In Vivo BM Stem Cell Gene Transfer for MPS type I

针对 I 型 MPS 的体内 BM 干细胞基因转移

• 批准号: 6923451
• 负责人: Dao Pan
• 金额: $ 18.63万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6923451
• 关键词: Lentivirus; biotechnology; bone marrow; gene delivery system; gene therapy; hematopoietic stem cells; immunofluorescence technique; laboratory mouse; mucopolysaccharidosis type I; stromal cells; technology /technique development; tissue /cell culture; transfection /expression vector

DESCRIPTION (provided by applicant): HIV-based lentiviral vectors (LV) have demonstrated the capacity for in vivo gene transfer and gene expression in non-dividing cells after local injection. However, the potential of in vivo bone marrow stem cell gene transfer is virtually unexplored, except for results recently published by others and us and our preliminary data. The in vivo approach of bone marrow stem cell gene transfer could take full advantage of any source of stem cells present in bone cavity and avoid many of the difficulties encountered by ex vivo HSC gene transfer. The ultimate objective of this study is to determine if intra bone marrow injection of LV can efficiently transduce BM stem cells, and if the levels of gene transfer can achieve phenotypic correction for diseases such as Mucopolysaccharidosis type I (MPS I) that affects multiple organs including the brain, bone and hematopoietic system. A selectable marking SIN-LV containing eGFP and MGMT (P140K) will be used to inject normal adult mice (Specific Aim1). Gene transfer in HSC will be evaluated in BMT recipients and in vitro LTCIC cultures by CPU assay, FACS analysis, QPCR, clonality analysis and fluorescent microscopy. Transduction of MPC will be studied in stromal cell cultures using in vitro MPC differentiation assays and immuno-fluorescent microscopy, while systemic biodistribution will also be assessed including the gonad. We will also investigate the effects on transduction profiles and efficiency in BM stem cells by pretreatment of 5FU/SDF-1, myeloablation, tourniquet, re-administration of LV and MGMT-mediated in vivo selection. MPS I knock-out mice will be utilized to study the degrees of metabolic, neurological and skeletal corrections after intra bone marrow injection of a LV containing a human IDUA cDNA (Specific Aim 2). Three age groups of MPS I mice (young, adult and elderly) will be used to assess the aging effects on gene transfer patterns and the degree of correction on degree of correction on disease manifestations. Taken together, these data would not only open a door to a novel approach for treatment of human diseases, but also provide a new tool to study adult stem cell plasticity and the nature of hematopoiesis.

描述（由申请人提供）：基于HIV的慢病毒载体（LV）已证明局部注射后在非分裂细胞中进行体内基因转移和基因表达的能力。然而，除了其他人和我们最近发表的结果以及我们的初步数据外，体内骨髓干细胞基因转移的潜力实际上尚未被探索。骨髓干细胞基因转移的体内方法可以充分利用骨腔中存在的任何干细胞来源，并避免离体HSC基因转移遇到的许多困难。本研究的最终目的是确定骨髓内注射 LV 是否可以有效转导骨髓干细胞，以及基因转移水平是否可以实现对影响多个器官（包括 I 型粘多糖贮积症（MPS I））等疾病的表型校正。大脑、骨骼和造血系统。含有 eGFP 和 MGMT (P140K) 的选择性标记 SIN-LV 将用于注射正常成年小鼠（具体目标 1）。 HSC 中的基因转移将通过 CPU 测定、FACS 分析、QPCR、克隆分析和荧光显微镜在 BMT 受者和体外 LTCIC 培养物中进行评估。将使用体外 MPC 分化测定和免疫荧光显微镜在基质细胞培养物中研究 MPC 的转导，同时还将评估包括性腺在内的全身生物分布。我们还将研究 5FU/SDF-1 预处理、清髓、止血带、LV 重新给药和 MGMT 介导的体内选择对 BM 干细胞转导谱和效率的影响。 MPS I 敲除小鼠将用于研究骨髓内注射含有人 IDUA cDNA 的 LV 后代谢、神经和骨骼矫正的程度（具体目标 2）。 将使用三个年龄组的 MPS I 小鼠（年轻、成年和老年）来评估衰老对基因转移模式的影响以及对疾病表现的校正程度的校正程度。总而言之，这些数据不仅将为治疗人类疾病的新方法打开一扇大门，而且还为研究成体干细胞可塑性和造血性质提供了新工具。