[Show abstract][Hide abstract] ABSTRACT: Vector-associated side effects in clinical gene therapy have provided insights into the molecular mechanisms of hematopoietic regulation in vivo. Surprisingly, many retrovirus insertion sites (RIS) present in engrafted cells have been found to cluster nonrandomly in close association with specific genes. Our data demonstrate that these genes directly influence the in vivo fate of hematopoietic cell clones. Analysis of insertions thus far has been limited to individual clinical studies. Here, we studied >7,000 insertions retrieved from various studies. More than 40% of all insertions found in engrafted gene-modified cells were clustered in the same genomic areas covering only 0.36% of the genome. Gene classification analyses displayed significant overrepresentation of genes associated with hematopoietic functions and relevance for cell growth and survival in vivo. The similarity of insertion distributions indicates that vector insertions in repopulating cells cluster in predictable patterns. Thus, insertion analyses of preclinical in vitro and murine in vivo studies as well as vector insertion repertoires in clinical trials yielded concerted results and mark a small number of interesting genomic loci and genes that warrants further investigation of the biological consequences of vector insertions.
[Show abstract][Hide abstract] ABSTRACT: Gene-modified autologous hematopoietic stem cells (HSC) can provide ample clinical benefits to subjects suffering from X-linked chronic granulomatous disease (X-CGD), a rare inherited immunodeficiency characterized by recurrent, often life-threatening bacterial and fungal infections. Here we report on the molecular and cellular events observed in two young adults with X-CGD treated by gene therapy in 2004. After the initial resolution of bacterial and fungal infections, both subjects showed silencing of transgene expression due to methylation of the viral promoter, and myelodysplasia with monosomy 7 as a result of insertional activation of ecotropic viral integration site 1 (EVI1). One subject died from overwhelming sepsis 27 months after gene therapy, whereas a second subject underwent an allogeneic HSC transplantation. Our data show that forced overexpression of EVI1 in human cells disrupts normal centrosome duplication, linking EVI1 activation to the development of genomic instability, monosomy 7 and clonal progression toward myelodysplasia.
Nature medicine 02/2010; 16(2):198-204. · 27.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Over the last two decades gene therapy has moved from preclinical to clinical studies for many diseases ranging from single gene disorders such as cystic fibrosis and Duchenne muscular dystrophy, to more complex diseases such as cancer and cardiovascular disorders. Gene therapy for severe combined immunodeficiency (SCID) is the most significant success story to date, but progress in many other areas has been significant. We asked 20 leaders in the field succinctly to summarize and comment on clinical gene therapy research in their respective areas of expertise and these are published in two parts in the Progress and Prospect series.
[Show abstract][Hide abstract] 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 review the developments leading to a gene therapy approach for the treatment of Chronic Granulomatous Disease (CGD), a primary life threatening immunodeficiency caused by a defect in the oxidative antimicrobial activity of phagocytes. Although the disease can be cured by bone marrow transplantation, this treatment is only available to patients with HLA-identical sibling or matched unrelated donors. One therapeutic option for patients without suitable donor is the genetic modification of autologous hematopoietic stem cells. Although early attempts to correct CGD by gene therapy were unsuccessful, these studies demonstrated the safety and limitations of gene transfer into hematopoietic stem cells (HSC) of CGD patients using retroviral vectors. The recent development of advanced gene transduction protocols together with improved retroviral vectors, combined with low intensity chemotherapy conditioning, allowed partial correction of the granulocytic function with a significant clinical benefit in treated patients. These results may have important implications for future applications of gene therapy in myeloid disorders and inherited diseases using hematopoietic stem cells.
Current Gene Therapy 07/2007; 7(3):155-61. · 5.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic granuloniatous disease (CGD) is a rare inherited imnunodeficiency characterized by recurrent, often life threatening bacterial and fungal infections due to a functional defect in the microbial-killing activity of phagocytic neutrophils. If regular care and conventional therapy fail, tile disease can be cured by bone marrow transplantation. This treatment is, however, only available to patients with human leukocyte antigen-identical sibling or matched unrelated donors. One therapeutic option for patients lacking suitable donors is the genetic modification of autologous hematopoietic stem cells. This review discusses the developments that have led to the realization of a successful gene therapy protocol for the correction of CGD.
Current opinion in molecular therapeutics 11/2006; 8(5):415-22. · 3.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gene transfer into hematopoietic stem cells has been used successfully for correcting lymphoid but not myeloid immunodeficiencies. Here we report on two adults who received gene therapy after 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 resulting from mutations in gp91(phox). We detected substantial gene transfer in both individuals' neutrophils that lead to a large number of functionally corrected phagocytes and notable clinical improvement. Large-scale retroviral integration site-distribution analysis showed activating insertions in MDS1-EVI1, PRDM16 or SETBP1 that had influenced regulation of long-term hematopoiesis by expanding gene-corrected myelopoiesis three- to four-fold in both individuals. Although insertional influences have probably reinforced the therapeutic efficacy in this trial, our results suggest that gene therapy in combination with bone marrow conditioning can be successfully used to treat inherited diseases affecting the myeloid compartment such as CGD.
Nature Medicine 05/2006; 12(4):401-9. · 22.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Molecular Therapy (2006) 13, S279|[ndash]|S279; doi: 10.1016/j.ymthe.2006.08.803
723. In Vivo Expansion of MDS1/EVI1, PRDM16 and SETBP1 Integration Clones in Successful Chronic Granulomatous Disease (CGD) Gene Therapy Trial
Kerstin Schwarzwaelder1,2, Manfred Schmidt1,2,3,|[ast]|, Marion G. Ott4, Stefan Stein5, Hanno Glimm1,3, Annette Deichmann1,2, Ulrich Siler6, Dieter Hoelzer4, Reinhard Seger6, Manuel Grez5 and Christof von Kalle1,7,|[ast]|1Department of Translational Oncology, National Center for Tumor Diseaes, Heidelberg, Germany2Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany3Internal Medicine I, University of Freiburg, Freiburg, Germany4Hematology and Oncology, Medical School, Frankfurt, Germany5Institute for Biomedical Research, Georg-Speyer-Haus, Frankfurt, Germany6Division of Immunology/Hematology, University Children's Hospital, Zurich, Switzerland7Cincinnati Children's Research Foundation, Division of Experimental Hematology, Cincinnati, OH|[ast]|MS and CvK hold US-patent on LAM-PCR.
[Show abstract][Hide abstract] ABSTRACT: The symptomatology of Chronic Granulomatous Disease (CGD) is primarily caused by the inability of PMN to produce reactive oxygen species and thereby to kill phagocytosed micro-organisms. The patients suffer from life-threatening bacterial and fungal infections with an overall mortality of 2–5% per year.In a gene therapy trial, G-CSF mobilized CD34+ cells from two gp91phox-/- CGD patients were transduced with a monocistronic gp91phox retroviral vector. The patients were treated with liposomal busulphan for two days and transduced cells were re-infused after the busulphan treatment. A significant fraction of gene marked cells was detected in peripheral blood of both patients since day +21. The aim of our study was to verify whether NADPH oxidase activity in gene marked peripheral blood cells was therapeutically relevant.Results: Previous to gene therapy no gp91phox protein associated with p22phox (cytochrome b558 complex) was detected in protein extracts obtained from gp91phox-/- PMN as measured by UV/VIS absorption spectra. For both patients, gene therapy led to the re-appearance of the cytochrome b spectrum indicating correct assembly of the cytochrome b558-dependent NAD(P)H oxidase. Moreover, superoxide anion production in corrected PMN could be demonstrated intracellulary by the reduction of nitroblue tetrazolium and extracellulary by measuring the reduction of cytochrome C. The specificity of the raction was shown by the inhibition of NADPH oxidase activity by AEBSF or DPI, two specific NADPH oxidase inhibitors, and by superoxide dismutase mediated inhibition. In parallel we performed a functional E.coli killing assay, in which the activity of β-galactosidase released by perforated/killed E.coli ML-35 (laci-z+y-) is documented. For patient 1 the E.coli killing assay has shown that gene therapy significantly improved the ability of corrected PMN to perforate and kill bacteria . This was confirmed by electron microscopic analysis showing that a fraction of PMN from patient 1 contained degraded E.coli resembling wild type PMN. Similarly, fungal killing activity was also demonstrated in an Aspergillus fumigatus microbicidal assay.In parallel to the in vitro assays, both patients were monitored by positron emission tomography (PET) scans. Despite anti-microbial treatment patient 1 had two liver absesses due to Staph. Aureus infection before gene therapy while patient 2 suffered from a lesion in a lung cavity caused by A. fumigatus. Both lesions dissapeared after gene therapy.Conclusion: Our data suggest that CGD gene therapy can functionally improve the production of reactive oxygen species by PMN in an amount that is sufficient to reach therapeutic relevant levels.
[Show abstract][Hide abstract] ABSTRACT: Chronic Granulomatous Disease (CGD) is a primary immunodeficiency characterized by recurrent and life-threatening bacterial and fungal infections. CGD is caused by mutations in any one of four genes encoding for subunits of the phagocytic NADPH oxidase complex. Most patients contain mutations or deletions within the gene coding for gp91phox, the larger subunit of the complex. Based on our preclinical work, two X-CGD patients, 26 and 25 years old, were treated with gene modified cells. G-CSF mobilized CD34+ cells were transduced with a monocistronic gp91phox retroviral vector. The transduction efficiency was 45% for Pat.1 and 40% for Pat. 2. The number of CD34+ cells reinfused was 1.2|[times]|10e7 per kg for Pat.1 and 0.9|[times]|10e7 per kg for Pat. 2. Before reinfusion, patients were treated with liposomal busulphan given i.v. at a dose of 4 mg/kg at two consecutive days, starting at day -3. The treatment was well tolerated and no adverse effects have been observed. Neutrophil counts declined to less than 100 cells per |[mu]|l at day 15 post reinfusion for both patients and recovered to more than 500 cells per |[mu]|l at day 30 for Pat. 1 and day 20 for Pat. 2. A significant fraction of gene marked cells has been detected in peripheral blood of both patients since day +21. Similarly, therapeutic relevant levels of NADPH oxidase activity have been observed since day +21. Both patients are well and have been free of severe bacterial and fungal infections since transplantation. Our data suggests that gene therapy is an option for the treatment of CGD, despite the fact that gp91phox corrected cells will not have per se a proliferative advantage over non-transduced cells.
[Show abstract][Hide abstract] ABSTRACT: Molecular Therapy (2005) 11, S415|[ndash]|S415; doi: 10.1016/j.ymthe.2005.07.623
1076. Stable Polyclonal Hematopoietic Repopulation after Successful Clinical Gene Therapy of Chronic Granulomatous Disease (CGD)
Manfred Schmidt1,|[ast]|, Kerstin Schwarzwaelder2, Marion G. Ott3, Stefan Stein4, Hanno Glimm2, Annette Deichmann1, Ulrich Sieler5, Dieter Hoelzer3, Reinhard Seger5, Manuel Grez4 and Christof von Kalle6,|[ast]|1Internal Medicine I, University of Freiburg, Freiburg, Germany2Institute for Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany3Hematology and Oncology, Medical School, Frankfurt, Germany4Georg-Speyer-Haus, Frankfurt, Germany5Division of Immunology/Hematology, University-Children's Hospital, Zuerich, Switzerland6Experimental Hematology, Cincinnati Childrens Research Foundation, Cincinnati, OH|[ast]|MS and CvK hold US-patent on LAM-PCR.
[Show abstract][Hide abstract] ABSTRACT: Ein effizienter Gentransfer ist in vitro und im Maussystem möglich. Es ist gelungen, eine hohe Expression transduzierter Gene
in frühen Vorläuferzellen zu zeigen. Schwierigkeiten bereitet jedoch noch die niedrige oder nur kurz anhaltende Expression
der Gene im Primatensystem und beim Menschen. Die bisher durchgeführten Studien beim Menschen zeigten nur geringe Expressionen,
was für einen therapeutischen Erfolg noch nicht ausreichte. Nur bei Krankheiten wie der Adenosindesaminase-Defizienz (ADA)
oder der Severe Combined Immunodeficiency Typ X1 (schwerer, kombinierter Immundefekt, Typ X1, X1-SCID) zeigen sich schon gute
klinische Erfolge, da die transduzierten Zellen in vivo einen Selektionsvorteil aufweisen. Verbesserungen an den Vektoren,
der Technik und die Einführung einer In-vivo-Selektion und/oder Myelosuppression könnten in Zukunft zu entscheidenden Verbesserungen
beitragen. Insgesamt wird sich die Gentherapie immer mehr als eine Therapieform für lebensbedrohliche angeborene Krankheiten
Gene transfer into pluripotent hematopoietic stem cells (PHSCs) is one of the most promising alternatives for the curative
treatment of a variety of inherited and acquired disorders of blood cells. In murine syngeneic bone marrow transplantation
models, a significant proportion of cells participating in long term engraftment of lethally irradiated mice can be reproducibly
and stably transduced ex vivo by the current generation of retroviral vectors. However, transfer of this technology to humans, non-human primates and other
large out-bred animals has been much less successful. Only in those cases in which the transduced cells do have a proliferative
advantage over their non-transduced counterparts, like in X1-SCID or ADA, a significant correction of disease was observed.
Improvments in vector design and gene transfer conditions in combination with either a myelosuppresive therapy or an expansion
of transduced cells in vivo will certainly increase the success of gene therapy of blood disorders.
[Show abstract][Hide abstract] ABSTRACT: Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder which results from absence or malfunction of the respiratory burst oxidase normally expressed in neutrophils and other phagocytic leukocytes. Two-thirds of the patients are males hemizygous for mutations in the X-linked gene coding for gp91-phox. As a therapeutic approach towards the X-linked form of CGD bicistronic retroviral vectors containing the gp91-phox gene and a selectable marker gene were constructed. The ability of these vectors to restore NADPH oxidase activity was tested in a human myeloid leukemic cell line that is defective in superoxide production, as well as in primary CD34+ cells obtained from X-CGD patients. Under optimal conditions 80% of the CD34+ cells derived from bone marrow of one X-CGD patient were transduced. The level of superoxide production, in phagocytes derived from transduced cells was 68.9% of normal levels. Considering that low levels of superoxide generating activity are sufficient for normal host defense, the present experiments provide the basis for the development of a gene replacement therapy for the X-linked form of CGD.
[Show abstract][Hide abstract] ABSTRACT: Chronic granulomatous disease (CGD) is a disorder of the lymphohematopoietic system, whereby phagocytes of affected patients are unable to kill microorganisms. CGD is caused by a functional defect in the phagocytic nicotinamide adenine dinucleotide phosphatase (NADPH) oxidase (phox) enzyme complex, leading to a lack of microbicidal metabolites. As a therapeutic approach toward the predominant X-linked form of CGD, we have developed a bicistronic retroviral vector containing the coding sequences of gp91-phox and a cytoplasmically truncated version of the human low-affinity receptor for nerve growth factor (deltaLNGFR). Full reconstitution of superoxide-generating activity was achieved with this vector in a gp91-phox-deficient cell line. Using an optimized gene transfer protocol, up to 85% of the CD34+ cells obtained from the bone marrow of X-CGD patients were transduced. CD15+ cells differentiated in vitro from transduced X-CGD CD34+ cells showed correction of NADPH oxidase activity to 45-52% of normal levels whereas deltaLNGFR expression was found in 40-67% of the CD15+ cells. Moreover, immunoblots prepared from extracts of transduced CD15+ cells revealed gp91-phox protein levels similar to those found in neutrophils derived from normal CD34+ cells. Taking into consideration that superoxide production in only 5 to 10% of wild-type neutrophils is sufficient to protect X-CGD heterozygotes from serious infections, the results achieved in this study shows that for X-CGD patients a curative approach based on the genetic modification of hematopoietic stem/progenitor cells is feasible.
Human Gene Therapy 08/1998; 9(11):1561-70. · 4.02 Impact Factor