Journal: bioRxiv
Article Title: Circularization of Single-Stranded DNA Donor Template Unleashes the Power of Non-Viral Gene Delivery for Long-Term HSCs editing
doi: 10.1101/2025.02.19.638978
Figure Lengend Snippet: a . Representative schema of HSPCs editing protocol using an mRNA encoded TALEN targeting the B2M locus and cssDNA or AAV (MOI=350 vg/cell) as DNA donor templates to insert a reported gene (CSR 2 , 2.2 kb) via disruptive insertion. mRNAs encoding a viability enhancer and a HDR enhancer (Via-Enh01 and HDR-Enh01, respectively) were also incorporated in the first transfection. The timing is indicated in days (D0-D7). Edited HSPCs retrieved 7 days post thawing (D7) were characterized by flow cytometry to assess the level of knock-in (KI) of DNA donor templates and knock-out (KO) of B2M as well as their viability. Their differentiation capacity into erythroid and myeloid progenitors as well as their transcriptomics profile were also assessed by colony forming unit (CFU) assay (supplementary figure 4b) and CITE-seq, respectively. Edited HSPCs retrieved 4 days post thawing (D4), were also injected in NCG mice to assess their ability to engraft, differentiate and keep their editing events, 16 weeks after injection onset. b . In vivo experimental results illustrating the level of human CD45+ cells (hCD45) engraftment and of KI frequencies and KI/KO ratio determined either before mice injection (input), or in hCD45+ cells engrafted in the bone marrow (BM) of NCG mice, 16 weeks after cells injection onset (output). Two-way ANOVA followed by Bonferroni multi-comparison test. P-values are indicated. The product of the frequency of hCD45+ cells engraftment and frequency of KI is also shown to illustrate the overall efficiency of each HSPC editing process. Mann–Whitney two-tailed non-parametric unpaired test with a confidence interval of 95%. P-value is indicated. On each box plot, the central mark indicates the median, the bottom and top edges of the box indicate the interquartile range (IQR), and the whiskers represent the maximum and minimum data point. Each dot represents data obtained from one HSPCs donor. c . UMAP plots showing aggregated 5’scRNA CITE-Seq data obtained from HSPCs either untreated, edited with TALEN and AAV or cssDNA donor templates (AAV or cssDNA respectively), 4 days post thawing (D4) and at the time of NCG mice injection onset (n = 3 independent biological donors). The different cell subpopulations identified are illustrated by a color code indicated at the bottom of the graph. The Long-term HSC-enriched cell subpopulation is indicated as LT-HSCe and definition of each subpopulation is documented in the methods section d . UMAP plots aggregated from 5’scRNA CITE-Seq data obtained from all experimental groups showing the position of LTHSCe subpopulation. e and f . UMAP plots showing the cell cycle phases (G0/G1, G2M and S) of each cell identified in each experimental groups and in all groups, respectively. g. left panel , illustrate the frequency of LT-HSCe within all subpopulations. Two-way ANOVA with Bonferroni post-tests. P-values are indicated. g middle and right panels , illustrate the frequency of KI and KO within the LT-HSCe subpopulation, respectively. Paired t-test. P-values are indicated. Each dot represents data obtained from one HSPCs donor. h . plot showing the frequency of KI(+) and KO(+) in each subpopulation found in the CssDNA and AAV-edited HSPCs (n=3 donors aggregated, subpopulations identified with fewer than 100 cells are not displayed). i . Gene Set Enrichment Analysis (GSEA) obtained in LT-HSCe to compare the AAV and CssDNA experimental groups to the untreated reference group and to directly compare the cssDNA group to the AAV reference group. Normalized Enrichment Score (NES) as well as Log P value obtained for each donor (n=3 independent HSPC donors) are illustrated by a red/white/blue color code and size of the dots, respectively. The different pathways found to be significantly up (red) or down (blue) regulated in the different experimental group comparisons are identified as individual pathways and aggregated in subgroups for the sake of clarity.
Article Snippet: One hundred thousand cells from each organ were harvested, and chimerism was assessed by flow cytometry using the following antibodies: mouse CD45 perCPvio700, Clone# REA747(Miltenyi, #130-110-636), human CD45 BV650, Clone# HI30 (BD, #563717) and viability dye FVS780, (BD, #565388).
Techniques: Transfection, Flow Cytometry, Knock-In, Knock-Out, Colony-forming Unit Assay, Injection, In Vivo, Comparison, MANN-WHITNEY, Two Tailed Test
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