Journal: Cell reports

Article Title: Activation of GPR44 decreases severity of myeloid leukemia via specific targeting of leukemia initiating stem cells

doi: 10.1016/j.celrep.2023.112794

Figure Lengend Snippet:

Article Snippet: psPAX2 (a gift from Didier Trono), pMD2.G (a gift from Didier Trono), pCL-Eco (a gift from Inder Verma), and MSCV-human p210 BCR–ABL-IRES-GFP (a gift from Martine Roussel) were obtained from Addgene.

Techniques: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Binding Assay, Staining, Modification, Saline, Concentration Assay, Over Expression, Protein Extraction, Membrane, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Protease Inhibitor, CCK-8 Assay, Reverse Transcription, Selection, Plasmid Preparation, Knock-Out, Software, Real-time Polymerase Chain Reaction, Flow Cytometry

Journal: Nature Communications

Article Title: Large-scale discovery of potent, compact and erythroid specific enhancers for gene therapy vectors

doi: 10.1038/s41467-025-59235-x

Figure Lengend Snippet: A one-shot approach for screening and functionally validating erythroid specific enhancers for lineage-specific induction of transgene expression at therapeutic levels in gene therapy viral vectors. Briefly, 5393 DNase I hypersensitive sites (DHS) activated de novo during ex vivo human erythropoiesis were selected and broken down into ~3 198bp-long tiles each, comprising a library of total ~15 k elements. The tiles were then cloned into a clinically relevant GFP reporter lentiviral vector and HUDEP-2 cells were transduced at MOI (Multiplicity of infection) <1. The cells were then sorted by flow cytometry into 3 bins based on GFP expression. DNA libraries were constructed from each bin and read counts were assigned to each tile as function of GFP expression. Top performing tiles were then mapped to their full-size DHSs which were then in turn cloned into a therapeutic vector where the candidate elements were assessed based on their ability to achieve phenotypic correction of β-thalassemia patient donor derived erythroid cells. MFI: Mean Fluorescence Intensity.

Article Snippet: The screening vector was based on a lentiviral vector pRRLSIN.cPPT.PGK-GFP.WPRE (a gift from Didier Trono; Addgene plasmid # 12252), in which an enhancer blocker and barrier insulator element C1 was introduced 36 bp into the proximal portion of the 3′ LTR; from this position, the insulator element is copied into the 5′ LTR during proviral cDNA synthesis, thus flanking the expression cassette on both sides upon integration.

Techniques: Expressing, Ex Vivo, Clone Assay, Plasmid Preparation, Infection, Flow Cytometry, Construct, Derivative Assay, Fluorescence

Journal: Nature Communications

Article Title: Large-scale discovery of potent, compact and erythroid specific enhancers for gene therapy vectors

doi: 10.1038/s41467-025-59235-x

Figure Lengend Snippet: a GFP expression (log 10 MFI) of the 40 DHSs individually cloned in the same backbone vector and transduced in HUDEP2 cells at a MOI < 1. Full length β-globin HS2, HS3 and HS4 were used as positive controls and a no-enhancer vector (gray band) as negative control. Mean ± SD is shown. Points are colored by T -test Benjamini-Hochberg adjusted p -value. Transductions were performed in three independent experiments and time points. b Correlation between the GFP intensity (log 10 MFI) of the 40 selected elements after transduction in HUDEP-2 cells ( x -axis) and in the K562 cell line ( y -axis). Beta-globin HS2 and HS3 are annotated. Pearson’s r correlation coefficient is shown ( c ) Correlation between the GFP intensity (log 10 MFI) of the 40 selected elements after transduction in HUDEP-2 cells (x-axis) and in CD34 + differentiated erythroid cells ( y -axis). Pearson’s r correlation coefficient is shown. d Each of the 40 identified enhancers is individually transduced into CD34 + cells and each pool ( n = 42) is subjected to ex vivo erythroid (Ery), megakaryocytic (Mk) and granulocytic-monocytic (GM) differentiation. After 7 days of differentiation the percentage of GFP + cells are determined by flow cytometry. Median is shown as a thick horizontal line, boxes extend to IQR and whiskers extend to 1.5x IQR. e Mobilized peripheral blood CD34 + cells from healthy donors were transduced with a lentiviral library of the identified 40 vectors. The cells were transplanted into NBSGW mice and bone marrow was collected 16 weeks post transplantation. Percent of GFP + cells was assessed by flow cytometry in all engrafted human hematopoietic lineages. Bars extend to mean of n = 3 ± SEM. MFI: Mean Fluorescence Intensity. Ery: Erythroid. Mk: Megakaryocytes. GM: Granulocyte-Monocyte. Source data for all relevant panels are provided within the Source Data file.

Article Snippet: The screening vector was based on a lentiviral vector pRRLSIN.cPPT.PGK-GFP.WPRE (a gift from Didier Trono; Addgene plasmid # 12252), in which an enhancer blocker and barrier insulator element C1 was introduced 36 bp into the proximal portion of the 3′ LTR; from this position, the insulator element is copied into the 5′ LTR during proviral cDNA synthesis, thus flanking the expression cassette on both sides upon integration.

Techniques: Expressing, Clone Assay, Plasmid Preparation, Negative Control, Transduction, Ex Vivo, Flow Cytometry, Transplantation Assay, Fluorescence