Expression of ΔF508 CFTR in normal mouse lung after site-specific modification of CFTR sequences by SFHR

被引:64
作者
Goncz, KK
Colosimo, A
Dallapiccola, B
Gagné, L
Hong, K
Novelli, G
Papahadjopoulos, D
Sawa, T
Schreier, H
Wiener-Kronish, J
Xu, Z
Gruenert, DC
机构
[1] Univ Vermont, Dept Med, Burlington, VT 05405 USA
[2] Univ G dAnnunzio, Dept Biomed Sci, Chieti, Italy
[3] Univ Roma La Sapienza, Dept Expt Med & Pathol, I-00185 Rome, Italy
[4] CSS Mendel Inst, Rome, Italy
[5] Univ Calif San Francisco, Dept Biopharmaceut Sci, San Francisco, CA 94143 USA
[6] Univ Calif San Francisco, Dept Anesthesia & Perioperat Care, San Francisco, CA 94143 USA
[7] Univ Calif San Francisco, Dept Surg, San Francisco, CA 94143 USA
[8] Calif Pacific Med Ctr, Inst Res, Liposome Res Lab, San Francisco, CA 94115 USA
[9] Univ Roma Tor Vergata, Genet Sect, Dept Biopathol & Diagnost Imaging, I-00161 Rome, Italy
[10] Vectron Therapeut AG, Marburg, Germany
关键词
gene targeting; gene therapy; cystic fibrosis; DNA delivery vehicles;
D O I
10.1038/sj.gt.3301476
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The development of gene targeting strategies for specific modification of genomic DNA in human somatic cells has provided a potential gene therapy for the treatment of inherited diseases. One approach, small fragment homologous replacement (SFHR), directly targets and modifies specific genomic sequences with small fragments of exogenous DNA (400-800 bp) that are homologous to genomic sequences except for the desired modification. This approach has been effective for the in vitro modification of exon la in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in human airway epithelial cells. As another step in the development of SFHR for gene therapy studies were carried out to target and modify specific genomic sequences in exon 10 of the mouse CFTR (mCFTR) in vivo. Small DNA fragments (783 bp), homologous to mCFTR except for a 3-bp deletion (Delta F508) and a silent mutation which introduces a unique restriction site (Kpnl), were instilled into the lungs of normal mice using four different DNA vehicles (AVE, LipofectAMINE, DDAB, SuperFect). Successful modification was determined by PCR amplification of DNA or mRNA-derived cDNA followed by Kpnl digestion. The results of these studies showed that SFHR can be used as a gene therapy to introduce specific modifications into the cells of clinically affected organs and that the cells will express the new sequence.
引用
收藏
页码:961 / 965
页数:5
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