In vitro correction of cystic fibrosis epithelial cell lines by small fragment homologous replacement (SFHR) technique

BMC Medical Genetics

Giuseppe Novelli³  Dieter C Gruenert²  Monica Lais³  Emanuela Bonifazi³  Anna Maria Nardone⁴  Annalucia Serafino¹  Emanuela Bruscia²  Federica Sangiuolo³ 

[1] Institute of Experimental Medicine, CNR, Rome, Italy;Department of Medicine, Human Molecular Genetics Unit, University of Vermont, Burlington, VT, USA;Department of Biopathology, Human Genetics Unit, Tor Vergata University of Rome, Italy;Azienda Ospedaliera Universitaria Policlinico Tor Vergata, Italy

关键词: transfection;    transmission electron microscopy (TEM);    gene targeting;    cystic fibrosis transmembrane conductance regulator (CFTR);    gene therapy;   

Others  :  1178806
DOI  :  10.1186/1471-2350-3-8

 received in 2002-04-29, accepted in 2002-09-23,  发布年份 2002

【 摘 要 】

Background

SFHR (small fragment homologous replacement)-mediated targeting is a process that has been used to correct specific mutations in mammalian cells. This process involves both chemical and cellular factors that are not yet defined. To evaluate potential of this technique for gene therapy it is necessary to characterize gene transfer efficacy in terms of the transfection vehicle, the genetic target, and the cellular processing of the DNA and DNA-vehicle complex.

Methods

In this study, small fragments of genomic cystic fibrosis (CF) transmembrane conductance regulator (CFTR) DNA, that comprise the wild-type and ΔF508 sequences, were transfected into immortalized CF and normal airway epithelial cells, respectively. Homologous replacement was evaluated using PCR and sequence-based analyses of cellular DNA and RNA. Individual stages of cationic lipid-facilitated SFHR in cultured cell lines were also examined using transmission electron microscopy (TEM).

Results

We demonstrated that the lipid/DNA (+/-) ratio influences the mode of entry into the cell and therefore affects the efficacy of SFHR-mediated gene targeting. Lipid/DNA complexes with more negative ratios entered the cell via a plasma membrane fusion pathway. Transfer of the DNA that relies on an endocytic pathway appeared more effective at mediating SFHR. In addition, it was also clear that there is a correlation between the specific cell line transfected and the optimal lipid/DNA ratio.

Conclusions

These studies provide new insights into factors that underlie SFHR-mediated gene targeting efficacy and into the parameters that can be modulated for its optimization.

【 授权许可】

   
2002 Sangiuolo et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.

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【 参考文献 】

• [1]Gruenert DC: Gene correction with small DNA fragments. Curr Res Molec Ther 1998, 1:607-613.
• [2]Gruenert DC: Opportunities and challenges in targeting genes for therapy. Gene Ther 1999, 6:1347-1348.
• [3]Kmiec EB: Targeted gene repair. Gene Ther 1999, 6:1-3.
• [4]Lai LW, Lien YH: Homologous recombination based gene therapy. Exp Nephr 1999, 7:11-14.
• [5]Woolf TM: Therapeutic repair of mutated nucleic acid sequences. Nat Biotechnol 1998, 16:341-344.
• [6]Yanez RJ, Porter ACG: Therapeutic gene targeting. Gene Ther 1998, 5:149-159.
• [7]Goncz KK, Kunzelmann K, Xu Z, Gruenert DC: Targeted replacement of normal and mutant CFTR sequences in human airway epithelial cells using DNA fragments. Hum Mol Genet 1998, 7:1913-1919.
• [8]Goncz KK, Gruenert DC: Site-directed alteration of DNA by small fragment homologous replacement (SFHR). In Gene Targeting Vector Protocols. Edited by Kmiec EB, Totowa NJ. Humana Press; 1998:85-99.
• [9]Goncz KK, Colosimo A, Dallapiccola B, Gagne L, Hong K, Novelli G, D Papahadjopoulos, Sawa T, Schreier H, Wiener-Kronish J, et al.: Expression of ΔF508 CFTR in normal mouse lung after site-specific modification of CFTR sequences by SFHR. Gene Ther 2001, 8:961-965.
• [10]Kunzelmann K, Legendre JK, Knoell D, Escobar LC, Xu Z, Gruenert DC: Gene Targeting of CFTR DNA in CF epithelial cells. Gene Ther 1996, 3:859-867.
• [11]Kerem B, Rommens JM, Buchanan JA, Markiewicz D, Cox TK, Chakravarti A, Buchwald M, Tsui LC: Identification of the cystic fibrosis gene: genetic analysis. Science 1989, 245:1073-1080.
• [12]Tsui LC: The spectrum of cystic fibrosis mutations. Trends in Genet 1992, 8:392-398.
• [13]Cozens AL, Yezzi MJ, Chin L, Simon EM, Finkbeiner WE, Wagner JA, Gruenert DC: Characterization of immortal cystic fibrosis tracheobronchial gland epithelial cells. Proc Natl Acad Sci USA 1992, 89:5171-5175.
• [14]Gruenert DC, Basbaum CB, Welsh MJ, Li M, Finkbeiner WE, Nadel JA: Characterisation of human tracheal epithelial cells transformed by an origin-defective simian virus 40. Proc Natl Acad Sci USA 1988, 85:5951-5955.
• [15]Gruenert DC, Finkbeiner WE, Widdicombe JH: Culture and transformation of human airway epithelial cells. Am J Physiol 1995, 268:L347-360.
• [16]Cozens AL, Yezzi MJ, Kunzelmann K, Ohrui T, Chin L, Eng K, Finkbeiner WE, Widdicombe JH, Gruenert DC: CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells. Am J Respir Cell Mol Biol 1994, 10:38-47.
• [17]Gruenert DC, Basbaum CB, Widdicombe JH: Long-term culture of normal and cystic fibrosis epithelial cells grown under serum-free conditions. In Vitro Cell Dev Biol 1990, 26:411-418.
• [18]Colosimo A, Goncz KK, Holmes AR, Kunzelmann K, Novelli G, Malone RW, Benneth MJ, Gruenert DC: Transfer and expression of foreign genes in mammalian cells. Biotechniques 2000, 29:314-318.
• [19]Densomore CL, Giddings TH, Waldrep JC, Kinsey BM, Knight V: Gene transfer by guanidinium-cholesterol-dioleoylphosphatidyl-ethanolamine liposome-DNA complexes in aerosol. J Gene Med 1999, 1:251-264.
• [20]Felgner PL: Nonviral strategies for gene therapy. Sci Am 1997, 276:102-106.
• [21]Zabner J, Fasbender AJ, Moninger T, Poellinger KA, Welsh MJ: Cellular and molecular barriers to gene transfer by a cationic liposome. J Biol Chem 1995, 270:18997-19007.
• [22]Kapsa R, Quigley A, Lynch GS, Steeper K, Kornberg AJ, Gregorevic P, Austin L, Byrne E: In vivo and in vitro correction of the mdx dystrophin gene nonsense mutation by short-fragment homologous replacement. Hum Gene Ther 2001, 12:29-642.
• [23]Colosimo A, Sangiuolo F, Di Sario S, Amicucci P, Serafino AL, Novelli A, Sabati M, Castro M, Lucidi V, Mossa G, Dalla piccola B, Novelli G: Current status of cationic liposome-mediated gene therapy in cystic fibrosis. Acta Pharm 1998, 4:221-227.
• [24]Colosimo A, Goncz KK, Novelli G, Dallapiccola B, Gruenert DC: Targeted correction of a defective selectable marker gene in human epithelial cells by small DNA fragments. Mol Ther 2001, 3:178-185.
• [25]Scheule RK, Bagley RG, Erickson AL, Wang KX, Fang SL, Vaccaro C, O'Riordan CR, Cheng SH, Smith AE: Delivery of purified, functional CFTR to epithelial cells in vitro using influenza hemagglutinin. Am J Respir Cell Mol Biol 1995, 13:330-343.
• [26]Schreier H, Ausborn M, Gunther S, Weissig V, Chander R: (Patho)physiologic pathways to drug targeting: artificial viral envelopes. J Mol Recognit 1995, 8:59-62.
• [27]Schwarzenberg P, Huang W, Oliver P, Osidipe T, Theodossiou C, Kolls JK: Poly-L-Lisine based molecular conjugate vectors: a high efficiency gene transfer system for human progenitor and leukemia cells. Am J Med Sci 2001, 321:129-136.
• [28]Kafri T: Lentivirus vectors: difficulties and hopes before clinical trials. Curr Opin Mol Ther 2001, 3:316-26.
• [29]Evans JT, Cravens P, Gatlin J, Kelly PF, Lipsky PE, Garcia JV: Pre-clinical evaluation of an in vitro selection protocol for the enrichment of transduced CD34(+) cell-derived human dendritic cells. Gene Ther 2001, 8:1427-35.
• [30]Remy-Kristensen A, Clamme JP, Vuilleumier C, Kuhry JG, Mely Y: Role of endocytosis in the transfection of L929 fibroblasts by polyethylenimine/DNA complexes. Biochim Biophys Acta 2001, 1514:21-32.
• [31]Ma H, Diamnond SL: Nonviral gene therapy and its delivery systems. Curr Pharm Biotechnol 2001, 2:1-17.
• [32]Nishikawa M, Huang L: Nonviral vectors in the new millennium: delivery barriers in gene transfer. Hum Gene Ther 2001, 12:861-70.
• [33]Bruscia E, Sangiuolo F, Goncz KK, Novelli G, Gruenert DC: Isolation of CF cell lines corrected at ΔF508-CFTR locus by SFHR-mediated targeting. Gene Ther 2002, in press.
• [34]Caplen NJ, Kinrade E, Sorgi F, Gao X, Gruenert DC, Geddes D, Coutelle C, Huang L, Alton EW, Williamson R: In vitro liposome-mediated DNA transfection of epithelial cell lines using the cationic liposome DC-Chol/DOPE. Gene Ther 1995, 2:603-613.
• [35]Gao XA, Huang LA: A novel cationic liposome reagent for efficient transfection of mammalian cells. Biochem Biophys Res Commun 1991, 179:280-285.