ALTERNATIVE VECTORS FOR CFTR GENE THERAPY

CFTR 基因治疗的替代载体

• 批准号: 6242306
• 负责人: RICHARD J SAMULSKI
• 金额: $ 13.87万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6242306
• 关键词: Adenoviridae; adeno associated virus group; chemical conjugate; cystic fibrosis; cytogenetics; defective virus; gene expression; gene mutation; gene therapy; genetic promoter element; genetic regulatory element; genetic transduction; helper virus; in situ hybridization; latent virus infection; plasmids; polymerase chain reaction; recombinant DNA; reporter genes; respiratory epithelium; temperature sensitive mutant; tissue /cell culture; transfection; transfection /expression vector; virus genetics

Genetic correction of cystic fibrosis (CF) requires the development of safe and efficient vector systems capable of achieving direct in vivo gene delivery to the airway epithelium. An ideal vector for gene therapy of CF lung disease should possess the following features: 1) tissue tropism for respiratory epithelium; 2) ability to transfer genes efficiently into terminally differentiated cells; and 3) stable chromosomal integration for prolonged gene expression. Currently, a number of viral systems are available for somatic gene transfer, including the well-characterized retrovirus, adenovirus, and the relatively new adeno-associated virus (AAV). In addition, liposomes and molecular conjugates are also being explored as alternative gene delivery systems. Unfortunately, none of these current vectors possess all the features desired of an ideal gene transfer system. Two general strategies to develop clinically useful vectors for CF are proposed. First, we shall improve current transient (adenoviral) and stable (AAV) expression vectors. In Specific Aim 1, we shall improve the safety of recombinant adenovirus vectors, focussing on strategies to minimize vector replication/reactivation and to develop techniques (hexon marking) to monitor these events. In Specific Aim 2, we shall characterize AAV as a vector for CFTR delivery in immortalized and primary cultures of airway epithelial cells, focussing on integrative capacities (targeted versus random), promoters, and stability of expression. Second, our experience with the AAV- and molecular conjugate- mediated gene transfer systems indicates that a strategy based on the combined advantages of these vector systems may lead to novel approaches for CF gene therapy. In Specific Aim 3, we shall develop strategies and reagents to incorporate an integration mechanism derived from AAV into adenovirus-coupled molecular conjugates. Both technically and conceptually, these Specific Aims are closely related, providing us with an efficient, interactive approach for developing a clinically useful vector for CF gene therapy. Further, the reagents derived from this proposed study may be applicable to other inherited diseases.

囊性纤维化（CF）的遗传纠正需要发展 能够达到直接体内基因的安全有效矢量系统 传递到气道上皮。 CF基因治疗的理想载体 肺病应具有以下特征：1） 呼吸上皮； 2）能够有效地转移基因 末端分化的细胞； 3）稳定的染色体整合 长时间的基因表达。目前，许多病毒系统 可用于体细胞基因转移，包括良好的特征 逆转录病毒，腺病毒和相对新的腺相关病毒 （AAV）。另外，脂质体和分子结合物也在 作为替代基因输送系统探索。不幸的是，没有 这些当前的向量具有理想基因所需的所有特征 转移系统。两种开发临床有用的一般策略 提出了CF的向量。首先，我们将改善当前瞬态 （腺病毒）和稳定（AAV）表达向量。在特定目标1中，我们 应提高重组腺病毒载体的安全性，重点关注 最小化矢量复制/重新激活并发展的策略 用于监视这些事件的技术（己孔标记）。在特定的目标2中，我们 应将AAV作为cftr的载体，以永生化和 气道上皮细胞的原发性培养物，集中于综合 能力（目标与随机），启动子和稳定性 表达。其次，我们在AAV和分子结合的经验 介导的基因转移系统表明，基于 这些向量系统的联合优势可能导致新的方法 用于CF基因治疗。 在特定目标3中，我们将制定策略和 试剂将源自AAV得出的集成机制纳入 腺病毒耦合分子缀合物。 在技​​术上和 从概念上讲，这些特定目标密切相关，为我们提供了 一种有效的互动方法，用于开发临床上有用的 CF基因治疗的载体。 此外，源自此的试剂 拟议的研究可能适用于其他遗传疾病。