ABSTRACT
Sickle cell disease is a devastating blood disorder that originates from a single point mutation in the HBB gene coding for hemoglobin. Here, we develop a GMP-compatible TALEN-mediated gene editing process enabling efficient HBB correction via a DNA repair template while minimizing risks associated with HBB inactivation. Comparing viral versus non-viral DNA repair template delivery in hematopoietic stem and progenitor cells in vitro, both strategies achieve comparable HBB correction and result in over 50% expression of normal adult hemoglobin in red blood cells without inducing ß-thalassemic phenotype. In an immunodeficient female mouse model, transplanted cells edited with the non-viral strategy exhibit higher engraftment and gene correction levels compared to those edited with the viral strategy. Transcriptomic analysis reveals that non-viral DNA repair template delivery mitigates P53-mediated toxicity and preserves high levels of long-term hematopoietic stem cells. This work paves the way for TALEN-based autologous gene therapy for sickle cell disease.
Subject(s)
Anemia, Sickle Cell , Gene Editing , Genetic Therapy , Hematopoietic Stem Cells , Transcription Activator-Like Effector Nucleases , Anemia, Sickle Cell/therapy , Anemia, Sickle Cell/genetics , Gene Editing/methods , Animals , Hematopoietic Stem Cells/metabolism , Humans , Female , Mice , Genetic Therapy/methods , Transcription Activator-Like Effector Nucleases/metabolism , Transcription Activator-Like Effector Nucleases/genetics , Hematopoietic Stem Cell Transplantation , beta-Globins/genetics , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , DNA Repair , Mutation , beta-Thalassemia/therapy , beta-Thalassemia/genetics , Disease Models, Animal , Gene Transfer TechniquesABSTRACT
Thromboembolic events are the main cause of mortality in BCR-ABL1-negative myeloproliferative neoplasms (MPNs) but their underlying mechanisms are largely unrecognized. The Janus kinase 2 (JAK2)V617F mutation is the most frequent genetic alteration leading to MPN. Usually found in haematopoietic progenitors and stem cells, this mutation has also been described in endothelial cells (ECs) of MPN patients. In this study, we have questioned the impact of the JAK2V617F mutation on EC phenotype and functions. We developed an induced pluripotent stem cells strategy to compare JAK2 mutant and wild-type ECs. Transcriptomic assays showed that several genes and pathways involved in inflammation, cell adhesion and thrombotic events were over-represented in JAK2V617F ECs and expression levels of von Willebrand factor and P-selectin (CD62P) proteins were increased. Finally, we found that leucocytes from MPN patients adhere more tightly to JAK2V617F ECs. Our results show that JAK2V617F ECs have a pro-inflammatory and pro-thrombotic phenotype and were functionally pro-adherent.
Subject(s)
Blood Platelets/physiology , Endothelial Cells/physiology , Induced Pluripotent Stem Cells/physiology , Janus Kinase 2/genetics , Myeloid Progenitor Cells/physiology , Myeloproliferative Disorders/genetics , Thrombosis/genetics , Cell Adhesion/genetics , Cell Differentiation , Cells, Cultured , Fusion Proteins, bcr-abl/metabolism , Gene Expression Profiling , Humans , Mutation/genetics , Transgenes/geneticsSubject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/etiology , Primary Myelofibrosis/complications , Biomarkers , Cell Transformation, Neoplastic/genetics , Female , Humans , Janus Kinase 2/genetics , Leukemia, Myeloid, Acute/diagnosis , Middle Aged , Mutation , Primary Myelofibrosis/genetics , Remission InductionABSTRACT
TALEN is one of the most widely used tools in the field of genome editing. It enables gene integration and gene inactivation in a highly efficient and specific fashion. Although very attractive, the apparent simplicity and high success rate of TALEN could be misleading for novices in the field of gene editing. Depending on the application, specific TALEN designs, activity assessments and screening strategies need to be adopted. Here we report different methods to efficiently perform TALEN-mediated gene integration and inactivation in different mammalian cell systems including induced pluripotent stem cells and delineate experimental examples associated with these approaches.
