one of the ultimate goals of gene therapy. To mimic the state of stable gene expression, we grafted skin from wild-type Dsg3 þ/ þ mice onto Dsg3^/^ mice instead of injecting Dsg3 cDNA. In this model, production of anti-Dsg3 IgG was observed in all of the mice tested 2 wk after skin grafting, and the titers increased thereafter (Fig 3, n ¼ 7). The sera of these mice did not show any cross-

Table I. Production of anti-Dsg3 IgG in Dsg3^/^ mice after naked DNA injection of pcDNAmDsg3 Dose per injection per mouse

n

Schedule

Mice with anti-Dsg3 IgG

Total DNA injecteda

Time requiredb

50 mg 100 mg 100 mg 200 mg

2 4 2 2

Twice per week Once per week Twice per week Once every 2 wk

1 1 1 2

500 mg 500 mg 1000 mg 600/800 mg

32 d 32 d 32 d 35/56 d

a

Total DNA injected when anti-Dsg3 IgG production was observed. Time required for development of anti-Dsg3 IgG.

b

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di¡erence in the titers in the two groups was statistically signi¢cant (p ¼ 0.0005, Mann^Whitney analysis at day 28). Furthermore, direct immuno£uorescence of the skin grafts on MR1/control IgG-treated mice was used to look for IgG deposition on the keratinocyte cell surfaces (Fig 5a). Distinct IgG deposition on the surfaces of keratinocytes in the epidermis and telogen hair follicles was observed in the skin grafts on the control IgG-treated mice (Fig 5b, n ¼ 5), whereas no apparent in vivo IgG deposition was observed in the skin grafts of the MR1-treated mice (Fig 5c, n ¼ 7). These ¢ndings indicate that the Dsg3 þ/ þ skin graft induced anti-Dsg3 IgG production in Dsg3^/^ mice in an antigen-speci¢c way, and that the blockade of CD40L by MR1 e⁄ciently prevented the unwanted antiDsg3 IgG production in this model. DISCUSSION

Figure 3. Dsg3 þ/ þ skin grafting onto Dsg3^/^ mice elicited antiDsg3 IgG production. (a) A Dsg3^/^ mouse with a Dsg3 þ/ þ skin graft, at day 10 after the skin graft. Patchy hair loss and erosions are the normal characteristics of Dsg3^/^ mice. Dsg3^/^ mice with the Dsg3 þ/ þ skin graft (n ¼ 7) produced anti-Dsg3 IgG within 2 wk after the graft, and this showed no cross-reactivity against Dsg1, as con¢rmed by ELISA (b). Error bars indicate the mean7SEM.

reactivity with Dsg1. As the Dsg3^/^ mice used in this study had a mixed genetic background of 129/SV and C57BL/6 J, the grafted Dsg3 þ/ þ skin of the C57BL/6 J mice was rejected about 3 wk after grafting. This earlier production of anti-Dsg3 IgG observed in these Dsg3 þ/ þ skin grafted Dsg3^/^ mice, compared to that in mice with naked DNA injection, might be partly explained by an adjuvant e¡ect accompanying rejection of the skin graft. We used these skin-grafted Dsg3^/^ mice as a model for stable gene transfer of Dsg3 in Dsg3^/^ mice and examined whether blockade of the CD40L^CD40 interaction by anti-CD40L monoclonal antibody, MR1, could prevent unwanted anti-Dsg3 IgG production in Dsg3^/^ mice. Dsg3^/^ mice with Dsg3 þ/ þ skin were treated with either MR1 (n ¼ 8) or control hamster IgG (n ¼ 8) at days 0, 2, 4, 7, 14, 21, and 28 following the skin graft. Serial serum samples from these mice were analyzed with mouse Dsg3 ELISA (Table II, Fig 4). The production of antiDsg3 IgG was detected in seven of eight mice treated with control IgG at day 14 and in eight of eight mice at day 21 and was maintained as long as observed until day 42. No apparent cross-reactivity against mouse Dsg1 was detected in those mice (mean OD value ¼ 0.01970.006). In contrast, none of the mice injected with MR1 showed apparent anti-Dsg3 IgG production at day 14. Although some anti-Dsg3 IgG production was noted after day 28 in the mice injected with MR1, the titers were signi¢cantly lower than in mice injected with control IgG. The

In this study, we employed Dsg3^/^ mice as an animal model of recessive genodermatosis. To simulate gene delivery, we used naked DNA injection in order to evaluate the immune reaction against a transgene product in the absence of a vehicle, such as viral vectors. In this study, Dsg3 was successfully expressed in the epidermis of Dsg3^/^ mice with naked DNA injection of pcDNAmDsg3. Some of the injected Dsg3^/^ mice developed anti-Dsg3 IgG, which showed no cross-reactivity with Dsg1. In contrast, such IgG production was not observed in Dsg3 þ/ þ control mice. Furthermore, the resulting anti-Dsg3 IgG bound in vivo to the Dsg3 expressed by naked DNA injection, as IgG deposition was observed on the surfaces of keratinocytes at the sites of DNA injection. Although it is not clear whether induced anti-Dsg3 IgG blocks the function of Dsg3, these ¢ndings suggested that the immune response provoked by gene therapy might impair the function of the introduced gene product itself. Therefore, the establishment of an adequate way to prevent such an immune reaction would be bene¢cial to successful gene therapy for recessive genetic diseases with null mutation, where the absence of tolerance against the introduced gene product is likely to elicit an unfavorable immune response. Several approaches have been proposed as a means of overcoming the immune response against a transgene product or vector in gene therapy models using viral vectors. Such strategies include inducing central tolerance by intrathymic inoculation of adenoviral antigens (Ilan et al, 1996), inducing oral tolerance by feeding adenoviral antigens (Ilan et al, 1997), utilizing immunosuppressive agents, such as cyclosporine or cyclophosphamide (Dai et al, 1995), and blocking costimulatory signals between CD28B7 (Kay et al, 1995) or CD40CD40L (Yang et al, 1996; Stein et al, 1999; 2000). CD40 is a glycoprotein member of the nerve growth factor receptor/tumor necrosis factor receptor family, which is expressed mainly on B cells and antigen-presenting cells, whereas its counterpart, CD40L, is transiently expressed on activated CD4 þ T helper cells (Datta and Kalled, 1997). The CD40CD40L pathway, which upregulates B7 on antigen-presenting cells (Ranheim and Kipps, 1993; Roy et al, 1995), is one of the critical costimulatory signals for T cell activation, B cell proliferation, and immunoglobulin secretion (Datta and Kalled, 1997; Yamada and Sayegh, 2002). The blockade of this interaction with anti-CD40L antibody has been used to inhibit the immune response against gene therapy transgene products in lung, liver, or brain (Yang et al, 1996; Stein et al, 1999; 2000). Such blockade of this pathway, however, has never been performed in gene therapy for the skin. Considering the central role of antigen-presenting cells in provoking the immune response in DNA immunization (Tuting et al, 1998), and the functional expression of CD40 on human epidermal Langerhans cells (Peguet-Navarro et al, 1995) and keratinocytes (Denfeld et al, 1996), blockade of the CD40^CD40L interaction with a monoclonal antibody against CD40L seems to be an e¡ective way to prevent an undesirable immune response in cutaneous gene therapy. Indeed, in the Dsg3 þ/ þ skin graft model, anti-Dsg3 IgG production was successfully suppressed in

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Table II. Anti-Dsg3 IgG production (OD450) in MR1/control IgG treated Dsg3^/^ mice with Dsg3 þ/ þ skin graft Mouse

0

MR1-treated mice #1 0.014 #2 0.013 #3 0.013 #4 0.013 #5 0.011 #6 0.010 #7 0.010 #8 0.005 Mean þ SEM 0.011 þ0.001 Control IgG-treated mice #9 0.050 # 10 0.015 # 11 0.014 # 12 0.008 # 13 0.006 # 14 0.006 # 15 0.008 # 16 0.004 Mean þ SEM 0.014 þ 0.005

Time course (d) 28

7

14

21

35

42

0.011 0.016 0.013 0.007 0.005 0.014 0.005 0.003 0.004 þ 0.002

0.009 0.025 0.044 0.049 0.017 0.014 0.002 0.009 0.021 þ0.006

0.002 0.014 0.070 0.043 ND 0.014 0.027 0.098 0.038 þ 0.012

0.017 0.042 0.085 0.600 ND 0.140 0.017 0.600 0.210 þ 0.094

0.003 0.047 0.210 0.780 ND 0.450 0.049 0.600 0.310 þ 0.11

ND 0.620 0.130 0.110 ND 0.590 ND 0.007 0.290 þ 0.130

0.004 0.015 0.012 0.006 0.014 0.007 0.005 0.011 0.009 þ 0.002

1.200 0.580 0.530 0.330 1.160 1.160 0.920 0.084 0.750 þ 0.150

1.520 0.970 1.060 1.300 0.790 1.750 1.300 1.520 1.280 þ 0.110

1.420 0.430 1.300 2.140 0.570 1.760 1.380 1.170 1.270 þ 0.200

1.400 0.700 1.300 2.360 0.600 1.980 1.610 1.110 1.380 þ 0.210

ND 1.310 1.300 1.210 1.860 0.990 ND ND 1.330 þ 0.140

OD values above the cuto¡ values are in bold. ND indicates not determined.

Figure 5. IgG deposition on the surfaces of keratinocytes in Dsg3 þ/ þ grafts on Dsg3^/^ mice and its e⁄cient prevention with MR1. Dsg3^/^ mice with Dsg3 þ/ þ skin grafts were treated with control hamster IgG (a, left) or MR1 (a, right). IgG deposition on keratinocyte cell surfaces was detected in the grafts from control IgG-treated mice (b), whereas this deposition was prevented in the grafts from MR1-treated mice (c). Scale bars: 1 cm for (a); 50 mm for (b), (c).

Figure 4. MR1 e¡ectively suppressed the anti-Dsg3 IgG production provoked by grafting Dsg3 þ/ þ skin on Dsg3^/^ mice. All of the control IgG-treated mice developed anti-Dsg3 IgG about 2 wk after grafting and this IgG production was maintained at high levels for more than 6 wk. This immune reaction was e¡ectively suppressed when recipient mice were treated with MR1. Error bars indicate the mean7SEM. nMean was obtained with ¢ve mice at day 42.

MR1-treated mice, whereas all of the control IgG-treated mice developed the undesired IgG within 2^3 wk, and the titer of IgG was maintained at high levels for more than 6 wk. Furthermore, no in vivo IgG deposition on keratinocyte surfaces was observed in the epidermis of skin grafts on the mice treated with MR1. The advantages of anti-CD40L therapy are that it does not require prior knowledge of the structure of the autoantigen and that its e¡ect is relatively speci¢c because the CD40L molecule is expressed mainly on activated T cells (Datta and Kalled, 1997). Unlike other immunosuppressive agents, such as corticosteroids or cyclosporine, generalized immunosuppression was not observed

in previous murine studies of anti-CD40L antibody (Durie et al, 1993; 1994; Mohan et al, 1995; Early et al, 1996; Gerriset et al, 1996). Although additional evidence is needed before it can be applied in practice, the blockade of the CD40^CD40L interaction with monoclonal antibody appears to be a valuable way to prevent an unwanted immune response against a transgene product, a response that may well be provoked in gene therapy. Taking all these ¢ndings into consideration, we conclude that an unwanted immune response against a desired gene product may be provoked in gene therapy for recessive disease, and that adequate prevention of such an unfavorable immune reaction is required for the practical application of this promising therapy. We would like to thank Dr. ShigeruTanaka for preparing the MR1 and Dr. Jouni Uitto for providing the mouse Dsg3 cDNA.We would also like to thank Mr.Takashi Kimura and all of the technical assistants in the Research Laboratory at the Tokyo Electric Power Company Hospital for their general research support, including animal care, and Ms. Minae Suzuki for performing the immuno£uorescence sectioning. This work was supported by Health Science Research Grants for Research on Speci¢c Diseases from the Ministry of Health, Labor, and Welfare, and by Grants-in-Aid of Scienti¢c Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

VOL. 120, NO. 4 APRIL 2003

BLOCKING IMMUNE RESPONSE IN GENE THERAPY

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