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An ex vivo model for gene therapy of hemophilia B using cultured human oral mucosal epithelium
Materials Science and Engineering C 13 Ž2000. 45–48 www.elsevier.comrlocatermsec
An ex vivo model for gene therapy of hemophilia B using cultured human oral mucosal epithelium Hirokazu Mizuno a,) , Nobuhiko Emi b, Akihiko Abe b, Isao Takahashi c , Hidehiko Saito b, Megumi Matsuno a , Yukio Sumi a , Ken-Ichiro Hata a , Minoru Ueda a a
b
Department of Oral Surgery, Nagoya UniÕersity School of Medicine, 65 Tsuruma-cho, Shouwa-ku, Nagoya 466-8550, Japan First Department of Internal Medicine, Nagoya UniÕersity School of Medicine, 65 Tsuruma-cho, Shouwa-ku, Nagoya 466-8550, Japan c Japan Red Cross Aichi Blood Center, Seto, Japan
Abstract Human oral mucosal cells are important target tissues for regenetic engineering and are sources of epithelial sheets. In this study, we examine the possibility of mucosal epithelial sheet as a target tissue for gene therapy. Human oral mucosal cells were transduced with a retroviral vector carrying human factor IX gene and constructed epithelium after G418 selection with 3T3 cells in vitro. The cultured oral mucosal epithelium membrane was then grafted onto immunodeficient mice. Between 0.6 and 1.8 ng of human factor IX per milliliter was found in mouse plasma, and the production was continued for 23 days in vivo. These results demonstrate a model of ex vivo gene therapy for hemophilia B using gene-modified oral mucosal epithelium. q 2000 Elsevier Science S.A. All rights reserved. Keywords: Hemophilia; Factor IX; Mucosal epithelium; Gene transfer; Retrovirus
1. Introduction Hemophilia B is an inherited, X-linked, recessive hemorrhagic disorder that results from the absence or dysfunction of factor IX. The occurrence of the disease among the population is approximately 1:30,000 male births w1x. Recent advances in recombinant technology have allowed one to transfer the genes of interest into human somatic cells. Correction of genetic defects by gene therapy would be an ideal treatment for inherited disorders such as hemophilia. We have already established a technique producing a large amount of graft from a small segment of oral mucosal tissue and grafted cultural oral mucosal epithelium for more than 100 patients w2x. These cells were easy to manipulate and expand in culture system. Cultured epithelial sheet formed using mucosal cells could maintain cell viability without keratinization under long-term culture conditions w3x. These characteristics fulfill the important requirements for a cell to be the target of ex vivo gene therapy.
) Corresponding author. Tel.: q81-52-744-2348; fax: q81-52-7442352. E-mail address: [email protected] ŽH. Mizuno..
Factor IX gene transfer has been accomplished to various cells or tissues, such as keratinocyte w4–7x, fibroblast w8x or bone marrow cells w9x. We investigated gene transfer to the cultured oral mucosal epithelium as a novel method of hemophilia gene therapy.
2. Materials and methods 2.1. Cell culture Oral mucosa, which was superfluous tissue debrided during surgery, was obtained from the healthy patients treated in the Department of Oral Surgery, Nagoya University Hospital after informed consent by patients. Human mucosal epithelial cells were isolated as previously described w3x. These cells were suspended in keratinocyte growth medium ŽKGM.. It was Humedia-KG2 ŽKurabo, Osaka, Japan. containing 10 mgrml insulin, 0.5 mgrml hydrocortisone, 50 ngrml amphotericin B, 50 mgrml Gentamycin, 0.1 ngrml human recombinant epidermal growth factor ŽrEGF., and 4% vrv bovine pituitary gland extract. Purified mucosal cells were cultured at 378C in a 10% CO 2 incubator. The culture medium was changed every 2 days.
0928-4931r00r$ - see front matter q 2000 Elsevier Science S.A. All rights reserved. PII: S 0 9 2 8 - 4 9 3 1 Ž 0 0 . 0 0 1 7 5 - 2
46
H. Mizuno et al.r Materials Science and Engineering C 13 (2000) 45–48
2.2. Plasmid construction and generation of Õirus
2.5. Measurements of factor IX antigen
The MoMLV-based retroviral vector pLRNL contains the neo-resistance ŽNeor. gene under control of the promoter of Rous sarcoma virus ŽRSV. w10x. A PstI site exists immediately upstream of the RSV promoter. Into this PstI site, PstI fragments containing the entire coding region of human factor IX was inserted, giving rise to the construct pLIXRNL w11x. The plasmid pLIXRNL was transfected to the amphotropic packaging cell line PA317 by calcium phosphate precipitation as described previously w12x, and selected for expression of neomycin resistance gene with G418 Ž400 mgrml; Gibco, Grand Island, NY. for 2 weeks. This virus producer cell was designated as PA317rLIXRNL. The virus titers of each producing cell line to transform 208F cell were approximately 4 = 10 4rml.
Factor IX antigen Žimmunoreactive factor IX. was estimated by a specific ELISA as previously described w14x. A standard normal pooled plasma prepared from 30 healthy donors was arbitrarily defined to contain 100% factor IX activity and 100% factor IX antigen. This normal pooled plasma was estimated to contain 3.2 mgrml of factor IX antigen. The total amount of factor IX antigen secreted into the medium of 48-h cultivation was measured and expressed as nanogram per milliliter per 48 h. Secretion of human factor IX into the bloodstream of athymic mice was measured by the following method. Blood was collected by retro-orbital puncture at intervals into one-tenth volume of 3.8% sodium citrate. Cells were removed by centrifugation, the plasma samples were frozen at y808C, and levels of factor IX were measured by a specific ELISA. We used seven athymic mice for grafting of factor IX secreting epithelium, and showed representative data.
2.3. Transfection for oral mucosal cells and formation of gene modified mucosal epithelium The cultured oral mucosal cells were trypsinized and plated in 35-mm dish at a density of 5 = 10 4 cellsrwell. After overnight incubation, the medium was changed to 2 ml of viral supernatant that contained 5 mgrml of polybrene ŽSigma, St. Louis, MO. for 3 h. After 24 h, transduced cells were selected by growth in a selective medium containing G418 Ž150 mgrml. for 10–14 days. Selected colonies were treated with 0.25% trypsin–EDTA ŽGibco. to isolate single cells and co-cultured with 3T3-J2 cells, which were treated with 4 mgrml of mitomycin C ŽKyowa Hakko Kogyo, Tokyo, Japan.. Half of the medium was changed every 2–3 days with epithelium formation medium ŽEFM. to make stratified epithelial sheet. The EFM was a 3:1 mixture of Dulbecco’s modified Eagle’s medium ŽDMEM. and Ham’s F-12 medium ŽGibco. supplemented with 5% fetal bovine serum ŽHyclone, UT., 5 mgrml insulin, 5 mgrml transferrin, 2 = 10y9 M triiodothyronine, 1 = 10y9 M cholera toxin, 0.4 mgrml hydrocortisone, 100 Urml penicillin ŽSigma., 0.1 mgrml kanamycin, 0.25 mgrml amphotericin B and 10 ngrml rEGF.
3. Results 3.1. Production of factor IX antigen in conditioned medium According to our established method, human mucosal cells were transduced with LIXRNL and selected with G418 for 2 weeks. Then selected oral mucosal cells and 3T3-J2 were mixed and co-cultured in mixed medium of
2.4. Grafting to athymic mice Stratified gene-modified epithelial sheet was detached as an intact epithelium with 400 PUrml Dispase ŽGibco. and washed twice with isotonic sodium chloride solution. The grafting technique was based on half of the medium and was changed every 2–3 days with EFM to make stratified epithelial sheet. The grafting technique was based on Barrandon et al.’s w13x method. The graft was inserted under a full-thickness skin flap on the back of the athymic mouse Ž4–5 weeks old BALBrc nurnu; SLC, Hamamatsu, Shizuoka, Japan. over the silicone sheet. The basal side of the epithelium came in contact with the inner side of the mouse skin.
Fig. 1. Human factor IX in conditioned medium. Genetically modified mucosal cells were seeded at density of 1=10 5 cellsrwell on feeder layer in a 35-mm dish. Factor IX producing epithelial sheet was formed after 2 weeks of culture. Medium was collected every 2 or 3 days for 50 days. The total amount of factor IX antigen secreted into the medium was expressed as nanogram per milliliter per 48 h.
H. Mizuno et al.r Materials Science and Engineering C 13 (2000) 45–48 Table 1 Human factor IX in plasma from athymic mice Time after grafting Ždays.
Mouse 1 Žngrml.
Mouse 2 Žngrml.
3 7 14 23
1.8 1.1 0.8 0.68
1.5 0.84 0.72 0.6
Factor IX producing oral mucosal epithelial sheets was grafted on two athymic mice. Factor IX was quantified by ELISA of plasma samples taken from two mice at different time-points Ž3, 7, 14, 23 days. after grafting. These results are corrected for untreated mouse plasma background.
KGM and EFM Ž1:1.. Half of the medium was changed every 2–3 days with EFM. It took 2 weeks to form gene-modified epithelial sheet, which was thick enough for grafting. A time course of factor IX secretion was established by measuring factor IX accumulation in the media. The results plotted in Fig. 1 show detectable levels of factor IX production for 50 days. The peak factor IX production was at 30 days after seeding. This result indicated that the transduced gene is expressed for at least 7 weeks in culture. The structure of the epithelial sheet was lost, however, after long-time culture in vitro. Moreover, we have checked the activity of concentrated factor IX produced by gene-modified oral mucosal epithelial cells, and confirmed more than 90% of activity compared to that of a normal pooled plasma. 3.2. Serum concentration of factor IX in grafted mouse After 2 weeks of co-cultivation with feeder layer, cultured epithelium consisting of only the factor IX transduced mucosal cells was completed, and grafted on the athymic mice. The factor IX transduced epithelium was placed basal side up on a 3 = 3 Žcm. silicone sheet. Two sheets were inserted under a full-thickness skin flap of the athymic mouse. Mice were bled by retro-orbital puncture, and levels of factor IX were measured at intervals. The initial levels were in the range of 1.5–1.8 ngrml. Table 1 shows the time course serum level of factor IX of two mice. Factor IX was detected in serum for more than 3 weeks although it was gradually decreased.
4. Discussion We have generated factor IX producing mucosal epithelial sheet for the model of hemophilia treatment. Produced human factor IX was transported to the circulation of the mouse from the gene-modified mucosal epithelial sheet. Primary human mucosal cells were transduced with pLIXRNL retroviral vector as mentioned above. The secretion of factor IX by gene-modified mucosal epithelial sheet was continued for 50 days in vitro. The concentration of
47
expressed factor IX seemed to be proportionate to the number of gene-modified cells in the dish, because cultured epithelial sheet could maintain the shape of sheet for only about 4 weeks. Then the epithelial sheet degraded. It took about 2 weeks to grow full-thickness gene-modified mucosal epithelial sheet consisting of factor IX cDNA transduced mucosal cells after seeding on 3T3-J2 feeder layer. This was twice as long as for normal mucosal cells. We supposed it was due to the cell damage caused by transfection and selection. When generated factor IX producing mucosal epithelial sheet was grafted under a skin flap in nude mouse, the peak levels of human factor IX detected in the plasma were about 1.5–1.8 ngrml. Recently, White et al. w4x demonstrated the stable expression of factor IX from retrovirally transduced primary human keratinocytes. The levels of factor IX were between 0.1 and 2.75 ngrml. The pattern of plasma levels of factor IX are pretty similar to our result. It is necessary to detect the factor IX product in our system for a longer period to see the differences of target cell between keratinocyte and oral mucosal cell. Our results emphasize that human mucosal cells are attractive and practical target cells for gene therapy for hemophilia. If gene-modified oral mucosal epithelium proves to be practical for gene therapy of hemophilia, it could also be used for therapy of other protein deficiency.
Acknowledgements This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, and Grants-in-Aid for Gene Therapy from the Ministry of Health and Welfare of Japan.
References w1x C.D. Forbes, R. Madhok, Genetic disorders of blood coagulation: clinical presentation and management, in: O.D. Ratnoff, C.D. Forbes ŽEds.., Disorders of Hemostasis, Saunders, Philadelphia, PA, 1991, pp. 141–202. w2x M. Ueda, K. Hata, K. Horie, S. Torii, The potential of oral mucosal cells for cultured epithelium: a preliminary report, Ann. Plast. Surg. 35 Ž1995. 498–504. w3x K. Hata, H. Kagami, M. Ueda, M. Matsuyama, The characteristics of cultured mucosal cell sheet as a material for grafting; comparison with cultured epidermal cell sheet, Ann. Plast. Surg. 34 Ž1995. 530–538. w4x S.J. White, S.M. Page, P. Margaritis, G.G. Grownlee, Long-term expression of human clotting factor IX from retrovirally transduced primary human keratinocytes in vivo, Hum. Gene Ther. 9 Ž1998. 1187–1195. w5x E.S. Fenjves, P.M. Schwartz, R.M. Blaese, L.B. Taichman, Keratinocyte gene therapy for adenosine deaminase deficiency: a model approach for inherited metabolic disorders, Hum. Gene Ther. 8 Ž1997. 911–917. w6x S.M. Page, G.G. Brownlee, An ex vivo keratinocyte model for gene therapy of hemophilia B, J. Invest. Dermatol. 109 Ž1997. 139–145.
48
H. Mizuno et al.r Materials Science and Engineering C 13 (2000) 45–48
w7x K.A. Choate, P.A. Khavari, Sustainability of keratinocyte gene transfer and cell survival in vivo, Hum. Gene Ther. 8 Ž1997. 895–901. w8x J. Brauker, G.H. Frost, V. Dwarki, T. Nijiar, R. Chin, V. CarrBrendel, C. Jasunas, C. Hodgett, W. Stone, L.K. Cohen, R.C. Johnson, Sustained expression of high levels of human factor IX from human cells implanted within an immunoisolation device into athymic rodents, Hum. Gene Ther. 9 Ž1998. 879–888. w9x D.R. Hurwitz, M. Kirchgesser, W. Merrill, T. Galanopoulos, C.A. McDrath, S. Emami, M. Hansen, V. Cherington, J.M. Appel, C.B. Bizinkauskas, H.H. Brackmann, P.H. Levine, J.S. Greengerger, Systemic delivery of human growth hormone or human factor IX in dogs by reintroduced genetically modified autologous bone marrow stromal cells, Hum. Gene Ther. 8 Ž1997. 137–156. w10x X. Li, J.K. Yee, J.A. Wolff, T. Friedmann, Factors affecting longterm stability of Moloney murine leukemia virus-based vectors, Virology 171 Ž1989. 331–341.
w11x T. Matsushita, N. Emi, I. Takahashi, J. Takamatsu, H. Saito, Construction and its expression of a new retroviral vector containing a human blood coagulation factor IX cDNA, Thromb. Res. 69 Ž1993. 387–393. w12x N. Emi, T. Friedmann, J.K. Yee, Pseudotype formation of murine leukemia virus with the G protein of vesicular stomatitis, J. Virol. 65 Ž1991. 1202–1207. w13x Y. Barrandon, V. Li, H. Green, New techniques for the grafting of cultured human epidermal cells onto athymic animals, J. Invest. Dermatol. 91 Ž1998. 315–318. w14x I. Takahashi, K. Kato, I. Sugiura, J. Takamatsu, T. Kamiya, H. Saito, Activated factor IX–antithrombin complexes in human blood: quantification by an enzyme-linked differential antibody immunoassay and determination of the in vivo half-life, J. Lab. Clin. Med. 118 Ž1991. 317–325.
An ex vivo model for gene therapy of hemophilia B using cultured human oral mucosal epithelium Hirokazu Mizuno a,) , Nobuhiko Emi b, Akihiko Abe b, Isao Takahashi c , Hidehiko Saito b, Megumi Matsuno a , Yukio Sumi a , Ken-Ichiro Hata a , Minoru Ueda a a
b
Department of Oral Surgery, Nagoya UniÕersity School of Medicine, 65 Tsuruma-cho, Shouwa-ku, Nagoya 466-8550, Japan First Department of Internal Medicine, Nagoya UniÕersity School of Medicine, 65 Tsuruma-cho, Shouwa-ku, Nagoya 466-8550, Japan c Japan Red Cross Aichi Blood Center, Seto, Japan
Abstract Human oral mucosal cells are important target tissues for regenetic engineering and are sources of epithelial sheets. In this study, we examine the possibility of mucosal epithelial sheet as a target tissue for gene therapy. Human oral mucosal cells were transduced with a retroviral vector carrying human factor IX gene and constructed epithelium after G418 selection with 3T3 cells in vitro. The cultured oral mucosal epithelium membrane was then grafted onto immunodeficient mice. Between 0.6 and 1.8 ng of human factor IX per milliliter was found in mouse plasma, and the production was continued for 23 days in vivo. These results demonstrate a model of ex vivo gene therapy for hemophilia B using gene-modified oral mucosal epithelium. q 2000 Elsevier Science S.A. All rights reserved. Keywords: Hemophilia; Factor IX; Mucosal epithelium; Gene transfer; Retrovirus
1. Introduction Hemophilia B is an inherited, X-linked, recessive hemorrhagic disorder that results from the absence or dysfunction of factor IX. The occurrence of the disease among the population is approximately 1:30,000 male births w1x. Recent advances in recombinant technology have allowed one to transfer the genes of interest into human somatic cells. Correction of genetic defects by gene therapy would be an ideal treatment for inherited disorders such as hemophilia. We have already established a technique producing a large amount of graft from a small segment of oral mucosal tissue and grafted cultural oral mucosal epithelium for more than 100 patients w2x. These cells were easy to manipulate and expand in culture system. Cultured epithelial sheet formed using mucosal cells could maintain cell viability without keratinization under long-term culture conditions w3x. These characteristics fulfill the important requirements for a cell to be the target of ex vivo gene therapy.
) Corresponding author. Tel.: q81-52-744-2348; fax: q81-52-7442352. E-mail address: [email protected] ŽH. Mizuno..
Factor IX gene transfer has been accomplished to various cells or tissues, such as keratinocyte w4–7x, fibroblast w8x or bone marrow cells w9x. We investigated gene transfer to the cultured oral mucosal epithelium as a novel method of hemophilia gene therapy.
2. Materials and methods 2.1. Cell culture Oral mucosa, which was superfluous tissue debrided during surgery, was obtained from the healthy patients treated in the Department of Oral Surgery, Nagoya University Hospital after informed consent by patients. Human mucosal epithelial cells were isolated as previously described w3x. These cells were suspended in keratinocyte growth medium ŽKGM.. It was Humedia-KG2 ŽKurabo, Osaka, Japan. containing 10 mgrml insulin, 0.5 mgrml hydrocortisone, 50 ngrml amphotericin B, 50 mgrml Gentamycin, 0.1 ngrml human recombinant epidermal growth factor ŽrEGF., and 4% vrv bovine pituitary gland extract. Purified mucosal cells were cultured at 378C in a 10% CO 2 incubator. The culture medium was changed every 2 days.
0928-4931r00r$ - see front matter q 2000 Elsevier Science S.A. All rights reserved. PII: S 0 9 2 8 - 4 9 3 1 Ž 0 0 . 0 0 1 7 5 - 2
46
H. Mizuno et al.r Materials Science and Engineering C 13 (2000) 45–48
2.2. Plasmid construction and generation of Õirus
2.5. Measurements of factor IX antigen
The MoMLV-based retroviral vector pLRNL contains the neo-resistance ŽNeor. gene under control of the promoter of Rous sarcoma virus ŽRSV. w10x. A PstI site exists immediately upstream of the RSV promoter. Into this PstI site, PstI fragments containing the entire coding region of human factor IX was inserted, giving rise to the construct pLIXRNL w11x. The plasmid pLIXRNL was transfected to the amphotropic packaging cell line PA317 by calcium phosphate precipitation as described previously w12x, and selected for expression of neomycin resistance gene with G418 Ž400 mgrml; Gibco, Grand Island, NY. for 2 weeks. This virus producer cell was designated as PA317rLIXRNL. The virus titers of each producing cell line to transform 208F cell were approximately 4 = 10 4rml.
Factor IX antigen Žimmunoreactive factor IX. was estimated by a specific ELISA as previously described w14x. A standard normal pooled plasma prepared from 30 healthy donors was arbitrarily defined to contain 100% factor IX activity and 100% factor IX antigen. This normal pooled plasma was estimated to contain 3.2 mgrml of factor IX antigen. The total amount of factor IX antigen secreted into the medium of 48-h cultivation was measured and expressed as nanogram per milliliter per 48 h. Secretion of human factor IX into the bloodstream of athymic mice was measured by the following method. Blood was collected by retro-orbital puncture at intervals into one-tenth volume of 3.8% sodium citrate. Cells were removed by centrifugation, the plasma samples were frozen at y808C, and levels of factor IX were measured by a specific ELISA. We used seven athymic mice for grafting of factor IX secreting epithelium, and showed representative data.
2.3. Transfection for oral mucosal cells and formation of gene modified mucosal epithelium The cultured oral mucosal cells were trypsinized and plated in 35-mm dish at a density of 5 = 10 4 cellsrwell. After overnight incubation, the medium was changed to 2 ml of viral supernatant that contained 5 mgrml of polybrene ŽSigma, St. Louis, MO. for 3 h. After 24 h, transduced cells were selected by growth in a selective medium containing G418 Ž150 mgrml. for 10–14 days. Selected colonies were treated with 0.25% trypsin–EDTA ŽGibco. to isolate single cells and co-cultured with 3T3-J2 cells, which were treated with 4 mgrml of mitomycin C ŽKyowa Hakko Kogyo, Tokyo, Japan.. Half of the medium was changed every 2–3 days with epithelium formation medium ŽEFM. to make stratified epithelial sheet. The EFM was a 3:1 mixture of Dulbecco’s modified Eagle’s medium ŽDMEM. and Ham’s F-12 medium ŽGibco. supplemented with 5% fetal bovine serum ŽHyclone, UT., 5 mgrml insulin, 5 mgrml transferrin, 2 = 10y9 M triiodothyronine, 1 = 10y9 M cholera toxin, 0.4 mgrml hydrocortisone, 100 Urml penicillin ŽSigma., 0.1 mgrml kanamycin, 0.25 mgrml amphotericin B and 10 ngrml rEGF.
3. Results 3.1. Production of factor IX antigen in conditioned medium According to our established method, human mucosal cells were transduced with LIXRNL and selected with G418 for 2 weeks. Then selected oral mucosal cells and 3T3-J2 were mixed and co-cultured in mixed medium of
2.4. Grafting to athymic mice Stratified gene-modified epithelial sheet was detached as an intact epithelium with 400 PUrml Dispase ŽGibco. and washed twice with isotonic sodium chloride solution. The grafting technique was based on half of the medium and was changed every 2–3 days with EFM to make stratified epithelial sheet. The grafting technique was based on Barrandon et al.’s w13x method. The graft was inserted under a full-thickness skin flap on the back of the athymic mouse Ž4–5 weeks old BALBrc nurnu; SLC, Hamamatsu, Shizuoka, Japan. over the silicone sheet. The basal side of the epithelium came in contact with the inner side of the mouse skin.
Fig. 1. Human factor IX in conditioned medium. Genetically modified mucosal cells were seeded at density of 1=10 5 cellsrwell on feeder layer in a 35-mm dish. Factor IX producing epithelial sheet was formed after 2 weeks of culture. Medium was collected every 2 or 3 days for 50 days. The total amount of factor IX antigen secreted into the medium was expressed as nanogram per milliliter per 48 h.
H. Mizuno et al.r Materials Science and Engineering C 13 (2000) 45–48 Table 1 Human factor IX in plasma from athymic mice Time after grafting Ždays.
Mouse 1 Žngrml.
Mouse 2 Žngrml.
3 7 14 23
1.8 1.1 0.8 0.68
1.5 0.84 0.72 0.6
Factor IX producing oral mucosal epithelial sheets was grafted on two athymic mice. Factor IX was quantified by ELISA of plasma samples taken from two mice at different time-points Ž3, 7, 14, 23 days. after grafting. These results are corrected for untreated mouse plasma background.
KGM and EFM Ž1:1.. Half of the medium was changed every 2–3 days with EFM. It took 2 weeks to form gene-modified epithelial sheet, which was thick enough for grafting. A time course of factor IX secretion was established by measuring factor IX accumulation in the media. The results plotted in Fig. 1 show detectable levels of factor IX production for 50 days. The peak factor IX production was at 30 days after seeding. This result indicated that the transduced gene is expressed for at least 7 weeks in culture. The structure of the epithelial sheet was lost, however, after long-time culture in vitro. Moreover, we have checked the activity of concentrated factor IX produced by gene-modified oral mucosal epithelial cells, and confirmed more than 90% of activity compared to that of a normal pooled plasma. 3.2. Serum concentration of factor IX in grafted mouse After 2 weeks of co-cultivation with feeder layer, cultured epithelium consisting of only the factor IX transduced mucosal cells was completed, and grafted on the athymic mice. The factor IX transduced epithelium was placed basal side up on a 3 = 3 Žcm. silicone sheet. Two sheets were inserted under a full-thickness skin flap of the athymic mouse. Mice were bled by retro-orbital puncture, and levels of factor IX were measured at intervals. The initial levels were in the range of 1.5–1.8 ngrml. Table 1 shows the time course serum level of factor IX of two mice. Factor IX was detected in serum for more than 3 weeks although it was gradually decreased.
4. Discussion We have generated factor IX producing mucosal epithelial sheet for the model of hemophilia treatment. Produced human factor IX was transported to the circulation of the mouse from the gene-modified mucosal epithelial sheet. Primary human mucosal cells were transduced with pLIXRNL retroviral vector as mentioned above. The secretion of factor IX by gene-modified mucosal epithelial sheet was continued for 50 days in vitro. The concentration of
47
expressed factor IX seemed to be proportionate to the number of gene-modified cells in the dish, because cultured epithelial sheet could maintain the shape of sheet for only about 4 weeks. Then the epithelial sheet degraded. It took about 2 weeks to grow full-thickness gene-modified mucosal epithelial sheet consisting of factor IX cDNA transduced mucosal cells after seeding on 3T3-J2 feeder layer. This was twice as long as for normal mucosal cells. We supposed it was due to the cell damage caused by transfection and selection. When generated factor IX producing mucosal epithelial sheet was grafted under a skin flap in nude mouse, the peak levels of human factor IX detected in the plasma were about 1.5–1.8 ngrml. Recently, White et al. w4x demonstrated the stable expression of factor IX from retrovirally transduced primary human keratinocytes. The levels of factor IX were between 0.1 and 2.75 ngrml. The pattern of plasma levels of factor IX are pretty similar to our result. It is necessary to detect the factor IX product in our system for a longer period to see the differences of target cell between keratinocyte and oral mucosal cell. Our results emphasize that human mucosal cells are attractive and practical target cells for gene therapy for hemophilia. If gene-modified oral mucosal epithelium proves to be practical for gene therapy of hemophilia, it could also be used for therapy of other protein deficiency.
Acknowledgements This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, and Grants-in-Aid for Gene Therapy from the Ministry of Health and Welfare of Japan.
References w1x C.D. Forbes, R. Madhok, Genetic disorders of blood coagulation: clinical presentation and management, in: O.D. Ratnoff, C.D. Forbes ŽEds.., Disorders of Hemostasis, Saunders, Philadelphia, PA, 1991, pp. 141–202. w2x M. Ueda, K. Hata, K. Horie, S. Torii, The potential of oral mucosal cells for cultured epithelium: a preliminary report, Ann. Plast. Surg. 35 Ž1995. 498–504. w3x K. Hata, H. Kagami, M. Ueda, M. Matsuyama, The characteristics of cultured mucosal cell sheet as a material for grafting; comparison with cultured epidermal cell sheet, Ann. Plast. Surg. 34 Ž1995. 530–538. w4x S.J. White, S.M. Page, P. Margaritis, G.G. Grownlee, Long-term expression of human clotting factor IX from retrovirally transduced primary human keratinocytes in vivo, Hum. Gene Ther. 9 Ž1998. 1187–1195. w5x E.S. Fenjves, P.M. Schwartz, R.M. Blaese, L.B. Taichman, Keratinocyte gene therapy for adenosine deaminase deficiency: a model approach for inherited metabolic disorders, Hum. Gene Ther. 8 Ž1997. 911–917. w6x S.M. Page, G.G. Brownlee, An ex vivo keratinocyte model for gene therapy of hemophilia B, J. Invest. Dermatol. 109 Ž1997. 139–145.
48
H. Mizuno et al.r Materials Science and Engineering C 13 (2000) 45–48
w7x K.A. Choate, P.A. Khavari, Sustainability of keratinocyte gene transfer and cell survival in vivo, Hum. Gene Ther. 8 Ž1997. 895–901. w8x J. Brauker, G.H. Frost, V. Dwarki, T. Nijiar, R. Chin, V. CarrBrendel, C. Jasunas, C. Hodgett, W. Stone, L.K. Cohen, R.C. Johnson, Sustained expression of high levels of human factor IX from human cells implanted within an immunoisolation device into athymic rodents, Hum. Gene Ther. 9 Ž1998. 879–888. w9x D.R. Hurwitz, M. Kirchgesser, W. Merrill, T. Galanopoulos, C.A. McDrath, S. Emami, M. Hansen, V. Cherington, J.M. Appel, C.B. Bizinkauskas, H.H. Brackmann, P.H. Levine, J.S. Greengerger, Systemic delivery of human growth hormone or human factor IX in dogs by reintroduced genetically modified autologous bone marrow stromal cells, Hum. Gene Ther. 8 Ž1997. 137–156. w10x X. Li, J.K. Yee, J.A. Wolff, T. Friedmann, Factors affecting longterm stability of Moloney murine leukemia virus-based vectors, Virology 171 Ž1989. 331–341.
w11x T. Matsushita, N. Emi, I. Takahashi, J. Takamatsu, H. Saito, Construction and its expression of a new retroviral vector containing a human blood coagulation factor IX cDNA, Thromb. Res. 69 Ž1993. 387–393. w12x N. Emi, T. Friedmann, J.K. Yee, Pseudotype formation of murine leukemia virus with the G protein of vesicular stomatitis, J. Virol. 65 Ž1991. 1202–1207. w13x Y. Barrandon, V. Li, H. Green, New techniques for the grafting of cultured human epidermal cells onto athymic animals, J. Invest. Dermatol. 91 Ž1998. 315–318. w14x I. Takahashi, K. Kato, I. Sugiura, J. Takamatsu, T. Kamiya, H. Saito, Activated factor IX–antithrombin complexes in human blood: quantification by an enzyme-linked differential antibody immunoassay and determination of the in vivo half-life, J. Lab. Clin. Med. 118 Ž1991. 317–325.