Journal: Nature medicine

Article Title: Highly efficient therapeutic gene editing of human hematopoietic stem cells

doi: 10.1038/s41591-019-0401-y

Figure Lengend Snippet: a , Eight modified synthetic (MS) sgRNAs targeting BCL11A enhancer DHS h+58 functional core marked with blue arrows. GATA and Half E-box motifs marked respectively with red or green. b , Editing efficiency of Cas9 coupled with various sgRNAs (each targeting BCL11A enhancer with exception of AAVS1 ) in CD34 + HSPCs measured by TIDE analysis. c , β-like globin expression by RT-qPCR analysis in erythroid cells in vitro differentiated from RNP edited CD34 + HSPCs. d , Correlation of BCL11A mRNA expression determined by RT-qPCR versus HbF by HPLC. Black dots represent samples edited with Cas9 coupled with different sgRNAs. The Pearson correlation coefficient ( r ) is shown. e, Editing efficiency as measured by TIDE analysis of Cas9:sgRNA RNP targeting AAVS1 or BCL11A DHS h+58 functional core (Enh) with MS-sgRNA-1617 in CD34 + HSPCs from β-thalassemia patients or healthy donors (β A β A ) of indicated β-globin genotypes. f-h , β-like globin expression by RT-qPCR normalized by α-globin ( P = 0.00017 for BCL11A enhancer as compared to AAVS1 edited for all comparisons as determined by unpaired two-tailed Student’s t tests), and HbF induction by HPLC analysis in erythroid cells in vitro differentiated. i , Enucleation of in vitro differentiated erythroid cells. j , Cell size measured by relative forward scatter intensity. k , Representative microscopy image showing rounder and more uniform appearance of enucleated erythroid cells following BCL11A enhancer editing. Blue arrow indicates poikilocytes. Bar = 15 μm. l , m , Imaging flow cytometry was used to establish a circularity index (l) and then quantify (m) circularity of enucleated erythroid cells. Bar = 5 μm. In all panels, data are plotted as mean ± SD and analyzed using unpaired two-tailed Student’s t tests. Data are representative of three biologically independent replicates.

Article Snippet: CD34 + HSPCs were enriched using the Miltenyi CD34 Microbead kit (Miltenyi Biotec).

Techniques: Modification, Functional Assay, Expressing, Quantitative RT-PCR, In Vitro, Two Tailed Test, Microscopy, Imaging, Flow Cytometry

Journal: Nature medicine

Article Title: Highly efficient therapeutic gene editing of human hematopoietic stem cells

doi: 10.1038/s41591-019-0401-y

Figure Lengend Snippet: a) , Correlation of indel frequencies of input HSPCs to indel frequencies of engrafted human cells in mice BM after 16 weeks. Each dot represents average indel frequencies of mice transplanted with the same input HSPCs. Legend denoting transplant is same as in ( c ). The Pearson correlation coefficient ( r ) is shown. b , Indel spectrum of input cells from healthy donor β A β A #2 electroporated with 2xNLS-Cas9 (coupled with sgRNA-1617) supplemented with 2% glycerol and engrafted 16 week BM human cells. c , Relative loss of edited alleles repaired by MMEJ and gain of edited alleles repaired by NHEJ in mice BM 16 weeks after transplant. The indel spectrum was determined by deep sequencing analysis. Indel length from −8 to +6 bp was calculated as NHEJ, and from −9 to −20 bp as MMEJ. These data comprise 28 mice transplanted with 8 BCL11A enhancer edited inputs and 5 mice transplanted with 2 AAVS1 edited inputs. Median of each group is shown as line, ** P < 0.005, **** P < 0.0001 as determined by Kolmogorov–Smirnov test. d-e , Indel spectra of HSPCs stained and sorted 2h after RNP electroporation with 3xNLS-Cas9 with sgRNA-1617. HSPCs prestimulated for 24h prior to electroporation. HSPCs stained with CD34, CD38, CD90, CD45RA in ( d ) and with Pyronin Y, Hoechst 33342 in ( e ). Indels determined by Sanger sequencing with TIDE analysis after culturing cells for 4 days after sort. Relative loss of edited alleles repaired by MMEJ and gain of edited alleles repaired by NHEJ at BCL11A enhancer and AAVS1 in sorted enriched HSCs ( f ) or G0 phase cells ( g ) shown. Data are plotted as mean ± SD for (f, g) and analyzed using unpaired two-tailed Student’s t tests. Data are representative of three biologically independent replicates.

Article Snippet: CD34 + HSPCs were enriched using the Miltenyi CD34 Microbead kit (Miltenyi Biotec).

Techniques: Sequencing, Staining, Electroporation, Two Tailed Test

Journal: Nature medicine

Article Title: Highly efficient therapeutic gene editing of human hematopoietic stem cells

doi: 10.1038/s41591-019-0401-y

Figure Lengend Snippet: a , Off-target sites detected by CIRCLE-seq for MS-sgRNA-1617 targeting human BCL11A enhancer. b , Deep sequencing analysis of potential off-target sites detected by CIRCLE-seq or in silico computational prediction within human CD34 + HSPCs edited by 2xNLS-Cas9 or 3xNLS-Cas9 RNP (coupled with MS-sgRNA-1617) targeting BCL11A enhancer. On-target sequence is at the BCL11A enhancer. Dotted line at 0.1% denotes sensitivity of deep sequencing to detect indels. c , RT-qPCR analysis of p21 expression after gene editing. Relative expression to GAPDH is shown. Data are plotted as mean ± SD and representative of three biologically independent replicates.

Article Snippet: CD34 + HSPCs were enriched using the Miltenyi CD34 Microbead kit (Miltenyi Biotec).

Techniques: Sequencing, In Silico, Quantitative RT-PCR, Expressing

Journal: Cell reports

Article Title: Dual α-globin-truncated erythropoietin receptor knockin restores hemoglobin production in α-thalassemia-derived erythroid cells

doi: 10.1016/j.celrep.2024.115141

Figure Lengend Snippet: (A) Schematic of custom AAV6 DNA repair donors designed to integrate promoterless HBA-2A-YFP transgenes at the start codon of HBB . The table to the left indicates whether cassette integrated HBB , HBA1 , or HBA2 UTRs, as well as HBA1 or HBA2 transgene. (B) Schematic of Cas9/AAV6 genome editing workflow in primary HSPCs followed by in vitro erythroid differentiation. (C) Percentage of CD34 − /CD45 − HSPCs acquiring erythroid cell surface markers GPA and CD71 as determined by flow cytometry. Bars represent mean ± SEM. (D) Percentage of YFP + cells among CD34 − /CD45 − /CD71 + /GPA + cells was determined at day 14 of erythroid differentiation using flow cytometry. Bars represent mean ± SEM. ** p < 0.005 by unpaired two-tailed t test. (E) MFI of YFP + cells (from D) was determined by flow cytometry. Bars represent mean ± SEM. (F) At day 11 of erythroid cell differentiation, cells were stained for HSPC/erythroid markers and analyzed by flow cytometry. The percentage of YFP + cells are noted ( n = 1). (G) The percentage of CD34 − /CD45 − cells that acquired CD71 and GPA erythroid cell markers are plotted over the course of erythroid differentiation ( n = 1). (H) The percentage of YFP + or GFP + cells are plotted over the course of erythroid differentiation ( n = 1).

Article Snippet: Human CD34 + -enriched HSPCs derived from Plerixafor and/or G-CSF-mobilized peripheral blood from healthy donors , STEMCELL Technologies, AllCells, Fred Hutchinson Cancer Center Hematology Core , 70073.2.

Techniques: In Vitro, Flow Cytometry, Two Tailed Test, Cell Differentiation, Staining

Journal: Cell reports

Article Title: Dual α-globin-truncated erythropoietin receptor knockin restores hemoglobin production in α-thalassemia-derived erythroid cells

doi: 10.1016/j.celrep.2024.115141

Figure Lengend Snippet: (A) Schematic of custom AAV6 DNA repair donors designed to integrate promoterless HBA transgenes at the start codon of HBB . Table to the left indicates whether the cassette integrated HBB or HBA1 UTRs along with HBA1 transgene. (B) The percentage of edited alleles in WT HSPCs at day 14 of erythroid differentiation. Bars represent mean ± SEM. (C) Percentage of CD34 − /CD45 − αTM HSPCs acquiring erythroid cell surface markers as determined by flow cytometry. Bars represent mean ± SEM. (D) The percentage of edited alleles in αTM HSPCs at day 14 of erythroid differentiation. Bars represent mean ± SEM. (E) HPLC elution chromatogram displaying the hemoglobin tetramer profile from WT healthy control HSPCs following in vitro erythroid differentiation. Time displayed on the x axis represents retention time in minutes for each hemoglobin tetramer type to elute. Absorbance on the y axis indicates the concentration of a particular hemoglobin tetramer. (F–H) HPLC elution chromatograms displaying the hemoglobin tetramer profile from αTM HSPCs that have undergone editing and erythroid differentiation. Chromatograms represent two different donors (F and G) and a technical replicate from donor 2 (H) that was edited independently.

Article Snippet: Human CD34 + -enriched HSPCs derived from Plerixafor and/or G-CSF-mobilized peripheral blood from healthy donors , STEMCELL Technologies, AllCells, Fred Hutchinson Cancer Center Hematology Core , 70073.2.

Techniques: Flow Cytometry, Control, In Vitro, Concentration Assay

Journal: Cell reports

Article Title: Dual α-globin-truncated erythropoietin receptor knockin restores hemoglobin production in α-thalassemia-derived erythroid cells

doi: 10.1016/j.celrep.2024.115141

Figure Lengend Snippet: (A) Schematic of custom AAV6 donors designed to integrate promoterless HBA and HBA + tEPOR transgenes at the start codon of HBB . Both vectors are flanked by HBA1 UTRs. (B) The percentage of CD34 − /CD45 − WT HSPCs acquiring erythroid cell surface markers as determined by flow cytometry. Bars represent mean ± SEM. (C) The percentage of edited alleles in WT HSPCs over the course of erythroid differentiation. Bars represent mean ± SEM. * p = 0.01 comparing editing frequencies of HBA -edited cells at day 14 vs. HBA + tEPOR -edited cells at day 14 of differentiation by unpaired two-tailed t test; *** p = 0.0004 comparing editing frequencies of HBA + tEPOR -edited cells at day 0 vs. day 14 of differentiation by unpaired two-tailed t test. (D) The percentage of CD34 − /CD45 − αTM HSPCs acquiring GPA and CD71 as determined by flow cytometry. Bars represent mean ± SEM. (E) The percentage of edited alleles in αTM HSPCs over the course of erythroid differentiation. Bars represent mean ± SEM. * p = 0.02 comparing editing frequencies of HBA + tEPOR -edited cells at day 0 vs. day 14 of differentiation by unpaired two-tailed t test; ** p = 0.009 comparing editing frequencies of HBA -edited cells at day 14 vs. HBA + tEPOR -edited cells at day 14 of differentiation by unpaired two-tailed t test. (F) Cell count at day 14 of erythroid cell differentiation with fold change normalized to HBA . Bars represent mean ± SEM. ** p = 0.007 by unpaired two-tailed t test. (G–I) Hemoglobin tetramer HPLC plots from two different donors (G and H) and a technical replicate from donor 2 (I) that was edited independently. For comparison purposes, the HPLC plots for HBA -edited cells, originally shown in – , are also presented here. (J) Western blot of mock and edited αTM HSPCs at the end of erythroid cell differentiation compared to WT umbilical cord blood-derived erythroid cells. The western blot image was taken from a single gel that was cropped to place the WT control next to edited conditions, as indicated by the black line. Loading was standardized by using the same number of cells for input. (K) Ratio of α-globin to dimeric β-globin quantification from western blot. Bars represent mean ± SEM.

Article Snippet: Human CD34 + -enriched HSPCs derived from Plerixafor and/or G-CSF-mobilized peripheral blood from healthy donors , STEMCELL Technologies, AllCells, Fred Hutchinson Cancer Center Hematology Core , 70073.2.

Techniques: Flow Cytometry, Two Tailed Test, Cell Counting, Cell Differentiation, Comparison, Western Blot, Derivative Assay, Control

Journal: Cell reports

Article Title: Dual α-globin-truncated erythropoietin receptor knockin restores hemoglobin production in α-thalassemia-derived erythroid cells

doi: 10.1016/j.celrep.2024.115141

Figure Lengend Snippet:

Article Snippet: Human CD34 + -enriched HSPCs derived from Plerixafor and/or G-CSF-mobilized peripheral blood from healthy donors , STEMCELL Technologies, AllCells, Fred Hutchinson Cancer Center Hematology Core , 70073.2.

Techniques: Virus, Clinical Proteomics, Recombinant, Saline, Sequencing, Derivative Assay, Clone Assay, Purification, Software, Western Blot, Control, Flow Cytometry, Selection, Electroporation, Transfection, DNA Extraction

Journal: bioRxiv

Article Title: Inhibition of NLRP1 Inflammasome Activation by Tyrosine Kinase Inhibitors Restores Erythropoiesis in Diamond-Blackfan Anemia Syndrome

doi: 10.1101/2025.02.20.639294

Figure Lengend Snippet: (A, E) Primary human CD34 + /CD133 + HSPCs from healthy donors were cultured with EPO, and 0.1 µM nilotinib was added from days 3 to 7 (A) or days 7 to 11 (E). (B, F) CASP1 activity was measured by flow cytometry with FAM FLICA and normalized to untreated control cells. (D, G) Erythroid differentiation was assessed by flow cytometry after staining with anti-CD235A-APC (Glycophorin A) and anti-CD71-FITC (Transferrin Receptor). Representative dot plots of differentiation stages are shown. The differentiation score was calculated as the ratio between CD235A + /CD71 + (intermediate erythroid progenitors) and CD235A - /CD71 + (early erythroid progenitors). MFI, mean fluorescence intensity. Data are shown as the mean ± SEM.**p<0.01 and ****p<0.0001 according to a Student’s t -test.

Article Snippet: CD34 + HSPCs were either enriched by immunomagnetic bead selection from donated human cord blood using an AutoMACS instrument (Miltenyi Biotec) in accordance with the manufacturer’s instructions or purchased from ZenBio (#SER-CD34-F) or StemCell Technologies (#70008).

Techniques: Cell Culture, Activity Assay, Flow Cytometry, Control, Staining, Fluorescence

Journal: bioRxiv

Article Title: Inhibition of NLRP1 Inflammasome Activation by Tyrosine Kinase Inhibitors Restores Erythropoiesis in Diamond-Blackfan Anemia Syndrome

doi: 10.1101/2025.02.20.639294

Figure Lengend Snippet: (A) Primary human CD34 + cells were purified from human cord blood or purchased from from ZenBio or StemCell Technologies, edited with CRISPR/Cas9 and differentiated for 13 days with EPO in the presence of either DMSO or 0.1 µM nilotinib. (B-D) Cells were stained with anti-CD235A-APC (Glycophorin A) and anti-CD71-FITC (Transferrin Receptor), or FAM FLICA, and erythroid differentiation (B-D) and CASP1 activity (E) were then analyzed by flow cytometry. Representative dot plots at different differentiation times are shown in B. The differentiation score was calculated as the ratio between CD235A + /CD71 + (intermediate erythroid progenitors) and CD235A - /CD71 + (early erythroid progenitors) (C), and the percentage of CD235A + /CD71 high (erythroblasts) and CD235A + /CD71 low (reticulocytes) (D) and CASP1 activity were determined at 13 days of culture (E). Data are shown as the mean ± SEM. P values were calculated using one-way ANOVA and Tukey’s multiple range test (C, D) or a Student’s t -test (E). ns, non-significant; *p<0.05; **p<0.01; ***p<0.01 and ****p<0.0001.

Article Snippet: CD34 + HSPCs were either enriched by immunomagnetic bead selection from donated human cord blood using an AutoMACS instrument (Miltenyi Biotec) in accordance with the manufacturer’s instructions or purchased from ZenBio (#SER-CD34-F) or StemCell Technologies (#70008).

Techniques: Purification, CRISPR, Staining, Activity Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Inhibition of NLRP1 Inflammasome Activation by Tyrosine Kinase Inhibitors Restores Erythropoiesis in Diamond-Blackfan Anemia Syndrome

doi: 10.1101/2025.02.20.639294

Figure Lengend Snippet: Primary human CD34 + HSPCs from healthy donors were differentiated with EPO in the presence of 0.1 µM imatinib (A-D), or 10 (E-H) and 1 nM (I-K) dasatinib from 3 to 7 days of culture. Cells were stained with anti-CD235A-APC (Glycophorin A) and anti-CD71-FITC (Transferrin Receptor), and erythroid differentiation was then analyzed by flow cytometry. The transcript levels of GATA1-dependent genes (B), caspase-1 activity determined with FAM FLICA (D,G) and the differentiation score calculated as the ratio between CD235A + /CD71 + (intermediate erythroid progenitors) and CD235A - /CD71 + (early erythroid progenitors) (D, H, K) at 7 dpd are shown. Data are shown as the mean ± SEM. P values were calculated using one-way ANOVA and Tukey’s multiple range test (D, H, K) or a Student’s t -test (C, G). ns, non-significant; *p<0.05; **p<0.01; ***p<0.01 and ****p<0.0001.

Article Snippet: CD34 + HSPCs were either enriched by immunomagnetic bead selection from donated human cord blood using an AutoMACS instrument (Miltenyi Biotec) in accordance with the manufacturer’s instructions or purchased from ZenBio (#SER-CD34-F) or StemCell Technologies (#70008).

Techniques: Staining, Flow Cytometry, Activity Assay

Journal: bioRxiv

Article Title: Inhibition of NLRP1 Inflammasome Activation by Tyrosine Kinase Inhibitors Restores Erythropoiesis in Diamond-Blackfan Anemia Syndrome

doi: 10.1101/2025.02.20.639294

Figure Lengend Snippet: (A) Primary human CD34 + cells were purchased from from ZenBio or StemCell Technologies, edited with CRISPR/Cas9 and differentiated for 13 days with EPO in the presence of of 0.1 µM imatinib (A-D) or 10 nM dasatinib (F-J) from 3 to 13 days of culture. (B-D). The transcript levels of RPS19 were analyzed by RT-qPCR at 13 days of culture (A). Cells were stained with either FAM FLICA or anti-CD235A-APC (Glycophorin A) and anti-CD71-FITC (Transferrin Receptor), and CASP1 activity (B, H), erythroid differentiation (B-D, F, G, I, J) were then analyzed by flow cytometry. The differentiation score was calculated as the ratio between CD235A + /CD71 + (intermediate erythroid progenitors) and CD235A - /CD71 + (early erythroid progenitors) (C, F, I), and the percentage of CD235A + /CD71 high (erythroblasts) and CD235A + /CD71 low (reticulocytes) (D, G, J) at 13 days of culture (E). Data are shown as the mean ± SEM. P values were calculated using one-way ANOVA and Tukey’s multiple range test (C, D, F, G, I, J) or a Student’s t -test (A, E, H). ns, non-significant; *p<0.05; **p<0.01; ***p<0.01 and ****p<0.0001.

Article Snippet: CD34 + HSPCs were either enriched by immunomagnetic bead selection from donated human cord blood using an AutoMACS instrument (Miltenyi Biotec) in accordance with the manufacturer’s instructions or purchased from ZenBio (#SER-CD34-F) or StemCell Technologies (#70008).

Techniques: CRISPR, Quantitative RT-PCR, Staining, Activity Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Inhibition of NLRP1 Inflammasome Activation by Tyrosine Kinase Inhibitors Restores Erythropoiesis in Diamond-Blackfan Anemia Syndrome

doi: 10.1101/2025.02.20.639294

Figure Lengend Snippet: A) Peripheral blood mononuclear cells (PBMCs, red) and bone marrow mononuclear cells (BMMCs, grey) were isolated from 13 patients with DBAS. Cells were cultured for 2 weeks in human methylcellulose complete medium at 37 °C, with or without the indicated TKIs. Burst-forming unit-erythroid (BFU-E) and colony-forming unit-granulocyte macrophage (CFU-GM) colonies were counted based on standard morphological criteria. (B-M) Each dot represents data from an individual patient. The increase in erythroid colonies (BFU-E; panels B-G) and myeloid colonies (CFU-GM; panels H, M) upon treatment with TKIs is depicted. Data are shown as the mean ± SEM. ns, non-significant; **p<0.01, ***P<0.001 and ****P<0.0001 according to a Student’s t -test.

Article Snippet: CD34 + HSPCs were either enriched by immunomagnetic bead selection from donated human cord blood using an AutoMACS instrument (Miltenyi Biotec) in accordance with the manufacturer’s instructions or purchased from ZenBio (#SER-CD34-F) or StemCell Technologies (#70008).

Techniques: Isolation, Cell Culture