High dose of AAV9 particles targets bone marrow, including HSPCs. (A) Representative anti-EGFP immunohistochemistry (IHC) images of bone marrow corresponding to the tibia. Mice were injected with the AAV9-EGFP vector or AAV9-empty vector at a concentration of 3.5 × 10¹² viral genomes (vg) per mouse. EGFP-positive cells were mainly located toward the end of the bones. Bars represent 500 μm (left) and 50 μm (right); hematoxylin and eosin stain. (B) Percentage of EGFP-positive cells relative to the total number of cells. Cells were separately counted in joint adjacent areas and in the middle of the bone. (C-D) Tert mRNA expression level in total bone marrow isolated 2 weeks (C) and 8 months (D) after VI with 3.5 × 10¹² viral genomes per mouse. AAV9-Tert relative to the expression of mice injected with the same amount of AAV9-empty vector. (E) Δ Ct values (Tert minus Act1) of the quantitative real-time PCR shown in panel D. Quantitative real-time PCR determined relative Tert expression in HSPCs (HSCs) sorted by FACS (F) and lineage-committed cells (G). (H) Colony-forming assay in MethoCult with whole bone marrow cells isolated from mice injected with AAV9-Tert or AAV9-empty. For all experiments, n indicates number of mice. Data are mean ± SEM. Statistical analysis: 2-sided Student t test; P values are shown. SEM, standard error of the mean; VI, virus injection.