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. 2022 Apr 19;25:392–409. doi: 10.1016/j.omtm.2022.04.009

Figure 6.

Figure 6

Selection of modified cells decreases engraftability and therapeutic efficacy of genome-edited HSPCs in NSG-MPS I mice

(A) Schematic depicting experimental design, timeline, and the AAV6 vector used to express truncated nerve growth factor receptor (tNGFR) as a selectable marker downstream of IDUA. (B) Representative flow cytometry plots showing expression of tNGFR in edited HSPCs. (C) Fraction of edited alleles measured by ddPCR in bulk HSPCs edited with the IDUA (n = 3) or IDUA-tNGFR (n = 8) AAV6 donor templates. Each symbol represents a different human cell donor. (D) Engraftment of human cells (CD45+HLA+) in different hematopoietic tissues of mice transplanted with bulk cells edited with IDUA alone (n = 12), selected IDUA-tNGFR+ (n = 10, 10 and 8), and IDUA-tNGFR- (n = 5) cells. Each symbol represents a mouse. (E) Fraction of edited alleles in the bone marrow of transplanted mice (input). Dashed lines represent the median of edited alleles in the transplanted cells. Each symbol represents a mouse. (F) Edited alleles fraction in input cells (n = 6 different human cell donors) and in bone-marrow-engrafted cells (n = 7 mice). Lines link transplant recipient mice with their respective donors. (G) Engraftability of edited cells when transplanted as bulk population (n = 3) or as selected IDUA-tNGFR+ cells (n = 6). Each symbol represents a different HSPC donor. (H) Percentage of long-term HSCs (LT-HSCs), given by CD34+CD90+CD38-CD45RA- in mock and selected cells; each dot represents a donor. (I) Enzyme activity of IDUA in serum, liver, spleen, and brain from NSG-MPS I mice transplanted with IDUA-bulk (n = 8), IDUA-tNGFR+ (n = 7), and IDUA-tNGFR-cells (n = 3). Each symbol represents a mouse. (J) GAG storage measured in the liver, spleen, and brain using the DMB method. Liver: Normal, n = 15; MPS I, n = 5; IDUA-tNGFR+, n = 6; IDUA-tNGFR-, n = 3. Spleen: Normal, n = 8; MPS I, n = 8; IDUA-tNGFR+, n = 8; IDUA-tNGFR-, n = 6. Brain: Normal, n = 15; MPS I, n = 7; IDUA-tNGFR+, n = 7; IDUA-tNGFR-, n = 3. All numerical data is presented as median. (C and E–G) Two-tailed unpaired t test; (D and H–J) (liver) Kruskal-Wallis test with post-hoc Dunn test; (J) (spleen and brain) ordinary one-way ANOVA with post-hoc Tukey test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.