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Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells.

Yusa K, Rashid ST, Strick-Marchand H, Varela I, Liu PQ, Paschon DE, Miranda E, Ordóñez A, Hannan NR, Rouhani FJ, Darche S, Alexander G, Marciniak SJ, Fusaki N, Hasegawa M, Holmes MC, Di Santo JP, Lomas DA, Bradley A, Vallier L - Nature (2011)

Bottom Line: Genetic correction of human iPSCs restored the structure and function of A1AT in subsequently derived liver cells in vitro and in vivo.This approach is significantly more efficient than any other gene-targeting technology that is currently available and crucially prevents contamination of the host genome with residual non-human sequences.Our results provide the first proof of principle, to our knowledge, for the potential of combining human iPSCs with genetic correction to generate clinically relevant cells for autologous cell-based therapies.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

ABSTRACT
Human induced pluripotent stem cells (iPSCs) represent a unique opportunity for regenerative medicine because they offer the prospect of generating unlimited quantities of cells for autologous transplantation, with potential application in treatments for a broad range of disorders. However, the use of human iPSCs in the context of genetically inherited human disease will require the correction of disease-causing mutations in a manner that is fully compatible with clinical applications. The methods currently available, such as homologous recombination, lack the necessary efficiency and also leave residual sequences in the targeted genome. Therefore, the development of new approaches to edit the mammalian genome is a prerequisite to delivering the clinical promise of human iPSCs. Here we show that a combination of zinc finger nucleases (ZFNs) and piggyBac technology in human iPSCs can achieve biallelic correction of a point mutation (Glu342Lys) in the α(1)-antitrypsin (A1AT, also known as SERPINA1) gene that is responsible for α(1)-antitrypsin deficiency. Genetic correction of human iPSCs restored the structure and function of A1AT in subsequently derived liver cells in vitro and in vivo. This approach is significantly more efficient than any other gene-targeting technology that is currently available and crucially prevents contamination of the host genome with residual non-human sequences. Our results provide the first proof of principle, to our knowledge, for the potential of combining human iPSCs with genetic correction to generate clinically relevant cells for autologous cell-based therapies.

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Correction of the G290T mutation in the Tyr gene in mIPSCsa, The strategy for precise genome modification using the piggyBac transposon. Top line, structure of the Tyr gene; red line, 5′ external probe for Southern blot analysis; open arrow, piggyBac transposon carrying a PGK-puroΔtk cassette; P1, P2 and P3, PCR primers; B, BamHI; E, EcoNI. b, c, Southern blot (b) and PCR analyses (c) showing insertion (c/PB) and excision (c/Rev) of the piggyBac transposon. ES, mouse ESCs as a control. d, e, Sequence analyses revealed correction of the G290T mutation (d) and seamless excision of the piggyBac transposon (e). Note that two silent mutations (A and T indicated by arrowheads) introduced near the TTAA site were also detected. f, A chimeric mouse generated by injecting corrected Tyr c/Rev mIPSCs (left) displays black coat color. Right, a non-injected albino mouse.
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Figure 1: Correction of the G290T mutation in the Tyr gene in mIPSCsa, The strategy for precise genome modification using the piggyBac transposon. Top line, structure of the Tyr gene; red line, 5′ external probe for Southern blot analysis; open arrow, piggyBac transposon carrying a PGK-puroΔtk cassette; P1, P2 and P3, PCR primers; B, BamHI; E, EcoNI. b, c, Southern blot (b) and PCR analyses (c) showing insertion (c/PB) and excision (c/Rev) of the piggyBac transposon. ES, mouse ESCs as a control. d, e, Sequence analyses revealed correction of the G290T mutation (d) and seamless excision of the piggyBac transposon (e). Note that two silent mutations (A and T indicated by arrowheads) introduced near the TTAA site were also detected. f, A chimeric mouse generated by injecting corrected Tyr c/Rev mIPSCs (left) displays black coat color. Right, a non-injected albino mouse.

Mentions: To explore the use of piggyBac for the correction of point mutations, we designed a vector to correct an albino mutation (G290T substitution in the Tyr gene) in mouse induced pluripotent stem cells (mIPSCs) isolated from fibroblasts of the C57Bl6-Tyrc-Brd strain15. The targeting vector was constructed, carrying a wild-type 290G sequence and a PGK-puroΔtk cassette flanked by piggyBac repeats into the TTAA site (Fig. 1a). Following isolation of targeted clones, the selection cassette was excised from the mIPSCs genome by transient expression of the piggyBac transposase and subsequent FIAU selection. Genomic modification was verified by Southern blot and PCR analyses (Fig. 1b, c). The correction of the G290T mutation and seamless piggyBac excision were confirmed by sequence analyses (Fig. 1d, e). Two introduced silent mutations were observed, confirming that the T290G substitution was mediated by gene correction, not by spontaneous reversion (Fig. 1e). The function of the reverted allele was tested by injecting the corrected mIPSCs into albino mouse blastocysts. The resulting chimeric mice displayed a black coat color, indicating phenotypic correction of the albino mutation (Fig. 1f). These results collectively demonstrate that the piggyBac transposon can be used as a versatile tool for highly precise modification (e.g. correction or mutation) of the mammalian genome at a single base-pair level.


Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells.

Yusa K, Rashid ST, Strick-Marchand H, Varela I, Liu PQ, Paschon DE, Miranda E, Ordóñez A, Hannan NR, Rouhani FJ, Darche S, Alexander G, Marciniak SJ, Fusaki N, Hasegawa M, Holmes MC, Di Santo JP, Lomas DA, Bradley A, Vallier L - Nature (2011)

Correction of the G290T mutation in the Tyr gene in mIPSCsa, The strategy for precise genome modification using the piggyBac transposon. Top line, structure of the Tyr gene; red line, 5′ external probe for Southern blot analysis; open arrow, piggyBac transposon carrying a PGK-puroΔtk cassette; P1, P2 and P3, PCR primers; B, BamHI; E, EcoNI. b, c, Southern blot (b) and PCR analyses (c) showing insertion (c/PB) and excision (c/Rev) of the piggyBac transposon. ES, mouse ESCs as a control. d, e, Sequence analyses revealed correction of the G290T mutation (d) and seamless excision of the piggyBac transposon (e). Note that two silent mutations (A and T indicated by arrowheads) introduced near the TTAA site were also detected. f, A chimeric mouse generated by injecting corrected Tyr c/Rev mIPSCs (left) displays black coat color. Right, a non-injected albino mouse.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198846&req=5

Figure 1: Correction of the G290T mutation in the Tyr gene in mIPSCsa, The strategy for precise genome modification using the piggyBac transposon. Top line, structure of the Tyr gene; red line, 5′ external probe for Southern blot analysis; open arrow, piggyBac transposon carrying a PGK-puroΔtk cassette; P1, P2 and P3, PCR primers; B, BamHI; E, EcoNI. b, c, Southern blot (b) and PCR analyses (c) showing insertion (c/PB) and excision (c/Rev) of the piggyBac transposon. ES, mouse ESCs as a control. d, e, Sequence analyses revealed correction of the G290T mutation (d) and seamless excision of the piggyBac transposon (e). Note that two silent mutations (A and T indicated by arrowheads) introduced near the TTAA site were also detected. f, A chimeric mouse generated by injecting corrected Tyr c/Rev mIPSCs (left) displays black coat color. Right, a non-injected albino mouse.
Mentions: To explore the use of piggyBac for the correction of point mutations, we designed a vector to correct an albino mutation (G290T substitution in the Tyr gene) in mouse induced pluripotent stem cells (mIPSCs) isolated from fibroblasts of the C57Bl6-Tyrc-Brd strain15. The targeting vector was constructed, carrying a wild-type 290G sequence and a PGK-puroΔtk cassette flanked by piggyBac repeats into the TTAA site (Fig. 1a). Following isolation of targeted clones, the selection cassette was excised from the mIPSCs genome by transient expression of the piggyBac transposase and subsequent FIAU selection. Genomic modification was verified by Southern blot and PCR analyses (Fig. 1b, c). The correction of the G290T mutation and seamless piggyBac excision were confirmed by sequence analyses (Fig. 1d, e). Two introduced silent mutations were observed, confirming that the T290G substitution was mediated by gene correction, not by spontaneous reversion (Fig. 1e). The function of the reverted allele was tested by injecting the corrected mIPSCs into albino mouse blastocysts. The resulting chimeric mice displayed a black coat color, indicating phenotypic correction of the albino mutation (Fig. 1f). These results collectively demonstrate that the piggyBac transposon can be used as a versatile tool for highly precise modification (e.g. correction or mutation) of the mammalian genome at a single base-pair level.

Bottom Line: Genetic correction of human iPSCs restored the structure and function of A1AT in subsequently derived liver cells in vitro and in vivo.This approach is significantly more efficient than any other gene-targeting technology that is currently available and crucially prevents contamination of the host genome with residual non-human sequences.Our results provide the first proof of principle, to our knowledge, for the potential of combining human iPSCs with genetic correction to generate clinically relevant cells for autologous cell-based therapies.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

ABSTRACT
Human induced pluripotent stem cells (iPSCs) represent a unique opportunity for regenerative medicine because they offer the prospect of generating unlimited quantities of cells for autologous transplantation, with potential application in treatments for a broad range of disorders. However, the use of human iPSCs in the context of genetically inherited human disease will require the correction of disease-causing mutations in a manner that is fully compatible with clinical applications. The methods currently available, such as homologous recombination, lack the necessary efficiency and also leave residual sequences in the targeted genome. Therefore, the development of new approaches to edit the mammalian genome is a prerequisite to delivering the clinical promise of human iPSCs. Here we show that a combination of zinc finger nucleases (ZFNs) and piggyBac technology in human iPSCs can achieve biallelic correction of a point mutation (Glu342Lys) in the α(1)-antitrypsin (A1AT, also known as SERPINA1) gene that is responsible for α(1)-antitrypsin deficiency. Genetic correction of human iPSCs restored the structure and function of A1AT in subsequently derived liver cells in vitro and in vivo. This approach is significantly more efficient than any other gene-targeting technology that is currently available and crucially prevents contamination of the host genome with residual non-human sequences. Our results provide the first proof of principle, to our knowledge, for the potential of combining human iPSCs with genetic correction to generate clinically relevant cells for autologous cell-based therapies.

Show MeSH
Related in: MedlinePlus