One-Step Biallelic and Scarless Correction of a ?-Thalassemia Mutation in Patient-Specific iPSCs without Drug Selection.
ABSTRACT: Monogenic disorders (MGDs), which are caused by single gene mutations, have a serious effect on human health. Among these, ?-thalassemia (?-thal) represents one of the most common hereditary hematological diseases caused by mutations in the human hemoglobin ? (HBB) gene. The technologies of induced pluripotent stem cells (iPSCs) and genetic correction provide insights into the treatments for MGDs, including ?-thal. However, traditional approaches for correcting mutations have a low efficiency and leave a residual footprint, which leads to some safety concerns in clinical applications. As a proof of concept, we utilized single-strand oligodeoxynucleotides (ssODNs), high-fidelity CRISPR/Cas9 nuclease, and small molecules to achieve a seamless correction of the ?-41/42 (TCTT) deletion mutation in ? thalassemia patient-specific iPSCs with remarkable efficiency. Additionally, off-target analysis and whole-exome sequencing results revealed that corrected cells exhibited a minimal mutational load and no off-target mutagenesis. When differentiated into hematopoietic progenitor cells (HPCs) and then further to erythroblasts, the genetically corrected cells expressed normal ?-globin transcripts. Our studies provide the most efficient and safe approach for the genetic correction of the ?-41/42 (TCTT) deletion in iPSCs for further potential cell therapy of ?-thal, which represents a potential therapeutic avenue for the gene correction of MGD-associated mutants in patient-specific iPSCs.
Project description:?-Thalassemia (?-Thal) is one of the most common genetic diseases in the world. The generation of patient-specific ?-Thal-induced pluripotent stem cells (iPSCs), correction of the disease-causing mutations in those cells, and then differentiation into hematopoietic stem cells offers a new therapeutic strategy for this disease. Here, we designed a CRISPR/Cas9 to specifically target the Homo sapiens hemoglobin ? (HBB) gene CD41/42(-CTTT) mutation. We demonstrated that the combination of single strand oligodeoxynucleotides with CRISPR/Cas9 was capable of correcting the HBB gene CD41/42 mutation in ?-Thal iPSCs. After applying a correction-specific PCR assay to purify the corrected clones followed by sequencing to confirm mutation correction, we verified that the purified clones retained full pluripotency and exhibited normal karyotyping. Additionally, whole-exome sequencing showed that the mutation load to the exomes was minimal after CRISPR/Cas9 targeting. Furthermore, the corrected iPSCs were selected for erythroblast differentiation and restored the expression of HBB protein compared with the parental iPSCs. This method provides an efficient and safe strategy to correct the HBB gene mutation in ?-Thal iPSCs.
Project description:Beta-thalassemia is one of the most common recessive genetic diseases, caused by mutations in the HBB gene. Over 200 different types of mutations in the HBB gene containing three exons have been identified in patients with ?-thalassemia (?-thal) whereas a homozygous mutation in exon 1 causes sickle cell disease (SCD). Novel therapeutic strategies to permanently correct the HBB mutation in stem cells that are able to expand and differentiate into erythrocytes producing corrected HBB proteins are highly desirable. Genome editing aided by CRISPR/Cas9 and other site-specific engineered nucleases offers promise to precisely correct a genetic mutation in the native genome without alterations in other parts of the human genome. Although making a sequence-specific nuclease to enhance correction of a specific HBB mutation by homology-directed repair (HDR) is becoming straightforward, targeting various HBB mutations of ?-thal is still challenging because individual guide RNA as well as a donor DNA template for HDR of each type of HBB gene mutation have to be selected and validated. Using human induced pluripotent stem cells (iPSCs) from two ?-thal patients with different HBB gene mutations, we devised and tested a universal strategy to achieve targeted insertion of the HBB cDNA in exon 1 of HBB gene using Cas9 and two validated guide RNAs. We observed that HBB protein production was restored in erythrocytes derived from iPSCs of two patients. This strategy of restoring functional HBB gene expression will be able to correct most types of HBB gene mutations in ?-thal and SCD. Stem Cells Translational Medicine 2018;7:87-97.
Project description:The generation of personalized induced pluripotent stem cells (iPSCs) followed by targeted genome editing provides an opportunity for developing customized effective cellular therapies for genetic disorders. However, it is critical to ascertain whether edited iPSCs harbor unfavorable genomic variations before their clinical application. To examine the mutation status of the edited iPSC genome and trace the origin of possible mutations at different steps, we have generated virus-free iPSCs from amniotic cells carrying homozygous point mutations in ?-hemoglobin gene (HBB) that cause severe ?-thalassemia (?-Thal), corrected the mutations in both HBB alleles by zinc finger nuclease-aided gene targeting, and obtained the final HBB gene-corrected iPSCs by excising the exogenous drug resistance gene with Cre recombinase. Through comparative genomic hybridization and whole-exome sequencing, we uncovered seven copy number variations, five small insertions/deletions, and 64 single nucleotide variations (SNVs) in ?-Thal iPSCs before the gene targeting step and found a single small copy number variation, 19 insertions/deletions, and 340 single nucleotide variations in the final gene-corrected ?-Thal iPSCs. Our data revealed that substantial but different genomic variations occurred at factor-induced somatic cell reprogramming and zinc finger nuclease-aided gene targeting steps, suggesting that stringent genomic monitoring and selection are needed both at the time of iPSC derivation and after gene targeting.
Project description:?-thalassemias (?-thal) are a group of blood disorders caused by mutations in the ?-globin gene (HBB) cluster. ?-globin associates with ?-globin to form adult hemoglobin (HbA, ?2?2), the main oxygen-carrier in erythrocytes. When ?-globin chains are absent or limiting, free ?-globins precipitate and damage cell membranes, causing hemolysis and ineffective erythropoiesis. Clinical data show that severity of ?-thal correlates with the number of inherited ?-globin genes (HBA1 and HBA2), with ?-globin gene deletions having a beneficial effect for patients. Here, we describe a novel strategy to treat ?-thal based on genome editing of the ?-globin locus in human hematopoietic stem/progenitor cells (HSPCs). Using CRISPR/Cas9, we combined 2 therapeutic approaches: (1) ?-globin downregulation, by deleting the HBA2 gene to recreate an ?-thalassemia trait, and (2) ?-globin expression, by targeted integration of a ?-globin transgene downstream the HBA2 promoter. First, we optimized the CRISPR/Cas9 strategy and corrected the pathological phenotype in a cellular model of ?-thalassemia (human erythroid progenitor cell [HUDEP-2] ?0). 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 ?-thal HSPCs and achieved correction of ?/? 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 ?/? globin imbalance in thalassemic HSPCs, paving the way for novel therapeutic strategies for ?-thal.
Project description:<h4>Background</h4>Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. We now report genetic correction of the beta hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for hemoglobin E and ?-thalassemia (HbE/?-thalassemia), the most common thalassemia syndrome in Thailand and Southeast Asia.<h4>Methods</h4>We used the CRISPR/Cas9 system to target the hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a single-stranded DNA oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and hemoglobin expression.<h4>Results</h4>The hemoglobin E mutation of HbE/?-thalassemia iPSCs was seamlessly corrected by the CRISPR/Cas9 system. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein.<h4>Conclusions</h4>Our study provides a strategy to correct hemoglobin E mutation in one step and these corrected iPSCs can be differentiated into hematopoietic stem cells to be used for autologous transplantation in patients with HbE/?-thalassemia in the future.
Project description:The therapeutic use of patient-specific induced pluripotent stem cells (iPSCs) is emerging as a potential treatment of ?-thalassemia. Ideally, patient-specific iPSCs would be genetically corrected by various approaches to treat ?-thalassemia including lentiviral gene transfer, lentivirus-delivered shRNA, and gene editing. These corrected iPSCs would be subsequently differentiated into hematopoietic stem cells and transplanted back into the same patient. In this article, we present a proof of principle study for disease modeling and screening using iPSCs to test the potential use of the modified U7 small nuclear (sn) RNA to correct a splice defect in IVS2-654 ?-thalassemia. In this case, the aberration results from a mutation in the human ?-globin intron 2 causing an aberrant splicing of ?-globin pre-mRNA and preventing synthesis of functional ?-globin protein. The iPSCs (derived from mesenchymal stromal cells from a patient with IVS2-654 ?-thalassemia/hemoglobin (Hb) E) were transduced with a lentivirus carrying a modified U7 snRNA targeting an IVS2-654 ?-globin pre-mRNA in order to restore the correct splicing. Erythroblasts differentiated from the transduced iPSCs expressed high level of correctly spliced ?-globin mRNA suggesting that the modified U7 snRNA was expressed and mediated splicing correction of IVS2-654 ?-globin pre-mRNA in these cells. Moreover, a less active apoptosis cascade process was observed in the corrected cells at transcription level. This study demonstrated the potential use of a genetically modified U7 snRNA with patient-specific iPSCs for the partial restoration of the aberrant splicing process of ?-thalassemia. Stem Cells Translational Medicine 2017;6:1059-1069.
Project description:<h4>Background & aims</h4>Wilson's disease (WD) is an autosomal recessive disorder of copper metabolism caused by loss-of-function mutations in <i>ATP7B</i>, which encodes a copper-transporting protein. It is characterized by excessive copper deposition in tissues, predominantly in the liver and brain. We sought to investigate whether gene-corrected patient-specific induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps) could serve as an autologous cell source for cellular transplantation therapy in WD.<h4>Methods</h4>We first compared the <i>in vitro</i> phenotype and cellular function of ATP7B before and after gene correction using CRISPR/Cas9 and single-stranded oligodeoxynucleotides (ssODNs) in iHeps (derived from patients with WD) which were homozygous for the ATP7B R778L mutation (ATP7B<sup>R778L/R778L</sup>). Next, we evaluated the <i>in vivo</i> therapeutic potential of cellular transplantation of WD gene-corrected iHeps in an immunodeficient WD mouse model (<i>Atp7b</i> <sup><i>-/-</i></sup> <i>/ Rag2</i> <sup><i>-/-</i></sup> <i>/ Il2rg</i> <sup><i>-/-</i></sup> ; ARG).<h4>Results</h4>We successfully created iPSCs with heterozygous gene correction carrying 1 allele of the wild-type <i>ATP7B</i> gene (ATP7B<sup>WT/-</sup>) using CRISPR/Cas9 and ssODNs. Compared with ATP7B<sup>R778L/R778L</sup> iHeps, gene-corrected ATP7B<sup>WT/-</sup> iHeps restored <i>i</i> <i>n vitro</i> ATP7B subcellular localization, its subcellular trafficking in response to copper overload and its copper exportation function. Moreover, <i>in vivo</i> cellular transplantation of ATP7B<sup>WT/-</sup> iHeps into ARG mice via intra-splenic injection significantly attenuated the hepatic manifestations of WD. Liver function improved and liver fibrosis decreased due to reductions in hepatic copper accumulation and consequently copper-induced hepatocyte toxicity.<h4>Conclusions</h4>Our findings demonstrate that gene-corrected patient-specific iPSC-derived iHeps can rescue the <i>in vitro</i> and <i>in vivo</i> disease phenotypes of WD. These proof-of-principle data suggest that iHeps derived from gene-corrected WD iPSCs have potential use as an autologous <i>ex vivo</i> cell source for <i>in vivo</i> therapy of WD as well as other inherited liver disorders.<h4>Lay summary</h4>Gene correction restored ATP7B function in hepatocytes derived from induced pluripotent stem cells that originated from a patient with Wilson's disease. These gene-corrected hepatocytes are potential cell sources for autologous cell therapy in patients with Wilson's disease.
Project description:Thalassemia has been recognized by the World Health Organization as important inherited disorders principally impacting on the populations of low income countries. In this report, the prevalence of common ?-thalassemia mutations in India was defined in 126 ?-thalassemia carrier subjects in a western Indian population mainly from the south-western Maharashtra. The six most common ?-thalassemia mutations were detected, which included IVS I-5 (G-C), IVS I-1 (G-T), codon 8-9 (+G), codon 41/42 (-TCTT), Codon 15 (G-A), and 619 bp deletion at 3' end of ?-globin gene. These mutations accounted for 93.66 % in 126 ?-thalassemia carrier subjects and 6.34 % remained uncharacterized. Out of 126, 82 (65.07 %) showed the most common (prevalent) type of mutation, IVS I-5 (G-C), followed by IVS I-1 (G-T) showed by 12 (9.52 %) subjects. Three (2.38 %) subjects showed 619 bp deletion, codon 8/9 (+G) and codon 15 (G-A) mutations were present in eight subjects each (6.34 %). Only five (3.96 %) subjects showed codon 41/42 (-TCTT). There were eight (6.34 %) subjects where mutation was not any of the six mutations studied. This study provides the pattern of ? thalassemia mutations from south-western Maharashtra, which will help to prevent ?-thalassemia using prenatal diagnosis and proper counseling.
Project description:A lentiviral vector encoding ?-globin flanked by insulator elements has been used to treat ?-thalassemia (?-Thal) successfully in one human subject. However, a clonal expansion was observed after integration in the HMGA2 locus, raising the question of how commonly lentiviral integration would be associated with possible insertional activation. Here, we report correcting ?-Thal in a murine model using the same vector and a busulfan-conditioning regimen, allowing us to investigate efficacy and clonal evolution at 9.2 months after transplantation of bone marrow cells. The five gene-corrected recipient mice showed near normal levels of hemoglobin, reduced accumulation of reticulocytes, and normalization of spleen weights. Mapping of integration sites pretransplantation showed the expected favored integration in transcription units. The numbers of gene-corrected long-term repopulating cells deduced from the numbers of unique integrants indicated oligoclonal reconstitution. Clonal abundance was quantified using a Mu transposon-mediated method, indicating that clones with integration sites near growth-control genes were not enriched during growth. No integration sites involving HMGA2 were detected. Cells containing integration sites in genes became less common after prolonged growth, suggesting negative selection. Thus, ?-Thal gene correction in mice can be achieved without expansion of cells harboring vectors integrated near genes involved in growth control.
Project description:OBJECTIVE:The aim of the study was to explore genotype distribution thalassemia and G6PD deficiency in Meizhou city, China. METHODS:A total of 16 158 individuals were involved in thalassemia genetic testing. A total of 605 subjects were screened for common Chinese G6PD mutations by gene chip analysis. Genotypes and allele frequencies were analyzed. RESULTS:A total of 5463 cases carried thalassemia mutations were identified, including 3585 cases, 1701 cases, and 177 cases with ?-, ?-, and ? + ?-thalassemia mutations, respectively. --SEA (65.12%), -?3.7 (19.05%), and -?4.2 (8.05%) deletion were the main mutations of ?-thalassemia, while IVS-II-654(C ? T) (40.39%), CD41-42(-TCTT) (32.72%), -28(A ? G) (10.11%), and CD17(A ? T) (9.32%) mutations were the principal mutations of ?-thalassemia in Meizhou. There were significant differences in allele frequencies in some counties. Genetic testing for G6PD deficiency, six mutation sites, and one polymorphism were detected in our study. A total of 198 alleles with the mutation were detected among 805 alleles (24.6%). G6PD Canton (c.1376 G ? T) (45.96%), G6PD Kaiping (c.1388 G ? A) (39.39%), and G6PD Gaohe (c.95 A ? G) (9.09%) account for 94.44% mutations, followed by G6PD Chinese-5 (c.1024 C ? T) (4.04%), G6PD Viangchan (c.871G ? A) (1.01%), and G6PD Maewo (c.1360 C ? T) (0.51%). There were some differences of the distribution of G6PD mutations among eight counties in Meizhou. CONCLUSIONS:The --SEA , -?3.7 , and -?4.2 deletion were the main mutations of ?-thalassemia, while IVS-II-654(C ? T), CD41-42(-TCTT), -28(A ? G), and CD17(A ? T) mutations were the principal mutations of ?-thalassemia in Meizhou. G6PD c.1376 G ? T, c.1388 G ? A, and c.95 A ? G were the main mutations of G6PD deficiency. There were some differences of the distribution of thalassemia and G6PD mutations among eight counties in Meizhou.