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.

Sickle Cell Disease Hematopoietic Stem Cell gene therapy with globin gene addition is promising

2021 ◽  
Author(s):  
Moataz Dowaidar
Keyword(s):  
Gene Therapy ◽  
Stem Cell ◽  
Sickle Cell Disease ◽  
Hematopoietic Stem Cell ◽  
Sickle Cell ◽  
Globin Gene ◽  
Autologous Transplantation ◽  
Gene Repair ◽  
Cell Disease ◽  
Hematopoietic Stem

Autologous transplantation of gene-modified HSCs might be used to treat Sickle Cell Disease (SCD) once and for all. Hematopoietic Stem Cell (HSC) gene therapy with lentiviral-globin gene addition was optimized by HSC collection, vector constructs, lentiviral transduction, and conditioning in the current gene therapy experiment for SCD, resulting in higher gene marking and phenotypic correction. Further advancements over the next decade should allow for a widely approved gene-addition therapy. Long-term engraftment is crucial for gene-corrected CD34+ HSCs, which might be addressed in the coming years, and gene repair of the SCD mutation in the-globin gene can be achieved in vitro using genome editing in CD34+ cells. Because of breakthroughs in efficacy, safety, and delivery strategies, in vivo gene addition and gene correction in BM HSCs is advancing. Overall, further research is needed, but HSC-targeted gene addition/gene editing therapy is a promising SCD therapy with curative potential that might be widely available soon.

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 Reactivation of γ-globin in adult β-YAC mice after ex vivo and in vivo hematopoietic stem cell genome editing

Blood ◽  
2018 ◽  
Vol 131 (26) ◽  
pp. 2915-2928 ◽  
Author(s):  
Chang Li ◽  
Nikoletta Psatha ◽  
Pavel Sova ◽  
Sucheol Gil ◽  
Hongjie Wang ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Stem Cell ◽  
Binding Site ◽  
Genome Editing ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Key Points ◽  
Cell Genome ◽  
Hsc Transplantation

Key Points CRISPR/Cas9-mediated disruption of a BCL11A binding site in HSCs of β-YAC mice results in the reactivation of γ-globin in erythrocytes. Our approach for in vivo HSC genome editing that does not require HSC transplantation and myeloablation should simplify HSC gene therapy.

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

Methylguanine Methyltransferase-Based In Vivo Selection Results in Only Transient Improvement in Long-Term Marking after Autologous Transplantation of Transduced Hematopoietic Stem Cells in Rhesus Macaques.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3272-3272
Author(s):  
Andre Larochelle ◽  
Uimook Choi ◽  
Nora Naumann ◽  
Josh R. Clevenger ◽  
Harry L. Malech ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Steady State ◽  
Ex Vivo ◽  
Rhesus Macaques ◽  
Autologous Transplantation ◽  
Survival Advantage ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Selective Survival

Abstract In vivo selective survival advantage of transduced cells contributed to clinically beneficial levels of genetic correction of lymphocytes following X-SCID gene therapy. For most blood disorders there will be no constitutive selective advantage of the gene-corrected cells. Alternatively, a selectable gene incorporated into the vector may provide selective survival advantage. The P140K mutant of human O6-methylguanine-DNA methyltransferase (MGMT*) is a candidate mammalian selectable gene for hematopoietic stem cell (HSC) gene therapy. AMD3100-mobilized CD34+ cells from 5 rhesus macaques were transduced daily from day 2 to 4 of culture using oncoretroviral (n=2 animals) or lentiviral (n=3 animals) vectors encoding the gp91phox-IRES-MGMT* cassette or the GFP-MGMT* fusion protein, respectively. Transduced CD34+ cells were selected after (in vivo, n=4) or before (ex vivo, n=1) autologous transplantation in rhesus macaques using the BG (120mg/m2)/TMZ 400 mg/m2 combination for in vivo selection and the BG (5uM)/BCNU (7.5uM) combination for ex vivo selection. Marking of peripheral blood (PB) cells was evaluated by FACS and/or real-time PCR. Bulk CD34+ cells were marked at 27–58% after transduction with oncoretroviral or lentiviral vectors. Four animals were transplanted with transduced non-selected CD34+ cells. Small fractions of cultured cells not transplanted were exposed to BG/BCNU resulting in an increase of marking to 88–97% in each case, confirming the in vitro survival advantage. Cells from animals #1 and #2 were transduced with oncoretroviral vectors and steady-state marking of 3.5% was obtained in PB. Animal #1 received BG/TMZ infusions at 3 and 6 months post-transplant. Marking declined to 3.3% and 1.1% after BG/TMZ treatment 1 and 2, respectively. Animal #2 received one cycle of BG/TMZ at 4 months post-transplant. Full hematopoietic recovery was not achieved and the animal died of infectious complications one month after treatment. Marking of 2% was detected in the PB at the time of death. Cells from animals #3 and #4 were transduced with lentiviral vectors. Animal #3 received 4 monthly infusions of BG/TMZ starting 5 months after transplantation. Marking increased from 0.1% at steady-state to 1.8% in PB after the first cycle but rapidly declined to 0.2%. Despite significant myelosuppression, additional cycles of BG/TMZ resulted in no significant improvement in marking. Animal #4 received 4 monthly infusions of BG/TMZ starting 3 months after transplantation. Marking increased from 3.3% at steady-state to 29.2% after the first cycle but rapidly declined to 6.2%. Each additional cycle of BG/TMZ resulted in a transient increase in marking with a peak increase gradually declining with each cycle. Animal #5 was transplanted with CD34+ cells transduced with lentiviral vector expressing GFP-MGMT* and exposed to BG/BCNU ex vivo before transplantation. At the time of reinfusion, 55% of the cells were vector positive. Stable hematopoietic recovery required one month, compared to an average recovery of 2 weeks in animals transplanted with transduced cells without ex vivo selection. Steady state marking in PB of only 0.7% was detected. These data combined with the theoretic concern that the use of cytotoxic drugs could increase the risk of leukemogenesis in the setting of drug-resistance gene therapy, raise concerns for the clinical applicability of this approach.

Hematopoietic Stem Cell Therapy In Eight Patients with Metachromatic Leukodystrophy – Relevance of Post-Transplant Medication.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3727-3727
Author(s):  
Judith Böhringer ◽  
Birgit Kustermann-Kuhn ◽  
Friederike Gieseke ◽  
Annika Erbacher ◽  
Michaela Döring ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Metachromatic Leukodystrophy ◽  
Chronic Gvhd ◽  
Conditioning Regimen ◽  
Unrelated Donor ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Gvhd Prophylaxis

Abstract Abstract 3727 The lysosomal storage disease metachromatic leukodystrophy (MLD) is caused by mutations in the arylsulfatase A (ASA) gene leading to demyelination in the central nervous system (CNS). Children often present impaired motor skills and progress to an inability to walk, paresis and cognitive deterioration. Therapeutic options are limited and currently focus on enzyme replacement, gene therapy and hematopoietic stem cell transplantation (HSCT). Both, gene therapy and HSCT aim to establish a continuous endogenous supply of ASA, which can be taken up by somatic cells of the recipient to correct their own lysosomal metabolism. We analyzed 8 patients with MLD, who underwent HSCT between 7 months and 15 years of age, regarding toxicity of a reduced intensity condition regimen and GvHD prophylaxis. The mean follow up was 2.8 years (range 8 months to 9.5 years). All patients received conditioning with treosulfan (3 × 14 g/m2), fludarabin (4 × 40 mg/m2) and thiotepa (10 mg/kg). Six patients received bone marrow from a 10/10 matched unrelated donor, one patient from her brother and one patient with late infantile MLD was transplanted from her haploidentical mother. Patients received a mean dose of 7.74 × 106 CD34+ cells/kg BW and engrafted at day 12 (range d11 to d22) with permanent full donor chimerism. Organ toxicity reached mucositis grade III and skin grade I. Three patients developed transient acute graft-versus-host disease (GvHD) of the skin grade II, which responded well to steroids. There was no case of transplant-related mortality and no chronic GvHD. The relative ASA activity in peripheral blood mononuclear cells (PBMCs) pre-transplantation was between 0 and 0.11 A514nm/106 cells and after transplantation between 0.52 and 2.24 A514nm/106 cells. Patients who were asymptomatic prior to transplantation stayed asymptomatic, but those who presented with neurological symptoms showed various degrees of progression. As the conditioning regimen showed no immediate neurotoxicity, we asked if other drugs may account for the disease progression early after transplantation. Cyclosporine A (CsA) is commonly used as post-transplant GvHD prophylaxis and is known for neurotoxic side effects. Thus, we analyzed effects of CsA on the activity of ASA in comparison to tacrolimus or mycophenolic acid (MPA). PBMCs were cultured with and without 75 or 150 ng/ml CsA (pharmacological level) for 8 days. During this time period, the ASA activity decreased to 70% of the activity of PBMC without CsA treatment. By contrast, the ASA activity of PBMCs did not decrease under treatment with pharmacological levels of tacrolimus (7,5 or 15 ng/ml) and MPA (3 or 7,5 μg/ml). Cell viability and metabolic activity were comparable in the presence of CsA and tacrolismus as assessed by MTS assays. This implies that the decrease of the ASA activity was not due to direct cell toxicity of CsA. Taken together, the conditioning regimen was well tolerated with low toxicity and good engraftment. HSCT is an option for treatment of asymptomatic MLD patients, whereas further studies are needed to identify symptomatic patients who may still benefit from the procedure. Here, the choice of GvHD prophylaxis may be an important factor. In vitro data suggested that CsA should be reconsidered with regimens including tacrolimus or mycophenolate mofetil as an alternative. Disclosures: No relevant conflicts of interest to declare.

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.

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