CF GENE THERAPY WITH NOVEL NONVIRAL VECTORS

描述：基因治疗可以提供改善的治疗或治愈 囊性纤维化 然而，目前对这种疾病的基因治疗的尝试， 集中于病毒或常规质粒载体以引入CFTR基因 有严重的内在局限性。 卡洛斯博士和 他的同事们提出了一种新的方法， 复制载体以携带CFTR基因。 申请人的实验室拥有 开发了一类独特的DNA载体，可以自主复制， 在人类和其他哺乳动物细胞中长时间保留。 这些 载体缺乏病毒的免疫原性和大小限制 比传统的质粒DNA长得多 的基因表达 矢量相应地延长。 这些载体可以被引入 通过气雾剂或气管内给药有效地进入肺上皮细胞 滴注新脂质：DNA复合物。 稳定的自主复制载体将首先适应在 通过向原代和分化的细胞提供通用启动子， 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改造的CFTR表达盒。 基因表达和功能将在至少 两个月后在肺上皮组织培养细胞。 矢量将是 与新的脂质EDMPC复合，并引入动物体内， 临床前测试 如有必要，将在患者中进行临床试验。 安排好了 因此，本提案的具体目标是：1）扩大 用于原代细胞染色体外复制载体 培养气道上皮，2）采用染色体外复制 用于在气道上皮细胞中表达CFTR的载体，和3）使用 用于在啮齿动物模型中体内产生CFTR的染色体外载体 系统 这项建议将一种有效的新型载体与一种安全的 一种非病毒DNA递送方法，创造了一种急需的新方法， 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.