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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 Z mutation in A1ATD-hIPSCsa, The strategy for precise genome modification using ZFNs and the piggyBac transposon. Top line, structure of the A1AT gene; blue lines, Southern blot probes; thin and thick boxes, non-coding and coding exons, respectively; open arrow, piggyBac transposon; B, BamHI; A, AflIII. b, Sequences of wild-type (Reference), Z, PB, and Rev alleles. Amino acid position 342 (blue), recognition sites for ZFNs (green), piggyBac excision site (red) are shown. Sequence changes in Rev allele from Z allele were indicated by asterisks. c, Surveyor nuclease assay showing the cleavage of Z mutation in ZFNs-transfected K562 cells. Non-transfected cells were used as a control. d, Southern blot analysis showing bi-allelic piggyBac insertion (B-16) and bi-allelic excision (B-16-C2, -C3 and -C6) during correction of the A1ATD-hIPSCs line B. Genomic DNA was digested by BamHI (5′ and PB probes) or AlfIII (3′ probe). Genotype: ZZ, homozygous for Z allele; PP, homozygous for insertion of piggyBac; RR, homozygous for reverted allele. e, Sequence analysis showing correction of Z mutation in 3 corrected hIPSC lines. Wild-type sequence (top line) and A1ATD-hIPSC (second line).
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Figure 2: Correction of the Z mutation in A1ATD-hIPSCsa, The strategy for precise genome modification using ZFNs and the piggyBac transposon. Top line, structure of the A1AT gene; blue lines, Southern blot probes; thin and thick boxes, non-coding and coding exons, respectively; open arrow, piggyBac transposon; B, BamHI; A, AflIII. b, Sequences of wild-type (Reference), Z, PB, and Rev alleles. Amino acid position 342 (blue), recognition sites for ZFNs (green), piggyBac excision site (red) are shown. Sequence changes in Rev allele from Z allele were indicated by asterisks. c, Surveyor nuclease assay showing the cleavage of Z mutation in ZFNs-transfected K562 cells. Non-transfected cells were used as a control. d, Southern blot analysis showing bi-allelic piggyBac insertion (B-16) and bi-allelic excision (B-16-C2, -C3 and -C6) during correction of the A1ATD-hIPSCs line B. Genomic DNA was digested by BamHI (5′ and PB probes) or AlfIII (3′ probe). Genotype: ZZ, homozygous for Z allele; PP, homozygous for insertion of piggyBac; RR, homozygous for reverted allele. e, Sequence analysis showing correction of Z mutation in 3 corrected hIPSC lines. Wild-type sequence (top line) and A1ATD-hIPSC (second line).

Mentions: We next explored whether this approach could be used to correct a mutation in hIPSCs derived from individuals with α1-antitrypsin deficiency (A1ATD)16. A1ATD is an autosomal recessive disorder found in 1 out of 2000 individuals of North European descent and represents the most common inherited metabolic disease of the liver17,18. It results from a single point mutation in the A1AT gene (the Z allele; Glu342Lys) that causes the protein to form ordered polymers within the endoplasmic reticulum of hepatocytes17,18. The resulting inclusions cause cirrhosis for which the only current therapy is liver transplantation. The increasing shortage of donors and harmful effects of immunosuppressive treatments impose major limitations on organ transplantation, making the potential of hIPSC-based therapy highly attractive. Since homologous recombination is relatively inefficient in hESCs6, we employed ZFN technology, which stimulates gene targeting in hESCs as well as hIPSCs7,10,19. ZFN pairs were designed to specifically cleave the site of the Z mutation (Fig. 2a-c, Supplementary Table 1 and Supplementary Note). A targeting vector was constructed from isogenic DNA with piggyBac repeats flanking the PGK-puroΔtk cassette (Fig. 2a). To minimize the distance between the mutation and the piggyBac transposon, a CTG leucine codon, 10 bp upstream of the mutation, was altered to a TTA leucine codon, generating the TTAA sequence, which would be left in the genome following piggyBac excision (Fig. 2b).


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 Z mutation in A1ATD-hIPSCsa, The strategy for precise genome modification using ZFNs and the piggyBac transposon. Top line, structure of the A1AT gene; blue lines, Southern blot probes; thin and thick boxes, non-coding and coding exons, respectively; open arrow, piggyBac transposon; B, BamHI; A, AflIII. b, Sequences of wild-type (Reference), Z, PB, and Rev alleles. Amino acid position 342 (blue), recognition sites for ZFNs (green), piggyBac excision site (red) are shown. Sequence changes in Rev allele from Z allele were indicated by asterisks. c, Surveyor nuclease assay showing the cleavage of Z mutation in ZFNs-transfected K562 cells. Non-transfected cells were used as a control. d, Southern blot analysis showing bi-allelic piggyBac insertion (B-16) and bi-allelic excision (B-16-C2, -C3 and -C6) during correction of the A1ATD-hIPSCs line B. Genomic DNA was digested by BamHI (5′ and PB probes) or AlfIII (3′ probe). Genotype: ZZ, homozygous for Z allele; PP, homozygous for insertion of piggyBac; RR, homozygous for reverted allele. e, Sequence analysis showing correction of Z mutation in 3 corrected hIPSC lines. Wild-type sequence (top line) and A1ATD-hIPSC (second line).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198846&req=5

Figure 2: Correction of the Z mutation in A1ATD-hIPSCsa, The strategy for precise genome modification using ZFNs and the piggyBac transposon. Top line, structure of the A1AT gene; blue lines, Southern blot probes; thin and thick boxes, non-coding and coding exons, respectively; open arrow, piggyBac transposon; B, BamHI; A, AflIII. b, Sequences of wild-type (Reference), Z, PB, and Rev alleles. Amino acid position 342 (blue), recognition sites for ZFNs (green), piggyBac excision site (red) are shown. Sequence changes in Rev allele from Z allele were indicated by asterisks. c, Surveyor nuclease assay showing the cleavage of Z mutation in ZFNs-transfected K562 cells. Non-transfected cells were used as a control. d, Southern blot analysis showing bi-allelic piggyBac insertion (B-16) and bi-allelic excision (B-16-C2, -C3 and -C6) during correction of the A1ATD-hIPSCs line B. Genomic DNA was digested by BamHI (5′ and PB probes) or AlfIII (3′ probe). Genotype: ZZ, homozygous for Z allele; PP, homozygous for insertion of piggyBac; RR, homozygous for reverted allele. e, Sequence analysis showing correction of Z mutation in 3 corrected hIPSC lines. Wild-type sequence (top line) and A1ATD-hIPSC (second line).
Mentions: We next explored whether this approach could be used to correct a mutation in hIPSCs derived from individuals with α1-antitrypsin deficiency (A1ATD)16. A1ATD is an autosomal recessive disorder found in 1 out of 2000 individuals of North European descent and represents the most common inherited metabolic disease of the liver17,18. It results from a single point mutation in the A1AT gene (the Z allele; Glu342Lys) that causes the protein to form ordered polymers within the endoplasmic reticulum of hepatocytes17,18. The resulting inclusions cause cirrhosis for which the only current therapy is liver transplantation. The increasing shortage of donors and harmful effects of immunosuppressive treatments impose major limitations on organ transplantation, making the potential of hIPSC-based therapy highly attractive. Since homologous recombination is relatively inefficient in hESCs6, we employed ZFN technology, which stimulates gene targeting in hESCs as well as hIPSCs7,10,19. ZFN pairs were designed to specifically cleave the site of the Z mutation (Fig. 2a-c, Supplementary Table 1 and Supplementary Note). A targeting vector was constructed from isogenic DNA with piggyBac repeats flanking the PGK-puroΔtk cassette (Fig. 2a). To minimize the distance between the mutation and the piggyBac transposon, a CTG leucine codon, 10 bp upstream of the mutation, was altered to a TTA leucine codon, generating the TTAA sequence, which would be left in the genome following piggyBac excision (Fig. 2b).

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