Generation of a High Number of Healthy Erythroid Cells from Gene-Edited Pyruvate Kinase Deficiency Patient-Specific Induced Pluripotent Stem Cells.

Zita Garate,Laurent Poirot,Xabi Agirre,Emmanuel Olivier,Noemi Fusaki,Omaira Alberquilla,Shengfang Jin,Charles Kung,Israel Orman,Laura Cerrato,Guillermo Güenechea ,Colin Hill ,Penelope A Kosinski ,Brian R Davis ,Maria Letı́cia Ribeiro ,Román Galetto ,Óscar Quintana-Bustamante ,Joanne C Mountford ,Juan A Bueren ,Ana M Crane ,Agnès Gouble ,Julián Sevilla ,Fátima Rodríguez-Fornés ,Tabita Maia ,Felipe Prósper ,Félix García‐Sánchez ,José C Segovia

doi:10.1016/j.stemcr.2015.10.002

Abstract

SummaryPyruvate kinase deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene. Erythrocytes from PKD patients show an energetic imbalance causing chronic non-spherocytic hemolytic anemia, as pyruvate kinase defects impair ATP production in erythrocytes. We generated PKD induced pluripotent stem cells (PKDiPSCs) from peripheral blood mononuclear cells (PB-MNCs) of PKD patients by non-integrative Sendai viral vectors. PKDiPSCs were gene edited to integrate a partial codon-optimized R-type pyruvate kinase cDNA in the second intron of the PKLR gene by TALEN-mediated homologous recombination (HR). Notably, we found allele specificity of HR led by the presence of a single-nucleotide polymorphism. High numbers of erythroid cells derived from gene-edited PKDiPSCs showed correction of the energetic imbalance, providing an approach to correct metabolic erythroid diseases and demonstrating the practicality of this approach to generate the large cell numbers required for comprehensive biochemical and metabolic erythroid analyses.

Highlights

Pyruvate kinase deficiency (PKD; OMIM: 266200) is a rare metabolic erythroid disease caused by mutations in the PKLR gene, which codes the R-type pyruvate kinase (RPK) in erythrocytes and L-type pyruvate kinase (LPK) in hepatocytes
Pyruvate kinase deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene
High numbers of erythroid cells derived from gene-edited PKDiPSCs showed correction of the energetic imbalance, providing an approach to correct metabolic erythroid diseases and demonstrating the practicality of this approach to generate the large cell numbers required for comprehensive biochemical and metabolic erythroid analyses

Summary

Introduction

Pyruvate kinase deficiency (PKD; OMIM: 266200) is a rare metabolic erythroid disease caused by mutations in the PKLR gene, which codes the R-type pyruvate kinase (RPK) in erythrocytes and L-type pyruvate kinase (LPK) in hepatocytes. The only definitive cure for PKD is allogeneic bone marrow transplantation (Suvatte et al, 1998; Tanphaichitr et al, 2000). The low availability of compatible donors and the risks associated with allogeneic bone marrow transplantation limit its clinical application. Transplantation of gene-corrected autologous hematopoietic progenitors might solve these problems. We have developed different gamma-retroviral and lentiviral vectors to correct a mouse PKD model (Meza et al, 2009), and their efficacy is currently being tested in hematopoietic progenitors from PKD patients The main drawback of current gene therapy approaches based on retro-/lentiviral vectors is the random integration of transgenes, which can promote insertional mutagenesis by disrupting tumor suppressor genes or cis-activating proto-oncogenes (Cavazza et al, 2013)

Results

Discussion

Conclusion