Basic spproaches for gene therapy of hemoglobinopathy

(1)转基因在靶细胞中的位置非依赖性表达是基因治疗成功的关键课题之一。我们制备了含有人β-珠蛋白基因座控制区(LOR)的DNaseI高敏感位点2(HS2)、3(HS3)和4(HS4)核心元件的重组腺相关病毒(RAAV)，人的β-珠蛋白基因和疱疹病毒酵母酶启动子驱动的新霉素耐药基因(neo^R)(rHS432、rHS43、rHS42、rHS32和rHS2)，以及在HSr2两侧含有两个拷贝的鸡β-珠蛋白绝缘体(rLNS/HS2/2ins)的重组腺相关病毒(RAAV)。分离每株rAAV感染的新霉素耐药小鼠红白血病细胞(MEL)，分析rAAV前病毒基因组结构，确定人β-珠蛋白基因的表达水平。在感染缺乏绝缘子的重组病毒的克隆中，观察到表达水平的高度变异性。相比之下，在克隆inf…中与RINS/HS2/LNS比较，人β-珠蛋白基因的表达范围为52.8%-58.3%。因此，这些结果表明，绝缘子显著地降低了由于位置效应而导致的转基因表达的变异性。这种绝缘体-rAAV载体系统有望为基因治疗提供恒定水平的转基因表达，而不受染色质插入位置的影响。(2)Hb E是东南亚人群中最常见的Hb变异体。β-珠蛋白基因(β^E)第26位密码子(Gag到AAG)的突变导致选择性剪接，导致正常和异常剪接的β-珠蛋白mRNA的产生。β-地中海贫血和β^E复合杂合子患有严重的β-地中海贫血。抑制由BE突变引起的异常剪接可以改善此类患者的严重程度。我们发现针对异常5剪接位点的反义寡核苷酸处理的细胞中，异常剪接被部分抑制。在本实验中，反义寡核苷酸的最大作用浓度为0.4微米，作用时间为36小时。我们还分析了SF2/ASF的瞬时和稳定表达对表达β、E-珠蛋白基因的细胞异常剪接的影响。在两个表达系统中也观察到了对异常剪接的部分抑制。这些结果表明，反义寡核苷酸治疗和SF2/ASF的表达可能是治疗β-地中海贫血/HBe疾病的应用。较少

(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