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B Fehse

Publications and source records attributed to B Fehse.

32 records · Page 2Linked to original sources
PubMed2000

Amphotropic and VSV-G-pseudotyped retroviral vectors transduce human hematopoietic progenitor cells with similar efficiency.

One restriction of retroviral gene transfer into hematopoietic stem cells is the low level of amphotropic virus receptor. In the present study, we examined whether retroviral vectors pseudotyped with the G-protein of vesicular stomatitis virus (VSV) can overcome this restriction. Human progenitor cells purified by magnetic beads and cell sorting were transduced with an amphotropic or VSV-G-pseudotyped retroviral vector containing the truncated human nerve growth factor receptor as a marker gene. Cells were prestimulated with flt-3 ligand, stem cell factor, and interleukin-3 and transduced on fibronectin. Marker gene expression was analyzed by flow cytometry. Transduction efficiencies of amphotropic and VSV-G-pseudotyped virus for CD34+ cells did not differ significantly. Gene transfer into CD34+CD38- cells, which are enriched in more immature progenitors, was not restricted and transfer efficiencies for this subset were also similar for both pseudotypes. The addition of fibronectin improved gene transfer with the amphotropic vector considerably (5- to 19.3-fold, mean 12.6), while the effect on the VSV-G-pseudotype was far less pronounced (1- to 3.9-fold, mean 2.1, P = 0.04). In conclusion, high levels of gene transfer to human hematopoietic progenitors were achieved with an optimized transduction protocol, and transduction efficiencies could not be improved further by the use of VSV-G-pseudotypes.

ADP-ribosyl Cyclase↗
PubMed2000

Retroviral transduction of T lymphocytes for suicide gene therapy in allogeneic stem cell transplantation.

Transplantation of suicide gene modified allogeneic T lymphocytes is an approach to prevent T cell mediated GVHD while preserving the 'graft-versus-leukemia' (GVL) effect of an allograft. A prerequisite for such a therapy is the efficient transduction of T cells with suitable vectors. Since existing techniques allow only insufficient transduction of T cells, the development of more efficient gene transfer protocols into these cells is of great importance. We present here a protocol for the highly efficient transduction of human primary T cells at high densities (1 x 10(6) cells/ml) by retroviral infection. The presented protocol allowed us to obtain transduction rates of more than 70% of CD3+ cells after two cycles of infection. It is based on the use of FBS-free media for both the production of retrovirus-containing supernatant, as well as the cultivation of the primary T cells. Since the protocol presented here works just as efficiently under large scale conditions, it may easily be adapted to clinical needs and 'good manufacturing practice' (GMP) standards.

Fibronectins↗
PubMed2000

Efficient depletion of alloreactive donor T lymphocytes based on expression of two activation-induced antigens (CD25 and CD69).

T lymphocytes play an important role in allogeneic bone marrow/stem cell transplantation by supporting engraftment and immune recovery. Moreover, donor T cells have been shown to mediate the so-called graft-versus-leukaemia effect and are, therefore, increasingly used for adoptive immunotherapy. However, T-cell infusions are associated with the risk of a graft-versus-host reaction, which may lead to a life-threatening disease. To overcome this problem, we followed a new strategy for the exclusive depletion of alloreactive cells. We activated allogeneic T cells by cultivation on an adherent cell layer derived from peripheral blood. We then depleted activated cells based on the expression of CD25, CD69 or both activation-induced antigens using magnetic cell sorting. Mixed lymphocyte culture (MLC) reactions and helper T-lymphocyte precursor cell frequency (HTLP-f) assays demonstrated that this technique led to a significant decrease in alloreactivity of 'donor' cells, which at the same time preserved reactivity against third-party cells. The lowest level of alloreactivity was found when CD25 and CD69 antibodies were used together for depletion. This corresponds with our observation that expression of CD25 or CD69 may partially represent different activation pathways. We conclude that ex vivo depletion of CD25- and CD69-expressing alloreactive cells may help to overcome limitations of adoptive immunotherapy.

Adoptive Transfer↗
PubMed1999

Rapid and efficient cloning of proviral flanking fragments by kanamycin resistance gene complementation.

We have developed a technique for the rapid cloning of unknown flanking regions of transgenic DNA. We complemented a truncated kanamycin resistance gene of a bacterial plasmid with a neomycin resistance gene fragment from a gene transfer vector. Optimized transformation conditions allowed us to directly select for kanamycin-resistant bacteria. We cloned numerous proviral flanking fragments from growth factor-independent cell mutants that were obtained after infection with a replication incompetent retroviral vector and identified integrations into the cyclin D2 and several unknown genomic sequences. We anticipate that our method could be adapted to various vector systems that are used to tag and identify genes and to map genomes.

Cloning, Molecular↗
PubMed1999

Bicistronic retroviral vectors for combining myeloprotection with cell-surface marking.

We have developed a retroviral vector coexpressing the multidrug-resistance 1 (MDR1) cDNA for inducing cancer drug resistance and the truncated version of the low-affinity nerve growth factor receptor (DeltaLNGFR) for cell-surface marking of transduced cells. The vector is based on the FMEV backbone which mediates high levels of gene expression in hematopoietic cells. To achieve optimal expression levels of both cDNAs, untranslated regions from MDR1 and DeltaLNGFR were removed and three different connections were tested: retroviral splice signals, an internal ribosomal entry site (IRES) from encephalomyocarditis virus, and an internal promoter from the chicken beta-actin gene. As determined by two-color flow cytometry, the best correlation of the expression of both cDNAs was obtained using the vector SF1mSdelta which utilized retroviral splice signals for co-expression. Simultaneous expression of both cDNAs at the single cell level was also shown by confocal laser microscopy. Lymphoid and hematopoietic progenitor cells, including primary human CD34+ cells, transduced with SF1mSdelta acquired dominant multidrug resistance. Transduced primary CD34+ cells could be enriched in vitro based on expression of DeltaLNGFR, avoiding exposure to cytostatic agents. Thus, monitoring the selection of chemotherapy-resistant cells and analyzing their biological properties may be alleviated, both in vitro and in vivo.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Establishment of an optimised gene transfer protocol for human primary T lymphocytes according to clinical requirements.

Current gene therapeutic protocols directed towards the treatment of inherited disorders (eg ADA-SCID) and viral infections (eg AIDS), as well as adoptive immunotherapy approaches are based on the use of genetically modified lymphocytes. Since only insufficient transduction of T cells is obtained using existing techniques, the development of more efficient gene transfer protocols into these cells is of great importance. We present here a protocol for the highly efficient transduction of human primary T cells at high densities (1 x 106/ml) by retroviral infection. Using retroviral vectors encoding a truncated human low-affinity nerve growth factor receptor (DeltaLNGFR) as a gene transfer marker, we obtained transduction frequencies of more than 70% of CD3+ cells after two cycles of infection. Our protocol is based on the use of FBS-free media for both the production of retrovirus-containing supernatant and the cultivation of the primary T cells. Since the protocol presented here works just as efficiently under large-scale conditions, it may be easily adapted to clinical needs and 'good manufacturing practice' (GMP) standards.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Dominant selection of hematopoietic progenitor cells with retroviral MDR1 co-expression vectors.

When transferring the human multidrug resistance 1 (MDR1) cDNA, FMEV retroviral vectors mediate high-dose multidrug resistance and, thus, background-free selection in primary human hematopoietic progenitor cells. Here, we analyzed strategies for co-expression of a second gene from an FMEV:MDR1 vector. When linking the cDNAs with the internal ribosomal entry site (IRES) of poliovirus or retroviral splice signals, almost all multidrug-resistant hematopoietic colonies simultaneously coexpressed the 3' positioned second gene, neomycin-phosphotransferase (neoR). The IRES strategy allowed functional co-transfer of a 4.2-kb lacZ-neoR fusion gene, resulting in a total proviral genome size of 11 kb, corresponding to the packaging limit of retroviral vectors. Preselection based on multidrug resistance elevated the expression of the second gene in IRES constructs, but not in splice vectors. Moreover, three intriguing observations were made. First, up to 30% of cells preselected for functional transfer of the 3' positioned cDNA (neoR) showed infunctional MDR1; this occurred irrespective of the linking principle and was associated with instability of the MDR1 transcription unit. Second, the levels of multidrug resistance achieved with the co-expression vectors were moderately lower (15-30% reduced) than those mediated by the monocistronic counterpart. Third, transduction with FMEV:MDR1 co-expression vectors still resulted in high-dose cancer drug resistance and background-free selection of hematopoietic progenitor cells (including primary human CD34+ colony-forming units). Thus, for the first time, we describe MDR1 co-expression vectors that maintain their desired function in early and primary human hematopoietic cells. However, careful interpretation of the data reveals that further vector improvements are required to obtain clinically useful MDR1 co-expression vectors.

Antigens, CD34↗
PubMed1998

Purging and haemopoietic progenitor cell selection by CD34+ cell separation.

Tumour cell contamination of autologous peripheral blood stem cell samples (PBSC) and bone marrow (BM) is frequent. Enrichment of CD34+ stem cells is a promising approach to purging tumour cells from autografts without damaging progenitor cells. Breast cancer cells were seeded (10(-3)-10(-7)) into mononuclear cells from G-CSF-mobilised PBSC and BM harvests from patients without breast cancer. CD34+ cells were enriched from mixtures either by immunomagnetic separation (Isolex-50, and MiniMACS) or by biotin-streptavidin immunoaffinity columns (Ceprate-LC). CD34+ cell fractions were determined by FACS, cancer cells were detected immunocytochemically with an anti-pancytokeratin antibody. The CD34+ cells were enriched with a median purity of 92.2% (43.5-96.1) (n = 17) (Isolex-50), 96.5% (66.6-99.2) (n = 17) (MiniMACS) and 77.9% (31.4-93.6) (n = 15) (Ceprate-LC) from PBSC and BM harvests. The percentages of median recovery of CD34+ cells were 30.8% (18.6-71.8) (Isolex-50), 69.9% (39.1-100) (MiniMACS) and 42.9% (23.7-100) (Ceprate-LC). The median tumour cell reductions in log steps were 3.7 (2.9-4.3) (n = 13) (Isolex-50), 3.5 (2.6-4.3) (n = 13) (MiniMACS) and 1.5 (0.9-2.9) (n = 17) (Ceprate-LC). Results were compared statistically by univariate analysis. Purity was significantly (P < 0.05) better after MiniMACS selection. Recovery rates were significantly different between all devices tested. Tumour cell purging was superior after immunomagnetic separation (P < 0.001). Tumour cell purging is a main objective of CD34+ selection in the autologous setting. Our in vitro data clearly indicate that immunomagnetic separation is more efficient in the prevention of accidental reinfusion of contaminating tumour cells compared to immunoaffinity. However, it is not yet known if the same results can be obtained with fresh contaminating tumour cells.

Antigens, CD34↗
PubMed1998

Highly-efficient gene transfer with retroviral vectors into human T lymphocytes on fibronectin.

Genetically modified lymphocytes have been successfully used for correction of ADA deficiency in children and in controlling graft-versus-host disease (GvHD) after allogeneic bone marrow transplantation. Low transduction efficiencies are, however, limiting for gene therapeutic strategies based on lymphocytes. In this study we compared protocols for highly efficient gene transfer into human T cells using retroviral vector-containing supernatant. We showed that infection of both human primary T cells and CD4+ Jurkat cells is most efficient on the matrix component fibronectin. Transduction was carried out with a retroviral vector encoding both the human intracytoplasmatically truncated low-affinity nerve growth factor receptor (deltaLNGFR) as a gene transfer marker and the Herpes simplex virus thymidine kinase for negative selection. Based on LNGFR expression genetically modified cells were enriched to near purity by magnetic cell sorting (MACS). Enriched cells could be shown to be highly sensitive to ganciclovir.

Anti-Allergic Agents↗
PubMed1997

Selective immunoaffinity-based enrichment of CD34+ cells transduced with retroviral vectors containing an intracytoplasmatically truncated version of the human low-affinity nerve growth factor receptor (deltaLNGFR) gene.

Human hematopoietic stem cells remain one of the most promising target cells for gene therapeutic approaches to treat malignant and nonmalignant diseases. To rapidly characterize transduced cells and to isolate these from residual nontransduced, but biologically equivalent, cells, we have used a Moloney murine leukemia virus (Mo-MuLV)-based retroviral vector containing the intracytoplasmatically truncated human low-affinity nerve growth factor receptor (deltaLNGFR) cDNA as a marker gene. Supernatant transduction of CD34+ cells (mean purity 97%) in fibronectin-coated tissue culture flasks resulted in 5.5-45% (mean 26%) transduced cells expressing deltaLNGFR (LNGFR+ cells). After transduction, more than 65% of the transduced cells remained CD34+. Compared with control (mock- and nontransduced) CD34+ cells, transduction did not decrease the cloning efficiency of CD34+ cells. Immunomagnetic selection of the transduced cells with a monoclonal anti-LNGFR antibody resulted in >90% LNGFR+ cells. Further phenotypic characterization of these highly enriched LNGFR+ cells indicated that the majority co-expressed the CD34 and CD38 antigens. These results show that transduced cells expressing an ectopic cell-surface protein can be rapidly and conveniently quantitated and characterized by fluorescence-activated cell sorting (FACS) analysis and fast and efficiently enriched by immunoadhesion using magnetic beads. The use of cell-surface reporters should facilitate optimization of methods of gene transfer into more primitive hematopoietic progenitors.

Animals↗
PubMed1995

Excision of specific DNA-sequences from integrated retroviral vectors via site-specific recombination.

Vectors for gene transfer and gene therapy were developed which combine the advantages of the integrase and recombinase systems. This was achieved by inserting two loxP sites for specific DNA excision into an MESV based retroviral vector. We show that this 'retroviral lox system' allows the infection of cells and the expression of transferred genes. In addition, we constructed an efficient retrovirus-based expression system for a modified Cre recombinase. Functional tests for DNA excision from integrated retroviral lox vectors were performed by the use of a negative selectable marker gene (thymidine kinase). Cre expression in cells infected with retroviral lox vectors and subsequent BrdU selection for cells in which site-specific recombination has occurred results in large numbers of independent cell clones. These results were confirmed by detailed molecular analysis. In addition we developed retroviral suicide vectors in which the enhancer/promoter elements of both LTRs were replaced by lox sequences. We show that lox-sequences located in the LTRs of retroviral vectors are stable during retroviral replication. Potential applications of this system would be the establishment of revertants of retrovirus-infected cells by controlled excision of nearly the complete proviral DNA.

Animals↗