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

The position-independent expression of transgenes in target cells is an ess ential subject for determining effective gene therapies. The chicken beta-g lobin insulator blocks the effects of regulatory sequences on transcription al units at differential domains. We prepared a recombinant adeno-associate d virus (rAAV) containing various combinations of the DNase I-hypersensitiv e site 2 (HS2), 3 (HS3), and 4 (HS4) core elements from the human beta-glob in locus control region (LCR), the human beta-globin gene, and the herpes v irus thymidine kinase promoter driven neomycin-resistant gene (neo(R)) (rHS 432, 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 side s of the rHS2 (rIns/HS2/2Ins). After isolating neomycin-resistant mouse ery throleukemia (MEL) cells infected with each rAAV, we analyzed the rAAV geno me by Southern blots and polymerase chain reaction (PCR), using primers spe cific for IIS core elements and the human beta-globin gene. All clones cont ained 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 unrearr anged rAAV genome for each rAAV construct, we assayed the expression of the human b-globin gene relative to the endogenous mouse beta(maj)-globin gene , using quantitative reverse transcriptase (RT)-PCR, In clones infected wit h rHS432, the expression level of the human beta-globin gene ranged from 51 .6% to 765.6% of that in the mouse beta(maj)-globin gene, Likewise, in rHS4 3, the expression level ranged from 36.7% to 259.0%; in rHS42, from 47.8% t o 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 wi th rIns/HS2/Ins, the range of expression of the human beta-globin gene rang ed from 52.8% to 58.3% of that in the mouse beta(maj)-globin gene, These re sults indicate that the insulator functioned dramatically to reduce the var iability 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 chrom osome.