Didier Nègre

Efficient and stable transduction of resting B-lymphocytes and primary chronic lymphocyte leukemia cells using measles virus gp displaying lentiviral vectors

Up to now, no lentiviral vector (LV) tool existed to govern efficient and stable gene delivery into quiescent B lymphocytes, which hampers its application in gene therapy and immunotherapy areas. Here, we report that LVs incorporating measles virus (MV) glycoproteins, H and F, on their surface allowed transduction of 50% of quiescent B cells, which are not permissive to VSVG-LV transduction. This high transduction level correlated with B-cell SLAM expression and was not at cost of cell-cycle entry or B-cell activation. Moreover, the naive and memory phenotypes of transduced resting B cells were maintained. Importantly, H/F-LVs represent the first tool permitting stable transduction of leukemic cancer cells, B-cell chronic lymphocytic leukemia cells, blocked in G(0)/G(1) early phase of the cell cycle. Thus, H/F-LV transduction overcomes the limitations of current LVs by making B cell-based gene therapy and immunotherapy applications feasible. These new LVs will facilitate antibody production and the study of gene functions in these healthy and cancer immune cells.

Overproduction and characterization of the iclR gene product of Escherichia coli K-12 and comparison with that of Salmonella typhimurium LT2

The iclR gene of Escherichia coli K-12, which encodes a regulatory protein (repressor) for the aceBAK operon, is located between that operon and metH in the 91-min region of the chromosome. The iclR gene was cloned and expressed in a coupled T7 RNA polymerase/promoter system and the gene product was identified by specific binding to a fragment containing the aceBAK operator region. The iclR gene product is a polypeptide of 274 amino acids (aa) with a calculated Mr of 29,741. Comparison of the deduced IclR aa sequence to that of Salmonella typhimurium revealed that the two IclR repressors exhibit 89% identity. A possible helix-turn-helix motif characteristic of DNA-binding proteins was found within the IclR sequence. A search in protein data banks revealed that IclR has a score of similarity of 43.7% with GylR, a transcriptional regulator of the glycerol operon of Streptomyces coelicolor.

Specific interactions between the IclR repressor of the acetate operon of Escherichia coli and its operator.

The positions of interference points between the IclR repressor of the acetate operon of Escherichia coli and its specific operator were examined. The number and nature of nucleotides essential to repressor binding were determined by scanning populations of DNA previously methylated at guanine residues by dimethyl sulfate, or depurinated by treatment with formic acid, or depyrimidated by treatment with hydrazine. A total of 46 nucleotides, distributed almost equally between the two strands of the operator region, were found to be functionally important, although to a varying extent. These are clustered in two successive domains which expand from nucleotide -54 to nucleotide -27 and can organize in a palindrome-like structure containing a large proportion of A and T residues.

Primary structure of the intergenic region between aceK and icIR in the Escherichia coli chromosome

Abstract The complete nucleotide sequence (2386 bp) of the intergenic region between aceK and icIR within the acetate operon of Escherichia coli has been determined. This region contains an open reading frame of 1836 bp whose expression has been detected by protein fusion experiments.

The RNA-binding protein Mex3b regulates the spatial organization of the Rap1 pathway

The four related mammalian MEX-3 RNA-binding proteins are evolutionarily conserved molecules for which the in vivo functions have not yet been fully characterized. Here, we report that male mice deficient for the gene encoding Mex3b are subfertile. Seminiferous tubules of Mex3b-deficient mice are obstructed as a consequence of the disrupted phagocytic capacity of somatic Sertoli cells. In addition, both the formation and the integrity of the blood-testis barrier are compromised owing to mislocalization of N-cadherin and connexin 43 at the surface of Sertoli cells. We further establish that Mex3b acts to regulate the cortical level of activated Rap1, a small G protein controlling phagocytosis and cell-cell interaction, through the activation and transport of Rap1GAP. The active form of Rap1 (Rap1-GTP) is abnormally increased at the membrane cortex and chemically restoring Rap1-GTP to physiological levels rescues the phagocytic and adhesion abilities of Sertoli cells. Overall, these findings implicate Mex3b in the spatial organization of the Rap1 pathway that orchestrates Sertoli cell functions.

Promoter vectors with restriction-site banks

New vectors harboring the promoter for the chloramphenicol acetyl transferase gene (cat promoter) have been constructed. These vectors are all derived from pJRD184 [Heusterspreute et al., Gene 39 (1985) 299-304], which contains a restriction-site bank. The cat promoter has been inserted at various positions and in reverse orientations so that almost all the restriction sites originally present on JRD184 can be used in cloning experiments. The expression of the aceK gene of Escherichia coli cloned under the control of the cat promoter has been tested. A large increase in the synthesis of the isocitrate dehydrogenase kinase, the aceK gene product, has demonstrated the efficiency of the newly constructed vectors.

High fidelity of guanine translation in a plasmid-directed in vitro system

The extent of misreading of individual bases in the first or second codon position has been measured in vitro in a simplified plasmid-directed coupled system in which natural messenger translation is restricted to the formation of the N-terminal di- or tripeptide. Experiments were performed under conditions of competition between cognate and noncognate tRNAs in the presence of streptomycin to maximize the frequency of reading errors. A striking lack of susceptibility to mistranslation of guanine, as compared to the other 3 bases, was observed.

Murine granulocyte-macrophage colony-stimulating factor expressed from a bicistronic simian immunodeficiency virus-based integrase-defective lentiviral vector does not enhance T-cell responses in mice.

As a prelude to immunization studies in nonhuman primates, we compared in mice the immunogenicity of a simian immunodeficiency virus (SIV)-based integrase (IN)-defective lentiviral vector (IDLV) encoding the model antigen-enhanced green fluorescence protein (eGFP) in the presence or absence of the murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) expressed from an internal ribosomal entry site (IRES) sequence. BALB/c mice were immunized once intramuscularly with IDLV expressing eGFP alone or eGFP and mGM-CSF and immune responses were evaluated up to 90 days from the single intramuscular immunization. Results indicated that the mGM-CSF was unable to improve the magnitude and quality of the immune response against the eGFP transgene in the context of the SIV-based IDLV, as evaluated by enzyme-linked immunosorbent spot (ELISPOT) assays for interferon-γ (IFN-γ) and by intracellular cytokine staining for IFN-γ, interleukin-2 (IL-2), and tumor necrosis factor-alpha (TNF-α). These findings suggest that for vaccination purposes, the presence of mGM-CSF expressed after the IRES in a SIV-based IDLV system does not favor the improvement of the immunological response against the transgene of interest. Further studies should investigate whether the selection of a different cytokine gene might improve the immune response against the transgene.

290. Baboon Envelope Pseudo Typed Lentiviral Vectors Mediate High-Level Gene Transfer in Human B Cells Allowing Secretion of FIX at Therapeutic Levels in NSG Mouse

B lymphocytes are attractive targets for gene therapy of genetic diseases associated with B-cell dysfunction. In addition, long-lasting transgene expression in B cells is of particular interest for immunotherapy by its potential to induce specific immune activation or tolerance. Moreover, B cells are potent specialized protein secreting cells. One obstacle though is that primary B cells are poorly transduced by VSVG pseudotyped lentiviral vectors (LVs) even when stimulated through the BCR to induce proliferation. We demonstrated that this is due to the poor expression of the low density lipid (LDL) receptor, identified as the VSV receptor (Amirache et al, 2014).Since we recently showed that baboon retroviral envelope pseudotyped LVs (BaEV) outperform VSV-G-LVs for gene transfer into cytokine-stimulated and resting hematopoietic stem cells, we evaluated them here for gene transfer into primary human B cells. Upon B cell receptor stimulation, BaEV-LVs transduced up to 70% of the B cells. As expected, VSV-G-LVs were unable to transduce activated nor unstimulated resting human B cells efficiently, even at high vector doses (MOI= 100, VSV-G-LV < 5% transduction). Remarkably, BaEV-LVs permitted highly efficient transduction of 30% of resting B cells. Moreover, they transduced as well memory as naive B cells without inducing phenotypic changes. In addition, BaEV-LVs permitted up to 80% transduction of human plasmocytes. Adaptive transfer of mature BaEV-LV transduced human B cells into NSG mice allowed differentiation into plasmablasts and plasma B cells, both characterized by a high level gene marking in the lymphnodes, spleen and bone marrow. These results encouraged us to evaluate BaEV-LV mediated gene transfer of Factor IX into human B cells for treatment of heamophelia, a severe blood disorder. We produced high-titer BaEV-LVs carrying a codon optimized FIX with a hyperactivating FIX-R338L mutation. These BaEV-FIX-LVs efficiently transduced plasmocytic B cell lines leading to high-level FIX secretion (20-70% of normal levels in human serum). Transduction of primary human B cells followed by adaptive transfer into NSG mice resulted in therapeutic levels of FIX in the serum (7-25% of normal FIX levels) at 3 to 4 weeks of engraftment. Moreover, a faster coagulation of the blood proved that FIX was functional.Concluding, the BaEV-LVs might represent in the future valuable tools for therapeutic protein secretion from autologous B cells for treatment of heamophelia and other disorders in vivo.

Inaccurate protein synthesis in a mutant of Salmonella typhimurium defective in transfer RNA pseudouridylation.

Protein synthesis was studied comparatively in a wild‐type strain of Salmonella typhimurium and in hisT mutant cells defective in the pseudouridylation of transfer RNA. From a quantitative point of view, no significant difference between the two types of strain was observed when measuring the rate of protein synthesis during either exponential growth or starvation for histidine. In contrast, the qualitative analysis of proteins by two‐dimensional gel electrophoresis showed that histidine‐starved hisT cells mistranslate the genetic program at a higher frequency than exponentially growing hisT cells or either starved or unstarved hisT + cells.