Assessing the residual CFTR gene expression in human nasal epithelium cells bearing CFTR splicing mutations causing cystic fibrosis

Abstract

The major purpose of the present study was to quantify correctly spliced CFTR transcripts in human nasal epithelial cells from cystic fibrosis (CF) patients carrying the splicing mutations c.580-1G4T (712-1G4T) and c.2657 þ 5G4A (2789 þ 5G4A) and to assess the applicability of this model in CFTR therapeutic approaches. We performed the relative quantification of CFTR mRNA by reverse transcription quantitative PCR (RT-qPCR) of these splicing mutations in four groups (wild type, CF-F508del controls, CF patients and CF carriers) of individuals. In addition, in vitro assays using minigene constructs were performed to evaluate the effect of a new CF complex allele c.[2657 þ 5G4A; 2562T4G]. Ex vivo qPCR data show that the primary consequence of both mutations at the RNA level is the skipping of their neighboring exon (6 and 16, respectively). The CFTR minigenes results mimicked the ex vivo data, as exon 16 skipping is the main aberrant transcript, and the correctly spliced transcript level was observed in a similar proportion when the c.2657 þ 5G4A mutation is present. In summary, we provide evidence that ex vivo quantitative transcripts analysis using RT-qPCR is a robust technology that could be useful for measuring the efficacy of therapeutic approaches that attempt to achieve an increase in CFTR gene expression.

Restoration of CFTR function in patients with cystic fibrosis carrying the F508del-CFTR mutation

“…Primary respiratory cell cultures derived from CF patients are considered the gold standard to validate compound efficacy 27,62 and might help as an ex vivo test to predict the propensity of each individual patient to benefit from the in vivo administration of the candidate drug. We observed that cysteamine could rescue >70% of the expression of functional CFTR mutant in ex vivo cultures of freshly isolated patients' nasal epithelial cells.…”

Correction of a Cystic Fibrosis Splicing Mutation by Antisense Oligonucleotides

“…The CFTR minigenes were produced using a “sticky feet PCR” strategy to insert CFTR introns (IVS) IVS14, IVS15, and IVS16 consecutively into the pCDNA5/FRT/CFTR mammalian expression vector carrying the complete wt‐CFTR cDNA as described previously [Masvidal et al., ]. Thus, “triple minigenes” consist of the full‐length 4.5 kb CFTR cDNA plus relevant intronic regions (IVS14, IVS15, and IVS16), from herein referred as wt and 2657+5G>A mutant (Mut) CFTR splicing minigenes.…”

“…CFTR protein detection was performed by Western blot as previously [Masvidal et al., ]. Briefly, proteins were separated by SDS‐PAGE 7% (w/v) gel and CFTR was detected using the anti‐CFTR monoclonal antibody M3A7 (1:2,000 dilution) (EMD Millipore, Billerica, MA).…”

“…We have previously reported the molecular consequences of the CFTR c.2657+5G>A splicing mutation (legacy name: 2789+5G>A), located near the donor site in intron 16 (IVS16), and showed that it originates transcripts lacking exon 16 concomitantly with wild‐type (wt) transcripts [Masvidal et al., ; Sharma, et al., ]. Herein, we tested whether an RNA‐based antisense oligonucleotide (AON) approach would correct the skipping of exon 16.…”

“…Herein, we tested whether an RNA‐based antisense oligonucleotide (AON) approach would correct the skipping of exon 16. We used our previous mutant CFTR minigene model consisting of the full‐length CFTR cDNA with intronic sequences, stably expressed in HEK293 Flp‐In cells (hosting a single copy of the 2657+5G>A mutant minigene) [Masvidal et al., ] to assess splicing correction by two tailor‐designed AONs. Data shown here demonstrate that through this strategy, we were able to modulate splicing and rescue normal full‐length CFTR transcripts to 95% as well as restore normal levels of functional CFTR protein.…”