Retroviral Mediated Gene Transfer Into Primate Hematopoietic Cells

为了成功地将基因转移到原始造血细胞，需要达到几个要求。这些包括鉴定所需的靶细胞群，鉴定待使用的适当载体，以及实现所需的基因表达水平。迄今为止，在人类受试者中成功的基因转移仍然存在问题。为了解决这些问题以及重要的安全性问题，正在进行非人灵长类动物的研究，以在人类临床研究之前优化向非人灵长类动物造血细胞的基因转移。已经评估的载体包括自失活（SIN）逆转录病毒载体，最近构建的最佳恒河猴CD 34+细胞的慢病毒载体。这些载体已被构建以表达报告基因，如增强型绿色荧光蛋白（EGFP），或治疗基因，如血红蛋白。转导条件采用含RGD的纤连蛋白片段、RetroNectin（CH-296）和多种重组造血生长因子，如干细胞因子（SCF）、白细胞介素-6、巨核细胞生长和分化因子（MGDF或血小板生成素）和人Flt-3（fms样酪氨酸激酶）配体，在含血清或无血清培养基中。评价的病毒载体包括逆转录病毒载体，例如第三代嵌合人免疫缺陷病毒1型（HIV-1）-基慢病毒载体。我们在过去一年的努力已经导致出版物评估使用载体跟踪随着时间的推移谱系贡献和识别的遗传因素在有效的基因转导的CD 34+细胞的重要性。继续努力提高基因标记的水平，将基因表达靶向特定细胞类型，如红细胞，评估移植后的免疫重建和遗传标记细胞对恢复的贡献，以及从除BM和细胞因子动员的PB之外的其他组织衍生干细胞，如成体间充质干/祖细胞和诱导的多能干细胞，并评估它们在该体内模型系统中的安全性。在过去的一年里，在开发诱导多能干细胞（iPS细胞）以及间充质基质细胞（MSC）方面取得了最近的成功。还在努力改进动员和收集干细胞的方法和技术。正在评估替代方法，例如，在小型受试者中白细胞分离术的仪器和方法。尽管方法不断改进，但问题依然存在。如何使用治疗基因获得一致的高水平表达？这是通过改变红细胞中的珠蛋白表达来评估的。其他来源于骨髓或其他容易获得的组织的干细胞是否可以被靶向以帮助其他器官的贡献或修复？这是通过iPS细胞和MSC的生成进行评估的。如何最好地评估干细胞及其后代的治疗效果？未来的研究旨在评估治疗载体，提高造血干细胞的恢复和转导效率，进一步描绘的性质和克隆性的人口有助于重建使用遗传跟踪方法，并分离或诱导和表征原始细胞群体，可能有助于器官发生或修复受损组织。

For successful gene transfer to primitive hematopoietic cells several requirements need to be achieved. These include identification of the desired target cell population, identification of the appropriate vector to be used, and achieving desired levels of gene expression. To date, successful gene transfer in human subjects remain problematic. To address these problems as well as important safety issues, studies in non-human primates are being undertaken to optimize gene transfer to nonhuman primate hematopoietic cells prior to human clinical studies. Vectors that have been evaluated include self-inactivating (SIN) retroviral vectors, mst recently lentiviral vectors constructed to optimall transduce rhesus CD34+ cells. These vectors have been constructed to express reporter genes, such as the enhanced green fluorescent protein (EGFP), or therapeutic genes, such as hemoglobin. Transduction conditions employed the RGD-containing fibronectin fragment, RetroNectin (CH-296) and a variety of recombinant hematopoietic growth factors, such as stem cell factor (SCF), interleukin-6, megakaryocyte growth and differentiation factor (MGDF or thrombopoietin) and the human Flt-3 (fms-like tyrosine kinase) ligand in either serum containing or serum free media. Viral vectors evaluated include retroviral vectors, such as third generation chimeric human immunodeficiency virus type-1 (HIV-1)-based lentiviral vectors. Our efforts over the past year have resulted in publications evaluating the use of vectors in tracking lineage contributions of over time and the identification of genetic factors important in efficient gene trasduction of CD34+ cells. Efforts continue to be made to improve the level of gene marking, targeting gene expression to specific cell types, such as red blood cells, evaluate immune recostitution following transplant and the contribution of gentically marked cells to the recovery, and to derive stem cells from other tissues besides BM and cytokine mobilized PB, such as adult mesenchymal stem/progenitor cells and induced pluripotential stem cells and evaluate their safety in this in vivo model sytem. Recent success in developing induced pluripotential stem cells (iPS cells) as well as mesenchymal stromal cells (MSC) have been made this past year. Attempts are also being made to improve methodology and the technology behind stem cell mobilization and collection. Alternative approaches are being evaluated, for example, in both the instrumentation and methodology of leukapheresis procedures in small subjects. Despite continued improvements in methodology, questions remain. How can consistent high levels of expression be obtained using therapeutic genes? This is being evaluated by modifying globin expression in red blood cells. Can other stem cells either derived from bone marrow or other easily accessible tissues be targeted to assist in either the contribution or repair of other organs? This is being evaluated through the generation of iPS cells and MSC. How best to evaluate stem cells and their progeny therapeutically? Future studies are aimed to evaluate therapeutic vectors, improve hematopoietic stem cell recovery and transduction efficiency, further delineate the nature and clonality of populations contributing to the reconstitution using genetic tracking methodologies, and to isolate or induce and characterize primitive cell populations which may contribute to organogenesis or the repair of damaged tissues.