In Vivo BM Stem Cell Gene Transfer for MPS type I

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，体内骨髓干细胞基因转移的潜力实际上是未开发的，除了最近发表的结果和我们的初步数据。体内骨髓干细胞基因转移方法可以充分利用骨腔中存在的任何干细胞来源，避免了体外HSC基因转移所遇到的许多困难。本研究的最终目的是确定骨髓内注射LV是否可以有效地转导BM干细胞，以及基因转移水平是否可以实现对粘多糖病I型（MPS I）等疾病的表型纠正，这些疾病影响多个器官，包括大脑、骨骼和造血系统。将使用含有eGFP和MGMT（P140 K）的可选择标记SIN-LV注射正常成年小鼠（特异性Aim 1）。将通过CPU测定、FACS分析、QPCR、克隆性分析和荧光显微镜检查在BMT受体和体外LTCIC培养物中评价HSC中的基因转移。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。我们还将研究通过5 FU/SDF-1预处理、骨髓消融、止血带、LV再给药和MGMT介导的体内选择对BM干细胞中转导谱和效率的影响。将使用MPS I基因敲除小鼠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（特异性目的2）。 将使用三个年龄组的MPS I小鼠（年轻、成年和老年）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。总之，这些数据不仅为治疗人类疾病的新方法打开了大门，而且还为研究成体干细胞可塑性和造血的性质提供了新的工具。

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.