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

Gene Therapy volume 8, pages 961–965 (2001)Cite this article

Abstract

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 10 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 (ΔF508) and a silent mutation which introduces a unique restriction site (KpnI), 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 KpnI 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.

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Acknowledgements

We thank Weiwen Zheng for her technical assistance. The work was funded by grants AI 44101 and HL 59239 (JW-K and TS), NIH grants DK46002 and DK47766 as well as grants from the Cystic Fibrosis Foundation, Cystic Fibrosis Research, Inc., and Pennsylvania Cystic Fibrosis, Inc. (DCG, KKG, ZX, AC). We would also like to dedicate this manuscript to our colleague Dr Dimitri Papahadjopoulos, whose untimely death is a tragic loss that has left a void within the scientific community.

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  1. D Papahadjopoulos: Deceased

Authors and Affiliations

  1. Department of Medicine, Human Molecular Genetics, University of Vermont, Burlington, VT, USA

    KK Goncz, A Colosimo & DC Gruenert

  2. Department of Biomedical Sciences, University of Chieti ‘G D'Annunzio’, Chieti, Italy

    A Colosimo

  3. Department of Experimental Medicine and Pathology, University of Rome ‘La Sapienza’ and CSS-Mendel Institute, Rome, Italy

    B Dallapiccola

  4. Department of Biopharmaceutical Sciences, University of California, San Francisco, CA, USA

    L Gagné

  5. Liposome Research Laboratory, California Pacific Medical Center, Research Institute, San Francisco, CA, USA

    K Hong & D Papahadjopoulos

  6. Department of Biopathology and Diagnostic Imaging, Section of Genetics, University of Rome ‘Tor Vergata’, Rome, Italy

    G Novelli

  7. Department of Anesthesia and Perioperative Care, University of California, San Francisco, CA, USA

    T Sawa & J Wiener-Kronish

  8. Vectron Therapeutics AG, Marburg, Germany

    H Schreier

  9. Department of Surgery, University of California, San Francisco, CA, USA

    Z Xu

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Goncz, K., Colosimo, A., Dallapiccola, B. et al. Expression of ΔF508 CFTR in normal mouse lung after site-specific modification of CFTR sequences by SFHR. Gene Ther 8, 961–965 (2001). https://doi.org/10.1038/sj.gt.3301476

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  • DOI: https://doi.org/10.1038/sj.gt.3301476

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