Figure 3

Targeted integration of the therapeutic hFANCA donor into the Mbs85 locus and phenotypic correction of gene-edited FA-A MEF clones #7 and #76#. (a) Sequenced region of clone #76# displaying the chromatogram containing the SV40pA sequence, the left and right mHA, and the right location of the Mbs85 locus (3′ integration junction). (b) FISH analyses in gene-edited FA-A MEF indicating the integration of the therapeutic PGK-hFANCA donor (red spot signal and red arrows) into chromosome 7, (two green signals corresponding to the two chromatids indicated with a green arrow) where the Mbs85 locus is located. Co-localization of both signals is indicated with an orange circle. DAPI chromosome staining is shown in blue. Scale bar represents 10 μm for all the microphotographs. Targeted integration event in clone #7# (left) and targeted and non-targeted integration events in clone #76# (right). (c) Western blot analysis of hFANCA expression in the bulk gene-edited FA-A MEFs, as well as in the derived clones. Expression levels were calculated as a fold change with respect to β-ACTIN, used as a loading control, and then normalized against the hFANCA expression of lymphoblastic cells from a healthy human donor (HD LCL). FA LCL: lymphoblastic cells derived from a FA-A patient; FA-A: immortalized non-gene-edited FA-A MEFs; WT: immortalized non-gene-edited WT MEFs; FA-AB: bulk of edited FA-A MEFs (T2.5 + D0.75). Analysed clones: #7# and #76#. d) Survival curves in edited FA-A MEF clones together with untransfected WT,FA-A MEFs and FA-A MEFS transduced with a PGK-hFANCA-wPRE* LV (FANCA-LV) at MOI 1, after their exposure to MMC (0, 3, 10, 30, 100 nM). Star (★) indicates that no colonies were generated in this condition at a dose of MMC. The survival shown would correspond to the growth of a single colony in these cultures. ^(**)P-value < 0.01, ^(***)P-value < 0.001, ^(****)P-value < 0.0001 indicate significant differences with respect to FA-A group, with a two-way ANOVA followed by a post-hoc Bonferroni test. Asterisks indicate groups with differences (* WT MEFs; ♦ Clone #76#; ● FA-A MEFS + FANCA_LV) with respect to FA-A group. (e) Chromosomal aberrations per chromosome induced by MMC, analysed in metaphases of gene-edited FA-A MEF clones, in comparison with non-edited WT and FA-A MEFs.