IN VIVO GENE DELIVERY FOR LONG-TERM EXPRESSION

这个项目的总体目标是开发新的腺病毒- 基于基因转移载体，比那些更适合的 目前可用于人类囊性纤维化的基因转移治疗和 其他遗传性疾病。这一发展的具体目标包括：1) 辅助子依赖腺病毒载体，适应大分子 重量DNA插入；2)定点染色体整合(安全港) 通过合法重组；3)长期的组织特异性表达；以及 4)小分子诱导剂(药物)调控表达。这些 如果目标得以实现，将克服 腺病毒系统：缺乏有效的核整合，插入有限 包装能力，以及对特定细胞类型表达的控制。一个 带有最小顺式元件的腺病毒扩增粒包装 将构建腺病毒基因组。这个载体具有潜在的 构建插入容量高达37个的腺病毒载体 KB。这样的载体将消除对包含顺式-DNA的大小限制 监管元素，还将开启添加功能的新可能性 通过同源重组实现有效的核靶向。基因 腺病毒载体的靶向效率将在小鼠和 人类的染色体位置。体内靶向的尝试将在 老鼠和狒狒。我们建议引入一种药物反应启动子， 将Cypla-1基因导入腺病毒载体中，检测其体内诱导性。如果 如果成功，这些进展可能会导致能够长期表达 药物介导的调控。

The overall goal of this project is the development of new adenovirus- based gene transfer vectors that are better suited than those that are currently available for human gene transfer therapy of cystic fibrosis and other genetic diseases. The specific aims of this development include: 1) helper-dependent adenoviral vectors that accommodate a high molecular weight DNA insert; 2) site-specific chromosomal integration (safe harbor) by legitimate recombination; 3) long-term tissue-specific expression; and 4) control of expression by small molecular weight inducers (drugs). These objectives, if achieved, will overcome three major disadvantages of the adenovirus system: lack of efficient nuclear integration, limited insert packaging capacity, and control of expression in particular cell types. An adenovirus amplicon plasmid with a minimum cis-element for packaging of adenovirus genome will be constructed. This vector has the potential for creating an adenovirus-based vector with capacity of an insert of up to 37 kb. Such a vector will remove size limitations for inclusion of cis- regulatory elements and will also open a new possibility to add a feature of efficient nuclear targeting by homologous recombination. The gene targeting efficiency of adenoviral vectors will be studied in mouse and human chromosomal sites. Attempts to target in vivo will be performed in mice and baboons. We propose to introduce a drug-responsive promoter, Cypla-1, into adenoviral vectors and examine the inducibility in vivo. If successful, these advances could lead to a long-term expression capable of drug-mediated regulation.