Journal
JOURNAL OF GENE MEDICINE
Volume 11, Issue 11, Pages 1012-1019Publisher
WILEY
DOI: 10.1002/jgm.1382
Keywords
adeno-associated virus; Fanconi anemia; gene repair; hematopoietic; homologous recombination
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Funding
- Ministry of Education, Science, Sports and Culture
- Japan Society for the Promotion of Science (JSPS)
- Mitsubishi Pharma Research Foundation
- Sankyo Foundation of Life Science
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Background Adeno-associated virus (AAV) vectors have been shown to correct a variety of mutations in human cells by homologous recombination (HR) at high rates, which can overcome insertional mutagenesis and transgene silencing, two of the major hurdles in conventional gene addition therapy of inherited diseases. We examined an ability of AAV vectors to repair a mutation in human hematopoietic cells by HR. Methods We infected a human B-lymphoblastoid cell line (BCL) derived from a normal subject with an AAV, which disrupts the hypoxanthine phosphoribosyl transferase1 (HPRT1) locus, to measure the frequency of AAV-mediated HR in BCL cells. We subsequently constructed an AAV vector encoding the normal sequences from the Fanconi anemia group A (FANCA) locus to correct a mutation in the gene in BCL derived from a FANCA patient. Results Under optimal conditions, approximately 50% of BCL cells were transduced with an AAV serotype 2 (AAV-2) vector. In FANCA BCL cells, up to 0.016% of infected cells were gene-corrected by HR. AAV-mediated restoration of normal genotypic and phenotypic characteristics in FANCA-mutant cells was confirmed at the DNA, protein and functional levels. Conclusions The results obtained in the present study indicate that AAV vectors may be applicable for gene correction therapy of inherited hematopoietic disorders. Copyright (C) 2009 John Wiley & Sons, Ltd.
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