Zinc Finger Nucleases Targeting The β-Globin Locus Drive Efficient Correction Of The Sickle Mutation In CD34+ Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2904-2904 ◽  
Author(s):  
Megan D Hoban ◽  
Alok V Joglekar ◽  
David Gray ◽  
Michael L Kaufman ◽  
Fabrizia Urbinati ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Stem Cell ◽  
Zinc Finger Nucleases ◽  
Gene Correction ◽  
Hematopoietic Stem ◽  
Site Specific ◽  
Allogeneic Hsct ◽  
Cd34 Cells ◽  
Cell Source ◽  
Donor Template

Abstract Despite major improvements in clinical care and advances in understanding of its complex pathophysiology, sickle cell disease (SCD) continues to be a significant cause of morbidity and early mortality. Allogeneic hematopoietic stem cell transplant (HSCT) 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 the immunological complications of graft rejection and graft-versus-host disease that can occur, especially for the more than 80% of patients who lack an HLA-identical sibling donor. Gene therapy could provide a way to cure SCD; however, the current approaches use integrating lentiviral vectors, and therefore carry a risk of insertional oncogenesis. An alternative approach is to use site-specific nucleases to correct the patients’ own cells, obviating the need for allogeneic HSCT and the use of randomly integrating vectors. Zinc finger nucleases (ZFNs) offer a possible way to achieve successful gene therapy by site-specifically and permanently modifying the endogenous gene in hematopoietic stem cells (HSCs). These engineered nucleases create a site-specific, double strand break upon dimerization. If a homologous donor molecule is co-introduced which contains the normal β-globin sequence at the site of the sickle mutation, the cells may undergo homology-directed repair to correct the mutation and restore functional hemoglobin production. With this aim in mind, we have designed and tested ZFN pairs targeting the β-globin locus along with a donor template that restores the normal β-globin gene sequence while simultaneously introducing a silent base pair change that generates a restriction enzyme site for analysis. These components have led to high levels of site-specific base-pair modification in introducing the sickle mutation at the normal β-globin locus in K562 cells (upwards of 45%). Using electroporation, we delivered the ZFNs as mRNA to cord blood-derived (CB) CD34+ cells which resulted in up to 30% allelic disruption as measured by the Surveyor Nuclease assay. To achieve gene correction, the ZFNs were again delivered as mRNA and the donor template was delivered as an integrase defective lentiviral vector (IDLV). Based on pyrosequencing data, this delivery method resulted in up to 10% gene correction (the correct nucleotide replacing the sickle mutation in β-globin). Importantly, in the clinically relevant cell source, namely CD34+ cells isolated from SCD patient bone marrow, these gene modification frequencies were maintained, resulting in up to 7% correction using this multi-modal delivery strategy. These data set the stage for further investigations, including ongoing studies in a humanized mouse model. Efficient correction of the sickle mutation in HSC may provide an excellent stem cell source for autologous transplantation for SCD. Disclosures: Cost: Sangamo BioSciences: Employment, Equity Ownership. Reik:Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment.

scholarly journals Expanded circulating hematopoietic stem/progenitor cells as novel cell source for the treatment of TCIRG1 osteopetrosis

Haematologica ◽  
2020 ◽  
Vol 106 (1) ◽  
pp. 74-86 ◽  
Author(s):  
Valentina Capo ◽  
Sara Penna ◽  
Ivan Merelli ◽  
Matteo Barcella ◽  
Serena Scala ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Progenitor Cells ◽  
Cell Transplantation ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Cell Source

Allogeneic hematopoietic stem cell transplantation is the treatment of choice for autosomal recessive osteopetrosis caused by defects in the TCIRG1 gene. Despite recent progress in conditioning, a relevant number of patients are not eligible for allogeneic stem cell transplantation because of the severity of the disease and significant transplant-related morbidity. We exploited peripheral CD34+ cells, known to circulate at high frequency in the peripheral blood of TCIRG1-deficient patients, as a novel cell source for autologous transplantation of gene corrected cells. Detailed phenotypical analysis showed that circulating CD34+ cells have a cellular composition that resembles bone marrow, supporting their use in gene therapy protocols. Transcriptomic profile revealed enrichment in genes expressed by hematopoietic stem and progenitor cells (HSPCs). To overcome the limit of bone marrow harvest/ HSPC mobilization and serial blood drawings in TCIRG1 patients, we applied UM171-based ex-vivo expansion of HSPCs coupled with lentiviral gene transfer. Circulating CD34+ cells from TCIRG1-defective patients were transduced with a clinically-optimized lentiviral vector (LV) expressing TCIRG1 under the control of phosphoglycerate promoter and expanded ex vivo. Expanded cells maintained long-term engraftment capacity and multi-lineage repopulating potential when transplanted in vivo both in primary and secondary NSG recipients. Moreover, when CD34+ cells were differentiated in vitro, genetically corrected osteoclasts resorbed the bone efficiently. Overall, we provide evidence that expansion of circulating HSPCs coupled to gene therapy can overcome the limit of stem cell harvest in osteopetrotic patients, thus opening the way to future gene-based treatment of skeletal diseases caused by bone marrow fibrosis.

scholarly journals Eliminating SCID row: new approaches to SCID

Hematology ◽  
2014 ◽  
Vol 2014 (1) ◽  
pp. 475-480 ◽  
Author(s):  
Donald B. Kohn
Keyword(s):  
Gene Therapy ◽  
Stem Cell ◽  
Ex Vivo ◽  
Autologous Transplantation ◽  
Unrelated Donor ◽  
Primary Immune Deficiency ◽  
Gene Repair ◽  
Gene Correction ◽  
Hematopoietic Stem ◽  
New Approaches

Abstract Treatments for patients with SCID by hematopoietic stem cell transplantation (HSCT) have changed this otherwise lethal primary immune deficiency disorder into one with an increasingly good prognosis. SCID has been the paradigm disorder supporting many key advances in the field of HSCT, with first-in-human successes with matched sibling, haploidentical, and matched unrelated donor allogeneic transplantations. Nevertheless, the optimal approaches for HSCT are still being defined, including determining the optimal stem cell sources, the use and types of pretransplantation conditioning, and applications for SCID subtypes associated with radiosensitivity, for patients with active viral infections and for neonates. Alternatively, autologous transplantation after ex vivo gene correction (gene therapy) has been applied successfully to the treatment of adenosine deaminase–deficient SCID and X-linked SCID by vector-mediated gene addition. Gene therapy holds the prospect of avoiding risks of GVHD and would allow each patient to be their own donor. New approaches to gene therapy by gene correction in autologous HSCs using site-specific endonuclease-mediated homology-driven gene repair are under development. With newborn screening becoming more widely adopted to detect SCID patients before they develop complications, the prognosis for SCID is expected to improve further. This chapter reviews recent advances and ongoing controversies in allogeneic and autologous HSCT for SCID.

Cyclophosphamide-Busulfan Instead of Busulfan-Cyclophosphamide for Conditioning in Allogeneic Hematopoietic Stem Cell Transplantation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2268-2268
Author(s):  
Nathan Cantoni ◽  
Sabine Gerull ◽  
Dominik Heim ◽  
Joerg Halter ◽  
Dimitrios Tsakiris ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cumulative Incidence ◽  
Liver Toxicity ◽  
Conditioning Regimen ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Stem Cell Source ◽  
Cell Source ◽  
Donor Type ◽  
Total Body

Abstract Abstract 2268 Poster Board II-245 Busulfan-cyclophosphamide (BU-CY) is the established non total body irradiation based myeloablative conditioning regimen for allogeneic hematopoietic stem cell transplantation (HSCT). The introduction of intravenous busulfan has facilitated its application and reduced toxicity. Theoretical considerations and pharmacological data indicate that previous application of busulfan may trigger liver toxicity of subsequent cyclophosphamide. A reverse order of cyclophosphamide-busulfan (CY-BU) would be preferable. Recent animal data confirmed this hypothesis, showing less liver toxicity and better outcomes in mice treated with CY-BU. While CY-BU was not feasible in patients with oral busulfan, it has become a possibility with the introduction of i.v. busulfan. We were therefore interested in exploring this concept and changed the order of drug application to CY-BU in 2003 in those patients not on a multicentre standardized BU-CY protocol. We now retrospectively analyzed in this single centre cohort study liver toxicity and transplantation outcome in patients receiving BU and CY as conditioning regimen for allogeneic HSCT. We analyzed 93 consecutive patients between 1993 and 2008, 52 male (55.9%), median age 46 years (range 16 to 70) with hematological malignancies (AML 41 [44.1%], ALL 11 [11.8%], CML 12 [12.9%], myelodysplastic syndrome and myeloproliferative neoplasia 22 [23.7%], lymphoproliferative disorders 4 [4.3%]) or other diseases (3 [3.2%]), receiving an allogeneic HSCT from an HLA- identical sibling (52 [55.9%]), other family member (3 [3.2%]) or a matched unrelated donor (38 [40.9%]) after conditioning regimen with BU-CY (34 patients; 18 patients with oral, 16 patients since 2003 with i.v. busulfan) or CY-BU (59 patients). Outcomes were analyzed using a Cox regression model, adjusting for disease, stage, donor type, stem cell source, previous total body irradiation (TBI) and busulfan administration (oral vs. intravenous). Pretransplant patient characteristics were comparable in the two cohorts for age, gender, underlying disease, stem cell source, donor type and EBMT risk score, but differed in stage (advanced disease BU-CY 28 [84,8%] vs. CY-BU 40 [66.7%]) and previous TBI (BU-CY 16 [48.5%] vs. CY-BU 9 [15.0%]). Liver function as measured by levels of bilirubin and liver enzymes (aspartate amino transferase [AST], alanine amino transferase [ALT], gamma glutamyl transpeptidase [GGT] and alkaline phosphatase [AP]) was not different between the groups before starting conditioning regimen. In contrast liver function differed significantly at day 20, with higher levels of ALT (median 51.0 vs 27.0 IU/l; p=0.012) and a higher incidence of veno-occlusive disease (VOD) (5/34 vs. 1/59, p=0.036) in the BU-CY group (Figure 1A). The cumulative incidence of transplant-related mortality (TRM) at 2 years was significantly higher in patients receiving BU-CY (BU-CY 0.48, CY-BU 0.24, p=0.024; hazard ratio 4.594 for BU-CY, 95% CI 1.382-15.268, p=0.013) (Figure 1B). The cumulative incidence of TRM with BU i.v.-CY was lower (0.44) than with BU oral-CY (0.56) but still higher than CY-BU. This did translate into a higher overall survival in patients after conditioning regimen with CY-BU (hazard ratio for mortality 0.426 for CY-BU, 95% CI 0.184-0.987, p=0.047). Time to engraftment (BU-CY median 13 days vs. CY-BU median 14 days), cumulative incidence of acute GVHD and relapse were similar between patients receiving BU-CY or CY-BU. These data support the concepts derived from Sadeghi et al in their mouse model in favor of CY-BU compared to the traditional BU-CY. They form the basis for prospective controlled studies. Disclosures: No relevant conflicts of interest to declare.

A Non-Human Primate Model for Lentivirus-Mediated Anti-HIV RNAi Strategies.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3103-3103
Author(s):  
Karen Beagles ◽  
Brian Beard ◽  
John Rossi ◽  
Jiing-Kuan Yee ◽  
Shiu-lok Hu ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Stem Cell ◽  
Cell Line ◽  
Lentiviral Vector ◽  
Large Animal ◽  
Hematopoietic Stem ◽  
Hiv Tat ◽  
Cd34 Cells ◽  
Anti Hiv ◽  
Human Primate

Abstract AIDS remains a significant health problem worldwide despite the advent of highly active antiretroviral therapy (HAART). Although substantial efforts have been made to develop a vaccine there is still no cure and alternative strategies are needed to treat HIV infection and to control its spread. Our goal is to evaluate lentiviral vectors that inhibit HIV replication by RNA interference (RNAi) in a non-human primate SHIV model to develop a hematopoietic stem cell (HSC) gene therapy for AIDS. SHIV89.6 P is a chimeric virus comprised of an SIV genome that contains the tat, rev and env genes of HIV and infects both T lymphocytes and macrophages. Infection of non-human primates with SHIV89.6P results in significant decreases in CD4+ T cells as early as 4 weeks post infection, and is currently the best large animal model available to test gene therapy strategies for AIDS. We present here data showing efficient transduction of M. nemestrina CD34+ cells with an HIV-based lentiviral vector and RNAi-mediated inhibition of SHIV89.6 P replication in a hybrid T/B lymphocyte cell line (CEMx174). Although others reported a block to transduction of M. mulatta CD34+ cells with an HIV-based lentiviral vector, we observed efficient transduction rates (» 50%) of M. nemestrina CD34+ cells, comparable to transduction rates observed in human CD34+ cells (» 60%). To determine effectiveness of anti tat/rev shRNA to inhibit SHIV89.6P in vitro, a human T cell/B cell hybrid cell line (CEMx174) was transduced with a lentiviral vector expressing a short-hairpin RNA (shRNA) targeted to both HIV tat and rev sequences that also contained either a GFP reporter gene or a MGMT(G156A) resistance gene at MOIs of 1.3 and 3 respectively. Polyclonal populations of CEMx174 cells transduced with the GFP and MGMT(G156A) vectors were challenged with a 2.15x103 TCID50 dose of SHIV 89.6P. One week post challenge, expression of both tat and rev transcripts was reduced 88% and 97% respectively in these cultures as measured by real-time PCR. In summary, we have shown efficient HIV-based lentiviral transduction of M. nemestrina cells and efficient inhibition of SHIV infection by shRNA against HIV tat and rev thus providing a useful model to test lentiviral-mediated anti-HIV RNAi stem cell gene therapy in vivo.

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

Blood ◽  
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.

scholarly journals Bone Marrow as a Hematopoietic Stem Cell Source for Gene Therapy in Sickle Cell Disease: Evidence from Rhesus and SCD Patients

2017 ◽  
Vol 28 (3) ◽  
pp. 136-144 ◽  
Author(s):  
Naoya Uchida ◽  
Atsushi Fujita ◽  
Matthew M. Hsieh ◽  
Aylin C. Bonifacino ◽  
Allen E. Krouse ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Stem Cell ◽  
Sickle Cell Disease ◽  
Hematopoietic Stem Cell ◽  
Sickle Cell ◽  
Cell Disease ◽  
Hematopoietic Stem ◽  
Stem Cell Source ◽  
Cell Source

scholarly journals Challenges in Vector and Trial Design Using Retroviral Vectors for Long-Term Gene Correction in Hematopoietic Stem Cell Gene Therapy

2012 ◽  
Vol 20 (6) ◽  
pp. 1084-1094 ◽  
Author(s):  
Jacqueline Corrigan-Curay ◽  
Odile Cohen-Haguenauer ◽  
Marina O'Reilly ◽  
Susan R. Ross ◽  
Hung Fan ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Stem Cell ◽  
Trial Design ◽  
Retroviral Vectors ◽  
Gene Correction ◽  
Hematopoietic Stem ◽  
Cell Gene ◽  
Stem Cell Gene ◽  
Stem Cell Gene Therapy

Novel Lentiviral Vectors Displaying ‘Early-Acting-Cytokines’ Preferentially Promote the Survival and Transduction of NOD/SCID Repopulating Human Hematopoietic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2107-2107
Author(s):  
E.L.S. Verhoeyen ◽  
Maciej Wiznerowicz ◽  
Delphine Olivier ◽  
Brigitte Izac ◽  
Didier Trono ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Stem Cell ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Lentiviral Vectors ◽  
Hematopoietic Stem ◽  
Efficient Gene ◽  
Cd34 Cells

Abstract A major limitation of current generation lentiviral vectors (LVs) is their inability to govern efficient gene transfer into quiescent target cells which hampers their application for hematopoietic stem cell gene therapy. Human CD34+ cells that reside into G0 phase of the cell cycle and thus are quiescent, are indeed higly enriched in hematopoietic stem cells. Here, we designed novel lentiviral vectors that overcome this type of restriction by displaying early-acting-cytokines on their surface. Presentation of a single cytokine, thrombopoietin (TPO), or co-presentation of TPO and stem cell factor (SCF) on the lentiviral vector surface improved gene transfer into quiescent CD34+ cord blood cells by 45-fold and 77-fold, respectively, as compared to conventional lentiviral vectors. Moreover, these new LVs preferentially transduced and promoted the survival of immature resting cells rather than cycling CD34+ cells. Most importantly, the new early-cytokine-displaying lentiviral vectors allowed highly efficient gene transfer in CD34+ immature cells with long-term in vivo NOD/SCID mice repopulating capacity, a hallmark of bona fide HSCs. In conclusion, the novel ‘early-acting cytokines’ displaying LVs described here provide simplified, reproducible gene transfer protocols that ensure efficient gene transfer in hematopoietic stem cells. As such, these novel reagents bring us one step closer to selective in vivo gene therapy.

HLA-DRB4 Mismatch Is Associated with Worse Outcomes in Unrelated Allogeneic Hematopoietic Stem Cell Transplantation for Hematological Malignancies

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4540-4540 ◽  
Author(s):  
Marie Y. Detrait ◽  
Ibrahim Yakoub-Agha ◽  
Valerie Dubois ◽  
Françoise Dufossé ◽  
Myriam Labalette ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Acute Gvhd ◽  
Chronic Gvhd ◽  
Unrelated Donor ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Stem Cell Source ◽  
Cell Source

Abstract Abstract 4540 Introduction The impact of HLA DRB3 and DRB4 allele mismatch after allogeneic HSCT using unrelated donors is unclear. We therefore examined retrospectively the outcome of 35 patients who received HLA-10/10 unrelated hematopoietic stem cell transplantation with a DRB3 or DRB4 mismatch between 2005 and 2011. This cohort of 35 patients was a part of a cohort of 132 consecutive patients who underwent allogeneic HSCT between 2005–2011 with a 10/10-HLA matched donor. There were 18 males (51.4%) and 17 females (48.6%) with a median age of 48 years (range, 6–64), there were 13 (37%) AML, 9 (26%) ALL, 4 (11.5%) MDS, 3 (8.5%) multiple myeloma and 6 (5.7%) other (CML, CLL, NHL). Twenty patients (57%) received a myeloablative conditioning (MAC) and 15 (43%) received a reduced intensity conditioning (RIC). At transplantation, 21 patients (60%) were in complete remission (CR), 4 patients (11.5%) in partial remission (PR) and 10 (28.5%) in relapse; 13 (37%) patients received peripheral blood stem cell (PBSC) and 22 (63%) received bone marrow (BM). Twelve (34%) patients had a mismatched DRB4 donor and 23 (66%) patients had a mismatched DRB3 donor. In the remains of 97 patients, there were 55 male (57%) and 42 female (43%), 28 (29%) patients received a MAC and 69 (71%) a RIC as regimen before allogeneic HSCT. The stem cell source was BM for 32 (34%) patients and PBSC for 65 (66%). At transplantation, 34 (35%) patients are in CR and 63 (65%) were in PR. The distribution of diagnosis was acute leukaemia and MDS for 44 (45%), CLL for 2 (2.5%) and other diagnosis (aplastic anemia, NHL, CML, MPS) for 51 patients (52.5%). Results After HSCT, 124 (94%) patients engrafted. After a median follow-up of 11.5 months (range, 0–76), the cumulative incidence of acute GvHD≥2 at 3 months was 20% (95%CI,16.5–24) and the cumulative incidence of chronic GvHD at one year was 19 % (95%CI, 15–22). In univariate analysis, the mismatch DRB3 or DRB4 had no effect on engraftment and no effect on acute GvHD (p=0.08) or chronic GvHD (p=0.63). There was no impact of DRB3 or DRB4 mismatch on relapse (p=0.33 and p=0.53, respectively) and on PFS (p=0.63 and p=0.07, respectively). We found an impact of the DRB4 mismatching (p=0.016) on overall survival. The median survival for patient without DRB3 or DRB4 mismatch was 23 months (14-NR), for patients with DRB3 mismatch 32 months (12-NR), and for DRB4 mismatched patients 5 months (3-NR). The probability of survival at 24 months, for patients without mismatch DRB3 or DRB4 is 47% (36–61), for patients with DRB3 mismatch 51% (32–82) and for DRB4 mismatched patients 19% (6–66%). (figure1). The multivariate analysis that studied age, type of disease, DRB3 or DRB4 mismatch, sexmatching, TBI, ATG, disease status at transplantation and type of conditioning and stem cell source showed a significant impact of mismatch DRB4 on survival (HR= 2.5 [95%CI, 1.2–5.5] p=0.019); there was no impact for DRB3 mismatch (HR= 1.3 (95%CI,0.5–3.9 p=0.58). We found also an impact of the DRB4 mismatch on TRM (HR= 3.5; [95%CI, 1.6 –8] p= 0.026). The incidence of TRM at 24 months for patients without DRB3 or DRB4 mismatch is 29% (24–34), for patients with DRB3 mismatch 17% (9–26%) and for DRB4 mismatched patients 50% (34–66%). (figure 2). Conclusion The HLA DRB4 matching donor is relevant for survival of patients who undergo allo-HSCT from unrelated donor in the HLA-10/10 matching settings. In view of the important impact of these loci mismatches on clinical outcome, it seems to be important to consider this matching loci in the unrelated donor selection. Disclosures: No relevant conflicts of interest to declare.

Generation Of FANCA-/- Human CD34+ Hematopoietic Stem Cells By shRNA Knockdown

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2903-2903
Author(s):  
Zejin Sun ◽  
Rikki Enzor ◽  
Paula Rio ◽  
D. Wade Clapp ◽  
Helmut Hanenberg
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Lentiviral Vector ◽  
Human Fibroblasts ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Control Shrna ◽  
Cd34 Cells

Abstract Fanconi anemia (FA) is a recessive DNA repair disorder characterized by bone marrow (BM) failure, genomic instability, and a predisposition to malignancies. Natural gene therapy due to molecular self-correction of hematopoietic stem cells (HSCs) has been reported in a minority of FA patients, suggesting that due to the in vivo selection advantage of the corrected cells, FA is an excellent model disease for stem cell gene therapy. However, the scarcity of autologous HSCs from FA patients for research purposes is one of the major road blocks to preclinical studies with human cells. Here, we developed a lentiviral vector with EGFP as marker gene that co-expresses two distinct shRNA sequences against FANCA under two different human promoters (H1 and U6). In vitro analysis in primary human fibroblasts showed that stable integration of this construct was highly efficient to induce the typical FA cellular phenotypes as assessed by (1) FANCD2 ubiquitination deficiency and (2) a characteristic G2/M arrest upon DNA damage induced by DNA crosslinking reagent Mitomycin C (MMC). We then transduced human cord blood (CB) CD34+ cells with this lentiviral vector and demonstrated a reduced survival of clonogenic cells in progenitor assays at 20nM MMC: 70% (scrambled control shRNA) vs. 23% (FANCA shRNA). This vector pseudotyped with a foamyviral envelope was then used to transduce CD34+ CB cells on fibronectin CH296. The next day, genetically modified cells were transplanted into NOD.Cg---Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. When analyzing the percentage of EGFP+ cells in the human graft (hCD45+ cells), we noticed a progressive decline of EGFP+ cells from 29% on day 5 to 5% at 4 months after transplantation in the peripheral blood of the recipient mice, mimicking the progressive BM failure in FA patients. In contrast, engraftment over time was stable in CD34+ cells transduced with scrambled control shRNA vector (33% on day 5 vs. 34% at 4 months). The human progenitors isolated from the BM of NSG recipient mice at sacrifice 4 months after initial transduction and transplantation are still hypersensitive to MMC, with a much lower survival rate of 34% at 20nM MMC in the FANCA shRNA group as compared to 78% in the scrambled control shRNA group, thus confirming the knockdown by the lentiviral shRNA construct is stable. In summary, the novel double shRNA lentiviral vector is capable of inducing all major hallmarks of FA cells in normal human CB CD34+ cells, thus providing unlimited FA-like cellular materials including NSG mice-repopulating HSCs for preclinical gene therapy and basic stem cell biology research in FA. Disclosures: No relevant conflicts of interest to declare.

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