ARUP Consult® assists with test selection and interpretation. READ PAPER. provided scientific advice and financial support; G.F. provided β-thalassemia HSPCs; A.M. provided HUDEP-2 β0 cells, performed and analyzed HPLC, and edited the manuscript; and M.A. (B) Editing efficiency in HUDEP-2 cells is expressed as percentage of modified HBA alleles. This condition is caused by changes to the genes for haemoglobin. InDels (A), HBA2 copies (B), and βAS3 integrated copies (C) quantification in edited thalassemic HSPCs in erythroid liquid culture (●) or in BFU-E (■). HSPCs derived from 1 patient of each genotype were used for this figure. The authors gratefully acknowledge the Conseil Général de l’Essonne (ASTRES) and Genopole Research in Evry for financial help for the purchase of equipment and are grateful to consenting mothers and to L. Rigonnot and staff of the Maternity at the Centre Hospitalier Sud-Francilien (CHSF; Evry, France) for providing umbilical cord blood samples. (D) HBA2 copies in RNP-treated HSPCs at day 0 (injection) and in BM of engrafted mice at week 16. (E) GFP+ cells in PB of transplanted mice over time. Representative chromatograms (G) and HbA (α2β2) quantification (H) are shown (mean ± SD, n = 3; **P < .01; ANOVA, Tukey test). β-Thalassemia heterozygotes (minor) have mild anemia microcytic, hypochromic erythrocytes with increased HbA2 (<5-7%) and normal or mildly increased HbF (less than 5%) with the rest being HbA. 11/20/2020. 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). Test Iron deficiency ... which can lower HbA2 levels into the normal range. Cas9 nickase editing results in seamless HBA2 deletion and reduced HSPCs toxicity. (E) Representative HPLC chromatograms of globin tetramer analysis in edited HUDEP-2 β0. It is difficult to determine whether they are carriers of silent mutations or high normal HbA2 without genetic test. Editing was measured across HBA2 deletion junctions. The lower number of DSBs could also explain the reduced toxicity observed in CFC edited with Cas9 D10A (Figure 6F-G). Black lines represent mean. 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). 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. Overall, we described an innovative CRISPR/Cas9 approach to improve α/β globin imbalance in thalassemic HSPCs, paving the way for novel therapeutic strategies for β-thal. 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. Other names that describe the test. 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). Droplets were generated using AutoDG Droplet Generator and analyzed with a QX200 Droplet Reader; data analysis was performed with QuantaSoft (BioRad). To minimize the possibility of generating a α-globin KO, the sgRNA was designed to target the 5′UTR (HBA15) of HBA1 and HBA2 (Figure 1A), where the presence of InDels resulting from double-strand breaks (DSBs) does not affect α-globin production.37  As a β0-thalassemia cell model, we used immortalized HUDEP-2, which can differentiate and express adult hemoglobin (supplemental Figure 1A-B), and we knocked out β-globin genes (HUDEP-2 β0) (supplemental Figure 1C). and M.A. NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were purchased from The Jackson Laboratory, Bar Harbor, ME (strain 005557) and maintained in specific-pathogen-free conditions. A.F. (F) CFC number expressed as percentage of untreated control (UT). All reagents and detailed sequence information are available upon request. Both HBA2 deleted and βAS3 KI HSPCs were then transplanted in immunodeficient NOD/SCID/γ (NSG) mice43  to evaluate their in vivo homing, engraftment, and multilineage potential (Figure 4A). Red dashed line indicates the number of expected HBA2 alleles in normal cells. γ4, Hb Bart; acHbF, acetylated fetal hemoglobin; HbF, fetal hemoglobin (α2γ2); α-p, α-precipitates; HbA, adult hemoglobin (α2β2). A comprehensive report is provided. Absence of beta chain causes beta-zero-thalassemia. Haemoglobin is a … Usually, a "tumor-normal comparison" is performed in large scale genomic sequencing assays. 1/20/2020. All recombinant single-stranded AAV2/6 used in this study were produced using a triple transfection protocol and purified by 2 sequential cesium chloride density gradients or chromatography, as described earlier.23  The vector titer of each preparation was determined by real-time quantitative polymerase chain reaction (qPCR)–based titration method using primers and probe corresponding to the inverted terminal repeat region of the AAV genome.24. Black lines indicate mean. See supplemental Table 2 for primer sequences. Because nicked genomic DNA is corrected by the endogenous base-excision repair pathway,49  Cas9 D10A is expected to have minimal genotoxicty.50,51. A 1-kb scale bar is indicated at top. We then plated HSPCs in methylcellulose containing cytokines supporting erythroid and myeloid differentiation (CFC assay), and we confirm that modified progenitors retained their multilineage potential, although some toxicity was observed (supplemental Figure 2D-E). The full-text version of this article contains a data supplement. (E-F) HPLC analysis of globin monomers in differentiated HUDEP-2 β0. Ferritin is not required as a routine test. HBA2 copies (C) and KI efficiency (D) in edited HSPCs in erythroid liquid culture (●) or in BFU-E (■). In addition, the percentage of InDels, as well as the extent of HBA2 deletion in RNP-treated HSPCs, remained similar ex vivo and in vivo, confirming a similar efficiency in both stem and progenitor cells (Figure 4C-D). Because edited HSPCs retained engraftment and multilineage differentiation potential in vivo, the proposed strategy has clear potential for future clinical testing. Adult hemoglobin consists of 2 pairs of globin subunits (α2β2), whose production is strictly regulated to ensure their balanced expression in erythroid cells. (E) Relative abundance of endogenous β and integrated βAS3 mRNA at day 12 of erythroid liquid culture. High Performance Liquid Chromatography/Electrophoresis/RBC Solubility/Polymerase Chain Reaction/Fluorescence Resonance Energy Transfer/Sequencing. HBA2-deleted and βAS3KI HSPCs engraft NSG mice and maintain their multilineage potential. The LV encoding the βAS3 gene under the control of the erythroid specific β-globin enhancer/promoter was already described.22. It will be particularly challenging to qualitatively and quantitatively assess all the possible on-target editing outcomes, especially low-frequency ones. To evaluate this strategy in clinically relevant cells, we transfected human umbilical cord blood HSPCs with RNP and then transduced with AAV, as described for HUDEP-2 cells (Figure 3A). Recommendations when to order or not order the test. Blood Adv 2021; 5 (5): 1137–1153. The sequencing reaction was performed with MiSeq sequencing system (Illumina) using an Illumina Miseq Reagent Kit V2 - 300 cycles (2 × 150 bp paired-end) and raw sequencing data (FASTQ files) were analyzed using the GUIDE-Seq computational pipeline.33  Briefly, after demultiplexing, putative PCR duplicates were consolidated into single reads and mapped to the human reference genome GrCh37. (A) Schematic representation of HSPC targeting experiments. Indicates test has been approved by the New York State Department of Health. Common occurring deletions are shown as gray bars and thin lines indicate regions of uncertainty of the breakpoints (adapted from Harteveld and Higgs98 ). HSPCs from 1 patient of each genotype were used. 12/20/2020. Patient History for Hemoglobinopathy/Thalassemia Testing. 83021. HPLC analysis was performed using a NexeraX2 SIL-30AC chromatograph (Shimadzu, Kyoto, Japan) and analyzed with LC Solution software. qPCR was performed using Maxima Syber Green/Rox (Life Scientific, Thermo Fisher Scientific, Waltham, MA). 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). 9/20/2020. To quantify on-target transgene integration events, primers and probes were designed spanning the donor DNA-genome 3′ junction. Hemoglobin A (Hb A), composed of both alpha and beta globin, is the type of hemoglobin that normally makes up 95% to 98% of hemoglobin in adults. Without any selection, we achieved robust genome cutting (Figure 3B), with an InDel pattern consisting mostly of 1 T nucleotide insertion (supplemental Figure 2A-C), together with efficient HBA2 deletion and donor DNA KI (Figure 3C-D). (B) Schematic representation of HUDEP-2 β0 targeting experiments. HbA2 is normal or slightly decreased, whereas HbF is increased to 5–20%. Are you an ARUP Client? Additional information related to the test. Optimal test for the initial and confirmatory diagnosis of any suspected hemoglobinopathy. Each cassette, followed by a green fluorescent protein (GFP) reporter gene under the control of the constitutive human phosphoglycerate kinase 1 promoter with an SV40 pA, was flanked by arms of homology (250 bp each) and cloned in a standard AAV vector backbone (AAV2) in sense orientation with respect to its inverted terminal repeat. Please follow the EQAS Online setup procedure on QCNet before accessing the mobile application. If you are a known carrier of a genetic condition please contact us, and we will provide a copy of the donor's test reports to your physician and/or genetic counselor. Red cell distribution width (RDW) is often normal ; Pre-pregnancy carrier testing of partner is important (Ensure parents have been tested if likely to have more children) Note: HbA2 may not be elevated in the presence of concomitant iron deficiency, therefore give iron treatment (if ferritin low) before ordering test. When β-globin chains are absent or limiting, free α-globins precipitate and damage cell membranes, causing hemolysis and ineffective erythropoiesis. If reflexed additional CPT codes may apply; refer to the reflexed test code for applicable codes. This analysis, together with our previous off-target analyses,37  confirms the target specificity of the selected gRNA HBA15 sequence. Reduced amounts of detectable beta globin causes beta-plus-thalassemia. One day after transfection, cells were replated and selected with puromycin (1 μg/mL) for 48 hours to enrich for transfected cells. By sequencing the tumor and e.g. Last, in bulk populations of RNP and RNP D10A edited HSPC, deep sequencing of 3 top-scoring off-targets identified by GUIDE-Seq32  in 293T cells (supplemental Figure 6C) showed low to undetectable off-target activity for both Cas9 and Cas9 D10A (supplemental Figure 6D). BM, bone marrow; PB, peripheral blood; SP, spleen. 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 … 10/19/2019. Sixteen weeks after transplantation, blood, bone marrow, and spleen were harvested and analyzed. When using RNP D10A, we observed that, without inducing any InDels (supplemental Figure 5E), both single and dual gRNA induced efficient HBA2 deletion and HDR integration, although to a lower extent compared with RNP (supplemental Figure 5F-H). mRNA globin imbalance (measured as α/β-like globins ratio) was ameliorated in all conditions (Figure 5D), with βAS3 KI cells performing better than HBA2-deleted erythroblasts because of βAS3 expression (Figure 5E; supplemental Figure 4C). 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. For live cell analysis, viability was assessed using Zombie Yellow dye (BioLegend, San Diego, CA) per manufacturers’ instructions to exclude dead cells from the analysis. BFU-E, burst-forming unit-erythroid; CFU-GM, CFU-granulocyte, macrophage. Black lines represent mean. (D) α/β-like globin mRNA ratios in edited thalassemic erythroblasts at day 12. 8/20/2019. There is an urgency to test the biological male partner concurrently when an at-risk woman who is a carrier is pregnant. For engraftment studies, an Fc Receptor Binding Inhibitor antibody was used to block unspecific binding of mouse antibody to human cells, as per manufacturers’ instructions. To optimize βAS3 expression, we designed 2 DNA donor cassettes: (1) a βAS3 cDNA followed by a posttranscriptionally regulatory element41  and SV40 polyadenylation signal (βAS3 cDNA); and (2) a βAS3 transgene that includes full-length HBB introns and the endogenous 3′UTR and polyadenylation signal (βAS3 full). (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). (I) Genotypes distribution of single KI-βAS3 BFU-E. Here, we describe a novel strategy to treat β-thal based on genome editing of the α-globin locus in human hematopoietic stem/progenitor cells (HSPCs). Normal range/expected value(s) for a specific disease state. HSPCs were transfected with RNP and transduced with AAV as described previously (Figure 3A). Most monosaccharides, including glucose, galactose and fructose, spontaneously (i.e. Conflict-of-interest disclosure: G.P. 12/1/2019. Red dashed line indicates the physiological α/β-like ratio of 1. laboratory, Marina Cavazzana, Anne Galy, and Ronzitti for fruitful discussion. Clinical data suggest that a reduction of α-globin to 75% to 25% of its physiological levels is safe and beneficial to patients with β-thalassemia.3,35  The most common natural mutations affecting α-globin synthesis are gene deletions that remove a single HBA gene from 1 or both chromosomes generating -α/αα or -α/-α genotypes (-α3.7 and -α4.2deletions36 ) (Figure 1A). Black lines represent mean; red line indicates the number of expected HBA2 alleles in untreated HSPCs.

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