Gene Transfer into Hematopoietic Stem Cells

基因转移至造血干细胞

• 批准号: 6967781
• 负责人: HANS-PETER KIEM
• 金额: $ 29.59万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6967781
• 关键词: NOD mouse; Retroviridae; SCID mouse; baboons; carcinogenesis; cell population study; cell transformation; gene delivery system; gene therapy; hematopoietic stem cells; hematopoietic tissue transplantation; hemoglobinopathy; human subject; iatrogenic disease; leukemia; longitudinal animal study; molecular biology information system; nonhuman therapy evaluation; patient oriented research; stem cell transplantation; therapy adverse effect; therapy design /development; transfection; transfection /expression vector; transposon /insertion element; virus integration

The overall goal of this project is to comprehensively analyze the safety of viral gene transfer and to explore strategies that reduce the risk of insertional mutagenesis in a clinically highly relevant large animal model. The development of leukemia in two patients enrolled in the French clinical trial of stem cell gene therapy for the treatment of X-linked SCID has led to a reassessment for the field of stem cell gene transfer. Up to this point, oncoretroviral vectors were considered safe, and the focus has therefore been on optimizing efficacy. To that end, we have developed stem cell transducfion conditions that result in long-term gene transfer levels of 20% to 30% in the baboon model. With the report of leukemia in the French trial three years after gene therapy, long-term follow-up data from clinically relevant large animal models with high-level marking has now become a unique and necessary resource to determine the safety of gene therapy utilizing integrating vector systems. Thus, in Specific Aim 1, we propose to monitor baboons with persistent high-level marking and gene expression for the development of monoclonality and leukemia. In Specific Aim 2, we will use linear amplification mediated (LAM)-PCR analysis to study and comprehensively characterize the proviral integration sites in these animals, and interrogate available human genome databases to determine the position of integration sites and the association to cancer-related genes. We will also develop and evaluate strategies to improve safety for future stem cell gene therapy studies. In Specific Aims 3 and 4 we propose to reduce the risk of insertional mutagenesis by decreasing the cell dose of gene-modified cells. We will first examine whether intra-marrow infusion will allow for more efficient engraftment of gene-modified stem cells. We will then determine whether more immature and thus smaller numbers ofhematopoietic stem/progenitor cells can be targeted for gene therapy applications. In Specific Aim 5, we will explore whether nonviral gene transfer strategies can be used to genetically modify stem cells, and we will evaluate their safety compared to viral vector systems. Results obtained in these nonhuman primate studies should be readily translatable and applicable to human gene therapy studies.

该项目的总体目标是全面分析病毒基因转移的安全性，并探索在临床高度相关的大动物模型中降低插入突变风险的策略。参加法国干细胞基因疗法治疗 X 连锁 SCID 临床试验的两名患者罹患白血病，引发了对干细胞基因转移领域的重新评估。到目前为止，肿瘤逆转录病毒载体被认为是安全的，因此重点是优化功效。为此，我们开发了干细胞转导条件，可实现 20% 至 30% 的长期基因转移水平 在狒狒模型中。随着基因治疗三年后在法国试验中报告白血病，来自具有高水平标记的临床相关大型动物模型的长期随访数据现已成为利用整合确定基因治疗安全性的独特且必要的资源。矢量系统。因此，在具体目标 1 中，我们建议通过持续高水平标记和基因表达来监测狒狒，以了解单克隆性和白血病的发展。在具体目标 2 中，我们将使用线性扩增介导 (LAM)-PCR 分析来研究和全面表征这些动物中的原病毒整合位点，并询问可用的人类基因组数据库以确定整合位点的位置和 与癌症相关基因的关联。我们还将制定和评估策略，以提高未来干细胞基因治疗研究的安全性。在具体目标 3 和 4 中，我们建议通过减少基因修饰细胞的细胞剂量来降低插入诱变的风险。我们将首先检查骨髓内输注是否可以更有效地植入基因修饰干细胞。然后我们将确定是否可以针对更不成熟且数量更少的造血干/祖细胞进行基因治疗应用。在 具体目标5，我们将探索非病毒基因转移策略是否可用于对干细胞进行基因修饰，并且我们将评估其与病毒载体系统相比的安全性。这些非人类灵长类动物研究中获得的结果应该很容易转化并适用于人类基因治疗研究。