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Biomedical subjects

Christopher Baum

Publications and source records attributed to Christopher Baum.

At least 19 recordsLinked to original sources
PubMed2006

Retroviral vector insertion sites associated with dominant hematopoietic clones mark "stemness" pathways.

Evidence from model organisms and clinical trials reveals that the random insertion of retrovirus-based vectors in the genome of long-term repopulating hematopoietic cells may increase self-renewal or initiate malignant transformation. Clonal dominance of nonmalignant cells is a particularly interesting phenotype as it may be caused by the dysregulation of genes that affect self-renewal and competitive fitness. We have accumulated 280 retrovirus vector insertion sites (RVISs) from murine long-term studies resulting in benign or malignant clonal dominance. RVISs (22.5%) are located in or near (up to 100 kb [kilobase]) to known proto-oncogenes, 49.6% in signaling genes, and 27.9% in other or unknown genes. The resulting insertional dominance database (IDDb) shows substantial overlaps with the transcriptome of hematopoietic stem/progenitor cells and the retrovirus-tagged cancer gene database (RTCGD). RVISs preferentially marked genes with high expression in hematopoietic stem/progenitor cells, and Gene Ontology revealed an overrepresentation of genes associated with cell-cycle control, apoptosis signaling, and transcriptional regulation, including major "stemness" pathways. The IDDb forms a powerful resource for the identification of genes that stimulate or transform hematopoietic stem/progenitor cells and is an important reference for vector biosafety studies in human gene therapy.

Animals↗
PubMed2006

Murine leukemia virus regulates alternative splicing through sequences upstream of the 5' splice site.

Alternative splicing of the primary transcript plays a key role in retroviral gene expression. In contrast to all known mechanisms that mediate alternative splicing in retroviruses, we found that in murine leukemia virus, distinct elements located upstream of the 5' splice site either inhibited or activated splicing of the genomic RNA. Detailed analysis of the first untranslated exon showed that the primer binding site (PBS) activates splicing, whereas flanking sequences either downstream or upstream of the PBS are inhibitory. This new function of the PBS was independent of its orientation and primer binding but associated with a particular destabilizing role in a proposed secondary structure. On the contrary, all sequences surrounding the PBS that are involved in stem formation of the first exon were found to suppress splicing. Targeted mutations that destabilized the central stem and compensatory mutations of the counter strand clearly validated the concept that murine leukemia virus attenuates its 5' splice site by forming an inhibitory stem-loop in its first exon. Importantly, this mode of splice regulation was conserved in a complete proviral clone. Some of the mutants that increase splicing revealed an opposite effect on translation, implying that the first exon also regulates this process. Together, these findings suggest that sequences upstream of the 5' splice site play an important role in splice regulation of simple retroviruses, directly or indirectly attenuating the efficiency of splicing.

5' Untranslated Regions↗
PubMed2006

LEF-1 is crucial for neutrophil granulocytopoiesis and its expression is severely reduced in congenital neutropenia.

We demonstrate here that lymphoid enhancer-binding factor 1 (LEF-1) mediates the proliferation, survival and differentiation of granulocyte progenitor cells. We initially documented the importance of this transcription factor in the bone marrow of individuals with severe congenital neutropenia (CN) with a 'differentiation block' at the promyelocytic stage of myelopoiesis. LEF-1 expression was greatly reduced or even absent in CN arrested promyelocytes, resulting in defective expression of the LEF-1 target genes CCND1, MYC and BIRC5, encoding cyclin D1 (ref. 2), c-Myc and survivin, respectively. In contrast, healthy individuals showed highest LEF-1 expression in promyelocytes. Reconstitution of LEF-1 in early hematopoietic progenitors of two individuals with CN corrected the defective myelopoiesis and resulted in the differentiation of these progenitors into mature granulocytes. Repression of endogenous LEF-1 by specific short hairpin RNA inhibited proliferation and induced apoptosis of CD34(+) progenitors from healthy individuals and of cells from two myeloid lines (HL-60 and K562). C/EBPalpha, a key transcription factor in granulopoiesis, was directly regulated by LEF-1. These observations indicate that LEF-1 is an instructive factor regulating neutrophilic granulopoiesis whose absence plays a critical role in the defective maturation program of myeloid progenitors in individuals with CN.

Antigens, CD↗
PubMed2006

Gene therapy: X-SCID transgene leukaemogenicity.

Gene therapy has been remarkably effective for the immunological reconstitution of patients with severe combined immune deficiency, but the occurrence of leukaemia in a few patients has stimulated debate about the safety of the procedure and the mechanisms of leukaemogenesis. Woods et al. forced high expression of the corrective therapeutic gene IL2RG, which encodes the gamma-chain of the interleukin-2 receptor, in a mouse model of the disease and found that tumours appeared in a proportion of cases. Here we show that transgenic IL2RG does not necessarily have potent intrinsic oncogenic properties, and argue that the interpretation of this observation with respect to human trials is overstated.

Animals↗
PubMed2006

Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity.

Retroviral vectors with long terminal repeats (LTRs), which contain strong enhancer/promoter sequences at both ends of their genome, are widely used for stable gene transfer into hematopoietic cells. However, recent clinical data and mouse models point to insertional activation of cellular proto-oncogenes as a dose-limiting side effect of retroviral gene delivery that potentially induces leukemia. Self-inactivating (SIN) retroviral vectors do not contain the terminal repetition of the enhancer/promoter, theoretically attenuating the interaction with neighboring cellular genes. With a new assay based on in vitro expansion of primary murine hematopoietic cells and selection in limiting dilution, we showed that SIN vectors using a strong internal retroviral enhancer/promoter may also transform cells by insertional mutagenesis. Most transformed clones, including those obtained after dose escalation of SIN vectors, showed insertions upstream of the third exon of Evi1 and in reverse orientation to its transcriptional orientation. Normalizing for the vector copy number, we found the transforming capacity of SIN vectors to be significantly reduced when compared with corresponding LTR vectors. Additional modifications of SIN vectors may further increase safety. Improved cell-culture assays will likely play an important role in the evaluation of insertional mutagenesis.

Animals↗
PubMed2006

In vivo selection of transduced hematopoietic stem cells and little evidence of their conversion into hepatocytes in vivo.

BACKGROUND/AIMS: FMEV-type retroviral vector provides high transgene expression in hepatocytes and hematopoietic stem cells (HSCs). Here, we examined whether these vectors could provide a sufficient drug-resistance gene expression in HSCs and whether transduced HSCs could differentiate into hepatocytes in vivo. METHODS: The CD45(+)/Lin(-) cells were transduced in vitro by FMEV-type vectors containing human O(6)-methylguanine-DNA methyltransferase (MGMT)/reporter genes and transferred into recipient mice. After the treatment with temozolomide and O(6)-benzylguanine (TMZ/BG) in vivo, we analyzed the transgene expression in peripheral blood cells by flow-cytometry. Immunohistochemistry was performed on the liver slices in partial hepatectomized recipient mice. RESULTS: After TMZ/BG treatment, transduced host cells were enriched in recipient mice. In the liver, we observed the efficient transgene expression in many small cells along sinusoids. However, only few large cells in hepatic lobules expressed albumin. They also expressed both a transgene and a recipient marker gene, suggesting the fusion of donor HSCs with recipient hepatocytes. CONCLUSIONS: This vector expressed a drug-resistance gene in HSCs highly enough to protect them from the drugs. But, the conversion of HSCs into hepatocytes in vivo might be a rare event in this model.

Animals↗
PubMed2006

Retrovirus vectors: toward the plentivirus?

Recombinant retroviral vectors based upon simple gammaretroviruses, complex lentiviruses, or potentially nonpathogenic spumaviruses represent relatively well characterized tools that are widely used for stable gene transfer. Different members of the Retroviridae family have developed distinct and potentially useful features related to their life cycle. These natural differences can be exploited for specialized applications in gene therapy and could conceivably be combined to create future retroviral hybrid vectors, ideally incorporating the following features: an efficient, noncytopathic packaging system with low likelihood of recombination; serum resistance; an ability to pseudotype with cell-specific envelopes; high-fidelity reverse transcription before cell entry; unrestricted cytoplasmic transport and nuclear import; an insulated expression cassette; specific chromosomal targeting; and physiologic or regulated levels of transgene expression. We envisage that, compared to contemporary vectors, a hybrid vector combining these properties would have increased therapeutic efficacy and an enhanced biosafety profile. Many of the above goals will require the inclusion of nonretroviral components into vector particles or transgenes.

Animals↗
PubMed2006

HOXB4 inhibits cell growth in a dose-dependent manner and sensitizes cells towards extrinsic cues.

Ectopic expression of the homeodomain transcription factor HOXB4 expands hematopoietic stem and progenitor cells in vivo and in vitro, making HOXB4 a highly interesting candidate for therapeutic stem cell expansion. However, when expressed at high levels, HOXB4 concomitantly perturbs differentiation and thus likely predisposes the manipulated cells for leukemogenesis. We therefore asked whether the expression level of HOXB4 may be a critical parameter that influences the growth and transformation properties of transduced cells. Using a set of retroviral vectors which covered a 40-fold range of expression levels, we studied the consequences of HOXB4 expression at different levels in the well established Rat-1 fibroblast cell system. HOXB4 transformed Rat-1 fibroblasts beyond a certain threshold level of expression. Further escalation of HOXB4 expression, however, did not enhance transformation. Instead, HOXB4 mediated a dose dependent anti-proliferative effect on Rat-1 and NIH3T3 fibroblasts. This effect was aggravated under reduced serum concentrations and was, at least partially, due to an enhanced sensitivity of HOXB4 overexpressing cells to induction of apoptosis. Based on these results we propose that HOXB4 affects cell growth in a dose-dependent manner by sensitizing cells towards extrinsic signals.

Animals↗
PubMed2006

Lentiviral vectors pseudotyped with murine ecotropic envelope: increased biosafety and convenience in preclinical research.

OBJECTIVE: Lentiviral vectors are increasingly used for preclinical models of gene therapy and other forms of experimental transgenesis. Due to the broad tropism and the ability for concentration by ultracentrifugation, most lentiviral vector preparations are produced using the vesicular stomatitis virus glycoprotein (VSV-g) protein as envelope. Recently, Hanawa and colleagues have demonstrated that the ecotropic envelope protein of murine leukemia viruses allows efficient pseudotyping of HIV-1-derived vector particles. However, this method has found little acceptance, despite potential advantages. MATERIALS AND METHODS: We produced lentiviral vectors pseudotyped with murine ecotropic envelope using a four-plasmid transient transfection system and evaluated their performance in murine fibroblasts and hematopoietic cells. RESULTS: Titers of lentiviral "ecotropic" supernatants were only slightly lower than those produced with VSV-g, could be concentrated by overnight centrifugation (13,000g), and efficiently transduced murine fibroblasts and hematopoietic cells but not human cells. Our Institutional Biosafety Committee agreed on the production and use of replication-defective lentiviral vectors pseudotyped with murine ecotropic envelope under biosafety level 1 (BL1) conditions with additional BL2 practices. We also obtained useful guidelines for the work with human infectious lentiviral vectors. CONCLUSIONS: For the researcher, "ecotropic" lentiviral vectors significantly improve the convenience of daily work, compared to the conditions required for lentiviral pseudotypes that are capable of infecting human cells. High efficiency and superior biosafety in combination with convenient handling will certainly boost the potential applicability of this important vector system.

Animals↗
PubMed2006

Mutagenesis and oncogenesis by chromosomal insertion of gene transfer vectors.

Increasing evidence reveals that random insertion of gene transfer vectors into the genome of repopulating hematopoietic cells may alter their fate in vivo. Although most insertional mutations are expected to have few if any consequences for cellular survival, clonal dominance caused by retroviral vector insertions in (or in the vicinity of) proto-oncogenes or other signaling genes has been described for both normal and malignant hematopoiesis. Important insights into these side effects were initially obtained in murine models. Results from ongoing clinical studies have revealed that similar adverse events may also occur in human gene therapy. However, it remains unknown to what extent the outcome of insertional mutagenesis induced by gene vectors is related to (1) the architecture and type of vector used, (2) intrinsic properties of the target cell, and (3) extrinsic and potentially disease-specific factors influencing clonal competition in vivo. This review discusses reports addressing these questions, underlining the need for models that demonstrate and quantify the functional consequences of insertional mutagenesis. Improving vector design appears to be the most straightforward approach to increase safety, provided all relevant cofactors are considered.

Animals↗
PubMed2006

Overexpression of MDR1 using a retroviral vector differentially regulates genes involved in detoxification and apoptosis and confers radioprotection.

Overexpression of P-glycoprotein (P-gp), the product of the MDR1 (multidrug resistance 1) gene, might complement chemotherapy and radiotherapy in the treatment of tumors. However, for safety and mechanistic reasons, it is important to know whether MDR1 overexpression influences the expression of other genes. Therefore, we analyzed differential gene expression in cells of the human lymphoblast cell line TK6 retrovirally transduced with MDR1 using the GeneChip Human Genome U133 Plus2.0 (Affymetrix). Sixty-one annotated genes showed a significant change in expression (P < 10(-4)) in MDR1-overexpressing cells compared to untransduced cells and cells transduced with a control virus expressing the neomycin phosphotransferase gene. Several genes coding for proteins involved in detoxification and exocytosis showed approximately 1.4- 4-fold increases in transcript levels (e.g. ALDH1A, UNC13). Additionally, pro-apoptosis genes were down-regulated (e.g. twofold for CASP1, 2.5-fold for NALP7) with concomitant increased expression of the potential anti-apoptosis gene AKT3. In functional assays the influence of MDR1 overexpression on apoptosis signaling was further corroborated by showing reduced rates of apoptosis in response to irradiation in TK6 cells transduced with MDR1. In conclusion, the resistant phenotype of MDR1-mediated P-gp-overexpressing cells is associated with differential expression of genes coding for metabolic and apoptosis-related proteins. These results have important implications for understanding the mechanisms by which MDR1 gene therapy can protect normal tissues from radiation- or chemotherapy-induced damage during tumor treatment.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Equal potency of gammaretroviral and lentiviral SIN vectors for expression of O6-methylguanine-DNA methyltransferase in hematopoietic cells.

Severe adverse events related to insertional mutagenesis have reinforced interest in self-inactivating (SIN) retroviral vectors lacking enhancer-promoter sequences in the long terminal repeats (LTRs). Here, we have compared the potency of gammaretroviral and lentiviral vectors expressing the P140K mutant of O(6)-methylguanine-DNA methyltransferase (MGMT). MGMT-P140K is a clinically relevant selection marker that mediates a strong survival advantage in hematopoietic cells exposed to alkylating agents. We designed gammaretroviral and lentiviral vectors that contained identical enhancer-promoter sequences located either in the LTR or downstream of the packaging region, for internal initiation of transcription from SIN backbones. Gammaretroviral vectors with intact LTRs containing enhancer-promoter sequences showed both higher titers and higher expression levels than the lentiviral counterparts, likely a result of suboptimal RNA processing of the lentiviral leader region. In the SIN context, gammaretroviral and lentiviral vectors with comparable internal cassettes had similar expression properties. Interestingly, gammaretroviral SIN vectors pseudotyped with RD114/TR had a higher transduction efficiency on proliferating human CD34(+) cells than lentiviral counterparts. These results encourage further investigations into the formation of retroviral hybrid vectors that combine the desired properties of high efficiency and increased biosafety.

Animals↗
PubMed2005

HOXB4 enforces equivalent fates of ES-cell-derived and adult hematopoietic cells.

Genetic manipulation of hematopoietic stem and progenitor cells is an important tool for experimental and clinical applied hematology. However, techniques that allow for gene targeting, subsequent in vitro selection, and expansion of genetically defined clones are available only for ES cells. Such molecularly defined and, hence, "safe" clones would be highly desirable for somatic gene therapy. Here, we demonstrate that in vitro differentiated ES cells completely recapitulate the growth and differentiation properties of adult bone marrow cells, in vitro and in vivo, when ectopically expressing HOXB4. Myeloid development was enforced and (T) lymphoid development suppressed over a wide range of expression levels, whereas only high expression levels of the transcription factor were detrimental for erythroid development. This indicates a close association between the amounts of ectopic HOXB4 present within a progenitor cell and and the decision to self renew or differentiate. Because HOXB4 mediates similar fates of ES-derived and bone marrow hematopoietic stem cells, the primitive embryonic cells can be considered a promising alternative for investigating hematopoietic reconstitution, in vivo, based on well defined clones. Provided that HOXB4 levels are kept within a certain therapeutic window, ES cells also carry the potential of efficient and safe somatic gene therapy.

Animals↗
PubMed2005

Clonal dominance of hematopoietic stem cells triggered by retroviral gene marking.

Gene marking with replication-defective retroviral vectors has been used for more than 20 years to track the in vivo fate of cell clones. We demonstrate that retroviral integrations themselves may trigger nonmalignant clonal expansion in murine long-term hematopoiesis. All 29 insertions recovered from clones dominating in serially transplanted recipients affected loci with an established or potential role in the self-renewal or survival of hematopoietic stem cells. Transcriptional dysregulation occurred in all 12 insertion sites analyzed. These findings have major implications for diagnostic gene marking and the discovery of genes regulating stem cell turnover.

Animals↗
PubMed2005

Leukemias following retroviral transfer of multidrug resistance 1 (MDR1) are driven by combinatorial insertional mutagenesis.

Previous studies have demonstrated leukemic complications in mice after high-copy retroviral gene transfer of the multidrug resistance 1 (MDR1) cDNA, encoding a membrane-located efflux pump expressed in hematopoietic stem cells. In contrast, no such complications or MDR1-associated alterations of hematopoiesis were observed in numerous other studies exploring MDR1 gene transfer into cell lines, mice, dogs, nonhuman primates, and human subjects. Here, we show that leukemias associated with retroviral expression of MDR1 depend on high vector dose, and involve the selection of clones with combinatorial insertional mutagenesis of proto-oncogenes or other signaling genes. Compared with insertion patterns in normal long-term repopulating hematopoietic cells, such hits were overrepresented in leukemic clones, pointing to a causal role. A similar constellation of insertion sites was also observed in a leukemia arising after high-copy retroviral gene transfer of a fluorescent protein. Spectral karyotyping demonstrated additional chromosomal translocations in a subset of cases, indicative of secondary genetic instability. We also show that insertional mutants can be amplified in vitro prior to transplantation. On the basis of these findings, we suggest the use of preclinical dose-escalation studies to define a therapeutic index for retroviral transgene delivery.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Gamma-glutamylcysteine synthetase and L-buthionine-(S,R)-sulfoximine: a new selection strategy for gene-transduced neural and hematopoietic stem/progenitor cells.

In most experimental gene therapy protocols involving stem/progenitor cells, only a small fraction of cells, often therapeutically inadequate, can be transduced and made to express the therapeutic gene. A promising strategy for overcoming this problem is the use of a dominant selection marker, such as a drug resistance gene. In this paper, we explore the potential of the heavy subunit of gamma-glutamylcysteine synthetase (gamma-GCSh) to act as a selection marker. We found that 3T3 fibroblasts transduced with the bicistronic retroviral vector SF91/GCSh-eGFP, encoding gamma-GCSh and the enhanced green fluorescent protein (eGFP), were highly resistant to L-buthionine-(S,R)-sulfoximine (BSO), a gamma-GCS inhibitor with a low clinical toxicity profile. The level of resistance was not proportional to the increase in intracellular glutathione. In fact, cells overexpressing both heavy and light gamma-GCS subunits had higher intracellular GSH levels, and a lower level of resistance to the cytotoxic activity of BSO, compared with cells overexpressing gamma-GCSh alone. 3T3 fibroblasts overexpressing gamma-GCSh could be selected from cultures containing both naive and gene-modified cells by application of exogenous BSO selection pressure for 4 days. Also, primary neural stem/progenitor cells derived from the lateral ventricles of mouse neonatal brains and primary hematopoietic stem/progenitor cells (HSCs/HPCs) from mouse bone marrow, transduced with the gamma-GCSh-eGFP vector, could be selected by BSO treatment in vitro. On ex vivo BSO selection and reimplantation into a syngeneic myeloablated host, donor HSCs/HPCs repopulated the marrow and continued to express the transgene(s). These results provide proof of principle that somatic stem/progenitor cells, transduced simultaneously with a potentially curative gene and gamma-GCSh, can be selected by treatment with BSO before in vivo transplantation.

3T3 Cells↗
PubMed2005

Expression of the p75 neurotrophin receptor in acute leukaemia.

The dLNGFR is a cytoplasmically deleted form of the low-affinity nerve growth factor receptor (LNGFR, also known as p75NTR). Recently, we observed a myeloid leukaemia in mice transplanted with dLNGFR-modified bone marrow cells. Retroviral-mediated expression of dLNGFR was suspected to contribute to the murine leukaemia. This led us to investigate the expression of p75NTR in human leukaemia. Expression of p75NTR was observed in nine of 119 (8%) adult patients with acute leukaemia by flow cytometry analysis, particularly in acute lymphoblastic leukaemia (26%). These results support further detailed analyses of neurotrophin receptors and downstream signalling pathways in haematological malignancies.

Adult↗