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  1. Article ; Online: Recent advances in lentiviral vector development and applications.

    Mátrai, Janka / Chuah, Marinee K L / VandenDriessche, Thierry

    Molecular therapy : the journal of the American Society of Gene Therapy

    2010  Volume 18, Issue 3, Page(s) 477–490

    Abstract: Lentiviral vectors (LVs) have emerged as potent and versatile vectors for ex vivo or in vivo gene transfer into dividing and nondividing cells. Robust phenotypic correction of diseases in mouse models has been achieved paving the way toward the first ... ...

    Abstract Lentiviral vectors (LVs) have emerged as potent and versatile vectors for ex vivo or in vivo gene transfer into dividing and nondividing cells. Robust phenotypic correction of diseases in mouse models has been achieved paving the way toward the first clinical trials. LVs can deliver genes ex vivo into bona fide stem cells, particularly hematopoietic stem cells, allowing for stable transgene expression upon hematopoietic reconstitution. They are also useful to generate induced pluripotent stem cells. LVs can be pseudotyped with distinct viral envelopes that influence vector tropism and transduction efficiency. Targetable LVs can be generated by incorporating specific ligands or antibodies into the vector envelope. Immune responses toward the transgene products and transduced cells can be repressed using microRNA-regulated vectors. Though there are safety concerns regarding insertional mutagenesis, their integration profile seems more favorable than that of gamma-retroviral vectors (gamma-RVs). Moreover, it is possible to minimize this risk by modifying the vector design or by employing integration-deficient LVs. In conjunction with zinc-finger nuclease technology, LVs allow for site-specific gene correction or addition in predefined chromosomal loci. These recent advances underscore the improved safety and efficacy of LVs with important implications for clinical trials.
    MeSH term(s) Animals ; Gene Transfer Techniques ; Genetic Therapy/methods ; Genetic Vectors ; Hematopoietic Stem Cells/cytology ; Humans ; Lentivirus/genetics ; Ligands ; Models, Genetic ; Mutagenesis ; Phenotype ; Pluripotent Stem Cells/cytology ; Retroviridae/genetics ; Risk ; Transgenes
    Chemical Substances Ligands
    Language English
    Publishing date 2010-01-19
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Review
    ZDB-ID 2010592-7
    ISSN 1525-0024 ; 1525-0016
    ISSN (online) 1525-0024
    ISSN 1525-0016
    DOI 10.1038/mt.2009.319
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  2. Article ; Online: Preclinical and clinical progress in hemophilia gene therapy.

    Mátrai, Janka / Chuah, Marinee K L / VandenDriessche, Thierry

    Current opinion in hematology

    2010  Volume 17, Issue 5, Page(s) 387–392

    Abstract: Purpose of review: Hemophilia A and B are attractive target diseases for gene therapy, as stable expression of coagulation factor VIII and IX may correct the bleeding diathesis. This review focuses on the recent progress in preclinical and clinical ... ...

    Abstract Purpose of review: Hemophilia A and B are attractive target diseases for gene therapy, as stable expression of coagulation factor VIII and IX may correct the bleeding diathesis. This review focuses on the recent progress in preclinical and clinical studies in gene therapy for hemophilia A and B.
    Recent findings: Hepatic gene delivery using vectors derived from adeno-associated virus (AAV) resulted in therapeutic but transient functional clotting factor IX (FIX) expression levels in severe hemophilia B patients. Although T-cell-mediated immune responses eliminated the transduced hepatocytes, transient immunosuppression may potentially overcome this limitation. Alternatively, vectors are being developed that result in higher FIX expression levels at lower vector doses. Lentiviral vectors are being explored for in-vivo hepatic gene delivery and for ex-vivo transduction of hematopoietic stem cells. This resulted in stable correction of the bleeding diathesis in hemophilic mice. Finally, nonviral vectors derived from transposons result in sustained clotting-factor expression in rodent models. Translational studies in large animal models are required to move these new approaches forward into the clinic.
    Summary: New insights from clinical trials and advances in preclinical studies may ultimately pave the way toward a cure in patients suffering from hemophilia.
    MeSH term(s) Animals ; Disease Models, Animal ; Genetic Therapy/methods ; Genetic Vectors ; Hemophilia A/therapy ; Hemophilia B/therapy ; Hepatocytes ; Humans ; Mice ; T-Lymphocytes, Regulatory
    Language English
    Publishing date 2010-09
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Review
    ZDB-ID 1153887-9
    ISSN 1531-7048 ; 1065-6251
    ISSN (online) 1531-7048
    ISSN 1065-6251
    DOI 10.1097/MOH.0b013e32833cd4bd
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  3. Article ; Online: PiggyBac toolbox.

    Di Matteo, Mario / Mátrai, Janka / Belay, Eyayu / Firdissa, Tewodros / Vandendriessche, Thierry / Chuah, Marinee K L

    Methods in molecular biology (Clifton, N.J.)

    2012  Volume 859, Page(s) 241–254

    Abstract: The PiggyBac (PB) transposon system was originally derived from the cabbage looper moth Trichoplusia ni and represents one of the most promising transposon systems to date. Engineering of the PB transposase enzyme (PBase) and its cognate transposon DNA ... ...

    Abstract The PiggyBac (PB) transposon system was originally derived from the cabbage looper moth Trichoplusia ni and represents one of the most promising transposon systems to date. Engineering of the PB transposase enzyme (PBase) and its cognate transposon DNA elements resulted in a substantial increase in transposition activities. Consequently, this has greatly enhanced the versatility of the PB toolbox. It is now widely used for stable gene delivery into a broad variety of cell types from different species, including mammalian cells. This opened up new perspectives for potential therapeutic applications in the fields of gene therapy and regenerative medicine. In particular, we have recently demonstrated that PB transposons could be used to stably deliver genes into human CD34(+) hematopoietic stem cells (HSCs) resulting in sustained transgene expression in its differentiated progeny. The PB transposon system is particularly attractive for the generation of induced pluripotent stem cells (iPS). Typically, this can be accomplished by stable gene transfer of genes encoding one or more reprogramming factors (i.e., c-MYC, KLF-4, OCT-4, and/or SOX-2). We have generated a PB-based nonviral reprogramming toolbox that contains different combinations of these reprogramming genes. The main advantage of using this PB toolbox for iPS generation is that the reprogramming cassette can be excised by de novo transposase expression, without leaving any molecular trace in the target cell genome. This "traceless excision" paradigm obviates potential risks associated with inadvertent re-expression of reprogramming factors in the iPS progeny. These various applications in gene therapy, stem cell engineering, and regenerative medicine underscore the emerging versatility of the PB toolbox.
    MeSH term(s) Animals ; Cell Culture Techniques ; DNA Transposable Elements ; Genetic Therapy/methods ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells/cytology ; Humans ; Induced Pluripotent Stem Cells/cytology ; Induced Pluripotent Stem Cells/transplantation ; Mutagenesis, Insertional ; Transfection ; Transgenes
    Chemical Substances DNA Transposable Elements
    Language English
    Publishing date 2012
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Review
    ISSN 1940-6029
    ISSN (online) 1940-6029
    DOI 10.1007/978-1-61779-603-6_14
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Understanding the role of defective invertases in plants: tobacco Nin88 fails to degrade sucrose.

    Le Roy, Katrien / Vergauwen, Rudy / Struyf, Tom / Yuan, Shuguang / Lammens, Willem / Mátrai, Janka / De Maeyer, Marc / Van den Ende, Wim

    Plant physiology

    2013  Volume 161, Issue 4, Page(s) 1670–1681

    Abstract: Cell wall invertases (cwINVs), with a high affinity for the cell wall, are fundamental enzymes in the control of plant growth, development, and carbon partitioning. Most interestingly, defective cwINVs have been described in several plant species. Their ... ...

    Abstract Cell wall invertases (cwINVs), with a high affinity for the cell wall, are fundamental enzymes in the control of plant growth, development, and carbon partitioning. Most interestingly, defective cwINVs have been described in several plant species. Their highly attenuated sucrose (Suc)-hydrolyzing capacity is due to the absence of aspartate-239 (Asp-239) and tryptophan-47 (Trp-47) homologs, crucial players for stable binding in the active site and subsequent hydrolysis. However, so far, the precise roles of such defective cwINVs remain unclear. In this paper, we report on the functional characterization of tobacco (Nicotiana tabacum) Nin88, a presumed fully active cwINV playing a crucial role during pollen development. It is demonstrated here that Nin88, lacking both Asp-239 and Trp-47 homologs, has no invertase activity. This was further supported by modeling studies and site-directed mutagenesis experiments, introducing both Asp-239 and Trp-47 homologs, leading to an enzyme with a distinct Suc-hydrolyzing capacity. In vitro experiments suggest that the addition of Nin88 counteracts the unproductive and rather aspecific binding of tobacco cwINV1 to the wall, leading to higher activities in the presence of Suc and a more efficient interaction with its cell wall inhibitor. A working model is presented based on these findings, allowing speculation on the putative role of Nin88 in muro. The results presented in this work are an important first step toward unraveling the specific roles of plant defective cwINVs.
    MeSH term(s) Amino Acid Sequence ; Biocatalysis ; Cloning, Molecular ; DNA, Complementary/genetics ; Models, Molecular ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Mutant Proteins/chemistry ; Mutant Proteins/metabolism ; Pichia/metabolism ; Plant Proteins/chemistry ; Plant Proteins/metabolism ; Recombinant Proteins/metabolism ; Sequence Alignment ; Substrate Specificity ; Sucrose/metabolism ; Nicotiana/enzymology ; beta-Fructofuranosidase/chemistry ; beta-Fructofuranosidase/metabolism
    Chemical Substances DNA, Complementary ; Mutant Proteins ; Plant Proteins ; Recombinant Proteins ; Sucrose (57-50-1) ; beta-Fructofuranosidase (EC 3.2.1.26)
    Language English
    Publishing date 2013-02-27
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 208914-2
    ISSN 1532-2548 ; 0032-0889
    ISSN (online) 1532-2548
    ISSN 0032-0889
    DOI 10.1104/pp.112.209460
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  5. Article: Simulation of the activation of alpha-chymotrypsin: analysis of the pathway and role of the propeptide.

    Mátrai, Janka / Verheyden, Gert / Krüger, Peter / Engelborghs, Yves

    Protein science : a publication of the Protein Society

    2004  Volume 13, Issue 12, Page(s) 3139–3150

    Abstract: Alpha-chymotrypsin undergoes a reversible conformational change from an inactive chymotrypsinogen-like structure at high pH to an active conformation at neutral pH. In order to gain insight into this process on a structural level, we applied molecular ... ...

    Abstract Alpha-chymotrypsin undergoes a reversible conformational change from an inactive chymotrypsinogen-like structure at high pH to an active conformation at neutral pH. In order to gain insight into this process on a structural level, we applied molecular dynamics and targeted molecular dynamics simulations in aqueous environment on the activation and inactivation processes of three different types of chymotrypsin. These are the wild-type bovine chymotrypsin containing the propeptide and the bovine and rat chymotrypsin lacking the propeptide. From these simulations, the importance of the propeptide and of the sequence differences between the rat and bovine variants from the viewpoint of activation could be evaluated and compared with previous fluorescence stopped flow results. The obtained results show the unambiguous influence of the propeptide on the explored conformational space, whereas the sequence differences between bovine and rat chymotrypsin play a minor role. The main features of activation are present in both the wild type and the variant lacking the propeptide, despite the fact that different parts of the conformational space were explored. The comparison of all trajectories shows that particular amino acid residues, such as 17, 18, 19, 187, 217, 218, and 223, undergo large dihedral transitions during the activation process, suggesting a role as hinge residues during the conformational change.
    MeSH term(s) Amino Acid Sequence ; Animals ; Cattle ; Chymotrypsin/metabolism ; Computer Simulation ; Enzyme Activation ; Enzyme Precursors/physiology ; Hydrogen-Ion Concentration ; Models, Molecular ; Molecular Sequence Data ; Protein Conformation ; Rats ; Sequence Alignment ; Signal Transduction ; Time Factors
    Chemical Substances Enzyme Precursors ; Chymotrypsin (EC 3.4.21.1) ; alpha-chymotrypsin (EC 3.4.21.1)
    Language English
    Publishing date 2004-12
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 1106283-6
    ISSN 1469-896X ; 0961-8368
    ISSN (online) 1469-896X
    ISSN 0961-8368
    DOI 10.1110/ps.04825004
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  6. Article: Understanding the Role of Defective Invertases in Plants: Tobacco Nin88 Fails to Degrade Sucrose

    Le Roy, Katrien / Janka Mátrai / Marc De Maeyer / Rudy Vergauwen / Shuguang Yuan / Tom Struyf / Willem Lammens / Wim Van den Ende

    Plant physiology. 2013 Apr., v. 161, no. 4

    2013  

    Abstract: An inactive invertase may indirectly stimulate the activity of active cell wall invertases. ...

    Abstract An inactive invertase may indirectly stimulate the activity of active cell wall invertases.
    Keywords beta-fructofuranosidase ; cell walls ; sucrose ; tobacco
    Language English
    Dates of publication 2013-04
    Size p. 1670-1681.
    Publishing place American Society of Plant Biologists
    Document type Article
    ZDB-ID 208914-2
    ISSN 1532-2548 ; 0032-0889
    ISSN (online) 1532-2548
    ISSN 0032-0889
    DOI 10.1104/pp.112.209460
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  7. Article: A fluorescence stopped-flow kinetic study of the conformational activation of alpha-chymotrypsin and several mutants.

    Verheyden, Gert / Matrai, Janka / Volckaert, Guido / Engelborghs, Yves

    Protein science : a publication of the Protein Society

    2004  Volume 13, Issue 9, Page(s) 2533–2540

    Abstract: The kinetic activation parameters (activation free energy, activation free enthalpy, and activation free entropy change) of the conformational change of alpha-chymotrypsin from an inactive to the active conformation were determined after a pH jump from ... ...

    Abstract The kinetic activation parameters (activation free energy, activation free enthalpy, and activation free entropy change) of the conformational change of alpha-chymotrypsin from an inactive to the active conformation were determined after a pH jump from pH 11.0 to pH 6.8 by the fluorescence stopped-flow method. The conformational change was followed by measuring changes in the protein fluorescence. For the bovine wild-type protein, the same kinetic parameters are obtained as in the study of proflavin binding. Several mutants were made with the goal to accelerate or decelerate this conformational transition. The inspiration for the choice of the mutants came from a previous modelling study done on the bovine wild-type chymotrypsin. The results of the fluorescence stopped flow experiments show that several mutants behaved as was expected based on the information provided by the modeling study on the wild-type variant. For some mutants our assumptions were not correct, and therefore additional modeling studies of the activation pathways of these mutant proteins are necessary to be able to explain the observed kinetic behavior.
    MeSH term(s) Animals ; Cattle ; Chymotrypsin/chemistry ; Chymotrypsin/genetics ; Chymotrypsin/metabolism ; Enzyme Activation ; Fluorescence ; Hydrogen-Ion Concentration ; Kinetics ; Models, Molecular ; Mutation ; Protein Conformation ; Rats ; Structure-Activity Relationship ; Tryptophan/chemistry
    Chemical Substances Tryptophan (8DUH1N11BX) ; Chymotrypsin (EC 3.4.21.1) ; alpha-chymotrypsin (EC 3.4.21.1)
    Language English
    Publishing date 2004-09
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 1106283-6
    ISSN 1469-896X ; 0961-8368
    ISSN (online) 1469-896X
    ISSN 0961-8368
    DOI 10.1110/ps.04709604
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  8. Article ; Online: International Comparison of Enumeration-Based Quantification of DNA Copy-Concentration Using Flow Cytometric Counting and Digital Polymerase Chain Reaction.

    Yoo, Hee-Bong / Park, Sang-Ryoul / Dong, Lianhua / Wang, Jing / Sui, Zhiwei / Pavšič, Jernej / Milavec, Mojca / Akgoz, Muslum / Mozioğlu, Erkan / Corbisier, Philippe / Janka, Mátrai / Cosme, Bruno / de V Cavalcante, Janaina J / Flatshart, Roberto Becht / Burke, Daniel / Forbes-Smith, Michael / McLaughlin, Jacob / Emslie, Kerry / Whale, Alexandra S /
    Huggett, Jim F / Parkes, Helen / Kline, Margaret C / Harenza, Jo Lynne / Vallone, Peter M

    Analytical chemistry

    2016  Volume 88, Issue 24, Page(s) 12169–12176

    Abstract: Enumeration-based determination of DNA copy-concentration was assessed through an international comparison among national metrology institutes (NMIs) and designated institutes (DIs). Enumeration-based quantification does not require a calibration ... ...

    Abstract Enumeration-based determination of DNA copy-concentration was assessed through an international comparison among national metrology institutes (NMIs) and designated institutes (DIs). Enumeration-based quantification does not require a calibration standard thereby providing a route to "absolute quantification", which offers the potential for reliable value assignments of DNA reference materials, and International System of Units (SI) traceability to copy number 1 through accurate counting. In this study, 2 enumeration-based methods, flow cytometric (FCM) counting and the digital polymerase chain reaction (dPCR), were compared to quantify a solution of the pBR322 plasmid at a concentration of several thousand copies per microliter. In addition, 2 orthogonal chemical-analysis methods based on nucleotide quantification, isotope-dilution mass spectrometry (IDMS) and capillary electrophoresis (CE) were applied to quantify a more concentrated solution of the plasmid. Although 9 dPCR results from 8 laboratories showed some dispersion (relative standard deviation [RSD] = 11.8%), their means were closely aligned with those of the FCM-based counting method and the orthogonal chemical-analysis methods, corrected for gravimetric dilution factors. Using the means of dPCR results, the RSD of all 4 methods was 1.8%, which strongly supported the validity of the recent enumeration approaches. Despite a good overall agreement, the individual dPCR results were not sufficiently covered by the reported measurement uncertainties. These findings suggest that some laboratories may not have considered all factors contributing to the measurement uncertainty of dPCR, and further investigation of this possibility is warranted.
    MeSH term(s) DNA/analysis ; Electrophoresis, Capillary ; Flow Cytometry/methods ; Mass Spectrometry ; Nucleotides/analysis ; Plasmids/analysis ; Polymerase Chain Reaction/methods
    Chemical Substances Nucleotides ; DNA (9007-49-2)
    Language English
    Publishing date 2016-11-30
    Publishing country United States
    Document type Comparative Study ; Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 1508-8
    ISSN 1520-6882 ; 0003-2700
    ISSN (online) 1520-6882
    ISSN 0003-2700
    DOI 10.1021/acs.analchem.6b03076
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  9. Article ; Online: Hyperfunctional coagulation factor IX improves the efficacy of gene therapy in hemophilic mice.

    Cantore, Alessio / Nair, Nisha / Della Valle, Patrizia / Di Matteo, Mario / Màtrai, Janka / Sanvito, Francesca / Brombin, Chiara / Di Serio, Clelia / D'Angelo, Armando / Chuah, Marinee / Naldini, Luigi / Vandendriessche, Thierry

    Blood

    2012  Volume 120, Issue 23, Page(s) 4517–4520

    Abstract: Gene therapy may provide a cure for hemophilia and overcome the limitations of protein replacement therapy. Increasing the potency of gene transfer vectors may allow improvement of their therapeutic index, as lower doses can be administered to achieve ... ...

    Abstract Gene therapy may provide a cure for hemophilia and overcome the limitations of protein replacement therapy. Increasing the potency of gene transfer vectors may allow improvement of their therapeutic index, as lower doses can be administered to achieve therapeutic benefit, reducing toxicity of in vivo administration. Here we generated codon-usage optimized and hyperfunctional factor IX (FIX) transgenes carrying an R338L amino acid substitution (FIX Padua), previously associated with clotting hyperactivity and thrombophilia. We delivered these transgenes to hemophilia B mice by hepatocyte-targeted integration-competent and -defective lentiviral vectors. The hyperfunctional FIX transgenes increased FIX activity reconstituted in the plasma without detectable adverse effects, allowing correction of the disease phenotype at lower vector doses and resulting in improved hemostasis in vivo. The combined effect of codon optimization with the hyperactivating FIX-R338L mutation resulted in a robust 15-fold gain in potency and therefore provides a promising strategy to improve the efficacy, feasibility, and safety of hemophilia gene therapy.
    MeSH term(s) Amino Acid Substitution ; Animals ; Blood Coagulation/genetics ; Blood Coagulation/physiology ; Dogs ; Factor IX/genetics ; Factor IX/physiology ; Feasibility Studies ; Genetic Therapy/methods ; Genetic Vectors/genetics ; Hemophilia B/genetics ; Hemophilia B/therapy ; Humans ; Lentivirus/genetics ; Mice ; Mutation ; Partial Thromboplastin Time ; Treatment Outcome
    Chemical Substances Factor IX (9001-28-9)
    Language English
    Publishing date 2012-10-04
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 80069-7
    ISSN 1528-0020 ; 0006-4971
    ISSN (online) 1528-0020
    ISSN 0006-4971
    DOI 10.1182/blood-2012-05-432591
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  10. Article ; Online: An alternate sucrose binding mode in the E203Q Arabidopsis invertase mutant: an X-ray crystallography and docking study.

    Mátrai, Janka / Lammens, Willem / Jonckheer, Abel / Le Roy, Katrien / Rabijns, Anja / Van den Ende, Wim / De Maeyer, Marc

    Proteins

    2008  Volume 71, Issue 2, Page(s) 552–564

    Abstract: In the present study, we report on the X-ray crystallographic structure of a GH32 invertase mutant, (i.e., the Arabidopsis thaliana cell-wall invertase 1-E203Q, AtcwINV1-mutant) in complex with sucrose. This structure was solved to reveal the features of ...

    Abstract In the present study, we report on the X-ray crystallographic structure of a GH32 invertase mutant, (i.e., the Arabidopsis thaliana cell-wall invertase 1-E203Q, AtcwINV1-mutant) in complex with sucrose. This structure was solved to reveal the features of sugar binding in the catalytic pocket. However, as demonstrated by the X-ray structure the sugar binding and the catalytic pocket arrangement is significantly altered as compared with what was expected based on previous X-ray structures on GH-J clan enzymes. We performed a series of docking and molecular dynamics simulations on various derivatives of AtcwINV1 to reveal the reasons behind this modified sugar binding. Our results demonstrate that the E203Q mutation introduced into the catalytic pocket triggers conformational changes that alter the wild type substrate binding. In addition, this study also reveals the putative productive sucrose binding modus in the wild type enzyme.
    MeSH term(s) Amino Acid Substitution ; Arabidopsis/enzymology ; Computer Simulation ; Crystallization ; Crystallography, X-Ray ; Models, Molecular ; Protein Conformation/drug effects ; Sucrose/metabolism ; beta-Fructofuranosidase/chemistry ; beta-Fructofuranosidase/genetics ; beta-Fructofuranosidase/metabolism
    Chemical Substances Sucrose (57-50-1) ; beta-Fructofuranosidase (EC 3.2.1.26)
    Language English
    Publishing date 2008-05-01
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 806683-8
    ISSN 1097-0134 ; 0887-3585
    ISSN (online) 1097-0134
    ISSN 0887-3585
    DOI 10.1002/prot.21700
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