Original Article

Journal of Human Genetics (1999) 44, 152–162; doi:10.1007/s100380050133

Position-independent human bold beta-globin gene expression mediated by a recombinant adeno-associated virus vector carrying the chicken bold beta-globin insulator

Takahito Inoue1, Haruyoshi Yamaza1, Yasuyoshi Sakai1, Shin-ichi Mizuno1, Mizuki Ohno1, Naotaka Hamasaki2 and Yasuyuki Fukumaki1

  1. 1Division of Disease Genes, Institute of Genetic Information, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
  2. 2Department of Clinical Chemistry and Laboratory Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan

Correspondence: Yasuyuki Fukumaki, Division of Disease Genes, Institute of Genetic Information, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Fax: +81-92-632-2375. E-mail: yfukumak@gen.kyushu-u.ac.jp

Received 20 November 1998; Accepted 26 December 1998.

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Abstract

The position-independent expression of transgenes in target cells is an essential subject for determining effective gene therapies. The chicken beta-globin insulator blocks the effects of regulatory sequences on transcriptional units at differential domains. We prepared a recombinant adeno-associated virus (rAAV) containing various combinations of the DNase I-hypersensitive site 2 (HS2), 3 (HS3), and 4 (HS4) core elements from the human beta-globin locus control region (LCR), the human beta-globin gene, and the herpes virus thymidine kinase promoter driven neomycin-resistant gene (neoR) (rHS432, rHS43, rHS42, rHS32, and rHS2), and also rAAV containing two copies of the 250-bp core sequence of the chicken beta-globin insulator on both sides of the rHS2 (rIns/HS2/2Ins). After isolating neomycin-resistant mouse erythroleukemia (MEL) cells infected with each rAAV, we analyzed the rAAV genome by Southern blots and polymerase chain reaction (PCR), using primers specific for HS core elements and the human beta-globin gene. All clones contained a single copy of the rAAV genome in the chromosome, however, some of them had a rearranged proviral genome. In five clones with a single unrearranged rAAV genome for each rAAV construct, we assayed the expression of the human b-globin gene relative to the endogenous mouse betamaj-globin gene, using quantitative reverse transcriptase (RT)-PCR. In clones infected with rHS432, the expression level of the human beta-globin gene ranged from 51.6% to 765.6% of that in the mouse betamaj-globin gene. Likewise, in rHS43, the expression level ranged from 36.7% to 259.0%; in rHS42, from 47.8% to 207.0%; in rHS32, from 47.9% to 105.4%; and in rHS2, from 6.1% to 172.1%, indicating a high variability of expression level in clones infected with recombinant virus lacking the insulator. In contrast, in clones infected with rIns/HS2/Ins, the range of expression of the human beta-globin gene ranged from 52.8% to 58.3% of that in the mouse betamaj-globin gene. These results indicate that the insulator functioned dramatically to reduce the variability of transgene expression due to the position effect. This insulator-rAAV vector system holds promise to provide a constant level of transgene expression for gene therapy, regardless of the insertion sites on the chromosome.

Keywords:

Gene therapy, beta-Globin gene, Adeno-associated virus, Position effect, Insulator