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Joseph Rosenecker - One of the best experts on this subject based on the ideXlab platform.
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Formation of solid lipid nanoparticle (SLN)-Gene Vector complexes for transfection of mammalian cells in vitro.
Cold Spring Harbor protocols, 2012Co-Authors: Carsten Rudolph, Joseph RoseneckerAbstract:Solid lipid nanoparticles (SLNs) offer several technological advantages over standard DNA carriers such as cationic lipids or cationic polymers. However, in the absence of endosomolytic agents such as chloroquine, Gene-transfer efficiency mediated by SLN-derived Gene Vectors consisting of optimized lipid composition remains lower compared to those achieved with standard transfection agents. This protocol describes the incorporation of a dimeric human immunodeficiency virus type-1 (HIV-1) TAT peptide into SLN Gene Vectors to increase Gene-transfer efficiency. This results in higher transfection rates than for standard transfection agents in vitro; the ternary SLN-Gene Vector complexes usually result in transfection levels equal to or higher than those observed with Gene Vector complexes formulated with branched polyethylenimine (PEI) 25 kDa. One significant advantage of using this method is the low cytotoxicity of the SLN Gene Vectors. The application of the Gene-transfer technique is limited to relatively low plasmid DNA (pDNA) concentrations of the resulting complexes (10 µg/mL). At higher concentrations, the particles tend to aggregate and precipitate. Therefore, their use for in vivo application, which Generally requires high pDNA concentrations, is limited.
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510. In Vivo-Gene Expression of Aerosolized PEI Gene Vector Complexes Is Solvent-Dependent
Molecular Therapy, 2004Co-Authors: Carsten Rudolph, Aurora Ortiz, Ulrike Schillinger, Christian Plank, Joseph RoseneckerAbstract:It has previously been shown that the in vitro -Gene transfer efficiency of nebulized polyethylenimine (PEI) 25 kDa based Gene Vector complexes was affected by the solvent (distilled water, glucose 5%, or hepes buffered saline (HBS)) which was used for complex formulation. When PEI polyplexes were formulated in distilled water neither the complex parameters such as particle size and zeta potential nor the transfection efficiency were markedly affected by the nebulization process using a standard jet nebulizer (PARI GmbH, Starnberg, Germany). These data suggested that water would be the best choice of solvent for the in vitro application. In this study we examined the effect of the solvent on PEI-mediated Gene transfer in vitro. PEI-based Gene Vector complexes (1 mg of pCMVLuc) were formulated either in distilled water, glucose 5%, HBS, or PBS at an N/P-ratio of N/P=10 using an optimized whole body nebulization device and luciferase Gene expression in the lungs was measured after 24 hours. Luciferase Gene expression mediated by complexes which were formulated in distilled water was 57- and 185-fold higher as compared with complexes formulated in 5% glucose or HBS, respectively. No luciferase Gene expression was observed when complexes were formulated in PBS. In contrast to complexes formulated in PBS which resulted in strong particle aggregation (847±142 nm), complexes formulated in distilled water (98±2 nm), glucose 5% (89±10 nm), or HBS (153±10 nm) were within the same particle size which could not explain the differences in Gene expression. In addition, solvent-dependent differences in Gene expression could not be explained by different deposition rates of Gene Vectors. Quantification of lung deposition of radioactively-labeled plasmid DNA did not result in a different pattern with respect to the solvent used. A possible reason to explain the solvent-dependent differences of Gene expression could be the effect of nebulized distilled water on the airway epithelium. Nebulized distilled water induces airway swelling, widening of the intercellular spaces, and increased cell permeability which could lead to facilitated uptake of the complexes by the airway epithelium. Such effects can be inhibited by pretreatment of mice with nebulized indomethacin. Indeed pretreatment of mice with nebulized indomethacin reduced Gene expression 2.9-fold. Histological analyses demonstrated deposition of FITC-labeled PEI Gene Vectors both in the alveolar epithelium and in the bronchial epithelium. In particular, Gene Vector uptake by alveolar macrophages was observed. Reporter Gene expression of β-galactosidase was detected in the epithelium of the large airways as well as in isolated alveolar macrophages. Taken together these data demonstrate that PEI-mediated aerosol Gene delivery is strongly solvent-dependent and distilled water represents the most efficient solvent for complex formulation. Increased cell permeability and paracellular diffusion which facilitates cellular Gene Vector uptake in the presence of water could be suggested as a possible mechanism for such solvent-dependent effect.
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Application of novel solid lipid nanoparticle (SLN)-Gene Vector formulations based on a dimeric HIV-1 TAT-peptide in vitro and in vivo.
Pharmaceutical research, 2004Co-Authors: Carsten Rudolph, Aurora Ortiz, Ulrike Schillinger, Christian Plank, Kerstin Tabatt, Rainer H. Müller, Joseph RoseneckerAbstract:Purpose. To optimize Gene delivery of SLN-based Gene Vectors by incorporation of a dimeric HIV-1 TAT peptide (TAT2) into SLN Gene Vectors. Methods. Plasmid DNA was complexed with two SLN preparations either with or without pre-compaction of DNA by TAT2, poly-l-arginine, or the mutant TAT2-M1. DNA complexed with polyethylenimine (PEI) served as a standard. Gene expression was analyzed upon transfection of bronchial epithelial cells in vitro and after intratracheal instillation or aerosol application to the lungs of mice in vivo. Stability of DNA was analyzed by agarose gel electrophoresis. Results. Incorporation of TAT2 into SLN Gene Vectors induced an up to 100-fold sequence-dependent increase of Gene expression as compared with the mutant TAT2-M1 and was 4- to 8-times higher as compared with PEI in vitro. In vivo application of TAT2-SLN Gene Vectors via jet nebulization increased SLN-based Gene expression but was accompanied with DNA degradation. DNA degradation was not observed when an innovative device operating on the principle of a perforated vibrating membrane was used. Conclusions. Incorporation of TAT2 into SLN Gene Vectors is suitable to optimize Gene transfer in vitro. The use of a mild nebulization technology avoids DNA degradation and offers the opportunity for further studies in large animal models.
David A. O'brochta - One of the best experts on this subject based on the ideXlab platform.
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Tn5 as an insect Gene Vector.
Insect biochemistry and molecular biology, 2004Co-Authors: Kathryn H Rowan, Peter W. Atkinson, Jamison Orsetti, David A. O'brochtaAbstract:The purpose of this study was to explore alternatives to insect-derived transposable elements as insect Gene Vectors with the intention of improving existing insect transGenesis methods. The mobility properties of the bacterial transposon, Tn5, were tested in mosquitoes using a transient transposable element mobility assay and by attempting to create transgenic insects. Tn5 synaptic complexes were assembled in vitro in the absence of Mg(2+) and co-injected with a target plasmid into developing yellow fever mosquito, Aedes aegypti, embryos. Target plasmids recovered from embryos a day later were screened for the presence of Tn5. Recombinants (transposition events) were found at a frequency of 1.2 x 10(-3). Some transposition events did not appear to be associated with canonical 9 bp direct duplications at the site of insertion and also were associated with either deletions or rearrangements. A Tn5 element containing the brain-specific transGene, 3 x P3DsRed, was assembled into synaptic complexes in vitro and injected into pre-blastoderm embryos of Ae. aegypti. Of the approximately 900 embryos surviving injection and developing into adults, two produced transgenic progeny. Both transgenic events involved the co-integrations of approximately five elements resulting in nested and tandem arrayed Tn5::3 x P3DsRed elements. This study extends the known host range of Tn5 to insects and makes available to insect biologists and others another eukaryotic genome-manipulation tool. The hyperactivity of synaptic complexes may be responsible for the unusual clustering of elements and managing this aspect of the element's behavior will be important in future applications of this technology to insects.
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Gene Vector and transposable element behavior in mosquitoes.
Journal of Experimental Biology, 2003Co-Authors: David A. O'brochta, Nagaraja Sethuraman, Raymond Wilson, Robert H. Hice, A. C. Pinkerton, Cynthia S. Levesque, Dennis K. Bideshi, Nijole Jasinskiene, Craig J. Coates, Anthony A. JamesAbstract:SUMMARY The development of efficient germ-line transformation technologies for mosquitoes has increased the ability of entomologists to find, isolate and analyze Genes. The utility of the currently available systems will be determined by a number of factors including the behavior of the Gene Vectors during the initial integration event and their behavior after chromosomal integration. Post-integration behavior will determine whether the transposable elements being employed currently as primary Gene Vectors will be useful as Gene-tagging and enhancer-trapping agents. The post-integration behavior of existing insect Vectors has not been extensively examined. Mos1 is useful as a primary germ-line transformation Vector in insects but is inefficiently remobilized in Drosophila melanogaster and Aedes aegypti. Hermes transforms D. melanogaster efficiently and can be remobilized in this species. This element is also useful for creating transgenic A. aegypti , but its mode of integration in mosquitoes results in the insertion of flanking plasmid DNA. Hermes can be remobilized in the soma of A. aegypti and transposes using a common cut-and-paste mechanism; however, the element does not remobilize in the germ line. piggyBac can be used to create transgenic mosquitoes and occasionally integrates using a mechanism other than a simple cut-and-paste mechanism. Preliminary data suggest that remobilization is infrequent. Minos also functions in mosquitoes and, like the other Gene Vectors, appears to remobilize inefficiently following integration. These results have implications for future Gene Vector development efforts and applications.
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Transformation of Stomoxys calcitrans with a Hermes Gene Vector.
Insect molecular biology, 2000Co-Authors: David A. O'brochta, Peter W. Atkinson, Michael J. LehaneAbstract:The ability of the Hermes transposable element to function as a germ line transformation Vector was tested in the stable fly, Stomoxys calcitrans. Plasmid-based transposable element mobility assays indicated moderate mobility of Hermes in this species. Germline transformants were created using a Hermes element containing the enhanced green fluorescent protein (EGFP) under the regulatory control of the promoter from Actin5C Gene of Drosophila melanogaster. Approximately 4% of the fifty-five adults that developed from the 1903 G(0) embryos injected with the Vector produced transgenic progeny. In the four transgenic lines established, the EGFP expression pattern was distinctly nonuniform and levels of expression were low. Promoters other than the one from the Actin5C Gene of D. melanogaster should be considered for widespread, constitutive expression. All transgenic lines contained multiple (2-4) integrated Hermes elements. Hermes integration events occurred through a canonical cut-and-paste mechanism.
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Transposable element interactions in insects: crossmobilization of hobo and Hermes.
Insect molecular biology, 1999Co-Authors: P. Sundararajan, Peter W. Atkinson, David A. O'brochtaAbstract:There are four non-drosophilid insect Gene Vector systems available that have been constructed from the short inverted repeat-type transposable elements Minos, piggyBac, mariner and Hermes. These elements (with the possible exception of piggyBac) are members of transposable element families that appear to be widespread in nature. Because these transposable element families are large it is possible that an insect species targeted for transformation will contain related transposable elements. The data presented here begin to address directly the question of interaction between diverged but related members of transposable element families. We tested the ability of the hAT elements hobo and Hermes to interact and cause crossmobilization. Using plasmid-based and chromosome-based element mobility assays we found that the terminal sequences of hobo and Hermes were almost equally good substrates for hobo transposase. However, this ability to crossmobilize was not reciprocal. Hermes transposase was only rarely able to cause the excision of hobo elements from plasmids and was never observed from germline chromosomes. These results have important implications for transgenic insect studies in the future.
Carsten Rudolph - One of the best experts on this subject based on the ideXlab platform.
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Formation of solid lipid nanoparticle (SLN)-Gene Vector complexes for transfection of mammalian cells in vitro.
Cold Spring Harbor protocols, 2012Co-Authors: Carsten Rudolph, Joseph RoseneckerAbstract:Solid lipid nanoparticles (SLNs) offer several technological advantages over standard DNA carriers such as cationic lipids or cationic polymers. However, in the absence of endosomolytic agents such as chloroquine, Gene-transfer efficiency mediated by SLN-derived Gene Vectors consisting of optimized lipid composition remains lower compared to those achieved with standard transfection agents. This protocol describes the incorporation of a dimeric human immunodeficiency virus type-1 (HIV-1) TAT peptide into SLN Gene Vectors to increase Gene-transfer efficiency. This results in higher transfection rates than for standard transfection agents in vitro; the ternary SLN-Gene Vector complexes usually result in transfection levels equal to or higher than those observed with Gene Vector complexes formulated with branched polyethylenimine (PEI) 25 kDa. One significant advantage of using this method is the low cytotoxicity of the SLN Gene Vectors. The application of the Gene-transfer technique is limited to relatively low plasmid DNA (pDNA) concentrations of the resulting complexes (10 µg/mL). At higher concentrations, the particles tend to aggregate and precipitate. Therefore, their use for in vivo application, which Generally requires high pDNA concentrations, is limited.
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510. In Vivo-Gene Expression of Aerosolized PEI Gene Vector Complexes Is Solvent-Dependent
Molecular Therapy, 2004Co-Authors: Carsten Rudolph, Aurora Ortiz, Ulrike Schillinger, Christian Plank, Joseph RoseneckerAbstract:It has previously been shown that the in vitro -Gene transfer efficiency of nebulized polyethylenimine (PEI) 25 kDa based Gene Vector complexes was affected by the solvent (distilled water, glucose 5%, or hepes buffered saline (HBS)) which was used for complex formulation. When PEI polyplexes were formulated in distilled water neither the complex parameters such as particle size and zeta potential nor the transfection efficiency were markedly affected by the nebulization process using a standard jet nebulizer (PARI GmbH, Starnberg, Germany). These data suggested that water would be the best choice of solvent for the in vitro application. In this study we examined the effect of the solvent on PEI-mediated Gene transfer in vitro. PEI-based Gene Vector complexes (1 mg of pCMVLuc) were formulated either in distilled water, glucose 5%, HBS, or PBS at an N/P-ratio of N/P=10 using an optimized whole body nebulization device and luciferase Gene expression in the lungs was measured after 24 hours. Luciferase Gene expression mediated by complexes which were formulated in distilled water was 57- and 185-fold higher as compared with complexes formulated in 5% glucose or HBS, respectively. No luciferase Gene expression was observed when complexes were formulated in PBS. In contrast to complexes formulated in PBS which resulted in strong particle aggregation (847±142 nm), complexes formulated in distilled water (98±2 nm), glucose 5% (89±10 nm), or HBS (153±10 nm) were within the same particle size which could not explain the differences in Gene expression. In addition, solvent-dependent differences in Gene expression could not be explained by different deposition rates of Gene Vectors. Quantification of lung deposition of radioactively-labeled plasmid DNA did not result in a different pattern with respect to the solvent used. A possible reason to explain the solvent-dependent differences of Gene expression could be the effect of nebulized distilled water on the airway epithelium. Nebulized distilled water induces airway swelling, widening of the intercellular spaces, and increased cell permeability which could lead to facilitated uptake of the complexes by the airway epithelium. Such effects can be inhibited by pretreatment of mice with nebulized indomethacin. Indeed pretreatment of mice with nebulized indomethacin reduced Gene expression 2.9-fold. Histological analyses demonstrated deposition of FITC-labeled PEI Gene Vectors both in the alveolar epithelium and in the bronchial epithelium. In particular, Gene Vector uptake by alveolar macrophages was observed. Reporter Gene expression of β-galactosidase was detected in the epithelium of the large airways as well as in isolated alveolar macrophages. Taken together these data demonstrate that PEI-mediated aerosol Gene delivery is strongly solvent-dependent and distilled water represents the most efficient solvent for complex formulation. Increased cell permeability and paracellular diffusion which facilitates cellular Gene Vector uptake in the presence of water could be suggested as a possible mechanism for such solvent-dependent effect.
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Application of novel solid lipid nanoparticle (SLN)-Gene Vector formulations based on a dimeric HIV-1 TAT-peptide in vitro and in vivo.
Pharmaceutical research, 2004Co-Authors: Carsten Rudolph, Aurora Ortiz, Ulrike Schillinger, Christian Plank, Kerstin Tabatt, Rainer H. Müller, Joseph RoseneckerAbstract:Purpose. To optimize Gene delivery of SLN-based Gene Vectors by incorporation of a dimeric HIV-1 TAT peptide (TAT2) into SLN Gene Vectors. Methods. Plasmid DNA was complexed with two SLN preparations either with or without pre-compaction of DNA by TAT2, poly-l-arginine, or the mutant TAT2-M1. DNA complexed with polyethylenimine (PEI) served as a standard. Gene expression was analyzed upon transfection of bronchial epithelial cells in vitro and after intratracheal instillation or aerosol application to the lungs of mice in vivo. Stability of DNA was analyzed by agarose gel electrophoresis. Results. Incorporation of TAT2 into SLN Gene Vectors induced an up to 100-fold sequence-dependent increase of Gene expression as compared with the mutant TAT2-M1 and was 4- to 8-times higher as compared with PEI in vitro. In vivo application of TAT2-SLN Gene Vectors via jet nebulization increased SLN-based Gene expression but was accompanied with DNA degradation. DNA degradation was not observed when an innovative device operating on the principle of a perforated vibrating membrane was used. Conclusions. Incorporation of TAT2 into SLN Gene Vectors is suitable to optimize Gene transfer in vitro. The use of a mild nebulization technology avoids DNA degradation and offers the opportunity for further studies in large animal models.
Kenneth Cornetta - One of the best experts on this subject based on the ideXlab platform.
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The National Gene Vector Biorepository: Eleven Years of Providing Resources to the Gene Therapy Community.
Human gene therapy, 2020Co-Authors: Kenneth Cornetta, Lorraine Matheson, Ryan Long, Lisa DuffyAbstract:The National Gene Vector Biorepository (NGVB) program has been highly accessed by Gene therapy investigators. The reagent repository has distributed over 1,000 reagents to 397 investigators. The Ph...
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screening clinical cell products for replication competent retrovirus the national Gene Vector biorepository experience
Molecular therapy. Methods & clinical development, 2018Co-Authors: Kenneth Cornetta, Lisa Duffy, Steven A Feldman, Crystal L Mackall, Marco L Davila, Kevin J Curran, Richard P Junghans, Jean Yuh Tang, James N Kochenderfer, Roisin E OcearbhaillAbstract:Replication-competent retrovirus (RCR) is a safety concern for individuals treated with retroviral Gene therapy. RCR detection assays are used to detect RCR in manufactured Vector, transduced cell products infused into research subjects, and in the research subjects after treatment. In this study, we reviewed 286 control (n = 4) and transduced cell products (n = 282) screened for RCR in the National Gene Vector Biorepository. The transduced cell samples were submitted from 14 clinical trials. All Vector products were previously shown to be negative for RCR prior to use in cell transduction. After transduction, all 282 transduced cell products were negative for RCR. In addition, 241 of the clinical trial participants were also screened for RCR by analyzing peripheral blood at least 1 month after infusion, all of which were also negative for evidence of RCR infection. The majority of Vector products used in the clinical trials were Generated in the PG13 packaging cell line. The findings suggest that screening of the retroviral Vector product Generated in PG13 cell line may be sufficient and that further screening of transduced cells does not provide added value.
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Screening Clinical Cell Products for Replication Competent Retrovirus: The National Gene Vector Biorepository Experience
Elsevier, 2018Co-Authors: Kenneth Cornetta, Lisa Duffy, Steven A Feldman, Crystal L Mackall, Marco L Davila, Kevin J Curran, Richard P Junghans, Jean Yuh Tang, James N Kochenderfer, Roisin O’cearbhaillAbstract:Replication-competent retrovirus (RCR) is a safety concern for individuals treated with retroviral Gene therapy. RCR detection assays are used to detect RCR in manufactured Vector, transduced cell products infused into research subjects, and in the research subjects after treatment. In this study, we reviewed 286 control (n = 4) and transduced cell products (n = 282) screened for RCR in the National Gene Vector Biorepository. The transduced cell samples were submitted from 14 clinical trials. All Vector products were previously shown to be negative for RCR prior to use in cell transduction. After transduction, all 282 transduced cell products were negative for RCR. In addition, 241 of the clinical trial participants were also screened for RCR by analyzing peripheral blood at least 1 month after infusion, all of which were also negative for evidence of RCR infection. The majority of Vector products used in the clinical trials were Generated in the PG13 packaging cell line. The findings suggest that screening of the retroviral Vector product Generated in PG13 cell line may be sufficient and that further screening of transduced cells does not provide added value. Keywords: retrovirus, safety testing, replicating virus, lentiviru
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The National Gene Vector Biorepository's Pharm/Tox Database
Molecular therapy : the journal of the American Society of Gene Therapy, 2009Co-Authors: Kenneth Cornetta, Lorraine MathesonAbstract:The National Center for Research Resources has funded a new program to support preclinical and clinical Gene therapy efforts. The National Gene Vector Biorepository (NGVB) will bring a variety of programs online during the next year. One of the resources will be a continuation of the Pharmacology/Toxicology (Pharm/Tox) Database formerly maintained by the National Gene Vector Laboratory. The purpose of the database is to provide Gene therapy investigators with a catalog of Gene therapy biodistribution and toxicology studies on file with the US Food and Drug Administration (FDA).
Lisa Duffy - One of the best experts on this subject based on the ideXlab platform.
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The National Gene Vector Biorepository: Eleven Years of Providing Resources to the Gene Therapy Community.
Human gene therapy, 2020Co-Authors: Kenneth Cornetta, Lorraine Matheson, Ryan Long, Lisa DuffyAbstract:The National Gene Vector Biorepository (NGVB) program has been highly accessed by Gene therapy investigators. The reagent repository has distributed over 1,000 reagents to 397 investigators. The Ph...
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screening clinical cell products for replication competent retrovirus the national Gene Vector biorepository experience
Molecular therapy. Methods & clinical development, 2018Co-Authors: Kenneth Cornetta, Lisa Duffy, Steven A Feldman, Crystal L Mackall, Marco L Davila, Kevin J Curran, Richard P Junghans, Jean Yuh Tang, James N Kochenderfer, Roisin E OcearbhaillAbstract:Replication-competent retrovirus (RCR) is a safety concern for individuals treated with retroviral Gene therapy. RCR detection assays are used to detect RCR in manufactured Vector, transduced cell products infused into research subjects, and in the research subjects after treatment. In this study, we reviewed 286 control (n = 4) and transduced cell products (n = 282) screened for RCR in the National Gene Vector Biorepository. The transduced cell samples were submitted from 14 clinical trials. All Vector products were previously shown to be negative for RCR prior to use in cell transduction. After transduction, all 282 transduced cell products were negative for RCR. In addition, 241 of the clinical trial participants were also screened for RCR by analyzing peripheral blood at least 1 month after infusion, all of which were also negative for evidence of RCR infection. The majority of Vector products used in the clinical trials were Generated in the PG13 packaging cell line. The findings suggest that screening of the retroviral Vector product Generated in PG13 cell line may be sufficient and that further screening of transduced cells does not provide added value.
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Screening Clinical Cell Products for Replication Competent Retrovirus: The National Gene Vector Biorepository Experience
Elsevier, 2018Co-Authors: Kenneth Cornetta, Lisa Duffy, Steven A Feldman, Crystal L Mackall, Marco L Davila, Kevin J Curran, Richard P Junghans, Jean Yuh Tang, James N Kochenderfer, Roisin O’cearbhaillAbstract:Replication-competent retrovirus (RCR) is a safety concern for individuals treated with retroviral Gene therapy. RCR detection assays are used to detect RCR in manufactured Vector, transduced cell products infused into research subjects, and in the research subjects after treatment. In this study, we reviewed 286 control (n = 4) and transduced cell products (n = 282) screened for RCR in the National Gene Vector Biorepository. The transduced cell samples were submitted from 14 clinical trials. All Vector products were previously shown to be negative for RCR prior to use in cell transduction. After transduction, all 282 transduced cell products were negative for RCR. In addition, 241 of the clinical trial participants were also screened for RCR by analyzing peripheral blood at least 1 month after infusion, all of which were also negative for evidence of RCR infection. The majority of Vector products used in the clinical trials were Generated in the PG13 packaging cell line. The findings suggest that screening of the retroviral Vector product Generated in PG13 cell line may be sufficient and that further screening of transduced cells does not provide added value. Keywords: retrovirus, safety testing, replicating virus, lentiviru