Globin LCR containing Adeno-AAV Vectors

含有腺-AAV 载体的球蛋白 LCR

• 批准号: 7275414
• 负责人: ANDRE Michael LIEBER
• 金额: $ 27.72万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7275414
• 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.

授予=6967773;P01HL 镰状细胞病的基因治疗需要基因转移载体，以相对高的水平赋予γ-珠蛋白基因稳定的、受调节的、谱系特异性的表达，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项最新发现：I) 含有 Ad 血清型 35 纤维 (Ad5/35) 特别是具有潜在干细胞能力的子集的腺病毒 (Ad) 载体，以及 ii) 将 AAV ITR 掺入缺乏所有纤维的 Ad 载体中。病毒基因 (Ad.AAV) 介导有效的随机载体整合，并允许将基因稳定转移到人类造血干细胞中。为了实现足够的珠蛋白表达，我们将产生含有 26 kb β-珠蛋白 LCR（5?HS1 至 5?HS5，β-珠蛋白启动子，3 HS1）的辅助依赖性 Ad5/35；其中 LCR 盒的两侧是 AAV ITR (Ad,AAV-LCR)。在具体目标 1 中，将使用最近构建的 Ad.AAV-LCR 载体来优化载体生产，该载体在 β 启动子/LCR 的控制下表达 GFP 基因。为了最大限度地降低肿瘤发生的风险，我们将进一步修改 Ad.AAV-LCR 载体，以允许选择载体整合到活性基因中，并通过 AAV Rep78 的瞬时共表达来增加位点特异性整合的频率。在具体目标 2 中，我们将研究 GFP 表达的持久性以及我们的载体在转导的红白血病细胞克隆中的整合模式。在具体目标 3 中，将使用半固体培养物中生长的转导 CD34+ 细胞集落进行表达研究和整合位点监测，然后在 NOD-SCID 小鼠中进行研究。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起始细胞库和 NOD-SCID 小鼠的表达谱。为了评估载体诱导的肿瘤发生，我们将使用 DNA 阵列将转导的长期培养起始细胞和 NOD-SCID 再生细胞库的表达谱与模拟感染细胞的表达谱进行比较。最后，如果我们确认新载体的安全性和有效性，在特定目标 4 中，我们将构建表达 γ-球蛋白的 Ad.AAV-LCR 载体，并在源自健康供体和镰状细胞病患者的 CD34+ 细胞中进行转导研究。 γ-珠蛋白表达将在祖细胞测定中进行分析。