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

Federica Sangiuolo 2 Emanuela Bruscia 1 2 Annalucia Serafino 0 Anna Maria Nardone 3 Emanuela Bonifazi 2 Monica Lais 2 Dieter C Gruenert 1 Giuseppe Novelli 2 0 Institute of Experimental Medicine, CNR , Rome , Italy 1 Department of Medicine, Human Molecular Genetics Unit, University of Vermont , Burlington, VT , USA 2 Department of Biopathology, Human Genetics Unit, Tor Vergata University of Rome , Italy 3 Azienda Ospedaliera Universitaria Policlinico Tor Vergata , Italy 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 F508 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. - Background Homologous replacement is a technology that can be used to modify specific genes within chromosomal DNA [16]. The potential of this type of strategy has obvious implications for maintaining genomic integrity and cellspecific expression. The direct conversion of mutant genomic sequences to a wild-type genotype, restoring the normal phenotype, has clear advantages over therapeutic cDNA. By preserving the integrity of the targeted gene, the relationship between the coding sequences and regulatory elements remains intact. Consequently, cell-specific expression is not altered. Small fragment homologous replacement (SFHR) involves the introduction of small fragments (~500-bp) of DNA into cells. After entering the cells, the fragment pairs with its genomic homologue and replaces the endogenous sequence with the exogenous fragment through an, as yet, undefined mechanism [1,2,6]. SFHR has already been successfully used to correct F508 mutation in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene [1,2,710]. The F508 is the most common mutation associated with CF [11,12] and demonstration of its correction has significant therapeutic implications. The studies showed that SFHR-mediated replacement of mutant or wild-type genomic sequences after the introduction of small DNA fragment (491/488 nt) into cultured epithelial cells [7,10]. In some studies, the fragment, also carried a silent mutation that introduced a unique XhoI restriction site that could be used as a secondary marker of SFHR-mediated correction [7,8]. Recent in vivo studies have also indicated that SFHR could be used to modify endogenous mouse CFTR in the lung and in the intestine [9]. Not only was it possible to demonstrate SFHR-mediated modification of the DNA, but there was also a clear indication that the cells expressing CFTR were modified [9]. However, there has been no detailed SFHR optimization analysis as it relates to the behaviour of the DNA during and after SFHR uptake, the intracellular distribution of the DNA, and long-term stability of SFHR using non-viral vehicles. In this study, different parameters that influence SFHR in human epithelial cells were evaluated to determine whether SFHR could be an effective strategy for gene therapy. These include the type of transfected cells, DNA fragment to lipid ratio (+/-, respectively) and the time of harvest after initiation of transfection (incubation time). Different DNA transfection conditions were evaluated with respect to their ability to modulate SFHR-mediated correction. SFHR-mediated replacement at the appropriate genomic locus and expression of the exogenous sequences was assayed using polymerase chain reaction (PCR) amplification, restriction fragment length polymorphic (RFLP) analysis and DNA sequencing. The intracellular fate of transfected gold-labelled DNA fragments was monitored by transmission electron microscopy (TEM). The results presented here provide insight into the mechanisms underlying SFHR-mediated correction of the most common CF mutation, the F508. Methods Cell cultures Studies were carried out in CF tracheobronchial cells transformed with an origin of replication defective simian virus 40 (SV40) containing plasmid (pSVori-) [1315]. The cell line, CFBE41o-, is homozygous for the F508 mutation ( F508/ F508). A wild-type airway epithelial cell line 16HBE14o-, also transformed with the pSVori- plasmid was used as representative of the normal cells [14 16]. Cells were grown in Eagle's Minimal Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics under humidified conditions at 37 C in 5% CO2. Stock cultures were grown in T75 flasks coated with an extracellular matrix of collagen/fibronectin/bovine serum albumin and subculture by trypsinization as described previously [17]. Synthesis of DNA fragments DNA fragments, 491-bp and 488-bp, that comprised exon 10 as well as the 3' and 5' flanking intron regions of the wild-type (wt) and mutant ( F508) CFTR gene respectively, were generated by PCR as previously described [7,8,10]. Fragments were column purified (Qiagen) and ethanol precipitated for subsequent use. Preparation of lipid/fragment complexes DNA-cationic lipid complexes were generated using the GENEPORTER (Gene Therapy Systems, San Diego, USA) liposome. The complexes were made at different charge ratios (+/-), by increasing the concentration of the double stranded DNA fragments and mixing with a constant quantity of LIPID (22.5 l) according to manufacturer's specifications. The mixture was then incubated at room temperature for 45 min and diluted to a final volume of 2 ml with serum-free MEM. DNA without lipid was used as the control in all experiments. Transfection protocol Approximately 2.5 106 cells were seeded in T75 flask 24 h before treatment. Cells were incuba (...truncated)