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Targeted correction and restored function of the CFTR gene in cystic fibrosis induced pluripotent stem cells.

Crane AM, Kramer P, Bui JH, Chung WJ, Li XS, Gonzalez-Garay ML, Hawkins F, Liao W, Mora D, Choi S, Wang J, Sun HC, Paschon DE, Guschin DY, Gregory PD, Kotton DN, Holmes MC, Sorscher EJ, Davis BR - Stem Cell Reports (2015)

Bottom Line: Starting with skin fibroblasts from patients diagnosed with cystic fibrosis, we derived and characterized induced pluripotent stem cell (iPSC) lines.We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele versus the other.The corrected cystic fibrosis iPSCs, when induced to differentiate in vitro, expressed the corrected CFTR gene; importantly, CFTR correction resulted in restored expression of the mature CFTR glycoprotein and restoration of CFTR chloride channel function in iPSC-derived epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Center for Stem Cell and Regenerative Medicine, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center, Houston, TX 77030, USA.

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Cre-Mediated Excision of puroTK Cassette from Corrected CF WT/ΔF508 iPSCs(A) Schematic of the modified allele, before and after Cre-mediated excision, and the unmodified allele. The location of PCR primers (arrows marked 3 and 3′), both located outside of donor sequences, used in verification by amplification are shown. Also indicated are the expected sizes of Cla I digestion products.(B) (Top) The PCR amplicons for the original targeted clones (17-9 and -16), the Cre-excised clones (17-9-C1 and -C2; 17-16-C1 and -C2), and the original clone 17 CF iPSCs. The presence of only the 1.8-kb band for Cre-excised clones is consistent with successful excision. (Bottom) The results of Cla I digestion of the PCR amplicons. The size of bands for the Cre-excised clones is consistent with successful excision.(C) RT-PCR analysis of CFTR expression for two targeted CF iPSCs (17-9 and -16) as well as their derived Cre-excised clones. Also shown is CFTR expression by the original clone 17 CF iPSCs and CFTR-expressing Calu-3 cells; mouse embryo fibroblasts (MEFs) and HEK293TN cells are negative controls.(D) Sequencing of CFTR RT-PCR product from mutant ΔI507/ΔF508 CFTR iPSCs (clone 17) revealed a mixture of ΔI507 and ΔF508 CFTR sequences.(E) Sequencing of CFTR RT-PCR product from corrected WT/ΔF508 CFTR iPSCs (clones 17-9 and 17-16), together with their Cre-excised derivatives, revealed a mixture of WT and ΔF508 CFTR sequences.See also Figure S2 and Tables S1 and S2.
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fig2: Cre-Mediated Excision of puroTK Cassette from Corrected CF WT/ΔF508 iPSCs(A) Schematic of the modified allele, before and after Cre-mediated excision, and the unmodified allele. The location of PCR primers (arrows marked 3 and 3′), both located outside of donor sequences, used in verification by amplification are shown. Also indicated are the expected sizes of Cla I digestion products.(B) (Top) The PCR amplicons for the original targeted clones (17-9 and -16), the Cre-excised clones (17-9-C1 and -C2; 17-16-C1 and -C2), and the original clone 17 CF iPSCs. The presence of only the 1.8-kb band for Cre-excised clones is consistent with successful excision. (Bottom) The results of Cla I digestion of the PCR amplicons. The size of bands for the Cre-excised clones is consistent with successful excision.(C) RT-PCR analysis of CFTR expression for two targeted CF iPSCs (17-9 and -16) as well as their derived Cre-excised clones. Also shown is CFTR expression by the original clone 17 CF iPSCs and CFTR-expressing Calu-3 cells; mouse embryo fibroblasts (MEFs) and HEK293TN cells are negative controls.(D) Sequencing of CFTR RT-PCR product from mutant ΔI507/ΔF508 CFTR iPSCs (clone 17) revealed a mixture of ΔI507 and ΔF508 CFTR sequences.(E) Sequencing of CFTR RT-PCR product from corrected WT/ΔF508 CFTR iPSCs (clones 17-9 and 17-16), together with their Cre-excised derivatives, revealed a mixture of WT and ΔF508 CFTR sequences.See also Figure S2 and Tables S1 and S2.

Mentions: The overall strategy for correction of CFTR exon 10 (CFTR legacy exon notation) mutations consisted of delivering CFTR-specific ZFNs together with a selectable CFTR donor DNA (Figure 1A). We designed ZFNs targeting CFTR exon 10, recognizing DNA sequences approximately 110 bp upstream of either the ΔI507 or ΔF508 deletions, to facilitate the correction of either mutant allele by HDR. The CFTR ZFNs were co-delivered with a plasmid encoding the CFTR donor to CF iPSCs. Puromycin-resistant colonies were initially screened via PCR and then sequenced to confirm that CFTR exon 10 was corrected via HDR. Southern blot analysis confirmed that four clones (17-1, 17-9, 17-14, and 17-16) exhibited the expected genomic organization in the corrected CFTR allele without any additional integration of pgk-puroTK sequences (Figure 1B). Sequencing of CFTR genomic DNA exon 10 sequences at targeted (wild-type [WT]) and unmodified (ΔF508) alleles for each of the four corrected clones demonstrated correction of one CFTR allele (ΔI507) per clone (Figure 1C). Transient delivery of a Cre-recombinase expression plasmid resulted in numerous puroTK-excised clones from each of the four successfully edited clones; successful excision was confirmed via PCR analysis with subsequent Cla I digestion (Figures 2A and 2B) and Southern blot analysis (Figure 1B).


Targeted correction and restored function of the CFTR gene in cystic fibrosis induced pluripotent stem cells.

Crane AM, Kramer P, Bui JH, Chung WJ, Li XS, Gonzalez-Garay ML, Hawkins F, Liao W, Mora D, Choi S, Wang J, Sun HC, Paschon DE, Guschin DY, Gregory PD, Kotton DN, Holmes MC, Sorscher EJ, Davis BR - Stem Cell Reports (2015)

Cre-Mediated Excision of puroTK Cassette from Corrected CF WT/ΔF508 iPSCs(A) Schematic of the modified allele, before and after Cre-mediated excision, and the unmodified allele. The location of PCR primers (arrows marked 3 and 3′), both located outside of donor sequences, used in verification by amplification are shown. Also indicated are the expected sizes of Cla I digestion products.(B) (Top) The PCR amplicons for the original targeted clones (17-9 and -16), the Cre-excised clones (17-9-C1 and -C2; 17-16-C1 and -C2), and the original clone 17 CF iPSCs. The presence of only the 1.8-kb band for Cre-excised clones is consistent with successful excision. (Bottom) The results of Cla I digestion of the PCR amplicons. The size of bands for the Cre-excised clones is consistent with successful excision.(C) RT-PCR analysis of CFTR expression for two targeted CF iPSCs (17-9 and -16) as well as their derived Cre-excised clones. Also shown is CFTR expression by the original clone 17 CF iPSCs and CFTR-expressing Calu-3 cells; mouse embryo fibroblasts (MEFs) and HEK293TN cells are negative controls.(D) Sequencing of CFTR RT-PCR product from mutant ΔI507/ΔF508 CFTR iPSCs (clone 17) revealed a mixture of ΔI507 and ΔF508 CFTR sequences.(E) Sequencing of CFTR RT-PCR product from corrected WT/ΔF508 CFTR iPSCs (clones 17-9 and 17-16), together with their Cre-excised derivatives, revealed a mixture of WT and ΔF508 CFTR sequences.See also Figure S2 and Tables S1 and S2.
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fig2: Cre-Mediated Excision of puroTK Cassette from Corrected CF WT/ΔF508 iPSCs(A) Schematic of the modified allele, before and after Cre-mediated excision, and the unmodified allele. The location of PCR primers (arrows marked 3 and 3′), both located outside of donor sequences, used in verification by amplification are shown. Also indicated are the expected sizes of Cla I digestion products.(B) (Top) The PCR amplicons for the original targeted clones (17-9 and -16), the Cre-excised clones (17-9-C1 and -C2; 17-16-C1 and -C2), and the original clone 17 CF iPSCs. The presence of only the 1.8-kb band for Cre-excised clones is consistent with successful excision. (Bottom) The results of Cla I digestion of the PCR amplicons. The size of bands for the Cre-excised clones is consistent with successful excision.(C) RT-PCR analysis of CFTR expression for two targeted CF iPSCs (17-9 and -16) as well as their derived Cre-excised clones. Also shown is CFTR expression by the original clone 17 CF iPSCs and CFTR-expressing Calu-3 cells; mouse embryo fibroblasts (MEFs) and HEK293TN cells are negative controls.(D) Sequencing of CFTR RT-PCR product from mutant ΔI507/ΔF508 CFTR iPSCs (clone 17) revealed a mixture of ΔI507 and ΔF508 CFTR sequences.(E) Sequencing of CFTR RT-PCR product from corrected WT/ΔF508 CFTR iPSCs (clones 17-9 and 17-16), together with their Cre-excised derivatives, revealed a mixture of WT and ΔF508 CFTR sequences.See also Figure S2 and Tables S1 and S2.
Mentions: The overall strategy for correction of CFTR exon 10 (CFTR legacy exon notation) mutations consisted of delivering CFTR-specific ZFNs together with a selectable CFTR donor DNA (Figure 1A). We designed ZFNs targeting CFTR exon 10, recognizing DNA sequences approximately 110 bp upstream of either the ΔI507 or ΔF508 deletions, to facilitate the correction of either mutant allele by HDR. The CFTR ZFNs were co-delivered with a plasmid encoding the CFTR donor to CF iPSCs. Puromycin-resistant colonies were initially screened via PCR and then sequenced to confirm that CFTR exon 10 was corrected via HDR. Southern blot analysis confirmed that four clones (17-1, 17-9, 17-14, and 17-16) exhibited the expected genomic organization in the corrected CFTR allele without any additional integration of pgk-puroTK sequences (Figure 1B). Sequencing of CFTR genomic DNA exon 10 sequences at targeted (wild-type [WT]) and unmodified (ΔF508) alleles for each of the four corrected clones demonstrated correction of one CFTR allele (ΔI507) per clone (Figure 1C). Transient delivery of a Cre-recombinase expression plasmid resulted in numerous puroTK-excised clones from each of the four successfully edited clones; successful excision was confirmed via PCR analysis with subsequent Cla I digestion (Figures 2A and 2B) and Southern blot analysis (Figure 1B).

Bottom Line: Starting with skin fibroblasts from patients diagnosed with cystic fibrosis, we derived and characterized induced pluripotent stem cell (iPSC) lines.We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele versus the other.The corrected cystic fibrosis iPSCs, when induced to differentiate in vitro, expressed the corrected CFTR gene; importantly, CFTR correction resulted in restored expression of the mature CFTR glycoprotein and restoration of CFTR chloride channel function in iPSC-derived epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Center for Stem Cell and Regenerative Medicine, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center, Houston, TX 77030, USA.

Show MeSH
Related in: MedlinePlus