An in Vivo Model of Somatic Cell Gene Therapy for Human Severe Combined Immunodeficiency

Ferrari, G.; Rossini, Silvano; Giavazzi, Raffaella; Maggioni, Daniela; Nobili, Nadia; Soldati, Monica; Ungers, Grace; Mavilio, Fulvio; Gilboa, Eli; Bordignon, Claudio

doi:10.1126/science.1848369

Cited by 124 publications

(52 citation statements)

References 31 publications

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“…Southern blot analysis showed multiplicity of random proviral integration in single epidermal stem cells, as previously shown for human myogenic cells (31,37). The rate of secretion of the exogenous protein by cultures generated by single clones was proportional to the number of integrations per progenitor cell.…”

Section: Resultssupporting

confidence: 72%

Clonal analysis of stably transduced human epidermal stem cells in culture.

Mathor

Ferrari

Dellambra

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

156

147

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We have transduced normal human keratinocytes with retroviral constructs expressing a bacterial .8-galactosidase (,B-gal) gene or a human cDNA under control of a long terminal repeat. Efficiency of gene transfer averaged approximately 50%Yo and 95% of clonogenic keratinocytes for ,B-gal and hIL-6, respectively. Both genes were stably integrated and expressed for more than 150 generations. Clonal analysis showed that both holoclones and their transient amplifying progeny expressed the transgene permanently. Southern blot analysis on isolated clones showed that many keratinocyte stem cells integrated multiple proviral copies in their genome and that the synthesis of the exogenous gene product in vitro was proportional to the number of proviral integrations. When cohesive epidermal sheets prepared from stem cells transduced with hIL-6 were grafted on athymic animals, the serum levels of hIL-6 were strictly proportional to the rate of secretion in vitro and therefore to the number of proviral integrations. The possibility of specifying the level of transgene expression and its permanence in a homogeneous clone of stem cell origin opens new perspectives in the long-term treatment of genetic disorders.Cultured human keratinocytes generate cohesive sheets of epithelium (1), which maintain the characteristics of the original donor site (2, 3) and retain stem cells, namely cells that upon division replace their own number and also give rise to cells that differentiate further into one or more specialized types (4-6). Cultured epithelial sheets are routinely used to make autologous grafts for patients suffering from large skin and mucosal defects; the engrafted stem cells assure the persistence of the regenerated epidermis during the patient's lifetime (7-9). Epidermal keratinocytes synthesize and secrete several gene products that might accomplish autocrine, paracrine, and even endocrine functions (10). Indeed, it has been unambiguously demonstrated that keratinocyte-derived polypeptides cross the basement membrane and are secreted into the bloodstream (11, 12). Basal keratinocytes are also sources of adhesion molecules and matrix proteins, which form the anchoring structures of the epidermis, and some of these proteins are affected in genetic skin diseases (13). Therefore, epidermal cells are very attractive targets for the gene therapy of skin diseases, as well as for systemic delivery of recombinant proteins for the treatment of a number of genetic disorders.Ex vivo transduced mammalian epidermal cells, including human keratinocytes, have been successfully generated (14-16). Protein production in vitro and/or secretion into the circulation by keratinocytes grafted onto animals has been shown for exogenous growth hormone (17-20), neomycin phosphotransferase (21), steroid sulfatase (22), factor IX (23), al-anti-trypsin (24), 13-chorionic gonadotropin (15), and ,B-galactosidase (13-gal) (19,(24)(25)(26) after transduction with replication-deficient retroviral or adenoviral vectors. However, none of these approa...

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Section: Resultssupporting

confidence: 72%

Clonal analysis of stably transduced human epidermal stem cells in culture.

Mathor

Ferrari

Dellambra

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

156

147

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“…ADA levels are normally highest in thymic T lymphocytes, and it has been postulated that absence of ADA has its most critical impact at the production stage of T lymphopoiesis 13,15 . However, a study by Ferrari et al 16 demonstrated that ADA genecorrected peripheral blood T lymphocytes had prolonged survival after injection into SCIDmice, compared with non-corrected, ADA-deficient T lymphocytes. Thus, ADA expression may also be essential for the survival of mature peripheral blood T lymphocytes.…”

Section: Discussionmentioning

confidence: 99%

T lymphocytes with a normal ADA gene accumulate after transplantation of transduced autologous umbilical cord blood CD34+ cells in ADA-deficient SCID neonates

Kohn

Carbonaro

et al. 1998

Nat Med

312

153

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Adenosine deaminase-deficient severe combined immunodeficiency was the first disease investigated for gene therapy because of a postulated production or survival advantage for genecorrected T lymphocytes, which may overcome inefficient gene transfer. Four years after three newborns with this disease were given infusions of transduced autologous umbilical cord blood CD34 + cells, the frequency of gene-containing T lymphocytes has risen to 1-10%, whereas the frequencies of other hematopoietic and lymphoid cells containing the gene remain at 0.01-0.1%. Cessation of polyethylene glycol-conjugated adenosine deaminase enzyme replacement in one subject led to a decline in immune function, despite the persistence of gene-containing T lymphocytes. Thus, despite the long-term engraftment of transduced stem cells and selective accumulation of gene-containing T lymphocytes, improved gene transfer and expression will be needed to attain a therapeutic effect.Adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID) became an early disease candidate for studies of gene therapy based on the results of allogeneic bone marrow transplant. Transplantation of bone marrow from a fully HLA-matched, unaffected sibling donor allows SCID to be cured completely 1,2 . Patients develop a protective immune © 1998 Nature Publishing Group Correspondence should be addressed to D.B.K. NIH Public AccessAuthor Manuscript Nat Med. Author manuscript; available in PMC 2013 September 19.Published in final edited form as:Nat Med. 1998 July ; 4(7): 775-780. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript system of donor-derived T lymphocytes, despite the absence of significant numbers of donor-derived cells in other hematopoietic lineages 3 . This observation has been interpreted as indicating that the normal T-lymphoid cells have a selective advantage in SCID patients and repopulate the immune system from a small number of engrafted, genetically-normal donor hematopoietic stem cells. The selective advantage for normal T and B cells in SCID patients has been recently re-confirmed in two SCID patients (one with ADA-deficient SCID and one with the X-linked form) who had spontaneous improvements in their immune function [4][5] . Gene-correction of a small number of autologous hematopoietic stem cells could similarly result in the development of a functional immune system in SCID gene therapy recipients.In May 1993, we treated three newborns with ADA-deficient SCID with a single infusion for each of retroviral vector-transduced autologous umbilical cord blood CD34 + cells. Initial observations during the first 18 months after this intervention showed the persistent presence of leukocytes in their peripheral blood and bone marrow which contained and expressed the inserted normal human ADA cDNA (ref.6). This observation was important because it provided convincing evidence that human hematopoietic stem cells could be transduced with retroviral vectors and re-engrafted in their hosts without administration of...

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“…G ene therapy represents a potentially novel approach to cancer treatment, in which the transfer of genetic material into a specific cell type alters the phenotype of the target cells [1][2][3] in a way that improves the outcome of therapy. Adenoviral vectors have become the most widely used vector for the delivery of prodrug activation transcription units in the field of cancer gene therapy.…”

mentioning

confidence: 99%

Use of L-plastin promoter to develop an adenoviral system that confers transgene expression in ovarian cancer cells but not in normal mesothelial cells

et al. 1999

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The objective of this study was to develop an adenoviral vector system that would generate a pattern of expression of exogenous therapeutic genes appropriate for the treatment of ovarian cancer. For this purpose, we have generated a replication-deficient recombinant adenoviral vector, AdLPLacZ, which contains the human L-plastin (LP) promoter (LP-P) driving the Escherichia coli LacZ gene. LP is constitutively expressed at high levels in malignant epithelial cells but is not expressed in normal tissues, except at low levels in mature hematopoietic cells. Because adenoviral vectors infect early hematopoietic multilineage precursor cells only poorly or not at all, this vector would be of use in the peritoneal cavity and in vitro for marrow purging. We first analyzed the expression of the LacZ reporter gene in ovarian and breast cancer cell lines, normal fibroblasts, and leukemia cell lines using the adenoviral vector in which the LacZ gene is governed by the LP-P promoter (AdLPLacZ) or in which the LacZ gene is governed by the cytomegalovirus (CMV) promoter (AdCMVLacZ). We found equivalent and high levels of expression of ␤-galactosidase (␤-gal) by AdLPLacZ and AdCMVLacZ vectors in the breast or ovarian cancer cell lines as well as in a fibrosarcoma cell line, indicating that the adenoviral vectors infected these cells and expressed their transgenes equally with the LP and CMV promoters. Expression of the LacZ gene with the CMV vector but not with the LP-P vector was observed in experiments with normal fibroblasts, indicating that the vectors infected the cells, but that the LP-P was not active within them. In hematopoietic cells such as U937 cells, no measurable ␤-gal activity was detected in cells infected either by AdLPLacZ or by AdCMVLacZ, indicating that the adenoviral vectors were not infecting the cells. Although ␤-gal activity was observed in fresh ascitic ovarian cancer cells after infection with adenoviral vectors containing CMV or the LP promoters, ␤-gal activity was detected in a portion of a biopsy of normal peritoneum when the tissues were exposed to the AdCMVLacZ vector, but not when tissues were exposed to the AdLPLacZ vector. These results suggest that the transcription of therapeutic genes in cells infected by the AdLP vectors would be restricted to LP expression-positive ovarian carcinoma cells but would not be seen in the normal mesothelial cells of the peritoneal cavity. This possibility implies that adenoviral vectors carrying therapeutic genes driven by the LP-P would be of use for the intracavitary treatment ovarian cancer.

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An in Vivo Model of Somatic Cell Gene Therapy for Human Severe Combined Immunodeficiency

Cited by 124 publications

References 31 publications

Clonal analysis of stably transduced human epidermal stem cells in culture.

Clonal analysis of stably transduced human epidermal stem cells in culture.

T lymphocytes with a normal ADA gene accumulate after transplantation of transduced autologous umbilical cord blood CD34+ cells in ADA-deficient SCID neonates

Use of L-plastin promoter to develop an adenoviral system that confers transgene expression in ovarian cancer cells but not in normal mesothelial cells

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