CF GENE THERAPY WITH NOVEL NONVIRAL VECTORS

描述：基因疗法可以提供更好的治疗或治愈 囊性纤维化。然而，目前针对这种疾病的基因治疗尝试， 以病毒载体或常规载体为载体导入CFTR基因 进入靶细胞，具有严重的先天局限性。卡洛斯博士和 同事们提出了一种使用稳定的染色体外染色体的新方法 复制载体携带cftr基因。申请人的实验室有 开发了一种独特的DNA载体，它可以自主复制，并且 在人类和其他哺乳动物细胞中保留很长时间。这些 载体缺乏病毒的免疫原性和大小限制，并被保留 在细胞中的长度比传统的质粒DNA要长得多。基因表达来自 向量相应地延长。这些向量可以被引入 雾化吸入或气管内高效导入肺上皮细胞 注入新的脂类：DNA复合体。 稳定的自主复制载体将首先适应于在 通过提供通用启动子对原代和分化细胞 EBNA-1基因。为了纠正囊性纤维化的突变CFTR基因， 他们将在载体上放置一个经过特殊调整的cftr表达盒。 基因的表达和功能将在至少一段时间内进行分析 在肺上皮组织培养细胞中培养2个月。向量将是 与新型脂质EDMPC络合，引入动物体内 临床前测试。如果有必要，将在患者中进行临床试验 安排好的。 因此，这项建议的具体目的是：1)扩大 应用于原代细胞的染色体外复制载体 呼吸道上皮细胞培养，2)染色体外复制 在呼吸道上皮细胞中表达CFTR的载体，以及3)使用 染色体外载体在啮齿动物体内产生CFTR的实验研究 系统。该建议将一种有效的新型载体与一种安全的 非病毒DNA传递方法，创造了一种亟需的新方法 囊性纤维化的基因治疗。

DESCRIPTION: Gene therapy could offer improved treatment or a cure of cystic fibrosis. However current attempts at gene therapy for the disease, focusing on viral or conventional plasmid vectors to introduce the CFTR gene into target cells, have serious inherent limitations. Dr. Calos and colleagues propose a new approach that uses stable extrachromosomal replicating vectors to carry the CFTR gene. The applicant's laboratory has developed a unique class of DNA vectors that replicate autonomously and are retained for long periods of time in human and other mammalian cells. These vectors lack the immunogenicity and size limits of viruses and are retained in cells much longer than conventional plasmid DNA. Gene expression from the vectors is correspondingly prolonged. These vectors can be introduced efficiently into lung epithelial cells in vivo by aerosol or intratracheal instillation of novel lipid:DNA complexes. Stable autonomously replicating vectors will be first adapted to function in primary and differentiated cells by provision of a universal promoter to the EBNA-1 gene. In order to correct the mutant CFTR gene of cystic fibrosis, they will place a specially adapted CFTR expression cassette on the vectors. Gene expression and function will be assayed over a time course of at least two months in lung epithelial tissue culture cells. Vectors will be complexed with the novel lipid EDMPC and introduced into animals for pre-clinical testing. If warranted, clinical trial in patients will be arranged. The Specific Aims of this proposal are thus to: 1) extend the utility of the extrachromosomal replicating vectors for application to primary cell cultures of airway epithelium, 2) to employ the extrachromosomal replicating vectors for expressing CFTR in airway epithelial cells, and 3) to employ the extrachromosomal vector for CFTR production in vivo in a rodent model system. This proposal pairs an effective new type of vector with a safe method for non-viral DNA delivery, creating a much needed new approach for gene therapy of cystic fibrosis.