BTK gene targeting by homologous recombination using a helper-dependent adenovirus/adeno-associated virus hybrid vector.
X-linked agammaglobulinemia (XLA) is one of the most common humoral immunodeficiencies, which is caused by mutations in Bruton's tyrosine kinase (BTK) gene. To examine the possibility of using gene therapy for XLA, we constructed a helper-dependent adenovirus/adeno-associated virus BTK targeting vector (HD-Ad.AAV BTK vector) composed of a genomic sequence containing BTK exons 6-19 and a green fluorescence protein-hygromycin cassette driven by a cytomegalovirus promoter. We first used NALM-6, a human male pre-B acute lymphoblastic leukemia cell line, as a recipient to measure the efficiency of gene targeting by homologous recombination. We identified 10 clones with the homologous recombination of the BTK gene among 107 hygromycin-resistant stable clones isolated from two independent experiments. We next used cord blood CD34⁺ cells as the recipient cells for the gene targeting. We isolated colonies grown in medium containing cytokines and hygromycin. We found that the targeting of the BTK gene occurred in four of the 755 hygromycin-resistant colonies. Importantly, the gene targeting was also observed in CD19⁺ lymphoid progenitor cells that were differentiated from the homologous recombinant CD34⁺ cells during growth in selection media. Our study shows the potential for the BTK gene therapy using the HD-Ad.AAV BTK vector via homologous recombination in hematopoietic stem cells.
Department of Pediatrics, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
H
H
Yamamoto
Department of Pediatrics, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
M
M
Ishimura
Department of Pediatrics, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
M
M
Ochiai
Department of Pediatrics, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
H
H
Takada
Department of Pediatrics, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan.
K
K
Kusuhara
Department of Medical Biophysics and Radiation Biology, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan.
Y
Y
Nakatsu
Department of Medical Biophysics and Radiation Biology, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan.
T
T
Tsuzuki
Division of Gene Therapy, Research Center for Genomic Medicine, Saitama Medical University, Hidaka, Saitama, Japan.
K
K
Mitani
Department of Pediatrics, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
T
T
Hara
D000077329:Agammaglobulinaemia Tyrosine Kinase; D000361:Agammaglobulinemia / Q000235:genetics / Q000628:therapy; D045744:Cell Line, Tumor; D000229:Dependovirus / Q000235:genetics*; D018390:Gene Targeting; D040181:Genetic Diseases, X-Linked / Q000235:genetics / Q000628:therapy; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics*; D006378:Helper Viruses / Q000235:genetics*; D059765:Homologous Recombination; D006801:Humans; D008297:Male; D009154:Mutation; D011505:Protein-Tyrosine Kinases / Q000235:genetics*
D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human