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。这些步骤包括识别所需的目标细胞群，识别要使用的适当载体，以及实现所需的基因表达水平。到目前为止，在人类受试者中成功的基因转移仍然是个问题。为了解决这些问题和重要的安全问题，在人类临床研究之前，正在进行非人类灵长类动物的研究，以优化向非人类灵长类造血细胞的基因转移。已评估的载体包括自失活(SIN)逆转录病毒载体和腺相关病毒载体。这些载体被构建来表达报告基因，如增强型绿色荧光蛋白(EGFP)，或治疗基因，如耐药基因，促红细胞生成素。转导条件采用含RGD的纤维连接蛋白片段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。评估的病毒载体包括基于Moloney小鼠白血病的载体、腺相关病毒(AAV)载体和基于第三代嵌合人类免疫缺陷病毒1型(HIV-1)的慢病毒载体。第三代慢病毒载体与水疱性口炎病毒G蛋白进行假分型。在过去的一年里，我们的努力导致发表了比较细胞因子动员疗法以改善造血干细胞/祖细胞动员的文章，涉及使用AAV载体靶向向唾液腺进行基因转移的新方法的研究，基于插入位点分析确定载体的相对安全性，以及对新型慢病毒载体的评估，这些载体可以改善造血干细胞的基因转导并有助于去髓动物的长期造血恢复。此外，继续对逆转录病毒载体有效转导的细胞以及其他组织(如脑血管周围巨噬细胞)进行基因跟踪，以确定它们对造血谱系恢复的贡献。使用临床可行方案的持久多谱系标记已经实现了5%-15%的范围。目前正在努力将这一水平的标记扩展到来自骨髓和动员的细胞因子外的其他组织的干细胞，如成体间充质干细胞/祖细胞和诱导的多潜能干细胞。正在对造血重建后的细胞进行表型和功能分析。尽管取得了这些成功，但问题依然存在。如何利用治疗性基因获得一致的高水平表达？从骨髓或其他容易获取的组织中获得的其他干细胞是否可以被靶向帮助其他器官的贡献或修复？未来的研究旨在评估治疗载体，提高造血干细胞的回收和转导效率，利用基因跟踪方法进一步描述有助于重建的群体的性质和克隆性，并随着新研究的启动，分离或诱导和表征可能有助于器官发生或受损组织修复的原始细胞群体。

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 and adeno-associated viral vectors. These vectors have been constructed to express reporter genes, such as the enhanced green fluorescent protein (EGFP), or therapeutic genes, such as drug resistance genes, erythropoietin. 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 Moloney murine leukemia-based vectors, adeno-associated virus (AAV) vectors, and third generation chimeric human immunodeficiency virus type-1 (HIV-1)-based lentiviral vectors. Third generation lentiviral vectors were pseudotyped with the vesicular stomatitis virus G-protein. Our efforts over the past year have resulted in publications comparing cytokine mobilization therapies to improve hematopoietic stem/progenitor cell mobilization, studies involving novel approaches using AAV vectors to target genetic transfer to the salivary gland, determination of the relative safety of vectors based on insertion site analysis, and the evaluation of novel lentiviral vectors that improve gene transduction of hematopoietic stem cells and contribute to long term hematopoietic recovery of a myeloablated animal. In addition, genetic tracking continues to be performed on cells that have been effectively transduced with a retroviral vector in order to determine their contributions to hematopoietic lineage recovery as well as other tissues such as brain perivascular macrophages. Persistent multiple lineage marking using clinically feasible protocols has been achieved ranging from 5-15%. Efforts are being made to extend this level of marking to stem cells derived from other tissues besides BM and cytokine mobilized PB, such as adult mesenchymal stem/progenitor cells and induced pluripotential stem cells. Phenotypic and functional analyses of cells following hematopoietic reconstitution are being evaluated. Despite these successes, questions remain. How can consistent high levels of expression be obtained using therapeutic genes? 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? 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, with the initiation of new studies, to isolate or induce and characterize primitive cell populations which may contribute to organogenesis or the repair of damaged tissues.