ADENOVECTORS FOR DELIVERY OF HEMATOPOIETIC GROWTH FACTORS AND RECEPTORS

用于输送造血生长因子和受体的腺病毒载体

• 批准号: 6258920
• 负责人: MALCOLM A. MOORE
• 金额: $ 12.87万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6258920
• 关键词: Adenoviridae; SCID mouse; cell adhesion; cell differentiation; cell migration; cytokine; developmental genetics; gene expression; hematopoiesis; hematopoietic growth factor; hematopoietic stem cells; immunogenetics; laboratory mouse; method development; protein isoforms; protein structure function; protooncogene; transfection /expression vector; virus receptors

Adenovectors have proved highly efficient in transferring and expressing genes in murine and human marrow stromal and endothelial cells. At high multiplicities of infection, Ad vector expression can be obtained in immature hematopoietic cells (CD34+) and a variety of differentiated cell populations (erythroid, myeloid, dendritic, megakaryocyte). In vitro and in vivo murine studies will evaluate Ad vector delivery of cytokine genes (erythropoietin, G-CSF, GM-CSF c-kit ligand, Flk-2 ligand), singly or in combination, for augmentation of hematopoiesis and acceleration of recovery following chemotherapy, radiation, or bone marrow transplantation. Mutations at the 51 locus point to the importance of its product, the c-kit ligand and, specifically, the transmembrane isoform (KL-2) without a proteolytic cleavage site as important for stem cell migration, proliferation, and differentiation, and for optimal erythropoiesis. Soluble ligand administered systemically or produced in the Sl-Dicke mouse cannot substitute for the transmembrane form. Transmembrane isoforms of Flk-2 ligand may also play an important role in local regulation of stem cell self-renewal. Transmembrane forms of c-kit and Flk-2 ligands will be expressed by Ad vectors in stromal cells and marrow endothelium, in vitro or by locoregional administration in vivo, and compared to vectors expressing soluble ligand with emphasis on improved stem cell homing, stromal/endothelial adhesion and self-renewal. We have evidence that murine Notch expressed by primitive hematopoietic cells, and its ligand DLL1 expressed by stromal and endothelial cells, influence developmental fate decisions in hematopoiesis. This will be evaluated in hematopoietic systems where Ad vectors permit expression of high levels of Notch or its ligand. Transient overexpression of c-kit and Flk-2 on hematopoietic stem cells will be evaluated, and the impact on stem cell migration, adherence. self-renewal, proliferative status, and resistance to TGFbeta inhibition determined. Flk-l is expressed as a very primitive hematopoietic precursor (hemoblast/hemangioblast) that first appears in the yolk sac blood islands or their equivalent in embryonic stem cell differentiation, but is not expressed in later fetal or adult stem cells. We shall use Ad vectors to express Flk-l in adult stem cells and evaluate their proliferative response to VEGF alone or in combination with KL. Ad vector expression of the murine ecotropic retroviral receptor will be evaluated as a method for improving retroviral gene transduction of human stem cells. Introduction of a marker gene (mutated nerve growth factor) and of a methotrexate drug-resistance gene (mutated dihydrofolate reductase) will permit evaluation of stem cell transduction efficiency in a long-term culture assay and in vivo in SCID mice.

事实证明，Adenovectors在转移和 基因在小鼠和人骨髓基质和内皮细胞中的表达 细胞。在高度感染的情况下，Ad载体表达可以 是从未成熟的造血细胞(CD34+)和多种 分化的细胞群(红系、髓系、树突状、 巨核细胞)。体外和体内的小鼠研究将评估 细胞因子基因(促红细胞生成素、粒细胞集落刺激因子、粒细胞集落刺激因子)的腺病毒载体 C-KIT配体，Flk-2配体)，单独或组合，用于 增强造血功能，加速康复 化疗、放疗或骨髓移植。突变 在51位点上，它的产品c-kit的重要性 配体，特别是跨膜异构体(KL-2)，没有 一个对干细胞迁移很重要的蛋白水解性裂解位点， 增殖和分化，以及最佳的红细胞生成。 系统给药或在SL-Dicke中产生的可溶性配体 小鼠不能替代跨膜形式。跨膜 Flk-2配体的亚型也可能在局灶性脑损伤中发挥重要作用 干细胞自我更新的调控。C-kit的跨膜形式 Flk-2配体将通过Ad载体在基质细胞中表达 和骨髓内皮细胞，体外或局部给药 并与表达可溶性配体的载体进行比较。 强调改善干细胞归巢、基质/内皮细胞黏附 和自我更新。我们有证据表明小鼠的Notch通过 原始造血细胞及其配体DLL1的表达 基质和内皮细胞，影响发育命运 在造血方面的决定。这将在造血学中进行评估 其中Ad载体允许高水平Notch表达的系统 或者它的配体。C-kit和Flk-2在卵巢癌细胞中的瞬时过表达 将对造血干细胞进行评估，以及对干细胞的影响 细胞迁移，黏附。自我更新、增殖状态，以及 测定对转化生长因子β抑制的抵抗力。卡斯特罗-L被表达为 一种非常原始的造血祖细胞(血母细胞/血管母细胞) 最早出现在卵黄囊血岛或与之相当的地方 在胚胎干细胞分化中，但在 后来的胎儿或成体干细胞。我们将使用广告载体来表达 成体干细胞中Flk-L的表达及其增殖反应的评价 对血管内皮生长因子单独或与KL联合应用。的AD向量表达 小鼠亲生性逆转录病毒受体将作为一种方法进行评估 用于改善人类干细胞的逆转录病毒基因转导。 标记基因(突变型神经生长因子)的导入 甲氨蝶呤耐药基因(突变的二氢叶酸 还原酶)将允许对干细胞转导进行评估 长期培养试验和在SCID小鼠体内的效率。