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 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.
FA lymphoblasts had a normal sensitivity to the cytostatic effect of hyperoxia, while in both control and FA lymphoblasts FAC mRNA levels were unaffected by oxygen.
The Fanconi Anaemia Complementation Group C (FACC) gene is mutated in patients of complementation group C. Several different forms of FACC mRNA that share the same coding region have been isolated.
The therapeutic potential of this system was established by stably transducing B-lymphoblastoid cells from a Fanconi anaemia group C (FA-C) patient with a mini-EBV constitutively expressing the normal FACC cDNA and showing in vitro correction of the FA phenotype.
Both the cross-linking defect and the enhanced cytotoxicity of cross-linkers on Fanconi anemia group C cells are corrected completely by cytoplasmic isoforms of the FAC protein, but not by an isoform targeted to the nucleus.
Overexpression of wild-type FAC cDNA in FA-C lymphoblasts (HSC536N cell line) prevented HN2-induced growth inhibition, G2 arrest, and DNA fragmentation that is characteristic of apoptosis.
Clinical variability of Fanconi anemia (type C) results from expression of an amino terminal truncated Fanconi anemia complementation group C polypeptide with partial activity.
The cloning of the gene for FA complementation group C [FAC] provides an opportunity to test the validity of the "DEB test' which in recent times has become the main arbiter as to whether a patient is classified as FA or non-FA.
To address this possibility, nonadherent low density T-lymphocyte depleted (NALT-) cells from fresh or cryopreserved cord blood were sorted for CD34 phenotype, transduced with a recombinant retroviral vector encoding Fanconi anemia complementation C (FACC) gene, and cells expanded ex vivo in suspension culture for 7 days with growth factors.
To test the effect of heterologous expression of FAC cDNA on drug-induced cytotoxicity, G2 accumulation, and p53 induction in FA cells, we compared two isogenic FA cell lines: HSC536N (mock), a FA type C cell line sensitive to mitomycin C (MMC), and the same cell line transfected (corrected) with wild-type FAC cDNA (HSC536N [+FAC]).