Adenine base editing reduces misfolded protein accumulation and toxicity in alpha-1 antitrypsin deficient patient iPSC-hepatocytes

Alpha-1 antitrypsin deficiency (AATD) is most commonly caused by the Z mutation, a single-base substitution that leads to AAT protein misfolding and associated liver and lung disease. In this study, we apply adenine base editors to correct the Z mutation in patient induced pluripotent stem cells (iPSCs) and iPSC-derived hepatocytes (iHeps). We demonstrate that correction of the Z mutation in patient iPSCs reduces aberrant AAT accumulation and increases its secretion. Adenine base editing (ABE) of differentiated iHeps decreases ER stress in edited cells, as demonstrated by single-cell RNA sequencing. We find ABE to be highly efficient in iPSCs and do not identify off-target genomic mutations by whole-genome sequencing. These results reveal the feasibility and utility of base editing to correct the Z mutation in AATD patient cells. Alpha-1 antitrypsin deficiency (AATD) is most commonly caused by the Z mutation, a single-base substitution that leads to AAT protein misfolding and associated liver and lung disease. In this study, we apply adenine base editors to correct the Z mutation in patient induced pluripotent stem cells (iPSCs) and iPSC-derived hepatocytes (iHeps). We demonstrate that correction of the Z mutation in patient iPSCs reduces aberrant AAT accumulation and increases its secretion. Adenine base editing (ABE) of differentiated iHeps decreases ER stress in edited cells, as demonstrated by single-cell RNA sequencing. We find ABE to be highly efficient in iPSCs and do not identify off-target genomic mutations by whole-genome sequencing. These results reveal the feasibility and utility of base editing to correct the Z mutation in AATD patient cells. IntroductionAlpha-1 antitrypsin deficiency (AATD) is a common heritable cause of both lung and liver disease. AATD results from mutations in the SERPINA1 gene, which encodes the antiprotease alpha-1 antitrypsin (AAT). AAT is produced in high abundance by hepatocytes1Schultz H.E. Heremans J.F. Synthesis of the Plasma Proteins. Elsevier, New York1966: 321-349Google Scholar and functions primarily to neutralize neutrophil elastase. The most prevalent disease-causative mutation is a single-base substitution from guanine (G) to adenine (A), which results in a glutamic acid-to-lysine substitution (Glu342Lys) and production of a mutant protein prone to misfolding and aggregation, termed “Z-AAT.”2Brantly M. Nukiwa T. Crystal R.G. Molecular basis of alpha-1-antitrypsin deficiency.Am. J. Med. 1988; 84: 13-31Abstract Full Text PDF PubMed Scopus (396) Google Scholar Reduced Z-AAT secretion and associated diminished circulating AAT levels3Wewers M.D. Casolaro M.A. Sellers S.E. Swayze S.C. McPhaul K.M. Wittes J.T. Crystal R.G. Replacement therapy for alpha 1-antitrypsin deficiency associated with emphysema.N. Engl. J. Med. 1987; 316: 1055-1062Crossref PubMed Scopus (494) Google Scholar result in a protease/antiprotease imbalance in the lungs that over time predisposes affected individuals to injury, most commonly manifested as emphysema.4Brantly M.L. Paul L.D. Miller B.H. Falk R.T. Wu M. Crystal R.G. Clinical features and history of the destructive lung disease associated with alpha-1-antitrypsin deficiency of adults with pulmonary symptoms.Am. Rev. Respir. Dis. 1988; 138: 327-336Crossref PubMed Scopus (264) Google Scholar In addition, accumulation of polymerized Z-AAT in the liver can result in toxic gain-of-function effects in hepatocytes, leading to liver disease in both neonates and adults.5Lomas D.A. Evans D.L. Finch J.T. Carrell R.W. The mechanism of Z alpha 1-antitrypsin accumulation in the liver.Nature. 1992; 357: 605-607Crossref PubMed Scopus (886) Google Scholar,6Eriksson S. Carlson J. Velez R. Risk of cirrhosis and primary liver cancer in alpha 1-antitrypsin deficiency.N. Engl. J. Med. 1986; 314: 736-739Crossref PubMed Scopus (475) Google ScholarAlthough infusion of pooled human AAT protein (“augmentation therapy”) has been shown to slow progression of lung disease in AATD patients,7Chapman K.R. Burdon J.G. Piitulainen E. Sandhaus R.A. Seersholm N. Stocks J.M. Stoel B.C. Huang L. Yao Z. Edelman J.M. McElvaney N.G. RAPID Trial Study GroupIntravenous augmentation treatment and lung density in severe α1 antitrypsin deficiency (RAPID): a randomised, double-blind, placebo-controlled trial.Lancet. 2015; 386: 360-368Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar no specific treatments are available for AATD-associated liver disease. To address both lung and liver disease in AATD, emerging treatment strategies have focused on correction of the Z mutation. Among these, early human clinical trials delivering a normal copy of the SERPINA1 gene to skeletal muscle have achieved long-term but sub-therapeutic production of normal “M-AAT” protein.8Brantly M.L. Chulay J.D. Wang L. Mueller C. Humphries M. Spencer L.T. Rouhani F. Conlon T.J. Calcedo R. Betts M.R. et al.Sustained transgene expression despite T lymphocyte responses in a clinical trial of rAAV1-AAT gene therapy.Proc. Natl. Acad. Sci. U S A. 2009; 106: 16363-16368Crossref PubMed Scopus (254) Google Scholar,9Mueller C. Gernoux G. Gruntman A.M. Borel F. Reeves E.P. Calcedo R. Rouhani F.N. Yachnis A. Humphries M. Campbell-Thompson M. et al.5 Year expression and neutrophil defect repair after gene therapy in alpha-1 antitrypsin deficiency.Mol. Ther. 2017; 25: 1387-1394Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar Although promising, this strategy does not rectify toxic gain-of-function effects in Z-AAT-expressing hepatocytes. Attractive alternatives include direct correction of the SERPINA1 mutation in patient cells in vivo or delivery of engraftable, patient-specific cells containing a normal copy of SERPINA1. ZFN, TALEN, and CRISPR editing strategies have previously been used to correct the Z mutation in human iPSCs in vitro and mice in vivo10Shen S. Sanchez M.E. Blomenkamp K. Corcoran E.M. Marco E. Yudkoff C.J. Jiang of alpha-1 antitrypsin deficiency with editing in Ther. PubMed Scopus Google K. E. A. Rouhani et gene correction of deficiency in induced pluripotent stem PubMed Scopus Google K. M. C. M. et highly of alpha-1 antitrypsin deficiency pluripotent stem Full Text Full Text PDF PubMed Scopus Google J.T. Wang C. Z. and gene correction for liver disease patient-specific stem PubMed Scopus Google Scholar but the of in cells and associated M. K. 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J. et of a human liver disease with of alpha-1 2015; Full Text Full Text PDF PubMed Scopus Google Scholar for the is available for editing we delivery of to iHeps and of the with in as iHeps by to the cells for with for the of of iHeps achieved by the cells with for and the cells, in with and These iHeps on the as to and to To cells in on with and with with the cells with for in with the AAT from and with and To by cells for with the as on cells a and AAT in the human AAT as the Z-AAT by this to a from F. Gernoux G. C. Wang Z. A. M.A. L.D. D.L. et of both human and hepatocytes for treatment of antitrypsin deficiency.Mol. Ther. 2017; 25: Full Text Full Text PDF PubMed Scopus Google the of base in of the cells with for with with normal with primary used from and and cells and with and with a and in and to of the with and cells on a the and cells pooled in a to cells and as the The with The produced the and The cells with and the cells with and the cells with and the cells with and used to and the with The to a of or to cells a or in in the or The for gene expression by a of with a and The of the in this is for are in the used to or with a used to or of to and in are as and are as with IntroductionAlpha-1 antitrypsin deficiency (AATD) is a common heritable cause of both lung and liver disease. AATD results from mutations in the SERPINA1 gene, which encodes the antiprotease alpha-1 antitrypsin (AAT). AAT is produced in high abundance by hepatocytes1Schultz H.E. Heremans J.F. Synthesis of the Plasma Proteins. Elsevier, New York1966: 321-349Google Scholar and functions primarily to neutralize neutrophil elastase. The most prevalent disease-causative mutation is a single-base substitution from guanine (G) to adenine (A), which results in a glutamic acid-to-lysine substitution (Glu342Lys) and production of a mutant protein prone to misfolding and aggregation, termed “Z-AAT.”2Brantly M. Nukiwa T. Crystal R.G. Molecular basis of alpha-1-antitrypsin deficiency.Am. J. Med. 1988; 84: 13-31Abstract Full Text PDF PubMed Scopus (396) Google Scholar Reduced Z-AAT secretion and associated diminished circulating AAT levels3Wewers M.D. Casolaro M.A. Sellers S.E. Swayze S.C. McPhaul K.M. Wittes J.T. Crystal R.G. Replacement therapy for alpha 1-antitrypsin deficiency associated with emphysema.N. Engl. J. Med. 1987; 316: 1055-1062Crossref PubMed Scopus (494) Google Scholar result in a protease/antiprotease imbalance in the lungs that over time predisposes affected individuals to injury, most commonly manifested as emphysema.4Brantly M.L. Paul L.D. Miller B.H. Falk R.T. Wu M. Crystal R.G. Clinical features and history of the destructive lung disease associated with alpha-1-antitrypsin deficiency of adults with pulmonary symptoms.Am. Rev. Respir. Dis. 1988; 138: 327-336Crossref PubMed Scopus (264) Google Scholar In addition, accumulation of polymerized Z-AAT in the liver can result in toxic gain-of-function effects in hepatocytes, leading to liver disease in both neonates and adults.5Lomas D.A. Evans D.L. Finch J.T. Carrell R.W. The mechanism of Z alpha 1-antitrypsin accumulation in the liver.Nature. 1992; 357: 605-607Crossref PubMed Scopus (886) Google Scholar,6Eriksson S. Carlson J. Velez R. Risk of cirrhosis and primary liver cancer in alpha 1-antitrypsin deficiency.N. Engl. J. Med. 1986; 314: 736-739Crossref PubMed Scopus (475) Google ScholarAlthough infusion of pooled human AAT protein (“augmentation therapy”) has been shown to slow progression of lung disease in AATD patients,7Chapman K.R. Burdon J.G. Piitulainen E. Sandhaus R.A. Seersholm N. Stocks J.M. Stoel B.C. Huang L. Yao Z. Edelman J.M. McElvaney N.G. RAPID Trial Study GroupIntravenous augmentation treatment and lung density in severe α1 antitrypsin deficiency (RAPID): a randomised, double-blind, placebo-controlled trial.Lancet. 2015; 386: 360-368Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar no specific treatments are available for AATD-associated liver disease. To address both lung and liver disease in AATD, emerging treatment strategies have focused on correction of the Z mutation. Among these, early human clinical trials delivering a normal copy of the SERPINA1 gene to skeletal muscle have achieved long-term but sub-therapeutic production of normal “M-AAT” protein.8Brantly M.L. Chulay J.D. Wang L. Mueller C. Humphries M. Spencer L.T. Rouhani F. Conlon T.J. Calcedo R. Betts M.R. et al.Sustained transgene expression despite T lymphocyte responses in a clinical trial of rAAV1-AAT gene therapy.Proc. Natl. Acad. Sci. U S A. 2009; 106: 16363-16368Crossref PubMed Scopus (254) Google Scholar,9Mueller C. Gernoux G. Gruntman A.M. Borel F. Reeves E.P. Calcedo R. Rouhani F.N. Yachnis A. Humphries M. Campbell-Thompson M. et al.5 Year expression and neutrophil defect repair after gene therapy in alpha-1 antitrypsin deficiency.Mol. Ther. 2017; 25: 1387-1394Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar Although promising, this strategy does not rectify toxic gain-of-function effects in Z-AAT-expressing hepatocytes. Attractive alternatives include direct correction of the SERPINA1 mutation in patient cells in vivo or delivery of engraftable, patient-specific cells containing a normal copy of SERPINA1. ZFN, TALEN, and CRISPR editing strategies have previously been used to correct the Z mutation in human iPSCs in vitro and mice in vivo10Shen S. Sanchez M.E. Blomenkamp K. Corcoran E.M. Marco E. Yudkoff C.J. Jiang of alpha-1 antitrypsin deficiency with editing in Ther. PubMed Scopus Google K. E. A. Rouhani et gene correction of deficiency in induced pluripotent stem PubMed Scopus Google K. M. C. M. et highly of alpha-1 antitrypsin deficiency pluripotent stem Full Text Full Text PDF PubMed Scopus Google J.T. Wang C. Z. and gene correction for liver disease patient-specific stem PubMed Scopus Google Scholar but the of in cells and associated M. K. A. of induced by leads to and PubMed Scopus Google Scholar editing that be in the single-base Z mutation in the editing of mutations In this a is to a to its the of a RNA the base of base editors and adenine base editors have been base editing of to in genomic 2017; PubMed Scopus Google editing of a base in genomic PubMed Scopus Google Scholar we to correct the mutation in the SERPINA1 gene in patient induced pluripotent stem cells We base editors to the Z mutation and demonstrated efficient editing in both iPSCs and differentiated iPSC-derived hepatocytes that we have previously to AATD disease in K. M. C. M. et highly of alpha-1 antitrypsin deficiency pluripotent stem Full Text Full Text PDF PubMed Scopus Google L. M. J. et of a human liver disease with of alpha-1 2015; Full Text Full Text PDF PubMed Scopus Google Scholar correction of the Z mutation the aberrant accumulation of AAT protein and AAT secretion. single-cell RNA that iHeps from These the of to in patient hepatocytes.