A New Step in Understanding of Fanconi Patients Peripheral Stem Cell Harvesting, a Bridge to Gene Therapy

Fanconi anemia (FA) is an inherited disorder, clinically characterized by congenital abnormalities, a fatal progressive bone marrow failure (BMF), and a predisposition to develop malignancies. Gene therapy by infusion of FA-corrected autologous hematopoietic stem cells (HSCs) may offer a potential alternative cure and to get around the problems of the Hematopoietic stem cell transplantation toxicity or the donor restriction. For gene therapy, an adequate number of HSC collected is a key point to a successful engraftment. However, the HSC collection in FA patients implies particular challenges because of their reduced BM stem cells numbers and implies a theorical risk of an inner depletion in stem cell reserve following collection.The main objective of this pilot study was to evaluate the feasibility and the safety of co-administration of G-CSF and plerixafor in patients with FA for the mobilization and collection of peripheral HSC for potential use in a GT trial. We present the results of this open-label phase I/II trial (N°EUDRACT 2014-005264-14) from 4 selected FANCA mutated patients (FA-A) with a weight >10 Kg and an age between 2 to 18 years old. A systematic combination of G-CSF (12μg/kg twice a day) plus plerixafor (Mozobil® 0.240 mg/kg/d ) was used to maximise the CD34+ cells mobilization. CD34+ cells and white blood cells (WBC) blood counts were monitored tightly along the mobilization protocol. No short-term adverse events linked to the mobilization and the collection procedures were observed. The combination of G-CSF and Plerixafor allowed crossing the PB mobilization threshold (≥5 CD34+cells/μL) for 2 patients. Interestingly, CD34+cells were mobilized quickly but transitionally after plerixafor injection. One patient mobilization had more than 100 CD34+cells μ/L with a early peak 2h after injection. The peak disappeared 11 hours after injection. We adapted the time of collection to the C34+ cells mobilization. No CD34+ blood cell rebound was observed after the apheresis was stopped. Our new datas suggest that mobilization of FA patients with G-CSF and plerixafor is safe. However, the age of the patient, a potential cytopenia or the lack of bone marrow progenitor cell may heavely compromise the collection. Nevertheless, the datas show a stable cytopenia despite the stimulation and collection of stem cells during the following months. This study underlines that a very cautious collection of stem cell in the Fanconi anemia to consider gene therapy is a necessity. These results also confirm that the kinetic of CD34+ cells mobilization is one of the key point to a successful stem cell harvesting for gene therapy trial. Disclosures Cavazzana: Smartimmune: Other: Founder of Smartimmune.

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ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION FOR ADULT PATIENTS WITH FANCONI ANEMIA.

Mediterranean Journal of Hematology and Infectious Diseases ◽

10.4084/mjhid.2016.054 ◽

2016 ◽

Vol 8 ◽

pp. 2016054 ◽

Cited By ~ 3

Author(s):

Hosein Kamranzadeh fumani ◽

Mohammad Zokaasadi ◽

Amir Kasaeian ◽

Kamran Alimoghaddam ◽

Asadollah Mousavi ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Hematopoietic Stem Cell ◽

Fanconi Anemia ◽

Bone Marrow Failure ◽

Term Survival ◽

Hematopoietic Stem

Background & objectives: Fanconi anemia (FA) is a rare genetic disorder caused by an impaired DNA repair mechanism which leads to an increased tendency toward malignancies and progressive bone marrow failure. The only curative management available for hematologic abnormalities in FA patients is hematopoietic stem cell transplantation (HSCT). This study aimed to evaluate the role of HSCT in FA patients.Methods: Twenty FA patients with ages of 16 or more who underwent HSCT between 2002 and 2015 enrolled in this study. All transplants were allogeneic and the stem cell source was peripheral blood and all patients had a full HLA-matched donor.Results: Eleven patients were female and 9 male (55% and 45%). Mean age was 24.05 years. Mortality rate was 50% (n=10) and the main cause of death was GVHD. Survival analysis showed an overall 5-year survival of 53.63% and 13 year survival of 45.96 % among patients.Conclusion: HSCT is the only curative management for bone marrow failure in FA patients and despite high rate of mortality and morbidity it seems to be an appropriate treatment with an acceptable long term survival rate for adolescent and adult group.

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Purified T Depleted Peripheral Blood and Bone Marrow Cd34 Transplantation from Haploidentical Mother to Child with Thalassemia.

Blood ◽

10.1182/blood.v108.11.3106.3106 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3106-3106

Author(s):

Pietro Sodani ◽

Buket Erer ◽

Javid Gaziev ◽

Paola Polchi ◽

Andrea Roveda ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

T Cell ◽

Peripheral Blood ◽

Therapeutic Option ◽

B Cell Lymphoma ◽

Marrow Transplant ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Approximately 60% of thalassemic patients can not apply to “gene therapy today” which the insertion of one allogenic HLA identical stem cell into the empty bone marrow as the vector of the normal gene for beta globin chain synthesis. We studied the use of the haploidentical mother as the donor of hematopoietic stem cells assuming that the immuno-tollerance established during the pregnancy will help to bypass the HLA disparity and allow the hemopoietic allogeneic reconstitution in the thalassemic recipient of the transplant. We have employed a new preparative regimen for the transplant in fourteen thalassemic children aged 3 to 12 years (median age 5 years) using T cell depleted peripheral blood stem cell (PBSCTs) plus bone marrow (BM) stem cells. All patients received hydroxyurea (OHU) 60 mg/kg and azathioprine 3 mg/kg from day -59 until day-11, fludarabine (FLU) 30 mg/m 2 from day -17 to day -11, busulphan (BU) 14 mg/kg starting on day -10, and cyclophosphamide(CY) 200mg/kg, Thiotepa 10 mg/kg and ATG Sangstat 2.5 mg/kg, followed by a CD34 + t cell depleted (CliniMacs system), granulocyte colony stimulating factor (G-csf) mobilized PBSC from their HLA haploidentical mother. The purity of CD34+ cells after MACS sorting was 98–99%, the average number of transplanted CD34+ cells was 15, 4 x 10 6/kg and the average number of infused T lymphocytes from BM was 1,8 x 10 5/Kg.The patients received cyclosporin after transplant for graft versus host disease(GVHD) prophylaxis during the first two months after the bone marrow transplantation. Results. Thirteen patients are alive. Four patients rejected the transplant and are alive with thalassemia One patients died six months after bone marrow transplant for central nervous system diffuse large B cell lymphoma EBV related. Nine patients are alive disease free with a median follow up of 30 months (range12–47). None of the seven patients showed AGVHD and CGVHD. This preliminary study suggest that the transplantation of megadose of haploidentical CD34+ cell from the mother is a realistic therapeutic option for those thalassemic patients without genotipically or phenotipically HLA identical donor.

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Non-Random Lentiviral Vector Insertions in Bone Marrow Progenitors from Fanconi Anemia Patients

Blood ◽

10.1182/blood.v112.11.2358.2358 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2358-2358

Author(s):

Ali Nowrouzi ◽

Africa Gonzales-Murillo ◽

Anna Paruzynski ◽

Ariana Jacome ◽

Paula Rio ◽

...

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Fanconi Anemia ◽

Large Scale ◽

Random Distribution ◽

Ex Vivo ◽

Hematopoietic Stem ◽

In Cis

Abstract Improved protocols using lentiviral vectors have been established with minimal cytokine exposure and short transduction times proving more suitable for overcoming the disease-specific challenge in correcting functionally defective hematopoietic stem cells (HSCs) of Fanconi Anemia (FA) patients. Bone marrow (BM) cells from FA patients were transduced ex vivo with lentiviral vectors (LVs) expressing FANCA and/or EGFP using optimized conditions to preserve the repopulating properties of the primitive hematopoietic stem cells (manuscript submitted). In a forward preclinical screening of possible LV-induced side effects we analyzed the insertional inventory in colonies generated by FA BM cells previously transduced with the LVs. We have established and optimized DNA and RNA isolation procedures for minimal cell numbers, suitable for large scale screening of colony forming cell (CFC) derived colonies by linear amplification-mediated PCR (LAM-PCR) and massive parallel pyrosequencing (454 GS Flx system; Roche). This approach is applicable for detecting early indicators of clonal selection, and is based on the analysis of common integration sites (CIS) and non-random distribution of vector insertions in particular genomic loci. From a total of 180 CFC-derived colonies expressing the EGFP LV marker gene, 298 vector insertions could be sequenced and mapped to the human genome. The analysis of vector targeted gene coding regions showed a non-random genomic distribution of LV insertions, with a significant overrepresentation of RefSeq genes that are part of distinct functional categories. Accordingly vector associated genes are predominantly involved in cellular signal cascades regulated by the MAP Kinase family known to be involved in a wide variety of cellular processes such as proliferation, differentiation, transcription regulation and development. Apart from the observed high integration frequency in genes (>80%), partial loss of vector LTR nucleotides was detected in >10% of the integrants (3–25bp). Notably, >20% of the lentiviral insertions were found to be located in CIS of predominantly 2nd order. Further screening assays of LV transduced CFC-derived colonies will allow a deeper investigation in the functional consequences of such CIS targeting in gene therapy protocols of FA. However our results suggest that the LV transduction of FA BM progenitors leads to a relatively high frequency of insertions in CIS which may be indicative of an insertion based (specific) selection mechanism. We herby show that the ex vivo large scale integration site analyses of CFC-derived colonies from patients considered to undergo gene therapeutic treatments constitutes a robust approach, which combined with mouse preclinical biosafety studies will help to improve the safety of clinical gene therapy protocols. The non-random distribution of LV integrations in CIS associated genes and in genes involved in particular cellular pathways may be indicative for the altered biochemical pathways characteristic of FA stem cells, with reported defects in DNA repair and self-renewal.

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Regulation of Hematopoietic Stem Cells by Interferons and by DNA Methylation: Implications for Bone Marrow Failure

Blood ◽

10.1182/blood.v124.21.sci-20.sci-20 ◽

2014 ◽

Vol 124 (21) ◽

pp. SCI-20-SCI-20

Author(s):

Margaret A. Goodell

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Bone Marrow Failure ◽

Stem Cell Differentiation ◽

Primary Myelofibrosis ◽

Differentiation Potential ◽

Hematopoietic Stem

Bone marrow failure (BMF), the inability to regenerate the differentiated cells of the blood, has a number of genetic and environmental etiologies, such as mutation of telomere-associated protein genes and immune-related aplastic anemia. Recently, mutations in DNA methyltransferase 3A (DNMT3A) have been found to be associated with approximately 15% of cases of primary myelofibrosis (MF), which can be a cause of BMF. The role of DNMT3A more broadly in hematopoiesis, and specifically in BMF, is currently poorly understood. DNMT3A is one of two de novo DNA methylation enzymes important in developmental fate choice. We showed that Dnmt3a is critical for normal murine hematopoiesis, as hematopoietic stem cells (HSCs) from Dnmt3a knockout (KO) mice displayed greatly diminished differentiation potential while their self-renewal ability was markedly increased1, in effect, leading to failure of blood regeneration or BMF. Combined with loss of Dnmt3b, HSCs exhibited a profound differentiation block, mediated in part by an increase of stabilized b-catenin. While we did not initially observe bone marrow pathology or malignancy development in mice transplanted with Dnmt3a KO HSCs, when we aged a large cohort of mice, all mice succumbed to hematologic disease within about 400 days. Roughly one-third of mice developed frank leukemia (acute lymphocytic leukemia or acute myeloid leukemia), one-third developed MDS, and the remainder developed primary myelofibrosis or chronic myelomonocytic leukemia. The pathological characteristics of the mice broadly mirror those of patients, suggesting the Dnmt3a KO mice can serve as a model for human DNMT3A-mutation associated disease. Strikingly, bone marrow of mice with different disease types exhibit distinct DNA methylation features. These will findings and the implications for disease development will be discussed. We are currently investigating the factors that drive different outcomes in the mice, including stressors such as exposure to interferons. We have hypothesized that HSC proliferation accelerates the Dnnmt3a-associated disease phenotypes. We have previously shown that interferons directly impinge on HSCs in the context of infections. Interferons activate HSCs to divide, generating differentiated progeny and cycling HSCs. Repeated interferon stimulation may permanently impair HSC function and bias stem cell output. When combined with loss of Dnmt3a, interferons may promote BMF. We will discuss broadly how external factors such as aging and infection may collaborate with specific genetic determinants to affect long-term hematopoiesis and malignancy development. Reference: Challen GA, Sun D, Jeong M, et al. Dnmt3a is essential for hematopoietic stem cell differentiation. Nat Genet 2012; 44: 23-31 Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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UM171 Regulates the Hematopoietic Differentiation of Human Acquired Aplastic Anemia-Derived Induced Pluripotent Stem Cells

Blood ◽

10.1182/blood-2019-127466 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2500-2500

Author(s):

Tellechea Maria Florencia ◽

Flavia S. Donaires ◽

Tiago C. Silva ◽

Lilian F. Moreira ◽

Yordanka Armenteros ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Pluripotent Stem Cells ◽

Patient Specific ◽

Hematopoietic Differentiation ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Induced Pluripotent

Aplastic anemia (AA) is characterized by a hypoplastic bone marrow associated with low peripheral blood counts. In acquired cases, the immune system promotes hematopoietic stem and progenitor cell (HSPC) depletion by the action of several pro-inflammatory Th1 cytokines. The current treatment options for severe cases consist of sibling-matched allogeneic hematopoietic stem cell transplantation (HSCT) and immunosuppressive therapy (IST) with anti-thymocyte globulin, cyclosporine, and eltrombopag. However, most patients are not eligible for HSCT and, although about 85% of patients respond to IST with eltrombopag, a proportion of patients eventually relapse, requiring further therapies. Failure to respond adequately to immunosuppression may be attributed to the scarcity of HSPCs at the time of diagnosis. Induced pluripotent stem cells (iPSCs) are potentially an alternative source of patient-specific hematopoietic cells. Patient-specific HSPCs derived from in vitro iPSC differentiation may serve as a tool to study the disease as well as a source of hematopoietic tissue for cell therapies. The pyrimidoindole molecule UM171 induces ex vivo expansion of HSCs of human cord and peripheral blood and bone marrow, but the pathways modulated by this molecule are not well understood. Here we evaluated the hematopoietic differentiation potential of iPSCs obtained from patients with acquired AA. We further determined the effects of UM171 on this differentiation process. First, we derived iPSCs from 3 patients with acquired AA after treatment (1 female; average age, 31 years; 2 partial responders, 1 complete responder) and 3 healthy subjects (3 females; average age, 61 years) and induced differentiation in vitro through the embryoid body system in cell feeder and serum-free medium supplemented with cytokines. The hematopoietic differentiation of healthy-iPSCs yielded 19% ± 8.1% (mean ± SEM) of CD34+cells after 16 days in culture, in contrast with 11% ± 4.9% of CD34+cells obtained from the differentiation of AA-iPSCs, which corresponds to a 1.7-fold reduction in CD34+cell yield. The total number of erythroid and myeloid CFUs was lower in the AA-iPSC group as compared to healthy-iPSCs (12±4.2 vs.24±7.2; respectively; p<0.03). These findings suggest that erythroid-derived AA-iPSC have an intrinsic defect in hematopoietic differentiation. Next, we tested whether UM171 modulated hematopoietic differentiation of AA-iPSCs. We found that UM171 significantly stimulated the differentiation of both healthy and AA-iPSCs. In the healthy-iPSC group, the percentage of CD34+cells was 1.9-fold higher when treated with UM171 compared to controls treated with DMSO (37% ± 7.8% vs.19% ± 8.1%; respectively; p<0.03) and in AA-iPSCs the increase was 3.9-fold (45% ± 11% vs. 11% ± 4.9%; p<0.07). The clonogenic capacity of progenitors to produce erythroid and myeloid colonies also was augmented in both groups in comparison to DMSO (28±11 vs. 23±7.2) for healthy-iPSCs and for AA-iPSCs (23±8.5 vs. 12±4.2, p<0.06). We then investigated the molecular pathways influenced by UM171. The transcriptional profile of differentiated CD34+cells showed that UM171 up-regulated genes involved in early hematopoiesis from mesoderm (BRACHYURY and MIXL1) and primitive streak specification (APELA and APLNR), to hemangioblasts and primitive hematopoietic progenitor commitment (TDGF1, SOX17, and KLF5). We also observed the up-regulation of pro-inflammatory NF-kB activators (MAP4K1, ZAP70, and CARD11) and the anti-inflammatory gene PROCR, a marker of cultured HSCs and an NF-kB inhibitor. This balanced network has been previously suggested to be modulated by UM171 (Chagraoui et. al. Cell Stem Cell 2019). Taken together, our results showed that acquired AA-iPSCs may have intrinsic defects that impair hematopoietic differentiation in vitro. This defect may be atavic to the cell or, alternatively, the consequence of epigenetic changes in erythroid precursors provoked by the immune attack. In addition, our findings demonstrate that UM171 significantly stimulate the hematopoietic differentiation of AA-iPSCs and identified a novel molecular mechanism for UM171 as an enhancer of early hematopoietic development programs. These observations may be valuable for improving the achievement of de novo hematopoietic cells. Disclosures No relevant conflicts of interest to declare.

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From Bone Marrow to Mobilized Peripheral Blood Stem Cells: The Circuitous Path to Clinical Gene Therapy for Fanconi Anemia

Blood ◽

10.1182/blood-2018-99-120278 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2208-2208

Author(s):

Pamela S Becker ◽

Jennifer Adair ◽

Grace Choi ◽

Anne Lee ◽

Ann Woolfrey ◽

...

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Bone Marrow ◽

Fanconi Anemia ◽

Research Funding ◽

Hematopoietic Stem ◽

Post Infusion ◽

Clinical Gene Therapy

Abstract For decades, it has remained challenging to achieve long-term engraftment and correction of blood counts using gene-modified hematopoietic stem cells for Fanconi anemia. Toward this goal, our group conducted preclinical studies using a safety modified lentiviral vector encoding full-length cDNA for FANCA in normal and affected patient hematopoietic progenitor cells, and in a mutant mouse model that supported the IND for a gene therapy clinical trial for Fanconi anemia, complementation group A (NCT01331018). These studies led us to incorporate methods such as addition of N-acetylcysteine and hypoxic incubation during transduction. Because of the low stem cell numbers of Fanconi patients and initial difficulty with using plerixafor off-label for mobilization, we began our study with bone marrow as the source of stem cells. Due to concerns regarding secondary cancers, no conditioning was administered prior to infusion of gene-modified cells. The US Food and Drug Administration approved adult patients initially, but later permitted pediatric patient enrollment with a minimum age of 4 years. The primary objective of our phase I trial was safety. Secondary objectives included in vitro correction of mitomycin C (MMC) sensitivity, procurement of sufficient cell numbers, and ultimately, long-term correction of blood counts in recipients. Eligibility included absolute neutrophil count ≥0.5, hemoglobin ≥8, platelet count ≥20,000, lack of matched family donor, adequate organ function, and not meeting criteria for diagnosis of MDS. Our three enrolled patients were ages 22, 10, and 5 years. All demonstrated defects in the FANCA gene, with two patients sequenced and one patient diagnosed by complementation. Due to in-process learning and the later addition of plerixafor mobilization to the protocol, three different laboratory procedures were used to prepare the gene-modified product for each patient. Cell products were CD34+ selected bone marrow, bone marrow mononuclear cells depleted of red cells by hetastarch, and G-CSF and plerixafor mobilized cells depleted of red blood cells and cells bearing lineage markers, respectively. Transduction efficiencies were 17.7, 42.7 and 26.3% of colony forming cells (CFC) in 0 nM MMC, and 80, 100, and 100% of CFC in 10 nM MMC. Growth of hematopoietic colonies in MMC indicated functional correction of the FANCA defect. The 1st patient received 6.1×10e4, the 2nd 2.9×10e5, and the 3rd 4.3×10e6 CD34+ cells/kg. Serious adverse events included cytopenias in all patients, and hospital admission for fever due to viral upper respiratory infection in one patient. The patients remain alive at 46, 38, and 12 months after receipt of gene-modified cells. Due to worsening cytopenias, the third patient underwent hematopoietic cell transplant from an unrelated donor 10 months after infusion of gene-modified cells. To date, he has done well with transplant, and no indication that prior gene therapy impacted the outcome. The blood counts for the first 2 patients who have not undergone allogeneic transplant remain stable at 1,111 and 1,077 days post infusion compared to the first blood counts when they arrived at our center. For the 1st patient, vector was detectable in white blood cells (WBC) up to 21 days, in the 2nd up to 582 days, and the 3rd up to 81 days post infusion. Thus, in these patients, despite dramatic improvement in cell dose during the study, there was lack of persistence in detection of gene-modified WBCs beyond 1.5 years. A number of factors may have contributed, including lack of conditioning, in vitro cell manipulation including cytokine exposure, inability to transduce primitive hematopoietic stem cells, and paucity of long-term repopulating cells at the ages of the patients, suggesting earlier collection may be beneficial. This study is now closed to enrollment. Valuable information gained as a result of this study will contribute to future clinical gene therapy trials. Current work focuses on how to evaluate stem cell fitness prior to attempting gene therapy, minimizing manipulation required for gene correction and/or in vivo genetic correction and non-chemotherapy-based conditioning to facilitate engraftment. We would like to personally thank each patient and their families for participating in this study, as we could not have learned these lessons without their support. Disclosures Becker: GlycoMimetics: Research Funding; Abbvie: Research Funding; Amgen: Research Funding; BMS: Research Funding; CVS Caremark: Consultancy; Trovagene: Research Funding; Rocket Pharmaceuticals: Research Funding; Novartis: Research Funding; Pfizer: Consultancy; JW Pharmaceuticals: Research Funding. Adair:Miltenyi Biotec: Honoraria; RX Partners: Honoraria; Rocket Pharmaceuticals: Patents & Royalties: PCT/US2017/037967 and PCT/US2018/029983. Kiem:Rocket Pharmaceuticals: Consultancy; Homology Medicine: Consultancy; Magenta: Consultancy.

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Mobilization and collection of peripheral blood CD34+ cells from patients with Fanconi anemia

Blood ◽

10.1182/blood.v98.10.2917 ◽

2001 ◽

Vol 98 (10) ◽

pp. 2917-2921 ◽

Cited By ~ 37

Author(s):

James M. Croop ◽

Ryan Cooper ◽

Christine Fernandez ◽

Vicki Graves ◽

Susan Kreissman ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Fanconi Anemia ◽

Peripheral Blood ◽

Bone Marrow Failure ◽

Peripheral Blood Stem Cells ◽

Target Weight ◽

Peripheral Blood Cd34 ◽

Cd34 Cells ◽

Blood Stem Cells

Abstract A potential therapeutic option for patients with Fanconi anemia is collection of peripheral blood stem cells prior to the development of severe pancytopenia. These hematopoietic cells potentially could be infused when symptomatic bone marrow failure develops, as autologous rescue after chemotherapy in the event of leukemic transformation, or as targets for gene therapy. Eight patients with Fanconi anemia were mobilized with 10 μg/kg per day of granulocyte colony-stimulating factor (median, 10 ± 4 days) to determine the feasibility of collecting peripheral blood stem cells for future use. Six patients achieved a peripheral blood CD34+ count of ≥ 6/μL and underwent apheresis. The collection goal was 2 × 106 CD34+ cells/kg based on a predicted weight 5 years from the date of collection. A mean of 2.6 ± 0.9 × 106 CD34+ cells/kg of the weight at the time of collection were collected, which corresponded to 1.9 ± 0.4 × 106 CD34+cells/kg of the target weight. The collections required a mean of 4 ± 3 days (range, 2-8 days) of apheresis. Six of the 8 subjects had ≥ 1 × 106 CD34+ cells/kg cryopreserved based on both actual and target weights, and 4 subjects had ≥ 2 × 106 CD34+ cells/kg cryopreserved based on the target weight. These results suggest that some patients with Fanconi anemia can have adequate numbers of CD34+ cells mobilized and collected from the peripheral blood prior to the onset of severe bone marrow failure, but they may require an extended mobilization and multiple days of collection.

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DNA Microarray Analysis of GPI-Negative Hematopoietic Stem Cells in Paroxysmal Nocturnal Hemoglobinuria Patients.

Blood ◽

10.1182/blood.v106.11.1048.1048 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1048-1048

Author(s):

Kazuhiko Ikeda ◽

Tsutomu Shichishima ◽

Yoshihiro Yamashita ◽

Yukio Maruyama ◽

Hiroyuki Mano

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem Cell ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Bone Marrow Failure ◽

Data Set ◽

Hematopoietic Stem ◽

Pnh Clone

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematological disorder which is manifested by complement-mediated hemolysis, venous thrombosis, and bone marrow failure. Deficiencies of glycosylphosphatidylinositol (GPI)-anchored proteins, due to mutations in the phosphatidylinositol glycan-class A (PIG-A) gene, contribute to complement-mediated hemolysis and affect all hematopoietic lineages in PNH. However, it is unclear how a PNH clone with a PIG-A gene mutation expands in bone marrow. Although some genes, including the Wilms’ tumor gene (Shichishima et al, Blood, 2002), the early growth response gene, anti-apoptosis genes, and the gene localized at breakpoints of chromosome 12, have been reported as candidate genes that may associate with proliferations of a GPI-negative PNH clone, previous studies were not intended for hematopoietic stem cell, indicating that the differences in gene expressions between GPI-negative PNH clones and GPI-positive cells from PNH patients remain unclear at the level of hematopoietic stem cell. To identify genes contributing to the expansion of a PNH clone, here we compared the gene expression profiles between GPI-negative and GPI-positive fractions among AC133-positive hematopoietic stem cells (HSCs). By using the FACSVantage (Becton Dickinson, San Jose, CA) cell sorting system, both of CD59+AC133+ and CD59− AC133+ cells were purified from bone marrow mononuclear cells obtained from 11 individuals with PNH. Total RNA was isolated from each specimen with the use of RNeasy Mini column (Qiagen, Valencia, CA). The mRNA fractions were amplified, and were used to generate biotin-labeled cDNAs by the Ovation Biotin system (NuGEN Technologies, San Carlos, CA). The resultant cDNAs were hybridized with a high-density oligonucleotide microarray (HGU133A; Affymetrix, Santa Clara, CA). A total of >22,000 probe sets (corresponding to >14,000 human genes) were assayed in each experiment, and thier expression intensities were analyzed by GeneSpring 7.0 software (Silicon Genetics, Redwood, CA). Comparison between CD59-negative and CD59-positive HSCs has identified a number of genes, expression level of which was statistically different (t-test, P <0.001) between the two fractions. Interestingly, one of the CD59− -specific genes isolated in our data set turned out to encode a key component of the proteasome complex. On the other hand, a set of transcriptional factors were specifically silenced in the CD59− HSCs. These data indicate that affected CD59-negative stem cells have a specific molecular signature which is distinct from that for the differentiation level-matched normal HSCs. Our data should pave a way toward the molecular understanding of PNH.

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

Blood ◽

10.1182/blood.v110.11.1677.1677 ◽

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.

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Generation of Dyskeratosis Congenita-like Hematopoietic Stem Cells through the Stable Inhibition of DKC1

10.21203/rs.3.rs-26927/v3 ◽

2021 ◽

Author(s):

Carlos Carrascoso-Rubio ◽

Hidde A. Zittersteijn ◽

Laura Pintado-Berninches ◽

Beatriz Fernández-Varas ◽

M. Luz Lozano ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Failure ◽

Clinical Manifestations ◽

Dyskeratosis Congenita ◽

Bone Marrow Failure Syndrome ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cd34 Cells

Abstract Dyskeratosis congenita (DC) is a rare telomere biology disorder, which results in different clinical manifestations, including severe bone marrow failure. To date, the only curative treatment for bone marrow failure in DC patients is allogeneic hematopoietic stem cell transplantation. However due to the toxicity associated to this treatment, improved therapies are recommended for DC patients. Here we aimed at generating DC-like human hematopoietic stem cells in which the efficacy of innovative therapies could be investigated. Because X-linked DC is the most frequent form of the disease and is associated with an impaired expression of DKC1, we have generated DC-like hematopoietic stem cells based on the stable knock-down of DKC1 in human CD34 + cells with lentiviral vectors encoding for DKC1 short hairpin RNAs. At a molecular level, DKC1 -interfered CD34 + cells showed a decreased expression of TERC, as well as a diminished telomerase activity and increased DNA damage, cell senescence and apoptosis. Moreover, DKC1 -interfered human CD34 + cells showed defective clonogenic ability and were incapable of repopulating the hematopoiesis of immunodeficient NSG mice. The development of DC-like hematopoietic stem cells will facilitate the understanding of the molecular and cellular basis of this inherited bone marrow failure syndrome, and will serve as a platform to evaluate the efficacy of new hematopoietic therapies for DC.

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