An RPS19-edited model for Diamond-Blackfan anemia reveals TP53-dependent impairment of hematopoietic stem cell activity

Abstract

Diamond-Blackfan anemia (DBA) is a genetic blood disease caused by heterozygous loss-offunction mutations in ribosomal protein (RP) genes, most commonly RPS19. The signature feature of DBA is hypoplastic anemia occurring in infants, although some older patients develop multi-lineage cytopenias with bone marrow hypocellularity. The mechanism of anemia in DBA is not fully understood and even less is known about the pancytopenia that occurs later in life, in part because patient hematopoietic stem and progenitor cells (HSPCs) are difficult to obtain, and the current experimental models are suboptimal. We modeled DBA by editing healthy human donor CD34 + HSPCs with CRISPR/Cas9 to create RPS19 haploinsufficiency. In vitro differentiation revealed normal myelopoiesis and impaired erythropoiesis, as observed in DBA.After transplantation into immunodeficient mice, bone marrow repopulation by RPS19 +/− HSPCs was profoundly reduced, indicating hematopoietic stem cell (HSC) impairment. The erythroid and HSC defects resulting from RPS19 haploinsufficiency were partially corrected by transduction with an RPS19-expressing lentiviral vector or by Cas9 disruption of TP53. Our results define a tractable, biologically relevant experimental model of DBA based on genome-editing of primary human HSPCs and they identify an associated HSC defect that emulates the pan-hematopoietic defect of DBA.

Hematopoietic cell transplantation and gene therapy for Diamond-Blackfan anemia: state of the art and science

“…Majority of the patients in this study received a melphalan-based RIC therapy ( 45 ). This is consistent with laboratory observations where wildtype hematopoietic stem/progenitor cells had a competitive advantage over RPS19 haploinsufficient cells in mouse and human cell models of DBA, providing rationale for use of RIC regimens ( 18 , 59 ). Moreover, in the context of poor outcomes in older patients with DBA, use of RIC therapy could potentially be an attractive option for this patient population ( 27 ).…”

“…In addition to a specific erythroid failure seen in younger patients, bone marrow hypocellularity, progressive multilineage cytopenias and immunodeficiency are noted in older patients with DBA, suggesting impairment of hematopoietic stem and progenitor cells (HSPCs) ( 15 – 17 ). Mechanisms of DBA HSPCs dysfunction are not clear, although TP53 activation appears to play a critical role ( 18 ). Additionally, systematic review of published case reports suggested an increased risk of cancer in DBA, which was subsequently confirmed by pioneering studies lead by the DBA Registry of North America (DBAR) ( 19 – 21 ).…”

“…One of the major challenges has been limited access to DBA CD34 + HSPCs for clinical development, as typically millions of CD34 + cells are required for these studies. We recently developed a CRISPR/Cas9-based approach to model RPS19- mutated DBA using CD34 + HSPCs from healthy donors ( 18 ). Utilizing this model, we demonstrated the efficacy of an RPS19- expressing SIN lentiviral vector ( 17 , 18 ).…”

“…We recently developed a CRISPR/Cas9-based approach to model RPS19- mutated DBA using CD34 + HSPCs from healthy donors ( 18 ). Utilizing this model, we demonstrated the efficacy of an RPS19- expressing SIN lentiviral vector ( 17 , 18 ). Recently, reduced translation of the erythroid transcription factor GATA1 was suggested to cause the characteristic erythroid defect of DBA, regardless of the mutated RP gene ( 11 ).…”

Gene therapy for congenital marrow failure syndromes – no longer grasping at straws?

“…Taking advantage of the efficiency of CRISPR/Cas9 editing of RPS19 , the authors knocked in a GFP reporter to the RPS19 locus, enabling tracking of RPS19 disrupted clones by the presence of the GFP signal. They found significant cellular toxicity related to ribonucleoprotein delivery of CRISPR/Cas9 components, unlike what was recently described in the RPS19 CRISPR model by Bhoopalan et al , 17 perhaps owing to differences in electroporation conditions. Nonetheless, to avoid some of this toxicity, the authors optimized mRNA delivery of CRISPR components by nanostraws and demonstrated the efficacy of this approach in primary human cells for the first time.…”