scholarly journals Outcomes of Gene Therapy for Severe Sickle Cell Disease (SCD) via Transplantation of Autologous Hematopoietic Stem Cells Transduced Ex Vivo with a Lentiviral ßAT87Q-Globin Vector

2016 ◽  
Vol 22 (3) ◽  
pp. S63 ◽  
Author(s):  
Sandeep Soni ◽  
John F. Tisdale ◽  
Matthew Hsieh ◽  
Julie Kanter ◽  
Philippe Leboulch ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Sickle Cell Disease ◽  
Hematopoietic Stem Cells ◽  
Sickle Cell ◽  
Ex Vivo ◽  
Cell Disease ◽  
Hematopoietic Stem

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

Blood ◽  
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.

scholarly journals Plerixafor enables safe, rapid, efficient mobilization of hematopoietic stem cells in sickle cell disease patients after exchange transfusion

Haematologica ◽  
2018 ◽  
Vol 103 (5) ◽  
pp. 778-786 ◽  
Author(s):  
Chantal Lagresle-Peyrou ◽  
François Lefrère ◽  
Elisa Magrin ◽  
Jean-Antoine Ribeil ◽  
Oriana Romano ◽  
...  
Keyword(s):  
Stem Cells ◽  
Sickle Cell Disease ◽  
Hematopoietic Stem Cells ◽  
Sickle Cell ◽  
Exchange Transfusion ◽  
Cell Disease ◽  
Hematopoietic Stem

scholarly journals Anti-CD117 Antibody Synergizes with 5-Azacytidine to Augment Engraftment of Hematopoietic Stem Cells in Mice with Sickle Cell Disease

2021 ◽  
Vol 27 (3) ◽  
pp. S16
Author(s):  
Andriyana K. Bankova ◽  
Wendy W. Pang ◽  
Brenda J. Velasco ◽  
Jessica Poyser ◽  
Janel Long-Boyle ◽  
...  
Keyword(s):  
Stem Cells ◽  
Sickle Cell Disease ◽  
Hematopoietic Stem Cells ◽  
Sickle Cell ◽  
Cell Disease ◽  
Hematopoietic Stem

Gene Therapy for Sickle Cell Disease - Moving from the Bench to the Bedside

Blood ◽  
2021 ◽  
Author(s):  
Allistair A Abraham ◽  
John F Tisdale
Keyword(s):  
Clinical Trial ◽  
Stem Cells ◽  
Gene Therapy ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Single Point Mutation ◽  
Full Potential ◽  
Cell Disease ◽  
Gene Modification ◽  
Hematopoietic Stem

Gene therapy as a potential cure for sickle cell disease (SCD) has long been pursued given that this hemoglobin disorder results from a single point mutation. Advances in genomic sequencing, increased understanding of hemoglobin regulation and discoveries of molecular tools for genome modification of hematopoietic stem cells have made gene therapy for SCD possible. Gene addition strategies using gene transfer vectors have been optimized over the last few decades to enable expression of normal or anti-sickling globins as strategies to ameliorate SCD. Many hurdles had to be addressed prior to clinical translation including collection of sufficient stem cells for gene-modification, increasing expression of transferred genes to a therapeutic level and conditioning patients in a safe manner that enabled adequate engraftment of gene-modified cells. The discovery of genome editors that make precise modifications has further advanced the safety and efficacy of gene therapy and a rapid movement to clinical trial has undoubtedly been supported by lessons learned from optimizing gene addition strategies. Current gene therapies being tested in clinical trial require significant infrastructure and expertise given the needs to harvest cells from and administer chemotherapy to patients who often have significant organ dysfunction and that gene-modification takes place ex vivo in specialized facilities. For these therapies to realize their full potential they would need to be portable, safe and efficient making an in-vivo based approach attractive. Additionally, adequate resources for SCD screening and access to standardized care are critically important for gene therapy to be a viable treatment option for SCD.

scholarly journals Editing the Sickle Cell Disease Mutation in Human Hematopoietic Stem Cells: Comparison of Endonucleases and Homologous Donor Templates

2019 ◽  
Vol 27 (8) ◽  
pp. 1389-1406 ◽  
Author(s):  
Zulema Romero ◽  
Anastasia Lomova ◽  
Suzanne Said ◽  
Alexandra Miggelbrink ◽  
Caroline Y. Kuo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Sickle Cell Disease ◽  
Hematopoietic Stem Cells ◽  
Sickle Cell ◽  
Cell Disease ◽  
Hematopoietic Stem ◽  
Disease Mutation

scholarly journals Investigational curative gene therapy approaches to sickle cell disease

2021 ◽  
Vol 5 (23) ◽  
pp. 5452-5452
Author(s):  
David A. Williams ◽  
Erica Esrick
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Clinical Manifestations ◽  
Fetal Hemoglobin ◽  
Unrelated Donor ◽  
Cell Disease ◽  
Hematopoietic Stem ◽  
Sickle Hemoglobin

Abstract Sickle cell disease (SCD) is an inherited blood condition resulting from abnormal hemoglobin production. It is one of the most common genetic diseases in the world. The clinical manifestations are variable and range from recurrent acute and debilitating painful crises to life-threatening pulmonary, cardiovascular, renal, and neurologic complications. The only curative treatment of SCD at this time is bone marrow transplantation (also called hematopoietic stem cell transplantation) using healthy blood stem cells from an unaffected brother or sister or from an unrelated donor if one can be identified who is a match in tissue typing. Unfortunately, only a minority of patients with sickle cell has such a donor available. The use of autologous hematopoietic stem cells and alternative types of genetic modifications is currently under study in clinical research trials for this disease. The approaches include the use of viral vectors to express globin genes that are modified to prevent sickle hemoglobin polymerization or to express interfering RNAs to “flip the switch” in adult red cells from adult β-sickle hemoglobin to fetal hemoglobin using a physiologic switch, and several gene editing approaches with the goal of inducing fetal hemoglobin or correcting/modifying the actual sickle mutation. In this audio review, we will discuss these different approaches and review the current progress of curative therapy for SCD using gene therapy.

Increasing Transduction Efficiency in Human Hematopoietic Stem Cells for Gene Therapy

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2052-2052
Author(s):  
Kismet M Baldwin ◽  
Fabrizia Urbinati ◽  
Zulema Romero-Garcia ◽  
Donald B. Kohn
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Clinical Trials ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Lentiviral Vector ◽  
Transduction Efficiency ◽  
Cell Disease ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 2052 Background: Sickle cell disease (SCD) is a multisystem disease, associated with severe episodes of acute illness and progressive organ damage. Currently, the only curative treatment is allogeneic hematopoietic stem cell transplant (HSCT); however, this is limited by availability of HLA compatible donors and by immunological complications of graft rejection or graft-versus-host disease. Autologous stem cell gene therapy for SCD has the potential to treat this illness without the immune suppression needed for 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 (Levasseuer Blood 102:4312–9, 2003). Initial evidence for the efficacy of the modification of human SCD BM CD34+ cells with the βAS3lentiviral (LV) vector for gene therapy of sickle cell disease has been demonstrated in our lab. However, this complex lentiviral vector is produced at a sub-optimal titer and large production batches would be needed to supply clinical trials. Hypothesis: Although, it has been proven that the βAS3 gene can be transduced into CD34+ hematopoietic stem/progenitor cells (HSPC), the transduction efficiency is still not optimal. The CD34+ cell population includes rare long-lived stem cells but also more abundant progenitors, which would be short-lived after transplant. We hypothesize that isolating the more primitive HSPC population (CD34+/CD38− cells approximately 1% of all CD34+ cells) and transducing them with the βAS3 lentiviral vector will increase transduction efficiency and greatly reduce vector needs. Methods: CD34+/CD38− cells were isolated from cord blood (CB) CD34+ cells obtained from healthy donors by fluorescence activated cell sorting (FACS) and transduced with the CCL.βAS3.FB LV vector. After 14 days in culture, vector copy number (VCN) was determined by qPCR. Isolation of a more primitive cell was confirmed via long term culture (LTC) assay for 90 days. At 2–3 weeks intervals, non-adherent cell number was obtained, VCN was analyzed and CFU assays were performed to assess their capability to fully maintain their hematopoietic potential after transduction. Results: CD34+/CD38− cells were effectively isolated using FACS (n=7; 6,329–33,742 cells; 34–99% theoretical yield). The isolated CD34+/CD38- cells were able to generate progeny over an extended period of LTC compared to the CD34+ cells whose cell expansion declined ∼60 days in culture. CFU assays demonstrated that βAS3 gene-modified CB CD34+/CD38- cells were fully capable of maintaining their hematopoietic potential. The isolated CD34+/CD38- cells required 3–40 fold less vector for transduction compared to an equivalent number of these cells contained within the larger, non-fractionated CD34+ preparations. Transduction of CD34+/CD38- cells measured at day 14, by qPCR, was improved relative to CD34+ cells, mean VCN 2.5, +/− SEM 0.33 (range 2–3.5) vs. VCN 1.3, +/− 0.40 (range 0.5–2), respectively (p=0.03). In LTC, VCN remained higher over time in the CD34+/CD38- cells compared to the CD34+ cells, mean VCN 2.0, +/− SEM 0.13 (range 1.6–2.7) vs. VCN 0.5, +/− 0.09 (range 0.2–0.9) respectively. In vivo studies are ongoing to investigate the transduction efficiency of stem/progenitor cells engrafting from CD34+ and CD34+/CD38- cells transplanted in the NSG mouse model. Immunomagnetic isolation of CD34+/CD38- cells using columns is underway in anticipation of potential use in future clinical trials. Further investigations into the mechanisms for increased transduction in the CD34+/CD38- cells are ongoing. Conclusions: This work provides initial evidence for the beneficial effects from isolating human CB CD34+/CD38− cells to improve transduction with the βAS3LV vector for gene therapy of sickle cell disease. Disclosures: No relevant conflicts of interest to declare.

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