Abstract
Retrovirus-mediated gene transfer into adult skin fibroblasts has provided measurable amounts of therapeutic proteins in animal models. However, the major problem emerging from these experiments was a limited time of vector encoded gene expression once transduced cells were engrafted. We hypothesized that sustained transduced gene expression in quiescent fibroblasts in vivo might be obtained by using a fibronectin (Fn) promoter. Fibronectin plays a key role in cell adhesion, migration and wound healing and is up-regulated in quiescent fibroblasts. Retroviral vectors containing human adenosine deaminase (ADA) cDNA linked to rat fibronectin promoter (LNFnA) or viral LTR promoter (LASN) were compared for their ability to express ADA from transduced primary rat skin fibroblasts in vivo. Skin grafts formed from fibroblasts transduced with LNFnA showed strong human ADA enzyme activity from 1 week to 3 months. In contrast, skin grafts containing LASN-transduced fibroblasts tested positive for human ADA for weeks 1 and 2, were faintly positive at week 3 and showed no human ADA expression at 1, 2 and 3 months. Thus, a fibronectin promoter provided sustained transduced gene expression at high levels for at least 3 months in transplanted rat skin fibroblasts, perhaps permitting the targeting of this tissue for human gene therapy.
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References
Miller AD . Human gene therapy comes of age Nature 1992 357: 455–460
Morgan RA, Anderson WF . Human gene therapy Annu Rev Biochem 1993 62: 191–217
Mulligan RC . The basic science of gene therapy Science 1993 260: 926–932
Kohn DB . The current status of gene therapy using hematopoietic stem cells Curr Opin Ped 1995 7: 56–63
Muzyczka N . Use of adeno-associated virus as a general transduction vector for mammalian cells. In: Muzyczka N (ed). Current Topics in Microbiology and Immunology. Viral Expression Vectors, vol 158 Springer-Verlag: New York, 1992, pp97–129
Samulski RJ . Adeno-asssociated virus: integration at a specific chromosomal locus Curr Opin Genet Dev 1993 3: 74–80
Kaplitt MG et al. Long-term gene expression and phenotypic correction using adeno-associated virus vectors in mammalian brain Nat Genet 1994 8: 148–154
Snyder RO et al. Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors Nat Genet 1997 16: 270–276
Adam MA et al. Internal initiation of translation in retroviral vectors carrying picornavirus 5′ nontranslated regions J Virol 1991 65: 4985–4990
Morgan RA et al. Retroviral vectors containing putative internal ribosome entry sites: development of a polycistronic gene transfer system and applications to human gene therapy Nucleic Acids Res 1992 20: 1293–1299
Ramesh N et al. High-titer bicistronic vectors employing foot-and-mouth disease virus internal ribosome entry site Nucleic Acids Res 1996 24: 2697–2700
Barr E, Tripathy S, Leiden JM . Genetically modified myoblasts for the treatment of erythropoietin-responsive anemias. J Cell Biochem 1994; (Suppl 18A): DZ012
Hamamori Y et al. Myoblast transfer of human erythropoietin gene in a mouse model of renal failure J Clin Invest 1995 95: 1808–1813
Naffakh N et al. Long-term secretion of therapeutic proteins from genetically modified skeletal muscles Hum Gene Ther 1996 7: 11–21
Shull RM et al. Myoblast gene therapy in canine mucopolysaccharidosis. I: abrogation by an immune response to alpha-L-iduronidase Hum Gene Ther 1996 7: 1595–1603
Clowes MM et al. Long-term biological response of injured rat carotid artery seeded with smooth muscle cells expressing retrovirally introduced human genes J Clin Invest 1994 93: 644–651
Geary RL et al. Gene transfer in baboons using prosthetic vascular grafts seeded with retrovirally transduced smooth muscle cells: a model for local and systemic gene therapy Hum Gene Ther 1994 5: 1213–1218
Osborne WRA et al. Gene therapy for long-term expression of erythropoietin in rats Proc Natl Acad Sci USA 1995 92: 8055–8058
Ohno T et al. Gene therapy for vascular smooth muscle cell proliferation after arterial injury Science 1994 265: 781–784
Plautz G, Nabel EG, Nabel GJ . Introduction of vascular smooth muscle cells expressing recombinant genes in vivo Circulation 1991 83: 578–583
Lejnieks DV et al. Granulocyte colony-stimulating factor expression from transduced vascular smooth muscle cells provides sustained neutrophil increases in rats Hum Gene Ther 1996 7: 1431–1436
Lynch CM et al. Long-term expression of human adenosine deaminase in vascular smooth muscle cells of rats: a model for gene therapy Proc Natl Acad Sci USA 1992 89: 1138–1142
Fenjves ES et al. Systemic distribution of apolipoprotein E secreted by grafts of epidermal keratinocytes: implications for epidermal function and gene therapy Proc Natl Acad Sci USA 1989 86: 8803–8807
Flowers MED et al. Long-term transplantation of canine keratinocytes made resistant to G418 through retrovirus-mediated gene transfer Proc Natl Acad Sci USA 1990 87: 2349–2353
Deng H, Lin Q, Khavari PA . Sustainable cutaneous gene therapy Nature Biotechnol 1997 15: 1388–1391
Ramesh N et al. High-level human adenosine deaminase expression in dog skin fibroblasts is not sustained following transplantation Hum Gene Ther 1993 4: 3–7
Palmer TD et al. Genetically modified skin fibroblasts persist long after transplantation but gradually inactivate introduced genes Proc Natl Acad Sci USA 1991 88: 1330–1334
Palmer TD, Thompson AR, Miller AD . Production of human factor IX in animals by genetically modified skin fibroblasts: potential therapy for hemophilia B Blood 1989 73: 438–445
St Louis D, Verma I . An alternative approach to somatic cell gene therapy Proc Natl Acad Sci USA 1988 85: 3150–3154
Moullier P et al. Correction of lysosomal storage in the liver and spleen of MPS VII mice by implantation of genetically modified skin fibroblasts Nat Genet 1993 4: 154–159
Moullier P et al. Long-term delivery of a lysosomal enzyme by genetically modified fibroblasts in dogs Nature Med 1995 1: 353–357
Naffakh N et al. Sustained delivery of erythropoietin in mice by genetically modified skin fibroblasts Proc Natl Acad Sci USA 1995 92: 3194–3198
Fenjves ES et al. Loss of expression of a retrovirus-transduced gene in human keratinocytes J Invest Dermatol 1996 106: 576–578
Kaleko M et al. Expression of human adenosine deaminase in mice after transplantation of genetically modified bone marrow Blood 1990 75: 1733–1741
Osborne WRA et al. Long-term expression of human adenosine deaminase in mice after transplantation of bone marrow infected with amphotropic retroviral vectors Hum Gene Ther 1990 1: 31–41
Scharfmann R, Axelrod JH, Verma IM . Long-term in vivo expression of retrovirus-mediated gene transfer in mouse skin fibroblasts Proc Natl Acad Sci USA 1991 88: 4626–4630
Naughton BA et al. Long-term expression of a retrovirally introduced beta-galactosidase gene in rodent cells implanted in vivo using biodegradable polymer meshes Somat Cell Mol Genet 1992 18: 451–462
Moullier P et al. Continuous systemic secretion of a lysosomal enzyme by genetically modified mouse skin fibroblasts Transplantation 1993 56: 427–432
Hynes RO . Fibronectins Springer-Verlag: New York 1990
Khandjian EW et al. Fibronectin gene expression in proliferating, quiescent and SV-40 infected mouse kidney cells Exp Cell Res 1992 202: 464–470
Kamazaki T, Kobayashi M, Mitsui Y . Enhanced expression of fibronectin during in vivo cellular aging of human endothelial cells and fibroblasts Exp Cell Res 1993 205: 396–402
Patel RS et al. Organization of the fibronectin gene provides evidence for exon shuffling during evolution EMBO J 1987 6: 2565–2572
Dean DC, Bowles CL, Bourgeois S . Cloning and analysis of the promoter region of the human fibronectin gene Proc Natl Acad Sci USA 1987 84: 1876–1880
Nakajima T et al. E1A-responsive elements for repression of rat fibronectin gene transcription Mol Cell Biol 1992 12: 2837–2846
Taylor RM, Wolfe JH . Decreased lysosomal storage in the adult MPS VII mouse brain in the vicinity of grafts of retroviral vector-corrected fibroblasts secreting high levels of B-glucuronidase Nature Med 1997 3: 771–774
Challita PM, Kohn DB . Lack of expression from a retroviral vector after transduction of murine hematopoietic stem cells is associated with methylation in vivo Proc Natl Acad Sci USA 1994 91: 2567–2571
Tapscott SJ et al. Gene therapy of rat 9L gliosarcoma tumors by transduction with selectable genes does not require drug selection Proc Natl Acad Sci USA 1994 91: 8185–8189
Riddel SR et al. T cell-mediated rejection of gene-modified HIV-specific cytotoxic T lymphocytes in HIV patients Nature Med 1996 2: 216–223
Palmer TD et al. Efficient retrovirus-mediated transfer and expression of a human adenosine deaminase gene in diploid skin fibroblasts from an adenosine deaminase-deficient human Proc Natl Acad Sci USA 1987 84: 1055–1059
Hershfield MS, Mitchell BS . Immunodeficiency diseases caused by adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds) . The Metabolic and Molecular Bases of Inherited Disease McGraw-Hill: New York 1995 1725–1768
Hock RA, Miller AD, Osborne WRA . Expression of human adenosine deaminase from various strong promoters after gene transfer into human hematopoietic cell lines Blood 1989 74: 876–881
Osborne WRA, Miller AD . Design of vectors for efficient expression of human purine nucleoside phosphorylase in skin fibroblasts from enzyme-deficient humans Proc Natl Acad Sci USA 1988 85: 6851–6855
Miller AD, Rosman GJ . Improved retroviral vectors for gene transfer and expression BioTechniques 1989 7: 980–990
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Wei, M., Lejnieks, D., Ramesh, N. et al. Sustained gene expression in transplanted skin fibroblasts in rats. Gene Ther 6, 840–844 (1999). https://doi.org/10.1038/sj.gt.3300878
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DOI: https://doi.org/10.1038/sj.gt.3300878