HDAd-PE5max vector and schematic of the experiment. (A) Top panel: expected prime editing of SCD mutation. In addition to fixing the sickle mutation (T > A conversion), a G > A silent mutation is introduced to prevent the prime editor from continuous editing by destroying the PAM. New nucleotides are shown as green letters. Bottom panel: the HDAd-PE5max vector contains engineered prime editor guide RNA (epegRNA), codon–optimized editing enzyme, dominant negative MLH1 gene, and MGMT^P140K cassette for in vivo selection. EF1α, elongation factor 1α promoter; miR RNA/β-3’UTR, miR-183-5p and miR-218-5p target sites embedded into β-globin to suppress nCas9-RT expression in HDAd producer cells; pA, polyadenylation signals (pA1, BGH pA; pA2, SV40 pA; pA3, rabbit β-globin pA); U6, U6 RNA polymerase III promoter; UbC, human ubiquitin C promoter. (B) Mobilization regimens. Left panel: G-CSF + AMD3100 regimen. G-CSF (250 μg/kg) was given SC for 4 days, followed by 1 SC injection of AMD3100 (5 mg/kg) on the 5th day. Dexamethasone (10 mg/kg) was injected IP 16 hours and 1.75 hours before the first HDAd dosing to blunt cytokine responses. HDAd-PE5max was injected IV in 2 doses (each 4 × 10¹⁰ vp per animal) at 45 and 75 minutes after AMD3100. Right panel: WU-106 + AMD3100 regimen. WU-106 (10 mg/kg, SC) and AMD3100 (5 mg/kg, SC) were injected together. HDAd was administered IV 2 and 2.5 hours after WU-106 + AMD3100 (in 2 doses each 4 × 10¹⁰ vp per animal). Cytokine prophylaxis with dexamethasone was done 16 hours and 2.5 hours before the first HDAd injection. (C) Mobilized CD46/Townes mice were transduced with HDAd-PE5max via IV injection. Three rounds of selection were conducted on days 5, 19, and 33 after transduction by IP injection with the indicated doses of O⁶BG/BCNU. The primary mice were euthanized at week 16 after transduction. BM Lin^– cells were isolated from primary mice and infused into lethally irradiated C57BL/6 mice. The secondary transplanted mice were followed for 16 weeks for terminal analyses.