RESUMEN
Sickle cell disease (SCD) is a monogenic disorder that affects millions worldwide. Allogeneic hematopoietic stem cell transplantation is the only available cure. Here, we demonstrate the use of CRISPR/Cas9 and a short single-stranded oligonucleotide template to correct the sickle mutation in the ß-globin gene in hematopoietic stem and progenitor cells (HSPCs) from peripheral blood or bone marrow of patients with SCD, with 24.5 ± 7.6% efficiency without selection. Erythrocytes derived from gene-edited cells showed a marked reduction of sickle cells, with the level of normal hemoglobin (HbA) increased to 25.3 ± 13.9%. Gene-corrected SCD HSPCs retained the ability to engraft when transplanted into non-obese diabetic (NOD)-SCID-gamma (NSG) mice with detectable levels of gene correction 16-19 weeks post-transplantation. We show that, by using a high-fidelity SpyCas9 that maintained the same level of on-target gene modification, the off-target effects including chromosomal rearrangements were significantly reduced. Taken together, our results demonstrate efficient gene correction of the sickle mutation in both peripheral blood and bone marrow-derived SCD HSPCs, a significant reduction in sickling of red blood cells, engraftment of gene-edited SCD HSPCs in vivo and the importance of reducing off-target effects; all are essential for moving genome editing based SCD treatment into clinical practice.
Asunto(s)
Anemia de Células Falciformes/terapia , Edición Génica/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/metabolismo , Globinas beta/genética , Anemia de Células Falciformes/genética , Animales , Sistemas CRISPR-Cas , Línea Celular Tumoral , Células Cultivadas , Eritrocitos/metabolismo , Terapia Genética/métodos , Humanos , Células K562 , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Resultado del TratamientoRESUMEN
Induction of red blood cell (RBC) fetal hemoglobin (HbF; α2γ2) ameliorates the pathophysiology of sickle cell disease (SCD) by reducing the concentration of sickle hemoglobin (HbS; α2ßS2) to inhibit its polymerization. Hydroxyurea (HU), the only US Food and Drug Administration (FDA)-approved drug for SCD, acts in part by inducing HbF; however, it is not fully effective, reflecting the need for new therapies. Whole-exome sequence analysis of rare genetic variants in SCD patients identified FOXO3 as a candidate regulator of RBC HbF. We validated these genomic findings through loss- and gain-of-function studies in normal human CD34+ hematopoietic stem and progenitor cells induced to undergo erythroid differentiation. FOXO3 gene silencing reduced γ-globin RNA levels and HbF levels in erythroblasts, whereas overexpression of FOXO3 produced the opposite effect. Moreover, treatment of primary CD34+ cell-derived erythroid cultures with metformin, an FDA-approved drug known to enhance FOXO3 activity in nonerythroid cells, caused dose-related FOXO3-dependent increases in the percentage of HbF protein and the fraction of HbF-immunostaining cells (F cells). Combined HU and metformin treatment induced HbF additively and reversed the arrest in erythroid maturation caused by HU treatment alone. HbF induction by metformin in erythroid precursors was dependent on FOXO3 expression and did not alter expression of BCL11A, MYB, or KLF1. Collectively, our data implicate FOXO3 as a positive regulator of γ-globin expression and identify metformin as a potential therapeutic agent for SCD.
Asunto(s)
Células Eritroides/efectos de los fármacos , Células Eritroides/metabolismo , Hemoglobina Fetal/biosíntesis , Proteína Forkhead Box O3/genética , Regulación de la Expresión Génica/efectos de los fármacos , Metformina/farmacología , Anemia de Células Falciformes/sangre , Anemia de Células Falciformes/genética , Biomarcadores , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Niño , Preescolar , Células Eritroides/citología , Femenino , Hemoglobina Fetal/genética , Proteína Forkhead Box O3/metabolismo , Expresión Génica , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Masculino , Modelos Biológicos , Transducción Genética , gamma-Globinas/genética , gamma-Globinas/metabolismoRESUMEN
In human prostate cancer, the microRNA biogenesis machinery increases with prostate cancer progression. Here, we show that deletion of the Dgcr8 gene, a critical component of this complex, inhibits tumor progression in a Pten-knockout mouse model of prostate cancer. Early stages of tumor development were unaffected, but progression to advanced prostatic intraepithelial neoplasia was severely inhibited. Dgcr8 loss blocked Pten null-induced expansion of the basal-like, but not luminal, cellular compartment. Furthermore, while late-stage Pten knockout tumors exhibit decreased senescence-associated beta-galactosidase activity and increased proliferation, the simultaneous deletion of Dgcr8 blocked these changes resulting in levels similar to wild type. Sequencing of small RNAs in isolated epithelial cells uncovered numerous miRNA changes associated with PTEN loss. Consistent with a Pten-Dgcr8 association, analysis of a large cohort of human prostate tumors shows a strong correlation between Akt activation and increased Dgcr8 mRNA levels. Together, these findings uncover a critical role for microRNAs in enhancing proliferation and enabling the expansion of the basal cell compartment associated with tumor progression following Pten loss.