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Y. Liu et al.
cline-induced D/D mice were regarded as the mock group. The setting of the WT group was the same as described above. Doxycycline was administrated to all the recipients directly after transplantation. After induction with doxycy- cline for 2-3 weeks, the majority of mice in the mock group died (13 out of 16 animals) due to severe anemia or BM failure (Figure 5B, Online Supplementary Figure S7). The few surviving mice exhibited significantly decreased levels of endogenous Rps19 expression in donor-derived BM cells at 16 weeks after doxycycline administration (Online Supplementary Figure S8) with the concomitant develop- ment of a severe anemia phenotype in the mock group (Figure 5C-G). In contrast, all recipients in the EFS-RPS19 group survived with normal blood cellularity compared to the WT group. The vector copy number was on average 8.3±4.0 and 10.9±3.9 in gene-corrected Rps19-deficient cells isolated from peripheral blood and BM, respectively (Figure 6A, B). By analyzing the fraction of myeloid-ery- throid compartments at 16 weeks, we observed almost complete donor-derived hematopoiesis in the EFS-RPS19
A
group, which was significantly higher than in the mock group (Figure 6C-I). Taken together, our results demon- strate that the EFS-RPS19 vector-treated group obtained full correction of anemia and BM failure.
Gene-corrected bone marrow cells showed polyclonal hematopoiesis and had a typical lentiviral insertion profile
The risk of insertional mutagenesis is a major concern for future applications of gene therapy in the clinic. To assess the safety of the EFS-RPS19 vector integration pro- file, as well as the clonal dynamics of the transduced cells, insertion site analysis was performed using the INSPI- IRED workflow.24 BM cells from uninduced donors (Figure 3A) of cohort 1 (animals 5-8) and cohort 2 (ani- mals 13-16), or those from doxycycline-induced donors (Figure 5A) of cohorts 3 (animals 7-11) and cohort 4 (ani- mals 17-20) were isolated 16 weeks after disease induc- tion in the recipients. Detailed information on pool size estimation and sequence diversity in each sample is
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Figure 3. Effective long-term correction of the anemia and bone marrow failure in mice treated with the EFS-RPS19 vector. (A) The scheme of the induced gene- corrected cell transplantation model and the plan for examining long-term therapeutic effects. (B) Surviral rate analysis. (C-G) Blood cellularity at 16 weeks after doxy- cycline induction (n=13-16, error bars represent the standard deviation, *P<0.05, **P<0.01, ***P<0.005 by one-way analysis of variance). 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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haematologica | 2022; 107(2)