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为治疗先天性严重血红蛋白病如β-地中海贫血提供了一种潜在的手段。出于这个原因，我们已经构建了一个慢病毒载体（TNS 9）携带人类β-珠蛋白基因，并证明，用这个载体，我们可以获得长期的校正受β地中海贫血影响的小鼠模型中间。此外，该载体拯救了一种新的受重型β地中海贫血影响的致死小鼠模型。然而，在这些小鼠中，产生的贫血和血红蛋白的校正水平还不是最佳的。我们相信，为了完全揭示这种基因治疗方法的潜力，我们需要在这种新的库利氏贫血小鼠模型中研究（目的1）慢病毒转导的造血干细胞（HSC）的分数、BM嵌合体的程度和相应的贫血校正水平之间的相关性。为此目的，我们将产生一种新的慢病毒载体，其结合了报告基因的表达，如人源化红移绿色荧光蛋白（hrGFP），在所有造血谱系中和人β-珠蛋白基因在红系细胞中的表达（TNS 9 +GFP）。为了增加人类β-珠蛋白表达（目的2），我们提出了新的慢病毒载体，可能会增加血红蛋白的生产。我们相信，我们可以通过将β-珠蛋白启动子延长1 Kb并插入对应于LCR的HS 1的1 Kb基因组区域来提高TNS 9的血红蛋白产量。另一个可以通过减少不同基因组整合位点表达的变异性来提高血红蛋白产生水平的基因组元件是cHS 4绝缘子元件。新的治疗载体的生产需要有效的策略来比较和鉴定最好的β-珠蛋白编码慢病毒。为此目的，我们提出（目标3）研究BM嵌合体中TNS 9相对于新慢病毒载体（目标2中提出）的平均表达水平。此外，我们提出（目的4）从慢病毒转导的单拷贝ES细胞产生转基因小鼠，以研究每种载体表达的水平和变异性。最后，将评估选择性耐药基因的插入，二氢叶酸还原酶（DHFR）与甲基鸟嘌呤-DNA-甲基转移酶（MGMT）的比较（目的5），以增强表达人β-珠蛋白基因的转导干细胞的竞争性再增殖。

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.