Journal: bioRxiv
Article Title: Base editing of β 0 thalassemia mutations as a therapeutic strategy for β-hemoglobinopathies: efficacy and genotoxicity studies
doi: 10.1101/2025.08.21.671453
Figure Lengend Snippet: A. Overview of the experimental protocol of HSPC xenotransplantation. B. Engraftment of human cells in NBSGW mice represented as percentage of human CD45 + cells in the total murine and human CD45 + cell population in PB, BM and spleen (SP) 16 weeks post-transplantation. Mice were transplanted with BT corrected HSPCs (BT-cor) or HD/BT HSPCs electroporated with TE buffer or only with NRCH-ABE8e mRNA as controls (TE and BE conditions) (BT-TE/BE: n=3; BT-cor: n=4; HD-TE/BE: n=4). Each data point represents an individual mouse. (Unpaired T-test or Mann-Whitney test, BT-TE vs BT-cor, BT-BE vs BT-cor). C. Frequency of human T (CD3) and B (CD19) lymphoid, myeloid (CD33), erythroid (GpA, CD71) and HSPC (CD34) cells in BM 16 weeks post-transplantation (BT-TE/BE: n=3; BT-cor: n=4; HD-TE/BE: n=4). (Unpaired T-test or Mann-Whitney test, BT-TE vs BT-cor, BT-BE vs BT-cor). D. Hematopoietic progenitor content in BM human CD45 + cells derived from mice transplanted with control and edited HSPCs (BT-TE/BE: n=3; BT-cor: n=3). ns = non-significant (Unpaired T-test or Mann-Whitney test, BT-TE vs BT-cor, BT-BE vs BT-cor). E. Base editing efficiency, calculated by the EditR software, in input, PB-, BM- and SP- derived BT human samples subjected to Sanger sequencing. BT-cor: n=4. The frequency of base editing in the input was calculated in cells cultured in the HSPC medium (▾), in liquid erythroid cultures (▴), and in pooled CFU (◆) colonies. For PB, BM and SP data, each mouse is identified by a specific color. F-H. Number of variants ( F ), relative percentage of variants types ( G ) and relative percentage of SNV types ( H ) in the human xenograft of mice engrafted with control (TE) and corrected (cor) BT HSPCs. We subtracted the variants that were shared between untreated and treated conditions in the in vitro input population (TE: n=3, cor: n=4). Data are expressed as mean±SEM. Unpaired T-test or Mann-Whitney test, BT-TE vs BT-cor.
Article Snippet: Cells from PB and organs were harvested and analyzed for the presence of human CD45 + (using an anti-human CD45-VioBlue antibody, Miltenyi Biotec) cells and the expression of different lineage specific surface markers (using an anti-human CD235/GpA FITC antibody (Biolegend); an anti-human CD33-APC antibody (Miltenyi Biotec); an anti-human CD19-PECy7 antibody (BD); an anti-human CD3-BV510 (Biolegend); an anti-human CD34-PE antibody (BD); an anti-human CD71 APC antibody (BD Pharmingen); and analyzed by CANTO II flow-cytometer (BD) and FCS Express v7 Software (De Novo Software).
Techniques: Transplantation Assay, MANN-WHITNEY, Derivative Assay, Control, Software, Sequencing, Cell Culture, In Vitro
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