Foamy Virus Vectors for Stem Cells

将基因转移到造血干细胞（HSC）中具有治疗许多疾病的潜力，包括血红蛋白病、免疫缺陷、感染性疾病，以及如果HSC形成不同组织类型，甚至其他器官系统的病症。然而，插入突变的致癌风险和人HSC的低转导率仍然是主要问题。在这里，我们建议开发基于泡沫病毒（FV）的逆转录病毒载体，以克服这些问题。野生型FV是圈养灵长类动物中常见的非致病性病毒，从未与肿瘤发生相关，这表明FV载体可能比其他整合载体更安全。为了确定其致癌风险，我们将研究插入突变 通过FV载体以多种方式，包括在人CD 34+细胞中定位整合位点;将前病毒位点与染色体基因位置、表达和构象相关联;以及确定在具有定位的前病毒的一组细胞中相邻基因是否以及如何被激活。将开发几种测定来观察载体激活邻近基因的潜力，包括用克隆的前病毒进行质粒转染测定;基于前病毒依赖性激活细胞因子独立性的细胞转化测定;以及 在癌症易感小鼠中进行骨髓移植试验以直接证明肿瘤发生。这些试验将用于比较不同的FV载体以及基于慢病毒和鼠白血病病毒的载体。将设计更安全的FV载体，其通过掺入具有更低增强子活性的内部启动子、改变载体骨架元件以防止通读转录和剪接以及包括遗传绝缘子元件而具有更低的激活邻近基因的潜力。在先前的工作中，我们发现FV载体在动物移植和异种移植中有效地转化小鼠和人HSC 这表明它们可能上级其他类型的干细胞载体。这里我们将 在更接近模拟人造血的大型动物模型（狗和非人灵长类动物）中测定HSC转导率。在这些移植研究中，将FV载体与tentivirus和鼠白血病病毒载体进行比较，以确定哪种载体类型更有效，并比较潜在的副作用。这些研究将建立有用的方法来评估任何整合载体的风险，改进现有的FV载体，并直接比较所有3种类型的逆转录病毒载体的安全性和有效性。

Gene transfer into hematopoietic stem cells (HSCs) has the potential to treat many diseases, including hemoglobinopathies, immunodeficiencies, infectious diseases, and even disorders of other organ systems if HSCs form different tissue types. However the oncogenic risks of insertional mutagenesis and low transduction rates of human HSCs remain major problems. Here we propose to develop retroviral vectors based on foamy virus (FV) in order to overcome these problems. Wild-type FV is a non-pathogenic virus common in captive primates that has never been linked to oncogenesis, suggesting that FV vectors may be safer than other integrating vectors. In order to establish their oncogenic risk, we will study insertional mutagenesis by FV vectors in a variety of ways, including mapping of integration sites in human CD34+ cells; correlating provirus sites with chromosomal gene location, expression, and conformation; and determining if and how neighboring genes are activated in a panel of cells with mapped proviruses. Several assays will be developed to look at a vector s potential for activating neighboring genes, including plasmid transfection assays with cloned proviruses; celt transformation assays based on provirus-dependent activation of cytokine independence; and bone marrow transplantation assays in cancer-prone mice to directly demonstrate oncogenesis. These assays will be used to compare different FV vectors and also vectors based on lentiviruses and murine leukemia viruses. Safer FV Vectors will be designed that have less potential for activating neighboring genes by incorporating internal promoters with less enhancer activity, changing vector backbone elements to prevent read-through transcription and splicing, and including genetic insulator elements. In prior work we showed that FV vectors efficiently transform mouse and human HSCs in animal transplantation and xenotransplantation models, suggesting that they may be superior to other types of stem cell vectors. Here we will determine HSC transduction rates in large animal models that more closely simulate human hematopoiesis (dog and non-human primate). FV vectors will be compared to tentivirus and murine leukemia virus vectors in these transplantation studies to determine which vector type is more effective, and to compare potential side effects. These studies will establish useful methods for assessing the risks of any integrating vector, improve existing FV vectors, and direcdy compare the safety and efficacy of all 3 types of retroviral vectors.