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: 6501114
• 负责人: MALCOLM A. MOORE
• 金额: $ 12.63万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6501114
• 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.

腺载体已被证明在转移和 在小鼠和人类骨髓基质和内皮细胞中表达基因 细胞。在高感染复数下，Ad 载体表达可以 在未成熟造血细胞（CD34+）和多种 分化细胞群（红细胞、髓细胞、树突细胞、 巨核细胞）。体外和体内小鼠研究将评估 Ad 载体递送细胞因子基因（促红细胞生成素、G-CSF、GM-CSF c-kit配体、Flk-2配体)，单独或组合，用于 增强造血功能并加速术后恢复 化疗、放疗或骨髓移植。突变 第 51 个位点表明了其产品的重要性，c-kit 配体，特别是跨膜亚型（KL-2），没有 蛋白水解切割位点对于干细胞迁移很重要， 增殖和分化，以及最佳红细胞生成。 全身给药或在 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部发挥重要作用 干细胞自我更新的调节。 c-kit 的跨膜形式 Flk-2配体将由基质细胞中的Ad载体表达 和骨髓内皮细胞，体外或局部给药 体内，并与表达可溶性配体的载体相比 强调改善干细胞归巢、基质/内皮粘附 和自我更新。我们有证据表明小鼠Notch表达为 原始造血细胞及其表达的配体DLL1 基质细胞和内皮细胞，影响发育命运 造血过程中的决定。这将在造血系统中进行评估 Ad 载体允许表达高水平 Notch 的系统 或其配体。 c-kit 和 Flk-2 的瞬时过表达 将评估造血干细胞，以及对干细胞的影响 细胞迁移、粘附。自我更新、增殖状态和 确定对TGFbeta抑制的抗性。 Flk-l 表示为 非常原始的造血前体（成血细胞/成血管细胞） 首先出现在卵黄囊血岛或类似物中 参与胚胎干细胞分化，但不表达于 后来的胎儿或成人干细胞。我们将使用Ad向量来表达 成体干细胞中的 Flk-l 并评估其增殖反应 单独使用VEGF或与KL联合使用。广告载体表达 鼠亲嗜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。 引入标记基因（突变神经生长因子）并 甲氨蝶呤耐药基因（突变的二氢叶酸 还原酶）将允许评估干细胞转导 长期培养测定和 SCID 小鼠体内的效率。