Figure 5.

Selection of BCL11A-Edited NHP CD34⁺ Cells In Vitro by O6BG/BCNU Treatment

(A) Schematic representation of the BCL11A AAV donor construct and position of the forward (F) and reverse (R) primers used in PCR analysis. 2A, Thosea asigna virus 2A self-cleaving peptide. (B) Representative flow cytometry results showing targeted integration events based on the frequency of GFP⁺ cells at 5 days post treatment using the conditions described on top. (C) PCR validation of targeted integration events described in (B) using primers designed inside and outside the donor cassette as described in (A). * denotes unspecific amplicons and the arrow shows the specific amplicon validated by sequencing. (D) Frequency of GFP⁺ cells following in vitro treatment with O6BG/BCNU (day 1) of NHP CD34⁺ cells treated with the indicated conditions. (E) HbF response to two rounds of O6BG/BCNU chemoselection in three transplanted pigtailed macaques. Animals Z13030, Z12344, and Z12365 underwent autologous BMT with CCR5 gene-edited CD34⁺ cells (subjects of separate study) and were allowed to recover for 221, 236, or 250 days, respectively. Two rounds of chemotherapy with O6BG/BCNU were subsequently administered in these animals at day 0 and 183 days thereafter (arrowheads).