RESUMO
Alternative polyadenylation (APA) contributes to transcriptome complexity by generating mRNA isoforms with varying 3' UTR lengths. APA leading to 3' UTR shortening (3' US) is a common feature of most cancer cells; however, the molecular mechanisms are not understood. Here, we describe a widespread mechanism promoting 3' US in cancer through ubiquitination of the mRNA 3' end processing complex protein, PCF11, by the cancer-specific MAGE-A11-HUWE1 ubiquitin ligase. MAGE-A11 is normally expressed only in the male germline but is frequently re-activated in cancers. MAGE-A11 is necessary for cancer cell viability and is sufficient to drive tumorigenesis. Screening for targets of MAGE-A11 revealed that it ubiquitinates PCF11, resulting in loss of CFIm25 from the mRNA 3' end processing complex. This leads to APA of many transcripts affecting core oncogenic and tumor suppressors, including cyclin D2 and PTEN. These findings provide insights into the molecular mechanisms driving APA in cancer and suggest therapeutic strategies.
Assuntos
Regiões 3' não Traduzidas/genética , Antígenos de Neoplasias/metabolismo , Neoplasias Pulmonares/patologia , Proteínas de Neoplasias/metabolismo , Neoplasias Ovarianas/patologia , RNA Mensageiro/metabolismo , Ubiquitina/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Animais , Antígenos de Neoplasias/genética , Apoptose , Biomarcadores Tumorais , Carcinogênese , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proliferação de Células , Fator de Especificidade de Clivagem e Poliadenilação/genética , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Poliadenilação , Splicing de RNA , RNA Mensageiro/genética , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
Stress granules (SGs) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SGs act as a protective mechanism to cope with acute stress, but persistent SGs have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Knockout of the MAGE-B2 mouse ortholog or overexpression of G3BP1 confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermosensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress and could aid in the development of male fertility therapies.
Assuntos
Grânulos Citoplasmáticos/genética , DNA Helicases/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Biossíntese de Proteínas , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , Estresse Fisiológico/genética , Regiões 5' não Traduzidas , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/patologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA Helicases/metabolismo , Feminino , Células HCT116 , Células HeLa , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Espermatogônias/citologia , Espermatogônias/patologia , Testículo/citologia , Testículo/metabolismoRESUMO
Sickle cell disease (SCD) is caused by a mutation in the ß-globin gene HBB1. We used a custom adenine base editor (ABE8e-NRCH)2,3 to convert the SCD allele (HBBS) into Makassar ß-globin (HBBG), a non-pathogenic variant4,5. Ex vivo delivery of mRNA encoding the base editor with a targeting guide RNA into haematopoietic stem and progenitor cells (HSPCs) from patients with SCD resulted in 80% conversion of HBBS to HBBG. Sixteen weeks after transplantation of edited human HSPCs into immunodeficient mice, the frequency of HBBG was 68% and hypoxia-induced sickling of bone marrow reticulocytes had decreased fivefold, indicating durable gene editing. To assess the physiological effects of HBBS base editing, we delivered ABE8e-NRCH and guide RNA into HSPCs from a humanized SCD mouse6 and then transplanted these cells into irradiated mice. After sixteen weeks, Makassar ß-globin represented 79% of ß-globin protein in blood, and hypoxia-induced sickling was reduced threefold. Mice that received base-edited HSPCs showed near-normal haematological parameters and reduced splenic pathology compared to mice that received unedited cells. Secondary transplantation of edited bone marrow confirmed that the gene editing was durable in long-term haematopoietic stem cells and showed that HBBS-to-HBBG editing of 20% or more is sufficient for phenotypic rescue. Base editing of human HSPCs avoided the p53 activation and larger deletions that have been observed following Cas9 nuclease treatment. These findings point towards a one-time autologous treatment for SCD that eliminates pathogenic HBBS, generates benign HBBG, and minimizes the undesired consequences of double-strand DNA breaks.
Assuntos
Adenina/metabolismo , Anemia Falciforme/genética , Anemia Falciforme/terapia , Edição de Genes , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Globinas beta/genética , Animais , Antígenos CD34/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Modelos Animais de Doenças , Feminino , Terapia Genética , Genoma Humano/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/patologia , Humanos , Masculino , CamundongosRESUMO
Z-DNA-binding protein 1 (ZBP1) is an innate immune sensor of nucleic acids that regulates host defense responses and development. ZBP1 activation triggers inflammation and pyroptosis, necroptosis, and apoptosis (PANoptosis) by activating receptor-interacting Ser/Thr kinase 3 (RIPK3), caspase-8, and the NLRP3 inflammasome. ZBP1 is unique among innate immune sensors because of its N-terminal Zα1 and Zα2 domains, which bind to nucleic acids in the Z-conformation. However, the specific role of these Zα domains in orchestrating ZBP1 activation and subsequent inflammation and cell death is not clear. Here we generated Zbp1ΔZα2/ΔZα2 mice that express ZBP1 lacking the Zα2 domain and demonstrate that this domain is critical for influenza A virus-induced PANoptosis and underlies perinatal lethality in mice in which the RIP homotypic interaction motif domain of RIPK1 has been mutated (Ripk1mRHIM/mRHIM). Deletion of the Zα2 domain in ZBP1 abolished influenza A virus-induced PANoptosis and NLRP3 inflammasome activation. Furthermore, deletion of the Zα2 domain of ZBP1 was sufficient to rescue Ripk1mRHIM/mRHIM mice from perinatal lethality caused by ZBP1-driven cell death and inflammation. Our findings identify the essential role of the Zα2 domain of ZBP1 in several physiological functions and establish a link between Z-RNA sensing via the Zα2 domain and promotion of influenza-induced PANoptosis and perinatal lethality.
Assuntos
Perda do Embrião/patologia , Desenvolvimento Embrionário , Necroptose , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/patologia , Piroptose , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Inflamassomos/metabolismo , Influenza Humana/metabolismo , Influenza Humana/patologia , Camundongos Endogâmicos C57BL , Domínios Proteicos , Deleção de SequênciaRESUMO
Fusion-positive rhabdomyosarcoma (FP-RMS) driven by the expression of the PAX3-FOXO1 (P3F) fusion oncoprotein is an aggressive subtype of pediatric rhabdomyosarcoma. FP-RMS histologically resembles developing muscle yet occurs throughout the body in areas devoid of skeletal muscle highlighting that FP-RMS is not derived from an exclusively myogenic cell of origin. Here we demonstrate that P3F reprograms mouse and human endothelial progenitors to FP-RMS. We show that P3F expression in aP2-Cre expressing cells reprograms endothelial progenitors to functional myogenic stem cells capable of regenerating injured muscle fibers. Further, we describe a FP-RMS mouse model driven by P3F expression and Cdkn2a loss in endothelial cells. Additionally, we show that P3F expression in TP53-null human iPSCs blocks endothelial-directed differentiation and guides cells to become myogenic cells that form FP-RMS tumors in immunocompromised mice. Together these findings demonstrate that FP-RMS can originate from aberrant development of non-myogenic cells driven by P3F.
Assuntos
Rabdomiossarcoma Alveolar , Rabdomiossarcoma , Animais , Criança , Humanos , Camundongos , Linhagem Celular Tumoral , Células Endoteliais/metabolismo , Proteína Forkhead Box O1/metabolismo , Regulação Neoplásica da Expressão Gênica , Músculo Esquelético/metabolismo , Proteínas de Fusão Oncogênica/genética , Fatores de Transcrição Box Pareados/genética , Fator de Transcrição PAX3/genética , Fator de Transcrição PAX3/metabolismo , Rabdomiossarcoma/genética , Rabdomiossarcoma/patologia , Rabdomiossarcoma Alveolar/genéticaRESUMO
Gene editing the BCL11A erythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for ß-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here we compared combined CRISPR-Cas9 endonuclease editing of the BCL11A +58 and +55 enhancers with leading gene modification approaches under clinical investigation. We found that combined targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two sgRNAs resulted in superior HbF induction, including in engrafting erythroid cells from sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers. We corroborated prior observations that double strand breaks (DSBs) could produce unintended on- target outcomes in hematopoietic stem and progenitor cells (HSPCs) such as long deletions and centromere-distal chromosome fragment loss. We show these unintended outcomes are a byproduct of cellular proliferation stimulated by ex vivo culture. Editing HSPCs without cytokine culture bypassed long deletion and micronuclei formation while preserving efficient on-target editing and engraftment function. These results indicate that nuclease editing of quiescent hematopoietic stem cells (HSCs) limits DSB genotoxicity while maintaining therapeutic potency and encourages efforts for in vivo delivery of nucleases to HSCs.
RESUMO
Inducing fetal hemoglobin (HbF) in red blood cells can alleviate ß-thalassemia and sickle cell disease. We compared five strategies in CD34+ hematopoietic stem and progenitor cells, using either Cas9 nuclease or adenine base editors. The most potent modification was adenine base editor generation of γ-globin -175A>G. Homozygous -175A>G edited erythroid colonies expressed 81 ± 7% HbF versus 17 ± 11% in unedited controls, whereas HbF levels were lower and more variable for two Cas9 strategies targeting a BCL11A binding motif in the γ-globin promoter or a BCL11A erythroid enhancer. The -175A>G base edit also induced HbF more potently than a Cas9 approach in red blood cells generated after transplantation of CD34+ hematopoietic stem and progenitor cells into mice. Our data suggest a strategy for potent, uniform induction of HbF and provide insights into γ-globin gene regulation. More generally, we demonstrate that diverse indels generated by Cas9 can cause unexpected phenotypic variation that can be circumvented by base editing.
Assuntos
Anemia Falciforme , Talassemia beta , Camundongos , Animais , gama-Globinas/genética , gama-Globinas/metabolismo , Edição de Genes , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Anemia Falciforme/genética , Antígenos CD34/metabolismo , Talassemia beta/genéticaRESUMO
Zinc finger nucleases (ZFNs) have been used successfully to create genome-specific double-strand breaks and thereby stimulate gene targeting by several thousand fold. ZFNs are chimeric proteins composed of a specific DNA-binding domain linked to a non-specific DNA-cleavage domain. By changing key residues in the recognition helix of the specific DNA-binding domain, one can alter the ZFN binding specificity and thereby change the sequence to which a ZFN pair is being targeted. For these and other reasons, ZFNs are being pursued as reagents for genome modification, including use in gene therapy. In order for ZFNs to reach their full potential, it is important to attenuate the cytotoxic effects currently associated with many ZFNs. Here, we evaluate two potential strategies for reducing toxicity by regulating protein levels. Both strategies involve creating ZFNs with shortened half-lives and then regulating protein level with small molecules. First, we destabilize ZFNs by linking a ubiquitin moiety to the N-terminus and regulate ZFN levels using a proteasome inhibitor. Second, we destabilize ZFNs by linking a modified destabilizing FKBP12 domain to the N-terminus and regulate ZFN levels by using a small molecule that blocks the destabilization effect of the N-terminal domain. We show that by regulating protein levels, we can maintain high rates of ZFN-mediated gene targeting while reducing ZFN toxicity.
Assuntos
Desoxirribonucleases/metabolismo , Desoxirribonucleases/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Engenharia de Proteínas , Bibliotecas de Moléculas Pequenas/farmacologia , Linhagem Celular , Desoxirribonucleases/química , Desoxirribonucleases/genética , Marcação de Genes , Humanos , Inibidores de Proteassoma , Proteína 1A de Ligação a Tacrolimo/genética , Proteína 1A de Ligação a Tacrolimo/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Dedos de ZincoRESUMO
We previously reported a specific inverse agonist (SPA70) of the nuclear receptor pregnane X receptor (PXR). However, derivatization of SPA70 yielded only agonists and neutral antagonists, suggesting that inverse agonism of PXR is difficult to achieve. Therefore, we sought to design proteolysis targeting chimeras (PROTACs) aimed at inducing PXR degradation. Conjugation of a SPA70 derivative to ligands of the E3 substrate receptor cereblon (CRBN) resulted in one molecule, SJPYT-195, that reduced PXR protein level in an optimized degradation assay described here. Further analysis revealed that SJPYT-195 was a molecular glue degrader of the translation termination factor GSPT1 and that GSPT1 degradation resulted in subsequent reduction of PXR protein. GSPT1 has recently gained interest as an anticancer target, and our results give new insights into chemical determinants of drug-induced GSPT1 degradation. Additionally, we have developed assays and cell models for PXR degrader discovery that can be applied to additional protein targets.
RESUMO
Chronic lymphoproliferative disorder of natural killer cells (CLPD-NK) is characterized by clonal expansion of natural killer (NK) cells where the underlying genetic mechanisms are incompletely understood. In the present study, we report somatic mutations in the chemokine gene CCL22 as the hallmark of a distinct subset of CLPD-NK. CCL22 mutations were enriched at highly conserved residues, mutually exclusive of STAT3 mutations and associated with gene expression programs that resembled normal CD16dim/CD56bright NK cells. Mechanistically, the mutations resulted in ligand-biased chemokine receptor signaling, with decreased internalization of the G-protein-coupled receptor (GPCR) for CCL22, CCR4, via impaired ß-arrestin recruitment. This resulted in increased cell chemotaxis in vitro, bidirectional crosstalk with the hematopoietic microenvironment and enhanced NK cell proliferation in vivo in transgenic human IL-15 mice. Somatic CCL22 mutations illustrate a unique mechanism of tumor formation in which gain-of-function chemokine mutations promote tumorigenesis by biased GPCR signaling and dysregulation of microenvironmental crosstalk.
Assuntos
Quimiocina CCL22 , Células Matadoras Naturais , Transtornos Linfoproliferativos , Animais , Quimiocina CCL22/genética , Células Matadoras Naturais/patologia , Ativação Linfocitária , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/metabolismo , Transtornos Linfoproliferativos/patologia , Camundongos , MutaçãoRESUMO
Hereditary persistence of fetal hemoglobin (HPFH) ameliorates ß-hemoglobinopathies by inhibiting the developmental switch from γ-globin (HBG1/HBG2) to ß-globin (HBB) gene expression. Some forms of HPFH are associated with γ-globin promoter variants that either disrupt binding motifs for transcriptional repressors or create new motifs for transcriptional activators. How these variants sustain γ-globin gene expression postnatally remains undefined. We mapped γ-globin promoter sequences functionally in erythroid cells harboring different HPFH variants. Those that disrupt a BCL11A repressor binding element induce γ-globin expression by facilitating the recruitment of nuclear transcription factor Y (NF-Y) to a nearby proximal CCAAT box and GATA1 to an upstream motif. The proximal CCAAT element becomes dispensable for HPFH variants that generate new binding motifs for activators NF-Y or KLF1, but GATA1 recruitment remains essential. Our findings define distinct mechanisms through which transcription factors and their cis-regulatory elements activate γ-globin expression in different forms of HPFH, some of which are being recreated by therapeutic genome editing.
Assuntos
Fator de Ligação a CCAAT/genética , Hemoglobina Fetal/genética , Fator de Transcrição GATA1/genética , gama-Globinas/genética , Animais , Sítios de Ligação , Células COS , Sistemas CRISPR-Cas , Linhagem Celular , Chlorocebus aethiops , Células Eritroides , Edição de Genes/métodos , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
Chimeric antigen receptor (CAR) T-cell therapy has had limited success in early-phase clinical studies for solid tumors. Lack of efficacy is most likely multifactorial, including a limited array of targetable antigens. We reasoned that targeting the cancer-specific extra domain B (EDB) splice variant of fibronectin might overcome this limitation because it is abundantly secreted by cancer cells and adheres to their cell surface. In vitro, EDB-CAR T cells recognized and killed EDB-positive tumor cells. In vivo, 1 × 106 EDB-CAR T cells had potent antitumor activity in both subcutaneous and systemic tumor xenograft models, resulting in a significant survival advantage in comparison with control mice. EDB-CAR T cells also targeted the tumor vasculature, as judged by IHC and imaging, and their antivascular activity was dependent on the secretion of EDB by tumor cells. Thus, targeting tumor-specific splice variants such as EDB with CAR T cells is feasible and has the potential to improve the efficacy of CAR T-cell therapy.
Assuntos
Fibronectinas/antagonistas & inibidores , Imunoterapia Adotiva/métodos , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/terapia , Linfócitos T/transplante , Animais , Antígenos de Neoplasias , Linhagem Celular Tumoral , Técnicas de Cocultura , Estudos de Viabilidade , Fibronectinas/genética , Fibronectinas/imunologia , Fibronectinas/metabolismo , Voluntários Saudáveis , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismo , Neoplasias/imunologia , Neoplasias/patologia , Cultura Primária de Células , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Splicing de RNA , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pediatric sarcomas represent a heterogeneous group of malignancies that exhibit variable response to DNA-damaging chemotherapy. Schlafen family member 11 protein (SLFN11) increases sensitivity to replicative stress and has been implicated as a potential biomarker to predict sensitivity to DNA-damaging agents (DDA). SLFN11 expression was quantified in 220 children with solid tumors using IHC. Sensitivity to the PARP inhibitor talazoparib (TAL) and the topoisomerase I inhibitor irinotecan (IRN) was assessed in sarcoma cell lines, including SLFN11 knock-out (KO) and overexpression models, and a patient-derived orthotopic xenograft model (PDOX). SLFN11 was expressed in 69% of pediatric sarcoma sampled, including 90% and 100% of Ewing sarcoma and desmoplastic small round-cell tumors, respectively, although the magnitude of expression varied widely. In sarcoma cell lines, protein expression strongly correlated with response to TAL and IRN, with SLFN11 KO resulting in significant loss of sensitivity in vitro and in vivo Surprisingly, retrospective analysis of children with sarcoma found no association between SLFN11 levels and favorable outcome. Subsequently, high SLFN11 expression was confirmed in a PDOX model derived from a patient with recurrent Ewing sarcoma who failed to respond to treatment with TAL + IRN. Selective inhibition of BCL-xL increased sensitivity to TAL + IRN in SLFN11-positive resistant tumor cells. Although SLFN11 appears to drive sensitivity to replicative stress in pediatric sarcomas, its potential to act as a biomarker may be limited to certain tumor backgrounds or contexts. Impaired apoptotic response may be one mechanism of resistance to DDA-induced replicative stress.
Assuntos
Dano ao DNA/genética , Genômica/métodos , Proteínas Nucleares/metabolismo , Sarcoma de Ewing/genética , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Camundongos , Camundongos Nus , Adulto JovemRESUMO
USP7, which encodes a deubiquitylating enzyme, is among the most frequently mutated genes in pediatric T-ALL, with somatic heterozygous loss-of-function mutations (haploinsufficiency) predominantly affecting the subgroup that has aberrant TAL1 oncogene activation. Network analysis of > 200 T-ALL transcriptomes linked USP7 haploinsufficiency with decreased activities of E-proteins. E-proteins are also negatively regulated by TAL1, leading to concerted down-regulation of E-protein target genes involved in T-cell development. In T-ALL cell lines, we showed the physical interaction of USP7 with E-proteins and TAL1 by mass spectrometry and ChIP-seq. Haploinsufficient but not complete CRISPR knock-out of USP7 showed accelerated cell growth and validated transcriptional down-regulation of E-protein targets. Our study unveiled the synergistic effect of USP7 haploinsufficiency with aberrant TAL1 activation on T-ALL, implicating USP7 as a haploinsufficient tumor suppressor in T-ALL. Our findings caution against a universal oncogene designation for USP7 while emphasizing the dosage-dependent consequences of USP7 inhibitors currently under development as potential cancer therapeutics.
Assuntos
Oncogenes/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteína 1 de Leucemia Linfocítica Aguda de Células T/genética , Peptidase 7 Específica de Ubiquitina/genética , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Linhagem da Célula/genética , Proliferação de Células/genética , Regulação Leucêmica da Expressão Gênica/genética , Haploinsuficiência/genética , Humanos , Pediatria , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Ativação Transcricional/genéticaRESUMO
With advancements in gene editing technologies, our ability to make precise and efficient modifications to the genome is increasing at a remarkable rate, paving the way for scientists and clinicians to uniquely treat a multitude of previously irremediable diseases. CRISPR-Cas9, short for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9, is a gene editing platform with the ability to alter the nucleotide sequence of the genome in living cells. This technology is increasing the number and pace at which new gene editing treatments for genetic disorders are moving toward the clinic. The ß-hemoglobinopathies are a group of monogenic diseases, which despite their high prevalence and chronic debilitating nature, continue to have few therapeutic options available. In this review, we will discuss our existing comprehension of the genetics and current state of treatment for ß-hemoglobinopathies, consider potential genome editing therapeutic strategies, and provide an overview of the current state of clinical trials using CRISPR-Cas9 gene editing.
RESUMO
Fetal and adult hematopoietic stem cells (HSCs) have distinct proliferation rates, lineage biases, gene expression profiles, and gene dependencies. Although these differences are widely recognized, it is not clear how the transition from fetal to adult identity is coordinated. Here we show that murine HSCs and committed hematopoietic progenitor cells (HPCs) undergo a gradual, rather than precipitous, transition from fetal to adult transcriptional states. The transition begins prior to birth and is punctuated by a late prenatal spike in type I interferon signaling that promotes perinatal HPC expansion and sensitizes progenitors to the leukemogenic FLT3ITD mutation. Most other changes in gene expression and enhancer activation are imprecisely timed and poorly coordinated. Thus, heterochronic enhancer elements, and their associated transcripts, are activated independently of one another rather than as part of a robust network. This simplifies the regulatory programs that guide neonatal HSC/HPC ontogeny, but it creates heterogeneity within these populations.
Assuntos
Células-Tronco Adultas , Análise de Célula Única , Animais , Hematopoese , Células-Tronco Hematopoéticas , Camundongos , Transdução de Sinais , TranscriptomaRESUMO
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.
Assuntos
Edição de Genes , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Proteínas Repressoras , Animais , Humanos , Macaca mulatta , Camundongos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transplante AutólogoRESUMO
Genome engineering using programmable nucleases is a rapidly evolving technique that enables precise genetic manipulations within complex genomes. Although this technology first surfaced with the creation of meganucleases, zinc finger nucleases, and transcription activator-like effector nucleases, CRISPR-Cas9 has been the most widely adopted platform because of its ease of use. This comprehensive review presents a basic overview of genome engineering and discusses the major technological advances in the field. In addition to nucleases, we discuss CRISPR-derived base editors and epigenetic modifiers. We also delve into practical applications of these tools, including creating custom-edited cell and animal models as well as performing genetic screens. Finally, we discuss the potential for therapeutic applications and ethical considerations related to employing this technology in humans. © 2019 American Physiological Society. Compr Physiol 9:665-714, 2019.
Assuntos
Desoxirribonucleases , Edição de Genes , Animais , Sistemas CRISPR-Cas , Quebras de DNA de Cadeia Dupla , HumanosRESUMO
MLL rearrangements are translocation mutations that cause both acute lymphoblastic leukemia and acute myeloid leukemia (AML). These translocations can occur as sole clonal driver mutations in infant leukemias, suggesting that fetal or neonatal hematopoietic progenitors may be exquisitely sensitive to transformation by MLL fusion proteins. To test this possibility, we used transgenic mice to induce one translocation product, MLL-ENL, during fetal, neonatal, juvenile and adult stages of life. When MLL-ENL was induced in fetal or neonatal mice, almost all died of AML. In contrast, when MLL-ENL was induced in adult mice, most survived for >1 year despite sustained transgene expression. AML initiation was most efficient when MLL-ENL was induced in neonates, and even transient suppression of MLL-ENL in neonates could prevent AML in most mice. MLL-ENL target genes were induced more efficiently in neonatal progenitors than in adult progenitors, consistent with the distinct AML initiation efficiencies. Interestingly, transplantation stress mitigated the developmental barrier to leukemogenesis. Since fetal/neonatal progenitors were highly competent to initiate MLL-ENL-driven AML, we tested whether Lin28b, a fetal master regulator, could accelerate leukemogenesis. Surprisingly, Lin28b suppressed AML initiation rather than accelerating it. This may explain why MLL rearrangements often occur before birth in human infant leukemia patients, but transformation usually does not occur until after birth, when Lin28b levels decline. Our findings show that the efficiency of MLL-ENL-driven AML initiation changes through the course of pre- and postnatal development, and developmental programs can be manipulated to impede transformation.
Assuntos
Transformação Celular Neoplásica/genética , Predisposição Genética para Doença , Leucemia Mieloide Aguda/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Fusão Oncogênica/genética , Animais , Comorbidade , Feminino , Estudos de Associação Genética , Hemorragia/etiologia , Humanos , Leucemia Mieloide Aguda/complicações , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/epidemiologia , Masculino , Camundongos , Razão de ChancesRESUMO
Induction of fetal hemoglobin (HbF) via clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of DNA regulatory elements that repress γ-globin gene (HBG1 and HBG2) expression is a promising therapeutic strategy for sickle cell disease (SCD) and ß-thalassemia, although the optimal technical approaches and limiting toxicities are not yet fully defined. We disrupted an HBG1/HBG2 gene promoter motif that is bound by the transcriptional repressor BCL11A. Electroporation of Cas9 single guide RNA ribonucleoprotein complex into normal and SCD donor CD34+ hematopoietic stem and progenitor cells resulted in high frequencies of on-target mutations and the induction of HbF to potentially therapeutic levels in erythroid progeny generated in vitro and in vivo after transplantation of hematopoietic stem and progenitor cells into nonobese diabetic/severe combined immunodeficiency/Il2rγ-/-/KitW41/W41 immunodeficient mice. On-target editing did not impair CD34+ cell regeneration or differentiation into erythroid, T, B, or myeloid cell lineages at 16 to 17 weeks after xenotransplantation. No off-target mutations were detected by targeted sequencing of candidate sites identified by circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), an in vitro genome-scale method for detecting Cas9 activity. Engineered Cas9 containing 3 nuclear localization sequences edited human hematopoietic stem and progenitor cells more efficiently and consistently than conventional Cas9 with 2 nuclear localization sequences. Our studies provide novel and essential preclinical evidence supporting the safety, feasibility, and efficacy of a mechanism-based approach to induce HbF for treating hemoglobinopathies.