Lentiviral Gene Therapy for the Correction of Congenital Dyserythropoietic Anemia Type II

Abstract Congenital dyserythropoietic anemias (CDAs) are a group of inherited anemias that affect the development of the erythroid lineage. CDA type II is the most common one: it accounts for around 60% of all cases, and more than 600 cases have been reported so far. CDA II is caused by biallelic mutations in the SEC23B gene and is characterized by ineffective erythropoiesis with morphologic abnormalities of erythroblasts, hemolysis, and secondary iron overload, which is the most frequent complication. Patients usually suffer from variable degrees of jaundice, splenomegaly, and absolute reticulocyte count inadequate depending on the degree of anemia. Hydrops fetalis, aplastic crisis and gallstones are other associated clinical signs. CDA II bone marrow is characterized by the presence of more than 10% mature binucleated erythroblasts. Another distinctive feature of CDA II erythrocytes is hypoglycosylation of membrane proteins. The management of CDA II is generally limited to blood transfusion and iron chelation. Splenectomy has proved to reduce the number of transfusions in CDA II patients. However, allogenic hematopoietic stem cell transplant (HSCT) represents the only curative option for this disease. Autologous HSCT of genetically corrected cells will mean a definitive treatment for CDA II, overcoming the limitations of allogeneic HSCT, such as limited availability of HLA-matched donors, infections linked to immunosuppression or development of graft versus host disease. This strategy has been used to treat many inherited hematological diseases, including red blood cell diseases such as β-thalassemia, sickle cell disease or pyruvate kinase deficiency. Therefore, we have addressed a similar strategy to be applied to CDAII patients. Two different lentiviral vectors carrying either wild type or codon optimized versions of SEC23B cDNA (wtSEC23B LV or coSEC23B LV, respectively) under the control of human phosphoglycerate kinase promoter (PGK) have been developed. Taking advantage of a CDA II model, in which SEC23B knock-out was done in human hematopoietic progenitors through gene editing, we have determined the most effective SEC23B LV version and the most suitable multiplicity of infection (MOI) to compensate protein deficiency. SEC23B knock out human hematopoietic progenitors (CD34 + cells; 80% frame shift mutations; SEC23BKO) showed a sharp reduction in SEC23B protein level. Those SEC23BKO hematopoietic progenitors were transduced with both lentiviral vectors at MOIs ranged from 3 to 25. We observed that SEC23B protein reached physiological or even supraphysiological levels. In addition, the reduction in the number of erythroid colony forming units (CFUs) identified in SEC23BKO CD34 + cells, was partially restored in the LV transduced SEC23BKO progenitors. Significantly, we observed a clear correlation between the used MOI and the vector copy number (VCN) in the CFUs derived from transduced SEC23BKO CD34 + cells. Furthermore, SEC23BKO hematopoietic progenitors were subjected to an in vitro erythroid differentiation protocol. A sharp decrease in the cell growth throughout erythroid differentiation was observed in SEC23BKO condition. However, the transduction with any of SEC23B LVs at MOIs above 10 was able to recover cell expansion to values equal to wild type cells. Interestingly, total level of protein glycosylation during erythroid differentiation was enhanced after SEC23B LV transduction. Glycosylation level in wtSEC23B LV transduced SEC23BKO cells was most similar to the level in wild type cells. Then, we transduced peripheral blood-derived hematopoietic progenitors (PB-CD34 + cells) from a CDA II patient with wtSEC23B LV at MOI 25 and differentiated in vitro to erythroid cells. A complete restauration of SEC23B protein expression and a cell growth increase of wtSEC23B transduced CDAII was observed with vector copy numbers of 0.3 after 14 days under erythroid conditions. More importantly, we could find a decrease in the percentage of bi-/multinucleated erythroid cells generated in vitro after wtSEC23B LV transduction. In summary, SEC23B LV compensate the SEC23B deficiency in SEC23BKO and in CDAII hematopoietic progenitor cells, paving the way for gene therapy of autologous hematopoietic stem and progenitor cell as an alternative and feasible treatment for CDA II. Disclosures Bianchi: Agios pharmaceutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Sanchez: Bloodgenetics: Other: Co-Founder and promoter; UIC: Current Employment. Ramirez: VIVEBiotech: Current Employment. Segovia: Rocket Pharmaceuticals, Inc.: Consultancy, Research Funding. Quintana Bustamante: Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company.

Download Full-text

Modelling Congenital Dyserythropoietic Anemia Type II through Gene Editing in Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood-2020-139207 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 27-27

Author(s):

Mercedes Dessy-Rodriguez ◽

Sara Fañanas-Baquero ◽

Veronica Venturi ◽

Salvador Payán-Pernía ◽

Cristian Tornador ◽

...

Keyword(s):

Gene Editing ◽

Erythroid Differentiation ◽

Therapeutic Approaches ◽

Hematopoietic Stem ◽

Knock Out ◽

New Therapeutic Approaches ◽

Multinucleated Cells ◽

Cda Ii

Congenital dyserythropoietic anemias (CDAs) are a group of inherited anemias that affect the development of the erythoid lineage. They are characterized by ineffective erythropoiesis with distinct morphologic abnormalities of erythroblasts, a degree of hemolysis, and secondary hemochromatosis. Patients usually present with congenital anemia, jaundice, splenomegaly, and an absolute reticulocyte count inadequate for the degree of anemia. CDA type II (CDAII) is the most frequent type. CDAII patients show anemia of variable degrees, and 20% are transfusion dependent. The most specific finding of CDAII marrow is the presence of more than 10% mature binucleated erythroblasts with two nuclei at the same erythroid maturation stage. Treatment of CDAII patients may involve blood transfusions, iron chelation therapy and splenectomy. The only described definitive therapy is allogeneic bone marrow transplantation, which implies additional side effects for these patients. Therefore, new therapeutic approaches are needed. CDA II is caused by mutations in the SEC23B gene. SEC23B is part of coat protein complex II (COPII). COPII is involved in protein processing and Golgi-reticulum trafficking. However, how mutations in SEC23B cause CDA II is not known yet. Therefore, studying the role of SEC23B in the erythropoiesis will help to elucidate the underlying mechanism of CDA II and to develop new therapeutic approaches for the disease. We have developed a CDA II model in human cells through the introduction of genomic mutations in the gene using the CRISPR/Cas9 gene editing system. Different single guides RNAs (sgRNA) targeting the start of the coding sequence of human SEC23B gene were designed and tested in human erythroleukemia K562 cell line and in healthy human cord blood hematopoietic stem and progenitors (hCB-CD34+). The gene editing outcome at SEC23B gene was assessed at: i) genomic level through Sanger sequencing, Inference of CRISPR Edits (ICE) and Next-Generation Sequencing (NGS). ii) Protein level through Western-blot analysis. iii) Functional level through morphological analysis and erythroid differentiation either in vitro or in vivo in human hematopoietic chimeras in NOD.Cg-KitW-41JTyr+PrkdcscidIl2rgtm1Wjl/ThomJ (NBSGW) mice. K562 cells were nucleofected with three different sgRNAs, as ribonucleoprotein (RNP), independently or in combination. Afterwards, seventy five K562 clones were established from the cells nucleofected with the most efficient sgRNA or with the combination of the three sgRNAs. Forty per cent of them showed a high efficiency of knock-out (higher than 50% of alleles). Eight SEC23BKO clones were selected for further analysis. All of those eight clones showed a reduction in SEC23B protein and six of them had a lower proliferation than control cells and morphological abnormalities, such as presence of bi/multinucleated cells. Moreover, when CB-CD34+ cells were nucleofected with the most efficient sgRNA or with the combination of the three sgRNAs, up to 80% of knock-out efficiency and close to 90% reduction of SEC23B protein were obtained. Interestingly, when those gene edited hematopoietic progenitors were differentiated in vitro to erythroid cells, their terminal differentiation was hampered, with a reduce percentage of enucleated cells and the presence of high number of bi/multinucleated cells. Similarly, the in vivo erythroid differentiation of these gene edited progenitors two months after HSPC transplant into NBSGW mice showed again an impairment of terminal erythroid differentiation with an increment in the percentage of erythroid bi/multinucleated cells without altering other human hematopoietic lineages. In summary, CRISPR/Cas9 system has been used to model CDA II in a human cell line and in human hematopoietic progenitors through the knock-out of SEC23B gene. Our system reproduced the most relevant feature characteristic of CDA II pathology. This gene editing based CDA II model will allow the study of how mutations in SEC23B cause CDA II and the development of new therapeutic strategies to cure this disease. Disclosures Tornador: Bloodgenetics: Current Employment. Sanchez:Bloodgenetics: Current Employment. Segovia:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from the Company., Patents & Royalties, Research Funding.

Download Full-text

Development of Megakaryocyte-Specific Lentiviral Vectors for Gene Therapy of Platelet Disorders.

Blood ◽

10.1182/blood.v110.11.5143.5143 ◽

2007 ◽

Vol 110 (11) ◽

pp. 5143-5143

Author(s):

Liesbeth De Waele ◽

Kathleen Freson ◽

Chantal Thys ◽

Christel Van Geet ◽

Désiré Collen ◽

...

Keyword(s):

Gene Therapy ◽

Transgene Expression ◽

Ex Vivo ◽

Phosphoglycerate Kinase ◽

Lentiviral Vectors ◽

Wild Type ◽

Hematopoietic Stem ◽

Platelet Disorders

Abstract The prevalence of congenital platelet disorders has not been established but for some life-threatening bleeding disorders the current therapies are not adequate, justifying the development of alternative strategies as gene therapy. In the case of platelet dysfunction and thrombocytopenia as described for GATA1 deficiency, potentially lethal internal bleedings can occur. The objective of the study is to develop improved lentiviral vectors for megakaryocyte(MK)-specific long term gene expression by ex vivo transduction of hematopoietic stem cells (HSC) to ultimately use for congenital thrombopathies as GATA1 deficiency. Self-inactivating lentiviral vectors were constructed expressing GFP driven by the murine (m) or human (h) GPIIb promoter. These promoters contain multiple Ets and GATA binding sites directing MK-specificity. To evaluate the cell lineage-specificity and transgene expression potential of the vectors, murine Sca1+ and human CD34+ HSC were transduced in vitro with Lenti-hGPIIb-GFP and Lenti-mGPIIb-GFP vectors. After transduction the HSC were induced to differentiate in vitro along the MK and non-MK lineages. The mGPIIb and hGPIIb promoters drove GFP expression at overall higher levels (20% in murine cells and 25% in human cells) than the ubiquitous CMV (cytomegalovirus) or PGK (phosphoglycerate kinase) promoters, and this exclusively in the MK lineage. Interestingly, in both human and murine HSC the hGPIIb promoter with an extra RUNX and GATA binding site, was more potent in the MK lineage compared to the mGPIIb promoter. Since FLI1 and GATA1 are the main transcription factors regulating GPIIb expression, we tested the Lenti-hGPIIb-GFP construct in GATA1 deficient HSC and obtained comparable transduction efficiencies as for wild-type HSC. To assess the MK-specificity of the lentiviral vectors in vivo, we transplanted irradiated wild-type C57Bl/6 mice with Sca1+ HSC transduced with the Lenti-hGPIIb-GFP constructs. Six months after transplantation we could detect 6% GFP positive platelets without a GFP signal in other cell lineages. Conclusion: In vitro and in vivo MK-specific transgene expression driven by the hGPIIb and mGPIIb promoters could be obtained after ex vivo genetic engineering of HSC by improved lentiviral vectors. Studies are ongoing to study whether this approach can induce phenotypic correction of GATA1 deficient mice by transplantation of ex vivo Lenti-hGPIIb-GATA1 transduced HSC.

Download Full-text

Loss of ASXL1 Triggers an Apoptotic Response in Human Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood.v126.23.4107.4107 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4107-4107

Author(s):

Susan Hilgendorf ◽

Hendrik Folkerts ◽

Jan Jacob Schuringa ◽

Edo Vellenga

Keyword(s):

Progenitor Cells ◽

Colony Formation ◽

Erythroid Differentiation ◽

Lentiviral Vectors ◽

Apoptotic Response ◽

Double Positive ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Stem And Progenitor Cells

Abstract In recent clinical studies, it has been shown that ASXL1 is frequently mutated in myelodysplastic syndrome (MDS), in particular in high-risk MDS patients who have a significant chance to progress to acute myeloid leukemia (AML). The majority of ASXL1 mutations leads to truncation of the protein and thereby to loss of its chromatin interacting and modifying domain, possibly facilitating malignant transformation. However, the functions of ASXL1 in human hematopoietic stem and progenitor cells are not well understood. In this study, we addressed whether manipulation of ASXL1-expression in the hematopoietic system in vitro mimics the changes observed in MDS-patients. We downregulated ASXL1 in CD34+ cord blood (CB) cells using lentiviral vectors containing several independent shRNAs and obtained a 40-50% reduction of ASXL1 expression. Colony Forming Cell (CFC) assays revealed that erythroid colony formation was significantly impaired (p<0.01) and, to some extent, granulocytic and macrophage colony formation as well (p<0.09, p<0.05 respectively). In myeloid suspension culture assays, we observed a modest reduction in expansion (two-fold at week 1) upon ASXL1 knockdown under myeloid conditions. In erythroid conditions, shASXL1 CB CD34+ cells showed a strong four-fold growth disadvantage, with a more than two-fold delay in erythroid differentiation. The reduced expansion was partly due to a significant increase in apoptosis (5.9% in controls vs. 14.0% shASXL1, p<0.02). The increase in cell death was restricted to differentiating cells, defined as CD71 bright- and CD71/GPA-double positive. In addition, we tested whether HSCs were affected by ASXL1 loss. Long-term culture-initiating cell (LTC-IC) assays revealed a two-fold decrease in stem cell frequency. To test dependency of shASXL1 CB 34+ cells on the microenvironment, transduced cells were cultured on MS5 bone marrow stromal cells with or without additional cytokines. shASXL1 CB CD34+ cells cultured on MS5 showed a modest two-fold reduction in cell growth at week 4. In the presence of EPO and SCF, we detected a growth disadvantage (three-fold at week 2) and a delay in erythroid differentiation, similar to what was observed in liquid culture. ASXL1 has been proposed to be an epigenetic modifier by recruiting/stabilizing the polycomb repressive complex 2 (PRC2). Active PRC2 can lead to trimethylation of H3K27 and silencing of certain loci. It has been proposed that perturbed ASXL1 activity may disturb PRC2 function, leading to reduced H3K27me3 and increased gene expression. Using an erythroid leukemic cell line, we downregulated ASXL1 and as a positive control EZH2, one of the core subunits of PRC2. We then performed ChIP and did PCR for several loci. Upon knockdown of ASXL1, we did not observe changes in H3K27me3 on any of he investigated loci. However, upon knockdown of EZH2 we observed more than 50% loss of the H3k27m3 mark for many of the loci. This implies that our observed phenotypes may not be conveyed via the PRC2 complex but maybe via an alternative pathway. Preliminary data revealed an increase in H2AK119ub, suggesting that the BAP1-ASXL1 complex may be involved. In patients, mutations in ASXL1 are frequently accompanied by a mutation of TP53. Possibly, this additional mutation is necessary to allow ASXL1-mutant induced transformation thereby bypassing the apoptotic response. Therefore, we modeled simultaneous loss of ASXL1 and TP53 using shRNA lentiviral vectors. Our data showed that while in primary CFC cultures shASXL1/shTP53 did not give rise to more colonies, an increase in colony-forming activity was observed upon replating of the cells. Furthermore, shASXL1/shTP53 transduced cells grown in erythroid liquid conditions revealed a decrease in apoptosis compared to the ASXL1 single mutation and an outgrowth of these double positive cells. Nevertheless, no transformation occurred in vitro. We therefore injected shASXL/TP53 transduced CB CD34+ in a humanized scaffold model in mice to determine whether transformation can occur in vivo. In conclusion, our data indicate that mutations in ASXL1 trigger an apoptotic response in CB CD34+ cells with a delay in differentiation, which leads to reduced stem and progenitor output in vitro without affecting H3K27me3. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Azacitidine and Ascorbate Inhibit the Competitive Outgrowth of Human TET2 Mutant HSPCs in a Xenograft Model of Pre-Leukemia

Blood ◽

10.1182/blood-2018-99-113907 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 887-887

Author(s):

Yusuke Nakauchi ◽

Daniel Thomas ◽

Rajiv Sharma ◽

M. Ryan Corces ◽

Andreas Reinisch ◽

...

Keyword(s):

Colony Formation ◽

Erythroid Differentiation ◽

High Dose ◽

Myeloid Lineage ◽

Hematopoietic Stem ◽

Nsg Mice ◽

Base Pair Deletion ◽

Cd34 Cells

Abstract The TET2 gene is frequently mutated in pre-leukemic hematopoietic stem cells in human acute myeloid leukemia (AML) and encodes for an enzyme that catalyzes the conversion of DNA 5-methylcytosine to 5-hydroxymethylcytosine. Recent studies suggest that (i) the product of this reaction can be enhanced using high dose ascorbate, and (ii) formation of the substrate 5-methylcytosine can be blocked with azacitidine. To understand the mechanisms of TET2 mutation-driven leukemogenesis, we developed two CRISPR/Cas9 approaches to disrupt the TET2 gene in primary human CD34+ HSPCs to mimic TET2-mutated pre-leukemia. First, in "Hit & Run," we use Cas9 with two single-guide RNAs (sgRNAs) to disrupt the TET2 gene within exon 3 (average indel frequencies=94.3%). Second, we using homology directed repair (HDR) of Cas9-mediated dsDNA breaks to disrupt the TET2 gene within exon 7 by inserting a GFP expression cassette to generate in vivo traceable cells. Thus, we have developed a tractable and cell-traceable model that recapitulates TET2-mutated pre-leukemia and clonal hematopoiesis. First, we examined the effects of TET2 disruption on human erythroid differentiation in vitro by culturing bulk CD34+ cells for 10 days under conditions that promote erythroid differentiation. Both Hit & Run and HDR (GFP+) TET2 disruption decreased CD71+CD235+ erythroid differentiation compared to control cells. Exposure to high dose ascorbate partially rescued the erythroid defect in TET2-disrupted cells (Hit & Run, n=3 independent experiments, p<0.02). This underscores the importance of TET2 in promoting erythroid differentiation and suggests TET2 mutations can exert a myeloid lineage skewing sensitive to ascorbate. Next, we investigated the effects of TET2 disruption on hematopoietic colony formation in methylcellulose. Both methods resulted in increased numbers of TET2-disrupted colonies compared to control (Hit & Run, n=4 independent experiments, p<0.0001; HDR, n=3 independent experiments, p<0.0001) and absence of erythroid BFU-E. Interestingly, analysis of indels in Hit & Run colonies showed that serial replating enriched for a 65 base pair deletion that results in a null allele, suggesting that TET2-disrupted cells outcompete normal HSPCs in vitro. Next, we transplanted control or TET2-disrupted Hit & Run CD34+ cells into NSG mice. Primary transplantation at 4 months showed no statistical differences in either engraftment rate (human CD45+) or differentiation (T/ B/ Myeloid cells), although the frequency of TET2 indels increased gradually in CD33+ cells. Intriguingly, 36 weeks after secondary transplantation, we detected a marked expansion of human myeloid lineage cells (lymphoid=22.1%, myeloid=73.0%, Mann-Whitney U, p=0.0485) and a particular increase in a CMML-like CD33highCD14+CD16- population. Furthermore, preliminary data from tertiary transplantation (8 weeks after transplantation) indicates persistent myeloid skewing in the bone marrow in some mice and expansion of TET2-mutant cells, suggesting a CMML-like disease. Finally, we used in vivo competition studies to determine if TET2-disrupted HSPCs are selectively targeted by azacitidine or ascorbate treatment compared to controls. NSG mice were intrafemorally transplanted with a one-to-one ratio of control and TET2-disrupted HSPCs, and 4 months later, these mice were treated with azacitidine (2.5mg/kg/dose, i.p. daily on days 1-5 of a 14-day cycle for 2 cycles) or ascorbate (4g/kg/dose, i.p. twice daily for a month). In PBS control treated mice, the percentage of TET2-disrupted cells increased from 29.3 to 71.6 over 4 weeks. Intriguingly, azacitidine slowed the expansion of TET2-disrupted cells in evaluable mice (delta increase of 42% in PBS vs 5% in azacitidine, p=0.036), but did not eradicate established TET2 pre-leukemia in all evaluable mice. Similarly, high dose ascorbate treatment slowed the rate of expansion to a lesser degree (delta increase of 42% in PBS vs 18.3% in ascorbate, p=0.14). Our data show that TET2 disruption in primary human HSPCs blocked erythroid differentiation, increased colony formation and replating, and caused myeloid skewing and a CMML-like disease in vivo after an extended period of time. In this model, azacitidine or ascorbate treatment slowed expansion of TET2-mutant human pre-leukemic clones raising the intriguing possibility of preventing CHIP progression to de novo AML. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Ezrin Regulates Hematopoietic Stem/Progenitor Cell Motility

Blood ◽

10.1182/blood.v118.21.1282.1282 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1282-1282

Author(s):

Yi Zeng ◽

Karl Staser ◽

Keshav Mohan Menon ◽

Su-jung Park ◽

Muithi Mwanthi ◽

...

Keyword(s):

Progenitor Cell ◽

Conflicts Of Interest ◽

Cell Contact ◽

Wild Type ◽

Hematopoietic Stem ◽

Knock Out ◽

Actin Organization ◽

Stromal Cell Derived Factor ◽

Cell Mobilization

Abstract Abstract 1282 Ezrin is a member of the ERM (ezrin, moesin and radixin) protein family that links plasma membrane proteins to the actin cytoskeleton. Ezrin in other in vitro cell systems has been hypothesized to participate in cell-cell contact and could have a role in stem/ progenitor cell mobilization and adhesion. To test this hypothesis, we crossed ezrinflox/flox mice with Mx1 cre transgenic mice to generate an inducible ezrin knock out mouse model. Inducible disruption of the ezrin gene in hematopoietic cells was achieved by the administration of polyIC. Ezrin knock out HSPCs exhibited a 30–40% decrease in baseline and chemokine stromal cell-derived factor-1 (SDF-1) stimulated motility in transwell migration assays in vitro. In addition, loss of ezrin led to a 60% decrease in the homing capacity of HSPCs in lethally irradiated recipient mice following transplantation. There was a 40–55% decrease in colony forming cells in peripheral blood and spleen of the mice following ezrin knock out, suggesting that ezrin knock out HSPCs may be deficient in egressing out of the bone marrow. To further understand the cause of the impaired motility of ezrin knock out HSPCs, we examined F-actin level of HSPCs at baseline and in response to SDF-1. Ezrin knock out HSPCs displayed 1.5 to 2 fold higher level of F-actin at baseline when compared with wild type cells. Following stimulation with SDF-1, wild type HSPCs that migrated to the bottom compartment of the transwell demonstrated a 2 time greater decrease in F-actin level when compared with ezrin knock out cells, suggesting that ezrin may participate in the regulation of F-actin depolymerization in HSPCs. In summary, we demonstrate that ezrin modulates HSPC migration and homing likely through its regulation on F-actin organization. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Preclinical Studies for Sickle Cell Disease Gene Therapy Using Bone Marrow CD34+ Cells Modified with a βAS3-Globin Lentiviral Vector

Blood ◽

10.1182/blood.v118.21.3119.3119 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3119-3119

Author(s):

Fabrizia Urbinati ◽

Zulema Romero Garcia ◽

Sabine Geiger ◽

Rafael Ruiz de Assin ◽

Gabriela Kuftinec ◽

...

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Blood Cells ◽

Globin Gene ◽

Cell Disease ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Abstract 3119 BACKGROUND: Sickle cell disease (SCD) affects approximately 80, 000 Americans, and causes significant neurologic, pulmonary, and renal injury, as well as severe acute and chronic pain that adversely impacts quality of life. Because SCD results from abnormalities in red blood cells, which in turn are produced from adult hematopoietic stem cells, hematopoietic stem cell transplant (HSCT) from a healthy (allogeneic) donor can benefit patients with SCD, by providing a source for life-long production of normal red blood cells. However, allogeneic HSCT is limited by the availability of well-matched donors and by immunological complications of graft rejection and graft-versus-host disease. Thus, despite major improvements in clinical care, SCD continues to cause significant morbidity and early mortality. HYPOTHESIS: We hypothesize that autologous stem cell gene therapy for SCD has the potential to treat this illness without the need for immune suppression of current allogeneic HSCT approaches. Previous studies have demonstrated that addition of a β-globin gene, modified to have the anti-sickling properties of fetal (γ-) globin (βAS3), to bone marrow (BM) stem cells in murine models of SCD normalizes RBC physiology and prevents the manifestations of sickle cell disease (Levassuer Blood 102 :4312–9, 2003). The present work seeks to provide pre-clinical evidence of efficacy for SCD gene therapy using human BM CD34+ cells modified with the bAS3 lentiviral (LV) vector. RESULTS: The βAS3 globin expression cassette was inserted into the pCCL LV vector backbone to confer tat-independence for packaging. The FB (FII/BEAD-A) composite enhancer-blocking insulator was inserted into the 3' LTR (Ramezani, Stem Cells 26 :32–766, 2008). Assessments were performed transducing human BM CD34+ cells from healthy or SCD donors with βAS3 LV vectors. Efficient (1–3 vector copies/cell) and stable gene transmission were determined by qPCR and Southern Blot. CFU assays demonstrated that βAS3 gene modified SCD CD34+ cells are fully capable of maintaining their hematopoietic potential. To demonstrate the effectiveness of the erythroid-specific bAS3 gene in the context of human HSPC (Hematopoietic Stem and Progenitor Cells), we optimized an in vitro model of erythroid differentiation of huBM CD34+ cells. We successfully obtained an expansion up to 700 fold with >80% fully mature enucleated RBC derived from CD34+ cells obtained from healthy or SCD BM donors. We then assessed the expression of the βAS3 globin gene by isoelectric focusing: an average of 18% HbAS3 over the total globin present (HbS, HbA2) per Vector Copy Number (VCN) was detected in RBC derived from SCD BM CD34+. A qRT-PCR assay able to discriminate HbAS3 vs. HbA RNA, was also established, confirming the quantitative expression results obtained by isoelectric focusing. Finally, we show morphologic correction of in vitro differentiated RBC obtained from SCD BM CD34+ cells after βAS3 LV transduction; upon induction of deoxygenation, cells derived from SCD patients showed the typical sickle shape whereas significantly reduced numbers were detected in βAS3 gene modified cells. Studies to investigate risks of insertional oncogenesis from gene modification of CD34+ cells by βAS3 LV vectors are ongoing as are in vivo studies to demonstrate the efficacy of βAS3 LV vector in the NSG mouse model. CONCLUSIONS: This work provides initial evidence for the efficacy of the modification of human SCD BM CD34+ cells with βAS3 LV vector for gene therapy of sickle cell disease. This work was supported by the California Institute for Regenerative Medicine Disease Team Award (DR1-01452). Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Improving Hematopoietic Stem Cell Based Gene Therapy By Targeting CD133+ Cells

Blood ◽

10.1182/blood.v122.21.4202.4202 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4202-4202

Author(s):

Benjamin Goebel ◽

Christian Brendel ◽

Daniela Abriss ◽

Sabrina Kneissl ◽

Martijn Brugman ◽

...

Keyword(s):

Gene Therapy ◽

Stem Cell ◽

Gene Transfer ◽

Cell Population ◽

Cell Populations ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Transfer Vectors

Abstract Introduction Generally, CD34+ cells are used for genetic modification in gene therapy trials. CD34+ cells consist of a heterogeneous cell population with mostly limited long-term repopulating capabilities, resulting in low long-term engraftment levels in particular in those diseases in which gene modified cells lack a proliferative advantage over non-modified cells. Therefore, modifications in gene transfer vectors and gene transfer strategies are required to improve long-term clinical benefit in gene therapy patients. One particular attractive approach to solve this problem is the improvement of HSC based gene transfer by specifically targeting cells with long-term engraftment capabilities. Material and Methods We constructed lentiviral gene transfer vectors (LV) specifically targeting CD133+ cells, a cell population with recognized long-term repopulating capabilities. Targeting is achieved by pseudotyping with engineered measles virus (MV) envelope proteins. The MV glycoprotein hemagglutinin, responsible for receptor recognition, is blinded for its native receptors and displays a single-chain antibody specific for CD133 (CD133-LV). These vectors were compared to VSV-pseudotyped lentiviral vectors in in vitro and in vivocompetitive repopulation assays using mobilized peripheral blood CD34+ cells. Results Superior transduction of isolated human hematopoietic stem cell populations (CD34+CD38- or CD34+CD133+ cells) compared to progenitor cell populations (CD34+CD38+ or CD34+CD133-) could be shown using the newly developed CD133-LV. Transduction of total CD34+ cells with CD133-LV vectors resulted in stable gene expression and gene marked cells expanded in vitro, while the number of VSV-G-LV transduced CD34+ cells declined over time. Competitive repopulation experiments in NSG mice showed a significantly improved engraftment of CD133-LV transduced HSCs. At ∼12 weeks post-transplantation gene marked hematopoiesis was dominated by the progeny of CD133-LV transduced cells in 42 out of 52 transplanted animals in the bone marrow and 39 out of 45 transplanted animals in the spleen, respectively. Consistent with this data we could show that stem cell content in the CD133-LV transduced population is about five times higher compared to the VSV-transduced population using a limiting dilution competitive repopulation assay (LDA-CRU). Experiments showing proof of principle for the application of this technology for the correction of Chronic Granulomatous Disease (XCGD) using patient derived CD34+ cells are currently ongoing. Discussion In conclusions this new strategy may be promising to achieve improved long-term engraftment in patients treated by gene therapy. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Innovative Hematopoietic Gene-Therapy Concepts for Hereditary Pulmonary Alveolar Proteinosis Utilizing Hematopoietic Stem Cell Derived Macrophages

Blood ◽

10.1182/blood.v126.23.4417.4417 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4417-4417

Author(s):

Nico Lachmann ◽

Christine Happle ◽

Takuji Suzuki ◽

Miriam Hetzel ◽

Kevin A. Link ◽

...

Keyword(s):

Gene Therapy ◽

Colony Formation ◽

Pulmonary Alveolar Proteinosis ◽

Hematopoietic Stem ◽

Gm Csf ◽

Protein Levels ◽

Donor Cells ◽

Cd34 Cells ◽

Alveolar Proteinosis

Abstract Hereditary pulmonary alveolar proteinosis (herPAP) is a rare lung disease caused by mutations in the granulocyte/macrophage-colony-stimulating factor (GM-CSF) receptor genes (CSF2RA and CSF2RB), resulting in disturbed alveolar macrophage (AM) differentiation, massive alveolar proteinosis, and life-threatening respiratory insufficiency. We here introduce pulmonary transplantation of gene corrected hematopoietic stem cell (HSC)-derived macrophage progenitors (MP) as a novel, cause directed, and well-tolerated therapy for herPAP. In a Csf2rb-/- mouse-model, selective pulmonary engraftment of healthy donor cells upon pulmonary transplantation of MPs was demonstrated by flow- and chip cytometry. Profound reduction of alveolar-protein levels and significant improvement of clinical parameters such as lung function and lung densities on CT scans were observed for more than nine months. Subsequent in situ analysis of donor cells revealed in vivo differentiation towards an AM phenotype characterized by CD11chi, CD11blo, MHC-II+, CD14+, F4/80+ surface marker, poor antigen presentation capacity, high phagocytic activity and AM-typical morphology on electron microscopy. Similar results were obtained following pulmonary transplantation of MPs differentiated from lentivirally corrected Csf2rb -/- HSCs. In vitro these gene-corrected HSCs expanded up to 1045-fold while differentiating into typical alveolar macrophages with F4/80, CD11b, CD11c, CD68, as well as Csf2rb mRNA and protein (CD131) expression and reconstitution of GM-CSF receptor signaling. Transplanted herPAP mice displayed significant improvement of biomarkers in the bronchoalveolar fluid (cloudiness, turbidity, SP-D, MCP-1, M-CSF, and GM-CSF) and in AMs (mRNA for PU.1, PPARg and ABCG1). Moreover, administration of human CD34+ cell-derived MPs profoundly improved symptoms in a humanized herPAP mouse model. Here, transplantation of 1-2x106 human MPs led to long-term pulmonary engraftment and reduced alveolar protein levels by 50-70%. CT scans six months after transplantation revealed significant improvements in herPAP related signs, including marked reduction of expiratory lung densities and normalisation of inspiratory to expiratory lung volume ratio. Furthermore, to genetically correct human CSF2RA-patient derived CD34+ cells, we have generated SIN-lentiviral vectors expressing a codon-optimized human CSF2RA-cDNA in combination with EGFP (Lv.EFS.CSF2RA.EGFP) from an EFS1a-promoter. BaF3 cells transduced with this vector showed stable and longterm (>3 month) expression of CSF2RA (CD116) by flow cytometry and survived in hGM-CSF dependant assays even at low concentrations of GM-CSF (5 ng/ml) confirming the formation of functional hybrid receptors of the murine GM-CSF receptor ß-chain with the transgene. Characterization of GM-CSF receptor downstream signalling revealed 5-6-fold increased STAT5 phosphorylation by Western blot in response to hGM-CSF (over control cells). Conferring this vector to CD34+ cells of CSF2RA-deficient patients yielded efficient CD116 (CSF2RA) expression, and rescued hGM-CSF dependent colony formation as well as monocytoid differentiation. Of note, clonogenic growth by G-CSF control treatment revealed no differences in colony formation or differentiation capacity when compared to uncorrected patient samples. Furthermore, healthy Lv.EFS.CSF2RA.EGFP transduced CD34+ samples, while showing robust CD116 overexpression, exhibited no aberrations in biological functions such as colony formation or in vitro differentiation towards macrophages. Thus, we here describe an innovative, cause directed and highly effective hematopoietic gene therapy approach to herPAP. Given the longevity of the transplanted MP population in our model, the strategy also may serve as a proof-of-principle to incorporate long-lived differentiated, cell sources into current hematopoietic gene therapy concepts. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Targeted Gene Modification In Hematopoietic Stem Cells: A Potential Treatment For Thalassemia and Sickle Cell Anemia

Blood ◽

10.1182/blood.v122.21.434.434 ◽

2013 ◽

Vol 122 (21) ◽

pp. 434-434

Author(s):

Andreas Reik ◽

Kai-Hsin Chang ◽

Sandra Stehling-Sun ◽

Yuanyue Zhou ◽

Gary K Lee ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Target Gene ◽

Ex Vivo ◽

Globin Gene ◽

Erythroid Differentiation ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Beta-thalassemia (β-thal) and sickle cell disease (SCD) are monogenic diseases caused by mutations in the adult β-globin gene. A bone marrow transplant (BMT) is the only curative treatment, but its application is limited since (i) HLA-matched donors can be found for <20% of cases, and (ii) the allogeneic nature of the transplant involves the significant risk of graft vs host disease (GvHD). Elevated levels of fetal γ-globin proteins observed in a subset of individuals carrying β-thal and SCD mutations ameliorate the clinical picture or prevent the development of disease complications. Thus, strategies for the selective and persistent upregulation of γ-globin represent an attractive therapeutic approach. Recent insights into the regulation of γ-globin transcription by a network of transcription factors and regulatory elements both inside and outside the β-globin locus have revealed a set of new molecular targets, the modulation of which is expected to elevate γ-globin levels for potential therapeutic intervention. To this end, we and others have established that designed zinc finger nucleases (ZFNs) transiently introduced into stem cells ex vivo provide a safe and efficient way to permanently ablate the expression of a specific target gene in hematopoietic stem cells (HSC) by introduction of mutations following target site cleavage and error-prone DNA repair. Here we report the development and comparison of different ZFNs that target various regulators of γ-globin gene transcription in human HSCs: Bcl11a, Klf1, and specific positions in the γ-globin promoters that result in hereditary persistence of fetal hemoglobin (HPFH). In all cases these target sites / transcription factors have previously been identified as crucial repressors of γ-globin expression in humans, as well as by in vitro and in vivo experiments using human erythroid cells and mouse models. ZFN pairs with very high genome editing activity in CD34+ HSCs were identified for all targeted sites (>75% of alleles modified). In vitro differentiation of these ZFN-treated CD34+ HSCs into erythroid cells resulted in potent elevation of γ-globin mRNA and protein levels without significant effects on erythroid development. Importantly, a similar and specific elevation of γ-globin levels was observed with RBC progeny of genome-edited CD34+ cells obtained from SCD and β-thal patients. Notably, in the latter case a normalization of the β-like to α-globin ratio to ∼1.0 was observed in RBCs obtained from genome-edited CD34s from two individuals with β-thalassemia major. To deploy this strategy in a clinical setting, we developed protocols that yielded comparably high levels of target gene editing in mobilized adult CD34+ cells at large scale (>108 cells) using a clinical-grade electroporation device to deliver mRNA encoding the ZFN pair. Analysis of modification at the most likely off-target sites based on ZFN binding properties, combined with the maintenance of target genome editing observed throughout erythroid differentiation (and in isolated erythroid colonies) demonstrated that the ZFNs were both highly specific and well-tolerated when deployed at clinical scale. Finally, to assess the stemness of the genome-edited CD34+ HSCs we performed transplantation experiments in immunodeficient mice which revealed long term engraftment of the modified cells (>16 weeks, ∼25% human chimerism in mouse bone marrow) with maintenance of differentiation in vivo. Moreover, ex vivo erythroid differentiation of human precursor cells isolated from the bone marrow of transplanted animals confirmed the expected elevation of γ-globin. Taken together, these data suggest that a therapeutic level of γ-globin elevation can be obtained by the selective disruption, at the genome level, of specific regulators of the fetal to adult globin developmental switch. The ability to perform this modification at scale, with full retention of HSC engraftment and differentiation in vivo, provides a foundation for advancing this approach to a clinical trial for the hemoglobinopathies. Disclosures: Reik: Sangamo BioSciences: Employment. Zhou:Sangamo BioSciences: Employment. Lee:Sangamo BioSciences: Employment. Truong:Sangamo BioSciences: Employment. Wood:Sangamo BioSciences: Employment. Zhang:Sangamo BioSciences: Employment. Luong:Sangamo BioSciences: Employment. Chan:Sangamo BioSciences: Employment. Liu:Sangamo BioSciences: Employment. Miller:Sangamo BioSciences: Employment. Paschon:Sangamo BioSciences: Employment. Guschin:Sangamo BioSciences: Employment. Zhang:Sangamo BioSciences: Employment. Giedlin:Sangamo BioSciences: Employment. Rebar:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment. Urnov:Sangamo BioSciences: Employment.

Download Full-text

Loss of ASXL1 Triggers an Apoptotic Response in Human Hematopoetic Stem and Progenitor Cells

Blood ◽

10.1182/blood.v124.21.4619.4619 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4619-4619

Author(s):

Susan Hilgendorf ◽

Hendrik Folkerts ◽

Jan Jacob Schuringa ◽

Edo Vellenga

Keyword(s):

Cell Death ◽

Stem Cell ◽

Progenitor Cells ◽

Liquid Culture ◽

Erythroid Differentiation ◽

Apoptotic Response ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Stem And Progenitor Cells

Abstract In recent clinical studies, it has been demonstrated that ASXL1 is frequently mutated in myelodysplastic syndrome (MDS), in particular in high-risk MDS patients who have a significant chance to progress to acute myeloid leukemia (AML). Mutation of ASXL1 leads to truncation of the protein and thereby to a loss of its chromatin interacting and modifying domain, possibly facilitating malignant transformation. However, the functions of ASXL1 in human hematopoietic stem and progenitor cells are not well understood. In this study, we addressed whether manipulation of ASXL1-expression in hematopoietic system in vitro mimics the changes observed in MDS-patients. We down regulated ASXL1 in CD34+ cord blood (CB) cells using a lentiviral approach and obtained a 40-50% reduction of ASXL1 expression. Colony forming (CFC) assays revealed that erythroid colony formation was significantly impaired (p=0.01) and, to some extent, granulocytic and macrophage colony formation (p=0.09, p=0.05 respectively). As MDS can affect all hematopoietic lineages, we first stimulated cell differentiation along the myeloid or erythroid lineage in liquid culture. Upon culturing shASXL1 CB CD34+ cells in suspension, we observed a modest reduction in expansion (two-fold at week1) under myeloid conditions. In erythroid conditions, shASXL1 CB CD34+ cells showed a strong four-fold growth disadvantage, with a more than two-fold delay in erythroid differentiation. The reduced expansion was partly due to a significant increase in apoptosis (5.9% in controls vs. 14.0% shASXL1, p=0.02). The increase of cell death was restricted to differentiating cells, defined as CD71 bright- and CD71/GPA-double positive. This phenotype is similar to what has been observed in patients, where increased cell death of progenitors occurs, and suggests that ASXL1 loss may reflect an MDS-like phenotype in this culture setting. Furthermore, as MDS is considered a hematopoietic stem cell (HSC)-driven disorder, we tested whether HSCs were affected by ASXL1 loss. Long-term culture initiating cell (LTC-IC) assays revealed a two-fold decrease in stem cell frequency. To test dependency of shASXL1 CB 34+ cells on the microenvironment, we performed cultures on stromal layers with or without cytokines. shASXL1 CB CD34+ cells cultured on MS5 stromal layer showed a modest, two-fold reduction in cell growth at week 4. In the presence of EPO and SCF, we detected a growth disadvantage (three-fold at week 2) and a delay in erythroid differentiation, similar to what was observed in liquid culture. In patients, mutations in ASXL1 are frequently accompanied by a loss of p53. Possibly, loss of p53 is necessary to allow ASXL1-mutant induced transformation thereby bypassing the apoptotic response. Therefore, we modeled simultaneous loss of ASXL1 and TP53 using shRNA lentiviral vectors. Our first data showed that while in primary CFC cultures shASXL1/shTP53 did not give rise to more colonies compared to shASXL1/shSCR cells, an increase in colony-forming activity was observed upon replating of the cells. Furthermore, when using erythroid liquid conditions, a decrease in apoptosis compared to the ASXL1 single mutation could be observed. Nevertheless, no transformation occurred and ASXL1 mutated cells were eventually lost in the double hit model despite reduced apoptosis, suggesting that the p53 axis might not be sufficient as the second hit for full transformation. In conclusion, our data indicate that mutations in ASXL1 may lead to an increase in cell death and reduced progenitor output in vitro, which may reflect disease development and progression as seen in patients. Unexpectedly, MS5 stromal did not alter the negative phenotype caused by ASXL1 knock down. Therefore, studies are ongoing to investigate whether an already established MDS microenvironment will influence ASXL1 mutation positively. To this end, we are using healthy human mesenchymal stem cells (MSC) and patient derived MDS MSCs. Disclosures No relevant conflicts of interest to declare.

Download Full-text