scholarly journals Disease Modeling and Phenotype Rescue Using Inducible Pluripotent Stem Cells from Patients with Diamond-Blackfan Anemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2496-2496
Author(s):  
Senthil Velan Bhoopalan ◽  
Min-Joon Han ◽  
Steven Ellis ◽  
Harry Lesmana ◽  
Jeremie H. Estepp ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Stem Cell ◽  
Research Funding ◽  
Bone Marrow Failure ◽  
Wild Type ◽  
Dna Breaks ◽  
Hematopoietic Stem ◽  
Diamond Blackfan Anemia ◽  
Bone Marrow Failure Syndromes

Diamond-Blackfan anemia (DBA) is a congenital ribosomopathy and bone marrow failure syndrome manifesting typically in infancy with erythroid hypoplasia. Approximately half of affected individuals also have developmental anomalies. Over time, additional cytopenias can develop, including reduced hematopoietic stem and progenitor cells (HSPC). Heterozygous loss-of-function mutations in over 20 ribosomal protein (RP) genes cause approximately 70% of DBA cases, although only 7 genes (RPS19, RPL5, RPS26, RPL11, RPL35a, RPS24 and RPS7) account for over 90% of patients with a known DBA genotype. Medical therapies including steroids, chronic transfusions are partially effective but have considerable side effects. Hematopoietic stem cell transplantation (HSCT) from matched related or unrelated donors is curative with recently reported good outcomes, although many patients lack a suitable donor and/or have serious treatment-related comorbidities that increase HSCT-related toxicities. Case reports of spontaneous genetic reversion in DBA suggest that RP gene-corrected HSPC have competitive advantage over RP-deficient cells, thus providing the rationale for gene therapy as a feasible therapeutic approach. Induced pluripotent stem cell (iPSC) technology provides a robust model of human disease and can recapitulate hematopoietic defects encountered in bone marrow failure syndromes. The goals of this study was to establish a culture system from patient-derived iPSCs that can recapitulate key aspects of DBA pathophysiology and provide a preclinical model for gene manipulation to correct the abnormal phenotype (Figure 1). We developed iPSCs from individuals with DBA who were enrolled on INSIGHT (NCT02720679), an IRB-approved, prospective study that includes biobanking of peripheral blood mononuclear cells (PBMNC) from patients with bone marrow failure syndromes. We first reprogrammed these DBA PBMNC into iPSCs using non-integrating Sendai virus to establish lines with pathogenic mutations in RPS19 (c.191>T, p.Leu64Pro), RPS19 (c.184C>T, p.Arg62Trp), RPL11 (c.61dupT, p.Cys21Leufs*13), and a variant of uncertain significance (VUS) in RPS7 (c.277_279delGTC, pVal93del). Undifferentiated iPSC lines exhibited abnormal ribosomal biogenesis revealed by polysome profiling and pre-rRNA analysis. Upon in vitrodifferentiation to hematopoietic lineages, the mutant iPSCs recapitulated DBA phenotypes with reduced CD34+ HSPCs, near absence of erythroid colonies (BFU-E and CFU-E) colonies and failure to produce erythroid cells in liquid culture. We used two methods to correct single nucleotide RP mutations in DBA iPSCs (Figure 1): i) CRISPR/Cas9-mediated homology-directed repair, and ii) base-editing, which utilizes catalytically inactive Cas9 fused to a deaminase that interconverts nucleotides directly in the absence of double-stranded DNA breaks. Corrected "isogenic" lines showed phenotype similar to wild type controls, with restored erythroid differentiation, and normal polysome maturation and pre-rRNA ratios. Because some patients carry large intragenic or whole RP gene deletions that are not amenable to gene correction, we also explored the feasibility of gene rescue by inserting a wild type copy of the defective gene (Figure 1). Using zinc-finger nuclease (ZFN), we inserted wild type RP cDNA constructs into the "safe harbor" AAVS1 locus on chromosome 19, thereby rescuing abnormal phenotypes of patient-derived iPSC lines with RPS19(p.Arg62Trp) and RPL11(p.Cys21Leufs*34) mutations. Additionally, we explored lentiviral gene delivery as an alternative method for RP gene replacement. We compared different promoters including MND, PGK and EF1a and found that the latter was most effective at rescuing RP gene expression in iPSC cells. Transduction of lentiviral vectors with wild type RPS19or RPL11fused to the EF1a promoter into three iPSC lines with RPS19or RPL11mutations resulted in stable transgene expression of RPS19or RPL11genes and phenotypic rescue. This study supports the feasibility of establishing iPSCs from DBA subjects with different genotypes. These iPSC lines provide a useful resource for numerous studies of DBA including preclinical approaches to gene therapy, evaluating the pathogenicity of RP gene variants of unknown significance and examining the pathophysiology of RP haploinsufficiency. Disclosures Estepp: Esperion: Consultancy; Forma Therapeutics: Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; Pfizer: Research Funding; Eli Lilly and Co: Research Funding; Daiichi Sankyo: Consultancy. Weiss:GlaxoSmithKline: Consultancy; Rubius Inc.: Consultancy; Cellarity Inc.: Consultancy; Beam Therapeutics: Consultancy; Esperion: Consultancy.

Gene Therapy Corrects the Lethal Bone Marrow Failure in a Mouse Model for RPS19-Deficient Diamond-Blackfan Anemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 513-513
Author(s):  
Pekka Jaako ◽  
Shubhranshu Debnath ◽  
Karin Olsson ◽  
Axel Schambach ◽  
Christopher Baum ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Mouse Model ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Bone Marrow Failure ◽  
Hematopoietic Stem ◽  
Diamond Blackfan Anemia ◽  
Stem And Progenitor Cells

Abstract Abstract 513 Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia associated with physical abnormalities and predisposition to cancer. Mutations in genes that encode ribosomal proteins have been identified in approximately 60–70 % of the patients. Among these genes, ribosomal protein S19 (RPS19) is the most common DBA gene (25 % of the cases). Current DBA therapies involve risks for serious side effects and a high proportion of deaths are treatment-related underscoring the need for novel therapies. We have previously demonstrated that enforced expression of RPS19 improves the proliferation, erythroid colony-forming potential and differentiation of patient derived RPS19-deficient hematopoietic progenitor cells in vitro (Hamaguchi, Blood 2002; Hamaguchi, Mol Ther 2003). Furthermore, RPS19 overexpression enhances the engraftment and erythroid differentiation of patient-derived hematopoietic stem and progenitor cells when transplanted into immunocompromised mice (Flygare, Exp Hematol 2008). Collectively these studies suggest the feasibility of gene therapy in the treatment of RPS19-deficient DBA. In the current project we have assessed the therapeutic efficacy of gene therapy using a mouse model for RPS19-deficient DBA (Jaako, Blood 2011; Jaako, Blood 2012). This model contains an Rps19-targeting shRNA (shRNA-D) that is expressed by a doxycycline-responsive promoter located downstream of Collagen A1 gene. Transgenic animals were bred either heterozygous or homozygous for the shRNA-D in order to generate two models with intermediate or severe Rps19 deficiency, respectively. Indeed, following transplantation, the administration of doxycycline to the recipients with homozygous shRNA-D bone marrow results in an acute and lethal bone marrow failure, while the heterozygous shRNA-D recipients develop a mild and chronic phenotype. We employed lentiviral vectors harboring a codon-optimized human RPS19 cDNA driven by the SFFV promoter, followed by IRES and GFP (SFFV-RPS19). A similar vector without the RPS19 cDNA was used as a control (SFFV-GFP). To assess the therapeutic potential of the SFFV-RPS19 vector in vivo, transduced c-Kit enriched bone marrow cells from control and homozygous shRNA-D mice were injected into lethally irradiated wild-type mice. Based on the percentage of GFP-positive cells, transduction efficiencies varied between 40 % and 60 %. Three months after transplantation, recipient mice were administered doxycycline in order to induce Rps19 deficiency. After two weeks of doxycycline administration, the recipients transplanted with SFFV-RPS19 or SFFV-GFP control cells showed no differences in blood cellularity. Remarkably, at the same time-point the recipients with SFFV-GFP homozygous shRNA-D bone marrow showed a dramatic decrease in blood cellularity that led to death, while the recipients with SFFV-RPS19 shRNA-D bone marrow showed nearly normal blood cellularity. These results demonstrate the potential of enforced expression of RPS19 to reverse the severe anemia and bone marrow failure in DBA. To assess the reconstitution advantage of transduced hematopoietic stem and progenitor cells with time, we performed similar experiments with heterozygous shRNA-D bone marrow cells. We monitored the percentage of GFP-positive myeloid cells in the peripheral blood, which provides a dynamic read-out for bone marrow activity. After four months of doxycycline administration, the mean percentage of GFP-positive cells in the recipients with SFFV-RPS19 heterozygous shRNA-D bone marrow increased to 97 %, while no similar advantage was observed in the recipients with SFFV-RPS19 or SFFV-GFP control bone marrow, or SFFV-GFP heterozygous shRNA-D bone marrow. Consistently, SFFV-RPS19 conferred a reconstitution advantage over the non-transduced cells in the bone marrow. Furthermore, SFFV-RPS19 reversed the hypocellular bone marrow observed in the SFFV-GFP heterozygous shRNA-D recipients. Taken together, using mouse models for RPS19-deficient DBA, we demonstrate that the enforced expression of RPS19 rescues the lethal bone marrow failure and confers a strong reconstitution advantage in vivo. These results provide a proof-of-principle for gene therapy in the treatment of RPS19-deficient DBA. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Treatment of inherited bone marrow failure syndromes beyond transplantation

Hematology ◽  
2017 ◽  
Vol 2017 (1) ◽  
pp. 96-101 ◽  
Author(s):  
Rodrigo T. Calado ◽  
Diego V. Clé
Keyword(s):  
Bone Marrow ◽  
Liver Toxicity ◽  
Bone Marrow Failure ◽  
Dyskeratosis Congenita ◽  
Hematopoietic Stem ◽  
Diamond Blackfan Anemia ◽  
Bone Marrow Failure Syndromes ◽  
Thrombopoietin Receptor ◽  
Cell Sources ◽  
Stimulating Factor

Abstract Despite significant progress in transplantation by the addition of alternative hematopoietic stem cell sources, many patients with inherited bone marrow failure syndromes are still not eligible for a transplant. In addition, the availability of sequencing panels has significantly improved diagnosis by identifying cryptic inherited cases. Androgens are the main nontransplant therapy for bone marrow failure in dyskeratosis congenita and Fanconi anemia, reaching responses in up to 80% of cases. Danazol and oxymetholone are more commonly used, but virilization and liver toxicity are major adverse events. Diamond-Blackfan anemia is commonly treated with corticosteroids, but most patients eventually become refractory to this treatment and toxicity is limiting. Growth factors still have a role in inherited cases, especially granulocyte colony-stimulating factor in congenital neutropenias. Novel therapies are warranted and thrombopoietin receptor agonists, leucine, quercetin, and novel gene therapy approaches may benefit inherited cases in the future.

Lentiviral FANCA Vectors Pseudotyped with a Modified Prototype Foamy Viral Envelope Corrects Murine Fanca−/− Hematopoietic Stem Cells with Reduced Toxicity.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1677-1677
Author(s):  
Zejin Sun ◽  
Yanzhu Yang ◽  
Yan Li ◽  
Daisy Zeng ◽  
Jingling Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Gene Transfer ◽  
Ex Vivo ◽  
Bone Marrow Failure ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Ex Vivo Culture

Abstract Fanconi anemia (FA) is a recessive DNA repair disorder characterized by congenital abnormalities, bone marrow failure, genomic instability, and a predisposition to malignancies. As the majority of FA patients ultimately acquires severe bone marrow failure, transplantation of stem cells from a normal donor is the only curative treatment to replace the malfunctioning hematopoietic system. Stem cell gene transfer technology aimed at re-introducing the missing gene is a potentially promising therapy, however, prolonged ex vivo culture of cells, that was utilized in clinical trials with gammaretroviruses, results in a high incidence of apoptosis and at least in mice predisposes the surviving reinfused cells to hematological malignancy. Consequently, gene delivery systems such as lentiviruses that allow a reduction in ex vivo culture time are highly desirable. Here, we constructed a lentiviral vector expressing the human FANCA cDNA and tested the ability of this construct pseudotyped with either VSVG or a modified prototype foamyvirus (FV) envelope to correct Fanca−/− stem and progenitor cells in vitro and in vivo. In order to minimize genotoxic stress due to extended in vitro manipulations, an overnight transduction protocol was utilized where in the absence of prestimulation, murine Fanca−/− bone marrow cKit+ cells were co-cultured for 16h with FANCA lentivirus on the recombinant fibronectin fragment CH296. Transduction efficiency and transfer of lentivirally expressed FANCA was confirmed functionally in vitro by improved survival of consistently approximately 60% of clonogenic progenitors in serial concentrations of mitomycin C (MMC), irregardless of the envelope that was utilized to package the vector. Transduction of fibroblasts was also associated with complete correction of MMC-induced G2/M arrest and biochemically with the restoration of FancD2 mono-ubiquitination. Finally, to functionally determine whether gene delivery by the recombinant lentivirus during such a short transduction period is sufficient to correct Fanca−/− stem cell repopulation to wild-type levels, competitive repopulation experiments were conducted as previously described. Follow-up of up to 8 months demonstrated that the functional correction were also achieved in the hematopoietic stem cell compartment as evidenced by observations that the repopulating ability of Fanca−/− stem cells transduced with the recombinant lentivirus encoding hFANCA was equivalent to that of wild-type stem cells. Importantly, despite the fact that the gene transfer efficiency into cells surviving the transduction protocol were similar for both pseudotypes, VSVG was associated with a 4-fold higher toxicity to the c-kit+ cells than the FV envelope. Thus, when target cell numbers are limited as stem cells are in FA patients, the foamyviral envelope may facilitate overall greater survival of corrected stem cells. Collectively, these data indicate that the lentiviral construct can efficiently correct FA HSCs and progenitor cells in a short transduction protocol overnight without prestimulation and that the modified foamy envelope may have less cytotoxicity than the commonly used VSVG envelope.

Comprehensive Analysis of Telomere Biology in Patients with Aplastic Anemia and Hypoplastic Myelodysplastic Syndrome: Further Evidence for a Common Mechanism

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2858-2858
Author(s):  
Anne-Sophie Bouillon ◽  
Monica S. Ferreira ◽  
Benjamin Werner ◽  
Sebastian Hummel ◽  
Jens P. Panse ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Myelodysplastic Syndrome ◽  
Aplastic Anemia ◽  
Bone Marrow Failure ◽  
Common Mechanism ◽  
Telomere Shortening ◽  
Hematopoietic Stem ◽  
Bone Marrow Failure Syndromes ◽  
The Individual

Abstract Introduction: Acquired aplastic anemia (AA) is typically characterized by pancytopenia and bone marrow (BM) failure mostly due to an autoimmune attack against the hematopoietic stem cell compartment. Thus, AA patients frequently respond to immunosuppressive therapy (IST). Hypoplastic myelodysplastic syndrome (hMDS) frequently mimics clinical and morphological features of AA and proper clinical discrimination of hMDS from AA sometimes remains difficult. Interestingly, some cases of hMDS respond at least partially to IST and on the other hand, AA can clonally evolve to hMDS. Telomeres shorten with each cell division and telomere length (TL) reflects the replicative potential of somatic cells. Whereas it is proposed that TL can to some degree discriminate hereditary subtypes of bone marrow failure syndromes from classical acquired forms, the role of TL for disease pathogenesis in hMDS remains unclear. In this study, we therefore aimed to investigate accelerated TL shortening as a possible (bio-)marker to distinguish hMDS from AA. Patients and Methods: TL of BM biopsies at diagnosis of 12 patients with hMDS and 15 patients with AA treated in the University Hospital Düsseldorf were analyzed. Mean age was 45.2 years in AA patients and 65.2 years in patients with hMDS. Confocal Q-FISH protocol was used for TL measurement as published previously (Blood, 2012). TL analysis was performed in single-blinded fashion. 28 patients (range 18-80 years) with newly diagnosed M. Hodgkin without BM affection were used as controls for linear regression and calculation of age-adapted TL difference. For the analysis of the data, we made use of a recently developed mathematical model of TL distribution on the stem cell level allowing us to extrapolate mean TL shortening per year (TS/y) based on the individual TL distributions of captured BM biopsies. Results: Using the controls to adjust for age, we found that age-adapted TL was significantly shortened both in patients with AA (median: -2.96 kb, range -4.21 to 0.26, p=0.001) and patients with hMDS (median: -2.26, range -3.85 to -0.64, p=0.005). In direct comparison, telomere shortening was more accelerated in patients with AA as compared to hMDS (p=0.048). Next, we analyzed the TL shortening per year (TS/y) based on the individual telomere distribution. Calculating the extrapolated TL shortening per year (TS/y), we found significant increased TS/y in AA patients (mean±SD: 235,8 bp/y±202,9, p=0.001) and hMDS patients (120,5±41,7 bp/y, p=0.0001) compared to controls (37,5±18,9 bp/y). Interestingly, the extrapolated rate of TS/y remained stable at different ages in hMDS patients as observed in healthy controls. In contrast, TS/y in AA patients showed a strong age-dependence with a maximum of TS/y in patients younger than 30 years (R²=0.42, p=0.008). Finally, we set to test whether TS/y can be used to identify AA or hMDS patients. Using 150 bp TS/y as a cut-off (4-fold the mean of controls), we found significantly more AA patients (10/15, 66.7%) had accelerated TL shortening compared to hMDS (1/12, 8.3% p=0.005). Conclusion: We provide first retrospective data on TL in patients with hMDS using confocal Q-FISH. Age-adapted TL is significantly shorter in patients with AA compared to hMDS. Interestingly, telomere shortening per year is both significantly increased in AA as compared to hMDS patients as well as in both groups compared to controls. The rate of telomere shortening TS/y might represent a new marker in patients with bone marrow failure syndromes that allows to discriminate AA from hMDS patients pending prospective validation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Inherited bone marrow failure syndromes: considerations pre- and posttransplant

Hematology ◽  
2017 ◽  
Vol 2017 (1) ◽  
pp. 88-95 ◽  
Author(s):  
Blanche P. Alter
Keyword(s):  
Bone Marrow ◽  
Birth Defects ◽  
Myeloid Leukemia ◽  
Bone Marrow Failure ◽  
Dyskeratosis Congenita ◽  
Hematopoietic Stem ◽  
Diamond Blackfan Anemia ◽  
Bone Marrow Failure Syndromes ◽  
Shwachman Diamond Syndrome ◽  
Generation Sequencing

Abstract Patients with inherited bone marrow failure syndromes are usually identified when they develop hematologic complications such as severe bone marrow failure, myelodysplastic syndrome, or acute myeloid leukemia. They often have specific birth defects or other physical abnormalities that suggest a syndrome, and sequencing of specific genes or next-generation sequencing can determine or confirm the particular syndrome. The 4 most frequent syndromes are Fanconi anemia, dyskeratosis congenita, Diamond Blackfan anemia, and Shwachman Diamond syndrome. This review discusses the major complications that develop as the patients with these syndromes age, as well as additional late effects following hematopoietic stem cell transplantation. The most common complications are iron overload in transfused patients and syndrome-specific malignancies in untransplanted patients, which may occur earlier and with higher risks in those who have received transplants.

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