Restoration of the cystic fibrosis transmembrane conductance regulator function by splicing modulation

Abstract

A significant fraction of disease-causing mutations affects pre-mRNA splicing. These mutations can generate both aberrant and correct transcripts, the level of which varies among different patients. An inverse correlation was found between this level and disease severity, suggesting a role for splicing regulation as a genetic modifier. Overexpression of splicing factors increased the level of correctly spliced RNA, transcribed from minigenes carrying disease-causing splicing mutations. However, whether this increase could restore the protein function was unknown. Here, we demonstrate that overexpression of Htra2-beta1 and SC35 increases the level of normal cystic fibrosis transmembrane conductance regulator (CFTR) transcripts in cystic-fibrosis-derived epithelial cells carrying the 3849+10 kb C --> T splicing mutation. This led to activation of the CFTR channel and restoration of its function. Restoration was also obtained by sodium butyrate, a histone deacetylase inhibitor, known to upregulate the expression of splicing factors. These results highlight the therapeutic potential of splicing modulation for genetic diseases caused by splicing mutations.

Figure 1

Effect of overexpression of splicing factors on the splicing pattern of CFTR exons in CFP15a cells carrying the 3849+10 kb C → T splicing mutation. Analysis of the splicing pattern of the cryptic 84 bp exon (A) and exon 9 (B). The top panels show examples of Genescan analysis of RT–PCR products from untransfected cells (left) and cells transfected (right) with Htra2-β1 (A) and ASF/SF2 (B). Percentages denote relative levels of correct and aberrant transcripts and numbers denote sizes of RT–PCR products (304 and 388 bp in (A), and 447 and 264 bp in (B)). The bottom panels show the effect of splicing factors on the level of aberrant transcripts. The grey columns represent a significant effect (P<0.01) using the Mann–Whitney U-test, adjusted with Bonferroni correction for eight comparisons. The white columns represent no effect. Error bars, standard deviation; UT, untransfected.

Figure 2

Increase of the protein level of splicing factors transfected into CFP15a cells. The top panels show tagged plasmids (endogenous and tagged of different sizes) and the bottom panels show untagged plasmids (endogenous and exogenous of same size). UT, untransfected.

Figure 3

Activation of CFTR Cl⁻ efflux by overexpression of splicing factors. CFTR activity in (A) T84, CFP15a, CFP22a and IB3 cells, (B) CFP15a cells following transfection with Htra2-β1, SC35 and ASF/SF2, (C) CFP22a and IB3 cells following transfection with Htra2-β1 and (D) CFP15a cells following transfection with SRp20, hnRNP A1, E4-ORF6 and E4-ORF3. The filled symbols indicate activation of the CFTR Cl⁻ efflux and the open symbols indicate no effect. The arrows indicate the time of forskolin administration.

Figure 4

Effect of NaBu on the splicing pattern of CFTR exons in CFP15a cells. Analysis of the splicing pattern of the cryptic 84 bp exon (A) and exon 9 (B). The top panels show examples of Genescan analysis of RT–PCR products from cells treated with 50 nM (left) and 50 μM (right) NaBu. Percentages and numbers in the top panels are as in Fig 1. The bottom panels show the effect of different NaBu concentrations on the level of aberrant transcripts. The grey bars show significant effect (P<0.01, Mann–Whitney U-test, adjusted with Bonferroni correction for seven comparisons) and the white bars show no effect. Error bars, standard deviation; UT, untreated.

Figure 5

Activation of CFTR Cl⁻ efflux by NaBu. Concentration-dependent activation in CFP15a cells (A) and CFP22a and IB3 cells (B). The arrows, filled and open symbols are as in Fig 3.

Figure 6

Effect of NaBu on the CFTR RNA level in CFP15a cells. The concentrations used are those that restored the CFTR function. Relative levels of CFTR RNA were quantified by real-time PCR, normalized to RPS9 for RNA loading, relative to untreated cells.