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Gene therapy of DBA
EFS-RPS19 groups after 16 weeks of doxycycline admin- istration (Online Supplementary Figure S4). Strikingly, all the mice in the EFS-RPS19 group survived without any signs of anemia and with normal BM cellularity com- pared to the WT group. These results indicate that the lethal BM failure can be prevented by the vector. The vec- tor copy number at 16 weeks after transplantation was 5.2±1.6 and 4.7±1.0 on average in gene-corrected cells isolated from peripheral blood and BM, respectively (Figure 4A, B). We also analyzed the fraction of myeloid- erythroid compartments by flow cytometry (Online Supplementary Figure S5). Donor-derived hematopoiesis was observed in the EFS-RPS19 group, and the mean per- centage of donor cells (CD45.2) in every progenitor pop- ulation was significantly higher in the EFS-RPS19 group than in the mock group (Figure 4C-I). Unlike in the EFS- RPS19 group, the transplanted cells in the mock group had limited reconstituting ability. In addition, we observed a significantly higher reconstitution of resident
A
BCD
recipient cells (CD45.1/CD45.2) in the few surviving mice in the mock group than in the EFS-RPS19 group, per- haps explaining why these animals did not develop severe BM failure.
EFS-RPS19 vector-treated Rps19-deficient bone marrow cells provide long-term reconstitution
We next investigated whether the EFS-RPS19 vector- treated Rps19-deficient BM cells could generate long-term engraftment and reconstitution in doxycycline-induced lethally irradiated WT recipients (Figure 5A). To this end, D/D mice were induced with doxycycline for 1 week and red blood cell counts and hemoglobin levels were meas- ured to confirm the DBA phenotype (Online Supplementary Figure S6). The isolated Lin– BM cells from doxycycline- induced mice were transduced with the vector and trans- planted into the doxycycline-induced lethally irradiated WT recipients (EFS-RPS19 group). Recipients receiving untransduced Rps19-deficient Lin– BM cells from doxycy-
EF
Figure 2. Effective correction of anemia by the EFS-RPS19 vector at 2 weeks after induction of the Diamond-BLackfan pheno- type. (A) The scheme of the uninduced gene- corrected cell transplantation model and plan for examining short-term therapeutic effects. (B-F) Blood cellularity at 2 weeks after doxycycline induction (n=13-16, error bars represent the standard deviation, *P<0.05, **P<0.01, ***P<0.005 ****P<0.001 by one-way analysis of vari- ance). BM: bone marrow; MOI: multiplicity of infection; WT: wild-type; RBC: red blood cells; MCV: mean corpuscular volume; WBC: white blood cells.
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