Gene Transfer into Hematopoietic Stem Cells

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型动物模型中降低插入突变风险的策略。在法国进行的干细胞基因疗法治疗X连锁SCID的临床试验中，两名患者发生白血病，这导致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，因此重点是优化疗效。为此，我们开发了干细胞转导条件，导致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法国试验中白血病的报告，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确定利用整合载体系统进行基因治疗的安全性的独特且必要的资源。因此，在具体目标1中，我们建议监测狒狒持续高水平标记和基因表达的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前病毒整合位点，并查询可用的人类基因组数据库以确定整合位点的位置和整合位点的表达。 与癌症相关的基因。我们还将开发和评估策略，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，我们提出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会使基因修饰的干细胞更有效地植入。然后我们将确定是否有更多的未成熟的，从而更少数量的造血干/祖细胞可以为基因治疗应用的目标。在 具体目标5，我们将探讨非病毒基因转移策略是否可以用于遗传修饰干细胞，我们将评估其安全性相比，病毒载体系统。在这些非人灵长类动物研究中获得的结果应易于翻译并适用于人类基因治疗研究。

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.