[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: The trifunctional antibody catumaxomab is a targeted immunotherapy for the intraperitoneal treatment of malignant ascites. In a Phase II/III trial in cancer patients (n = 258) with malignant ascites, catumaxomab showed a clear clinical benefit vs. paracentesis and had an acceptable safety profile. Human antimouse antibodies (HAMAs), which could be associated with beneficial humoral effects and prolonged survival, may develop against catumaxomab as it is a mouse/rat antibody. This post hoc analysis investigated whether there was a correlation between the detection of HAMAs 8 days after the fourth catumaxomab infusion and clinical outcome. HAMA-positive and HAMA-negative patients in the catumaxomab group and patients in the control group were analyzed separately for all three clinical outcome measures (puncture-free survival, time to next puncture and overall survival) and compared to each other. There was a strong correlation between humoral response and clinical outcome: patients who developed HAMAs after catumaxomab showed significant improvement in all three clinical outcome measures vs. HAMA-negative patients. In the overall population in HAMA-positive vs. HAMA-negative patients, median puncture-free survival was 64 vs. 27 days (p < 0.0001; HR 0.330), median time to next therapeutic puncture was 104 vs. 46 days (p = 0.0002; HR 0.307) and median overall survival was 129 vs. 64 days (p = 0.0003; HR 0.433). Similar differences between HAMA-positive and HAMA-negative patients were seen in the ovarian, nonovarian and gastric cancer subgroups. In conclusion, HAMA development may be a biomarker for catumaxomab response and patients who developed HAMAs sooner derived greater benefit from catumaxomab treatment.
International Journal of Cancer 06/2011; 130(9):2195-203. · 6.20 Impact Factor
[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: 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: 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: 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.