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1.
Cell Stem Cell ; 28(5): 833-845.e5, 2021 05 06.
Artículo en Inglés | MEDLINE | ID: mdl-33513358

RESUMEN

Severe congenital neutropenia (SCN) is a life-threatening disorder most often caused by dominant mutations of ELANE that interfere with neutrophil maturation. We conducted a pooled CRISPR screen in human hematopoietic stem and progenitor cells (HSPCs) that correlated ELANE mutations with neutrophil maturation potential. Highly efficient gene editing of early exons elicited nonsense-mediated decay (NMD), overcame neutrophil maturation arrest in HSPCs from ELANE-mutant SCN patients, and produced normal hematopoietic engraftment function. Conversely, terminal exon frameshift alleles that mimic SCN-associated mutations escaped NMD, recapitulated neutrophil maturation arrest, and established an animal model of ELANE-mutant SCN. Surprisingly, only -1 frame insertions or deletions (indels) impeded neutrophil maturation, whereas -2 frame late exon indels repressed translation and supported neutrophil maturation. Gene editing of primary HSPCs allowed faithful identification of variant pathogenicity to clarify molecular mechanisms of disease and encourage a universal therapeutic approach to ELANE-mutant neutropenia, returning normal neutrophil production and preserving HSPC function.


Asunto(s)
Elastasa de Leucocito , Neutropenia , Animales , Síndromes Congénitos de Insuficiencia de la Médula Ósea , Edición Génica , Humanos , Elastasa de Leucocito/genética , Mutación/genética , Neutropenia/genética , Virulencia
2.
J Clin Invest ; 130(12): 6677-6687, 2020 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-32897878

RESUMEN

Gene editing of the erythroid-specific BCL11A enhancer in hematopoietic stem and progenitor cells (HSPCs) from patients with sickle cell disease (SCD) induces fetal hemoglobin (HbF) without detectable toxicity, as assessed by mouse xenotransplant. Here, we evaluated autologous engraftment and HbF induction potential of erythroid-specific BCL11A enhancer-edited HSPCs in 4 nonhuman primates. We used a single guide RNA (sgRNA) with identical human and rhesus target sequences to disrupt a GATA1 binding site at the BCL11A +58 erythroid enhancer. Cas9 protein and sgRNA ribonucleoprotein complex (RNP) was electroporated into rhesus HSPCs, followed by autologous infusion after myeloablation. We found that gene edits persisted in peripheral blood (PB) and bone marrow (BM) for up to 101 weeks similarly for BCL11A enhancer- or control locus-targeted (AAVS1-targeted) cells. Biallelic BCL11A enhancer editing resulted in robust γ-globin induction, with the highest levels observed during stress erythropoiesis. Indels were evenly distributed across PB and BM lineages. Off-target edits were not observed. Nonhomologous end-joining repair alleles were enriched in engrafting HSCs. In summary, we found that edited HSCs can persist for at least 101 weeks after transplant and biallelic-edited HSCs provide substantial HbF levels in PB red blood cells, together supporting further clinical translation of this approach.


Asunto(s)
Edición Génica , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Proteínas Represoras , Animales , Humanos , Macaca mulatta , Ratones , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Trasplante Autólogo
3.
Nat Med ; 26(4): 535-541, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32284612

RESUMEN

Base editing by nucleotide deaminases linked to programmable DNA-binding proteins represents a promising approach to permanently remedy blood disorders, although its application in engrafting hematopoietic stem cells (HSCs) remains unexplored. In this study, we purified A3A (N57Q)-BE3 base editor for ribonucleoprotein (RNP) electroporation of human-peripheral-blood-mobilized CD34+ hematopoietic stem and progenitor cells (HSPCs). We observed frequent on-target cytosine base edits at the BCL11A erythroid enhancer at +58 with few indels. Fetal hemoglobin (HbF) induction in erythroid progeny after base editing or nuclease editing was similar. A single therapeutic base edit of the BCL11A enhancer prevented sickling and ameliorated globin chain imbalance in erythroid progeny from sickle cell disease and ß-thalassemia patient-derived HSPCs, respectively. Moreover, efficient multiplex editing could be achieved with combined disruption of the BCL11A erythroid enhancer and correction of the HBB -28A>G promoter mutation. Finally, base edits could be produced in multilineage-repopulating self-renewing human HSCs with high frequency as assayed in primary and secondary recipient animals resulting in potent HbF induction in vivo. Together, these results demonstrate the potential of RNP base editing of human HSPCs as a feasible alternative to nuclease editing for HSC-targeted therapeutic genome modification.


Asunto(s)
Anemia de Células Falciformes/patología , Edición Génica , Terapia Genética/métodos , Células Madre Hematopoyéticas/metabolismo , Proteínas Represoras/genética , gamma-Globinas/genética , Anemia de Células Falciformes/terapia , Animales , Antígenos CD34/metabolismo , Sistemas CRISPR-Cas , Células Cultivadas , Estudios de Factibilidad , Femenino , Edición Génica/métodos , Marcación de Gen/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/patología , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Cultivo Primario de Células , Proteínas Represoras/metabolismo , Talasemia beta/patología , Talasemia beta/terapia , gamma-Globinas/metabolismo
4.
Nat Med ; 25(5): 776-783, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30911135

RESUMEN

Re-expression of the paralogous γ-globin genes (HBG1/2) could be a universal strategy to ameliorate the severe ß-globin disorders sickle cell disease (SCD) and ß-thalassemia by induction of fetal hemoglobin (HbF, α2γ2)1. Previously, we and others have shown that core sequences at the BCL11A erythroid enhancer are required for repression of HbF in adult-stage erythroid cells but are dispensable in non-erythroid cells2-6. CRISPR-Cas9-mediated gene modification has demonstrated variable efficiency, specificity, and persistence in hematopoietic stem cells (HSCs). Here, we demonstrate that Cas9:sgRNA ribonucleoprotein (RNP)-mediated cleavage within a GATA1 binding site at the +58 BCL11A erythroid enhancer results in highly penetrant disruption of this motif, reduction of BCL11A expression, and induction of fetal γ-globin. We optimize conditions for selection-free on-target editing in patient-derived HSCs as a nearly complete reaction lacking detectable genotoxicity or deleterious impact on stem cell function. HSCs preferentially undergo non-homologous compared with microhomology-mediated end joining repair. Erythroid progeny of edited engrafting SCD HSCs express therapeutic levels of HbF and resist sickling, while those from patients with ß-thalassemia show restored globin chain balance. Non-homologous end joining repair-based BCL11A enhancer editing approaching complete allelic disruption in HSCs is a practicable therapeutic strategy to produce durable HbF induction.


Asunto(s)
Edición Génica/métodos , Células Madre Hematopoyéticas/metabolismo , Secuencia de Aminoácidos , Anemia de Células Falciformes/sangre , Anemia de Células Falciformes/genética , Anemia de Células Falciformes/terapia , Secuencia de Bases , Sistemas CRISPR-Cas , Proteínas Portadoras/genética , Elementos de Facilitación Genéticos , Células Precursoras Eritroides/metabolismo , Hemoglobina Fetal/biosíntesis , Hemoglobina Fetal/genética , Trasplante de Células Madre Hematopoyéticas , Humanos , Mutación INDEL , Proteínas Nucleares/genética , ARN Guía de Kinetoplastida/genética , Proteínas Represoras , Talasemia beta/sangre , Talasemia beta/genética , Talasemia beta/terapia , gamma-Globinas/biosíntesis , gamma-Globinas/genética
5.
Blood ; 133(21): 2255-2262, 2019 05 23.
Artículo en Inglés | MEDLINE | ID: mdl-30704988

RESUMEN

The thalassemias are compelling targets for therapeutic genome editing in part because monoallelic correction of a subset of hematopoietic stem cells (HSCs) would be sufficient for enduring disease amelioration. A primary challenge is the development of efficient repair strategies that are effective in HSCs. Here, we demonstrate that allelic disruption of aberrant splice sites, one of the major classes of thalassemia mutations, is a robust approach to restore gene function. We target the IVS1-110G>A mutation using Cas9 ribonucleoprotein (RNP) and the IVS2-654C>T mutation by Cas12a/Cpf1 RNP in primary CD34+ hematopoietic stem and progenitor cells (HSPCs) from ß-thalassemia patients. Each of these nuclease complexes achieves high efficiency and penetrance of therapeutic edits. Erythroid progeny of edited patient HSPCs show reversal of aberrant splicing and restoration of ß-globin expression. This strategy could enable correction of a substantial fraction of transfusion-dependent ß-thalassemia genotypes with currently available gene-editing technology.


Asunto(s)
Edición Génica , Regulación de la Expresión Génica , Células Madre Hematopoyéticas , Sitios de Empalme de ARN , Empalme del ARN , Globinas beta , Talasemia beta , Sistemas CRISPR-Cas , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Mutación Puntual , Globinas beta/biosíntesis , Globinas beta/genética , Talasemia beta/genética , Talasemia beta/metabolismo , Talasemia beta/terapia
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