ABCMdb

PMID: 12825202

de Semir D, Nadal M, Gonzalez JR, Larriba S, Avinyo A, Nunes V, Casals T, Estivill X, Aran JM
Suitability of oligonucleotide-mediated cystic fibrosis gene repair in airway epithelial cells.
J Gene Med. 2003 Jul;5(7):625-39., [PubMed]

Sentences

No. Mutations Sentence Comment

10 ABCC7 p.Trp1282* In addition, a methodology allowing the relative quantification of the percentage of W1282X mutation repair in a heterozygous background using the PCR/oligonucleotide ligation assay (PCR/OLA) was developed. Login to comment 14 ABCC7 p.Trp1282* ABCC7 p.Trp1282* Moreover, regardless of the corrector oligonucleotide structure applied to our CF bronchial epithelial cells, of compound heterozygous genotype (F508del/W1282X), the percentage of their resulting wild-type allele in the W1282X (exon 20) locus of the CFTR gene was not significantly different from that of the control untreated cells by our PCR/OLA assay (confidence interval at 95% ± 4 allele wild-type). Login to comment 17 ABCC7 p.Trp1282* Copyright  2003 John Wiley & Sons, Ltd. Keywords W1282X mutation correction; chimeraplasts; non-viral vectors; PCR/OLA Introduction Cystic fibrosis (CF) is the most common autosomal recessive inherited disease in Caucasian populations. Login to comment 20 ABCC7 p.Trp1282* However, other minority pathologic mutations may occur at higher frequencies in selected populations, as exemplified by the W1282X mutation on 60% of Ashkenazic CF chromosomes. Login to comment 34 ABCC7 p.Trp1282* In this study, we further sought to validate the chimeraplasty and the use of short single-stranded oligonucleotide technologies for the correction of the W1282X nonsense mutation in CF airway epithelial cells. Login to comment 46 ABCC7 p.Trp1282* Chimeric RNA/DNA oligonucleotides (CSO-1, CSO-1C, CSO-2 and CSO-3) (A) and short single-stranded oligonucleotides (CSO-4 and CSO-4C) (B) were designed to target either the transcribed or the non-transcribed strand of the W1282X mutation locus within exon 20 of the CFTR gene (C). Login to comment 47 ABCC7 p.Trp1282* Correction of the point mutation W1282X requires replacement of the mutant A residue with a G residue in the transcribed strand, or of the corresponding complementary residues in the non-transcribed strand. Login to comment 91 ABCC7 p.Trp1282* Three oligonucleotide probes were used for the analysis of the W1282X mutation in the IB3.1 cells. Login to comment 92 ABCC7 p.Trp1282* ABCC7 p.Trp1282* A combination of two upstream (allelic) probes: WT (5 -TATCACTCCAAAGGCTTTCCTC-3 ) for detection of the wild-type allele, and M (5 -TATCACTCCAAAGGCT- TTCCTT-3 ) for detection of the W1282X allele, in which non-complementary 5 -poly(A) extensions (ranging from none to 8A) were added for sizing, plus one common, downstream, 5 -phosphorylated and 3 -FAM-labeled reporter probe: W1282X-CR (5 - CACTGTTGCAAAGTTATTGAATCC-3 ). Login to comment 95 ABCC7 p.Trp1282* A portion of the OLA reaction was run on an ABI PRISM 310 genetic analyzer (Applied Biosystems, Foster City, CA, USA), and the fluorescent OLA products from W1282X or wild-type alleles were automatically quantified by recording the corresponding peak areas or peak heights of the electropherograms with the GeneScan software, v. Login to comment 110 ABCC7 p.Trp1282* Statistical analysis General linear models were used to estimate the relationship between peak areas corresponding to wild-type and W1282X mutant alleles and the percentage of wild-type allele within exon 20 of the CFTR gene from IB3.1 cells. Login to comment 118 ABCC7 p.Trp1282* This widely used and physiologically relevant human CF bronchial epithelial cell line has been well characterized at the molecular level as a compound heterozygote with genotype F508del/W1282X. Login to comment 119 ABCC7 p.Trp1282* Our initial genetic screening of the CFTR gene confirmed the above genotype but revealed a consistent imbalance between both alleles, the F508del allele being slightly more frequent than the W1282X allele (Figure 2A). Login to comment 126 ABCC7 p.Trp1282* (A) Genomic DNA isolated from IB3.1 cells was screened for 31 of the most common CFTR gene mutations (together accounting for 77% of the CF chromosomes worldwide), including the deletion F508 (within exon 10), and the transversion W1282X (within exon 20), by multiplex PCR/OLA and sequence-coded separation using the Genotyper software (CF multiplex PCR/OLA kit from PE Applied Biosystems). Login to comment 127 ABCC7 p.Trp1282* The unbalanced heterozygous nature of the F508del and W1282X mutations is clearly visible in the CFTR gene fingerprint from IB3.1 cells. Login to comment 130 ABCC7 p.Trp1282* Thus, the above findings are relevant when attempting to undertake and quantify oligonucleotide-mediated correction of the W1282X nonsense mutation in our cellular model. Login to comment 146 ABCC7 p.Trp1282* ABCC7 p.Trp1282* Adaptation of the PCR/OLA assay for the assessment of the W1282X mutation correction frequency on IB3.1 cells To assess the percentage of oligonucleotide-directed W1282X mutation correction in the IB3.1 cells, of heterozygous genotype in that locus, we adapted a molecular diagnostic assay, previously employed for the analysis of CFTR gene mutations [28,29], which uses fluorescence-based oligonucleotide ligation technology (OLA). Login to comment 147 ABCC7 p.Trp1282* As a first step, we optimized the detection of allelic W1282X (G-3978 → A) or wild-type variants within exon 20 of the CFTR gene from genomic DNA of IB3.1 cells, on the exponential phase of their amplification. Login to comment 152 ABCC7 p.Trp1282* Adjustment of two parameters was found to be essential to allow a reliable quantification of the OLA products reflecting the percentage of W1282X and wild-type alleles from the exon 20 locus of the CFTR gene in the treated and untreated IB3.1 DNA samples: the nature of the thermostable DNA ligase and the size of the allelic OLA probes. Login to comment 153 ABCC7 p.Trp1282* Specificity of the ligation reaction proved to be critical for a correct quantification of the relative percentage of both the wild-type and the W1282X alleles in a given IB3.1 DNA sample. Login to comment 156 ABCC7 p.Trp1282* Optimization of the PCR/OLA assay for the relative quantification of the W1282X mutation repair in IB3.1 airway epithelial cells (I). Login to comment 158 ABCC7 p.Trp1282* Electropherogram displays represent fluorescence detection of oligonucleotide ligation products (single signal, or dual signal with peaks differing at least 2 bp, depending on the poly-A tails appended to the discriminating allelic probes) formed after hybridization over normal or W1282X mutant CFTR target sequences, and analyzed by capillary electrophoresis in an entangled polymer network in 8 M urea. Login to comment 161 ABCC7 p.Trp1282* Up to 16% of non-specific OLA product resulting from cross-hybridization ligation was detected using Taq DNA ligase but not Tsc DNA ligase W1282X mutation (data not shown). Login to comment 163 ABCC7 p.Trp1282* This DNA ligase proved to be highly specific for discriminating the wild-type and the W1282X alleles and gave no or insignificant cross-ligation background of mutant allele when a normal DNA from a non-CF individual was tested (Figure 4). Login to comment 164 ABCC7 p.Trp1282* ABCC7 p.Trp1282* To analyze whether the above PCR/OLA assay could be used for an accurate assessment of the proportion of either W1282X or wild-type allele present in the exon 20 locus of the CFTR gene from a DNA sample, we mixed different ratios of genomic DNA isolated from a normal non-CF individual, and from a CF homozygous patient for the W1282X mutation. Login to comment 169 ABCC7 p.Trp1282* The optimized extensions ''5 -AAAA`` for the wild-type discriminating OLA probe (WT + 4A), and ''5 -AAAAAA`` for the W1282X mutant discriminating OLA probe (M + 6A), gave the best regression (R2 = 0.996) in our PCR/OLA assay. Login to comment 170 ABCC7 p.Trp1282* Thus, this optimized methodology proved to be suitable for the assessment of the W1282X mutation correction frequency on IB3.1 cells. Login to comment 171 ABCC7 p.Trp1282* Lack of appreciable oligonucleotide-mediated repair on IB3.1 CF airway epithelial cells To ascertain whether chimeraplasts and/or short single-stranded oligonucleotides would mediate the permanent correction of the W1282X mutation at physiologically relevant frequencies, capable of yielding a measurable reversion of the CF phenotype [30], we investigated the percentage of oligonucleotide-mediated wild-type allele augmentation on chimeraplast- or short single-stranded oligonucleotide-treated IB3.1 cells with respect to the background wild-type allele already present in the heterozygous untreated IB3.1 cells. Login to comment 172 ABCC7 p.Trp1282* We tested different chimeraplast structures (see Figure 1), ranging from the standard 68 nt initial design (CSO-1) [31], and its homologous counterpart targeting the complementary strand of the W1282X DNA locus (CSO-1C), to a 80 nt, CSO-1-related, chimeraplast with an extended targeting region comprising two runs of 12nt of 2 -O-methyl RNA, separated by a 7 nt stretch of DNA (CSO-2). Login to comment 176 ABCC7 p.Trp1282* However, none of the above chimeraplast designs seemed to elicit targeted W1282X point mutation repair on the IB3.1 chromosomes, at least at a level above the sensitivity of our PCR/OLA method of detection (see standard curve from Figure 5B), because, in all cases, the difference between the percentage of the wild-type allele in the transfected IB3.1 cells and that of the wild-type allele present in either untransfected cells or transfected with an irrelevant chimeraplast was not statistically significant (Table 2, and see Figure 6 as an example). Login to comment 178 ABCC7 p.Trp1282* Therefore, we tested two additional 25 nt single-stranded oligonucleotide structures (CSO-4 and CSO-4C) complementary to both template strands of the W1282X locus, to discriminate any strand bias for gene correction. Login to comment 182 ABCC7 p.Trp1282* Again, no significant W1282X mutation correction in the IB3.1 cells was obtained using CSO-3, CSO-4 or CSO-4C oligonucleotides under the different conditions tested (Table 2). Login to comment 183 ABCC7 p.Trp1282* We conclude that the oligonucleotide-mediated targeted repair of the W1282X nonsense mutation within the CFTR gene is an inefficient process in the IB3.1 CF bronchial epithelial cells. Login to comment 185 ABCC7 p.Trp1282* Lack of appreciable oligonucleotide-mediated CFTR (W1282X) mutation repair on IB3.1 airway epithelial cells Treatment Oligo conc. Login to comment 187 ABCC7 p.Trp1282* of the W1282X chromosomal point mutation in CF airway epithelial cells. Login to comment 192 ABCC7 p.Trp1282* Although these cells have a compound heterozygous genotype for the CFTR gene (F508del/W1282X), the presence of a G → A transversion in nearly half of their chromosomes makes them amenable to attempt chimeraplast- and short single-stranded oligonucleotide-directed gene repair. Login to comment 194 ABCC7 p.Trp1282* ABCC7 p.Trp1282* Notably, it has been shown in the latter case that aminoglycoside antibiotics were able to suppress the W1282X premature stop mutation from IB3.1 cells because of the reappearance of cAMP-activated chloride currents, restoration of CFTR protein at the apical plasma membrane, and increase in the abundance of CFTR mRNA levels from the W1282X allele. Login to comment 202 ABCC7 p.Trp1282* Optimization of the PCR/OLA assay for the relative quantification of the W1282X mutation repair in IB3.1 airway epithelial cells (II). Login to comment 203 ABCC7 p.Trp1282* Sensitivity assessment of the PCR/OLA methodology for the relative quantification of W1282X mutant and wild-type alleles within exon 20 locus of the CFTR gene, from DNA of treated or untreated IB3.1 cells. Login to comment 204 ABCC7 p.Trp1282* (A) OLA electropherograms are shown using DNAs from a CF-affected individual homozygous for the W1282X mutation (1), from a normal non-CF individual (7), and from graded mixtures of the above DNAs (2 to 6). Login to comment 205 ABCC7 p.Trp1282* (B) Standard curves from these samples using different allelic probe combinations such as those shown allowed optimization of the 5 allelic probe length for the best accurate estimation of the frequency of the W1282X mutation correction as a function of the peak areas from both alleles. Login to comment 208 ABCC7 p.Trp1282* ABCC7 p.Trp1282* The tables below the standard curves represent the predicted values of % wild-type allele matching the corresponding ratios of non-CF normal DNA (allele wt) to W1282X homozygous DNA (allele W1282X), according to the linear regression model obtained, and their respective lower and upper limits of confidence Figure 6. Login to comment 209 ABCC7 p.Trp1282* Oligonucleotide-mediated W1282X mutation repair is inefficient in IB3.1 airway epithelial cells. Login to comment 210 ABCC7 p.Trp1282* ABCC7 p.Trp1282* Example of the outcome of the repair experiments performed: genomic DNA from IB3.1 cells either untransfected (control) or transfected with a chimeraplast (CSO-3), or a short single-stranded oligonucleotide (CSO-4), both targeted towards accomplishment of the W1282X CFTR A to G transversion, was subjected to the PCR/OLA assay for the relative quantification of the percentage of W1282X mutation correction. Login to comment 211 ABCC7 p.Trp1282* The electropherograms obtained show well-defined, independent peaks differing in 2 bp, which correspond to the wild-type and W1282X mutant alleles, respectively. Login to comment 216 ABCC7 p.Trp1282* Thus, our results indicate that CF airway epithelial cells are competent to support chimeraplast-mediated targeted repair, and that it is not inconsistent to attempt in vivo oligonucleotide-mediated correction of the W1282X mutation in our cellular model since the limits of targeted gene repair are not yet known. Login to comment 226 ABCC7 p.Trp1282* The heterozygous nature of our CF airway epithelial cell model for the W1282X mutation required a special effort to optimize a quantitative methodology to determine the frequencies of targeted gene repair. Login to comment 227 ABCC7 p.Trp1282* Because our results showed more than 50% of wild-type exon 20 from the F508del- containing chromosomes in the IB3.1 cells, this high background of the 'corrected` allele in the W1282X locus previous to the repair process prevented the use of classical quantitative gene mutation detection such as allele-specific hybridization techniques [14,15,47]. Login to comment 228 ABCC7 p.Trp1282* Thus, we took advantage of a powerful DNA diagnostic technology, the fluorescent PCR/OLA, already employed for identification of normal and mutant CF alleles [28,29], to develop an easy, fast, sensitive and specific molecular assay for the relative quantification of the W1282X mutation repair at the genomic level in a diploid setting. Login to comment 229 ABCC7 p.Trp1282* Nevertheless, the sensitivity of our relative W1282X single-nucleotide allele discrimination/quantification method (confidence interval at 95% near ±4% allele wild-type) may be lower than that achieved by the above indicated allele-specific hybridization-based assays, although it would allow the reliable detection of the minimum percentage of corrected, wild-type allele in lung epithelial cells (5-10%) that seems to be required for at least partial phenotypic correction of CF, e.g., to favorably increase CFTR-mediated chloride conductance overall [48]. Login to comment 230 ABCC7 p.Trp1282* To our knowledge, our adapted PCR/OLA assay is unique in our attempts for a relative quantification of the percentages of the W1282X mutation repair in the heterozygous background of IB3.1 cells. Login to comment 232 ABCC7 p.Trp1282* Although oligonucleotide synthesis and delivery, and the mutation detection assay, have all been optimized for an effective oligonucleotide-mediated repair, we have not been able to detect any significant oligonucleotide-induced in vivo repair activity of the W1282X chromosomal mutation in the CFTR gene from IB3.1 cells. Login to comment 245 ABCC7 p.Trp1282* Nevertheless, we have not observed any significant repair activity for the W1282X mutation above the background levels of our detection assay with any of the corrective-oligonucleotides tested. Login to comment 248 ABCC7 p.Trp1282* However, using this methodology, we have been unable to validate oligonucleotide-mediated targeted gene repair as an effective approach for the correction of the W1282X mutation within the CFTR gene in the repair-competent IB3.1 CF airway epithelial cells. Login to comment