Gene therapy for Cooley's anemia in a new mouse model

描述(由申请人提供)： 基于干细胞的基因治疗提供了一种潜在的方法来治愈先天性严重的血红蛋白疾病，如β-地中海贫血。为此，我们构建了携带人β-珠蛋白基因的慢病毒载体(TNS9)，并证明利用该载体可以获得对受中间型β-地中海贫血影响的小鼠模型的长期纠正。此外，该载体挽救了一种新的受严重β-地中海贫血影响的致命小鼠模型。然而，在这些小鼠中，贫血和产生的血红蛋白的纠正水平尚未达到最佳水平。我们认为，为了完全揭示这种基因治疗方法的潜力，我们需要在这个新的库利氏贫血小鼠模型中(目的1)研究慢病毒转导的造血干细胞(HSC)比例、骨髓嵌合体程度和相应的贫血纠正水平之间的相关性。为此，我们将构建一种新的慢病毒载体，它结合了报告基因，如人源化红移绿色荧光蛋白(HrGFP)在所有造血系中的表达和人β-珠蛋白基因在红系细胞(TNS9+GFP)中的表达。为了增加人类β-珠蛋白的表达(目标2)，我们建议创造新的慢病毒载体，可能会增加血红蛋白的产生。我们认为，通过将β-珠蛋白启动子延长1kb，并插入与LCR的HS1相对应的1kb的基因组区域，我们可以提高TNS9的血红蛋白产量。另一个可以通过降低不同基因组整合位点表达的变异性来提高血红蛋白产量水平的基因组元件是cHS4绝缘体元件。生产新的治疗载体需要有效的策略来比较和确定最佳的β-珠蛋白编码慢病毒。为此，我们建议(目标3)研究TnS9相对于新慢病毒载体(在目标2中提出)在BM嵌合体中的平均表达水平。此外，我们建议(目标4)从慢病毒转导的单拷贝ES细胞中产生转基因小鼠，以研究每个载体的表达水平和变异性。最后，将对插入选择性耐药基因，二氢叶酸还原酶(DHFR)和甲基鸟嘌呤-DNA-甲基转移酶(MGMT)进行评估(目标5)，以促进表达人β-珠蛋白基因的转导干细胞的竞争性再繁殖。

DESCRIPTION (provided by applicant): Stem cell-based gene therapy offers a potential means to cure congenital severe hemoglobinopathies such as beta-thalassemia. For this reason we have constructed a lentiviral vector (TNS9) carrying the human beta-globin gene and demonstrated that with this vector we can obtain long-term correction of a mouse model affected by beta-thalassemia intermedia. Furthermore, this vector rescues a new lethal mouse model affected by beta-thalassemia major. However, in these mice the level of correction of the anemia and hemoglobin produced are not yet optimal. We believe that in order to unveil completely the potential of this gene therapy approach, we need to investigate, in this new mouse model of Cooley's anemia, (Aim 1) the correlation between the fraction of lentiviral transduced hematopoietic stem cells (HSC), the degree of BM chimerism and the corresponding level of anemia correction. For this purpose, we will generate a new lentiviral vector that combines expression of a reporter gene, such as the humanized red-shifted green fluorescent protein (hrGFP), in all the hematopoietic lineages and expression of the human beta-globin gene in erythroid cells (TNS9+GFP). To increase human beta-globin expression (Aim 2) we propose to generate new lentiviral vectors that could potentially increase hemoglobin production. We believe that we can raise hemoglobin production from TNS9 by extending the beta-globin promoter by 1 Kb and inserting a 1 Kb genomic region corresponding to the HS1 of the LCR. Another genomic element that could raise the level of hemoglobin production by diminishing the variability of expression at different genomic integration sites is the cHS4 insulator element. The production of new therapeutic vectors requires efficient strategies to compare and identify the best beta-globin encoding lentivirus. For this purpose we propose (Aim 3) to investigate the average level of expression of TNS9 versus the new lentiviral vectors (proposed in Aim 2) in BM chimeras. In addition, we propose (Aim 4) to generate transgenic mice from lentiviral transduced single copy ES cells to study the level of and variability in expression for each vector. Finally, insertion of selective drug resistance genes, dihydrofolate reductase (DHFR) versus methylguanine-DNA-methyltransferase (MGMT), will be evaluated (Aim 5) to enhance competitive repopulation of transduced stem cells expressing the human beta-globin gene.