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B Fehse

Publications and source records attributed to B Fehse.

At least 19 recordsLinked to original sources
PubMed2006

Retroviral vector insertions in T-lymphocytes used for suicide gene therapy occur in gene groups with specific molecular functions.

Graft-versus-host disease (GvHD) is a severe complication in the context of allogeneic stem cell transplantation and adoptive immunotherapy. The transfer of a suicide gene into donor T-lymphocytes (TLCs) allows selective elimination of GvHD-causing cells. As retroviral gene transfer into hematopoietic stem cells can induce leukaemia, there is an urgent need also to analyze retroviral integration sites in TLCs. We examined suicide gene-transduced TLCs in four grafts and from four transplanted patients. One-hundred and fifteen integration sites were detected in vitro. Of these 90 could be mapped to the human genome; 50% (45) were located in genes and 32% (29) were detected 10 kb upstream or downstream of transcription start sites. We found a significant overrepresentation of genes encoding for proteins with receptor activity, signal transducer activity, transcription regulator activity, nucleic acid binding activity and translation regulator activity. Similar data were obtained from patient samples. Our results point to preferred vector integration patterns, which are specific for the target cell population and probably independent of selection processes. Thus, future preclinical analysis of the integration repertoire with abundant amounts of transduced cells could allow a prediction also for the in vivo situation, where target cells are scarce.

Animals↗
PubMed2006

Allogeneic stem-cell transplantation in patients with refractory acute leukemia: a long-term follow-up.

We examined retrospectively 44 patients with refractory acute leukemia (acute myeloid leukemia (AML)/acute lymphoblastic leukemia=25/19) who underwent allogeneic transplantation at our center between 11/1990 and 04/2004. The median leukemic blasts was 25% and age 28 years (range, 3-56). Twenty-one patients had untreated relapse, 13 failed reinduction, eight in partial remission and two aplastic. Conditioning was myeloablative using cyclophosphamide, busulfan, total-body irradiation and etoposide (Bu/Cy/VP, n=22; TBI/Cy/VP, n=17; others, n=5) followed by marrow or peripheral blood transplant (n=23/21) from unrelated or related donors (n=28/16). All patients had graft-versus-host disease (GVHD) prophylaxis with cyclosporin and methotrexate. One patient experienced late graft failure. Severe acute-GVHD and chronic-GVHD appeared in eight and 14 patients, respectively. Thirteen patients (30%) remain alive after a median of 25.3 months (range, 2.4-134.1); with 31 deaths, mostly from relapse (n=15) and infections (n=12). Overall survival (OS) and progression-free survival (PFS) at 5 years was 28 and 26%, respectively. OS and PFS were significantly better with blasts < or =20% and time to transplant < or =1 year while transplant-related mortality was less with the use of TBI. We conclude that patients with refractory leukemia can benefit from allogeneic BMT, especially with < or =20% marrow blast.

Adolescent↗
PubMed2006

CD34(+)-selected stem cell boost for delayed or insufficient engraftment after allogeneic stem cell transplantation.

BACKGROUND: Poor graft function without rejection may occur after stem cell transplantation (SCT). CD34(+) stem cell boost (SCB) can restore marrow function but may induce or exacerbate GvHD. We therefore investigated the feasibility and efficacy of CD34(+)-selected SCB in some patients with poor graft function. We present the results for eight patients (median age 46 years) transplanted initially for myelofibrosis, acute leukemia, myeloma and NHL. Six patients had received HLA-matched and two mismatched grafts (PB, BM; n=5, 3). After a median of 128 days post-transplant, the median leukocyte and platelet counts were, respectively, 2.05/nL and 18/nL. None had achieved platelet counts >50/nL even though donor chimerism was >95% in seven recipients. METHODS: Positive selection of CD34(+) stem cells was performed on a CliniMACS device, observing GMP and achieving a median of 98.5% purity. The patients received a median of 1.7 x 10(6)/kg CD34(+) cells and 2.5 x 10(3)/kg CD3(+) T lymphocytes. RESULTS: Hemograms at days +30, +60 and +90, respectively, showed steadily increasing median leukocyte (2.55, 3.15 and 4.20/nL) and platelet (29, 39 and 95/nL) counts. After a median follow-up of 144 days, five patients remained alive. No patient had developed acute or chronic GvHD. One patient died of leukemic relapse and two others of systemic mycosis. DISCUSSION: These preliminary results point to the possibility of safely improving graft function using CD34(+) positively selected stem cells without necessarily increasing the incidence of GvHD in patients with poor graft function post-SCT. Experience with more patients and longer follow-up should clarify the optimal role for this procedure.

Adolescent↗
PubMed2005

Aldehyde dehydrogenase activity as a marker for the quality of hematopoietic stem cell transplants.

Taking advantage of fluorescent substrates for their metabolic marker aldehyde dehydrogenase (ALDH), hematopoietic stem cells (HSC) were defined as SSC(lo)ALDH(br) - reflecting their low orthogonal light scattering and bright fluorescence intensity in flow cytometry. Based thereon, we investigated the usefulness of ALDH activity for characterizing HSC graft quality, particularly under stress conditions. We first compared the expression of ALDH vs CD34 in bone marrow and peripheral blood stem cell (PBSC) samples over 7 days. We noted that (i) only ALDH activity but not CD34 expression strongly reflected colony-forming ability over time, and that (ii) PBSC grafts stored at room temperature lost most of their progenitor cells within just 48 h. We then retrospectively related ALDH and CD34 expression as well as granulocyte-macrophage colony-forming units (CFU-GM) potential for 19 cryopreserved allogeneic PBSC grafts to engraftment data. Strikingly, in all six patients who received markedly decreased numbers of SSC(lo)ALDH(br) cells, this was associated not only with almost complete loss of CFU-GM potential but also with delayed establishment/permanent absence of full hematopoietic donor cell chimerism, whereas all other patients showed early complete donor chimerism. In conclusion, we suggest to measure ALDH activity as a surrogate marker for HSC activity, and to transport and store PBSC under controlled cooling conditions.

Aldehyde Dehydrogenase↗
PubMed2005

Improving the ex vivo retroviral-mediated suicide-gene transfer process in T lymphocytes to preserve immune function.

The retroviral-mediated transfer of a suicide gene into donor T cells has been proposed as a method to control alloreactivity after hematopoietic stem cell (HSC) transplantation. Gene-modified cells (GMC) may be infused into the patient either at the time of transplantation, together with a T-cell depleted HSC graft, or after transplantation, as a donor lymphocyte infusion. Administration of a so-called pro-drug activating the "suicide" mechanism only after occurrence of GvHD should selectively destroy the alloreactive GMC in vivo, eventually leading to GvHD abrogation. Although phase I-II clinical trials provided vital proof of the principle of GvHD control by suicide-gene therapy, this approach is still suboptimal. Indeed, current gene transfer strategies rely on gamma-retroviral vectors that require extensive T-cell activation and expansion for efficient transduction. Both in vitro and in vivo studies have shown that the activation, cell expansion, transduction and selection steps lead to TCR repertoire alterations and impairment of crucial T-cell functions, such as alloreactivity and anti-EBV reactivity. Thus, improvements of the suicide-gene transfer processes are required in order to preserve T-cell function. This could be achieved by using CD3/CD28 co-stimulation and immunomagnetic selection of transduced cells. In future clinical trials, lentiviral vectors may prove to be a better alternative to gamma-retroviral-mediated gene transfer, by reducing the need for prolonged ex vivo culture.

Animals↗
PubMed2004

Self-inactivating retroviral vectors with improved RNA processing.

Three RNA features have been identified that elevate retroviral transgene expression: an intron in the 5' untranslated region (5'UTR), the absence of aberrant translational start codons and the presence of the post-transcriptional regulatory element (PRE) of the woodchuck hepatitis virus in the 3'UTR. To include such elements into self-inactivating (SIN) vectors with potentially improved safety, we excised the strong retroviral promoter from the U3 region of the 3' long terminal repeat (LTR) and inserted it either downstream or upstream of the retroviral RNA packaging signal (Psi). The latter concept is new and allows the use of an intron in the 5'UTR, taking advantage of retroviral splice sites surrounding Psi. Three LTR and four SIN vectors were compared to address the impact of RNA elements on titer, splice regulation and transgene expression. Although titers of SIN vectors were about 20-fold lower than those of their LTR counterparts, inclusion of the PRE allowed production of more than 10(6) infectious units per ml without further vector optimizations. In comparison with state-of-the-art LTR vectors, the intron-containing SIN vectors showed greatly improved splicing. With regard to transgene expression, the intron-containing SIN vectors largely matched or even exceeded the LTR counterparts in all cell types investigated (embryonic carcinoma cells, fibroblasts, primary T cells and hematopoietic progenitor cells).

Animals↗
PubMed2004

Restoration of SHIP activity in a human leukemia cell line downregulates constitutively activated phosphatidylinositol 3-kinase/Akt/GSK-3beta signaling and leads to an increased transit time through the G1 phase of the cell cycle.

The inositol 5-phosphatase SHIP (SHIP-1) is a negative regulator of signal transduction in hematopoietic cells and targeted disruption of SHIP in mice leads to a myeloproliferative disorder. We analyzed the effects of SHIP on the human leukemia cell line Jurkat in which expression of endogenous SHIP protein is not detectable. Restoration of SHIP expression in Jurkat cells with an inducible expression system caused a 69% reduction of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and a 65% reduction of Akt kinase activity, which was associated with reduced phosphorylation of glycogen synthase kinase 3beta (GSK-3beta) (Ser-9) without changing the phosphorylation of Bad (Ser-136), FKHR (Ser-256) or MAPK (Thr-202/Tyr-204). SHIP-expressing Jurkat cells showed an increased transit time through the G1 phase of the cell cycle, but SHIP did not cause a complete cell cycle arrest or apoptosis. Extension of the G1 phase was associated with an increased stability of the cell cycle inhibitor p27(Kip1) and reduced phosphorylation of the retinoblastoma protein Rb at serine residue 780. Our data indicate that restoration of SHIP activity in a human leukemia cell line, which has lost expression of endogenous SHIP, downregulates constitutively activated phosphatidylinositol 3-kinase/Akt/GSK-3beta signaling and leads to an increased transit time through the G1 phase of the cell cycle.

Apoptosis↗
PubMed2004

Stem cell clonality and genotoxicity in hematopoietic cells: gene activation side effects should be avoidable.

Two serious adverse events involving activation of the LMO2 oncogene through retrovirus vector insertion in the otherwise extremely successful first gene therapy trial for X-linked severe combined immunodeficieny type 1 (SCID-X1) had initially caused widespread concern in the patient and research communities. Careful consideration 1 year after diagnosis of the second case still finds 12 of the treated patients clearly benefiting from gene therapy (freedom from treatment failure, 80%; survival 100%), a situation that should not portend the end of gene therapy for this disease, and is, in fact encouraging. While current approaches are justified to treat patients with otherwise life-threatening disorders, a broad consensus has developed that systematic basic research is required to further understand the pathophysiology of these serious adverse events and to provide new insights, enabling safer and more effective gene therapy strategies. With the continued success of SCID-X1 gene therapy in the majority of patients treated, it is of even greater importance to understand exactly which vector element or combination of elements predispose to toxicity. An in-depth study of the mechanisms behind the activation of the LMO2 and gammac genes will be highly instructive for the development of safer procedures and vectors. We summarize the central observations, ongoing experimental approaches, new concepts, and developments relevant to understanding, interpreting, and eventually overcoming the real and perceived obstacles posed by insertional mutagenesis due to gene transfer vectors.

Adaptor Proteins, Signal Transducing↗
PubMed2002

A novel 'sort-suicide' fusion gene vector for T cell manipulation.

Retroviral suicide gene vectors have successfully been used in clinical studies to improve the safety of adoptive immunotherapy with allogeneic T lymphocytes in the treatment of malignant and viral diseases. At the same time these studies have revealed several problems that are yet to be resolved including impaired T cell function due to long ex vivo culture. Here we present new retroviral vectors co-expressing truncated CD34, a gene transfer marker which ensures rapid enrichment of transduced cells using commercially available GMP-approved devices, and a splice-corrected variant of Herpes simplex virus thymidine kinase (scHSVtk) which confers high sensitivity to the prodrug ganciclovir. We show that a retroviral hybrid vector, MP71, based on the myeloproliferative sarcoma virus (MPSV) and the murine embryonic stem cell virus (MESV), encoding a tCD34/scHSVtk fusion protein mediates high expression of the 'sort-suicide' selection marker, thereby allowing for highly efficient purification and selective elimination of transduced cells.

Antigens, CD34↗
PubMed2002

Persisting multilineage transgene expression in the clonal progeny of a hematopoietic stem cell.

Many applications of hematopoietic gene therapy require selection for clones with active transgene expression. However, it was unclear whether the clonal progeny of a retrovirally transduced hematopoietic stem cell would be capable of maintaining transgene expression through serial repopulation and multilineage differentiation. Such investigations require simultaneous analyses of clonality, multilineage activity and transgene copy numbers. Using a mouse model, the present study demonstrates that a single hematopoietic stem cell expressing a marker gene from one or two insertions of a simple retroviral vector actively maintains multilineage transgene expression in the vast majority (80-99%) of bone marrow and peripheral blood cells. Gene expression persisted through serial transplantations for at least 97 weeks post gene transfer and was observed in the lymphoid (B, T and NK cells), myeloid (CD11b(+), Gr-1(+)), erythroid (Ter119(+), mature red blood cells) and megakaryocytic (as indicated by platelets) progeny. Therefore, a single immunoselection for hematopoietic stem cells expressing the transgene in vivo was sufficient to establish a completely chimeric hematopoiesis. These observations imply that the retroviral vectors used in this study contain cis-elements that mediate expression through massive clonal expansion and multilineage differentiation, provided the insertion occurred in genetic loci permissive for expression in hematopoietic stem cells.

Animals↗
PubMed2002

Molecular methods for detection and quantification of myeloma cells after bone marrow transplantation: comparison between real-time quantitative and nested PCR.

Multiple myeloma is characterized by malignant plasma cell-infiltration of bone marrow. Treatment with high-dose therapy results in a high rate of clinical remissions, but almost all patients ultimately relapse. Clinical staging and detection of relapse are limited in sensitivity. Therefore, we established molecular methods based on the highly clone-specific CDR regions of the immunoglobulin VH locus for sensitive and specific detection of residual myeloma cells after bone marrow transplantation. VDJ rearrangements were identified using a set of VH primers and a JH primer. Clone-specific rearrangements were detected by comparison with germ-line sequences. With the nested PCR approach, first-round amplification with the consensus primers was done followed by second amplification with myeloma-specific primers. The real-time quantitative PCR was performed using a myeloma-specific forward primer in combination with a JH consensus TaqMan probe and reverse primer. Sensitivity was tested using dilutions of myeloma cell lines into mononuclear cells. Nested PCR had a sensitivity of 10(-6) and TaqMan PCR of 10(-4) to 10(-5). Specificity was determined by testing different cell lines and patients' probes. These results were confirmed by follow up of 2 patients after allogeneic transplantation with dose-reduced conditioning. Molecular methods are very sensitive and specific tools for follow up of myeloma patients after allogeneic transplantation. By using the quantitative approach, it is possible to see kinetics of bone marrow tumor load, which can be used to guide therapeutic decisions like donor leukocyte infusions (DLI).

Bone Marrow Transplantation↗
PubMed2001

The inositol 5-phosphatase SHIP is expressed as 145 and 135 kDa proteins in blood and bone marrow cells in vivo, whereas carboxyl-truncated forms of SHIP are generated by proteolytic cleavage in vitro.

The inositol polyphosphate 5-phosphatase SHIP plays an important role in negative signalling in B cells and mast cells and in the down-regulation of cytokine receptor-mediated signals in myeloid cells. SHIP is expressed as a 145 kDa full-length protein and an isoform of 135 kDa due to alternative splicing. Additional smaller forms of SHIP which are truncated at the carboxy terminus have been described in bone marrow and peripheral blood mononuclear cells (PBMC). Our data demonstrate that human bone marrow cells and PBMC from healthy donors and patients with acute myeloid leukemia express the 145 kDa form of SHIP and low amounts of a 135 kDa form of SHIP in vivo whereas C-terminal-truncated SHIP proteins are generated by a PMSF-sensitive protease during the preparation of cell lysates in vitro. We have further characterized this protease and identified a proteolytic cleavage site in the human SHIP protein C-terminal to tryptophan residue 941. These data support a physiological role for the 145 and 135 kDa forms of SHIP in bone marrow and peripheral blood cells from normal donors and patients with acute myeloid leukemia.

Alternative Splicing↗
PubMed2001

Real-time quantitative Y chromosome-specific PCR (QYCS-PCR) for monitoring hematopoietic chimerism after sex-mismatched allogeneic stem cell transplantation.

Y chromosome-specific sequences can be used to detect remaining male cells after sex-mismatched allogeneic blood stem cell transplantation (HSCT) involving a male patient and female donor, which represents approximately 25% of all cases. We developed a quantitative Y chromosome-specific PCR assay (QYCS-PCR) based on the DFFRY gene for the determination of hematopoietic donor chimerism. We analyzed blood and marrow samples from more than 40 patients at various time points after both standard and nonmyeloablative allogeneic HSCT. We found that real-time PCR combines extreme sensitivity, with a detection level of less than 1 male in 100,000 female cells (<0.001%), with very good reproducibility, especially in the important range of minor host chimerism. QYCS-PCR results were in close agreement with data from other techniques as bcr/abl-PCR and/or fluorescent in situ hybridization (FISH) analysis. In two relapsed patients, increasing numbers of Y-positive hematopoietic cells indicated recurrence of malignant disease prior to clinical confirmation. In conclusion, quantitative Y chromosome-specific PCR is a promising approach for monitoring the extent of chimerism in blood and other tissues after sex-mismatched hematopoietic stem cell transplantation (HSCT) or organ transplantation.

Adolescent↗
PubMed2001

Membrane-anchored peptide inhibits human immunodeficiency virus entry.

Peptides derived from the heptad repeats of human immunodeficiency virus (HIV) gp41 envelope glycoprotein, such as T20, can efficiently inhibit HIV type 1 (HIV-1) entry. In this study, replication of HIV-1 was inhibited more than 100-fold in a T-helper cell line transduced with a retrovirus vector expressing membrane-anchored T20 on the cell surface. Inhibition was independent of coreceptor usage.

Amino Acid Sequence↗
PubMed2001

T lymphocytes as targets of gene transfer with Moloney-type retroviral vectors.

Peripheral T lymphocytes are a target of choice for many gene therapeutic strategies. Retrovirus-mediated transduction allows genomic integration and long-term expression of transgenes in target cells. Over many years, low transduction efficiency into primary T lymphocytes has limited clinical application of existing protocols. Recently, gene transfer rates > 50% have been achieved facilitating clinical studies. More attention is thus being focused on the ability of gene-modified cells to carry out innate as well as conferred functions in vivo and the influence of culture conditions, retroviral vector and host response thereon.

Animals↗
PubMed2000

CD34 splice variant: an attractive marker for selection of gene-modified cells.

This study presents a promising selection system for gene-modified cells other than human hematopoietic progenitor and endothelial cells based on transgenic expression of human CD34. Three retrovirally transduced variants of CD34 were compared, differing in the length of their cytoplasmic domains. These were the full-length transmembrane protein (flCD34), a truncated form (tCD34) that is found as a naturally occurring splice variant and has a partial deletion of the cytoplasmic domain for signal transduction, and an engineered variant which is completely deprived of its cytoplasmic tail (dCD34). All three variants allowed selection of gene-modified cells using commercially available immunoaffinity technology. However, examination by flow cytometry as well as by Southern, Northern, and Western blot revealed that dCD34, as opposed to tCD34, is not stably anchored in the membrane and thus is expressed at low levels on the surface of transduced cells. Therefore, tCD34 was chosen as the more promising candidate for a clinically applicable cell surface marker. We show that gene-modified human primary T lymphocytes expressing tCD34 can be enriched to high purity (>95%) using clinically approved immunoaffinity columns. In addition, we demonstrate the utility of tCD34 for surface marking of murine hematopoietic cells in vivo, including primary T lymphocytes detected 9 weeks after bone marrow transplantation.

Animals↗
PubMed2000

Depletion of alloreactive donor T cells using immunomagnetic cell selection.

Donor T cells support both engraftment and immune reconstitution after allogeneic BMT. Moreover, they may exert potent anti-tumor effects (graft-versus-leukemia, GVL), which are used for adoptive immunotherapy. On the other hand, infusion of allogeneic T cells is frequently associated with the manifestation of immune reactions against healthy tissue, which may lead to life-threatening graft-versus-host disease (GVHD). To overcome this problem, we developed a new strategy for the exclusive depletion of alloreactive cells from donor leukocytes. We activated donor T cells by co-cultivation with a stroma layer of recipient cells and analyzed activation kinetics of CD3+, CD4+ and CD8+ T cells. Based on these data, activated cells were then depleted based on expression of activation-induced antigens using magnetic cell sorting (MACS). Alloreactivity of donor T cells was remarkably decreased after depletion of cells expressing either CD25 or CD69, as was shown in suitable in vitro assays. The lowest level of alloreactivity was found when both CD25- and CD69-positive cells were depleted. Importantly, depleted cell fractions preserved reactivity against third-party cells. In summary, we found that MACS-based ex vivo depletion of alloreactive cells may be a suitable way to prevent GVHD and therefore improve allogeneic BMT and adoptive immunotherapy.

Antigens, CD↗