Long-Term Follow-Up of Patients Treated by Gene Therapy for X-Linked Chronic Granulomatous Disease.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 194-194 ◽  
Author(s):  
Marion G. Ott ◽  
Manuel Grez ◽  
Stefan Stein ◽  
Ulrich Siler ◽  
Ulrike Koehl ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Chronic Granulomatous Disease ◽  
Ex Vivo ◽  
Therapeutic Option ◽  
High Morbidity ◽  
Granulomatous Disease ◽  
Phagocytic Cells

Abstract Chronic granulomatous disease (CGD) is a primary immunodeficiency in which phagocytic cells of affected patients have impaired antimicrobial activity due to a defect in the production of reactive oxygen species (ROS). CGD is caused by mutations in any one of four genes encoding for the subunits of the NADPH oxidase complex. Although curable by HSC transplantation, this strategy is usually limited only to patients with HLA-matched sibling or unrelated donors, as mismatched transplantation is associated with high morbidity and mortality due to graft failure and slow immune reconstitution. A therapeutic alternative for CGD patients is the genetic modification of autologous HSC. In January 2004 we initiated a Phase I/II clinical trial for X-CGD patients including conditioning with busulfan (8 mg/kg/total dose) prior to infusion of genetically modified HSC. G-CSF mobilized CD34+ cells from 2 adult patients (25 and 26 years) were transduced ex-vivo with a monocistronic gp91phox retroviral vector. Therapeutically significant gene marking levels were detected in neutrophils of both patients with up to 60% functionally corrected phagocytes 14 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 levels in B-cells has remained constant at a level of 20%, while gene marking in T-cells is below 5%. Gene marking in bone marrow was detected at levels between 30% and 40% one year after transplantation of gene modified cells. Killing assays in vitro have demonstrated antibacterial and antifungal activity in gene transduced phagocytes and both patients recovered of Staph. aureus and Aspergillus fumigatus infections after gene therapy. Our results suggest that gene therapy in combination with bone marrow conditioning is a therapeutic option for inherited diseases affecting the myeloid compartment and can be successfully used to treat CGD.

scholarly journals Enhanced Host Defense After Gene Transfer in the Murine p47phox-Deficient Model of Chronic Granulomatous Disease

Blood ◽  
1997 ◽  
Vol 89 (7) ◽  
pp. 2268-2275 ◽  
Author(s):  
Michael Mardiney ◽  
Sharon H. Jackson ◽  
S. Kaye Spratt ◽  
Fei Li ◽  
Steven M. Holland ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Chronic Granulomatous Disease ◽  
Host Defense ◽  
Ex Vivo ◽  
Pseudomonas Cepacia ◽  
Granulomatous Disease ◽  
Human Immune System ◽  
Human Form

Abstract The p47phox−/− mouse exhibits a phenotype similar to that of human chronic granulomatous disease (CGD) and, thus, is an excellent model for the study of gene transfer technology. Using the Moloney murine leukemia virus–based retroviral vector MFG-S encoding the human form of p47phox, we performed ex vivo gene transfer into Sca-1+ p47phox−/− marrow progenitor cells without conditioning of donors with 5-fluorouracil. Transduced progenitors were transplanted into moderately irradiated (500 cGy), G-CSF preconditioned sibling p47phox−/− mice. Using the fluorescent probe dihydrorhodamine 123 (DHR), in vivo biochemical correction of the superoxide-generating NADPH oxidase system was detected by flow cytometry in 12.3% ± 0.9% of phorbol myristate acetate–stimulated peripheral blood neutrophils at 4 weeks and 2.6% ± 1.0% at 14 weeks after transplantation. Following gene therapy, mice were challenged with the CGD pathogen Burkholderia (formerly Pseudomonas) cepacia and bacteremia levels were assessed at 24 hours and 7 days after inoculation. At both time points, bacteremia levels in gene corrected p47phox−/− mice were significantly lower than untreated p47phox−/− mice (0.89 ± 0.30 colonies v 237.7 ± 83.6 colonies at 24 hours, P < .02; 4.0 ± 2.0 colonies v 110.2 ± 26.5 colonies at 7 days, P < .0014). More importantly, Kaplan-Meier survival analysis showed a significant survival advantage of gene corrected versus untreated p47phox−/− mice (P < .001). Thus, stem-cell–directed ex vivo gene therapy is capable of restoring phagocyte oxidant-dependent host-defense function in this mouse model of a human immune-system disorder.

scholarly journals Chronic granulomatous disease. Expression of the metabolic defect by in vitro culture of bone marrow progenitors.

1980 ◽  
Vol 66 (3) ◽  
pp. 599-602 ◽  
Author(s):  
P E Newburger ◽  
M S Kruskall ◽  
J M Rappeport ◽  
S H Robinson ◽  
M E Chovaniec ◽  
...  
Keyword(s):  
Bone Marrow ◽  
In Vitro Culture ◽  
Chronic Granulomatous Disease ◽  
Granulomatous Disease ◽  
Disease Expression ◽  
Metabolic Defect

scholarly journals A novel BCMA/CD3 bispecific T-cell engager for the treatment of multiple myeloma induces selective lysis in vitro and in vivo

Leukemia ◽  
2016 ◽  
Vol 31 (8) ◽  
pp. 1743-1751 ◽  
Author(s):  
S Hipp ◽  
Y-T Tai ◽  
D Blanset ◽  
P Deegen ◽  
J Wahl ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
T Cell ◽  
Plasma Cells ◽  
Ex Vivo ◽  
Xenograft Model ◽  
Selective Lysis ◽  
Bispecific T Cell Engager

Abstract B-cell maturation antigen (BCMA) is a highly plasma cell-selective protein that is expressed on malignant plasma cells of multiple myeloma (MM) patients and therefore is an ideal target for T-cell redirecting therapies. We developed a bispecific T-cell engager (BiTE) targeting BCMA and CD3ɛ (BI 836909) and studied its therapeutic impacts on MM. BI 836909 induced selective lysis of BCMA-positive MM cells, activation of T cells, release of cytokines and T-cell proliferation; whereas BCMA-negative cells were not affected. Activity of BI 836909 was not influenced by the presence of bone marrow stromal cells, soluble BCMA or a proliferation-inducing ligand (APRIL). In ex vivo assays, BI 836909 induced potent autologous MM cell lysis in both, newly diagnosed and relapsed/refractory patient samples. In mouse xenograft studies, BI 836909 induced tumor cell depletion in a subcutaneous NCI-H929 xenograft model and prolonged survival in an orthotopic L-363 xenograft model. In a cynomolgus monkey study, administration of BI 836909 led to depletion of BCMA-positive plasma cells in the bone marrow. Taken together, these results show that BI 836909 is a highly potent and efficacious approach to selectively deplete BCMA-positive MM cells and represents a novel immunotherapeutic for the treatment of MM.

Ex vivo-expanded bone marrow CD34+ for acute myocardial infarction treatment: in vitro and in vivo studies

Cytotherapy ◽  
2011 ◽  
Vol 13 (9) ◽  
pp. 1140-1152 ◽  
Author(s):  
Monica Gunetti ◽  
Alessio Noghero ◽  
Fabiola Molla ◽  
Lidia Irene Staszewsky ◽  
Noeleen de Angelis ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Acute Myocardial Infarction ◽  
Bone Marrow ◽  
Ex Vivo ◽  
In Vivo Studies

Abstract 229: The Novel Angiogenic Cytokine Secretoneurin promotes Angiogenesis, Arteriogenesis and Vasculogenesis in the Hind-Limb Ischemia Model

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Wilfried Schgoer ◽  
Margot Egger ◽  
Arno Peer ◽  
Johannes Jeschke ◽  
Ivan Tancevski ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Green Fluorescence Protein ◽  
Limb Ischemia ◽  
Promoter Sequence ◽  
The Novel ◽  
Gene Therapy Vector ◽  
Adductor Muscles

Introduction - Secretoneurin (SN) represents a sensory, inflammatory neuropeptide which was recently demonstrated to act as an angiogenic and vasculogenic cytokine in vitro and in vivo. The present study was conducted to test the hypothesis that SN may be implicated in reparative angiogenesis. Furthermore, we challenged the healing potential of SN applied as a newly generated SN gene therapy vector in the setting of limb ischemia. Methods and Results - We cloned the human SN coding sequence into the pAAV plasmid containing a cytomegalovirus enhancer/promoter sequence. Bioactivity of recombinant SN was shown by proliferative and chemotactic activity on endothelial cells in vitro. Unilateral limb ischemia was induced in C57/bl mice by femoral artery resection. By Real Time PCR, Western Blotting, SN-specific RIA and Immunhistochemistry, we documented that SN is up-regulated in ischemic muscles. Next, we tested whether SN gene therapy may exert curative effects in this ischemia model. Injection of the SN plasmid into ischemic adductor muscles increased capillary (0.67 vs. 0.35, n = 24, p = 0.02) and arteriole (0.16 vs. 0.8, n = 24, p = 0.04) density, reduced endothelial cell apoptosis, and accelerated perfusion recovery as shown by Laser Doppler Perfusion Index (LDPI ratio ischemic/control leg after 28 days of ischemia 1.1 vs. 0.7, n = 24, p < 0.01) in comparson to pAAV-GFP (green-fluorescence protein) treated mice. Furthermore, SN gene therapy significantly reduced toe necrosis of ischemic limbs compared to control animals (26% vs. 50%, n = 24, p < 0.05). In bone marrow transplantation models, increased vascularity of ischemic hind-limbs after SN gene therapy was shown to be mediated, at least in part, by enhanced recruitment of bone marrow-derived endothelial progenitor cells. Conclusions -These results suggest that the novel angiogenic cytokine Secretoneurin is up-regulated by ischemia in skeletal muscle cells. Furthermore, results from gene therapy in this ischemia model suggest that Secretoneurin represent a promising new substance for therapeutic angiogenesis.

scholarly journals Delivery of human factor IX in mice by encapsulated recombinant myoblasts: a novel approach towards allogeneic gene therapy of hemophilia B

Blood ◽  
1996 ◽  
Vol 87 (12) ◽  
pp. 5095-5103 ◽  
Author(s):  
G Hortelano ◽  
A Al-Hendy ◽  
FA Ofosu ◽  
PL Chang
Keyword(s):  
Gene Therapy ◽  
Human Factor ◽  
Ex Vivo ◽  
Cost Effective ◽  
Factor Ix ◽  
Hemophilia B ◽  
Therapy Strategy ◽  
Human Factor Ix

A potentially cost-effective strategy for gene therapy of hemophilia B is to create universal factor IX-secreting cell lines suitable for implantation into different patients. To avoid graft rejection, the implanted cells are enclosed in alginate-polylysine-alginate microcapsules that are permeable to factor IX diffusion, but impermeable to the hosts' immune mediators. This nonautologous approach was assessed by implanting encapsulated mouse myoblasts secreting human factor IX into allogeneic mice. Human factor IX was detected in the mouse plasma for up to 14 days maximally at approximately 4 ng/mL. Antibodies to human factor IX were detected after 3 weeks at escalating levels, which were sustained throughout the entire experiment (213 days). The antibodies accelerated the clearance of human factor IX from the circulation of the implanted mice and inhibited the detection of human factor IX in the mice plasma in vitro. The encapsulated myoblasts retrieved periodically from the implanted mice up to 213 days postimplantation were viable and continued to secrete human factor IX ex vivo at undiminished rates, hence suggesting continued factor IX gene expression in vivo. Thus, this allogeneic gene therapy strategy represents a potentially feasible alternative to autologous approaches for the treatment of hemophilia B.

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.

Hematopoietic Repopulation, In Vivo, with Genetically Defined Clones Derived from HOXB4 Expressing ES-Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 196-196
Author(s):  
Sandra Pilat ◽  
Sebastian Carotta ◽  
Bernhard Schiedlmeier ◽  
Kenji Kamino ◽  
Andreas Mairhofer ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Es Cells ◽  
Es Cell ◽  
Somatic Gene Therapy ◽  
Hematopoietic System ◽  
Somatic Gene

Abstract In the context of somatic gene therapy of the hematopoietic system, transplantation of molecularly defined and, hence, “safe” clones would be highly desirable. However, techniques which allow gene targeting, subsequent in vitro selection and clonal expansion are only available for embryonic stem (ES) cells. After in vitro differentiation, some of their progeny cells are capable of mediating long term hematopoietic repopulation after transplantation into immunodeficient recipient mice, in vivo. This is especially efficient when the homeodomain transcription factor HOXB4 is ectopically expressed (1). We have recently shown that HOXB4-ES-cell derivatives behave similar to bone marrow cells also expressing this transcription factor ectopically, both in vitro and in vivo (2). Here we demonstrate that long term repopulation (>6 months) in Rag2(−/−)γ C(−/−) mice can be achieved with ES-cell derived hematopoietic cells (ES-HCs) obtained from single, molecularly characterized ES-clones, in which the insertion sites of the retroviral expression vector had been defined. Clones expressing HOXB4 above a certain level showed a high extent of chimerism in the bone marrow of transplanted mice (average 75%; range 45–95%, n=4) whereas ES-HC clones expressing lower levels only repopulated with very low efficiency (average 2.5% chimerism, range 1–4%, n=6 mice). These results suggest that the capability of long-term repopulation, in vivo, is highly dependent on the expression levels of HOXB4 in the transplanted clones. Only mice reconstituted with ES-HC clones expressing high amounts of HOXB4 and thus showing substantial chimerism, recapitulated the morphohistological phenotype observed in polyclonally reconstituted mice. This included the bias towards myelopoiesis, “benign” myeloid proliferation in spleen and the incompatibility of HOXB4 expression with T-cell poiesis (2). In summary, we demonstrate that repopulation of the hematopoietic system can be achieved with preselected clones of genetically manipulated stem cells in which a) the insertion site of the retroviral (gene therapy) vector has been characterized prior to transplantation and b) in which ectopic HOXB4 has to be expressed above a certain threshold level. Thus, ES cells carry the potential for performing safe somatic gene therapy when using integrating gene therapy vectors. Nevertheless, advanced cell therapy will certainly require the expression of HOXB4 in a regulated manner to avoid unwanted effects such as disturbed lineage differentiation.

Clonal Analyses of Retroviral Vector Integrations in ADA-SCID Patients Treated with Stem Cell Gene Therapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3249-3249
Author(s):  
Barbara Cassani ◽  
Grazia Andolfi ◽  
Massimiliano Mirolo ◽  
Luca Biasco ◽  
Alessandra Recchia ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Human Genome ◽  
Ex Vivo ◽  
Cd34 Cells

Abstract Gene transfer into hematopoietic stem/progenitor cells (HSC) by gammaretroviral vectors is an effective treatment for patients affected by severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA)-deficiency. Recent studied have indicated that gammaretroviral vectors integrate in a non-random fashion in their host genome, but there is still limited information on the distribution of retroviral insertion sites (RIS) in human long-term reconstituting HSC following therapeutic gene transfer. We performed a genome-wide analysis of RIS in transduced bone marrow-derived CD34+ cells before transplantation (in vitro) and in hematopoietic cell subsets (ex vivo) from five ADA-SCID patients treated with gene therapy combined to low-dose busulfan. Vector-genome junctions were cloned by inverse or linker-mediated PCR, sequenced, mapped onto the human genome, and compared to a library of randomly cloned human genome fragments or to the expected distribution for the NCBI annotation. Both in vitro (n=212) and ex vivo (n=496) RIS showed a non-random distribution, with strong preference for a 5-kb window around transcription start sites (23.6% and 28.8%, respectively) and for gene-dense regions. Integrations occurring inside the transcribed portion of a RefSeq genes were more represented in vitro than ex vivo (50.9 vs 41.3%), while RIS <30kb upstream from the start site were more frequent in the ex vivo sample (25.6% vs 19.4%). Among recurrently hit loci (n=50), LMO2 was the most represented, with one integration cloned from pre-infusion CD34+ cells and five from post-gene therapy samples (2 in granulocytes, 3 in T cells). Clone-specific Q-PCR showed no in vivo expansion of LMO2-carrying clones while LMO2 gene overexpression at the bulk level was excluded by RT-PCR. Gene expression profiling revealed a preference for integration into genes transcriptionally active in CD34+ cells at the time of transduction as well as genes expressed in T cells. Functional clustering analysis of genes hit by retroviral vectors in pre- and post-transplant cells showed no in vivo skewing towards genes controlling self-renewal or survival of HSC (i.e. cell cycle, transcription, signal transduction). Clonal analysis of long-term repopulating cells (>=6 months) revealed a high number of distinct RIS (range 42–121) in the T-cell compartment, in agreement with the complexity of the T-cell repertoire, while fewer RIS were retrieved from granulocytes. The presence of shared integrants among multiple lineages confirmed that the gene transfer protocol was adequate to allow stable engraftment of multipotent HSC. Taken together, our data show that transplantation of ADA-transduced HSC does not result in skewing or expansion of malignant clones in vivo, despite the occurrence of insertions near potentially oncogenic genomic sites. These results, combined to the relatively long-term follow-up of patients, indicate that retroviral-mediated gene transfer for ADA-SCID has a favorable safety profile.

Development of Megakaryocyte-Specific Lentiviral Vectors for Gene Therapy of Platelet Disorders.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 5143-5143
Author(s):  
Liesbeth De Waele ◽  
Kathleen Freson ◽  
Chantal Thys ◽  
Christel Van Geet ◽  
Désiré Collen ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Transgene Expression ◽  
Ex Vivo ◽  
Phosphoglycerate Kinase ◽  
Lentiviral Vectors ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Platelet Disorders

Abstract The prevalence of congenital platelet disorders has not been established but for some life-threatening bleeding disorders the current therapies are not adequate, justifying the development of alternative strategies as gene therapy. In the case of platelet dysfunction and thrombocytopenia as described for GATA1 deficiency, potentially lethal internal bleedings can occur. The objective of the study is to develop improved lentiviral vectors for megakaryocyte(MK)-specific long term gene expression by ex vivo transduction of hematopoietic stem cells (HSC) to ultimately use for congenital thrombopathies as GATA1 deficiency. Self-inactivating lentiviral vectors were constructed expressing GFP driven by the murine (m) or human (h) GPIIb promoter. These promoters contain multiple Ets and GATA binding sites directing MK-specificity. To evaluate the cell lineage-specificity and transgene expression potential of the vectors, murine Sca1+ and human CD34+ HSC were transduced in vitro with Lenti-hGPIIb-GFP and Lenti-mGPIIb-GFP vectors. After transduction the HSC were induced to differentiate in vitro along the MK and non-MK lineages. The mGPIIb and hGPIIb promoters drove GFP expression at overall higher levels (20% in murine cells and 25% in human cells) than the ubiquitous CMV (cytomegalovirus) or PGK (phosphoglycerate kinase) promoters, and this exclusively in the MK lineage. Interestingly, in both human and murine HSC the hGPIIb promoter with an extra RUNX and GATA binding site, was more potent in the MK lineage compared to the mGPIIb promoter. Since FLI1 and GATA1 are the main transcription factors regulating GPIIb expression, we tested the Lenti-hGPIIb-GFP construct in GATA1 deficient HSC and obtained comparable transduction efficiencies as for wild-type HSC. To assess the MK-specificity of the lentiviral vectors in vivo, we transplanted irradiated wild-type C57Bl/6 mice with Sca1+ HSC transduced with the Lenti-hGPIIb-GFP constructs. Six months after transplantation we could detect 6% GFP positive platelets without a GFP signal in other cell lineages. Conclusion: In vitro and in vivo MK-specific transgene expression driven by the hGPIIb and mGPIIb promoters could be obtained after ex vivo genetic engineering of HSC by improved lentiviral vectors. Studies are ongoing to study whether this approach can induce phenotypic correction of GATA1 deficient mice by transplantation of ex vivo Lenti-hGPIIb-GATA1 transduced HSC.

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