Basic spproaches for gene therapy of hemoglobinopathy

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(1) Position-independent expression of transgenes in target cells is one of essential subjects for successful gene therapies. We prepared recombinant adeno-associated virus (rAAV) containing the combination of the DNaseI hypersensitive site 2 (HS2), 3 (HS3), and 4 (HS4) core elements from the human beta-globin locus control region (LOR), the human beta-globin gene, and the herpes virus tymidine kinase promoter driven neomycin-resistant gene (neo^R) (rHS432, rHS43, rHS42, rHS32, and rHS2), and also rAAV containing two copies of the 250-bp core fragment of the chicken beta-globin insulator on both sides of the rHS2 (rlns/HS2/2Ins). After isolating neomycin resistant mouse erythroleukemia (MEL) cells infected with each rAAV, we analyzed structure of proviral genome of rAAV and determined expression level of the human beta-globin gene. The high variability of the expression level was observed among clones infected with recombinant virus lacking the insulator. In contrast, in the clones inf … More ected with rIns/HS2/lns, the range of expression of the human beta-globin gene was from 52.8% to 58.3% of the mouse beta-major globin gene. Thus, these results indicate that the insulator dramatically functioned to reduce the variability oftransgene expression due to the position effect. This insulator-rAAV vectorsystem is a promising way to provide the constant level of the transgene expression for gene therapy regardless of insertion sites in chromatin.(2) Hemoglobin (Hb) E is the most common Hb variant among Southeast Asian populations. The mutation in codon 26 (GAG to AAG) of the beta-globin gene (beta^E) induces an alternative splicing, resulting in production of normally and aberrantly spliced beta-globin mRNA.Compound heterozygotes for beta-thalassemia and beta^E have a severe beta-thalassemia- disease. Repression of aberrant splicing due to the BE mutation could ameliorate severity of such patients. We showed that the aberrant splicing was partially repressed in the cells treated with antisense oligoribonucleotide targeted to the aberrant 5 splice site. Maximum effect of the antisense oligoribonucleotide was observed at concentration of 0.4 muM and 36 hrs after the treatment in our experiment. We also analyzed the effect of transient and stable expression of SF2/ASF on aberrant splicing in cells expressing the beta^E-globin gene. Partial repression of the aberrant splicing was also observed in both expression systems. These results imply that antisense oligoribonucleotide treatment and SF2/ASF expression are possible therapeutic applications for beta-thalassemia/HbE disease. Less