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1.
Blood Adv ; 5(5): 1137-1153, 2021 03 09.
Article in English | MEDLINE | ID: mdl-33635334

ABSTRACT

ß-thalassemias (ß-thal) are a group of blood disorders caused by mutations in the ß-globin gene (HBB) cluster. ß-globin associates with α-globin to form adult hemoglobin (HbA, α2ß2), the main oxygen-carrier in erythrocytes. When ß-globin chains are absent or limiting, free α-globins precipitate and damage cell membranes, causing hemolysis and ineffective erythropoiesis. Clinical data show that severity of ß-thal correlates with the number of inherited α-globin genes (HBA1 and HBA2), with α-globin gene deletions having a beneficial effect for patients. Here, we describe a novel strategy to treat ß-thal based on genome editing of the α-globin locus in human hematopoietic stem/progenitor cells (HSPCs). Using CRISPR/Cas9, we combined 2 therapeutic approaches: (1) α-globin downregulation, by deleting the HBA2 gene to recreate an α-thalassemia trait, and (2) ß-globin expression, by targeted integration of a ß-globin transgene downstream the HBA2 promoter. First, we optimized the CRISPR/Cas9 strategy and corrected the pathological phenotype in a cellular model of ß-thalassemia (human erythroid progenitor cell [HUDEP-2] ß0). Then, we edited healthy donor HSPCs and demonstrated that they maintained long-term repopulation capacity and multipotency in xenotransplanted mice. To assess the clinical potential of this approach, we next edited ß-thal HSPCs and achieved correction of α/ß globin imbalance in HSPC-derived erythroblasts. As a safer option for clinical translation, we performed editing in HSPCs using Cas9 nickase showing precise editing with no InDels. Overall, we described an innovative CRISPR/Cas9 approach to improve α/ß globin imbalance in thalassemic HSPCs, paving the way for novel therapeutic strategies for ß-thal.


Subject(s)
beta-Thalassemia , Animals , CRISPR-Cas Systems , Hematopoietic Stem Cells/metabolism , Humans , Mice , alpha-Globins/genetics , beta-Globins/genetics , beta-Thalassemia/genetics , beta-Thalassemia/therapy
2.
Nat Commun ; 11(1): 4287, 2020 08 27.
Article in English | MEDLINE | ID: mdl-32855419

ABSTRACT

Warsaw Breakage Syndrome (WABS) is a rare disorder related to cohesinopathies and Fanconi anemia, caused by bi-allelic mutations in DDX11. Here, we report multiple compound heterozygous WABS cases, each displaying destabilized DDX11 protein and residual DDX11 function at the cellular level. Patient-derived cell lines exhibit sensitivity to topoisomerase and PARP inhibitors, defective sister chromatid cohesion and reduced DNA replication fork speed. Deleting DDX11 in RPE1-TERT cells inhibits proliferation and survival in a TP53-dependent manner and causes chromosome breaks and cohesion defects, independent of the expressed pseudogene DDX12p. Importantly, G-quadruplex (G4) stabilizing compounds induce chromosome breaks and cohesion defects which are strongly aggravated by inactivation of DDX11 but not FANCJ. The DNA helicase domain of DDX11 is essential for sister chromatid cohesion and resistance to G4 stabilizers. We propose that DDX11 is a DNA helicase protecting against G4 induced double-stranded breaks and concomitant loss of cohesion, possibly at DNA replication forks.


Subject(s)
Abnormalities, Multiple/etiology , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , DNA Helicases/genetics , DNA Helicases/metabolism , G-Quadruplexes , Sister Chromatid Exchange , Abnormalities, Multiple/genetics , Abnormalities, Multiple/pathology , Cell Proliferation , DEAD-box RNA Helicases/chemistry , DNA Helicases/chemistry , Fanconi Anemia Complementation Group Proteins/genetics , Fanconi Anemia Complementation Group Proteins/metabolism , Humans , Male , Middle Aged , Mutation, Missense , Protein Stability , Pseudogenes , RNA Helicases/genetics , RNA Helicases/metabolism , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Syndrome , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism
3.
Nat Commun ; 11(1): 4146, 2020 08 13.
Article in English | MEDLINE | ID: mdl-32792546

ABSTRACT

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

4.
Nat Commun ; 11(1): 3778, 2020 07 29.
Article in English | MEDLINE | ID: mdl-32728076

ABSTRACT

Targeted genome editing has a great therapeutic potential to treat disorders that require protein replacement therapy. To develop a platform independent of specific patient mutations, therapeutic transgenes can be inserted in a safe and highly transcribed locus to maximize protein expression. Here, we describe an ex vivo editing approach to achieve efficient gene targeting in human hematopoietic stem/progenitor cells (HSPCs) and robust expression of clinically relevant proteins by the erythroid lineage. Using CRISPR-Cas9, we integrate different transgenes under the transcriptional control of the endogenous α-globin promoter, recapitulating its high and erythroid-specific expression. Erythroblasts derived from targeted HSPCs secrete different therapeutic proteins, which retain enzymatic activity and cross-correct patients' cells. Moreover, modified HSPCs maintain long-term repopulation and multilineage differentiation potential in transplanted mice. Overall, we establish a safe and versatile CRISPR-Cas9-based HSPC platform for different therapeutic applications, including hemophilia and inherited metabolic disorders.


Subject(s)
Cell Engineering/methods , Gene Editing , Hematopoietic Stem Cell Transplantation/methods , Hematopoietic Stem Cells/metabolism , Animals , CRISPR-Cas Systems/genetics , Cell Line , Female , Gene Expression Regulation , Hemophilia A/therapy , Humans , Metabolic Diseases/therapy , Mice , Promoter Regions, Genetic/genetics , Transplantation, Autologous/methods , Transplantation, Heterologous , alpha-Globins/genetics , alpha-Globins/metabolism
5.
Gastroenterology ; 147(5): 1064-72.e5, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25088490

ABSTRACT

BACKGROUND & AIMS: Lynch syndrome, a nonpolyposis form of hereditary colorectal cancer, is caused by inherited defects in DNA mismatch repair (MMR) genes. Most patients carry a germline mutation in 1 allele of the MMR genes MSH2 or MLH1. With spontaneous loss of the wild-type allele, cells with defects in MMR exist among MMR-proficient cells, as observed in healthy intestinal tissues from patients with Lynch syndrome. We aimed to create a mouse model of this situation to aid in identification of environmental factors that affect MMR-defective cells and their propensity for oncogenic transformation. METHODS: We created mice in which the MMR gene Msh2 can be inactivated in a defined fraction of crypt base columnar stem cells to generate MSH2-deficient intestinal crypts among an excess of wild-type crypts (Lgr5-CreERT2;Msh2(flox/-) mice). Intestinal tissues were collected; immunohistochemical analyses were performed for MSH2, along with allele-specific PCR assays. We traced the fate of MSH2-deficient crypts under the influence of different external factors. RESULTS: Lgr5-CreERT2;Msh2(flox/-) mice developed more adenomas and adenocarcinomas than control mice; all tumors were MSH2 deficient. Exposure of Lgr5-CreERT2;Msh2(flox/-) mice to the methylating agent temozolomide caused MSH2-deficient intestinal stem cells to proliferate more rapidly than wild-type stem cells. The MSH2-deficient intestinal stem cells were able to colonize the intestinal epithelium and many underwent oncogenic transformation, forming intestinal neoplasias. CONCLUSIONS: We developed a mouse model of Lynch syndrome (Lgr5-CreERT2;Msh2(flox/-) mice) and found that environmental factors can modify the number and mutability of the MMR-deficient stem cells. These findings provide evidence that environmental factors can promote development of neoplasias and tumors in patients with Lynch syndrome.


Subject(s)
Adenocarcinoma/chemically induced , Adenocarcinoma/genetics , Adenoma/chemically induced , Adenoma/genetics , Colorectal Neoplasms, Hereditary Nonpolyposis/chemically induced , Colorectal Neoplasms, Hereditary Nonpolyposis/genetics , DNA Mismatch Repair/drug effects , Dacarbazine/analogs & derivatives , Intestines/drug effects , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Adenoma/metabolism , Adenoma/pathology , Animals , Cell Proliferation/drug effects , Cell Transformation, Neoplastic/chemically induced , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Colorectal Neoplasms, Hereditary Nonpolyposis/metabolism , Colorectal Neoplasms, Hereditary Nonpolyposis/pathology , Dacarbazine/toxicity , Disease Models, Animal , Female , Intestinal Mucosa/metabolism , Intestines/pathology , Male , Mice, Knockout , MutS Homolog 2 Protein/deficiency , MutS Homolog 2 Protein/genetics , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Risk Factors , Temozolomide , Time Factors
6.
Mol Imaging Biol ; 15(2): 194-202, 2013 Apr.
Article in English | MEDLINE | ID: mdl-22777578

ABSTRACT

PURPOSE: TH-MYCN transgenic mice represent a valuable preclinical model of neuroblastoma. Current methods to study tumor progression in these mice are inaccurate or invasive, limiting the potential of this murine model. The aim of our study was to assess the potential of small animal positron emission tomography (SA-PET) to study neuroblastoma progression in TH-MYCN mice. PROCEDURE: Serial SA-PET scans using the tracer 2-deoxy-2-[(18)F]fluoro-D-glucose ((18)F-FDG) have been performed in TH-MYCN mice. Image analysis of tumor progression has been compared with ex vivo evaluation of tumor volumes and histological features. RESULTS: [(18)F]FDG-SA-PET allowed to detect early staged tumors in almost 100 % of TH-MYCN mice positive for disease. Image analysis of tumor evolution reflected the modifications of the tumor volume, histological features, and malignancy during disease progression. Image analysis of TH-MYCN mice undergoing chemotherapy treatment against neuroblastoma provided information on drug-induced alterations in tumor metabolic activity. CONCLUSIONS: These data show for the first time that [(18)F]FDG-SA-PET is a useful tool to study neuroblastoma presence and progression in TH-MYCN transgenic mice.


Subject(s)
Molecular Imaging/methods , Neuroblastoma/diagnostic imaging , Positron-Emission Tomography/methods , Proto-Oncogene Proteins/genetics , Animals , Antineoplastic Agents/therapeutic use , Disease Models, Animal , Disease Progression , Fluorodeoxyglucose F18 , Histocytochemistry , Linear Models , Mice , Mice, Transgenic , N-Myc Proto-Oncogene Protein , Neuroblastoma/drug therapy , Neuroblastoma/genetics , Neuroblastoma/pathology , Statistics, Nonparametric
7.
Mutat Res ; 715(1-2): 52-60, 2011 Oct 01.
Article in English | MEDLINE | ID: mdl-21801734

ABSTRACT

Short synthetic single-stranded oligodeoxyribonucleotides (ssODNs) can be used to introduce subtle modifications into the genome of mouse embryonic stem cells (ESCs). We have previously shown that effective application of ssODN-mediated gene targeting in ESC requires (transient) suppression of DNA mismatch repair (MMR). However, whereas transient down-regulation of the mismatch recognition protein MSH2 allowed substitution of 3 or 4 nucleotides, 1 or 2 nucleotide substitutions were still suppressed. We now demonstrate that single- or dinucleotide substitution can effectively be achieved by transient down-regulation of the downstream MMR protein MLH1. By exploiting highly specific real-time PCR, we demonstrate the feasibility of substituting a single basepair in a non-selectable gene. However, disabling the MMR machinery may lead to inadvertent mutations. To obtain insight into the mutation rate associated with transient MMR suppression, we have compared the impact of transient and constitutive MMR deficiency on the repair of frameshift intermediates at mono- and dinucleotide repeats. Repair at these repeats relied on the substrate specificity and functional redundancy of the MSH2/MSH6 and MSH2/MSH3 MMR complexes. MLH1 knockdown increased the level of spontaneous mutagenesis, but modified ESCs remained germ line competent. Thus, transient MLH1 suppression provides a valuable extension of the MSH2 knockdown strategy, allowing rapid generation of mice carrying single basepair alterations in their genome.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , DNA Mismatch Repair , DNA, Single-Stranded , Nuclear Proteins/metabolism , Oligonucleotides , Animals , Cell Line , Dinucleotide Repeats , Down-Regulation , Embryonic Stem Cells , Frameshift Mutation , Gene Knockdown Techniques , Gene Targeting , Mice , MutL Protein Homolog 1 , Mutagenesis
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