Globin LCR containing Adeno-AAV Vectors

含有腺-AAV 载体的球蛋白 LCR

• 批准号: 6967773
• 负责人: ANDRE Michael LIEBER
• 金额: $ 26.13万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6967773
• 关键词: Adenoviridae; NOD mouse; SCID mouse; adeno associated virus group; biotechnology; carcinogenesis; gene expression; gene expression profiling; gene therapy; genetic regulatory element; genetic transduction; globin; green fluorescent proteins; hematopoietic stem cells; human subject; microarray technology; patient oriented research; recombinant virus; sickle cell anemia; therapy adverse effect; therapy design /development; tissue /cell culture; transfection /expression vector; virus infection mechanism; virus integration

GRANT=6967773;P01HL Gene therapy of sickle cell disease requires gene transfer vectors that confer table, regulated, lineage-specific expression of the gamma-globin gene at relatively high levels without the risk of cellular transformation or tumorigenesis. Our proposal to achieve this is based on two recent findings: I) adenovirus (Ad) vectors containing the Ad serotype 35 fibers (Ad5/35) particularly subsets with potential stem cell capacity and ii) incorporation of AAV ITRs into Ad vectors devoid of all viral genes (Ad.AAV) mediates efficient random vector integration and allows for stable gene transfer into human hematopoietic stem cells. To achieve adequate globin expression, we will produce helper-dependent Ad5/35 containing the 26 kb beta-globin LCR (5?HS1 to 5?HS5, beta-globin promoter, 3 HS1); whereby the LCR cassette is flanked by AAV ITRs (Ad,AAV-LCR). In Specific Aim 1, vector production will be optimized with a recently constructed Ad.AAV-LCR vector expressing the GFP gene under the control of the beta-promoter/LCR. To minimize the risk of tumorigenesis, we will further modify Ad.AAV-LCR vectors to allow for selection against vector integration into active genes and to increase the frequency of site-specific integration by transient co-expression of AAV Rep78. In Specific Aim 2, we will study the persistence of GFP expression and the integration pattern of our vectors in clones of transduced erythroleukemic cells. In Specific Aim 3, expression studies and monitoring of integration sites will be performed with colonies of transduced CD34+ cells grown in semisolid cultures, followed by studies in NOD-SCID mice. In an attempt to assess vector-induced tumorigenesis, we will compare the expression profiles of the pool of transduced long-term culture initiating cells and NOD-SCID mice. In an attempt to assess vector-induced tumorigenesis, we will compare the expression profiles of the pool of transduced long-term culture initiating cells and NOD-SCID repopulating cells with that of mock-infected cells using DNA arrays. Finally, if we confirm safety and efficacy with our new vector, in Specific Aim 4, we will construct gamma-globin expressing Ad.AAV-LCR vectors and perform transduction studies in CD34+ cells derived from healthy donors and patients with sickle cell disease. Gamma-globin expression will be analyzed in progenitor assays.

格兰特= 696773; p01hl 镰状细胞疾病的基因疗法需要基因转移载体，以赋予表，调节表，脉络膜特异性表达γ-球蛋白基因在相对较高的水平上，而没有细胞转化或肿瘤发生的风险。我们实现这一目标的建议基于两个最新发现：i）包含AD血清型35纤维（AD5/35）的腺病毒（AD）载体，尤其是具有潜在干细胞容量的亚集，ii）将AAV ITR掺入没有所有病毒基因（AD.AAV）中的AD量构图，可以使整合性的腔室有效地转化为人类的sTee sene sene sene sene sene store store stot sene store stot sene store stot sene store stot sene store stot sene。为了获得足够的球蛋白表达，我们将产生含有26 kbβ-蛋白LCR（5？hs1至5？hs5，β-珠蛋白启动子，3 HS1）的辅助依赖性AD5/35； 3 HS1）;因此，LCR盒侧面是AAV ITR（AD，AAV-LCR）。在特定的目标1中，将通过最近构造的AD.AAV-LCR矢量来优化矢量产生，该AD在Beta启动器/LCR的控制下表达GFP基因。为了最大程度地减少肿瘤发生的风险，我们将进一步修改AD.AAV-LCR矢量，以使矢量整合到活性基因中进行选择，并通过AAV REP78的瞬态共表达来增加位点特异性整合的频率。在特定的目标2中，我们将研究GFP表达的持续性和在转导的红血球细胞克隆中的载体的整合模式。在特定的目标3中，将使用在半固体培养物中生长的转导的CD34+细胞的菌落对整合位点进行监测，然后对NOD-SCID小鼠进行研究。为了评估载体诱导的肿瘤发生，我们将比较转导的长期培养启动细胞和点头scID小鼠的表达谱。为了评估载体诱导的肿瘤发生，我们将比较转导的长期培养启动细胞的表达谱，并使用DNA阵列与模拟感染细胞的细胞点点头重塑细胞。最后，如果我们在特定的目标4中确认使用新载体的安全性和功效，我们将构建表达AD.AAV-LCR载体的γ-球蛋白，并对来自健康供体的CD34+细胞进行转导研究。将在祖细胞测定中分析γ-球蛋白的表达。