Wolfram Ostertag

Oncoretrovirus and Lentivirus Vectors Pseudotyped with Lymphocytic Choriomeningitis Virus Glycoprotein: Generation, Concentration, and Broad Host Range

ABSTRACT Lymphocytic choriomeningitis virus (LCMV) is a noncytopathic arenavirus shown to infect a broad range of different cell types. Here, we combined the beneficial characteristics of the LCMV glycoprotein (LCMV-GP) and those of retroviral vectors to generate a new, safe, and efficient gene transfer system. These LCMV-GP pseudotypes were systematically compared with vectors containing the widely used amphotropic murine leukemia virus envelope (A-MLVenv) or the vesicular stomatitis virus G protein (VSV-G). Production of LCMV-GP-pseudotyped oncoretroviral and lentiviral vectors by transient transfection resulted in vector titers similar to those with A-MLVenv or VSV-G. In contrast to A-MLVenv particles, LCMV-GP pseudotypes could be efficiently concentrated by ultracentrifugation without loss of vector titer. Unlike the cell-toxic VSV-G, a stable retroviral packaging cell line constitutively expressing LCMV-GP could be established. Vectors pseudotyped with LCMV-GP efficiently transduced many cell lines from different species and tissues relevant for gene therapy. Transduction of human glioma cells was studied in detail. These cells are a major target for cancer gene therapy and were transduced more efficiently with LCMV-GP-pseudotyped vectors than with the generally used A-MLVenv particles. The high stability, low toxicity, and broad host range make LCMV-GP-pseudotyped vectors attractive for gene transfer applications. The recombinant LCMV-GP-pseudotyped vectors will also allow functional characterization of naturally occurring and recombinant LCMV-GP variants.

HOXB4's road map to stem cell expansion

Homeodomain-containing transcription factors are important regulators of stem cell behavior. HOXB4 mediates expansion of adult and embryo-derived hematopoietic stem cells (HSCs) when expressed ectopically. To define the underlying molecular mechanisms, we performed gene expression profiling in combination with subsequent functional analysis with enriched adult HSCs and embryonic derivatives expressing inducible HOXB4. Thereby, we identified a set of overlapping genes that likely represent “universal” targets of HOXB4. A substantial number of loci are involved in signaling pathways important for controlling self-renewal, maintenance, and differentiation of stem cells. Functional assays performed on selected pathways confirmed the biological coherence of the array results. HOXB4 activity protected adult HSCs from the detrimental effects mediated by the proinflammatory cytokine TNF-α. This protection likely contributes to the competitive repopulation advantage of HOXB4-expressing HSCs observed in vivo. The concept of TNF-α inhibition may also prove beneficial for patients undergoing bone marrow transplantation. Furthermore, we demonstrate that HOXB4 activity and FGF signaling are intertwined. HOXB4-mediated expansion of adult and ES cell-derived HSCs was enhanced by specific and complete inhibition of FGF receptors. In contrast, the expanding activity of HOXB4 on hematopoietic progenitors in day 4–6 embryoid bodies was blunted in the presence of basic FGF (FGF2), indicating a dominant negative effect of FGF signaling on the earliest hematopoietic cells. In summary, our results strongly suggest that HOXB4 modulates the response of HSCs to multiple extrinsic signals in a concerted manner, thereby shifting the balance toward stem cell self-renewal.

Retroviral vector-mediated expression of HoxB4 in hematopoietic cells using a novel coexpression strategy.

Retroviral vector-mediated expression of the homeoboxgene, HoxB4, in hematopoietic cells has been reported to mediate a benign expansion of gene-modified hematopoietic stem and precursor cells in vivo. In the present study, we used a novel coexpression strategy for coordinated expression of HoxB4 along with a cytoplasmic protein from a retroviral vector. The novel coexpression strategy, based on cotranslational protein separation mediated by the 2A sequence of foot-and-mouth disease virus (FMDV), allows an indirect quantification of HoxB4 expression levels when inserting a reporter such as the enhanced green fluorescent protein (GFP) in the retroviral vector. Presence of the 2A sequence did not interfere with the correct subcellular localization of HoxB4 (nuclear) and GFP (cytoplasmic), nor with the titer of bicistronic vectors, and mediated functional long-term coexpression (at least 1 year) of GFP and HoxB4 after transplantation of transduced mouse bone marrow cells in mice.

EXPRESSION OF THE GM-CSF GENE AFTER RETROVIRAL TRANSFER IN HEMATOPOIETIC STEM CELL LINES INDUCES SYNCHRONOUS GRANULOCYTE-MACROPHAGE DIFFERENTIATION

Multipotent murine stem cell lines (FDC-Pmix) depend on IL-3 for self-renewal and proliferation and can be induced to differentiate into multiple hematopoietic lineages. Single FDC-Pmix cells infected with retroviral vectors expressing GM-CSF are induced to differentiate into granulocytes and macrophages. This results in a complete loss of clonogenic cells if IL-3 is not exogenously supplied; however, multipotent variants can be selected that do not terminally differentiate if cells are kept in the presence of IL-3. Unidirectional and synchronous granulocyte and macrophage differentiation accompanied with loss of self-renewal capacity is induced when IL-3 is removed. Our data indicate that activation of the GM-CSF receptor induces differentiation of stem cells by an instructive mechanism that can be blocked by the activated IL-3 receptor. A model of how receptors can induce proliferation and cell-specific differentiation by two separate pathways is discussed.

FMEV vectors: both retroviral long terminal repeat and leader are important for high expression in transduced hematopoietic cells

FMEV retroviral vectors combine the long terminal repeat of Friend mink cell focus-forming viruses with the 5′ untranslated leader region of the murine embryonic stem cells virus. These modules were connected to achieve high transgene expression in hematopoietic progenitor and stem cells. Here, we report the cloning of safety-improved and versatile FMEV vectors allowing module-wise exchange of crucial elements for comparative studies. By transfer and expression of four different marker genes (neomycin phosphotransferase, lacZ, enhanced green fluorescent protein and truncated low affinity nerve growth factor receptor), we formally demonstrate that both the long terminal repeat and the leader contribute to the high expression of FMEV in transduced hematopoietic cells. Most prominent are the data recorded in the absence of selection in myelo-erythroid progenitor cells. Here, FMEV vectors mediate up to two orders of magnitude increased transgene expression levels when compared with vectors based on the Moloney murine leukemia virus.

The fusion of TEL and ABL in human acute lymphoblastic leukaemia is a rare event

We have recently identified a common ALL patient which harboured a chromosomal fusion between the TEL gene on chromosome 12 and the ABL gene on chromosome 9. We designed an RT‐PCR assay to screen 186 adult ALL and 30 childhood ALL patients for this novel translocation. We were unable to identify any additional cases with a TEL/ABL fusion product.

Recombinant Expression of Lymphocytic Choriomeningitis Virus Strain WE Glycoproteins: a Single Amino Acid Makes the Difference

ABSTRACT Cytoplasmic vector systems are generally used for expression of lymphocytic choriomeningitis virus (LCMV) proteins. However, we achieved high levels of cell surface glycoproteins using a standard nuclear expression plasmid. Expression was independent of other LCMV proteins but was blocked by a missense mutation within the original LCMV(WE) glycoprotein cDNA.

Improved post-transcriptional processing of an MDR1 retrovirus elevates expression of multidrug resistance in primary human hematopoietic cells

we describe the functional analysis of a novel retroviral vector, sf91m3, which was designed for improved expression of the in vivo selectable marker, multidrug resistance 1 gene (mdr1), in hematopoietic cells. sf91m3 combines several promising features. the vector backbone lacks viral coding sequences and aug-start codons 5′ of the mdr1 cdna. a point mutation of a cryptic splice acceptor of the mdr1 cdna increases the probability of transferring an intact provirus. the titer of a pg13 packaging cell clone containing a single proviral integration is high (>2 × 106 particles/ml from frozen stocks of serum-free vector harvests). human hematopoietic cells transduced with sf91m3 reliably express mdr1 before and after passage through nod/scid mice, as shown by quantitative pcr and efflux assays with rhodamine 123 or hoechst 33342. finally, sf91m3 mediates resistance to escalated doses of cytotoxic agents, as shown by survival and differentiation of transduced colony-forming cells in the presence of colchicine at 48 ng/ml (>10 × IC50). Thus, SF91m3 may represent an interesting candidate for future trials addressing the safety and utility of MDR1 gene transfer; moreover, this study demonstrates that sequence alterations improving post-transcriptional processing of retroviral vectors have a substantial impact for gene expression in hematopoietic cells.

Transforming genes and target cells of murine spleen focus-forming viruses.

Publisher Summary This chapter outlines the factors that determine leukemogenesis induced by acutely transforming retroviruses by the interaction with either myeloid stem or progenitor cells of the mammalian system. The acutely transforming retroviruses described will be limited to the spleen focus-forming viruses, defined as those retroviruses that induce a proliferative hematopoietic disease upon the intravenous injection of adult animals. The chapter discusses the molecular features of the three known groups of acutely transforming murine viruses: (1) viruses with recombinant env genes, (2) viruses with the mos oncogene, and (3) viruses with the ras oncogene. Data pertaining to the relevant proto-oncogenes and their products are also presented. It outlines the critical factors of the viral genome or transforming gene, based on structure-function analysis, which determine the oncogenicity of the retroviruses and its target cell specificity. It also discusses retrovirus-target cell interaction and outlines the experiments that are still necessary to fully understand the oncogenesis induced by spleen focus-forming viruses in the myeloid system.

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 (ΔLNGFR) 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 ΔLNGFR 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 β-actin gene. As determined by two-color flow cytometry, the best correlation of the expression of both cDNAs was obtained using the vector SF1mSδ 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 SF1mSδ acquired dominant multidrug resistance. Transduced primary CD34+ cells could be enriched in vitro based on expression of ΔLNGFR, 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.