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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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Allele-Preferred Targeting of CF iPSCs(A) Schematic of donor 1 and donor 2 engineered with respective A or G base change at intron 9; results from a total of two independent targeted-integration experiments with either donor 1 or donor 2 are shown.(B) Schematic of uncorrected CFTR alleles ΔI507 and ΔF508, highlighting A or G base within intron 9.
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fig4: Allele-Preferred Targeting of CF iPSCs(A) Schematic of donor 1 and donor 2 engineered with respective A or G base change at intron 9; results from a total of two independent targeted-integration experiments with either donor 1 or donor 2 are shown.(B) Schematic of uncorrected CFTR alleles ΔI507 and ΔF508, highlighting A or G base within intron 9.

Mentions: In our targeted correction of the ΔI507/ΔF508 clone 17 iPSCs, we observed a strong preference for targeting the ΔI507 allele versus the ΔF508 allele; sequencing of the unmodified allele yielded either the ΔI507 mutant allele (present in 3 of 21 clones) or ΔF508 mutant allele (in 18 of 21 clones) (a ΔI507/ΔF508 targeting ratio of 6:1) (Figure 4A). Although we initially speculated that perhaps a greater level of chromatin accessibility or transcriptional activity for one allele versus the other may have been responsible, our sequencing of CFTR cDNA from the original ΔI507/ΔF508 iPSCs, as shown previously, revealed transcriptional activity from both mutant alleles (Figure 2D).


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)

Allele-Preferred Targeting of CF iPSCs(A) Schematic of donor 1 and donor 2 engineered with respective A or G base change at intron 9; results from a total of two independent targeted-integration experiments with either donor 1 or donor 2 are shown.(B) Schematic of uncorrected CFTR alleles ΔI507 and ΔF508, highlighting A or G base within intron 9.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400651&req=5

fig4: Allele-Preferred Targeting of CF iPSCs(A) Schematic of donor 1 and donor 2 engineered with respective A or G base change at intron 9; results from a total of two independent targeted-integration experiments with either donor 1 or donor 2 are shown.(B) Schematic of uncorrected CFTR alleles ΔI507 and ΔF508, highlighting A or G base within intron 9.
Mentions: In our targeted correction of the ΔI507/ΔF508 clone 17 iPSCs, we observed a strong preference for targeting the ΔI507 allele versus the ΔF508 allele; sequencing of the unmodified allele yielded either the ΔI507 mutant allele (present in 3 of 21 clones) or ΔF508 mutant allele (in 18 of 21 clones) (a ΔI507/ΔF508 targeting ratio of 6:1) (Figure 4A). Although we initially speculated that perhaps a greater level of chromatin accessibility or transcriptional activity for one allele versus the other may have been responsible, our sequencing of CFTR cDNA from the original ΔI507/ΔF508 iPSCs, as shown previously, revealed transcriptional activity from both mutant alleles (Figure 2D).

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