FETAL MODEL FOR GENE TRANSFER FOR SICKLE CELL DISEASE

镰状细胞病基因转移胎儿模型

• 批准号: 6650012
• 负责人: Alice F Tarantal
• 金额: $ 13.59万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6650012
• 关键词: Lentivirus; Macaca mulatta; adeno associated virus group; biotechnology; clinical research; embryo /fetus; erythropoietin; gene therapy; globin; green fluorescent proteins; growth factor receptors; hematopoietic stem cells; human subject; in utero transplantation; murine leukemia virus; sickle cell anemia; stem cell transplantation; transfection /expression vector

The long-term goal of the Project is to explore fetal gene transfer for sickle cell disease (SCD) in a fetal rhesus monkey model. Gene therapy strategies that target hematopoietic stem cells (HSC) have been proposed as a long-term treatment for hemoglobinopathies such as SCD. However, traditional ex vivo gene therapy approaches for transducing CD34+ HSC in humans and large animal models have proven disappointing due to low gene transfer efficiencies, poor expression of the introduced gene, and technical difficulties associated with the transplant procedures. In this Project we are proposing direct in vivo gene transfer into pre-immune fetal rhesus monkeys because this approach eliminates the many problems and limitations associated with removal of HSC, in vitro transduction, and subsequent re-introduction in vivo, and avoids significant immune responses by administering the transgene while the fetus is immunologically immature. Initial studies will compare viral vector systems in the transgene while the fetus is immunologically immature. Initial studies will compare viral vector systems in order to determine the best method for transduction of fetal HSC in utero (Specific Aim 1). The vector systems we will explore include Moloney murine leukemia virus (MLV), HIV-1-derived lentivirus, and adeno-associated the vectors to early gestation rhesus fetuses either by the intrahepatic or intraportal routes in order to determine the most efficient method for targeting fetal HSC in utero. Fetal samples will be collected in utero )blood, liver) then at birth and monthly thereafter (blood, marrow) until 1 year of age for assessments of transduction and transgene expression (flow cytometry, PCR, hematopoietic progenitor assays). Once we have determined the best vector system and approach for the efficient marking of fetal HSC, we will introduce the human beta-gamma globin gene into the HSC of preimmune fetal rhesus monkeys (Specific Aim 2). We will assess MND (which enhances expression in HSC) or the beta-globin LCR (which has been shown to increase the frequency of globin-expressing erythroid cells) to determine which is most efficient in achieving stable gene expression. Fetal samples will be collected in utero transduction and gene expression. Finally, to test whether human HSC with the erythropoietin receptor (EpoR) insert have a proliferative advantage, we will use our human: rhesus xenograft model to perform a competitive repopulation study by administering a 50:50 mixture of HSC expressing the EpoR (marked with EGFP) and HSC without the inset (marked with EYFP) (Specific Aim 3). These studies will allow us to assess whether the addition of EpoR to the HSC provides a method for expanding targeted populations of genetically altered cells in vivo, which could facilitate gene therapy techniques. Thus, we propose to explore methods for safely transferring healthy genes into fetal monkeys that can be persistently expressed in hematopoietic cells as a model for the human fetus diagnosed with hemoglobinopathies such as SCD.

该项目的长期目标是在恒河猴胎儿模型中探索镰状细胞病（SCD）的胎儿基因转移。针对造血干细胞 (HSC) 的基因治疗策略已被提议作为血红蛋白病（如 SCD）的长期治疗方法。然而，由于基因转移效率低、导入基因表达差以及与移植程序相关的技术困难，在人类和大型动物模型中转导 CD34+ HSC 的传统离体基因治疗方法已被证明令人失望。在这个项目中，我们建议将体内基因直接转移到免疫前的恒河猴胎儿中，因为这种方法消除了与去除 HSC、体外转导和随后重新引入体内相关的许多问题和限制，并避免了显着的免疫反应通过在胎儿免疫学不成熟时施用转基因。初步研究将比较胎儿免疫学不成熟时转基因中的病毒载体系统。初步研究将比较病毒载体系统，以确定在子宫内转导胎儿 HSC 的最佳方法（具体目标 1）。我们将探索的载体系统包括莫洛尼鼠白血病病毒 (MLV)、HIV-1 衍生的慢病毒和通过肝内或门静脉内途径将载体腺相关到早期妊娠恒河猴胎儿，以确定最有效的靶向方法子宫内胎儿 HSC。将在子宫内收集胎儿样本（血液、肝脏），然后在出生时收集，此后每月收集（血液、骨髓）直至 1 岁，以评估转导和转基因表达（流式细胞术、PCR、造血祖细胞测定）。一旦我们确定了有效标记胎儿 HSC 的最佳载体系统和方法，我们将把人类 β-γ 珠蛋白基因引入到免疫前胎儿恒河猴的 HSC 中（具体目标 2）。我们将评估 MND（增强 HSC 中的表达）或 β-珠蛋白 LCR（已被证明可以增加表达珠蛋白的红系细胞的频率），以确定哪一个最有效地实现稳定的基因表达。胎儿样本将在子宫内转导和基因表达中收集。最后，为了测试带有促红细胞生成素受体 (EpoR) 插入的人类 HSC 是否具有增殖优势，我们将使用我们的人类：恒河猴异种移植模型，通过施用 50:50 表达 EpoR 的 HSC 混合物（标记为EGFP）和没有插图的 HSC（用 EYFP 标记）（具体目标 3）。这些研究将使我们能够评估将 EpoR 添加到 HSC 是否提供了一种在体内扩大转基因细胞目标群体的方法，这可以促进基因治疗技术。因此，我们建议探索将健康基因安全转移到胎猴体内的方法，这些基因可以在造血细胞中持续表达，作为诊断患有血红蛋白病（如 SCD）的人类胎儿的模型。