Long-Term Safety and Efficacy of Stem Cell Gene Therapy for ADA-SCID.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 200-200
Author(s):  
Alessandro Aiuti ◽  
Ulrike Benninghoff ◽  
Barbara Cassani ◽  
Federica Cattaneo ◽  
Luciano Callegaro ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Severe Combined Immunodeficiency ◽  
Hematopoietic Stem ◽  
Enzyme Replacement ◽  
Cell Functions ◽  
Cd34 Cells

Abstract Severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA) deficiency is a fatal congenital disorder of the immune system associated with systemic toxicity due to accumulation of purine metabolites. We previously showed that retroviral-mediated ADA gene transfer into autologous hematopoietic stem/progenitor cells (HSC) allowed restoration of immune and metabolic functions. We have now enrolled eight ADA-SCID children (age: 7–67 months) in our phase I/II gene therapy trial in which HSC are combined with low intensity conditioning with busulfan (total dose 4 mg/Kg i.v.). Previous treatment included haploidentical bone marrow transplant (n=3) or long-term (>1 year) enzyme replacement therapy (PEG-ADA) (n=4) associated with insufficient immune reconstitution or severe autoimmunity. In the latter case, PEG-ADA was discontinued to favour the growth advantage for gene corrected cells. The patients received a median dose of 8.8x106/Kg bone marrow CD34+ cells (range 0.9–10.8), containing on average 26.2±9.6% transduced CFU-C. Five patients experienced ANC <0.5x109/L, which was extended beyond day +30 in two patients. With a median follow up of 3.1 years (range 0.4–5.9), no adverse events related to gene transfer have been observed. Long-term engraftment of transduced HSC was demonstrated by stable multilineage marking, persisting more than 5 years from gene therapy. The average proportion of transduced cells in the peripheral blood at one year post-gene therapy (n=6) was 5% for granulocytes, 95% for T cells, 56% for B cells and 62% for NK cells. Comparison of the insertion sites retrieved ex vivo from patients with those identified in pre-transplant transduced CD34+ cells showed no skewing in the profile of genome distributions or in the gene families hit by the vector, and no clonal expansion. In the six children with a follow-up >1 year after gene therapy, we observed a progressive increase in lymphocyte counts which was sustained over time (median at 1.5 years 1.6x109/L), polyclonal thymopoiesis and normalization of T-cell functions in vitro. Serum Ig levels improved and evidence of antigen-specific antibodies was obtained, leading to IVIG discontinuation in five patients. All the children are currently healthy and thriving, and none of them showed severe infections. Sustained ADA activity in lymphocytes and RBC resulted in a dramatic reduction of RBC purine toxic metabolites (dAXP<30 nmoles/ml in 5 patients) and amelioration of children’s growth and development. In summary, these data confirm that gene therapy is safe and efficacious in correcting both the immune and metabolic defect in ADA-SCID, with proven clinical benefit.

Long-term Outcomes after Gene Therapy for Adenosine Deaminase Severe Combined Immune Deficiency (ADA SCID)

Blood ◽  
2021 ◽  
Author(s):  
Bryanna C. Reinhardt ◽  
Omar Habib ◽  
Kit L. Shaw ◽  
Elizabeth K Garabedian ◽  
Denise Ann Carbonaro-Sarracino ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Replacement Therapy ◽  
Adenosine Deaminase ◽  
Immune Reconstitution ◽  
Severe Combined Immunodeficiency ◽  
Hematopoietic Stem ◽  
Enzyme Replacement ◽  
Cd34 Cells ◽  
Adenosine Deaminase Activity

Patients lacking functional adenosine deaminase activity suffer from severe combined immunodeficiency (ADA SCID), which can be treated with ADA enzyme replacement therapy (ERT), allogeneic hematopoietic stem cell transplantation (HSCT), or autologous HSCT with gene-corrected cells (gene therapy-GT). A cohort of 10 ADA SCID patients, aged 3 months to 15 years, underwent GT in a Phase II clinical trial between 2009 and 2012. Autologous bone marrow CD34+ cells were transduced ex vivo with the MND-ADA gamma-retroviral vector (gRV) and infused following busulfan reduced intensity conditioning. These patients were monitored in a long-term follow-up protocol over 8-11 years. Nine of ten patients have sufficient immune reconstitution to protect against serious infections, and have not needed to resume ERT or proceed to secondary allogeneic HSCT. ERT was restarted 6 months after GT in the oldest patient who had no evidence of benefit from GT. Four of nine evaluable patients with the highest gene marking and B cell numbers remain off immunoglobulin replacement therapy and responded to vaccines. There were broad ranges of responses in normalization of ADA enzyme activity and adenine metabolites in blood cells, and levels of cellular and humoral immune reconstitution. Outcomes were generally better in younger patients and those receiving higher doses of gene-marked CD34+ cells. No patient experienced a leukoproliferative event after GT, despite persisting prominent clones with vector integrations adjacent to proto-oncogenes. These long-term findings demonstrate enduring efficacy of GT for ADA SCID, but risks of genotoxicity with gRVs. (Clinicaltrials.gov #NCT00794508)

ADA-deficient SCID is associated with a specific microenvironment and bone phenotype characterized by RANKL/OPG imbalance and osteoblast insufficiency

Blood ◽  
2009 ◽  
Vol 114 (15) ◽  
pp. 3216-3226 ◽  
Author(s):  
Aisha V. Sauer ◽  
Emanuela Mrak ◽  
Raisa Jofra Hernandez ◽  
Elena Zacchi ◽  
Francesco Cavani ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Severe Combined Immunodeficiency ◽  
Intrinsic Defect ◽  
Combined Immunodeficiency ◽  
Hematopoietic Stem ◽  
Enzyme Replacement ◽  
Bone Phenotype

Abstract Adenosine deaminase (ADA) deficiency is a disorder of the purine metabolism leading to combined immunodeficiency and systemic alterations, including skeletal abnormalities. We report that ADA deficiency in mice causes a specific bone phenotype characterized by alterations of structural properties and impaired mechanical competence. These alterations are the combined result of an imbalanced receptor activator of nuclear factor-κB ligand (RANKL)/osteoprotegerin axis, causing decreased osteoclastogenesis and an intrinsic defect of osteoblast function with subsequent low bone formation. In vitro, osteoblasts lacking ADA displayed an altered transcriptional profile and growth reduction. Furthermore, the bone marrow microenvironment of ADA-deficient mice showed a reduced capacity to support in vitro and in vivo hematopoiesis. Treatment of ADA-deficient neonatal mice with enzyme replacement therapy, bone marrow transplantation, or gene therapy resulted in full recovery of the altered bone parameters. Remarkably, untreated ADA–severe combined immunodeficiency patients showed a similar imbalance in RANKL/osteoprotegerin levels alongside severe growth retardation. Gene therapy with ADA-transduced hematopoietic stem cells increased serum RANKL levels and children's growth. Our results indicate that the ADA metabolism represents a crucial modulatory factor of bone cell activities and remodeling. The trials were registered at www.clinicaltrials.gov as #NCT00598481 and #NCT00599781.

Gene Therapy for Fanconi Anemia in Seattle: Clinical Experience and Next Steps

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3510-3510 ◽  
Author(s):  
Jennifer E Adair ◽  
Pamela S. Becker ◽  
Devikha Chandrasekaran ◽  
Grace Choi ◽  
Ann E Woolfrey ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Blood Cell ◽  
Phase I ◽  
Fanconi Anemia ◽  
Magnetic Bead ◽  
Cd34 Cells ◽  
Equity Ownership

Abstract One hallmark of the rare, monogenic disorder called Fanconi anemia (FA) is an accelerated decline in hematopoietic stem cells (HSCs) leading to bone marrow (BM) failure. Long-term treatment requires successful bone marrow transplant (BMT) from an unaffected donor. However, BMT success is limited if the donor is not a matched sibling and ~70% of FA patients lack such donors. Gene therapy could be an alternative, correcting the genetic defect in the patient's own HSCs, and negating the need for a BMT donor. Based on lessons learned in previous FA gene therapy studies, we developed an optimized protocol for lentivirus (LV)-mediated FANCA gene transfer into HSCs from FA-A patients. This phase I clinical trial incorporates vector recommendations from the International FA Gene Therapy Working Group. Two patients have been treated on this trial to date (National Clinical Trials Registry ID: NCT01331018). This protocol harvests BM to collect unmanipulated HSC and does not include conditioning prior to cell infusion. Patient 1 is a 22-year old male with confirmed FA-A resulting from a splice variant in exon 22 of the FANCA gene (c. 1827-1 G>A). Baseline ANC averaged 0.5-1.0 K/mcL and baseline platelet counts averaged 40 K/mcL. A total of 3.2 x 107 CD34+ cells were present in 1.1L of BM, but only 9.4 x 106 total CD34+ cells were successfully isolated by magnetic bead separation, due to low level CD34 expression. LV transduction at 10 infectious units (IU)/cell resulted in a vector copy number (VCN) of 0.33 per cell and 18.4% of colony-forming cells transduced. Patient 2 is a 10-year old male with confirmed FA-A resulting from gross deletion of exons 6-31 of the FANCA gene. Baseline ANC and platelets declined over a 4.5-year interval prior to gene therapy and were 0.67 K/mcL and 82 K/mcL, respectively, in the 6 months prior to treatment. A total of 400mL of BM was collected, containing a total of 30.6 x 106 CD34+ cells. To avoid excessive CD34+ cell loss, the CD34+ cell magnetic bead purification step was omitted and the entire red blood cell depleted BM product was subjected to LV transduction at 10 IU/cell. We observed a VCN of 1.83 per cell and 43% of colony-forming cells transduced, suggesting more efficient transduction of the mixed cell population. Both patients tolerated the harvest and infusion procedures but displayed low and declining levels of transduced cells in peripheral blood after infusion. For future subjects, use of pre-infusion conditioning may be required to achieve long-term engraftment in vivo. Interestingly, both patients have maintained stable blood cell counts since gene therapy. These data demonstrate that LV gene therapy in FA patients is safe and suggest that avoidance of direct CD34 selection is advantageous for transduction and gene transfer. However, one complicating factor is the volume of concentrated LV vector required to transduce the non-purified cell product infused in Patient 2. To address these barriers, we developed a modified clinical protocol which utilizes depletion of mature cell lineages including CD3+, CD14+, CD16+ and CD19+ cells. Using healthy donor bone marrow we demonstrate that this protocol efficiently depletes >85% of cells expressing each lineage marker, reducing the volume of LV vector required for gene transfer by 60-70%. Most importantly, we demonstrate that this protocol preserves >90% of CD34+ cells present in the starting bone marrow product, and that these cells are efficiently transduced and capable of engrafting in a xenotransplant model. This protocol is currently being implemented for subjects in the ongoing phase I trial. Disclosures Adair: Rocket Pharmaceuticals: Consultancy, Equity Ownership. Kiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Research Funding.

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 Stromal support enhances cell-free retroviral vector transduction of human bone marrow long-term culture-initiating cells

Blood ◽  
1992 ◽  
Vol 79 (6) ◽  
pp. 1393-1399 ◽  
Author(s):  
KA Moore ◽  
AB Deisseroth ◽  
CL Reading ◽  
DE Williams ◽  
JW Belmont
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Transduction Efficiency ◽  
Hematopoietic Stem ◽  
Long Term Culture ◽  
Stromal Support

Gene transfer into hematopoietic stem cells by cell-free virions is a goal for gene therapy of hematolymphoid disorders. Because the hematopoietic microenvironment provided by the stroma is required for stem cell maintenance both in vivo and in vitro, we reasoned that cell- free transduction of bone marrow cells (BMC) may be aided by stromal support. We used two high-titer replication-defective retroviral vectors to differentially mark progenitor cells. The transducing vector was shown to be a specific DNA fragment by polymerase chain reaction of colony-forming cells derived from progenitors maintained in long-term culture (LTC). BMC were infected separately by cell-free virions with or without pre-established, irradiated, allogeneic stromal layers, and in the presence or absence of exogenous growth factors (GF). The GF assessed were interleukin-3 (IL-3) and IL-6 in combination, leukemia inhibitory factor (LIF), mast cell growth factor (MGF), and LIF and MGF in combination. In addition, we developed a competitive LTC system to directly assess the effect of infection conditions on the transduction of clonogenic progenitors as reflected by the presence of a predominate provirus after maintenance in the same microenvironment. The results show gene transfer into human LTC-initiating cells by cell-free retroviral vector and a beneficial effect of stromal support allowing a transduction efficiency of 64.6% in contrast to 15.8% without a supporting stromal layer. A high transduction rate was achieved independent of stimulation with exogenous GF. We propose that autologous marrow stromal support during the transduction period may have application in clinical gene therapy protocols.

scholarly journals Stromal support enhances cell-free retroviral vector transduction of human bone marrow long-term culture-initiating cells

Blood ◽  
1992 ◽  
Vol 79 (6) ◽  
pp. 1393-1399 ◽  
Author(s):  
KA Moore ◽  
AB Deisseroth ◽  
CL Reading ◽  
DE Williams ◽  
JW Belmont
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Transduction Efficiency ◽  
Hematopoietic Stem ◽  
Long Term Culture ◽  
Stromal Support

Abstract Gene transfer into hematopoietic stem cells by cell-free virions is a goal for gene therapy of hematolymphoid disorders. Because the hematopoietic microenvironment provided by the stroma is required for stem cell maintenance both in vivo and in vitro, we reasoned that cell- free transduction of bone marrow cells (BMC) may be aided by stromal support. We used two high-titer replication-defective retroviral vectors to differentially mark progenitor cells. The transducing vector was shown to be a specific DNA fragment by polymerase chain reaction of colony-forming cells derived from progenitors maintained in long-term culture (LTC). BMC were infected separately by cell-free virions with or without pre-established, irradiated, allogeneic stromal layers, and in the presence or absence of exogenous growth factors (GF). The GF assessed were interleukin-3 (IL-3) and IL-6 in combination, leukemia inhibitory factor (LIF), mast cell growth factor (MGF), and LIF and MGF in combination. In addition, we developed a competitive LTC system to directly assess the effect of infection conditions on the transduction of clonogenic progenitors as reflected by the presence of a predominate provirus after maintenance in the same microenvironment. The results show gene transfer into human LTC-initiating cells by cell-free retroviral vector and a beneficial effect of stromal support allowing a transduction efficiency of 64.6% in contrast to 15.8% without a supporting stromal layer. A high transduction rate was achieved independent of stimulation with exogenous GF. We propose that autologous marrow stromal support during the transduction period may have application in clinical gene therapy protocols.

Long-Term Clinical Follow-Up and Safety/Toxicity Analysis in 3 X-CGD Patients Treated by Gene Therapy and Non-Myeloablative Conditioning.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 199-199 ◽  
Author(s):  
Marion G. Ott ◽  
Manfred Schmidt ◽  
Stefan Stein ◽  
Kerstin Schwarzwaelder ◽  
Ulrich Siler ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Myeloid Cells ◽  
Adult Patients ◽  
Hematopoietic Stem

Abstract Gene transfer into hematopoietic stem cells has been successfully used to correct immunodeficiencies affecting the lymphoid compartment. However, similar results have not been reported for diseases affecting myeloid cells, mainly due to low engraftment levels of gene-modified cells observed in unconditioned patients. Here we report on two adult patients (P1 and P2, follow up >24 months) and one child (P3, 6 years, follow up 15 months) who received gene-transduced hematopoietic stem cells in combination with nonmyeloablative bone marrow conditioning for the treatment of X-linked Chronic Granulomatous Disease (X-CGD), a primary immunodeficiency caused by a defect in the oxidative antimicrobial activity of phagocytes. Therapeutically significant gene marking was detected in neutrophils of both adult patients (P1 and P2) leading to large numbers (up to 60%) of functionally corrected phagocytes 24 months after gene therapy. This high correction resulted from an unexpected but temporarily restricted expansion of gene transduced myeloid cells in vivo. In contrast gene marking and functionally reconstitution levels in P3 have been low (1–2%). Both adult patients suffered from active infections prior to gene therapy (P1 of bacterial liver abscesses and P2 of lung aspergillosis) and were free of severe bacterial and fungal infections until 24 months after transplantation. P3 suffered from an Aspergillus infection of the spinal cord with paraparesis before transplantation and recovered after gene therapy despite low numbers of functionally corrected cells in the peripheral blood. Large-scale mapping of retroviral integration site distribution revealed that activating insertions in the zinc finger transcription factor homologs MDS1/EVI1, PRDM16, or in SETBP1 have expanded gene-corrected long term myelopoiesis 3- to 4-fold in both adults, providing direct evidence in humans that these genes may influence regulation of normal long-term hematopoiesis. The hematopoietic repopulation in P1 was polyclonal until 18 months after therapy. P1 died of a severe bacterial sepsis after colon perforation 27 months after gene therapy. No evidence of malignant transformation was found in peripheral blood or bone marrow aspirates from this patient. Gene marking at death was still 60%; however the function of gene transduced cells, the number of corrected cell clones and the activity of a predominant clone was greatly decreased. P2 has been free of infections since transplantation (last monitoring: month 26). Hematopoietic repopulation was polyclonal in P2 until day 560. In conclusion, gene therapy in combination with bone marrow conditioning has provided a transitory therapeutic benefit for all 3 patients. Further improvements in vector design and conditioning regimes are under investigation to provide a stable and long term correction of the disease.

scholarly journals Fully Closed, Large-Scale, and Clinical Grade Cell Sorting of Hematopoietic Stem Cell (HSC)-Enriched CD90+ Cells for Transplantation and Gene Therapy

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3246-3246
Author(s):  
Stefan Radtke ◽  
Margaret Cui ◽  
Anai M Perez ◽  
Yan-Yi Chan ◽  
Stefanie Schmuck ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Stem Cell ◽  
Transduction Efficiency ◽  
Target Cells ◽  
Gene Modification ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Cell Fractions

Introduction: Hematopoietic stem cell (HSC) gene therapy/editing is a viable treatment option for various hematological diseases and disorders including hemoglobinopathies and HIV/AIDS. Most if not all currently available approaches target CD34-enriched cell fractions, a heterogeneous mix of mostly committed progenitor cells and only very few true HSCs with long-term multilineage engraftment potential. As a consequence, gene therapy/editing approaches are currently limited in their HSC targeting efficiency, very expensive consuming huge quantities of modifying reagents, and can lead to unwanted side-effects in non-target cells. We recently described a novel HSC-enriched CD34 subset (CD90+CD45RA-) that is exclusively responsible for rapid recovery onset, robust long-term multilineage engraftment, as well as entire reconstitution of the bone marrow stem cell compartment in the nonhuman primate (NHP) stem cell transplantation and gene therapy model (Radtke et al. 2017, STM). Most importantly, we demonstrate that this CD34 subset reduces the number of target cells, modifying reagents and costs by more than 10-fold without compromising the long-term efficiency of gene-modification in the NHP (Humbert and Radtke et al. 2019, STM). Here, we aimed to develop a clinical protocol to reliably purify and efficiently gene-modify human HSC-enriched CD90+ cell fractions. Methods: Large-scale enrichment of CD34+ cells from GCSF-mobilized leukapheresis products was initially performed on the Miltenyi CliniMACS Prodigy according to previously established protocols (Adair et al. 2017, Nat. Comm.). Yield, purity, quality, and feasibility of CD90 sorting was then comprehensively tested on two different commercially available cell sorting systems comparing the jet-in-air sorter FX500 from Sony and the cartridge-based closed-system sorter MACSQuant Tyto from Miltenyi Biotech with our clinically approved gold-standard CD34-mediated gene therapy approach. Sorted CD90+ and bulk CD34+ cells were transduced with a clinical-grade lentivirus encoding for GFP and the multilineage differentiation as well as engraftment potential tested using in vitro assays and the NSG mouse xenograft model, respectively. Results: Flow-cytometric sort-purification of CD90+ cells was similarly efficient in purity and yield using either the FX500 or Tyto (Figure A,B). Both approaches reliably reduced the overall target cell count by 10 to 15-fold without impacting the cells viability and in vitro colony-forming cell potential. Unexpectedly, the transduction efficiency of sort-purified CD90+ cells was significantly improved compared to bulk-transduced CD34+ cells and especially the CD34+CD90+ subset (Figure C). All cell fractions demonstrated robust mouse xenograft potential (Figure D). Most importantly, significantly higher levels of GFP+ expression in the peripheral blood, bone marrow, spleen and thymus were observed after transplantation of gene-modified CD90+ compared to bulk CD34+ cells in NSG mice (Figure E). Conclusion: Here, we show that sort-purification of our HSC-enriched CD34+CD90+ cell subset is technically feasible and highly reproducible in two different systems. Purification of human CD90+ cell fractions significantly increased the gene-modification efficiency of primitive human HSCs with multilineage mouse engraftment potential. These findings should have important implications for currently available as well as future HSC gene therapy and gene editing protocols. Isolation of an HSC-enriched phenotype will allow more targeted gene modification and thus likely reduce unwanted off target effects. Our approach further reduced the overall costs for gene modifying reagents, can be combined with a closed transduction system, increase the portability and ultimately make HSC gene therapy GMP-facility independent and affordable. Finally, this stem cell selection strategy may also allow efficient and effective depletion of donor T cells in the setting of allogeneic stem cell or organ transplantation. Figure: A) Purity and B) yield of CD90+ cells after sort-purification. C) Transduction efficiency of bulk-transduced CD34+CD90+ cells and sort-purified CD90+ cells. Frequency of D) human chimerism and E) GFP+ human CD45+ cells in the peripheral blood (PB), bone marrow, spleen and thymus after transplantation of gene-modified bulk CD34+ or sort-purified CD90+ cells. Figure Disclosures Kiem: CSL Behring: Consultancy; Rocket Pharma: Consultancy, Equity Ownership; Homology Medicines: Consultancy, Equity Ownership; Magenta Therapeutics: Consultancy.

Hematopoietic Stem Cell Gene Therapy Trial with Lentiviral Vector in X-Linked Adrenoleukodystrophy

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 821-821 ◽  
Author(s):  
Marina Cavazzana-Calvo ◽  
Nathalie Cartier ◽  
Salima Hacein-Bey Abina ◽  
Gabor Veres ◽  
Manfred Schmidt ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Stem Cell ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Cd34 Cells ◽  
Hsc Transplantation

Abstract We report preliminary results in 3 children with cerebral X-linked adrenoleukodystrophy (ALD) who received in September 2006, January 2007 and June 2008 lentiviral vector transduced autologous hematopoietic stem cell (HSC). We have previously demonstrated that cerebral demyelination associated with cerebral ALD can be stopped or reversed within 12–18 months by allogeneic HSC transplantation. The long term beneficial effects of HCT transplantation in ALD are due to the progressive turn-over of brain macrophages (microglia) derived from bone-marrow cells. For the current HSC gene therapy procedure, we used mobilized peripheral blood CD34+ cells that were transduced ex vivo for 18 hours with a non-replicative HIV1-derived lentiviral vector (CG1711 hALD) at MOI25 and expressing the ALD cDNA under the control of the MND (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site substituted) promoter, and in the presence of 4 human recombinant cytokines (Il- 3, Stem Cell Factor [SCF], Flt3-ligand and Megakaryocyte Growth and Differentiation Factor [MGDF]) and CH-296 retronectine. Transduced cells were frozen to perform the required (RCL) safety tests. After thawing and prior to reinjection, 50%, 30% and 40% of transduced CD34+ cells expressed the ALD protein with a mean of 0.7, 0.6 and 0.65 copies of integrated provirus per cell. Transduced CD34+ cells were infused to ALD patients after a conditioning regimen including full doses of cyclophosphamide and busulfan. Hematopoietic recovery occured at day 13–15 post-transplant and the procedure was uneventful. In patient P1 and P2, the percentage of lymphocytes and monocytes expressing the ALD protein declined from day 60 to 6 months after gene therapy (GT) and remained stable up to 16 months post-GT. In P1, 9 to 13% of CD14+, CD3+, CD19+ and CD15+ cells expressed ALD protein 16 months post-transplant. In P2 and at the same time-point after transplant, 10 to 18% of CD14+, CD3+, CD19+ and CD15+ cells expressed ALD protein. ALD protein was expressed in 18–20% of bone marrow CD34+ cells from patients P1 and P2, 12 months post-transplant. In patient P3, 20 to 23% of CD3+, CD14+ and CD15+ cells expressed ALD protein 2 months after transplant. Tests assessing vector-derived RCL and vector mobilization were negative up to the last followups in the 3 patients. Integration of the vector was polyclonal and studies of integration sites arein progress. At 16 months post-transplant, HSC gene therapy resulted in neurological effects comparable with allogeneic HSC transplantation in patient P1 and P2. These results support that: ex-vivo HSC gene therapy using HIV1-derived lentiviral vector is not associated with the emergence of RCL and vector mobilization; a high percentage of hematopoietic progenitors were transduced expressing ALD protein in long term; no early evidence of selective advantage of the transduced ALD cells nor clonal expansion were observed. (This clinical trial is sponsored by Institut National de la Santé et de la Recherche Médicale and was conducted in part under a R&D collaboration with Cell Genesys, Inc., South San Francisco, CA)

Expression vectors for human adenosine deaminase gene therapy

Genome ◽  
1989 ◽  
Vol 31 (2) ◽  
pp. 832-839 ◽  
Author(s):  
Kateri A. Moore ◽  
Frederick A. Fletcher ◽  
Raye Lynn Alford ◽  
Deborah K. Villalon ◽  
Dianne H. Hawkins ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Adenosine Deaminase ◽  
Retroviral Vectors ◽  
Expression Vectors ◽  
Mouse Bone Marrow ◽  
Human Genetic Disease ◽  
Hematopoietic Stem

Somatic gene transfer offers a possible new approach for treatment of human genetic disease. Defects affecting blood-forming tissues are candidates for therapies involving transfer of genetic information into hematopoietic stem cells. Adenosine deaminase (ADA) deficiency is being used as a model disease for which gene transfer techniques can be developed and evaluated. We describe here the construction and testing of 20 retroviral vectors for their ability to transfer and express human ADA in vitro and in vivo via a mouse bone marrow transplantation model. After infection of primary bone marrow with one of these vectors (pΔNN2ADA), human ADA was detected in 60–85% of spleen colonies at day 14 and maintained long term in the blood of fully reconstituted mice. This system offers the opportunity to assess methods for increasing efficiency of gene transfer, for regulation of expression of foreign genes in hematopoietic progenitors, and for long-term measurement of the stability of expression in these cells.Key words: gene therapy, adenosine deaminase, retrovirus vectors, immunodeficiency.

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