Figure 4.

In vivo performance of the double-edited cells. (A) Experimental procedure of the in vivo experiments. CD34⁺ cells from healthy donors were cultured in the presence of cytokines and a small molecule combination to maintain their HSC population for 2 days before electroporation with the respective RNPs or RNP combinations. The cells were transplanted into NBSGW mice 2 days posttransfection. The mice were euthanized 16 weeks posttransplantation and their bone marrow (BM) was collected for further analysis. (B) Engraftment and multilineage reconstitution of the human cells in the recipients 16 weeks posttransplantation. (C) HbF expression in BM-engrafted human erythroid (GlyA⁺) cells assayed by FACS. (D) HPLC data for γ-globin chain expression. The values are represented as a fold difference from mice receiving untransfected cells. (E) Indel frequency per loci in BM-engrafted human cells (dots) and in the input cells (bars). Each dot represents a different mouse. The indel drop is presented as a percentage on top of the graph. Values are represented as means ± SEM (n ≥ 3 mice). (F) Composition of colonies pre- (in vitro) and post- (in vivo) transplantation in terms of indel presence in 1 or both targeting sites.