RPS19-deficient Diamond-Blackfan Anemia Research Articles

Single-Cell-Multiomics Demonstrates Molecular Efficacy of a Clinical Lentiviral Vector for Gene Therapy of RPS19-Deficient Diamond-Blackfan Anemia

It has previously been demonstrated that lentiviral vector-mediated gene transfer of human codon-optimized RPS19 to hematopoietic stem cells by the gene therapy vector (CLIN-LV-EFS-coRPS19-PRE*) corrects the anemic phenotype of RPS19-deficient mice, supporting development of this vector for clinical gene therapy of RPS19-deficient Diamond-Blackfan Anemia (DBA). In this study, we evaluate the molecular efficacy of CLIN-LV-EFS-coRPS19-PRE* in CD34+ cells from RPS19-deficient DBA patients. CD34+ cells from two healthy donors and three DBA patients with confirmed heterozygous mutations in RPS19 were transduced using GMP-like reagents according to a protocol developed for clinical use. To evaluate the therapeutic effect of RPS19-gene transfer, transduced (GT) and untransduced (Mock) CD34+ cells were cultured under conditions supporting erythroid and myeloid progenitor proliferation and differentiation. In order to obtain a comprehensive molecular characterization of the therapeutic mechanisms, cells from day 9 of culture were subjected to CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing) analysis, a single-cell-multiomics method combining the advantages of FACS and scRNA-Seq for simultaneously analyzing transcriptomes alongside cell surface protein abundance at the single cell level. In GT cells, transduced (coRPS19-positive) cells were identified based on transgene expression. At day 9, 84-91% of erythroid progenitors in the GT-DBA samples expressed the coRPS19-transgene, while the frequency in myeloid progenitors was 29-56%. To reveal the molecular therapeutic effect of GT, we compared coRPS19-positive cells to Mock-treated cells from the same individuals. Expression of coRPS19 in DBA erythroid progenitor cells led to a significant induction of genes associated with terminal erythropoiesis (HEMGN, HBB, AHSP, EPB42 and GYPA) and down-regulation of genes associated with apoptosis and p53 activation (BAX, MDM2, ZMAT3 and MIR34AHG). GT also induced up-regulation of the large non-coding RNA LINC01133 and down-regulation of XACT. Interestingly, the two most-significantly changed genes in coRPS19-positive erythroid cells in all DBA samples were RPL22L1 and CD70. RPL22L1 is an RNA-binding component of the 60S ribosomal subunit that regulates pre-mRNA splicing but is not required for global cap-dependent translation. CD70 mRNA and protein exclusively expressed in erythroid progenitors in Mock-treated DBA samples and down-regulated in coRPS19-positive cells. CD70 is thus a new potential marker of DBA erythroid progenitor cells with a possible role in DBA pathogenesis. Myeloid progenitor DBA cells primarily responded to GT by up-regulation of ribosomal protein genes, suggesting RPS19-deficiency in myeloid progenitors leads to reduced ribosome biogenesis without the nucleolar stress observed in erythroid cells. To summarize, GT-induced changes in gene and protein expression agree with restoration of healthy ribosome biogenesis and elimination of nucleolar stress-induced p53 activation in erythroid progenitor cells responsible for the DBA phenotype, demonstrating molecular efficacy of CLIN-LV-EFS-coRPS19-PRE* supporting development for clinical gene therapy. In addition, the GT-induced reversal of RPS19-deficiency reveals several genes with potential relevance in DBA diagnostics and pathogenesis, such as RPL22L1 and CD70 for further investigation.

Bone marrow transplantation without myeloablative conditioning in a mouse model for Diamond-Blackfan anemia corrects the disease phenotype

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes coding for ribosomal proteins. Among these genes, the ribosomal protein S19 (RPS19) gene is the most frequently mutated. Previously, a mouse model deficient in RPS19 was developed by our laboratory, which recapitulates the hematopoietic disease phenotype by manifesting pathologic features and clinical symptoms of DBA. Characterization of this model revealed that chronic RPS19 deficiency leads to exhaustion of hematopoietic stem cells and subsequent bone marrow (BM) failure. In this study, we evaluated a nonmyeloablative conditioning protocol for BM transplants in RPS19-deficient mice by transplanting wild-type BM cells to RPS19-deficient recipients given no conditioning or sublethal doses of irradiation before transplant. We describe full correction of the hematopoietic phenotype in mice given sublethal doses of irradiation, as well as in animals completely devoid of any preceding irradiation. In comparison, wild-type animals receiving the same preconditioning regimen and number of transplanted cells exhibited significantly lower engraftment levels. Thus, robust engraftment and repopulation of transplanted cells can be achieved in reduced-intensity conditioned RPS19-deficient recipients. As gene therapy studies with autologous gene-corrected hematopoietic stem cells are emerging, we propose the results described here can guide determination of the level of conditioning for such a protocol in RPS19-deficient DBA. On the basis of our findings, a relatively mild conditioning strategy would plausibly be sufficient to achieve sufficient levels of engraftment and clinical success.

Successful gene therapy of Diamond-Blackfan anemia in a mouse model and human CD34+ cord blood hematopoietic stem cells using a clinically applicable lentiviral vector.

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure disorder in which pure red blood cell aplasia is associated with physical malformations and a predisposition to cancer. Twentyfive percent of patients with DBA have mutations in a gene encoding ribosomal protein S19 (RPS19). Our previous proof-of-concept studies demonstrated that DBA phenotype could be successfully treated using lentiviral vectors in Rps19-deficient DBA mice. In our present study, we developed a clinically applicable single gene, self-inactivating lentiviral vector, containing the human RPS19 cDNA driven by the human elongation factor 1αshort promoter, which can be used for clinical gene therapy development for RPS19-deficient DBA. We examined the efficacy and safety of the vector in a Rps19-deficient DBA mouse model and in human primary RPS19- deficient CD34+ cord blood cells. We observed that transduced Rps19-deficient bone marrow cells could reconstitute mice long-term and rescue the bone marrow failure and severe anemia observed in Rps19-deficient mice, with a low risk of mutagenesis and a highly polyclonal insertion site pattern. More importantly, the vector can also rescue impaired erythroid differentiation in human primary RPS19-deficient CD34+ cord blood hematopoietic stem cells. Collectively, our results demonstrate the efficacy and safety of using a clinically applicable lentiviral vector for the successful treatment of Rps19-deficient DBA in a mouse model and in human primary CD34+ cord blood cells. These findings show that this vector can be used to develop clinical gene therapy for RPS19-deficient DBA patients.

Oxidative DNA Damage, Inflammatory Signature, and Altered Erythrocytes Properties in Diamond-Blackfan Anemia.

Molecular pathophysiology of Diamond-Blackfan anemia (DBA) involves disrupted erythroid-lineage proliferation, differentiation and apoptosis; with the activation of p53 considered as a key component. Recently, oxidative stress was proposed to play an important role in DBA pathophysiology as well. CRISPR/Cas9-created Rpl5- and Rps19-deficient murine erythroleukemia (MEL) cells and DBA patients’ samples were used to evaluate proinflammatory cytokines, oxidative stress, DNA damage and DNA damage response. We demonstrated that the antioxidant defense capacity of Rp-mutant cells is insufficient to meet the greater reactive oxygen species (ROS) production which leads to oxidative DNA damage, cellular senescence and activation of DNA damage response signaling in the developing erythroblasts and altered characteristics of mature erythrocytes. We also showed that the disturbed balance between ROS formation and antioxidant defense is accompanied by the upregulation of proinflammatory cytokines. Finally, the alterations detected in the membrane of DBA erythrocytes may cause their enhanced recognition and destruction by reticuloendothelial macrophages, especially during infections. We propose that the extent of oxidative stress and the ability to activate antioxidant defense systems may contribute to high heterogeneity of clinical symptoms and response to therapy observed in DBA patients.

A CLINICALLY APPLICABLE LENTIVIRAL VECTOR CORRECTS THE ANEMIA AND BONE MARROW FAILURE IN A MOUSE MODEL FOR DIAMOND-BLACKFAN ANEMIA

Diamond-Blackfan Anemia (DBA) is a congenital erythroid hypoplasia caused by dysfunction of genes coding for ribosomal proteins. The ribosomal protein S19 (RPS19) gene is the most frequently mutated. Our previous studies showed that gene therapy with autologous HSCs modified to express RPS19 represents a potential therapeutic option. Here, we assessed the efficacy of a clinically applicable single gene lentiviral vector with a good safety profile for treatment of RPS19-deficient DBA. We designed a lentiviral vector harbouring a codon-optimized human RPS19 cDNA driven by the shortened version of the human elongation factor 1α (EFS) promoter. We transduced c-Kit+ bone marrow cells isolated from our established doxycycline inducible RPS19-deficient DBA mouse model and injected cells into lethally irradiated wild-type mice (Vector). The recipients transplanted with mock transduced c-Kit+ bone marrow cells from DBA mice were regarded as Mock group. After transplantation, cells were engrafted for 2 months, followed by doxycycline induction to recipients to induce DBA phenotype. The age matched wild-type mice receiving no irradiation and transplantation but same doxycycline induction concentration were regarded as control group (WT). The short-term effect of vector was analysed at 2 weeks after doxycycline induction. In Vector group, all recipients showed normal blood cellularity (RBC:10.79±1.05 × 109/L, Hb:162.75±5.26g/L) comparing with WT group (RBC:8.55±1.03 × 109/L, Hb:162±2.45g/L), while in Mock group, half of mice died due to dramatic BM failure and rest of mice showed DBA phenotype (RBC:4.74±2.2 × 109/L, Hb:79.13±34.24g/L). Our results demonstrate the feasibility and preclinical efficacy for treatment of DBA by using lentiviral vector. The long-term effects (16 weeks) and analysis of vector integration sites are in progress.

Enforced expression of RPS19 derived from human elongation factor 1A short (EFS) promoter corrects the anemia and lethal bone marrow failure in mouse model for RPS19-deficient diamond-blackfan anemia

Enforced expression of RPS19 derived from human elongation factor 1A short (EFS) promoter corrects the anemia and lethal bone marrow failure in mouse model for RPS19-deficient diamond-blackfan anemia

Disruption of the 5S RNP-Mdm2 interaction significantly improves the erythroid defect in a mouse model for Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by haploinsufficiency of genes encoding ribosomal proteins (RPs). Perturbed ribosome biogenesis in DBA has been shown to induce a p53-mediated ribosomal stress response. However, the mechanisms of p53 activation and its relevance for the erythroid defect remain elusive. Previous studies have indicated that activation of p53 is caused by the inhibition of mouse double minute 2 (Mdm2), the main negative regulator of p53, by the 5S ribonucleoprotein particle (RNP). Meanwhile, it is not clear whether this mechanism solely mediates the p53-dependent component found in DBA. To approach this question, we crossed our mouse model for RPS19-deficient DBA with Mdm2(C305F) knock-in mice that have a disrupted 5S RNP-Mdm2 interaction. Upon induction of the Rps19 deficiency, Mdm2(C305F) reversed the p53 response and improved expansion of hematopoietic progenitors in vitro, and ameliorated the anemia in vivo. Unexpectedly, disruption of the 5S RNP-Mdm2 interaction also led to selective defect in erythropoiesis. Our findings highlight the sensitivity of erythroid progenitor cells to aberrations in p53 homeostasis mediated by the 5S RNP-Mdm2 interaction. Finally, we provide evidence indicating that physiological activation of the 5S RNP-Mdm2-p53 pathway may contribute to functional decline of the hematopoietic system in a cell-autonomous manner over time.

Glucocorticoids Reduce Expression Of p53 Target Genes and Reverse Anemia In a Mouse Model Of Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a rare congenital disease of macrocytic anemia, increased cancer predisposition and progressive bone marrow failure. Mutations in ribosomal protein genes are responsible for most cases and the most commonly mutated gene, RPS19 is affected in around 25% of DBA patients. Although glucocorticoid (GC) administration has been used to stimulate erythropoiesis in DBA patients for decades, the therapeutic mechanism of GC treatment is poorly understood. Since it has been shown that bone marrow failure in DBA is caused by p53 activation we hypothesized the therapeutic mechanism of GC involves reduced p53 activity. To assess possible connections between p53 activity and GC administration in DBA we used a doxycycline-inducible mouse model for RPS19-deficient DBA (Jaako et al, Blood 2011). In these mice, doxycycline administration leads to reduced Rps19 levels, defective ribosomal biogenesis, proliferative arrest, increased apoptosis of erythroid progenitors and p53-dependent bone marrow failure.In this study we show that Rps19-deficient mice respond to treatment with the GC prednisolone, with a significant increase in red blood cell count as well as in hemoglobin concentration and hematocrit compared to Rps19-deficient controls (p-values<0.05). Also, prednisolone treated Rps19-deficient mice show a significant increase in the Ter119+ erythroid cell fraction in the bone marrow compared to Rps19-deficient controls. Gene expression analysis of c-Kit+ fetal liver cells from E14.5-15.5 of Rps19-deficient embryos cultured in vitro in the presence of doxycycline for 3 days, showed an increase in p53 responsive gene expression of multiple p53 targets such as p21, Bax, Ccng1, Phlda3 and Prl-3. However, upon administration of the synthetic GC dexamethasone all these genes significantly failed to up-regulate, even in the presence of doxycycline (p-values< 0.02). Also, dexamethasone treated RPS19-deficient c-Kit+ fetal liver cells show an increase in proliferation compared to RPS19-deficient controls (p-value< 0.05), that starts already after 2 days of in vitro culture, being more pronounced over time. These results indicate that administration of GC dampens p53 responsive gene expression of genes known to promote cell cycle arrest as well as apoptosis. Although GCs are known to regulate many functions within an organism, our results indicate that the therapeutic effect of GC in DBA patients at least in part is explained by inhibition of p53 responsive gene activation, leading to increased proliferation and survival of erythroid progenitors. Disclosures:No relevant conflicts of interest to declare.

Imbalance of p53 Family Members as a New Target of Therapeutics for Treatment of Diamond Blackfan Anemia.

Diamond-Blackfan anemia (DBA) is characterized by defective erythropoiesis, congenital anomalies and risk of malignancies. Ribosomal protein (RP) S19 is mutated in about 25% of patients and RPS24 in 2%. RPS19 deficiency impairs ribosomal biogenesis. It still remains unknown how impaired ribosome biogenesis and function lead to DBA. We found that RPS19 deficiency in zebrafish results in hematopoietic and developmental abnormalities resembling DBA. Our data suggest that the RPS19-deficient phenotype is mediated by upregulation of the p53 family genes. DeltaNp63 is BMP4 target and during gastrulation, required for specification of non-neural ectoderm. Its upregulation suppresses the development of neural structures and is likely to contribute to brain and craniofacial defects observed in RPS19 deficient zebrafish and DBA. In response to ribosomal stress from RPS19 deficiency, deltaNp63 expression is induced in erythroid progenitors suggesting a role for deltaNp63 in hematopoiesis. Similar to other vertebrates, zebrafish experience two waves of blood formation, primitive, producing mostly erythrocytes and macrophages and definitive producing all types of blood cells. Erythroid progenitors proliferate and differentiate into mature erythrocytes in the Intermediate Cell Mass (ICM), a region between the dorsal aorta and axial vein. We found that number of blood cells in the ICM was reduced in RPS19-deficient embryos and their differentiation was delayed. Morphologically, erythrocytes from morphants differed from wild-type cells. They varied in size and some cells retained a blast-like phenotype. The hemoglobin level was also decreased in morphants. The immature phenotype of RPS19-deficient erythroid cells and their inability to differentiate into mature erythrocytes may be explained by DNp63 induction. Importantly, downregulation of expression of both p53 and DNp63 can rescue the RPS19 deficient phenotype in zebrafish. Our findings suggest that suppression of p53 pathway might be beneficial for DBA patients with RPS19 mutations. Several drugs were reported to be effective in the treatment of DBA although the mechanism of their action is unknown. They include such dissimilar compounds as corticosteroids, cyclosporine A, and valproic acid. Metoxyclopramide was reported as effective by some authors but not others. We treated RPS19-deficient embryos with these drugs and found that dexamethasone, cyclosporine A, and valproic acid improve RPS19-deficient phenotype. We further found that treatment of embryos with dexamethasone and cyclosporine A influences p53 level. Metoxyclopramide was not effective in rescuing RPS19-deficient embryos and had no effect on p53 level. We next questioned if known compounds inhibiting p53 pathway can improve the phenotype and survival of RPS19-deficient zebrafish. Pifithrin m inhibits p53 binding to mitochondria by reducing its affinity to antiapoptotic proteins Bcl-xL and Bcl-2. It specifically suppresses the mitochondria branch of the p53 pathway but does not affect other p53 functions. Pifithrin has no effect of the p53 level in zebrafish embryos but was efficient in improving RPS19-deficient phenotype. These data suggest that suppression of p53 activity might be a mechanism of action of some drugs currently used for DBA treatment and open new directions for development of novel drugs.

Deficient Ribosomal Protein S19 in Diamond-Blackfan Anemia Causes a Reduction in bcl-2 and Bad and Leads to Apoptosis in Erythroid Progenitor Cells.

Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia in which 25% of the patients have a mutation in the ribosomal protein (RP) S19 gene. It is not known how the RPS19 deficiency impairs erythopoiesis and proliferation of hematopoietic progenitors. We have established an in vitro models for RPS19 deficient DBA using lentiviral vector mediated doxycycline (Dox) inducible small interfering RNA (siRNA) against RPS19 (Mol Ther. 11:627–637. 2005). Suppression of cell growth and erythroid colony formation correlated with the suppression level of RPS 19, indicating that these cell lines are useful to determine the mechanisms of RPS19 deficient DBA. To elucidate molecular mechanisms in RPS19 deficient DBA, we analyzed cell cycle of Dox induced RPS19 deficient TF-1 cells. RPS19 deficient TF-1 cells showed G0/G1 arrest (82% vs 58%, p<0.05) together with accumlation of p21 and p27, and apoptotic cells detected by Annexin-V analysis also increased compared to control Dox induced TF-1 cells (13% vs 3.1%, p<0.05). Increase of apoptotic cells in RPS19 deficient cells was confirmed by TUNEL assay. Western blot analysis of apoptotic related protein showed that the level of bcl-2 and Bad was decreased in RPS19 deficient TF1 cells compared to control cells. This down-regulation of apoptotic related protein was improved by transduction with lentiviral vector expressing modified RPS19, which is not affected by siRNA but produce normal functional RPS19 protein and rescues the DBA phenotype. Moreover, primary CD34 positive cells from DBA patients detected by Annexin-V analysis also generate a high number of apoptotic cells compared to normal CD34 positive cells during in vitro culture (38% vs 8.9%, n=5, p<0.001). These findings indicate that erythroid progenitor cells are more sensitive to apoptosis than other hematopoietic progenitors and that RPS19 deficiency causes apotosis and accelerated loss of erythroid progenitors in RPS19 deficient DBA.

Development of cellular models for ribosomal protein S19 (RPS19)-deficient diamond–blackfan anemia using inducible expression of siRNA against RPS19

Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia in which 25% of the patients have a mutation in the ribosomal protein S19 (RPS19) gene. No models exist for RPS19-deficient DBA and the molecular pathogenesis is unknown. To establish an in vitro inducible model for DBA, human erythroid leukemic cell lines, TF-1 and UT-7 cells, were cotransduced with a lentiviral vector expressing the green fluorescent protein (GFP) gene and small interfering RNA (siRNA) against RPS19 controlled by a tet operator regulatory element and another transactivator vector containing the red fluorescent protein (RFP) gene and the cDNA encoding a tetracycline-controllable transcriptional repressor. Following transduction, the RFP-positive and GFP-negative cell population was sorted by flow cytometry. Upon incubation with doxycycline (0.5 mug/ml), more than 98% of cells expressed GFP and the siRNA. Significant suppression of erythroid differentiation, cell growth, and colony formation was observed in cells treated with siRNA against RPS19 but not in cells treated with a control vector. These findings show that RPS19 plays an important role in the regulation of hematopoietic cell proliferation and erythroid differentiation. These novel cell lines represent models for RPS19-deficient DBA and can be used to identify the molecular mechanisms in RPS19-deficient DBA.

Apoptotic Change Is a Major Reason for Defect in Early Erythroid Development in Cell Line Models for Ribosomal Protein (RP) S19 Deficient Diamond-Blackfan Anemia.

Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia in which 25% of the patients have a mutation in the ribosomal protein (RP) S19 gene. It is not known how the RPS19 deficiency impairs erythopoiesis and proliferation of hematopoietic progenitors. The majority of cases appear to result from an intrinsic disorder of the erythroid progenitor that involves its inability to respond normally to inducers of erythroid proliferation and differentiation. Erythropoietin (Epo) controls the proliferation, differentiation and survival of the erythroid progenitors. However, so far, no receptor-ligand defects have yet been identified. We have established an in vitro models for RPS19 deficient DBA using lentiviral vector mediated doxycycline (Dox) inducible small interfering RNA (siRNA) against RPS19 (Blood 102:504a, 2003). To analyze the effect of suppression of RPS19 for erythroid differentiation, we used cytokine dependent TF-1 cell lines (CD34+, CD71+, GFAlow) together with new established cytokine independent K562 cell lines (CD34−, CD71+, GFAhigh). We established two types of cell lines (TF-1A, K562A and TF1B, K562B) using different siRNA against RPS19. Five days after Dox induction, RPS19 protein level was suppressed to 45–55% in TF-1A and K562A, 65–75% in TF1B and K562B compared to control scramble transduced cell lines (TF1S, K562S) by western blot analysis. Suppression of cell growth and colony formation correlated with the suppression level of RPS 19 in TF1A, B and K562A, B cells. In contrast, after stimulation with Epo, glycophorin A expression, measured by flow cytometry, and hemoglobin content (DAF staining) showed suppression of erythroid differentiation only in TF1A and TF1B but not in K562A and K562B. Since Epo induces the stimulation of Jak2 tyrosine kinase which leads to the tyrosine phosphorylation of several proteins including the Epo receptor itself. As a result, different intracellular pathways are activated such as Ras/MAP kinase, phosphatidylinositol 3-kinase and STAT transcription factors. Therefore, we analyzed Epo stimulated signal transduction in these cell lines. However, no abnormal signal transduction could be detected in any of the cell lines. Cell cycle analysis showed that the percentage of cells in the G0/G1 phase increased and apoptotic cells detected by Annexin-V analysis also increased in TF1 (A<B) and K562 (A<B) cells. Western blot analyzed p21 showed that the level of p21 was increased in TF1 (A<B) and K562 (A<B) cells. These results indicate that Epo triggered onset of terminal maturation is intact in RPS19 deficient DBA cell line models. We speculate that apoptotic change by suppressed RPS19 may be one of the major reason for the accelerated loss of erythroid progenitor clonogenicity in RPS19 deficient DBA. These cell lines are useful to determine the mechanisms of RPS19 deficient DBA.

900. Development of Models for RPS19 Deficient Diamond-Blackfan Anemia Using Regulatable Expression of siRNA Against RPS19

Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia in which 25% of the patients have a mutation in the ribosomal protein (RP) S19 gene. We have previously shown that RPS19 transgene overexpression improves erythroid development and proliferation in RPS19 deficient DBA cells (Blood 100: 2724–31, 2002; Mol Ther 7: 613–22, 2003). It is not known how the RPS19 deficiency impairs erythopoiesis and proliferation of hematopoietic progenitors and a model for RPS19 deficient DBA is not available for development of gene therapy. Therefore, to establish a model system for RPS19 deficient DBA, a lentiviral vector was generated where the GFP gene is controlled by an EF1a promoter and an expression cassette containing a small interfering RNA (siRNA) against RPS19 controlled by a polIII promoter and a tet operator element (siRPS19). The control vector is analogous except it expresses an siRNA against a random sequence. Suppression of expression of RPS19 was detected in siRPS19 transduced primary CD34+ cells by quantitative RT-PCR and Northern blot analysis. BFU-E and CFU-G/M colonies from siRPS19 transduced CD34+ cells were decreased compared to control vector transduced cells. Suppression of cell growth and erythroid differentiation was also observed in erythroid liquid cultures. The hematopoietic and erythropoietic phenotype of these knockdown BM cells mimics the that of BM cells from DBA patients. To establish a RPS19 deficient cell line model, inducible expression of the siRNA was achieved by co-transduction of another lentiviral vector containing the tetracycline repressor gene and red fluorescent protein (RFP) gene (J Virol 77, 8957–61, 2003). Human erythroid cell lines, TF-1 and UT-7 cells were co-transduced with siRPS19 or the control vector and the transactivator vector. To generate inducible cell lines, RFP positive and GFP negative populations were sorted. When these cells are incubated with doxycycline (Dox), more than 97% cells expressed GFP. We observed suppression of cell growth and colony formation in siRPS19 transduced cells but not in the control vector transduced cells. Significant suppression of erythroid differentiation was observed by analysis of glycohporin A and evaluation of hemoglobin content. Cell cycle analysis showed that the percentage of cells in the G0/G1 phase increased and apoptotic cells detected by Annexin-V binding analysis were also increased in siRPS19 transduced cells. These findings show that RPS19 plays an important role in the regulation of hematopoietic cell proliferation, apoptosis and erythroid differentiation. This regulatable expression system of siRNA has therefore been successfully used to generate a cell line model for DBA. We asked whether transgene overexpression of RPS19 can rescue growth suppression in these DBA model cell lines, using engineered RPS19 cDNA which is not affected by the siRNA knockdown but produces normal RPS19 protein (eRPS19). Improvement of cell growth and colony formation was detected in eRPS19 transduced cells. These model systems can be used to identify the molecular mechanism in RPS19 deficient DBA and for development of gene therapy.

Proliferation deficiency of multipotent hematopoietic progenitors in ribosomal protein S19 (RPS19)-deficient diamond–Blackfan anemia improves following RPS19 gene transfer

Diamond–Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by a specific deficiency in erythroid progenitors. Since some patients with DBA develop a reduction in thrombocytes and granulocytes with age, we asked whether multipotent hematopoietic progenitors from DBA patients had normal proliferative capacity in liquid expansion cultures. CD34+ cells derived from DBA patients showed deficient proliferation in liquid culture containing IL-3, IL-6, and SCF. Single CD34+ CD38− cells from DBA patients exhibited deficient proliferation recruitment in a limiting dilution assay containing IL-3, IL-6, SCF, Tpo, FL, and G-CSF or containing IL-3, IL-6, and SCF. Our findings suggest that the underlying hematopoietic defect in DBA may not be limited to the erythroid lineage. Since a fraction of DBA patients have a deficiency in ribosomal protein S19 (RPS19), we constructed lentiviral vectors containing the RPS19 gene for overexpression in hematopoietic progenitors from RPS19-deficient DBA patients. Enforced expression of the RPS19 transgene improved the proliferation of CD34+ cells from DBA patients with RPS19 mutation. Similarly, enforced expression of RPS19 improved erythroid development of RPS19-deficient hematopoietic progenitors as determined by colony assays and erythroid differentiation cultures. These findings suggest that gene therapy for RPS19-deficient DBA is feasible.

Gene transfer improves erythroid development in ribosomal protein S19–deficient Diamond-Blackfan anemia

Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by a specific deficiency in erythroid progenitors. Forty percent of the patients are blood transfusion-dependent. Recent reports show that the ribosomal protein S19 (RPS19) gene is mutated in 25% of all patients with DBA. We constructed oncoretroviral vectors containing the RPS19 gene to develop gene therapy for RPS19-deficient DBA. These vectors were used to introduce the RPS19 gene into CD34(+) bone marrow (BM) cells from 4 patients with DBA with RPS19 gene mutations. Overexpression of the RPS19 transgene increased the number of erythroid colonies by almost 3-fold. High expression levels of the RPS19 transgene improved erythroid colony-forming ability substantially whereas low expression levels had no effect. Overexpression of RPS19 had no detrimental effect on granulocyte-macrophage colony formation. Therefore, these findings suggest that gene therapy for RPS19-deficient patients with DBA using viral vectors that express the RPS19 gene is feasible.