Correction of mutant Fanconi anemia gene by homologous recombination in human hematopoietic cells using adeno-associated virus vector.
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.
Paiboonsukwong
Kittiphong
K
Gene Therapy Division, Research Center for Genomic Medicine, Saitama Medical University, Saitama, Japan.
Ohbayashi
Fumi
F
Shiiba
Haruka
H
Aizawa
Emi
E
Yamashita
Takayuki
T
Mitani
Kohnosuke
K
D002478:Cells, Cultured; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism; D052217:Fanconi Anemia Complementation Group A Protein / Q000235:genetics* / Q000378:metabolism; D018390:Gene Targeting / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism; D008957:Models, Genetic; D009154:Mutation; D011995:Recombination, Genetic; D014161:Transduction, Genetic
C498471:FANCA protein, human; D052217:Fanconi Anemia Complementation Group A Protein; D007041:Hypoxanthine Phosphoribosyltransferase