A functionally active retrovirus vector for gene therapy in Fanconi anemia group C

Christopher E. Walsh, Markus Grompe, Elio Vanin, Manuel Buchwald, Neal S. Young, Arthur W. Nienhuis, Johnson M. Liu

Research output: Contribution to journalArticle

74 Citations (Scopus)

Abstract

Fanconi anemia (FA) is a rare genetic disorder characterized by progressive pancytopenia, congenital abnormalities, and a predisposition to malignancy. Recently, mutation in a novel gene named FACC (Fanconi anemia C complementing) has been identified as causing one type of FA. Here, we report successful functional complementation of four FA(C) cell lines using a retroviral vector to transfer a copy of the normal FACC gene. The hallmark of the FA cell phenotype is extreme sensitivity to cross-linking agents such as mitomycin C (MMC). Cell lines transduced by FACC viral vectors were distinguished by their ability to grow at concentrations of MMC several orders of magnitude higher than those concentrations inhibitory of parental controls. The genetically corrected cell lines were analyzed for susceptibility to MMC-induced chromosomal breakage and were found to have been normalized. These two different assays confirmed that our retroviral vectors were capable of transferring a functional FACC gene to lymphoid cell lines established from FA(C) patients. We next analyzed the ability of our viral vectors to functionally correct hematopoietic progenitor cells from a patient bearing a splice donor mutation. Progenitor cells were purified by an immunoaffinity column to enrich for cells with high CD34 expression. Similar to FA lymphoid cell lines, this patient's CD34-enriched cells were extremely sensitive to MMC. After infection of these progenitor cells with viral vectors bearing normal FACC, increased numbers of colonies formed both in the absence and presence of ≤5 nmol/L MMC, but no colonies formed from uninfected cells, even in the absence of MMC. Polymerase chain amplification was used to confirm proviral DNA integration. Thus, retroviral vectors can be engineered to transfer a normal FACC gene to lymphoid cell lines and primary hematopoietic cells bearing four different FACC mutations. FA stem cells rescued by gene transduction should have a selective growth advantage within the hypoplastic FA marrow environment in vivo. These experiments suggest that gene therapy may be an effective treatment strategy for FA.

Original languageEnglish (US)
Pages (from-to)453-459
Number of pages7
JournalBlood
Volume84
Issue number2
StatePublished - Jul 15 1994
Externally publishedYes

Fingerprint

Fanconi Anemia
Gene therapy
Mitomycin
Retroviridae
Genetic Therapy
Bearings (structural)
Cells
Genes
Cell Line
Stem cells
Stem Cells
Amplification
Assays
Lymphocytes
Mutation
DNA
Chromosome Breakage
Pancytopenia
Inborn Genetic Diseases

ASJC Scopus subject areas

  • Hematology

Cite this

Walsh, C. E., Grompe, M., Vanin, E., Buchwald, M., Young, N. S., Nienhuis, A. W., & Liu, J. M. (1994). A functionally active retrovirus vector for gene therapy in Fanconi anemia group C. Blood, 84(2), 453-459.

A functionally active retrovirus vector for gene therapy in Fanconi anemia group C. / Walsh, Christopher E.; Grompe, Markus; Vanin, Elio; Buchwald, Manuel; Young, Neal S.; Nienhuis, Arthur W.; Liu, Johnson M.

In: Blood, Vol. 84, No. 2, 15.07.1994, p. 453-459.

Research output: Contribution to journalArticle

Walsh, CE, Grompe, M, Vanin, E, Buchwald, M, Young, NS, Nienhuis, AW & Liu, JM 1994, 'A functionally active retrovirus vector for gene therapy in Fanconi anemia group C', Blood, vol. 84, no. 2, pp. 453-459.
Walsh CE, Grompe M, Vanin E, Buchwald M, Young NS, Nienhuis AW et al. A functionally active retrovirus vector for gene therapy in Fanconi anemia group C. Blood. 1994 Jul 15;84(2):453-459.
Walsh, Christopher E. ; Grompe, Markus ; Vanin, Elio ; Buchwald, Manuel ; Young, Neal S. ; Nienhuis, Arthur W. ; Liu, Johnson M. / A functionally active retrovirus vector for gene therapy in Fanconi anemia group C. In: Blood. 1994 ; Vol. 84, No. 2. pp. 453-459.
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abstract = "Fanconi anemia (FA) is a rare genetic disorder characterized by progressive pancytopenia, congenital abnormalities, and a predisposition to malignancy. Recently, mutation in a novel gene named FACC (Fanconi anemia C complementing) has been identified as causing one type of FA. Here, we report successful functional complementation of four FA(C) cell lines using a retroviral vector to transfer a copy of the normal FACC gene. The hallmark of the FA cell phenotype is extreme sensitivity to cross-linking agents such as mitomycin C (MMC). Cell lines transduced by FACC viral vectors were distinguished by their ability to grow at concentrations of MMC several orders of magnitude higher than those concentrations inhibitory of parental controls. The genetically corrected cell lines were analyzed for susceptibility to MMC-induced chromosomal breakage and were found to have been normalized. These two different assays confirmed that our retroviral vectors were capable of transferring a functional FACC gene to lymphoid cell lines established from FA(C) patients. We next analyzed the ability of our viral vectors to functionally correct hematopoietic progenitor cells from a patient bearing a splice donor mutation. Progenitor cells were purified by an immunoaffinity column to enrich for cells with high CD34 expression. Similar to FA lymphoid cell lines, this patient's CD34-enriched cells were extremely sensitive to MMC. After infection of these progenitor cells with viral vectors bearing normal FACC, increased numbers of colonies formed both in the absence and presence of ≤5 nmol/L MMC, but no colonies formed from uninfected cells, even in the absence of MMC. Polymerase chain amplification was used to confirm proviral DNA integration. Thus, retroviral vectors can be engineered to transfer a normal FACC gene to lymphoid cell lines and primary hematopoietic cells bearing four different FACC mutations. FA stem cells rescued by gene transduction should have a selective growth advantage within the hypoplastic FA marrow environment in vivo. These experiments suggest that gene therapy may be an effective treatment strategy for FA.",
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AB - Fanconi anemia (FA) is a rare genetic disorder characterized by progressive pancytopenia, congenital abnormalities, and a predisposition to malignancy. Recently, mutation in a novel gene named FACC (Fanconi anemia C complementing) has been identified as causing one type of FA. Here, we report successful functional complementation of four FA(C) cell lines using a retroviral vector to transfer a copy of the normal FACC gene. The hallmark of the FA cell phenotype is extreme sensitivity to cross-linking agents such as mitomycin C (MMC). Cell lines transduced by FACC viral vectors were distinguished by their ability to grow at concentrations of MMC several orders of magnitude higher than those concentrations inhibitory of parental controls. The genetically corrected cell lines were analyzed for susceptibility to MMC-induced chromosomal breakage and were found to have been normalized. These two different assays confirmed that our retroviral vectors were capable of transferring a functional FACC gene to lymphoid cell lines established from FA(C) patients. We next analyzed the ability of our viral vectors to functionally correct hematopoietic progenitor cells from a patient bearing a splice donor mutation. Progenitor cells were purified by an immunoaffinity column to enrich for cells with high CD34 expression. Similar to FA lymphoid cell lines, this patient's CD34-enriched cells were extremely sensitive to MMC. After infection of these progenitor cells with viral vectors bearing normal FACC, increased numbers of colonies formed both in the absence and presence of ≤5 nmol/L MMC, but no colonies formed from uninfected cells, even in the absence of MMC. Polymerase chain amplification was used to confirm proviral DNA integration. Thus, retroviral vectors can be engineered to transfer a normal FACC gene to lymphoid cell lines and primary hematopoietic cells bearing four different FACC mutations. FA stem cells rescued by gene transduction should have a selective growth advantage within the hypoplastic FA marrow environment in vivo. These experiments suggest that gene therapy may be an effective treatment strategy for FA.

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