Figure 6.

Edited PKD-HSPCs engraft in immunodeficient mice and restore the energetic balance in erythroid derived cells

(A) ATP quantification of in vitro differentiated erythroid cells obtained from peripheral blood CD34⁺ cells of four different PKD patients. Cells were edited with the therapeutic AAV donor. Peripheral blood CD34⁺ cells from 6 different healthy donors were also in vitro differentiated and analyzed. Two replicates per healthy donor were performed. HD unedited, HSPCs from a healthy donor who had undergone in vitro erythroid differentiation; PKD control, HSPCs from a PKD patient who had undergone in vitro erythroid differentiation; PKD GFP or coRPK edited, HSPCs from a PKD patient who had undergone gene editing with either GFP-AAV or coRPK-AAV, respectively, and subsequent in vitro erythroid differentiation. Mean ± SD is shown. Dunn’s multiple comparison test was performed. The significance is represented by p values: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, and ∗∗∗∗p < 0.001. (B) Percentage of human CD45⁺ cells in BM of immunodeficient NBSGW animals transplanted with mobilized peripheral blood CD34⁺ (PB-CD34⁺) cells with or without gene editing correction from a single PKD patient, analyzed 2 mpt. (C) Percentage of hCD34⁺ cells within the human population (hCD45⁺) in animals engrafted with PKD edited human cells. (D) Percentage of the hCD19⁺ cells within the human population (hCD45⁺) in animals engrafted with PKD edited human cells. (E) Percentage of the hCD33⁺ cells within the human population (hCD45⁺) in animals engrafted with PKD edited human cells. (F) Percentage of the hCD235a⁺ cells in mouse BM in animals engrafted with PKD edited human cells. (G) Representative agarose gel of 5′ and 3′ PCRs in samples pre-transplant (liquid culture), after erythroid differentiation (Ery diff), in mice receiving edited cells (1.1, 1.2, 1.3, 1.4, 1.5, and 1.6) and in mice receiving unedited cells (2.1, 2.2, 2.3, and 2.4). Mock, unedited cells; Mock+EP, unedited electroporated cells; coRPK, cells edited with therapeutic donor. Arrows indicate amplification of the band showing homologous directed integration. (H) Percentage of HDR analyzed by ddPCR in BM from primary NBSGW mice transplanted with unedited and edited PKD-HSPCs. (I) Percentage of correct HDR CFUs derived from hCD34⁺ cells, which were sorted from BM of individual NBSGW mice transplanted with mPB-CD34⁺ cells from a single PKD patient with (n = 5 mice) or without (n = 4 mice)gene editing correction. Cell sorting was performed 2 mpt. Sorted cells were cultured in methylcellulose for 2 weeks, and the percentage of HDR was assessed by specific PCR in 5′ and 3′ junctions. From 3 to 23 CFUs were analyzed per animal. (J) ATP quantification in hCD235a⁺ cells sorted from BM of individual NBSGW mice transplanted with mPB-CD34⁺ cells from a single PKD patient with (n = 5 mice) or without (n = 4 mice) gene editing correction. Cell sorting was performed 2 mpt. Kruskal-Wallis multiple comparison test was performed. The significance is represented by p values: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, and ∗∗∗∗p < 0.001.