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J Clin Invest. 1994 October; 94(4): 1440–1448.
PMCID: PMC295276

Phenotypic correction of Fanconi anemia in human hematopoietic cells with a recombinant adeno-associated virus vector.

Abstract

Fanconi anemia (FA) is a recessive inherited disease characterized by defective DNA repair. FA cells are hypersensitive to DNA cross-linking agents that cause chromosomal instability and cell death. FA is manifested clinically by progressive pancytopenia, variable physical anomalies, and predisposition to malignancy. Four complementation groups have been identified, termed A, B, C, and D. The gene for the FA complementation group C, FACC, has been cloned. Expression of the FACC cDNA corrects the phenotypic defect of FA(C) cells, resulting in normalized cell growth in the presence of DNA cross-linking agents such as mitomycin C (MMC). Gene transfer of the FACC gene should provide a survival advantage to transduced hematopoietic cells, suggesting that FA might be an ideal candidate for gene therapy. We demonstrated efficient transduction, expression, and phenotypic correction in lymphoblastoid cell lines derived from FA (C) patients using a recombinant adeno-associated virus (rAAV) vector containing the FACC gene. Molecular characterization of the transduced FACC gene showed an intact unrearranged proviral genome with expression sufficient to normalize cell growth, cell cycle kinetics and chromosomal breakage in the presence of MMC. These observations were extended by testing rAAV transduction in hematopoietic progenitor cells. Peripheral blood CD34+ cells isolated from a FA (C) patient and transduced with rAAV/FACC virus yielded 5-10-fold more progenitor colonies than mock-infected cells, consistent with genetic "rescue" of corrected cells. This is the first demonstration of rAAV gene correction in primary human hematopoietic progenitor cells and has important implications for gene therapy of hematopoietic disorders, specifically FA.

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  • Schroeder TM, Anschütz F, Knopp A. Spontane Chromosomenaberrationen bei familiärer Panmyelopathie. Humangenetik. 1964;1(2):194–196. [PubMed]
  • Sasaki MS, Tonomura A. A high susceptibility of Fanconi's anemia to chromosome breakage by DNA cross-linking agents. Cancer Res. 1973 Aug;33(8):1829–1836. [PubMed]
  • Auerbach AD, Rogatko A, Schroeder-Kurth TM. International Fanconi Anemia Registry: relation of clinical symptoms to diepoxybutane sensitivity. Blood. 1989 Feb;73(2):391–396. [PubMed]
  • Kubbies M, Schindler D, Hoehn H, Schinzel A, Rabinovitch PS. Endogenous blockage and delay of the chromosome cycle despite normal recruitment and growth phase explain poor proliferation and frequent edomitosis in Fanconi anemia cells. Am J Hum Genet. 1985 Sep;37(5):1022–1030. [PubMed]
  • Ishida R, Buchwald M. Susceptibility of Fanconi's anemia lymphoblasts to DNA-cross-linking and alkylating agents. Cancer Res. 1982 Oct;42(10):4000–4006. [PubMed]
  • Strathdee CA, Duncan AM, Buchwald M. Evidence for at least four Fanconi anaemia genes including FACC on chromosome 9. Nat Genet. 1992 Jun;1(3):196–198. [PubMed]
  • Strathdee CA, Gavish H, Shannon WR, Buchwald M. Cloning of cDNAs for Fanconi's anaemia by functional complementation. Nature. 1992 Apr 30;356(6372):763–767. [PubMed]
  • Whitney MA, Saito H, Jakobs PM, Gibson RA, Moses RE, Grompe M. A common mutation in the FACC gene causes Fanconi anaemia in Ashkenazi Jews. Nat Genet. 1993 Jun;4(2):202–205. [PubMed]
  • Muzyczka N. Use of adeno-associated virus as a general transduction vector for mammalian cells. Curr Top Microbiol Immunol. 1992;158:97–129. [PubMed]
  • Walsh CE, Liu JM, Miller JL, Nienhuis AW, Samulski RJ. Gene therapy for human hemoglobinopathies. Proc Soc Exp Biol Med. 1993 Dec;204(3):289–300. [PubMed]
  • Kotin RM, Siniscalco M, Samulski RJ, Zhu XD, Hunter L, Laughlin CA, McLaughlin S, Muzyczka N, Rocchi M, Berns KI. Site-specific integration by adeno-associated virus. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2211–2215. [PubMed]
  • Samulski RJ, Zhu X, Xiao X, Brook JD, Housman DE, Epstein N, Hunter LA. Targeted integration of adeno-associated virus (AAV) into human chromosome 19. EMBO J. 1991 Dec;10(12):3941–3950. [PubMed]
  • Samulski RJ. Adeno-associated virus: integration at a specific chromosomal locus. Curr Opin Genet Dev. 1993 Feb;3(1):74–80. [PubMed]
  • Walsh CE, Liu JM, Xiao X, Young NS, Nienhuis AW, Samulski RJ. Regulated high level expression of a human gamma-globin gene introduced into erythroid cells by an adeno-associated virus vector. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):7257–7261. [PubMed]
  • Miller JL, Walsh CE, Ney PA, Samulski RJ, Nienhuis AW. Single-copy transduction and expression of human gamma-globin in K562 erythroleukemia cells using recombinant adeno-associated virus vectors: the effect of mutations in NF-E2 and GATA-1 binding motifs within the hypersensitivity site 2 enhancer. Blood. 1993 Sep 15;82(6):1900–1906. [PubMed]
  • Chatterjee S, Johnson PR, Wong KK., Jr Dual-target inhibition of HIV-1 in vitro by means of an adeno-associated virus antisense vector. Science. 1992 Nov 27;258(5087):1485–1488. [PubMed]
  • Zhou SZ, Broxmeyer HE, Cooper S, Harrington MA, Srivastava A. Adeno-associated virus 2-mediated gene transfer in murine hematopoietic progenitor cells. Exp Hematol. 1993 Jul;21(7):928–933. [PubMed]
  • Samulski RJ, Chang LS, Shenk T. Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression. J Virol. 1989 Sep;63(9):3822–3828. [PMC free article] [PubMed]
  • Rigaud G, Grange T, Pictet R. The use of NaOH as transfer solution of DNA onto nylon membrane decreases the hybridization efficiency. Nucleic Acids Res. 1987 Jan 26;15(2):857–857. [PMC free article] [PubMed]
  • Gibson RA, Hajianpour A, Murer-Orlando M, Buchwald M, Mathew CG. A nonsense mutation and exon skipping in the Fanconi anaemia group C gene. Hum Mol Genet. 1993 Jun;2(6):797–799. [PubMed]
  • Gavish H, dos Santos CC, Buchwald M. A Leu554-to-Pro substitution completely abolishes the functional complementing activity of the Fanconi anemia (FACC) protein. Hum Mol Genet. 1993 Feb;2(2):123–126. [PubMed]
  • Kaiser TN, Lojewski A, Dougherty C, Juergens L, Sahar E, Latt SA. Flow cytometric characterization of the response of Fanconi's anemia cells to mitomycin C treatment. Cytometry. 1982 Mar;2(5):291–297. [PubMed]
  • Berger R, Le Coniat M, Gendron MC. Fanconi anemia. Chromosome breakage and cell cycle studies. Cancer Genet Cytogenet. 1993 Aug;69(1):13–16. [PubMed]
  • Andrews RG, Singer JW, Bernstein ID. Human hematopoietic precursors in long-term culture: single CD34+ cells that lack detectable T cell, B cell, and myeloid cell antigens produce multiple colony-forming cells when cultured with marrow stromal cells. J Exp Med. 1990 Jul 1;172(1):355–358. [PMC free article] [PubMed]
  • Verfaillie C, Blakolmer K, McGlave P. Purified primitive human hematopoietic progenitor cells with long-term in vitro repopulating capacity adhere selectively to irradiated bone marrow stroma. J Exp Med. 1990 Aug 1;172(2):509–502. [PMC free article] [PubMed]
  • LaFace D, Hermonat P, Wakeland E, Peck A. Gene transfer into hematopoietic progenitor cells mediated by an adeno-associated virus vector. Virology. 1988 Feb;162(2):483–486. [PubMed]
  • Saunders EF, Freedman MH. Constitutional aplastic anaemia: defective haematopoietic stem cell growth in vitro. Br J Haematol. 1978 Oct;40(2):277–287. [PubMed]
  • Alter BP, Knobloch ME, He L, Gillio AP, O'Reilly RJ, Reilly LK, Weinberg RS. Effect of stem cell factor on in vitro erythropoiesis in patients with bone marrow failure syndromes. Blood. 1992 Dec 15;80(12):3000–3008. [PubMed]
  • Stark R, Thierry D, Richard P, Gluckman E. Long-term bone marrow culture in Fanconi's anaemia. Br J Haematol. 1993 Apr;83(4):554–559. [PubMed]
  • Bagby GC, Jr, Segal GM, Auerbach AD, Onega T, Keeble W, Heinrich MC. Constitutive and induced expression of hematopoietic growth factor genes by fibroblasts from children with Fanconi anemia. Exp Hematol. 1993 Oct;21(11):1419–1426. [PubMed]
  • Wevrick R, Clarke CA, Buchwald M. Cloning and analysis of the murine Fanconi anemia group C cDNA. Hum Mol Genet. 1993 Jun;2(6):655–662. [PubMed]
  • Dutrillaux B, Aurias A, Dutrillaux AM, Buriot D, Prieur M. The cell cycle of lymphocytes in Fanconi anemia. Hum Genet. 1982;62(4):327–332. [PubMed]
  • Hartwell LH, Weinert TA. Checkpoints: controls that ensure the order of cell cycle events. Science. 1989 Nov 3;246(4930):629–634. [PubMed]
  • Lau CC, Pardee AB. Mechanism by which caffeine potentiates lethality of nitrogen mustard. Proc Natl Acad Sci U S A. 1982 May;79(9):2942–2946. [PubMed]
  • Weinert TA, Hartwell LH. The RAD9 gene controls the cell cycle response to DNA damage in Saccharomyces cerevisiae. Science. 1988 Jul 15;241(4863):317–322. [PubMed]
  • Sabatier L, Dutrillaux B. Effect of caffeine in Fanconi anemia. I. Restoration of a normal duration of G2 phase. Hum Genet. 1988 Jul;79(3):242–244. [PubMed]
  • Frazelle JH, Harris JS, Swift M. Responses of Fanconi anemia fibroblasts to adenine and purine analogues. Mutat Res. 1981 Feb;80(2):373–380. [PubMed]
  • Pincheira J, Bravo M, López-Sáez JF. Fanconi's anemia lymphocytes: effect of caffeine, adenosine and niacinamide during G2 prophase. Mutat Res. 1988 May;199(1):159–165. [PubMed]

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