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

新小鼠模型中库利贫血的基因治疗

• 批准号: 7985265
• 负责人: STEFANO RIVELLA
• 金额: $ 5.4万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-10 至 2010-11-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7985265
• 关键词: Affect; Anemia; Cells; Chimera organism; Chimerism; Cooley' s anemia; Data; Dihydrofolate Reductase; Drug resistance; Elements; Erythroid Cells; Gene Expression; Genes; Genomics; Globin; Green Fluorescent Proteins; Hematopoietic; Hematopoietic stem cells; Hemoglobin; Hemoglobin concentration result; Hemoglobinopathies; Housekeeping; Human; Insulator Elements; Lentivirus Vector; Mus; O(6)-Methylguanine-DNA Methyltransferase; Phosphoglycerate Kinase; Production; Protein Isoforms; Reporter Genes; Residual state; Site; Stem cells; Subfamily lentivirinae; Surface; Thalassemia intermedia; Transgenic Mice; Transplantation; beta Globin; beta Thalassemia; cellular transduction; embryonic stem cell; gene therapy; mouse model; novel therapeutics; promoter; vector

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.

描述（由申请人提供）： 基于干细胞的基因疗法为治疗先天性严重血红蛋白病（如β地中海贫血）提供了一种潜在的方法。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，我们可以获得中间型β-地中海贫血小鼠模型的长期校正。此外，该载体拯救了一种受重型β地中海贫血影响的新的致死小鼠模型。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。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贫血小鼠模型中研究（目标 1）慢病毒转导的造血干细胞 (HSC) 分数、 BM嵌合程度和相应的贫血纠正水平。为此，我们将生成一种新的慢病毒载体，该载体结合了报告基因（例如人源化红移绿色荧光蛋白（hrGFP））在所有造血谱系中的表达以及人β-珠蛋白基因在红细胞中的表达(TNS9+GFP)。为了增加人类 β-珠蛋白表达（目标 2），我们建议生成新的慢病毒载体，该载体可能会增加血红蛋白的产生。我们相信，通过将 β-珠蛋白启动子延伸 1 Kb 并插入对应于 LCR HS1 的 1 Kb 基因组区域，可以提高 TNS9 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变异性来提高血红蛋白产量水平的基因组元件是 cHS4 绝缘子元件。新治疗载体的生产需要有效的策略来比较和鉴定最佳的β珠蛋白编码慢病毒。为此，我们建议（目标 3）研究 BM 嵌合体中 TNS9 与新慢病毒载体（目标 2 中提出的）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 细胞生成转基因小鼠，以研究每个载体的表达水平和变异性。最后，将评估选择性耐药基因、二氢叶酸还原酶 (DHFR) 与甲基鸟嘌呤-DNA-甲基转移酶 (MGMT) 的插入（目标 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。

项目成果

Protective role of calreticulin in HFE hemochromatosis.

钙网蛋白在 HFE 血色素沉着症中的保护作用。

• DOI: 10.1016/j.freeradbiomed.2007.09.014
• 发表时间: 2008
• 期刊: Free radical biology & medicine
• 影响因子: 7.4
• 作者: Pinto,JorgeP;Ramos,Pedro;deAlmeida,SérgioF;Oliveira,Susana;Breda,Laura;Michalak,Marek;Porto,Graça;Rivella,Stefano;deSousa,Maria
• 通讯作者: deSousa,Maria

Regulation of iron absorption in hemoglobinopathies.

• DOI: 10.2174/156652408786241401
• 发表时间: 2008-11
• 期刊: Current molecular medicine
• 影响因子: 2.5
• 作者: Rechavi G;Rivella S
• 通讯作者: Rivella S

Exploring the role of hepcidin, an antimicrobial and iron regulatory peptide, in increased iron absorption in beta-thalassemia.

探索铁调素（一种抗菌和铁调节肽）在增加 β 地中海贫血铁吸收中的作用。

• DOI: 10.1196/annals.1345.069
• 发表时间: 2005
• 期刊: Annals of the New York Academy of Sciences.
• 作者: Breda,Laura;Gardenghi,Sara;Guy,Ella;Rachmilewitz,EliezerA;Weizer-Stern,Orly;Adamsky,Konstantin;Amariglio,Ninette;Rechavi,Gideon;Giardina,PatriciaJ;Grady,RobertW;Rivella,Stefano
• 通讯作者: Rivella,Stefano

Ineffective erythropoiesis and thalassemias.

• DOI: 10.1097/moh.0b013e32832990a4
• 发表时间: 2009-05
• 期刊: Current opinion in hematology
• 影响因子: 3.2
• 作者: Rivella S