Targeted Integration of a Super-Exon into the <i>CFTR</i> Locus Leads to Functional Correction of a Cystic Fibrosis Cell Line Model

<div><p><i>In vitro</i> disease models have enabled insights into the pathophysiology of human disease as well as the functional evaluation of new therapies, such as novel genome engineering strategies. In the context of cystic fibrosis (CF), various cellular disease models have been established in recent years, including organoids based on induced pluripotent stem cell technology that allowed for functional readouts of CFTR activity. Yet, many of these <i>in vitro</i> CF models require complex and expensive culturing protocols that are difficult to implement and may not be amenable for high throughput screens. Here, we show that a simple cellular CF disease model based on the bronchial epithelial <i>ΔF508</i> cell line CFBE41o- can be used to validate functional CFTR correction. We used an engineered nuclease to target the integration of a super-exon, encompassing the sequences of <i>CFTR</i> exons 11 to 27, into exon 11 and re-activated endogenous <i>CFTR</i> expression by treating CFBE41o- cells with a demethylating agent. We demonstrate that the integration of this super-exon resulted in expression of a corrected mRNA from the endogenous <i>CFTR</i> promoter and used short-circuit current measurements in Ussing chambers to corroborate restored ion transport of the repaired CFTR channels. In conclusion, this study proves that the targeted integration of a large super-exon in <i>CFTR</i> exon 11 leads to functional correction of CFTR, suggesting that this strategy can be used to functionally correct all <i>CFTR</i> mutations located downstream of the 5’ end of exon 11.</p></div

Genotype analysis of targeted CFBE41o- clones.

<p>(a) PCR-based genotyping. Genetic analysis of the corrected CFBE41o- clones was carried out as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161072#pone.0161072.g002" target="_blank">Fig 2</a>. DD, donor detection; 5’, 5’ junction; 3’, 3’ junction; RI, random integration. RI was detected with primers R1/R2, amplifying parts of the donor backbone (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161072#pone.0161072.s004" target="_blank">S1 Table</a>) (b) Allelic discrimination. A three primer based PCR approach was employed to discriminate between parental allele (amplification by primer pair P3/P6) and targeted allele (P5/P6). Positions of PCR primers are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161072#pone.0161072.g002" target="_blank">Fig 2A</a>; pcl, polyclonal population. (c) Sequence of restored alleles. The CTT triplet is highlighted in gray and the sequence tag at the 5’ end of exon 11 is indicated. The single nucleotide polymorphism (SNP; G A, rs213950) present in CFBE41o- cells is highlighted in black. The sequence of 16HBE cells is indicated as a reference.</p

Targeted insertion of a therapeutic donor in <i>CFTR</i> exon 11.

<p>(a) Schematic. ZFN mediated cleavage of the target site stimulates homologous recombination (HR) with the donor template. The donor carries a <i>CFTR</i> cDNA comprising exons 11–27 and a puromycin selection cassette (PuroR), flanked by homology arms (HAL, homology arm left; HAR, homology arm right) of 750–850 bp. Positions of primer binding sites are indicated, <i>ΔF508</i> mutation is depicted as white box, vertical arrow indicates ZFN binding site and hatched box represents 30 bp modified region to prevent ZFN binding to the donor (also referred to as sequence tag). (b) Pre-selection analysis. CFBE41o- cells were transfected with different ZFN:donor ratios (0:3 indicates absence of ZFN expression vectors). The polyclonal cell population was subjected to PCR-based genotyping 3 days post-transfection with the indicated primers: Primer pairs P1/P2 for donor detection (DD), P3/P4 for detection of 5’ junction (5’), P5/P6 for detection of 3’ junction (3’). (c) Post-selection analysis. The transfected cells were subjected to puromycin selection and the polyclonal cell population assessed by PCR-based genotyping as in (b).</p

Functionality of <i>CFTR</i> specific ZFNs in human CFBE41o- bronchial epithelial cells.

<p>(a) Schematic of ZFN binding site in <i>CFTR</i> exon 11. <i>ΔF508</i> mutation is depicted as white box. The ZFN binds to two target half-sites separated by a 6 bp spacer. (b) ZFN expression levels. CFBE41o- cells were transfected with indicated amounts of ZFN expression vectors and harvested to determine ZFN expression levels by immunoblotting using HA tag specific antibodies. ß-actin was included as a loading control. (c) ZFN cleavage activity. CFBE41o- cells were transfected with 0.5 μg of ZFN expression plasmid, either left (L) or right (R) subunit alone, or in combination (L&R). A 728 bp PCR amplicon encompassing the ZFN cleavage site was subjected to digestion with the mismatch-sensitive T7 endonuclease I (T7EI). Expected positions of the cleavage products are indicated, an asterisk indicates an unspecific cleavage product. Representative gels of a total of four experiments (n = 4) are shown on top. Average cleavage activity is shown on the bottom, where error bars indicate standard error of the mean (SEM).</p

Functional restoration of CFTR channel activity.

<p>(a) CFTR activity. Representative measurements of short-circuit current (I_SC) upon application of the cAMP activation cocktail of parental CFBE41o- cells, uncorrected clone #7, corrected clone #9, and wild-type 16HBE cells in Ussing chamber experiments. (b, c) Statistical analysis. Transepithelial measurements of clone #7 (b) and corrected clone #9 (c). The number of experiments is indicated. Bars represent mean ± SEM.</p

Analysis of the cleavage activity of AvrBs3-PvuII fusion proteins.

<p>(<b>A</b>) <b>and </b>(<b>B</b>) Comparison of the cleavage rates of selected AvrBs3-PvuII fusion proteins (as indicated) under low ionic strength: 76 mM (20 mM Tris-Ac, 50 mM K-Ac, 2 mM Mg-Ac, pH 7.5). In the top row the cleavage of the addressed substrate (T3-6bp-P-6bp-T3) is shown, in the bottom row that of the unaddressed substrate (-P-). All cleavage experiments were done with 8 nM DNA and 8 nM enzyme. (<b>C</b>) Comparison of the cleavage rates of an unaddressed substrate by selected AvrBs3-PvuII fusion proteins (as indicated) under physiological ionic strength: 143 mM (20 mM Tris-Ac, 120 mM K-Ac, 1 mM Mg-Ac, pH 7.5). The experiments were done with an excess of enzyme, the TALE-scPvuII fusion protein (top, 60 nM enzyme, 6 nM DNA) shows a higher cleavage activity with an unaddressed substrate (-P-) than the homodimeric TALE-PvuII^T46G fusion protein (bottom, 80 nM enzyme, 8 nM DNA). See the appearance of nicked and linearized DNA with AvrBs3-28-L-scPvuII^T46G. There is no nicking or cleavage detectable of the unaddressed substrate with AvrBs3-28-L-PvuII^T46G. oc, open circle; lin, linearized; sc, supercoiled.</p

Engineered highly specific endonucleases that can be used for gene targeting by introducing a double-strand break into a complex genome and thereby stimulating homologous recombination.

<p>With the exception of engineered homing endonucleases (“meganucleases”) in which the function of DNA binding and DNA cleavage is present in the same polypeptide chain [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082539#B77" target="_blank">77</a>], the other engineered nucleases consist of separate DNA-binding (green) and DNA-cleavage (blue) modules. Zinc finger nucleases and TALE nucleases usually have the non-specific cleavage domain of the restriction endonuclease FokI as DNA-cleavage module, but as shown recently and in the present paper the restriction endonuclease PvuII can also be used for this purpose [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082539#B54" target="_blank">54</a>]. PvuII has also been employed in TFO-linked nucleases [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082539#B49" target="_blank">49</a>] and in protein fusions (with catalytically inactive I-SceI) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082539#B53" target="_blank">53</a>] as DNA-cleavage module. Zinc finger nucleases, TALE nucleases and TFO-linked nucleases are programmable, as are the RNA-mediated nucleases [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082539#B36" target="_blank">36</a>] [modified after [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082539#B3" target="_blank">3</a>]] .</p

Analysis of competition cleavage experiments with AvrBs3-PvuII fusion proteins.

<p>(<b>A</b>) Competition cleavage experiments with AvrBs3-28-L-PvuII^T46G under physiological ionic strength. Shown is the cleavage pattern with supercoiled plasmid DNA with an addressed site (8 nM) in competition with a PCR fragment (unP) with an unaddressed site (32 nM). The experiment was carried out with a variable excess of enzyme over plasmid substrate (0.25 to 40-fold). The enzyme shows complete cleavage of the addressed substrate but no cleavage of the unaddressed substrate, even in an overnight incubation with a 40-fold excess of enzyme over the addressed plasmid substrate (8 nM) and 10-fold excess over the unaddressed PCR substrate (32 nM). The brackets indicate the positions where one would expect the products of cleavage of the unaddressed PCR substrate. oc, open circle; lin, linearized; sc, supercoiled. (<b>B</b>) Quantitative determination of the preference of AvrBs3-28-L-PvuII^T46G for an addressed (T3-6bp-P-6bp-T3) over an unaddressed site (-P-). The reactions were performed in triplicate under physiological conditions with 20 nM enzyme and 20 nM addressed substrate (squares) and unaddressed substrate (circles), both PCR fragments were radioactively labelled with [α³²P]dATP. The insert shows the primary data: the electrophoretic analysis of the cleavage reaction products using an Instant Imager. From the fit, a cleavage preference of > 34,000-fold was determined. </p

Analysis of the cleavage activity of AvrBs3-PvuII fusion proteins on AvrBs3 and AvrBs4 substrates.

<p>(<b>A</b>) Specificity of cleavage analyzed with the T3-6bp-P-6bp-T3 substrate and the T4-6bp-P-6bp-T4 substrate which differ in 11 (8, respectively, considering the degeneracy of the TALE recognition code) out of 19 positions from the AvrBs3 target site. No nicking or cleavage of the AvrBs4 substrate (8 nM) by AvrBs3-28-L-PvuII^T46G (8 nM) could be detected. (<b>B</b>) Cleavage of a “half-site” substrate by AvrBs3-28-L-PvuII^T46G. The “half-site” substrate is a bipartite substrate consisting of an AvrBs3 recognition site and a PvuII recognition site (T3-6bp-P). The sc plasmid (8 nM) with the “half-site” was incubated with an equimolar concentration of AvrBs3-28-L-PvuII^T46G (8 nM). The assay was done under physiological ionic strength and in competition with a 32 nM PCR fragment (unP) with one unaddressed PvuII site (-P-). Whereas the “half-site” substrate is cleaved almost to completion, the unaddressed PCR fragment is not cleaved at all. (<b>C</b>) The effect of the distance of the AvrBs3 and the PvuII site on the rate of DNA cleavage by various AvrBs3-PvuII fusion proteins. 20 nM radioactively labelled PCR fragments with 2 (T3-2-P-2-T3), 4 (T3-4-P-4-T3), 6 (T3-6-P-6-T3) and 8 (T3-8-P-8-T3) bp between the AvrBs3 and the PvuII site were incubated with 20 nM AvrBs3-28-L-PvuII^T46G, AvrBs3-28-PvuII^T46G and AvrBs3-L-PvuII^T46G for 60 min.</p

TALE-PvuII fusion proteins.

<p>(<b>A</b>) Scheme of the architecture of TALE–PvuII fusion proteins. Left: wtPvuII, a homodimer in which the DNA-binding module of a TALE protein is fused via a linker of defined length. Right: scPvuII, a monomeric nuclease in which the DNA-binding module of a TALE protein is fused via a linker of defined length. (<b>B</b>) Model of a TALE–wtPvuII fusion protein. The fusion protein is a dimer of identical subunits, each composed of a PvuII subunit and a TALE protein. This model was constructed by aligning the structures of the individual proteins [pdb 1pvi [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082539#B74" target="_blank">74</a>] and pdb 3ugm [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082539#B76" target="_blank">76</a>]] on a DNA composed of the PvuII recognition site and two TALE target sites up- and downstream of the PvuII recognition site, separated by 6 bp. The C-termini of the PvuII subunits and the N-termini of the TALE protein are separated by about 3 nm. This distance must be covered by a peptide linker of suitable length. The image was generated with PyMol.</p