Escherichia coli-cloned CFTR loci relevant for human artificial chromosome therapy.
Escherichia coli-cloned CFTR loci relevant for human artificial chromosome therapy.
复制标题
大肠杆菌克隆的 CFTR 位点与人类人工染色体治疗相关。
作者：
L. Rocchi;C. Braz;S. Cattani;A. Ramalho;S. Christan;Marlene Edlinger;F. Ascenzioni;A. Laner;S. Kraner;M. Amaral;D. Schindelhauer
囊性纤维化的经典基因治疗由于针对病毒载体的免疫应答和基于cDNA的转基因的短期表达而具有有限的成功。这些限制可以通过将完整的基因组CFTR基因递送到非整合的人类人工染色体（HAC）上来克服。在这里，我们报告了基因组CFTR基因座的重建，并分析了三种基于P1噬菌体和F因子细菌的人工染色体掺入HAC的情况。（1）5A，含有延伸到两个相邻基因中的整个野生型CFTR基因座的大的、不可选择的BAC（296.8-kb插入片段，从kb -58.4到+51.4），包含所有调节子;（2）CGT 21，一种小的可选择的调聚PAC（134.7kb，从kb-60.7到+ 2），其含有连接外显子10、EGFP、外显子24和3'非翻译区的合成的最后一个外显子;和（3）CF 225，一种中等大小的非选择性PAC（225.3kb，从kb -60.7到+9.8），其连接自具有优化密码子和沉默XmaI限制性变体的两个PAC，以区分转基因和内源表达。将端粒化的、杀稻瘟菌素-S-可选择的、着丝粒-熟练的α-卫星构建体共转染到HT 1080细胞中，显示当使用CGT 21或5A时，可行的HAC形成率为1/1025株。在分析的122个细胞系中，CF225没有被整合到从头HAC中，但表达了整合体。稳定性分析表明，预制一个大的，标记的CFTR转基因，稳定复制的功能性着丝粒附近的可行性。虽然在这个阶段不能得出关于高效的HAC-proficient构建体构型的明确结论，但产生了重要的转移资源并进行了表征，证明了从头HAC作为潜在理想的基因治疗载体系统的前景。
Classical gene therapy for cystic fibrosis has had limited success because of immune response against viral vectors and short-term expression of cDNA-based transgenes. These limitations could be overcome by delivering the complete genomic CFTR gene on nonintegrating human artificial chromosomes (HACs). Here, we report reconstruction of the genomic CFTR locus and analyze incorporation into HACs of three P1 phage-based and F factor bacteria-based artificial chromosomes (PACs/BACs) of various sizes: (1) 5A, a large, nonselectable BAC containing the entire wild-type CFTR locus extending into both adjacent genes (296.8-kb insert, from kb -58.4 to +51.4) containing all regulators; (2) CGT21, a small, selectable, telomerized PAC (134.7 kb, from kb -60.7 to + 2) containing a synthetic last exon joining exon 10, EGFP, exon 24, and the 3' untranslated region; and (3) CF225, a midsized, nonselectable PAC (225.3 kb, from kb -60.7 to +9.8) ligated from two PACs with optimized codons and a silent XmaI restriction variant to discriminate transgene from endogenous expression. Cotransfection with telomerized, blasticidin-S-selectable, centromere-proficient α-satellite constructs into HT1080 cells revealed a workable HAC formation rate of 1 per ∼25 lines when using CGT21 or 5A. CF225 was not incorporated into a de novo HAC in 122 lines analyzed, but integrants were expressed. Stability analyses suggest the feasibility of prefabricating a large, tagged CFTR transgene that stably replicates in the proximity of a functional centromere. Although definite conclusions about HAC-proficient construct configurations cannot be drawn at this stage, important transfer resources were generated and characterized, demonstrating the promise of de novo HACs as potentially ideal gene therapy vector systems.
影响因子：
56.9
作者：
J. Riordan;J. Rommens;N. Alon;R. Rozmahel;Z. Grzelczak;J. Zieleński;N. Plavsic;Jia-Ling Chou
通讯作者：
J. Riordan;J. Rommens;N. Alon;R. Rozmahel;Z. Grzelczak;J. Zieleński;N. Plavsic;Jia-Ling Chou