Journal: Molecular Therapy. Nucleic Acids

Article Title: Forced enhancer-promoter rewiring to alter gene expression in animal models

doi: 10.1016/j.omtn.2023.01.016

Figure Lengend Snippet: Optimization of ZF-Ldb1 constructs for use in rhesus macaque transplantation (A) Schematic of ZF-Ldb1 constructs. (B) Experimental design for testing HbF induction of ZF constructs. Adult CD34 + human cells were transduced at an MOI of 20 or 100 on day 0, and HbF was measured by HPLC analysis on days 9 and 12 of in vitro erythroid differentiation culture. (C) HPLC analysis following transduction of ZF constructs in two independent CD34 + donor cells on day 12 of culture. HPLC elution tracings (left) and quantification of HPLC peaks (right) illustrate high levels of HbF induction. Vector copy number (VCN D12) and percent Venus/mEmerald positivity (%Fluor D12) are displayed for each vector tested. n = 2 independent donors.

Article Snippet: Flow cytometry of rhesus macaque mEmerald (with a 515/20 filter, Chroma Technology) and Venus (with a 525/LP filter, BD Biosciences) was performed using a CytoFlex (Beckman Coulter) instrument on days 5, 7, 9, and 12 of differentiation.

Techniques: Construct, Transplantation Assay, In Vitro, Transduction, Plasmid Preparation

Journal: Molecular Therapy. Nucleic Acids

Article Title: Forced enhancer-promoter rewiring to alter gene expression in animal models

doi: 10.1016/j.omtn.2023.01.016

Figure Lengend Snippet: Robust ex vivo γ-globin induction in rhesus macaque progenitor cells transduced with optimized ZF constructs (A) Hemoglobin electrophoresis (top) and RP-HPLC (bottom) in differentiated rhesus macaque CD34 + HSPCs; n = 1. (B) Flow cytometric analysis of mEmerald or Venus vector expression in differentiated rhesus macaque CD34 + HSPCs. Cells were transduced with a lentivirus (MOI = 50 for CD34 + and MOI = 10 for PBMCs) at high cell density (2 × 10 6 cells/mL) in XVIVO-10 medium + SFT (100 ng/mL each); n = 3. Statistical analyses were done using one-way ANOVA. Error bars represent standard deviation. ∗∗∗p < 0.001. (C and D) Ex vivo analysis of rhesus macaques receiving transplants, showing (C) hemoglobin electrophoresis and (D) RP-HPLC analysis of non-transduced cells (NTCs) and GG38-expressing lentivirus-transduced cells on day 14 of erythroid differentiation of infused products; n = 1. HbF and flow cytometry assays were performed on unsorted bulk erythroid cells. HbC, hemoglobin C; HbS, hemoglobin S/sickle hemoglobin; HbF, hemoglobin F/fetal hemoglobin; HbA, hemoglobin A/adult hemoglobin.

Article Snippet: Flow cytometry of rhesus macaque mEmerald (with a 515/20 filter, Chroma Technology) and Venus (with a 525/LP filter, BD Biosciences) was performed using a CytoFlex (Beckman Coulter) instrument on days 5, 7, 9, and 12 of differentiation.

Techniques: Ex Vivo, Transduction, Construct, Electrophoresis, Plasmid Preparation, Expressing, Standard Deviation, Flow Cytometry