1/20/2020. To reduce HSPC toxicity associated with nuclease-induced DSBs, we evaluated the use of Cas9 D10A nickase (Cas9 D10A). Both cassettes were cloned in an AAV6 vector with a GFP reporter gene under the control of a constitutive promoter and flanked by homology arms to favor homologous DNA recombination (HDR)42  (Figure 2A). (E) Representative HPLC chromatograms of globin tetramer analysis in edited HUDEP-2 β0. Mobilized peripheral blood (MPB) and UCB CD34+ were also purchased from Cliniscience (Nanterre, France). In addition, we performed Sanger sequencing of PCR products spanning deletion junctions and observed that, whereas Cas9 left a composite pattern of InDels, Cas9 D10A deletions were seamless, suggesting absence of exonuclease processing (Figure 6I). γ4, Hb Bart; acHbF, acetylated fetal hemoglobin; HbF, fetal hemoglobin (α2γ2); α-p, α-precipitates; HbA, adult hemoglobin (α2β2). Download PDF Black lines represent mean; red line indicates the number of expected HBA2 alleles in untreated HSPCs. HPLC analysis was performed using a NexeraX2 SIL-30AC chromatograph (Shimadzu, Kyoto, Japan) and analyzed with LC Solution software. 7/19/2019. 6/19/2019. The authors thank Chiara Antoniani for the generation of HUDEP-2 β0 cells, Anna De Cian for Cas9 protein production, Samantha Scaramuzza for help with patients HSPCs, Fanny Collaud, Genethon “Vector Core Facility” for AAV production, Genethon “Imaging and Cytometry Core Facility” for image analysis and fluorescence-activated cell sorting, Genethon “Functional Evaluation Facility” for help with mice experimentation, and the Imagine “Genomic Platform” for amplicon sequencing. Reference range Units ... Coagulation normal ranges for neonates are based on gestational age and postnatal age. Glycated hemoglobin (glycohemoglobin, HbA1c, hemoglobin A1c, A1c, or less commonly HbA 1c, HgbA1c, Hb1c, etc.) To establish a correlation between α-globin expression and number of HBA genes, we generated multiple cell clones with mono- or biallelic HBA2 deletions (-α/αα and -α/-α, respectively, n = 3 per genotype) and we showed a significant amelioration of the α/β-like globin imbalance upon deletion of HBA2, with the -α/-α clones being indistinguishable from wild-type HUDEP-2 cells (Figure 1D; supplemental Figure 1D). (F) α/β-like globin mRNA ratios in edited thalassemic BFU-E. β0/β+colonies (n = 71) are plotted on the left axis, β0/β0 (n = 63) on the right axis. ns, not significant. Hijacking the endogenous HBA promoter for the expression of βAS3 transgene has 2 main advantages: (1) it does not require the genomic insertion of an exogenous promoter, which could result in transactivation of neighboring genes; and (2) compared with HBB gene correction strategies, it is a viable approach also for point mutations and deletions that inactivate regulatory elements in the HBB promoter.58. The Hemoglobin Evaluation Reflexive Cascade begins with HPLC analysis. Alpha Thalassemia HBA1 and HBA2 Seq: 2005805: Hemoglobin Cascade Interpretation: 50398-7: 2008793: Hemoglobin, Capillary Electrophoresis: 13514-5: 2010236: Beta Globin (HBB) Del/Dup Result: 2011645: Alpha Globin (HBA1 and HBA2) Del/Dup Rst: 3003512: Gamma Globin … If reflexed additional CPT codes may apply; refer to the reflexed test code for applicable codes. This test was performed in a CLIA certified laboratory and is intended for clinical purposes. Because HBA1 and HBA2 are similarly expressed,54  removal of 1 or 2 HBA2 genes should reduce α-globin expression to ∼75% to 50% of its normal level, thus falling in the therapeutic window to benefit β-thal.3  This α-globin reduction strategy could be used to treat moderate β+-thalassemia or to complement pharmacological treatment aimed at increasing expression of β-like globin in severe β+ or β0 patients.55-57  To treat β0-thalassemia, where there is no residual β-globin expression, we combined α-globin reduction with β-globin replacement. HSPCs from 1 patient of each genotype were used. Streptococcus pyogenes Cas9 protein (with 2 SV40 nuclear localization signals) was provided by J.P. Concordet and was expressed and purified as previously described.28 Streptococcus pyogenes Cas9 D10A protein (Alt-R S.p. This test assess the type and relative amounts of hemoglobin present in red blood cells. 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) β-globin expression, by targeted integration of a β-globin transgene downstream the HBA2 promoter. Globin mRNA analysis of single BFU-E colonies derived from β0- and β+-edited HSPCs showed that α/β imbalance was improved in all conditions, in accordance with the reduced number of HBA2 genes (supplemental Figure 4G) and with the stronger effect resulting from the concomitant α downregulation and βAS3 expression (Figure 5F). 11/20/2020. (I) Frequency of different InDel patterns in HBA2 deleted BFU-E treated with Cas9 (RNP, red; n = 28) or Cas9 nickase (RNP D10A, gray; n = 96). In the absence of β-globin, a portion of the α-globin pool complexes with β-like globins, such as γ- and δ-globins (to form HbF and HbA2, respectively), whereas the excess precipitates in insoluble aggregates (α-precipitates). Overall, these data show that we can modify β-thalassemia HSPCs and reduce their α/β globin balance, without affecting HSPCs potential. HbA is usually < 90%. Reduced amounts of detectable beta globin causes beta-plus-thalassemia. (E) InDel quantification at gRNA target site in edited HSPCs. Black lines indicate mean. Indicates test has been approved by the New York State Department of Health. Depending on findings, one or more reflexive tests may be required in order to provide a clinical interpretation. Negative controls were obtained by staining cells with isotype control antibodies. This analysis, together with our previous off-target analyses,37  confirms the target specificity of the selected gRNA HBA15 sequence. β-thalassemias (β-thal) are a group of blood disorders caused by mutations in the β-globin gene (HBB) cluster. The same gRNA that deletes the HBA2 gene will facilitate the integration of the β-globin transgene at this locus, whereas the endogenous HBA promoter will provide strong erythroid β expression, as previously suggested.38-40  To discriminate exogenous vs endogenous β-globin expression, we used an HBB transgene containing 3 antisickling point mutations (βAS3).21. Other names that describe the test. Primers and probe were optimized using the standard curve method to reach 100% ± 5% efficiency. To mimic these beneficial deletions with Streptococcus pyogenes (Sp)Cas9 nuclease, we designed a single gRNA that cuts both HBA1 and HBA2 alleles removing 1 HBA2 gene per chromosome. (D) α/β-like globin mRNA ratios in edited thalassemic erythroblasts at day 12. HSPC-derived erythroblasts were lysed in water and globin chains were separated using a 250 × 4.6-mm, 3.6-µm Aeris Widepore column (Phenomenex, Torrance, CA). performed experiments and analyzed data on Cas9 (D10A); E.C. Search for other works by this author on: The challenge of haemoglobinopathies in resource-poor countries, α-Globin as a molecular target in the treatment of β-thalassemia, Effect of alpha-gene numbers on phenotype of HbE/beta thalassemia patients, Coinheritance of the different copy numbers of alpha-globin gene modifies severity of beta-thalassemia/Hb E disease, The correlation of α-globin gene mutations and the XmnI polymorphism with clinical severity of Hb E/β-thalassemia, Management of iron overload in hemoglobinopathies, On modeling human leukocyte antigen-identical sibling match probability for allogeneic hematopoietic cell transplantation: estimating the need for an unrelated donor source, Gene therapy for hemoglobinopathies: the state of the field and the future, Outcomes after matched unrelated donor versus identical sibling hematopoietic cell transplantation in adults with acute myelogenous leukemia, Therapeutic options for patients with severe beta-thalassemia: the need for 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efficiently corrects the human sickle cell disease phenotype, Production, purification and characterization of adeno-associated vectors, Fast and reliable titration of recombinant adeno-associated virus type-2 using quantitative real-time PCR, Optimization of CRISPR/Cas9 delivery to human hematopoietic stem and progenitor cells for therapeutic genomic rearrangements, Establishment of immortalized human erythroid progenitor cell lines able to produce enucleated red blood cells, Growing and genetically manipulating human umbilical cord blood-derived erythroid progenitor (HUDEP) cell lines, Homology-directed repair in rodent zygotes using Cas9 and TALEN engineered proteins, Plerixafor and G-CSF combination mobilizes hematopoietic stem and progenitors cells with a distinct transcriptional profile and a reduced, Cellular function reinstitution of offspring red blood cells cloned from the sickle cell disease patient blood post CRISPR genome editing, Easy quantitative assessment of genome 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editing specificity [published correction appears in, In trans paired nicking triggers seamless genome editing without double-stranded DNA cutting, Single-strand nicks induce homologous recombination with less toxicity than double-strand breaks using an AAV vector template, The differences in quantities of alpha 2- and alpha 1-globin gene variants in heterozygotes, Hydroxyurea can eliminate transfusion requirements in children with severe beta-thalassemia, Alpha globin gene mutation: a major determinant of hydroxyurea response in transfusion-dependent HbE-β-thalassaemia, The emerging role of fetal hemoglobin induction in non-transfusion-dependent thalassemia, Correction of β654-thalassaemia mice using direct intravenous injection of siRNA and antisense RNA vectors, siRNA-mediated reduction of alpha-globin results in phenotypic improvements in beta-thalassemic cells, A double-switch vector system positively regulates transgene expression by endogenous microRNA expression (miR-ON 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CRISPR-Cas9 enrichment and long read nanopore sequencing, The co-inheritance of alpha-thalassemia and sickle cell anemia is associated with better hematological indices and lower consultations rate in Cameroonian patients and could improve their survival, The interaction of alpha-thalassemia and homozygous sickle-cell disease, NHLBI Trans-Omics for Precision Medicine (TOPMed) Consortium, Hematology & Hemostasis, Diabetes, and Structural Variation TOPMed Working Groups, Common α-globin variants modify hematologic and other clinical phenotypes in sickle cell trait and disease, Variability of homozygous sickle cell disease: the role of alpha and beta globin chain variation and other factors, Correction of a mouse model of sickle cell disease: lentiviral/antisickling beta-globin gene transduction of unmobilized, purified hematopoietic stem cells, © 2021 by The American Society of Hematology, Copyright ©2020 by American Society of Hematology, https://doi.org/10.1182/bloodadvances.2020001996, http://www.ncbi.nlm.nih.gov/bioproject/676022. Here, we propose to use the CRISPR/Cas9 system to combine these 2 strategies: (1) α-globin chain reduction by recreating the natural α-thalassemia trait (-α3.7 deletion, -α/αα or -α/-α) and (2) targeted integration and expression of a HBB transgene under the control of the endogenous HBA promoter. Single gRNA generating 2 in cis nicks could induce genomic deletion by strand displacement, possibly facilitated by the homology of HBA1 and 2 genes, whereas nick induced AAV integration can proceed via genome-AAV alignment, followed by Holiday junction resolution and completion of homologous recombination.53  Because we did not observe any difference between HBA15 and dual gRNA RNP D10A, we decided to continue on HSPCs using only HBA15. PCR amplification was then performed using 1 ng of double-stranded ligation product and Kapa Taq polymerase reagents (KAPA HiFi HotStart ReadyMix PCR Kit; Sigma-Aldrich). Peripheral blood was stained and red blood cells lysed during sample fixation (VersaLyse Lysing Solution and IOTest3 Fixative Solution; Beckman Coulter). 16 Full PDFs related to this paper. A specific ddPCR confirmed on-target integration at molecular level (∼0.8 βAS3 on-target copies per cell), in good agreement with GFP expression (∼95% GFP positive cells after sorting) (Figure 2C). It is difficult to determine whether they are carriers of silent mutations or high normal HbA2 without genetic test. Human albumin (ALB) or ZNF843 genes were used as reference for copy number evaluation (assay ID: dHsaCP2506312, BioRad). 83021. Requests for original data should be sent to Giulia Pavani (gpavani@genethon.fr). Ferritin is not required as a routine test. The remaining authors declare no competing financial interests. (H) Genotypes of BFU-E (n = 202) derived from RNP D10A + AAV HSPCs. 12/1/2019. G.P. The LV encoding the βAS3 gene under the control of the erythroid specific β-globin enhancer/promoter was already described.22. HBA2 copies (C) and KI efficiency (D) in edited HSPCs in erythroid liquid culture (●) or in BFU-E (■). Expression is regulated by 4 erythroid-specific enhancers (multispecies conserved elements [MCS-R]) located 10 to 50 kb upstream. To treat β0-thalassemia patients, it is essential to express β-like globin chains; however, reaching expression levels to balance the endogenous α-globin has proven very challenging.14  Therefore, we aim to lower the therapeutic threshold of β-globin expression by reducing α-globin abundance. HBA1 and HBA2 genes are located in the subtelomeric region of chromosome (Chr) 16. Each dot represents a single colony. (A-B) HBA2 copies (A) and KI efficiency (B) in Cas9 (RNP) or Cas9 nickase (RNP D10A) edited HSPCs in erythroid liquid culture. Refrigerated. Overall, these results demonstrate that KI of βAS3 full cassette under the endogenous HBA promoter can restore HbA expression in β0 thalassemic cells. Importantly, our HBA2 deletion strategy reduced but did not abolish α-globin production, as observed in α-globin KO control cells obtained with a gRNA targeting the coding sequence within the first exon of HBA1 and HBA2 genes (Figure 1D). Normal range/expected value(s) for a specific disease state. In the newborn period, if the electrophoresis shows Hb Bart's or HbH, the … DNA testing is only available in specialised laboratories. The lower number of DSBs could also explain the reduced toxicity observed in CFC edited with Cas9 D10A (Figure 6F-G). MPB- or UCB-derived HSPCs were thawed and cultured in prestimulation media for 48 hours (StemSpan, Stemregenin-1 0.75 μM, UM171 0.35 μM; StemCell Technologies, Vancouver, BC, Canada; rhSCF 300 ng/mL, rhFlt3-L 300 ng/mL, rhTPO 100 ng/mL, and rhIL-3 20 ng/mL; CellGenix, Freiburg, Germany). Black lines represent mean. The codes reflect our interpretation of CPT coding requirements based upon AMA guidelines published annually. LV were titrated in HCT116 cells and HIV-1 Gag p24 content was measured by enzyme-linked immunosorbent assay (PerkinElmer, Waltham, MA) according to the manufacturer’s instructions. Tests added may include electrophoresis, solubility testing, mutational analysis and/or sequencing.Quantitation of hemoglobin by HPLC or electrophoresis is most definitive in individuals one year of age and older. equally contributed to this work. By genotyping single BFU-E colonies, we observed that most of RNP D10A-edited BFU-E had 1 HBA2 deletion, followed by 1 HBA2 deletion and 1 βAS3 KI (Figure 6H; supplemental Figure 6A-B). This is in sharp contrast with α-globin KO cells, where the predominant hemoglobin observed was the toxic HbBart (γ-globin tetramers), typical of severe forms of α-thalassemia (Figure 1E). (A) Schematic representation of engraftment experiments. Haemoglobin is a … A faculty hematopathologist personally directs and interprets each stage of testing to completion. Primer and probes for PCRs were purchased from Sigma-Aldrich (St Louis, MO) and Integrated DNA Technologies (Coralville, IA). As positive control, HSPCs were transduced with a LV encoding for a βAS3 transgene under the control of the β-globin gene promoter and its mini-locus control region, currently in clinical trial for thalassemia (LV βAS3).13,48. May also include abnormal ranges. non-enzymatically) bond with hemoglobin, when present in the bloodstream of humans.However, glucose is less likely to do so … May also include abnormal ranges. Correspondence: Mario Amendola, INTEGRARE, Genethon, UMR S951 INSERM, University Evry, University Paris-Saclay, 1 bis, Rue de I’Internationale, 91000 Evry, France; e-mail: mamendola@genethon.fr. Black lines indicate mean; gray line shows the range of α/β-like globin ratio in healthy donor MPB and UCB HSPCs (n = 5). DNA analysis should be offered to identify and confirm couples at risk, in prenatal testing and in pre-implantation genetic diagnosis [ 1 ] . In addition, the investigational test showed a statistically higher overall percent agreement with each of the t3 reference methods … Human umbilical cord blood (UCB) samples were provided by Centre Hospitalier Sud-Francilien (CHSF; Evry, France) and processed according to France bioethics laws (Declaration DC-2012-1655 to the French Ministry of Higher Education and Research). and AFM-Telethon, INSERM, Genopole Chaire Fondagen and the Agence Nationale de la Recherche (ANR-16-CE18 STaHR) (M.A.). Single BFU-E genotyping showed that the majority of colonies had HBA2 deletion (Figure 3G) and βAS3 KI (Figure 3H) and 41% of them harbored both modifications (Figure 3I; supplemental Figure 2F). After checking the correct amplification by Sanger sequencing (Genewiz), amplicons were purified using NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Hoerdt, France), and 500 ng of DNA were used for library preparation. This condition is caused by changes to the genes for haemoglobin. HSPCs were transfected with RNP or RNP D10A and then transduced with the AAV6 βAS3 donor DNA. Separate specimens must be submitted when multiple tests are ordered. A total of 2.5 × 105 hematopoietic stem/progenitor cells (HSPCs) per condition were transfected with ribonucleoprotein (RNP) using a P3 Primary Cell 4D-Nucleofector Kit (CA137 program).25  In knock-in experiments, 10 minutes after transfection, HSPCs were transduced with AAV6 for 6 hours (multiplicity of infection, 10 000-30 000), washed, and left in prestimulation media for additional 24 to 48 hours. (D) βAS3 transcript upregulation in targeted HUDEP-2 β0 upon erythroid differentiation (qPCR, n = 2, mean ± SD). Thalassaemia is the most common inherited blood condition in the world. By sequencing the tumor and e.g. 9/19/2019. Remarkably, the expression of 1 βAS3 transgene was sufficient to reestablish almost one-half of normal hemoglobin level in HUDEP-2 β0. A total of 200 ng of genomic DNA of RNP-edited HSPCs were used to amplify top 3 off-targets identified with Guide-Seq using Phusion High-Fidelity polymerase (New England Biolabs). Colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte; Bars represent mean ± SD (n = 2-4). (F) Relative abundance of endogenous β and KI-βAS3 mRNA at day 12 of erythroid liquid culture (n = 6, mean ± SD). This test was developed and its performance characteristics determined by ARUP Laboratories. Using genome editing tools, Mettananda et al elegantly demonstrated the possibility of reducing α-globin expression in HbE patients’ cells by deleting a powerful α-globin enhancer.20  Although promising, this modification is beneficial only for β+ patients and requires plasmid transfection combined with GFP-sorting enrichment, a strategy that is not suited for clinical translation.25  Finally, the need for 2 gRNAs to perform enhancer deletion increases the risk of on- and off-target side effects and can be technically challenging. This result clearly indicates that only a controlled reduction of α-globin results in a beneficial effect. Synonyms. To quantify HBA2 copy number, primers and probes were designed on the 3′UTR of HBA2 gene because it differs significantly from HBA1. In particular, we tested the HBA2 deletion approach in β+ cells and the combination of the α-deletion and βAS3 KI strategy in β+- and β0-thalassemic HSPCs (supplemental Figure 4A-B). In this study, we demonstrated the possibility of correcting α/β globin imbalance in β-thalassemia cells using the CRISPR/Cas9 system. The normal adult hemoglobin tetramer consists of two alpha chains and two beta chains. We designed 2 different cassettes for the expression of HBB (gene ID: 3043): (1) the first cassette contains a β-globin complementary DNA (cDNA), with 3 antisickling point mutations (βAS3),21  followed by a woodchuck posttranscriptional regulatory element and a SV40polyA; and (2) the second cassette contains the whole βAS3 gene with introns and its original 3′ untranslated region (UTR) and polyA (pA) site, with 3 antisickling point mutations. Mutant beta globin causes sickle cell anemia. EQAS Online is also accessible from any internet connected Smartphone. Of note, modified HSPCs retained proper erythroid differentiation (supplemental Figure 4D) and multilineage potential (CFC assay; supplemental Figure 4F), although we observed some toxicity associated with the editing procedure (supplemental Figure 4E).
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