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Boris Fehse

Publications and source records attributed to Boris Fehse.

30 records · Page 2Linked to original sources
PubMed2003

Side effects of retroviral gene transfer into hematopoietic stem cells.

Recent conceptual and technical improvements have resulted in clinically meaningful levels of gene transfer into repopulating hematopoietic stem cells. At the same time, evidence is accumulating that gene therapy may induce several kinds of unexpected side effects, based on preclinical and clinical data. To assess the therapeutic potential of genetic interventions in hematopoietic cells, it will be important to derive a classification of side effects, to obtain insights into their underlying mechanisms, and to use rigorous statistical approaches in comparing data. We here review side effects related to target cell manipulation; vector production; transgene insertion and expression; selection procedures for transgenic cells; and immune surveillance. We also address some inherent differences between hematopoiesis in the most commonly used animal model, the laboratory mouse, and in humans. It is our intention to emphasize the need for a critical and hypothesis-driven analysis of "transgene toxicology," in order to improve safety, efficiency, and prognosis for the yet small but expanding group of patients that could benefit from gene therapy.

Animals↗
PubMed2003

Predictable and efficient retroviral gene transfer into murine bone marrow repopulating cells using a defined vector dose.

OBJECTIVE: Current protocols of retroviral gene transfer into murine hematopoietic stem cells (HSC) result in variable gene transfer efficiency and involve various procedures that are not clinically applicable. We developed and evaluated a reliable transduction protocol that is more related to clinical methods. MATERIALS AND METHODS: HSC were enriched from steady-state bone marrow by magnetic cell sorting (lineage depletion) and cultured in defined serum-free medium containing an improved growth factor cocktail (Flt3-ligand, stem cell factor, interleukin-3, interleukin-11). Cell-free ecotropic retroviral vector particles, generated by transient transfection of human 293T-based packaging cells, were preloaded at defined titers on CH296-coated tissue culture plates, thus largely avoiding serum contamination. These conditions were evaluated in 17 experiments involving 29 transduction cultures and 185 recipient mice. RESULTS: After two rounds of infection, the gene marking rates in cultured mononuclear cells and stem/progenitor cells (Lin(-)c-Kit(+)) were 15 to 85% (53.7%+/-21.7%, n=23) and 30 to 95% (69.8%+/-20.4%, n=17), respectively. Even after one round of infection, gene transfer was efficient (31.2%+/-15.1%, n=12). Using identical conditions, gene transfer rates were highly reproducible. Average transgene expression in reconstituted animals correlated well with pretransplant data. Using a moderate multiplicity of infection, the majority of transduced cells carried less than three transgene copies. In addition, coinfection was possible to establish two different vectors in single cells. CONCLUSION: The protocol described here achieves efficient retroviral transduction of murine bone marrow repopulating cells with a defined gene dosage, largely avoiding procedures that decrease stem cell output and repopulating capacity. This protocol may help to improve the predictive value of preclinical efficiency/toxicity studies for gene therapeutic interventions and basic research.

Animals↗
PubMed2003

Characterization of cell types during rat liver development.

Hepatic stem cells have been identified in adult liver. Recently, the origin of hepatic progenitors and hepatocytes from bone marrow was demonstrated. Hematopoietic and hepatic stem cells share the markers CD 34, c-kit, and Thy1. Little is known about liver stem cells during liver development. In this study, we investigated the potential stem cell marker Thy1 and hepatocytic marker CK-18 during liver development to identify putative fetal liver stem cell candidates. Livers were harvested from embryonic and fetal day (ED) 16, ED 18, ED 20, and neonatal ED 22 stage rat fetuses from Sprague-Dawley rats. Fetal livers were digested by collagenase-DNAse solution and purified by percoll centrifugation. Magnetic cell sorting (MACS) depletion of fetal liver cells was performed using OX43 and OX44 antibodies. Cells were characterized by immunocytochemistry for Thy1, CK-18, and proliferating cell antigen Ki-67 and double labeling for Thy1 and CK-18. Thy1 expression was found at all stages of liver development before and after MACS in immunocytochemistry. Thy1 positive cells were enriched after MACS only in early developmental stages. An enrichment of CK-18 positive cells was found after MACS at all developmental stages. Cells coexpressing Thy1 and CK-18 were identified by double labeling of fetal liver cell isolates. In conclusion, hepatic progenitor cells (CK-18 positive) in fetal rat liver express Thy1. Other progenitors express only CK-18. This indicates the coexistence of different hepatic cell compartments. Isolation and further characterization of such cells is needed to demonstrate their biologic properties.

Animals↗
PubMed2003

Influence of anti-thymocyte globulin as part of the conditioning regimen on immune reconstitution following matched related bone marrow transplantation.

The aim of this work was to analyze the influence of anti-thymocyte globulin (ATG) as part of the conditioning regimen on immune reconstitution following matched related bone marrow transplantation. The rate and pattern of the recovery of total lymphocytes, natural killer (NK) cells, and several T and B cell subsets were determined in 38 patients for more than 2 years following BMT. We compared two patient groups: the first comprised 19 patients after matched related BMT without ATG prevention for graft-versus-host disease (GVHD) and the second contained 19 patients after matched related BMT with ATG treatment for GVHD prophylaxis. We observed impaired immune reconstitution in the ATG group in comparison with the non-ATG group, indicating a significant influence of ATG on immune recovery for several months after BMT.

Adolescent↗
PubMed2003

Liver-specific gene expression in cultured human hematopoietic stem cells.

Hematopoietic and hepatic stem cells share characteristic markers such as CD34, c-kit, and Thy1. Based on the recent observations that hepatocytes may originate from bone marrow, we investigated the potential of CD34(+) bone marrow cells to differentiate into hepatocytic cells in vitro. CD34(+) and CD34(-) human bone marrow cells were separated by magnetic cell sorting. Cells were cultured on a collagen matrix in a defined medium containing hepatocyte growth factor. Cell count and size were measured by flow cytometry, and reverse transcription polymerase chain reaction was carried out for the liver-specific markers CK-19 and albumin. During cell culture, CD34(+) cells showed an increasing cell number and proliferative activity as assessed by Ki-67 staining. Under the specified culture conditions, CD34(+) cells expressed albumin RNA and CK-19 RNA after 28 days, whereas CD34(-) cells did not show liver-specific gene expression. The results indicate that CD34(+) adult human bone marrow stem cells can differentiate into hepatocytic cells in vitro.

Adult↗
PubMed2003

Mutagenesis by retroviral transgene insertion: risk assessment and potential alternatives.

Focus on retroviral gene transfer into hematopoietic cells has previously led to a proposed classification of side effects and discussion of issues related to combinatorial toxicity (including oncogenicity) of genetic interventions. Recent data underline that this discussion has to be broadened to any type of stable transfer technology. This review summarizes insights obtained in cell culture and animal models that were specifically designed to evaluate side effects of transgene insertion and new approaches of insertion site targeting. Ongoing research aims to reduce side effects of therapeutic transgene insertion by dose optimization and definition of contributing risk factors.

Base Sequence↗
PubMed2002

Unrelated stem cell transplantation in multiple myeloma after a reduced-intensity conditioning with pretransplantation antithymocyte globulin is highly effective with low transplantation-related mortality.

We investigated the feasibility of unrelated stem cell transplantation in 21 patients with advanced stage II/III multiple myeloma after a reduced-intensity conditioning regimen consisting of fludarabine (150 mg/m(2)), melphalan (100-140 mg/m(2)), and antithymocyte globulin (ATG; 10 mg/kg on 3 days). The median patient age was 50 years (range, 32-61 years). All patients had received at least one prior autologous transplantation, in 9 cases as part of an autologous-allogeneic tandem protocol. No graft failure was observed. At day 40 complete donor chimerism was detected in all patients. Grade II to IV acute graft-versus-host disease (GVHD) was seen in 8 patients (38%), and severe grade III/IV GVHD was observed in 4 patients (19%). Six patients (37%) developed chronic GVHD, but only 2 patients (12%) experienced extensive chronic GVHD. The estimated probability of nonrelapse mortality at day 100 was 10% and at 1 year was 26%. After allografting, 40% of the patients achieved a complete remission, and 50% achieved a partial remission, resulting in an overall response rate of 90%. After a median follow-up of 13 months, the 2-year estimated overall and progression-free survival rates are 74% (95% CI, 54%-94%) and 53% (95% CI, 29%-87%), respectively. A shorter progression-free survival was seen in patients who already experienced relapse to prior autograft (26% versus 86%, P =.04). Dose-reduced conditioning with pretransplantation ATG followed by unrelated stem cell transplantation provides durable engraftment and donor chimerism, reduces substantially the risk of transplant-related organ toxicity, and induces high remission rates.

Adult↗
PubMed2002

Autologous stem cell transplantation followed by a dose-reduced allograft induces high complete remission rate in multiple myeloma.

We evaluated toxicity, engraftment, chimerism, graft-versus-host disease (GVHD), and response to a dose-reduced allograft after cytoreductive autografting in 17 patients with advanced stage II/III multiple myeloma (MM). After autografting with melphalan (200 mg/m2) the patients received after a median interval of 119 days (range 60-210) a dose-reduced regimen consisting of fludarabine (180 mg/m2), melphalan (100 mg/m2), and antithymocyte globulin (3 x 10 mg/kg) followed by allografting from related (n = 7), mismatched related (n = 2), or unrelated (n = 8) donors to induce a graft-versus-myeloma effect. After dose-reduced allografting all patients became neutropenic (< 0.2 x 10(9)/L) for at least 8 days. All patients engrafted with a median time for leukocyte (> 1 x 10(9)/L) and platelet (> 20 x 10(9)/L) counts of 16 (range, 11-24) and 23 days (range, 12-43), respectively. Complete donor chimerism was detected after a median of 30 days (range, 19-38). Acute GVHD stage II occurred in 4 patients (25%) and grade III GVHD in 2 patients (13%). Chronic GVHD developed in 40% of the patients, but only 1 patient experienced extensive chronic GVHD requiring further immunosuppressive therapy. Two patients died of alveolar hemorrhage and pneumonia, resulting in a day 100 mortality rate of 11%. The rate of complete remission with negative immunofixation increased from 18% after autografting to 73% after allografting. After a median follow-up of 17 months after autologous and 13 months after allogeneic transplantation 13 patients are alive and 12 of them free of relapse or progression. The tandem auto-allotransplant protocol is highly active and provides rapid engraftment with complete donor chimerism and tolerable toxicity.

Adult↗
PubMed2002

Highly efficient retroviral gene transfer based on centrifugation-mediated vector preloading of tissue culture vessels.

Efficient retroviral gene transfer into primary cells is a prerequisite for various gene therapeutic strategies. We have developed a transduction protocol based on the preloading of tissue culture vessels with retroviral particles by low-speed (1000g) centrifugation. We show that vector-preloaded tissue culture vessels allow highly efficient gene transfer into various target cells. We obtained transduction rates of up to 85% for primary T lymphocytes after just a single round of transduction. Under clinically relevant conditions using a vector developed for suicide gene therapy and produced under good manufacturing practice (GMP) conditions, the described method allowed generation of large numbers (>2x10(9)) of gene-modified T cells. The preloading concept ensures transduction of target cells in their optimal growth medium regardless of the medium used for vector production. This facilitated highly efficient gene transfer into quite different target cells such as CD34(+) and AC133(+) bone marrow progenitor as well as mesenchymal stem cells. The presented method combines high gene-transfer rates with a great potential for standardization in accordance with GMP guidelines and is consequently well suited for both research and clinical applications. (c)2002 Elsevier Science (USA).

Cell Culture Techniques↗
PubMed2002

Upstream conserved sequences of mouse leukemia viruses are important for high transgene expression in lymphoid and hematopoietic cells.

Highly conserved enhancer sequences located in the upstream part of the long terminal repeat (LTR) of murine leukemia retroviruses (MLV) were reported to compromise viral gene expression in multipotent embryonic cells in vitro and to reduce the likelihood for maintenance of retroviral gene expression in hematopoietic cells in vivo. We show that deletion of these sequences (nucleotides +37 to +95) attenuates rather than increases the transcriptional activity of retroviral vectors in hematopoietic cells almost independently of the developmental lineage (erythroid, myeloid, or lymphoid). Expression rates of modified vectors were reduced by as much as 34-65%, although the strong enhancer array located in the direct repeat of the LTR was preserved. Sequence analysis and electrophoretic mobility shift assays revealed the presence of a highly conserved binding site for NFAT (nuclear factor of activated T cells) proteins that immediately neighbors a known binding site for the transcription factor Yin-Yang1 (YY1) [corrected]. Specific inactivation of the NFAT site reduced transgene expression in all cell types investigated and had a similar effect as the destruction of a neighboring SP1 motif. Combined destruction of individual motifs for NFAT, SP1, and E twenty-six transcription factors (ETS) resulted in a severe attenuation (by 40-60%) of the retroviral enhancer. These results provide novel clues for the manipulation of retrovirus replication and vector tropism.

5' Flanking Region↗
PubMed2002

Human mesenchymal stem cells are not of donor origin in patients with severe aplastic anemia who underwent sex-mismatched allogeneic bone marrow transplant.

Stromal defects are part of the etiology of severe aplastic anemia (SAA), and hematopoietic engraftment is poor in unrelated and mismatched transplant. Therefore, we wanted to find out whether human mesenchymal stem cells (MSC) are partly of donor origin in patients with SAA years after successful bone marrow transplant (BMT). Three SAA patients 3, 5, and 8 years after BMT (cyclophosphamide, ATG) with bone marrow from an HLA-identical sibling donor of the opposite sex were investigated. MSC were grown from patients' bone marrow aspirates according to Caplan et al. The number of MSC that were isolated from SAA bone marrow post transplant was about 10 times lower than in normal controls. Primary cultures of adherent MSC and passage-one cells were analyzed by dual-color interphase fluorescence in situ hybridization (FISH) analysis using centromere-specific DNA probes for X and Y chromosome. FISH did not show any clear evidence of donor cells in the adherent MSC: In all cases, less than 0.5% of nuclei showed a donor-type signal pattern that is well within assay limits. In a female patient, the absence of male donor cells was confirmed by sensitive and quantitative, Y chromosome-specific TaqMan PCR (QYCS-PCR). In contrast, Ficoll-separated hematopoietic cells from the same aspirates were greater than 90% of donor origin, as expected. In SAA, as previously found in patients with lysosomal and peroxisomal storage disease, bone marrow MSC remain host-derived despite successful hematopoietic engraftment years after allogeneic BMT.

Adult↗