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Y. Liu et al.
transplanted recipients receiving D/D BM cells developed an acute and lethal BM failure, while recipients of D/+ BM cells developed a mild chronic anemia.15 As D/D mice develop lethal BM failure shortly after doxycycline induc- tion, the more severely affected D/D mice were used in the present study to investigate whether the lethal phe- notype could be fully rescued with the vector.
The clinically applicable single gene lentiviral vector was developed using a SIN lentiviral vector design har- boring the codon-optimized human RPS19 cDNA driven by an internal EFS promoter (named EFS-RPS19) (Figure 1C). Compared to the human codon-optimized RPS19 cDNA, there are six mismatches in the shRNA construct for generating the mouse model. Because of this, gene expression derived from the human codon-optimized RPS19 cDNA is not affected by the shRNA.
We first examined the transduction efficiency of the vector. In our previous study, we transduced cells at a MOI of 10-20 and demonstrated the rescue of the anemia and BM failure with a reduced risk of insertional mutage- nesis.12 To investigate the therapeutic effects of a lower average vector copy number per cell, we decided to use an MOI of 5-10. As shown in Online Supplementary Figure S1, the transduction efficiency was 75% on average in c- kit+ cells isolated from D/D mice. We next examined both endogenous Rps19 and vector-derived RPS19 mRNA expression in c--kit+ cells isolated from D/D and +/+ mice at a MOI of 5. As shown in Online Supplementary Figure S2, the endogenous Rps19 mRNA expression levels were significantly decreased in the cells isolated from D/D mice compared to the levels in cells from +/+ mice after doxycycline administration. Cells transduced with EFS- RPS19 exhibited a 2.5-fold higher level of expression of human RPS19 mRNA compared to the endogenous Rps19 expression in the D/D group. Interestingly, transduced cells isolated from +/+ mice showed a significantly lower level of human RPS19 mRNA expression than the trans- duced cells isolated from D/D mice at the same MOI, indicating the internal physiological regulation of excess RPS19 production as reported by others.29,30 The overall results indicated that cells transduced with the EFS-RPS19 vector could successfully express the human RPS19 trans- gene
Gene-corrected bone marrow cells can rescue the Diamond-Blackfan anemia phenotype in vivo
We next assessed the function of gene-corrected BM cells using the EFS-RSP19 vector in vivo. As shown in Figure 2A, uninduced (no doxycycline) c-kit+ BM cells from D/D mice (CD45.2) were transduced with the EFS- RPS19 vector (MOI=5-10), and then transplanted into lethally irradiated wild-type B6SJL recipient mice (CD45.1/CD45.2, named the EFS-RPS19 group). Mice receiving uninduced c-kit+ BM cells without vector trans- duction were regarded as the mock group (negative con- trol). Following engraftment and stable donor-derived reconstitution of the hematopoietic system, doxycycline was administrated to all recipients to induce the DBA phenotype. To determine whether the vector-treated cells could achieve a full correction, age-matched B6SJL wild- type (WT) mice receiving no irradiation and no transplan- tation but the same doxycycline administration were used as the control group.
Before doxycycline administration, both the mock and EFS-RPS19 groups showed high overall donor reconstitu- tion, indicating minimal to absent recipient-derived hematopoiesis (Online Supplementary Figure S3). After induction with doxycycline for 2 weeks, recipients in the mock group showed a dramatic decrease in red blood cell counts, mean corpuscular volume (MCV), and white blood cell and platelets counts, indicating that the mice developed BM failure shortly after doxycycline adminis- tration (Figure 2B-F). In contrast, recipients in the EFS- RPS19 vector-treated group showed normal blood cellu- larity compared to the WT group. To assess the long-term therapeutic effects, recipients were administered doxycy- cline for 16 weeks (Figure 3A). As shown in Figure 3B, most of the recipients in the mock group died (9 out of 16) due to severe anemia or BM failure (data not shown) at 2-3 weeks after doxycycline administration. The few remaining recipients exhibited a macrocytic anemia phe- notype with significantly reduced red blood cell counts and increased MCV at 16 weeks. The hemoglobin levels and platelet counts were also decreased compared to those in the WT group (Figure 3C-G). As expected, there was a significantly decreased expression of endogenous Rps19 in donor-derived BM cells from both the mock and
AB
C
Figure 1. The inducible Rps19-deficient mouse model and structure of the EFS-RPS19 self-inactivating lentiviral vector. (A) Overview of modified loci. Black arrow- heads indicate the transcriptional start sites. (B) Breeding strategy to adjust the level of Rps19 downregulation. Homozygous mice (D/D mice) are used in the project. (C) The self-inactivating lentiviral vector harboring a codon-optimized human RPS19 cDNA driven by human elongation factor 1a short (EFS) promoter. LTR: long ter- minal repeat; pA: polyadenylation signal; PPT: polypurine tract; RRE: Rev response element; SA: splice acceptor.
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