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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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Functional analysis of restored A1AT in c-hIPSCs-derived hepatocyte-like cellsa, Immunofluorescence showing the absence of polymeric A1AT protein in hepatocyte-like cells generated from c-hIPSCs. All forms of A1AT (left panels) and misfolded polymeric A1AT (middle panels). b, c, ELISA to assess the intracellular (b) and secreted (c) levels of polymeric A1AT protein in hepatocyte-like cells derived from A1ATD-hIPSCs (ZZ), c-hIPSCs (RR) and control hIPSCs (++). d, Endoglycosidase H (E) and peptide:N-glycosidase (P) digestion of A1AT immunoprecipitated from uncorrected (ZZ), corrected (RR) and control (++) hIPSC-derived hepatocyte-like cells (upper panels) and corresponding culture medium (lower panels). e, Chymotrypsin ELISA showing that corrected cells (RR) have A1AT enzymatic inhibitory activity that is superior to uncorrected cells (ZZ) and close to adult hepatocytes. f, g, Immunofluorescence of transplanted liver sections detecting human albumin (f) and A1AT (g). DNA was counterstained with DAPI. h, ELISA read-out of human albumin in the mouse serum longitudinally followed for each mouse. Asterisk, the mouse was subjected to histology analysis. Scale bars, 100 μm. Data in b, c and e are shown as mean ± s.d. (n=3). Student’s t-test was performed. NS, not significant.
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Figure 3: Functional analysis of restored A1AT in c-hIPSCs-derived hepatocyte-like cellsa, Immunofluorescence showing the absence of polymeric A1AT protein in hepatocyte-like cells generated from c-hIPSCs. All forms of A1AT (left panels) and misfolded polymeric A1AT (middle panels). b, c, ELISA to assess the intracellular (b) and secreted (c) levels of polymeric A1AT protein in hepatocyte-like cells derived from A1ATD-hIPSCs (ZZ), c-hIPSCs (RR) and control hIPSCs (++). d, Endoglycosidase H (E) and peptide:N-glycosidase (P) digestion of A1AT immunoprecipitated from uncorrected (ZZ), corrected (RR) and control (++) hIPSC-derived hepatocyte-like cells (upper panels) and corresponding culture medium (lower panels). e, Chymotrypsin ELISA showing that corrected cells (RR) have A1AT enzymatic inhibitory activity that is superior to uncorrected cells (ZZ) and close to adult hepatocytes. f, g, Immunofluorescence of transplanted liver sections detecting human albumin (f) and A1AT (g). DNA was counterstained with DAPI. h, ELISA read-out of human albumin in the mouse serum longitudinally followed for each mouse. Asterisk, the mouse was subjected to histology analysis. Scale bars, 100 μm. Data in b, c and e are shown as mean ± s.d. (n=3). Student’s t-test was performed. NS, not significant.

Mentions: To confirm that the genetic correction of hIPSCs resulted in the expected phenotypic correction, hIPSCs were differentiated in vitro into hepatocyte-like cells, the main cell type affected by the disease A1ATD. Differentiation of the corrected lines occurred as expected, resulting in a near homogenous population of hepatocyte-like cells (Supplementary Fig. 6a-c). Remarkably, CGH analysis of differentiated cells showed that hepatic differentiation neither increases the number of genetic abnormalities nor selects for cells with abnormal karyotype (Supplementary Table 2d). The resulting cells shared key functional attributes of their in vivo counterparts including glycogen storage, LDL-cholesterol uptake, albumin secretion and Cytochrome P450 activity (Supplementary Fig. 6d-g). Importantly, immunofluorescence and ELISA both confirmed the absence of mutant polymeric A1AT in c-hIPSCs-derived hepatocyte-like cells that instead efficiently secreted normal endoglycosidase–H-insensitive monomeric A1AT (Fig. 3a-d). In addition, secreted A1AT displayed an enzymatic inhibitory activity that was comparable to that obtained from normal adult hepatocytes (Fig. 3e), thereby suggesting that physiological restoration of enzyme inhibitory activity could be achieved.


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)

Functional analysis of restored A1AT in c-hIPSCs-derived hepatocyte-like cellsa, Immunofluorescence showing the absence of polymeric A1AT protein in hepatocyte-like cells generated from c-hIPSCs. All forms of A1AT (left panels) and misfolded polymeric A1AT (middle panels). b, c, ELISA to assess the intracellular (b) and secreted (c) levels of polymeric A1AT protein in hepatocyte-like cells derived from A1ATD-hIPSCs (ZZ), c-hIPSCs (RR) and control hIPSCs (++). d, Endoglycosidase H (E) and peptide:N-glycosidase (P) digestion of A1AT immunoprecipitated from uncorrected (ZZ), corrected (RR) and control (++) hIPSC-derived hepatocyte-like cells (upper panels) and corresponding culture medium (lower panels). e, Chymotrypsin ELISA showing that corrected cells (RR) have A1AT enzymatic inhibitory activity that is superior to uncorrected cells (ZZ) and close to adult hepatocytes. f, g, Immunofluorescence of transplanted liver sections detecting human albumin (f) and A1AT (g). DNA was counterstained with DAPI. h, ELISA read-out of human albumin in the mouse serum longitudinally followed for each mouse. Asterisk, the mouse was subjected to histology analysis. Scale bars, 100 μm. Data in b, c and e are shown as mean ± s.d. (n=3). Student’s t-test was performed. NS, not significant.
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Figure 3: Functional analysis of restored A1AT in c-hIPSCs-derived hepatocyte-like cellsa, Immunofluorescence showing the absence of polymeric A1AT protein in hepatocyte-like cells generated from c-hIPSCs. All forms of A1AT (left panels) and misfolded polymeric A1AT (middle panels). b, c, ELISA to assess the intracellular (b) and secreted (c) levels of polymeric A1AT protein in hepatocyte-like cells derived from A1ATD-hIPSCs (ZZ), c-hIPSCs (RR) and control hIPSCs (++). d, Endoglycosidase H (E) and peptide:N-glycosidase (P) digestion of A1AT immunoprecipitated from uncorrected (ZZ), corrected (RR) and control (++) hIPSC-derived hepatocyte-like cells (upper panels) and corresponding culture medium (lower panels). e, Chymotrypsin ELISA showing that corrected cells (RR) have A1AT enzymatic inhibitory activity that is superior to uncorrected cells (ZZ) and close to adult hepatocytes. f, g, Immunofluorescence of transplanted liver sections detecting human albumin (f) and A1AT (g). DNA was counterstained with DAPI. h, ELISA read-out of human albumin in the mouse serum longitudinally followed for each mouse. Asterisk, the mouse was subjected to histology analysis. Scale bars, 100 μm. Data in b, c and e are shown as mean ± s.d. (n=3). Student’s t-test was performed. NS, not significant.
Mentions: To confirm that the genetic correction of hIPSCs resulted in the expected phenotypic correction, hIPSCs were differentiated in vitro into hepatocyte-like cells, the main cell type affected by the disease A1ATD. Differentiation of the corrected lines occurred as expected, resulting in a near homogenous population of hepatocyte-like cells (Supplementary Fig. 6a-c). Remarkably, CGH analysis of differentiated cells showed that hepatic differentiation neither increases the number of genetic abnormalities nor selects for cells with abnormal karyotype (Supplementary Table 2d). The resulting cells shared key functional attributes of their in vivo counterparts including glycogen storage, LDL-cholesterol uptake, albumin secretion and Cytochrome P450 activity (Supplementary Fig. 6d-g). Importantly, immunofluorescence and ELISA both confirmed the absence of mutant polymeric A1AT in c-hIPSCs-derived hepatocyte-like cells that instead efficiently secreted normal endoglycosidase–H-insensitive monomeric A1AT (Fig. 3a-d). In addition, secreted A1AT displayed an enzymatic inhibitory activity that was comparable to that obtained from normal adult hepatocytes (Fig. 3e), thereby suggesting that physiological restoration of enzyme inhibitory activity could be achieved.

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