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Matxalen Llosa - One of the best experts on this subject based on the ideXlab platform.

  • The Conjugative Relaxase TrwC Promotes Integration of Foreign DNA in the Human Genome.
    Applied and Environmental Microbiology, 2017
    Co-Authors: Coral González-prieto, Richard Gabriel, Christoph Dehio, Manfred Schmidt, Matxalen Llosa
    Abstract:

    : Bacterial conjugation is a mechanism of horizontal DNA transfer. The relaxase TrwC of the conjugative plasmid R388 cleaves one strand of the transferred DNA at the oriT gene, covalently attaches to it, and leads the single-stranded DNA (ssDNA) into the recipient cell. In addition, TrwC catalyzes site-specific Integration of the transferred DNA into its target sequence present in the genome of the recipient bacterium. Here, we report the analysis of the efficiency and specificity of the integrase activity of TrwC in human cells, using the type IV secretion system of the human pathogen Bartonella henselae to introduce relaxase-DNA complexes. Compared to Mob relaxase from plasmid pBGR1, we found that TrwC mediated a 10-fold increase in the rate of plasmid DNA transfer to human cells and a 100-fold increase in the rate of chromosomal Integration of the transferred DNA. We used linear amplification-mediated PCR and plasmid rescue to characterize the Integration Pattern in the human genome. DNA sequence analysis revealed mostly reconstituted oriT sequences, indicating that TrwC is active and recircularizes transferred DNA in human cells. One TrwC-mediated site-specific Integration event was detected, proving that TrwC is capable of mediating site-specific Integration in the human genome, albeit with very low efficiency compared to the rate of random Integration. Our results suggest that TrwC may stabilize the plasmid DNA molecules in the nucleus of the human cell, probably by recircularization of the transferred DNA strand. This stabilization would increase the opportunities for Integration of the DNA by the host machinery.IMPORTANCE Different biotechnological applications, including gene therapy strategies, require permanent modification of target cells. Long-term expression is achieved either by extrachromosomal persistence or by Integration of the introduced DNA. Here, we studied the utility of conjugative relaxase TrwC, a bacterial protein with site-specific integrase activity in bacteria, as an integrase in human cells. Although it is not efficient as a site-specific integrase, we found that TrwC is active in human cells and promotes random Integration of the transferred DNA in the human genome, probably acting as a DNA chaperone until it is integrated by host mechanisms. TrwC-DNA complexes can be delivered to human cells through a type IV secretion system involved in pathogenesis. Thus, TrwC could be used in vivo to transfer the DNA of interest into the appropriate cell and promote its Integration. If used in combination with a site-specific nuclease, it could lead to site-specific Integration of the incoming DNA by homologous recombination.

Steven J Husson - One of the best experts on this subject based on the ideXlab platform.

  • the bet family of proteins targets moloney murine leukemia virus Integration near transcription start sites
    Cell Reports, 2013
    Co-Authors: Jan De Rijck, Frederic D Bushman, Christine De Kogel, Jonas Demeulemeester, Sofie Vets, Sara El Ashkar, Nirav Malani, Bart Landuyt, Steven J Husson
    Abstract:

    Summary A hallmark of retroviral replication is Integration of the viral genome into host cell DNA. This characteristic makes retrovirus-based vectors attractive delivery vehicles for gene therapy. However, adverse events in gene therapeutic trials, caused by activation of proto-oncogenes due to murine leukemia virus (MLV)-derived vector Integration, hamper their application. Here, we show that bromodomain and extraterminal (BET) proteins (BRD2, BRD3, and BRD4) and MLV integrase specifically interact and colocalize within the nucleus of the cell. Inhibition of the BET proteins' chromatin interaction via specific bromodomain inhibitors blocks MLV virus replication at the Integration step. MLV Integration site distribution parallels the chromatin binding profile of BET proteins, and expression of an artificial fusion protein of the BET integrase binding domain with the chromatin interaction domain of the lentiviral targeting factor LEDGF/p75 retargets MLV Integration away from transcription start sites and into the body of actively transcribed genes, conforming to the HIV Integration Pattern. Together, these data validate BET proteins as MLV Integration targeting factors.

Axel Schambach - One of the best experts on this subject based on the ideXlab platform.

  • Pre-clinical Development of a Lentiviral Vector Expressing the Anti-sickling βAS3 Globin for Gene Therapy for Sickle Cell Disease
    'Elsevier BV', 2018
    Co-Authors: Valentina Poletti, Axel Schambach, Fabrizia Urbinati, Sabine Charrier, Guillaume Corre, Roger P. Hollis, Beatriz Campo Fernandez, Samia Martin, Michael Rothe, Donald B. Kohn
    Abstract:

    Sickle cell disease (SCD) is caused by a mutation (E6V) in the hemoglobin (Hb) β-chain that induces polymerization of Hb tetramers, red blood cell deformation, ischemia, anemia, and multiple organ damage. Gene therapy is a potential alternative to human leukocyte antigen (HLA)-matched allogeneic hematopoietic stem cell transplantation, available to a minority of patients. We developed a lentiviral vector expressing a β-globin carrying three anti-sickling mutations (T87Q, G16D, and E22A) inhibiting axial and lateral contacts in the HbS polymer, under the control of the β-globin promoter and a reduced version of the β-globin locus-control region. The vector (GLOBE-AS3) transduced 60%–80% of mobilized CD34+ hematopoietic stem-progenitor cells (HSPCs) and drove βAS3-globin expression at potentially therapeutic levels in erythrocytes differentiated from transduced HSPCs from SCD patients. Transduced HSPCs were transplanted in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG)-immunodeficient mice to analyze biodistribution, chimerism, and transduction efficiency in bone marrow (BM), spleen, thymus, and peripheral blood 12–14 weeks after transplantation. Vector Integration site analysis, performed in pre-transplant HSPCs and post-transplant BM cells from individual mice, showed a normal lentiviral Integration Pattern and no evidence of clonal dominance. An in vitro immortalization (IVIM) assay showed the low genotoxic potential of GLOBE-AS3. This study enables a phase I/II clinical trial aimed at correcting the SCD phenotype in juvenile patients by transplantation of autologous hematopoietic stem cells (HSC) transduced by GLOBE-AS3. Keywords: gene therapy, sickle cell disease, lentiviral vector, Integration, genotoxicity, hematopoietic stem-progenitor cells, stem cell clonality, xenograft, NSG mic

  • alpharetroviral vectors from a cancer causing agent to a useful tool for human gene therapy
    Viruses, 2014
    Co-Authors: Julia D Suerth, Verena Labenski, Axel Schambach
    Abstract:

    Gene therapy using integrating retroviral vectors has proven its effectiveness in several clinical trials for the treatment of inherited diseases and cancer. However, vector-mediated adverse events related to insertional mutagenesis were also observed, emphasizing the need for safer therapeutic vectors. Paradoxically, alpharetroviruses, originally discovered as cancer-causing agents, have a more random and potentially safer Integration Pattern compared to gammaretro- and lentiviruses. In this review, we provide a short overview of the history of alpharetroviruses and explain how they can be converted into state-of-the-art gene delivery tools with improved safety features. We discuss development of alpharetroviral vectors in compliance with regulatory requirements for clinical translation, and provide an outlook on possible future gene therapy applications. Taken together, this review is a broad overview of alpharetroviral vectors spanning the bridge from their parental virus discovery to their potential applicability in clinical settings.

  • stem cell marking with promotor deprived self inactivating retroviral vectors does not lead to induced clonal imbalance
    Molecular Therapy, 2009
    Co-Authors: Martijn H Brugman, Christopher Baum, Axel Schambach, Kerstin Cornils, Claudia Lange, Regine Nowak, Michael Lioznov, Boris Fehse
    Abstract:

    Stable genetic modification of stem cells holds great promise for gene therapy and marking, but commonly used γ-retroviral vectors were found to influence growth/survival characteristics of hematopoietic stem cells (HSCs) by insertional mutagenesis. In this article, we show that promoter-deprived γ-retroviral self-inactivating (pd-SIN) vectors allow stable genetic marking of serially reconstituting murine HSC. In contrast to findings with γ-retroviral long terminal repeat (LTR) vectors, serial transplantation of pd-SIN-marked HSC in a sensitive mouse model was apparently not associated with induced clonal imbalance of gene-marked HSC. Furthermore, insertions of pd-SIN into protooncogenes, growth-promoting and signaling genes occurred significantly less frequent than in control experiments with LTR vectors. Also, transcriptional dysregulation of neighboring genes potentially caused by the pd-SIN insertion was rarely seen and comparatively weak. The Integration Pattern of promotor-deprived SIN vectors in reconstituting HSC seems to depend on the transcriptional activity of the respective gene loci reflecting the picture described for LTR vectors. In conclusion, our data strongly support the use of SIN vectors for gene-marking studies and suggest an increased therapeutic index for vectors lacking enhancers active in HSC.

Leandro Peña - One of the best experts on this subject based on the ideXlab platform.

  • accumulation of transgene derived sirnas is not sufficient for rnai mediated protection against citrus tristeza virus in transgenic mexican lime
    Molecular Plant Pathology, 2010
    Co-Authors: Carmelo Lopez, Pedro Moreno, Carmen Fagoaga, Luis Navarro, Magdalena Cervera, Leandro Peña
    Abstract:

    Mexican lime plants transformed with the 3'-terminal 549 nucleotides of the Citrus tristeza virus (CTV) genome in sense, antisense and intron-hairpin formats were analysed for transgene-derived transcript and short interfering RNA (siRNA) accumulation, and for CTV resistance. Propagations from all sense, antisense and empty-vector transgenic lines were susceptible to CTV, except for a single sense-line plant with a complex transgene Integration Pattern that showed transgene-derived siRNAs in association with low levels of the transgene-derived transcript. In contrast, nine of 30 intron-hairpin lines showed CTV resistance, with 9%-56% of bud-propagated plants, depending on the line, remaining uninfected on graft inoculation, and the others being susceptible. Although resistance was always associated with the presence of transgene-derived siRNAs, their level in different sense and intron-hairpin transformants was variable irrespective of the response to CTV infection. In intron-hairpin lines with single transgene Integration, CTV resistance was correlated with low accumulation of the transgene-derived transcript rather than with high accumulation of transgene-derived siRNAs.

  • efficient production of transgenic citrus plants expressing the coat protein gene of citrus tristeza virus
    Plant Cell Reports, 2000
    Co-Authors: Abel Casso Dominguez, Pedro Moreno, Jose Guerri, Mariano Cambra, Luis Navarro, Leandro Peña
    Abstract:

    The coat protein gene of citrus tristeza virus (CTV) has been introduced into Mexican lime (Citrus aurantifolia Swing.) plants by using an improved Agrobacterium-mediated genetic transformation system. Internodal stem segments from greenhouse-grown seedlings were co-cultivated with A. tumefaciens strain EHA 105 carrying the binary plasmid pBI 121/CTV-CP in a medium rich in auxins that provided the explant cells with the proper treatment to shift them to a competent state for transformation. The transformation frequency was enhanced, and this allowed us to recover 42 transgenic plants from 1200 explants. Regenerated shoots were identified as transformants by performing β-glucuronidase (GUS) assays and subsequently by PCR amplifications of the CTV-CP transgene. Southern analyses revealed that at least one copy of the CTV-CP gene was integrated in all PCR positive plants. Interestingly, 70% of them had linked T-DNAs arranged at one locus. Copy number of the CTV-CP gene varied from one to six among the transgenic lines. Half of them showed truncated T-DNAs in which the left border was lost. Expression of the CTV-CP transgene was demonstrated in 38 out of 42 plants by western analysis and DASI-ELISA. No correlation was found between coat protein expression and transgene copy number or Integration Pattern.

Paolo Colangelo - One of the best experts on this subject based on the ideXlab platform.

  • are developmental shifts the main driver of phenotypic evolution in diplodus spp perciformes sparidae
    BMC Evolutionary Biology, 2019
    Co-Authors: Daniele Ventura, Paolo Colangelo, Paolo Piras, Jacopo Pagani Guazzugli Bonaiuti, Giandomenico Ardizzone
    Abstract:

    Sparid fishes of the genus Diplodus show a complex life history. Juveniles have adaptations well suited to life in the water column. When fishes recruit into the adult population, individuals develop a radically differentiated shape that reflects their adaptation to the new benthic environment typical of the adult. A comparative analysis of ontogenetic trajectories was performed to assess the presence of divergence in the developmental Pattern. By using a geometric morphometric approach, we investigated the Pattern of shape variation across ontogenetic stages that span from early settlement to the adult stage in four species of the genus Diplodus. Landmarks were collected on the whole body of fishes to quantify the phenotypic variation along two well defined life stages, i.e. juvenile and adult. A comparative analysis of ontogenetic trajectories was performed to assess the presence of divergence in the developmental Pattern. Subsequently, we investigated the Patterns of Integration and modularity as proxy for the alteration of the developmental processes. This have allowed to give an insight in morphological developmental Patterns across ecologically and ontogenetically differentiated life stages and to investigate the process leading to the adult shape. Our results suggest that the origin of morphological novelties in Diplodus spp. arise from shifts of the ontogenetic trajectories during development. During the settlement phase, the juveniles’ morphological shapes converge towards similar regions of the morphospace. When the four species approach the transition between settlement and recruitment, we observe the lowest level of inter- and intra-specific disparity. After this transition we detect an abrupt shift of ontogenetic trajectories, i.e. the path taken by species during development, that led to highly divergent adult phenotypes. We suggest that the evolution of new ecomorphologies, better suited to exploit different niches (pelagic vs. benthonic) and reduce inter-specific competition in Diplodus spp., are related to the shift in the ontogenetic trajectory that in turn is associated to changes in modularity and Integration Pattern.

  • Are developmental shifts the main driver of phenotypic evolution in Diplodus spp. (Perciformes: Sparidae)?
    BMC Evolutionary Biology, 2019
    Co-Authors: Paolo Colangelo, Daniele Ventura, Paolo Piras, Jacopo Pagani Guazzugli Bonaiuti, Giandomenico Ardizzone
    Abstract:

    Background Sparid fishes of the genus Diplodus show a complex life history. Juveniles have adaptations well suited to life in the water column. When fishes recruit into the adult population, individuals develop a radically differentiated shape that reflects their adaptation to the new benthic environment typical of the adult. A comparative analysis of ontogenetic trajectories was performed to assess the presence of divergence in the developmental Pattern . By using a geometric morphometric approach, we investigated the Pattern of shape variation across ontogenetic stages that span from early settlement to the adult stage in four species of the genus Diplodus . Landmarks were collected on the whole body of fishes to quantify the phenotypic variation along two well defined life stages, i.e. juvenile and adult. A comparative analysis of ontogenetic trajectories was performed to assess the presence of divergence in the developmental Pattern. Subsequently, we investigated the Patterns of Integration and modularity as proxy for the alteration of the developmental processes. This have allowed to give an insight in morphological developmental Patterns across ecologically and ontogenetically differentiated life stages and to investigate the process leading to the adult shape. Result Our results suggest that the origin of morphological novelties in Diplodus spp. arise from shifts of the ontogenetic trajectories during development. During the settlement phase, the juveniles’ morphological shapes converge towards similar regions of the morphospace. When the four species approach the transition between settlement and recruitment, we observe the lowest level of inter- and intra-specific disparity. After this transition we detect an abrupt shift of ontogenetic trajectories, i.e. the path taken by species during development, that led to highly divergent adult phenotypes. Discussion We suggest that the evolution of new ecomorphologies, better suited to exploit different niches (pelagic vs. benthonic) and reduce inter-specific competition in Diplodus spp., are related to the shift in the ontogenetic trajectory that in turn is associated to changes in modularity and Integration Pattern.